**To Grow, Stop or Die? – Novel Tumor-Suppressive Mechanism Regulated by the Transcription Factor E2F**

Eiko Ozono, Shoji Yamaoka and Kiyoshi Ohtani

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54510

## **1. Introduction**

Proliferation of mammalian cells is strictly regulated by growth stimulation. Cell prolifera‐ tion is stimulated not only by normal growth stimulation but also by abnormal growth stim‐ ulation originated from oncogenic changes. Such abnormal growth stimulation leads to tumorigenesis, if not properly guarded by appropriate cellular response. Cells are endowed with intrinsic tumor suppressor pathways to protect cells from tumorigenesis upon such on‐ cogenic threat [1]. The tumor suppressor pathways halt cell proliferation either by restrain‐ ing cell cycle progression or by inducing apoptosis (programmed cell death) in case of being unable to stop aberrant cell cycle progression. Consequently, the cell-fate, whether to grow, stop growing or die, is dependent on the balance between growth-promoting effects origi‐ nated from oncogenic changes and growth-suppressive effects mediated by the tumor sup‐ pressor pathways upon oncogenic changes (Figure 1). When the tumor suppressor pathways are disabled by further oncogenic changes, the balance of cell-fate determination shifts from growth suppression to proliferation, and cells start deregulated proliferation, leading to tumorigenesis. Among the intrinsic tumor suppressor pathways, two major path‐ ways are the RB pathway and the p53 pathway. Both pathways are important for induction of cell cycle arrest or apoptosis [2]. In addition, accumulating evidence indicates that the tu‐ mor suppressor TAp73, a member of the p53 family, also plays crucial roles in tumor sup‐ pression by inducing apoptosis independent of p53 [3, 4].

The transcription factor E2F, the main target of the RB pathway, plays crucial roles in cell cycle progression by activating growth-promoting genes [5]. In this regard, E2F is thought to mediate growth-promoting effects originating from normal growth stimulation and oncogenic changes. Supporting this notion, E2F could be an oncoprotein [6]. On the other hand, recent studies indi‐ cate that E2F also plays crucial roles in activation of the major intrinsic tumor suppressor path‐

© 2013 Ozono et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Ozono et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ways by sensing oncogenic changes, halting cell proliferation by inducing cell cycle arrest or apoptosis. Supporting this notion, E2F could also be a tumor suppressor [7]. Taken together, these observations indicate that E2F is located at the center of the balance between cell prolifera‐ tion and cell cycle arrest or apoptosis, determining the cell-fate upon oncogenic changes (Figure 1). E2F can be regarded as a double-edged sword in cell growth control. In this chapter, we will describe the major intrinsic tumor suppressor pathways and activation of the tumor suppressor pathways by E2F upon oncogenic changes. We will focus on how E2F differentially regulates expression of target genes upon normal growth stimulation and oncogenic changes that have completely opposite roles in cell-fate determination, to grow, stop or die.

leading to expression of E2F target genes by releasing them from suppression by RB during G1 to S phase cell cycle progression (Figure 2) [13]. In contrast, growth-suppressive signals such as contact inhibition and DNA damage induce expression of CDK inhibitors, which are called brakes in cell cycle progression owing to their ability to inhibit activity of CDKs. Suppression of CDKs keeps RB in hypo-phosphorylated form, which binds to and inhibits E2F. Consequently, the activity of E2F to activate growth-promoting genes is controlled by the activity of RB, which is regulated by CDKs and CDK inhibitors. The pathway converging to RB, including CDKs and

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Whether a cell progresses one round of the cell cycle or not is determined at the restriction point, which is located at late G1 phase of the cell cycle. Once a cell passed through the re‐ striction point, it is programmed that the cell cycle automatically proceeds to the end of M phase. Thus, whether a cell proliferates or not is determined by whether the cell passes through the restriction point or not. Two major determinants whether a cell passes through the restriction point or not are E2F activity and cyclin dependent kinase activity, which is induced by E2F through activation of the *Cyclin E* (*CycE*) gene [14]. Since E2F plays essential roles in passing through the restriction point, RB can be regarded as a gatekeeper in cell cy‐ cle progression by controlling E2F activity. Hence disruption of the RB pathway and conse‐ quent activation of E2F is thought to be an essential event for tumorigenesis [2]. Actually, deletion or mutation of the *RB1* gene is observed in about 30% of cancers. Moreover, defects in the RB pathway upstream of RB such as overexpression of CycD and dysfunction of CDK inhibitors such as p16INK4a are frequently observed in other cancers retaining *RB1* [2]. It is

The RB pathway plays essential roles in tumor suppression by inducing cell cycle arrest or cellular senescence through suppression of the activity of E2F. As described below, disrup‐ tion of the RB pathway reinforces cell cycle arrest by induction of CDK inhibitor p21Cip1 ex‐ pression through the p53 pathway or p27Kip1 expression. When cells failed to induce cell cycle arrest, apoptosis is triggered through the p53 pathway or TAp73, a p53 family mem‐

predicted that all cancers have at least some defect in the RB pathway.

**Figure 2.** The regulatory mechanism of the transcription factor E2F by the RB pathway.

ber, which activates various pro-apoptotic genes.

CDK inhibitors, is referred to the RB pathway.

**Figure 1.** To grow, stop or die? The balance between the activity of growth-promoting pathways and tumor suppressor pathways determines the cell fate upon oncogenic changes. E2F is located at the center of the balance (see also Figure 6).

## **2. Major tumor-suppressor pathways**

## **2.1. The RB pathway (CDK inhibitor–Cyc/CDK–RB)**

The retinoblastoma gene (*RB1*) is the first identified tumor suppressor gene [8]. Individuals with heterozygous deletion or mutation of the *RB1* gene are susceptible to retinoblastoma in early life by additional deletion or mutation of the other allele. The *RB1* gene product pRB, to‐ gether with its relatives, p107 and p130, comprises a family of pocket proteins [9, 10] (Hereafter we refer all pocket proteins to RB, and the *RB1* gene product to pRB). The main target of RB is the transcription factor E2F, which plays central role in cell proliferation by activating growth-pro‐ moting genes including those required for DNA replication and cell cycle progression [9]. In quiescent phase, RB binds to E2F and suppresses transcriptional activity of E2F. Moreover, RB recruits various chromatin-modifying factors, such as histone deacetylases (HDACs) [11] and histone methyl transferase SUV39H1 [12] to actively suppress expression of E2F target genes. Hence the main role of RB in tumor suppression is thought to be suppression of cell prolifera‐ tion through suppression of E2F target gene expression. Growth stimulation induces expres‐ sion of cyclins and activates cyclin dependent kinases (CDKs), which are called accelerators and engines in cell cycle progression, respectively. CDKs, in turn, inactivate RB by phosphorylation, leading to expression of E2F target genes by releasing them from suppression by RB during G1 to S phase cell cycle progression (Figure 2) [13]. In contrast, growth-suppressive signals such as contact inhibition and DNA damage induce expression of CDK inhibitors, which are called brakes in cell cycle progression owing to their ability to inhibit activity of CDKs. Suppression of CDKs keeps RB in hypo-phosphorylated form, which binds to and inhibits E2F. Consequently, the activity of E2F to activate growth-promoting genes is controlled by the activity of RB, which is regulated by CDKs and CDK inhibitors. The pathway converging to RB, including CDKs and CDK inhibitors, is referred to the RB pathway.

ways by sensing oncogenic changes, halting cell proliferation by inducing cell cycle arrest or apoptosis. Supporting this notion, E2F could also be a tumor suppressor [7]. Taken together, these observations indicate that E2F is located at the center of the balance between cell prolifera‐ tion and cell cycle arrest or apoptosis, determining the cell-fate upon oncogenic changes (Figure 1). E2F can be regarded as a double-edged sword in cell growth control. In this chapter, we will describe the major intrinsic tumor suppressor pathways and activation of the tumor suppressor pathways by E2F upon oncogenic changes. We will focus on how E2F differentially regulates expression of target genes upon normal growth stimulation and oncogenic changes that have

**Figure 1.** To grow, stop or die? The balance between the activity of growth-promoting pathways and tumor suppressor pathways determines the cell fate upon oncogenic changes. E2F is located at the center of the balance (see also Figure 6).

The retinoblastoma gene (*RB1*) is the first identified tumor suppressor gene [8]. Individuals with heterozygous deletion or mutation of the *RB1* gene are susceptible to retinoblastoma in early life by additional deletion or mutation of the other allele. The *RB1* gene product pRB, to‐ gether with its relatives, p107 and p130, comprises a family of pocket proteins [9, 10] (Hereafter we refer all pocket proteins to RB, and the *RB1* gene product to pRB). The main target of RB is the transcription factor E2F, which plays central role in cell proliferation by activating growth-pro‐ moting genes including those required for DNA replication and cell cycle progression [9]. In quiescent phase, RB binds to E2F and suppresses transcriptional activity of E2F. Moreover, RB recruits various chromatin-modifying factors, such as histone deacetylases (HDACs) [11] and histone methyl transferase SUV39H1 [12] to actively suppress expression of E2F target genes. Hence the main role of RB in tumor suppression is thought to be suppression of cell prolifera‐ tion through suppression of E2F target gene expression. Growth stimulation induces expres‐ sion of cyclins and activates cyclin dependent kinases (CDKs), which are called accelerators and engines in cell cycle progression, respectively. CDKs, in turn, inactivate RB by phosphorylation,

completely opposite roles in cell-fate determination, to grow, stop or die.

**2. Major tumor-suppressor pathways**

18 Future Aspects of Tumor Suppressor Gene

**2.1. The RB pathway (CDK inhibitor–Cyc/CDK–RB)**

Whether a cell progresses one round of the cell cycle or not is determined at the restriction point, which is located at late G1 phase of the cell cycle. Once a cell passed through the re‐ striction point, it is programmed that the cell cycle automatically proceeds to the end of M phase. Thus, whether a cell proliferates or not is determined by whether the cell passes through the restriction point or not. Two major determinants whether a cell passes through the restriction point or not are E2F activity and cyclin dependent kinase activity, which is induced by E2F through activation of the *Cyclin E* (*CycE*) gene [14]. Since E2F plays essential roles in passing through the restriction point, RB can be regarded as a gatekeeper in cell cy‐ cle progression by controlling E2F activity. Hence disruption of the RB pathway and conse‐ quent activation of E2F is thought to be an essential event for tumorigenesis [2]. Actually, deletion or mutation of the *RB1* gene is observed in about 30% of cancers. Moreover, defects in the RB pathway upstream of RB such as overexpression of CycD and dysfunction of CDK inhibitors such as p16INK4a are frequently observed in other cancers retaining *RB1* [2]. It is predicted that all cancers have at least some defect in the RB pathway.

The RB pathway plays essential roles in tumor suppression by inducing cell cycle arrest or cellular senescence through suppression of the activity of E2F. As described below, disrup‐ tion of the RB pathway reinforces cell cycle arrest by induction of CDK inhibitor p21Cip1 ex‐ pression through the p53 pathway or p27Kip1 expression. When cells failed to induce cell cycle arrest, apoptosis is triggered through the p53 pathway or TAp73, a p53 family mem‐ ber, which activates various pro-apoptotic genes.

**Figure 2.** The regulatory mechanism of the transcription factor E2F by the RB pathway.

## **2.2. The p53 pathway (ARF–p53–cell cycle arrest or apoptosis related effectors)**

The tumor suppressor p53 is a transcription factor that is activated by a variety of stress sig‐ nals, including DNA damage, hypoxia and various oncogenic changes including aberrant activation of E2F [15]. In response to such stress signals, p53 induces either cell cycle arrest or apoptosis. Cell cycle arrest is mainly mediated through activation of the CDK inhibitor *p21Cip1* gene [16], whose product suppresses wide range of CDKs. Apoptosis is mainly medi‐ ated through activation of the *Bax* and BH3 only family genes, whose products destabilize mitochondrial membrane to facilitate cytochrome c release, which triggers apoptotic cas‐ cades of caspase activation.

partly overlap with those of p53, such as the *PUMA* and *NOXA* genes [28, 29] that are cru‐ cial for induction of apoptosis. Moreover, TAp73 can induce apoptosis in the absence of p53 [3, 4]. Therefore, TAp73 seems to back-up the important tumor suppressive function of p53.

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The transcription factor E2F was originally identified as a cellular DNA-binding protein, which mediate E1A-dependent activation of the adenovirus E2 promoter [30]. Members of E2F family are downstream targets of the tumor suppressor RB and make repressor com‐ plexes with RB that keep cells in quiescent state [9, 10]. Though E2F plays central roles in cell proliferation by activating growth-promoting genes, certain members of E2F can induce apoptosis [15]. In this paragraph, we will describe three points as follows (1) E2F family members, the role of them in cell cycle progression, cell cycle arrest and apoptosis. (2) Regu‐ latory mechanism of E2F. (3) E2F target genes, to better understand the regulatory mecha‐

E2F consists of eight family members (E2F1-E2F8). E2F1-E2F5 are bound by their repressor RB family proteins through RB binding domain. E2F1-E2F5 activate transcription when free

**Figure 3.** E2F activates major intrinsic tumor suppressor pathways.

nism of cell-fate determination mediated through E2F.

**3. The transcription factor E2F**

**3.1. E2F family members**

Since p53 plays crucial roles in induction of cell cycle arrest or apoptosis, the expression level of p53 is kept low by rapid ubiquitin/proteasome-dependent degradation, mainly caused by Mdm2 (mouse double minute 2, Hdm2 in humans), which is often overex‐ pressed in many cancers [15]. Mdm2 is E3 ubiquitin ligase, which directly binds to p53 and promotes p53 degradation. Mdm2 also inhibits *TP53* mRNA translation [17]. The *Mdm2* gene is a target of p53, forming a negative-feedback loop to control the level of p53 [18]. Another negative regulator of p53 is MdmX, also known as Mdm4. MdmX has recently emerged as a discrete critical negative regulator of p53 [19]. Though MdmX is not a direct target of p53, structure of MdmX is significantly similar to that of Mdm2. MdmX is reported to enhance p53 ubiquitination by altering the substrate preference of the Mdm2, thereby indirectly regulating p53 [20].

Regarding response to oncogenic stresses, a potent activator of p53 is the tumor suppressor ARF (alternative reading frame, known as p14 in humans and as p19 in rodents) [2]. ARF directly binds to and sequesters Mdm2 to nucleoli, stabilizing p53 that leads to the expres‐ sion of its target genes [21]. The pathway including upstream and downstream of p53 is re‐ ferred to the p53 pathway. Of note, the *ARF* gene is a direct target of E2F [22] and this E2F-ARF interaction connects the RB pathway and the p53 pathway, enabling efficient tumorsuppressive response. When the RB pathway is disrupted by oncogenic changes, E2F is activated to induce ARF gene expression, leading to activation of the p53 pathway, which inhibits oncogenic cell growth by inducing cell cycle arrest or apoptosis (Figure 3). There‐ fore, disruption of both of the tumor suppressor pathways powerfully shifts the cell-fate de‐ termination balance to proliferation (Figure 1) and is thought to be essential to induce deregulated cell proliferation that leads to tumorigenesis. Indeed, about 50% of cancers car‐ ry *TP53* mutations or deletion and most cancers have defects in the p53 pathway including those in upstream and downstream of p53.

## **2.3. The TAp73 pathway (E2F–TAp73–pro-apoptotic targets)**

The tumor suppressor TAp73 is a homologue of p53 and can induce apoptosis independent‐ ly of p53 [3, 4, 23]. The *TP73* gene encodes two isoforms, TAp73 and DNp73, which are driv‐ en by different promoters. DNp73 lacks the transactivation (TA) domain and counteracts TAp73 and p53 [24]. Thus DNp73 is anti-apoptotic. The *TAp73* gene is thought to be a tumor suppressor gene and is known to be a target of E2F1 [3, 4, 25]. Similarly to p53, TAp73 is activated by both oncogenic changes and DNA damage [26, 27] and TAp73 target genes partly overlap with those of p53, such as the *PUMA* and *NOXA* genes [28, 29] that are cru‐ cial for induction of apoptosis. Moreover, TAp73 can induce apoptosis in the absence of p53 [3, 4]. Therefore, TAp73 seems to back-up the important tumor suppressive function of p53.

**Figure 3.** E2F activates major intrinsic tumor suppressor pathways.

## **3. The transcription factor E2F**

**2.2. The p53 pathway (ARF–p53–cell cycle arrest or apoptosis related effectors)**

cades of caspase activation.

20 Future Aspects of Tumor Suppressor Gene

the Mdm2, thereby indirectly regulating p53 [20].

those in upstream and downstream of p53.

**2.3. The TAp73 pathway (E2F–TAp73–pro-apoptotic targets)**

The tumor suppressor p53 is a transcription factor that is activated by a variety of stress sig‐ nals, including DNA damage, hypoxia and various oncogenic changes including aberrant activation of E2F [15]. In response to such stress signals, p53 induces either cell cycle arrest or apoptosis. Cell cycle arrest is mainly mediated through activation of the CDK inhibitor *p21Cip1* gene [16], whose product suppresses wide range of CDKs. Apoptosis is mainly medi‐ ated through activation of the *Bax* and BH3 only family genes, whose products destabilize mitochondrial membrane to facilitate cytochrome c release, which triggers apoptotic cas‐

Since p53 plays crucial roles in induction of cell cycle arrest or apoptosis, the expression level of p53 is kept low by rapid ubiquitin/proteasome-dependent degradation, mainly caused by Mdm2 (mouse double minute 2, Hdm2 in humans), which is often overex‐ pressed in many cancers [15]. Mdm2 is E3 ubiquitin ligase, which directly binds to p53 and promotes p53 degradation. Mdm2 also inhibits *TP53* mRNA translation [17]. The *Mdm2* gene is a target of p53, forming a negative-feedback loop to control the level of p53 [18]. Another negative regulator of p53 is MdmX, also known as Mdm4. MdmX has recently emerged as a discrete critical negative regulator of p53 [19]. Though MdmX is not a direct target of p53, structure of MdmX is significantly similar to that of Mdm2. MdmX is reported to enhance p53 ubiquitination by altering the substrate preference of

Regarding response to oncogenic stresses, a potent activator of p53 is the tumor suppressor ARF (alternative reading frame, known as p14 in humans and as p19 in rodents) [2]. ARF directly binds to and sequesters Mdm2 to nucleoli, stabilizing p53 that leads to the expres‐ sion of its target genes [21]. The pathway including upstream and downstream of p53 is re‐ ferred to the p53 pathway. Of note, the *ARF* gene is a direct target of E2F [22] and this E2F-ARF interaction connects the RB pathway and the p53 pathway, enabling efficient tumorsuppressive response. When the RB pathway is disrupted by oncogenic changes, E2F is activated to induce ARF gene expression, leading to activation of the p53 pathway, which inhibits oncogenic cell growth by inducing cell cycle arrest or apoptosis (Figure 3). There‐ fore, disruption of both of the tumor suppressor pathways powerfully shifts the cell-fate de‐ termination balance to proliferation (Figure 1) and is thought to be essential to induce deregulated cell proliferation that leads to tumorigenesis. Indeed, about 50% of cancers car‐ ry *TP53* mutations or deletion and most cancers have defects in the p53 pathway including

The tumor suppressor TAp73 is a homologue of p53 and can induce apoptosis independent‐ ly of p53 [3, 4, 23]. The *TP73* gene encodes two isoforms, TAp73 and DNp73, which are driv‐ en by different promoters. DNp73 lacks the transactivation (TA) domain and counteracts TAp73 and p53 [24]. Thus DNp73 is anti-apoptotic. The *TAp73* gene is thought to be a tumor suppressor gene and is known to be a target of E2F1 [3, 4, 25]. Similarly to p53, TAp73 is activated by both oncogenic changes and DNA damage [26, 27] and TAp73 target genes

The transcription factor E2F was originally identified as a cellular DNA-binding protein, which mediate E1A-dependent activation of the adenovirus E2 promoter [30]. Members of E2F family are downstream targets of the tumor suppressor RB and make repressor com‐ plexes with RB that keep cells in quiescent state [9, 10]. Though E2F plays central roles in cell proliferation by activating growth-promoting genes, certain members of E2F can induce apoptosis [15]. In this paragraph, we will describe three points as follows (1) E2F family members, the role of them in cell cycle progression, cell cycle arrest and apoptosis. (2) Regu‐ latory mechanism of E2F. (3) E2F target genes, to better understand the regulatory mecha‐ nism of cell-fate determination mediated through E2F.

#### **3.1. E2F family members**

E2F consists of eight family members (E2F1-E2F8). E2F1-E2F5 are bound by their repressor RB family proteins through RB binding domain. E2F1-E2F5 activate transcription when free from RB and repress transcription when bound by RB. E2F1-E2F3a are induced at G1/S boundary and activate transcription free from RB. In contrast, E2F3b-E2F5 are expressed all through the cell cycle and play main roles in transcriptional repression in G0/G1 bound by RB. E2F6-E2F8 repress transcription independently of RB. Hence E2F family members are divided into two groups: activator E2Fs (E2F1-E2F3a) and repressor E2Fs (E2F3b-E2F8). Ac‐ tivator E2Fs play major roles in activation of target genes involved in growth promotion, growth suppression and induction of apoptosis. Repressor E2Fs are roughly divided into two groups; E2F3b-E2F5, which make repressor complex together with RB, and recently identified E2F6-E2F8, which function independently of RB.

senescence by activating the ARF-p53 pathway, when overexpressed in human normal fi‐ broblasts [36]. Moreover, E2F1 null mice resulted in tumorigenesis [7]. Taken together, E2F1 seems to play the most important roles in tumor suppression among activator E2Fs by acti‐

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E2F2 has 46% overall amino acid sequence similarity to E2F1 [37] and is thought to play roles in both cell proliferation and tumor suppression. E2F2 null mice exhibit increased pro‐ liferation of hematopoietic cells and frequently develop autoimmunity and tumors [38, 39]. In tumor suppression, E2F2 plays major roles in suppression of Myc-induced T cell lympho‐ magenesis. Inactivation of neither E2F1 nor E2F3 had no effect on tumor progression in T

E2F3 is thought to be the most important activator E2F in cell proliferation. Although E2F1 or E2F2 null mice are viable and tumor-prone, E2F3 null mice are typically embryonic lethal in pure background [41] or show partially penetrant embryonic lethality in mixed back‐ ground [42]. Although mouse embryonic fibroblasts (MEFs) with combined knockout of E2F1 and E2F2 proliferate, those with combined knockout of E2F1, E2F2 and E2F3 fail to proliferate and can not re-enter the cell cycle [5]. The E2F3 locus encodes two isoforms, E2F3a and E2F3b, a truncated variant of E2F3 in its N-terminus [43, 44]. Although both E2F3s partly overlaps their roles in cell cycle progression [45], E2F3a is expressed in G1/S to S phases and is thought to play crucial roles on cell proliferation, while E2F3b is expressed equivalently in quiescent and proliferating cells, and associates with pRB, representing the predominant E2F-pRB complex in quiescent cells [44, 46]. E2F3b also plays roles in myogen‐

**Figure 5.** Roles of activator E2Fs in cell-fate determination. E2F1 plays crucial roles in induction of apoptosis. E2F3 is thought be essential for cell proliferation. It is predicted that the character of E2F2 is in the middle of E2F1 and E2F3.

ic differentiation by promoting gene expression related to differentiation [47].

vating genes involved in apoptosis and cell cycle arrest.

cells, and only loss of E2F2 accelerated lymphomagenesis [40].

**Figure 4.** Structure of E2F family members. E2F1-E2F6 bind to their target promoter with binding partner DP proteins through dimerization domain. E2F7 and E2F8 have two DNA binding domains and make homodimers or E2F7/E2F8 heterodimer.

A structural characteristic of activator E2Fs (E2F1-E2F3a) is longer N terminal region, which does not exist in repressor E2Fs (E2F3b-E2F6). Expression of E2F1-E2F3a is induced by E2Fs themselves [31]. When growth stimulation inactivates p130 by phosphorylation through ac‐ tivation of CycD/Cdk4 or CycD/Cdk6, repressor E2Fs (E2F4 and E2F5) are released from p130 and activates the *E2F1*-*E2F3a* genes. E2F1-E2F3a in turn replace E2F4 and E2F5, and ac‐ tivate growth-promoting genes including E2F1-E2F3a themselves. Each activator E2F has preferential roles in cell cycle progression and induction of apoptosis. p107 preferentially make complexes with E2F4-E2F5 in G1/S to S phases.

E2F1 is generally thought to be the most powerful transcriptional activator of pro-apoptotic genes among activator E2Fs. Overexpression of E2F1 in tissue culture cells alone can induce cell cycle progression in otherwise quiescent fibroblasts [32]. Overexpression of E2F1 can be oncogenic *in vitro* [33, 34] and *in vivo* [6]. In contrast, E2F1 is dispensable for cell cycle pro‐ gression, since E2F1 knockout mice are viable. On the other hand, over expression of E2F1 in cancer cell lines leads to apoptosis [35]. E2F1 also plays a key role in induction of cellular senescence by activating the ARF-p53 pathway, when overexpressed in human normal fi‐ broblasts [36]. Moreover, E2F1 null mice resulted in tumorigenesis [7]. Taken together, E2F1 seems to play the most important roles in tumor suppression among activator E2Fs by acti‐ vating genes involved in apoptosis and cell cycle arrest.

from RB and repress transcription when bound by RB. E2F1-E2F3a are induced at G1/S boundary and activate transcription free from RB. In contrast, E2F3b-E2F5 are expressed all through the cell cycle and play main roles in transcriptional repression in G0/G1 bound by RB. E2F6-E2F8 repress transcription independently of RB. Hence E2F family members are divided into two groups: activator E2Fs (E2F1-E2F3a) and repressor E2Fs (E2F3b-E2F8). Ac‐ tivator E2Fs play major roles in activation of target genes involved in growth promotion, growth suppression and induction of apoptosis. Repressor E2Fs are roughly divided into two groups; E2F3b-E2F5, which make repressor complex together with RB, and recently

**Figure 4.** Structure of E2F family members. E2F1-E2F6 bind to their target promoter with binding partner DP proteins through dimerization domain. E2F7 and E2F8 have two DNA binding domains and make homodimers or E2F7/E2F8

A structural characteristic of activator E2Fs (E2F1-E2F3a) is longer N terminal region, which does not exist in repressor E2Fs (E2F3b-E2F6). Expression of E2F1-E2F3a is induced by E2Fs themselves [31]. When growth stimulation inactivates p130 by phosphorylation through ac‐ tivation of CycD/Cdk4 or CycD/Cdk6, repressor E2Fs (E2F4 and E2F5) are released from p130 and activates the *E2F1*-*E2F3a* genes. E2F1-E2F3a in turn replace E2F4 and E2F5, and ac‐ tivate growth-promoting genes including E2F1-E2F3a themselves. Each activator E2F has preferential roles in cell cycle progression and induction of apoptosis. p107 preferentially

E2F1 is generally thought to be the most powerful transcriptional activator of pro-apoptotic genes among activator E2Fs. Overexpression of E2F1 in tissue culture cells alone can induce cell cycle progression in otherwise quiescent fibroblasts [32]. Overexpression of E2F1 can be oncogenic *in vitro* [33, 34] and *in vivo* [6]. In contrast, E2F1 is dispensable for cell cycle pro‐ gression, since E2F1 knockout mice are viable. On the other hand, over expression of E2F1 in cancer cell lines leads to apoptosis [35]. E2F1 also plays a key role in induction of cellular

identified E2F6-E2F8, which function independently of RB.

22 Future Aspects of Tumor Suppressor Gene

make complexes with E2F4-E2F5 in G1/S to S phases.

heterodimer.

E2F2 has 46% overall amino acid sequence similarity to E2F1 [37] and is thought to play roles in both cell proliferation and tumor suppression. E2F2 null mice exhibit increased pro‐ liferation of hematopoietic cells and frequently develop autoimmunity and tumors [38, 39]. In tumor suppression, E2F2 plays major roles in suppression of Myc-induced T cell lympho‐ magenesis. Inactivation of neither E2F1 nor E2F3 had no effect on tumor progression in T cells, and only loss of E2F2 accelerated lymphomagenesis [40].

E2F3 is thought to be the most important activator E2F in cell proliferation. Although E2F1 or E2F2 null mice are viable and tumor-prone, E2F3 null mice are typically embryonic lethal in pure background [41] or show partially penetrant embryonic lethality in mixed back‐ ground [42]. Although mouse embryonic fibroblasts (MEFs) with combined knockout of E2F1 and E2F2 proliferate, those with combined knockout of E2F1, E2F2 and E2F3 fail to proliferate and can not re-enter the cell cycle [5]. The E2F3 locus encodes two isoforms, E2F3a and E2F3b, a truncated variant of E2F3 in its N-terminus [43, 44]. Although both E2F3s partly overlaps their roles in cell cycle progression [45], E2F3a is expressed in G1/S to S phases and is thought to play crucial roles on cell proliferation, while E2F3b is expressed equivalently in quiescent and proliferating cells, and associates with pRB, representing the predominant E2F-pRB complex in quiescent cells [44, 46]. E2F3b also plays roles in myogen‐ ic differentiation by promoting gene expression related to differentiation [47].

**Figure 5.** Roles of activator E2Fs in cell-fate determination. E2F1 plays crucial roles in induction of apoptosis. E2F3 is thought be essential for cell proliferation. It is predicted that the character of E2F2 is in the middle of E2F1 and E2F3.

E2F4 and E2F5 were cloned by their association with p107 and p130, and are significantly detected in quiescent cells [48, 49]. Knockout mice of either *E2F4* or E2F5 are viable [50-52]. E2F4 knockout mice are runted and display defects in late stage of maturation. In addition, these mice present reduced thickness of the gut epithelium and developmental craniofacial defects. E2F5 knockout mice develop hydrocephalus after birth apparently due to increased secretion of cerebrospinal fluid by the choroid plexus. E2F4 and E2F5 double knockout mice die before birth because of developmental defects, suggesting that E2F4 and E2F5 have some redundant functions during development [53]. Cells lacking E2F4 and E2F5 are unable to stop cell cycling upon growth-suppressive signals such as TGF-β treatment, suggesting that E2F4 and E2F5 are major repressor E2F in restraining cell cycle progression. These two E2Fs are thought to be important for cell cycle exit and terminal differentiation.

the growth related genes, including the *CycE* gene. CycE activates Cdk2, whose activity is essential for initiating DNA replication, and drive cells into S phase [31]. In late S phase, Cy‐ cA/Cdk2 complex represses the transcriptional activity of E2F/DP complex by phosphoryla‐

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Classical E2F targets are genes involved in DNA replication and cell cycle progression. In addition to these, recent studies with DNA microarray and chromatin immunoprecipitation (ChIP) identified a variety of E2F targets. These include genes involved in DNA repair, checkpoint, differentiation, development, metabolism, micro RNAs, apoptosis, cell cycle ar‐

E2F regulates expression of most of the genes involved in initiation of DNA replication: the *ORC1* (*origin recognition complex1*), *CDC6*, *MCM* (*maintenance of minichromosome*) *2-7*, *ASK* and *CDC45* genes. These factors are assembled into pre-replication complex, which is activated by CycE/Cdk2 to initiate DNA replication [31]. Cdt1 also plays crucial roles in initiation of DNA replication and is negatively regulated by geminin. The *Cdt1* and *geminin* genes are both E2F targets [62]. Genes, which code for machineries responsible for DNA replication, are classical E2F targets. These include the *DHFR* (*dihydrofolate re‐ ductase*), *DNA polymerase α*, *thymidine kinase, thymidylate synthase* and *PCNA (proliferating*

E2F target genes related to DNA repair are the *Rad51*, *MSH2* and *MLH1* genes, which are involved in homologous recombination repair and mismatch repair [63]. E2F target genes related to checkpoint are the *ATM*, *Chk1*, *Mad3*, *Bub1*, *Claspin* and *RanBP1* genes [53, 64-67]. It is expected that E2F regulates these DNA repair and checkpoint genes to prepare machi‐

E2F induces expression of genes, which play major roles in induction of S phase, such as the *CycE* and *activator E2F* (*E2F1*-*E2F3a*) genes themselves [31]. Also, the upstream negative reg‐ ulators of pRB, the *Emi1* and *Skp2* genes are E2F targets [68, 69]. The genes, which encode CycA, Cdc2 (CDK1), CycB and B-myb that are important for S and G2/M phase progression, are also E2F targets [53]. Repressor E2Fs, *E2F7* and *E2F8* genes are reported to be targets of E2F1 that promote embryonic development by suppressing E2F1-p53 induced apoptosis, forming a negative feedback loop. Although c-Myc is reported as a target of E2F [70], E2F may play a role in suppression of c-Myc expression upon negative growth signals [71].

E2F also regulates expression of genes involved in development and differentiation. The *Firizzled homologs1-3*, *Homeobox* and *TGF* genes are shown to be targets of E2F

tion, which releases the complex from the binding element [61].

**3.3. E2F target genes**

rest and others.

*DNA replication*

*cell nuclear antigen)* [53].

neries to quickly respond in case of emergency.

*DNA repair, checkpoint*

*Cell cycle progression*

*Development and differentiation*

E2F6 does not possess RB binding domain. As predicted from the structure, E2F6 contrib‐ utes gene silencing independent of RB [54] and is predicted to make repressor complex with polycomb proteins [55]. Consistent with this, *E2f6*-/- animals display overt homeotic trans‐ formations of the axial skeleton that are strikingly similar to the skeletal transformations ob‐ served in polycomb deficient mice [56]. E2F6 is reported to bind the same E2F-repressor site as E2F4, suggesting E2F6 may partly overlaps its function as transcriptional repressor with E2F4 in S phase [57].

E2F7 and E2F8 are thought to function as transcriptional repressors independently of RB proteins. An important target of E2F7 and E2F8 is the *E2F1* gene. E2F7 and E2F8 knockout mice are embryonic lethal, at least in part, due to apoptosis caused by inability to down reg‐ ulate expression of E2F1, leading to activation of p53 [58]. Recent report showed that E2F7 and E2F8 promote angiogenesis through transcriptional activation of the VEGFA promoter with hypoxia inducible factor 1 (HIF1) [59].

#### **3.2. Regulatory mechanism of E2F**

E2F1 through E2F6 associate with DP family proteins (DP1 or DP2) to form heterodimeric complexes that bind to DNA in a sequence-specific manner (consensus sequence: TTTC/G G/ CCGC). E2F7 and E2F8 have two DNA binding domains and do not require DP proteins for binding to DNA. E2F7 and E2F8 make homodimer or E2F7/E2F8 heterodimer to bind to the target [58].

Transcriptional activity of E2F1 through E2F5 is suppressed by binding of RB family pro‐ teins. pRB preferentially binds E2F1 through E2F3, whereas p107 and p130 preferentially bind E2F4 and E2F5. However, pRB can also bind E2F4, depending on the cellular circum‐ stances. E2F6 through E2F8 can repress transcription independently of pRB family proteins [10, 60]. In quiescent phase, E2F3b/pRB, E2F4/p130 and E2F5/p130 repress promoters of E2F target genes. Upon growth stimulation, activated CycD/Cdk4 and CycD/Cdk6 phosphory‐ late pRB and p130, inhibiting their binding to the E2Fs and allowing accumulation of the free E2Fs [31]. This release from repression conferred by the pRB family proteins is the pri‐ mary activation step for induction of E2F target genes. In this context, E2F3b, E2F4 and E2F5 mainly act as repressors together with the pRB family proteins during G0/G1 phases. Ex‐ pression of E2F1, E2F2 and E2F3a is induced at the G1/S boundary by E2F itself, and activate the growth related genes, including the *CycE* gene. CycE activates Cdk2, whose activity is essential for initiating DNA replication, and drive cells into S phase [31]. In late S phase, Cy‐ cA/Cdk2 complex represses the transcriptional activity of E2F/DP complex by phosphoryla‐ tion, which releases the complex from the binding element [61].

#### **3.3. E2F target genes**

E2F4 and E2F5 were cloned by their association with p107 and p130, and are significantly detected in quiescent cells [48, 49]. Knockout mice of either *E2F4* or E2F5 are viable [50-52]. E2F4 knockout mice are runted and display defects in late stage of maturation. In addition, these mice present reduced thickness of the gut epithelium and developmental craniofacial defects. E2F5 knockout mice develop hydrocephalus after birth apparently due to increased secretion of cerebrospinal fluid by the choroid plexus. E2F4 and E2F5 double knockout mice die before birth because of developmental defects, suggesting that E2F4 and E2F5 have some redundant functions during development [53]. Cells lacking E2F4 and E2F5 are unable to stop cell cycling upon growth-suppressive signals such as TGF-β treatment, suggesting that E2F4 and E2F5 are major repressor E2F in restraining cell cycle progression. These two E2Fs

E2F6 does not possess RB binding domain. As predicted from the structure, E2F6 contrib‐ utes gene silencing independent of RB [54] and is predicted to make repressor complex with polycomb proteins [55]. Consistent with this, *E2f6*-/- animals display overt homeotic trans‐ formations of the axial skeleton that are strikingly similar to the skeletal transformations ob‐ served in polycomb deficient mice [56]. E2F6 is reported to bind the same E2F-repressor site as E2F4, suggesting E2F6 may partly overlaps its function as transcriptional repressor with

E2F7 and E2F8 are thought to function as transcriptional repressors independently of RB proteins. An important target of E2F7 and E2F8 is the *E2F1* gene. E2F7 and E2F8 knockout mice are embryonic lethal, at least in part, due to apoptosis caused by inability to down reg‐ ulate expression of E2F1, leading to activation of p53 [58]. Recent report showed that E2F7 and E2F8 promote angiogenesis through transcriptional activation of the VEGFA promoter

E2F1 through E2F6 associate with DP family proteins (DP1 or DP2) to form heterodimeric complexes that bind to DNA in a sequence-specific manner (consensus sequence: TTTC/G

CCGC). E2F7 and E2F8 have two DNA binding domains and do not require DP proteins for binding to DNA. E2F7 and E2F8 make homodimer or E2F7/E2F8 heterodimer to bind to the

Transcriptional activity of E2F1 through E2F5 is suppressed by binding of RB family pro‐ teins. pRB preferentially binds E2F1 through E2F3, whereas p107 and p130 preferentially bind E2F4 and E2F5. However, pRB can also bind E2F4, depending on the cellular circum‐ stances. E2F6 through E2F8 can repress transcription independently of pRB family proteins [10, 60]. In quiescent phase, E2F3b/pRB, E2F4/p130 and E2F5/p130 repress promoters of E2F target genes. Upon growth stimulation, activated CycD/Cdk4 and CycD/Cdk6 phosphory‐ late pRB and p130, inhibiting their binding to the E2Fs and allowing accumulation of the free E2Fs [31]. This release from repression conferred by the pRB family proteins is the pri‐ mary activation step for induction of E2F target genes. In this context, E2F3b, E2F4 and E2F5 mainly act as repressors together with the pRB family proteins during G0/G1 phases. Ex‐ pression of E2F1, E2F2 and E2F3a is induced at the G1/S boundary by E2F itself, and activate

are thought to be important for cell cycle exit and terminal differentiation.

E2F4 in S phase [57].

24 Future Aspects of Tumor Suppressor Gene

target [58].

with hypoxia inducible factor 1 (HIF1) [59].

**3.2. Regulatory mechanism of E2F**

Classical E2F targets are genes involved in DNA replication and cell cycle progression. In addition to these, recent studies with DNA microarray and chromatin immunoprecipitation (ChIP) identified a variety of E2F targets. These include genes involved in DNA repair, checkpoint, differentiation, development, metabolism, micro RNAs, apoptosis, cell cycle ar‐ rest and others.

## *DNA replication*

E2F regulates expression of most of the genes involved in initiation of DNA replication: the *ORC1* (*origin recognition complex1*), *CDC6*, *MCM* (*maintenance of minichromosome*) *2-7*, *ASK* and *CDC45* genes. These factors are assembled into pre-replication complex, which is activated by CycE/Cdk2 to initiate DNA replication [31]. Cdt1 also plays crucial roles in initiation of DNA replication and is negatively regulated by geminin. The *Cdt1* and *geminin* genes are both E2F targets [62]. Genes, which code for machineries responsible for DNA replication, are classical E2F targets. These include the *DHFR* (*dihydrofolate re‐ ductase*), *DNA polymerase α*, *thymidine kinase, thymidylate synthase* and *PCNA (proliferating cell nuclear antigen)* [53].

#### *DNA repair, checkpoint*

E2F target genes related to DNA repair are the *Rad51*, *MSH2* and *MLH1* genes, which are involved in homologous recombination repair and mismatch repair [63]. E2F target genes related to checkpoint are the *ATM*, *Chk1*, *Mad3*, *Bub1*, *Claspin* and *RanBP1* genes [53, 64-67]. It is expected that E2F regulates these DNA repair and checkpoint genes to prepare machi‐ neries to quickly respond in case of emergency.

## *Cell cycle progression*

G/

E2F induces expression of genes, which play major roles in induction of S phase, such as the *CycE* and *activator E2F* (*E2F1*-*E2F3a*) genes themselves [31]. Also, the upstream negative reg‐ ulators of pRB, the *Emi1* and *Skp2* genes are E2F targets [68, 69]. The genes, which encode CycA, Cdc2 (CDK1), CycB and B-myb that are important for S and G2/M phase progression, are also E2F targets [53]. Repressor E2Fs, *E2F7* and *E2F8* genes are reported to be targets of E2F1 that promote embryonic development by suppressing E2F1-p53 induced apoptosis, forming a negative feedback loop. Although c-Myc is reported as a target of E2F [70], E2F may play a role in suppression of c-Myc expression upon negative growth signals [71].

#### *Development and differentiation*

E2F also regulates expression of genes involved in development and differentiation. The *Firizzled homologs1-3*, *Homeobox* and *TGF* genes are shown to be targets of E2F [53]. It is reported that overexpression of E2F1-3 induced various genes involved in development and differentiation [72]. Interestingly, although E2F7 and E2F8 are gener‐ ally regarded as transcriptional repressors, a recent study reported the roles of E2F7 and E2F8 in transcriptional activation of genes such as VEGFA as written before [59].

#### *Cellular metabolism*

Recent work reported that E2F1 and pRB are required for repression of genes implicated in oxidative metabolism [73]. E2F1 repressed key genes that regulated energy homeostasis and mitochondrial functions in muscle and brown adipose tissue, and E2F1 null mice had a marked oxidative phenotype. Their work suggests a metabolic switch from oxidative to gly‐ colytic metabolism that responds to stressful conditions.

#### *Micro RNAs*

Accumulating evidence indicates that expression of E2F is regulated by microRNAs and that E2F also induces expression of microRNAs [74]. One of the major microRNAs regulated by E2F is miR-17~92. miR-17~92 is a negative regulator of E2F1-E2F3 and is also a target of E2F1-E2F3, constructing a fail-safe mechanism to regulate E2F activity. E2F seems to be rig‐ orously controlled by many miRNAs, suggesting that E2F activity must be strictly control‐ led for appropriate cell cycle progression.

**Figure 6.** E2F activates both growth-promoting genes and growth- suppressive genes, including E2F itself. It is pre‐ dicted that the expression levels of E2F targets, related to growth- promotion or growth- suppression, decide the bal‐

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27

Since E2F1-E2F3a are activated by growth stimulation, it is generally thought that their tar‐ get genes are all activated by growth stimulation. However, E2F1-E2F3a also activate genes involved in cell cycle arrest or apoptotic, which are inconvenient for cell growth. It has yet to be elucidated how E2F regulates genes involved in cell cycle arrest or apoptosis, regarding

Since all E2F targets are thought to be activated by growth stimulation, it is surprising that our previous work identified three growth suppressive E2F targets, which were not activat‐ ed by growth stimulation at all in human normal fibroblasts [75-77]. In contrast, these E2F targets were activated by deregulated E2F activity induced by overexpression of E2F1 or forced inactivation of pRB. Overexpression of E2F1 generates exceeding amount of exoge‐ nously introduced E2F1, which becomes out control by RB proteins. Forced inactivation of pRB induces endogenous deregulated E2F activity out control by pRB. We refer these growth suppressive E2F targets, which are not activated by growth stimulation but are acti‐ vated by deregulated E2F, to 'atypical E2F targets'. These atypical E2F targets include the tumor suppressor *ARF* and *TAp73* genes and the CDK inhibitor *p27Kip1* gene These three atypical E2F target genes play major roles in tumor suppression. ARF is an upstream activa‐ tor of the tumor suppressor p53. CDK inhibitor p27Kip1 activates the RB pathway by inhibit‐ ing CDKs. TAp73 is the tumor suppressor, which can induce apoptosis in dependently of p53. Our observations suggest that E2F activity induced by RB dysfunction, one of major on‐

ance of cell-fate determination.

**4. Deregulated E2F**

normal cell growth and tumor suppression.

#### *Apoptosis and cell cycle arrest*

E2F can activate genes involved in apoptosis and cell cycle arrest, which are inconvenient for cell proliferation. Regulatory mechanism of these growth-suppressive genes by E2F is yet to be elucidated, especially regarding regulation of cell growth versus tumor suppres‐ sion in response to normal growth stimulation and oncogenic changes. The *caspase3, caspase7* and *Apaf1* genes, which code for apoptotic machineries, are direct targets of E2F [53]. Cas‐ pases are expressed as inactive precursors (procaspases), and expression of procaspases and Apaf-1 alone does not necessarily induce apoptosis. These apoptotic machineries require up‐ stream signals to be activated to induce apoptosis. Expression of these two pro-apoptotic E2F targets and all E2F targets mentioned above is induced by growth stimulation. Thus, ex‐ pression of the pro-apoptotic machinery by growth stimulation is thought to be fail-safe mechanism to induce apoptosis in case of emergency. We refer these E2F target genes, whose expression is induced by growth stimulation, to 'typical E2F targets'. Typical E2F tar‐ gets are activated by growth stimulation, which physiologically inactivates RB by phosphor‐ ylation and activates E2F by release from repression.

In contrast to typical E2F targets, we identified three E2F targets, which are not activated by growth stimulation. These include the tumor suppressor *ARF*, *TAp73* and CDK inhibitor *p27Kip1* genes. As described below, these genes are critically important in tumor suppression. We describe the regulatory mechanism of the tumor suppressor *ARF*, *TAp73* and CDK in‐ hibitor *p27Kip1* genes by E2F in next paragraph.

To Grow, Stop or Die? – Novel Tumor-Suppressive Mechanism Regulated by the Transcription Factor E2F http://dx.doi.org/10.5772/54510 27

**Figure 6.** E2F activates both growth-promoting genes and growth- suppressive genes, including E2F itself. It is pre‐ dicted that the expression levels of E2F targets, related to growth- promotion or growth- suppression, decide the bal‐ ance of cell-fate determination.

## **4. Deregulated E2F**

[53]. It is reported that overexpression of E2F1-3 induced various genes involved in development and differentiation [72]. Interestingly, although E2F7 and E2F8 are gener‐ ally regarded as transcriptional repressors, a recent study reported the roles of E2F7 and E2F8 in transcriptional activation of genes such as VEGFA as written before [59].

Recent work reported that E2F1 and pRB are required for repression of genes implicated in oxidative metabolism [73]. E2F1 repressed key genes that regulated energy homeostasis and mitochondrial functions in muscle and brown adipose tissue, and E2F1 null mice had a marked oxidative phenotype. Their work suggests a metabolic switch from oxidative to gly‐

Accumulating evidence indicates that expression of E2F is regulated by microRNAs and that E2F also induces expression of microRNAs [74]. One of the major microRNAs regulated by E2F is miR-17~92. miR-17~92 is a negative regulator of E2F1-E2F3 and is also a target of E2F1-E2F3, constructing a fail-safe mechanism to regulate E2F activity. E2F seems to be rig‐ orously controlled by many miRNAs, suggesting that E2F activity must be strictly control‐

E2F can activate genes involved in apoptosis and cell cycle arrest, which are inconvenient for cell proliferation. Regulatory mechanism of these growth-suppressive genes by E2F is yet to be elucidated, especially regarding regulation of cell growth versus tumor suppres‐ sion in response to normal growth stimulation and oncogenic changes. The *caspase3, caspase7* and *Apaf1* genes, which code for apoptotic machineries, are direct targets of E2F [53]. Cas‐ pases are expressed as inactive precursors (procaspases), and expression of procaspases and Apaf-1 alone does not necessarily induce apoptosis. These apoptotic machineries require up‐ stream signals to be activated to induce apoptosis. Expression of these two pro-apoptotic E2F targets and all E2F targets mentioned above is induced by growth stimulation. Thus, ex‐ pression of the pro-apoptotic machinery by growth stimulation is thought to be fail-safe mechanism to induce apoptosis in case of emergency. We refer these E2F target genes, whose expression is induced by growth stimulation, to 'typical E2F targets'. Typical E2F tar‐ gets are activated by growth stimulation, which physiologically inactivates RB by phosphor‐

In contrast to typical E2F targets, we identified three E2F targets, which are not activated by growth stimulation. These include the tumor suppressor *ARF*, *TAp73* and CDK inhibitor *p27Kip1* genes. As described below, these genes are critically important in tumor suppression. We describe the regulatory mechanism of the tumor suppressor *ARF*, *TAp73* and CDK in‐

colytic metabolism that responds to stressful conditions.

led for appropriate cell cycle progression.

ylation and activates E2F by release from repression.

hibitor *p27Kip1* genes by E2F in next paragraph.

*Apoptosis and cell cycle arrest*

*Cellular metabolism*

26 Future Aspects of Tumor Suppressor Gene

*Micro RNAs*

Since E2F1-E2F3a are activated by growth stimulation, it is generally thought that their tar‐ get genes are all activated by growth stimulation. However, E2F1-E2F3a also activate genes involved in cell cycle arrest or apoptotic, which are inconvenient for cell growth. It has yet to be elucidated how E2F regulates genes involved in cell cycle arrest or apoptosis, regarding normal cell growth and tumor suppression.

Since all E2F targets are thought to be activated by growth stimulation, it is surprising that our previous work identified three growth suppressive E2F targets, which were not activat‐ ed by growth stimulation at all in human normal fibroblasts [75-77]. In contrast, these E2F targets were activated by deregulated E2F activity induced by overexpression of E2F1 or forced inactivation of pRB. Overexpression of E2F1 generates exceeding amount of exoge‐ nously introduced E2F1, which becomes out control by RB proteins. Forced inactivation of pRB induces endogenous deregulated E2F activity out control by pRB. We refer these growth suppressive E2F targets, which are not activated by growth stimulation but are acti‐ vated by deregulated E2F, to 'atypical E2F targets'. These atypical E2F targets include the tumor suppressor *ARF* and *TAp73* genes and the CDK inhibitor *p27Kip1* gene These three atypical E2F target genes play major roles in tumor suppression. ARF is an upstream activa‐ tor of the tumor suppressor p53. CDK inhibitor p27Kip1 activates the RB pathway by inhibit‐ ing CDKs. TAp73 is the tumor suppressor, which can induce apoptosis in dependently of p53. Our observations suggest that E2F activity induced by RB dysfunction, one of major on‐ cogenic changes, has distinct function from that induced by growth stimulation in activating target genes (Figure 7).

the *ARF* gene is frequently observed in cancers. Moreover, *Arf* null mice are highly prone to tumorigenesis [81]. Various oncogenic signals are able to elicit the activation of the *ARF* gene. Overexpression of adenovirus E1a or E2F1 in primary mouse embryonic fibroblasts (MEFs) rapidly induces ARF gene expression and p53-dependent apoptosis [16]. Myc over‐ expression and oncogenic mutant Ras are also strong activators of the *ARF* gene and combi‐ nation of absence of Arf in mice severely impaired the tumor suppressive activity of p53 [82]. Arf promoter seems to monitor these oncogenic signals as shown by ARF promoter-*GFP* transgenic model, in which GFP expression was observed in tumors induced by Myc or Ras but not in normal growing tissues [83]. Other studies elucidated that ARF also restrains cell growth independently of p53, interacting with other factors [84]. Taken together, the *ARF* gene plays crucial roles in tumor suppression through p53-dependent and independent

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The CDK inhibitor p27Kip1 is an upstream regulator of the RB pathway and known to contribute to the ability of pRB to induce cell-cycle arrest, differentiation and senescence [85-87]. There is cross regulation between p27Kip1 and pRB. p27Kip1 enhances pRB growth suppressive function by inhibiting Cyc/CDK, keeping pRB in hypo-phosphorylated form. pRB increases the amount of p27Kip1 by sequestrating Skp2, a component of E3 ubiquitin ligase complex, which promotes degradation of p27Kip1 [86]. pRB also known to cooper‐ ate with APC/Ccdh1, another E3 ubiquitin ligase, to induces Skp2 degradation, stabilizing p27Kip1 [88]. Taken together, pRB and p27Kip1 seem to keep close relationship in the RB pathway to efficiently suppress aberrant cell proliferation. Indeed, our previous study showed that inactivation of RB by adenovirus E1a increased BrdU (bromodeoxyuridine) positive cells much earlier in *p27Kip1*-/- MEFs than in wild type MEFs [66]. These results support the notion that p27Kip1 plays important roles in the RB pathway to suppress cell

The tumor suppressor *TAp73* gene had been identified as a direct target of E2F using cancer cell lines [4]. We found that the *TAp73* gene was activated by deregulated E2F but not by physiological E2F in human normal fibroblasts [77]. TAp73 is a p53 family member and plays important roles in tumor suppression with its other family member p53 and p63. All of the three genes express differentially spliced isoforms [89, 90]. Two major isoforms are TA isoforms, which retain transactivation (TA) domain, and delta N (DN) isoforms, which lack TA domain. Since these family members activate their targets as tetramers and DN isoforms lack transactivation (TA) domain, DN isoforms have dom‐ inant-negative properties [24]. Although p53 is deleted or mutated in half of all human cancers, deletion or mutations of p73 and p63 occur rarely [91, 92]. Rather over expres‐ sion of DN form of p73 and p63 are commonly observed in many cancers, such as over expression of DNp73 isoform in gliomas and carcinomas of the breast and the colon [93, 94], and that of DNp63 isoform in bladder carcinomas [95]. Each DN isoform can sup‐ press all three types of TA forms [92, 96, 97]. Therefore, it is expected that tumor-sup‐ pressive TA isoforms are suppressed by DN isoforms in many cancers. This could explain why deletion or mutations of the *TP73* and *TP63* genes are rare. *TAp73* can in‐ duce apoptosis independently of p53. Moreover, TAp73 knockout mice are tumor prone,

cycle progression induced by oncogenic changes.

pathways.

E2F activity induced by RB dysfunction activates both typical and atypical E2F targets. In contrast, E2F activity induced by growth stimulation activates only typical E2F targets and interestingly, not atypical E2F targets. Both of the E2Fs are similar in the sense that they are released from repression by RB. However, it is shown that growth stimulation does not to‐ tally inactivate pRB and some portion of activator E2Fs is still in complex with pRB [78]. Moreover, the *RB1* gene is an E2F target [79] and expression of pRB is increased in G1/S to S phases [80]. Indeed, the amount of activator E2Fs/pRB complex rather increases in G1/S to S phases, as examined by gel mobility shift assay [78]. This observation indicates that E2F ac‐ tivity induced by growth stimulation is still under control of RB. In contrast, E2F activity in‐ duced by dysfunction of RB is thought to be out of control by RB. Here, we refer E2F activated by growth stimulation to 'physiological E2F' and that by dysfunction of RB to 'de‐ regulated E2F'. Our findings indicate that deregulated E2F is functionally different from physiological E2F.

In this paragraph, we describe the regulatory mechanism of atypical E2F targets by deregu‐ lated E2F and discuss about the characteristics of deregulated E2F activity regarding its role in tumor suppression.

**Figure 7.** Atypical E2F target genes are specifically activated by deregulated E2F activity through specific E2F respon‐ sive elements.

## **4.1. Atypical E2F targets**

The first atypical E2F target identified was the *ARF* gene [75]. ARF is the major activator of p53 pathway and links the RB and p53 tumor suppressor pathways [22], playing crucial roles in tumor suppression. Consistent with this notion, deletion, mutation or silencing of the *ARF* gene is frequently observed in cancers. Moreover, *Arf* null mice are highly prone to tumorigenesis [81]. Various oncogenic signals are able to elicit the activation of the *ARF* gene. Overexpression of adenovirus E1a or E2F1 in primary mouse embryonic fibroblasts (MEFs) rapidly induces ARF gene expression and p53-dependent apoptosis [16]. Myc over‐ expression and oncogenic mutant Ras are also strong activators of the *ARF* gene and combi‐ nation of absence of Arf in mice severely impaired the tumor suppressive activity of p53 [82]. Arf promoter seems to monitor these oncogenic signals as shown by ARF promoter-*GFP* transgenic model, in which GFP expression was observed in tumors induced by Myc or Ras but not in normal growing tissues [83]. Other studies elucidated that ARF also restrains cell growth independently of p53, interacting with other factors [84]. Taken together, the *ARF* gene plays crucial roles in tumor suppression through p53-dependent and independent pathways.

cogenic changes, has distinct function from that induced by growth stimulation in activating

E2F activity induced by RB dysfunction activates both typical and atypical E2F targets. In contrast, E2F activity induced by growth stimulation activates only typical E2F targets and interestingly, not atypical E2F targets. Both of the E2Fs are similar in the sense that they are released from repression by RB. However, it is shown that growth stimulation does not to‐ tally inactivate pRB and some portion of activator E2Fs is still in complex with pRB [78]. Moreover, the *RB1* gene is an E2F target [79] and expression of pRB is increased in G1/S to S phases [80]. Indeed, the amount of activator E2Fs/pRB complex rather increases in G1/S to S phases, as examined by gel mobility shift assay [78]. This observation indicates that E2F ac‐ tivity induced by growth stimulation is still under control of RB. In contrast, E2F activity in‐ duced by dysfunction of RB is thought to be out of control by RB. Here, we refer E2F activated by growth stimulation to 'physiological E2F' and that by dysfunction of RB to 'de‐ regulated E2F'. Our findings indicate that deregulated E2F is functionally different from

In this paragraph, we describe the regulatory mechanism of atypical E2F targets by deregu‐ lated E2F and discuss about the characteristics of deregulated E2F activity regarding its role

**Figure 7.** Atypical E2F target genes are specifically activated by deregulated E2F activity through specific E2F respon‐

The first atypical E2F target identified was the *ARF* gene [75]. ARF is the major activator of p53 pathway and links the RB and p53 tumor suppressor pathways [22], playing crucial roles in tumor suppression. Consistent with this notion, deletion, mutation or silencing of

target genes (Figure 7).

28 Future Aspects of Tumor Suppressor Gene

physiological E2F.

in tumor suppression.

sive elements.

**4.1. Atypical E2F targets**

The CDK inhibitor p27Kip1 is an upstream regulator of the RB pathway and known to contribute to the ability of pRB to induce cell-cycle arrest, differentiation and senescence [85-87]. There is cross regulation between p27Kip1 and pRB. p27Kip1 enhances pRB growth suppressive function by inhibiting Cyc/CDK, keeping pRB in hypo-phosphorylated form. pRB increases the amount of p27Kip1 by sequestrating Skp2, a component of E3 ubiquitin ligase complex, which promotes degradation of p27Kip1 [86]. pRB also known to cooper‐ ate with APC/Ccdh1, another E3 ubiquitin ligase, to induces Skp2 degradation, stabilizing p27Kip1 [88]. Taken together, pRB and p27Kip1 seem to keep close relationship in the RB pathway to efficiently suppress aberrant cell proliferation. Indeed, our previous study showed that inactivation of RB by adenovirus E1a increased BrdU (bromodeoxyuridine) positive cells much earlier in *p27Kip1*-/- MEFs than in wild type MEFs [66]. These results support the notion that p27Kip1 plays important roles in the RB pathway to suppress cell cycle progression induced by oncogenic changes.

The tumor suppressor *TAp73* gene had been identified as a direct target of E2F using cancer cell lines [4]. We found that the *TAp73* gene was activated by deregulated E2F but not by physiological E2F in human normal fibroblasts [77]. TAp73 is a p53 family member and plays important roles in tumor suppression with its other family member p53 and p63. All of the three genes express differentially spliced isoforms [89, 90]. Two major isoforms are TA isoforms, which retain transactivation (TA) domain, and delta N (DN) isoforms, which lack TA domain. Since these family members activate their targets as tetramers and DN isoforms lack transactivation (TA) domain, DN isoforms have dom‐ inant-negative properties [24]. Although p53 is deleted or mutated in half of all human cancers, deletion or mutations of p73 and p63 occur rarely [91, 92]. Rather over expres‐ sion of DN form of p73 and p63 are commonly observed in many cancers, such as over expression of DNp73 isoform in gliomas and carcinomas of the breast and the colon [93, 94], and that of DNp63 isoform in bladder carcinomas [95]. Each DN isoform can sup‐ press all three types of TA forms [92, 96, 97]. Therefore, it is expected that tumor-sup‐ pressive TA isoforms are suppressed by DN isoforms in many cancers. This could explain why deletion or mutations of the *TP73* and *TP63* genes are rare. *TAp73* can in‐ duce apoptosis independently of p53. Moreover, TAp73 knockout mice are tumor prone, infertile, sensitive for carcinogen-induced tumorigenesis and defective for maintenance of genomic stability [98]. The *TAp73* gene is activated by various stress signals including deregulated E2F and DNA damage. These observations suggest that TAp73 contributes to tumor suppression in addition to the p53 pathway in response to various oncogenic changes.

of E2F target genes to normal growth stimulation. To induce physiological E2F, we used se‐ rum stimulation, common growth stimulation for fibroblasts. To induce deregulated E2F ac‐ tivity, we used ectopically expressed E2F1 or forced inactivation of RB either by adenovirus E1a, which binds to and inactivates all RB family proteins, or shRNA against *RB1* (shRB), which represses the expression of pRB. The latter (adenovirus E1a and shRB) is expected to

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Ectopically expressed E2F1, adenovirus E1a and shRB induced ARF gene expression in RT-PCR, and activated ARF promoter in reporter assay. However, serum stimulation, which physiologically activates E2F, did not induce ARF gene expression or activate ARF promoter under the condition that CDC6 gene (one of typical E2F targets involved in DNA replica‐ tion) expression was significantly induced and CDC6 promoter was clearly activated. These results indicate that the *ARF* gene is specifically activated by deregulated E2F but not by physiological E2F. Promoter analyses identified the E2F responsive element of ARF promot‐ er (EREA), which specifically responds to deregulated E2F activity. Interestingly, the se‐ quence of EREA was composed of only GC repeat and lacked T stretch. This is in contrast to that of consensus E2F binding motif, which is composed of T stretch and GC repeat

C/GCGC) in typical E2F targets. In addition, the location of EREA was far upstream

from transcription start site compared to that of typical E2F sites, which is within 100 bp from transcription start site in most cases. Moreover, our gel mobility shift assay and ChIP assay showed that EREA specifically binds ectopically expressed E2F1 but not physiological E2F1 induced by serum stimulation, both *in vitro* and *in vivo*, respectively. Taken together, these observations suggest that the *ARF* gene is specifically activated through EREA by de‐ regulated E2F activity, triggered by ectopically expressed E2F1 or forced inactivation of RB,

A later study showed that ARF promoter lacking EREA was still activated by overexpression of E2F1 [99]. Our further analyses of ARF promoter identified multiples of EREA-like elements in ARF promoter. It seems that, although EREA is the major E2F responsive element in ARF pro‐ moter, it is not the sole responsive element and multiple EREA-like elements co-operate to spe‐

Defects in the RB pathway activate E2F out of control by RB, promoting abnormal cell growth. In response to such oncogenic insults, deregulated E2F activates the *ARF* gene, lead‐ ing to activation of p53 to protect cell from tumorigenesis. For induction of tumorigenesis, the p53 pathway must be disabled by further oncogenic changes. Indeed, defects in the p53 pathway are observed in almost all cancers. It is expected that the presence of deregulated E2F be tolerated in cancer cells by further inactivation of the p53 pathway. We thus exam‐ ined the existence of deregulated E2F activity in *RB1* deficient cancer cell lines and in nor‐ mal growing fibroblasts. When we introduced constitutively active form of pRB into *RB1* deficient cancer cell lines (5637, Saos-2 and C-33 A) and normal growing fibroblasts (WI-38 and HFF), activity of EREA and ARF promoter were decreased in *RB1* deficient cancer cell lines, but not in normal growing fibroblasts. These results showed that deregulated E2F ac‐ tivity specifically exists in *RB1* deficient cancer cell lines but not in normal growing fibro‐

but not by physiological E2F activity, induced by serum stimulation (Figure 7).

cifically respond to deregulated E2F activity (manuscript in preparation).

induce endogenous deregulated E2F activity.

(TTTG/C

Accumulating evidence indicates that the three atypical E2F targets explained above play crucial roles in tumor suppression, by activating the RB pathway, the p53 pathway and the p53 independent pathway. Taken together, deregulated E2F seems to be critically important for activating major intrinsic tumor-suppressor pathways in responding to oncogenic changes to suppress tumorigenesis (Figure 8).

**Figure 8.** Deregulated E2F plays important roles in activating major tumor suppressor pathways (RB, p53 and TAp73 pathways) by activating the *ARF*, *p27Kip1* and *TAp73* genes.

## **4.2. Distinct transcriptional regulatory mechanism mediated by deregulated E2F**

ARF, p27Kip1 and TAp73 exert its effects when expressed unlike pro-apoptotic targets, which are expressed as inactive precursors such as pro-caspases. Thus the regulation of expression of these genes is critically important for tumor suppression. The finding that these genes are specifically activated by deregulated E2F but not by physiologically activated E2F indicates that there is a mechanism to specifically respond to oncogenic changes to suppress tumori‐ genesis, while allowing normal cell growth upon normal growth stimulation.

We first identified the tumor suppressor *ARF* gene as an atypical E2F target [75]. In our studies, we used human normal fibroblasts (HFFs or WI-38) to examine the responsiveness of each growth-suppressive E2F target to physiological and deregulated E2F. This is because most of previous studies used cancer cell lines and were unable to examine responsiveness of E2F target genes to normal growth stimulation. To induce physiological E2F, we used se‐ rum stimulation, common growth stimulation for fibroblasts. To induce deregulated E2F ac‐ tivity, we used ectopically expressed E2F1 or forced inactivation of RB either by adenovirus E1a, which binds to and inactivates all RB family proteins, or shRNA against *RB1* (shRB), which represses the expression of pRB. The latter (adenovirus E1a and shRB) is expected to induce endogenous deregulated E2F activity.

infertile, sensitive for carcinogen-induced tumorigenesis and defective for maintenance of genomic stability [98]. The *TAp73* gene is activated by various stress signals including deregulated E2F and DNA damage. These observations suggest that TAp73 contributes to tumor suppression in addition to the p53 pathway in response to various oncogenic

Accumulating evidence indicates that the three atypical E2F targets explained above play crucial roles in tumor suppression, by activating the RB pathway, the p53 pathway and the p53 independent pathway. Taken together, deregulated E2F seems to be critically important for activating major intrinsic tumor-suppressor pathways in responding to oncogenic

**Figure 8.** Deregulated E2F plays important roles in activating major tumor suppressor pathways (RB, p53 and TAp73

ARF, p27Kip1 and TAp73 exert its effects when expressed unlike pro-apoptotic targets, which are expressed as inactive precursors such as pro-caspases. Thus the regulation of expression of these genes is critically important for tumor suppression. The finding that these genes are specifically activated by deregulated E2F but not by physiologically activated E2F indicates that there is a mechanism to specifically respond to oncogenic changes to suppress tumori‐

We first identified the tumor suppressor *ARF* gene as an atypical E2F target [75]. In our studies, we used human normal fibroblasts (HFFs or WI-38) to examine the responsiveness of each growth-suppressive E2F target to physiological and deregulated E2F. This is because most of previous studies used cancer cell lines and were unable to examine responsiveness

**4.2. Distinct transcriptional regulatory mechanism mediated by deregulated E2F**

genesis, while allowing normal cell growth upon normal growth stimulation.

changes.

30 Future Aspects of Tumor Suppressor Gene

changes to suppress tumorigenesis (Figure 8).

pathways) by activating the *ARF*, *p27Kip1* and *TAp73* genes.

Ectopically expressed E2F1, adenovirus E1a and shRB induced ARF gene expression in RT-PCR, and activated ARF promoter in reporter assay. However, serum stimulation, which physiologically activates E2F, did not induce ARF gene expression or activate ARF promoter under the condition that CDC6 gene (one of typical E2F targets involved in DNA replica‐ tion) expression was significantly induced and CDC6 promoter was clearly activated. These results indicate that the *ARF* gene is specifically activated by deregulated E2F but not by physiological E2F. Promoter analyses identified the E2F responsive element of ARF promot‐ er (EREA), which specifically responds to deregulated E2F activity. Interestingly, the se‐ quence of EREA was composed of only GC repeat and lacked T stretch. This is in contrast to that of consensus E2F binding motif, which is composed of T stretch and GC repeat (TTTG/C C/GCGC) in typical E2F targets. In addition, the location of EREA was far upstream from transcription start site compared to that of typical E2F sites, which is within 100 bp from transcription start site in most cases. Moreover, our gel mobility shift assay and ChIP assay showed that EREA specifically binds ectopically expressed E2F1 but not physiological E2F1 induced by serum stimulation, both *in vitro* and *in vivo*, respectively. Taken together, these observations suggest that the *ARF* gene is specifically activated through EREA by de‐ regulated E2F activity, triggered by ectopically expressed E2F1 or forced inactivation of RB, but not by physiological E2F activity, induced by serum stimulation (Figure 7).

A later study showed that ARF promoter lacking EREA was still activated by overexpression of E2F1 [99]. Our further analyses of ARF promoter identified multiples of EREA-like elements in ARF promoter. It seems that, although EREA is the major E2F responsive element in ARF pro‐ moter, it is not the sole responsive element and multiple EREA-like elements co-operate to spe‐ cifically respond to deregulated E2F activity (manuscript in preparation).

Defects in the RB pathway activate E2F out of control by RB, promoting abnormal cell growth. In response to such oncogenic insults, deregulated E2F activates the *ARF* gene, lead‐ ing to activation of p53 to protect cell from tumorigenesis. For induction of tumorigenesis, the p53 pathway must be disabled by further oncogenic changes. Indeed, defects in the p53 pathway are observed in almost all cancers. It is expected that the presence of deregulated E2F be tolerated in cancer cells by further inactivation of the p53 pathway. We thus exam‐ ined the existence of deregulated E2F activity in *RB1* deficient cancer cell lines and in nor‐ mal growing fibroblasts. When we introduced constitutively active form of pRB into *RB1* deficient cancer cell lines (5637, Saos-2 and C-33 A) and normal growing fibroblasts (WI-38 and HFF), activity of EREA and ARF promoter were decreased in *RB1* deficient cancer cell lines, but not in normal growing fibroblasts. These results showed that deregulated E2F ac‐ tivity specifically exists in *RB1* deficient cancer cell lines but not in normal growing fibro‐ blasts. The presence of deregulated E2F activity may serve as a useful marker to discriminate cancer cells from normal growing cells.

**4.3. Difference between deregulated E2F and physiologically activated E2F**

lation of typical E2F target promoter and that of atypical E2F target promoter.

(TTTG/C

distance.

which recognize the sequence and bind.

**1.** Although sequences of EREK and ERE73s resemble that of typical E2F binding sites, se‐ quence of EREA is different from that of typical E2F sites. The sequence of consensus E2F binding motif of typical E2F targets is composed of T stretch and GC repeat

C/GCGC). EREA is composed of only GC repeat and lacks T stretch. Difference in

binding sequence suggests the possibility that there may be a difference in factors,

**2.** In all three cases, location of E2F responsive elements of atypical E2F targets is far from the transcriptional start site compared to that of typical E2F binding sites in typical E2F targets. In the case of typical E2F targets, location of E2F binding sites is within 100 bp from transcriptional start site in most cases. Typical E2F targets are under repression by E2F/RB complex in quiescent phase and are released from repression upon growth stimulation. Close proximity of E2F binding sites to transcriptional start site may be re‐ quired for this mode of regulation. In contrast, atypical E2F targets are not under re‐ pression by E2F/RB complex and literally activated by deregulated E2F. E2F responsive elements of atypical E2F targets behave as enhancer elements, which can function from

**3.** Deregulated E2F1 bound to EREA, EREK and ERE73s and physiologically activated E2F1 did not bind to these elements as shown by ChIP assay. In the case of EREA, it is also shown that repressor type E2F4 does not bind to EREA. This observation is com‐ patible with the observation that these atypical E2F targets are not under repression by

Deregulated E2F and physiologically activated E2F are similar in a sense that both are 're‐ leased from RB'. However, there is a functional difference between deregulated E2F and physiologically activated E2F. Deregulated E2F activates both typical and atypical E2F tar‐ gets. In contrast, physiologically activated E2F activates typical E2F targets but not atypical E2F targets. Why atypical E2F targets are activated by deregulated E2F and not by physio‐ logically activated E2F? What is the difference between deregulated E2F and physiologically activated E2F? Deregulated E2F is totally 'out of control' by RB due to dysfunction of the RB pathway. In contrast, physiologically activated E2F is temporarily released from RB, predict‐ ed to be 'under control' of RB. During normal cell growth, activator E2Fs are induced at G1/S boundary of the cell cycle. At the point, it is generally believed that pRB is phosphory‐ lated and inactivated. However, pRB is not totally inactivated at the point. Previous studies indicate that the *RB1* gene is an E2F target [79] and expression of pRB is increased in G1/S to S phases [80]. Moreover, the amount of activator E2Fs/pRB complex rather increases at G1/S boundary as shown by gel mobility shift assay [78], indicating that some portion of pRB is still active and is regulating the activity of activator E2Fs. Thus, physiologically activated E2F is still under control by pRB. It is likely that activity of activator E2Fs is strictly control‐ led by degree of phosphorylation of pRB dependent on the activity of CDKs, reflecting the strength of growth stimulation. There must be difference between activation of E2F out of control by pRB and activation of E2F under control by pRB. Our studies of regulatory mech‐ anism of atypical target promoters by E2F elucidated the four different points between regu‐

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Our search for new E2F targets with subtraction method identified the CDK inhibitor *p27Kip1* gene as an atypical E2F target [66]. p27Kip1 plays important roles in cell cycle arrest by inhib‐ iting Cyc/CDKs. Using reporter assay, we showed that EREK (E2F responsive element of p27Kip1) was responsible for specifically sensing deregulated E2F activity in human normal fibroblasts and that EREK was specifically activated in the *RB1* deficient cancer cell lines [76]. Consistent with EREA, the location of EREK was far upstream compared to typical E2F binding sites. Interestingly, the sequence of EREK contained T in addition to GC repeat and is rather similar to that of typical E2F binging site. However, EREK bound deregulated E2F1 but not physiological E2F1 in ChIP assay, showing that its character was similar to EREA. These results suggest that not only the sequence of E2F responsive elements, but also the se‐ quence around the responsive elements may be important for discriminating deregulated E2F activity from physiological E2F activity. There is also a possibility that structure of the whole promoter might also affect the discrimination.

Third atypical E2F target is the tumor suppressor *TAp73* gene [77]. TAp73 promoter specifi‐ cally responded to deregulated E2F activity through four ERE73s (E2F responsive elements of TAp73), which were specifically activated by deregulated E2F activity. The sequences of ERE73s contained T stretch and were similar to that of typical E2F binding sites. Important‐ ly, our ChIP assay showed that bindings of ectopically expressed 'exogenous' E2F1 and de‐ regulated 'endogenous' E2F1 induced by adenovirus E1a were detected on ERE73s, but not that of physiological E2F1 induced by serum stimulation. Thus, although the sequences of ERE73s were similar to or almost same as that of typical E2F binding sites, the characters of both were completely different. ERE73s were specifically activated by deregulated E2F ac‐ tivity and specifically bound to both 'exogenous' and 'endogenous' deregulated E2F1. These results support the notion that both sequences of the E2F binding site and its flanking region may be important for discriminating deregulated E2F activity from physiological E2F activi‐ ty. Consistent with EREA and EREK, reporter assay showed that ERE73s were also activated in the *RB1* deficient cancer cell lines and not in normal fibroblasts. Moreover, reintroduction of the constitutive active from of pRB by recombinant adenovirus reduced the expression of the *TAp73* gene in all the cancer cell lines in RT-PCR, indicating that the cancer cell lines har‐ bor deregulated E2F activity that activates the endogenous *TAp73* gene.

Interestingly, our unpublished data suggest that not only the *RB1* deficient cancer cell lines but also cancer cell lines retaining pRB harbor deregulated E2F activity. Activity of ERE73s and expression of the *TAp73* gene were suppressed by introduction of the constitutive active form of pRB in cancer cell lines retaining pRB. These results suggest the possibility that E2Fmediated transcriptional program can sense defects in the RB pathway, not only pRB itself but also upstream regulators of pRB. Taken together, deregulated E2F activity might be‐ come a universal means to discriminate cancer cells (may be regardless of the presence of pRB) from normal growing cells.

## **4.3. Difference between deregulated E2F and physiologically activated E2F**

blasts. The presence of deregulated E2F activity may serve as a useful marker to

Our search for new E2F targets with subtraction method identified the CDK inhibitor *p27Kip1* gene as an atypical E2F target [66]. p27Kip1 plays important roles in cell cycle arrest by inhib‐ iting Cyc/CDKs. Using reporter assay, we showed that EREK (E2F responsive element of p27Kip1) was responsible for specifically sensing deregulated E2F activity in human normal fibroblasts and that EREK was specifically activated in the *RB1* deficient cancer cell lines [76]. Consistent with EREA, the location of EREK was far upstream compared to typical E2F binding sites. Interestingly, the sequence of EREK contained T in addition to GC repeat and is rather similar to that of typical E2F binging site. However, EREK bound deregulated E2F1 but not physiological E2F1 in ChIP assay, showing that its character was similar to EREA. These results suggest that not only the sequence of E2F responsive elements, but also the se‐ quence around the responsive elements may be important for discriminating deregulated E2F activity from physiological E2F activity. There is also a possibility that structure of the

Third atypical E2F target is the tumor suppressor *TAp73* gene [77]. TAp73 promoter specifi‐ cally responded to deregulated E2F activity through four ERE73s (E2F responsive elements of TAp73), which were specifically activated by deregulated E2F activity. The sequences of ERE73s contained T stretch and were similar to that of typical E2F binding sites. Important‐ ly, our ChIP assay showed that bindings of ectopically expressed 'exogenous' E2F1 and de‐ regulated 'endogenous' E2F1 induced by adenovirus E1a were detected on ERE73s, but not that of physiological E2F1 induced by serum stimulation. Thus, although the sequences of ERE73s were similar to or almost same as that of typical E2F binding sites, the characters of both were completely different. ERE73s were specifically activated by deregulated E2F ac‐ tivity and specifically bound to both 'exogenous' and 'endogenous' deregulated E2F1. These results support the notion that both sequences of the E2F binding site and its flanking region may be important for discriminating deregulated E2F activity from physiological E2F activi‐ ty. Consistent with EREA and EREK, reporter assay showed that ERE73s were also activated in the *RB1* deficient cancer cell lines and not in normal fibroblasts. Moreover, reintroduction of the constitutive active from of pRB by recombinant adenovirus reduced the expression of the *TAp73* gene in all the cancer cell lines in RT-PCR, indicating that the cancer cell lines har‐

discriminate cancer cells from normal growing cells.

32 Future Aspects of Tumor Suppressor Gene

whole promoter might also affect the discrimination.

bor deregulated E2F activity that activates the endogenous *TAp73* gene.

pRB) from normal growing cells.

Interestingly, our unpublished data suggest that not only the *RB1* deficient cancer cell lines but also cancer cell lines retaining pRB harbor deregulated E2F activity. Activity of ERE73s and expression of the *TAp73* gene were suppressed by introduction of the constitutive active form of pRB in cancer cell lines retaining pRB. These results suggest the possibility that E2Fmediated transcriptional program can sense defects in the RB pathway, not only pRB itself but also upstream regulators of pRB. Taken together, deregulated E2F activity might be‐ come a universal means to discriminate cancer cells (may be regardless of the presence of Deregulated E2F and physiologically activated E2F are similar in a sense that both are 're‐ leased from RB'. However, there is a functional difference between deregulated E2F and physiologically activated E2F. Deregulated E2F activates both typical and atypical E2F tar‐ gets. In contrast, physiologically activated E2F activates typical E2F targets but not atypical E2F targets. Why atypical E2F targets are activated by deregulated E2F and not by physio‐ logically activated E2F? What is the difference between deregulated E2F and physiologically activated E2F? Deregulated E2F is totally 'out of control' by RB due to dysfunction of the RB pathway. In contrast, physiologically activated E2F is temporarily released from RB, predict‐ ed to be 'under control' of RB. During normal cell growth, activator E2Fs are induced at G1/S boundary of the cell cycle. At the point, it is generally believed that pRB is phosphory‐ lated and inactivated. However, pRB is not totally inactivated at the point. Previous studies indicate that the *RB1* gene is an E2F target [79] and expression of pRB is increased in G1/S to S phases [80]. Moreover, the amount of activator E2Fs/pRB complex rather increases at G1/S boundary as shown by gel mobility shift assay [78], indicating that some portion of pRB is still active and is regulating the activity of activator E2Fs. Thus, physiologically activated E2F is still under control by pRB. It is likely that activity of activator E2Fs is strictly control‐ led by degree of phosphorylation of pRB dependent on the activity of CDKs, reflecting the strength of growth stimulation. There must be difference between activation of E2F out of control by pRB and activation of E2F under control by pRB. Our studies of regulatory mech‐ anism of atypical target promoters by E2F elucidated the four different points between regu‐ lation of typical E2F target promoter and that of atypical E2F target promoter.


E2F/RB. The fact that deregulated E2F bind to atypical E2F targets, while physiological E2F does not bind to atypical E2F targets, suggest that there is difference in binding be‐ havior between deregulated E2F and physiologically activated E2F.

**4.** Regulatory mechanism of promoters is different between typical E2F targets and atypi‐ cal E2F targets. Promoters of typical E2F targets are repressed by E2F/RB complex. Growth stimulation inactivates RB and releases promoters from the repression by RB. Thus, so-called activation of typical E2F targets by physiological E2F is 'release from re‐ pression by RB'. In contrast, activation of atypical E2F targets by deregulated E2F is lit‐ erally 'activation'. Mutation of EREA, EREK or ERE73s in corresponding full-length promoter did not enhance basal promoter activities, indicating that these three promot‐ ers are not under repression through the E2F responsive elements. This is consistent with the observation that binding of physiological E2F to promoters of atypical E2F tar‐

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35

gets was not observed in ChIP assay, including repressor type E2F4 (Figure 9).

ulation from physiological growth stimulation.

**5. Conclusion and further research**

It is generally accepted that the amount of free E2F is important for differential regula‐ tion of E2F targets, which have opposite roles in cell-fate determination, as proposed as threshold model [100]. According to this model, when the amount of free E2F (released from repression of RB) is below the threshold, E2F activates only growth-related target genes. When the amount of free E2F exceeds the threshold, E2F activates not only growth-related targets but also pro-apoptotic targets. However, molecular mechanism of how different amount of free E2F differentially regulates target genes is not yet elucidat‐ ed. The 'quantitative' difference of free E2F seems not sufficient to explain the four differ‐ ences between deregulated E2F and physiologically activated E2F. The above-mentioned differences strongly suggest the presence of qualitative difference between physiologically activated E2F by 'temporal release' from RB and deregulated E2F induced by 'dysfunc‐ tion' of the RB pathway [75-77] (Figure 9). The 'qualitative' difference between both of the E2Fs seems to be a useful cue to elucidate how cells discriminate oncogenic growth stim‐

Accumulating evidence indicates that E2F plays essential roles in cell-fate determination, to grow, stop or die. Together with G1/S gatekeeper RB, E2F governs control of the restriction point, deciding whether to grow or not. Upon normal growth stimulation, physiologically acti‐ vated E2F facilitates cell proliferation. Upon dysfunction of the RB pathway, deregulated E2F suppresses cell growth by inducing p27Kip1 to restrain cells from aberrant cell growth. p21Cip1, in‐ duced by p53 through activation of the *ARF* gene, may also contribute to suppression of cell growth. When the arrest mechanism failed to stop the aberrant cell cycle progression, E2F indu‐ ces apoptosis through activation of p53 and TAp73 to protect cells from tumorigenesis. E2F seems to sense and discriminate between normal growth signals and abnormal growth signals

originating from various oncogenic changes, asking cells whether to grow, stop or die.

Deregulated E2F activity specifically exists in cancer cell lines but not in normal growing fi‐ broblasts, suggesting that deregulated E2F activity may be a useful means to discriminate abnormally growing cancer cells from physiologically growing normal cells. Since the gen‐ eration of deregulated E2F activity is expected to be based on the mechanism of oncogene‐ sis, deregulated E2F activity could be a universal marker to discriminate cancer cells from

**Figure 9.** Differences in E2F regulation of target promoters between deregulated E2F and physiological E2F. (1) Se‐ quence of the atypical E2F responsive elements and surrounding regions are different from that of typical E2F targets. (2) E2F responsive elements of the atypical E2F targets locate far upstream from transcriptional start sites compared to that of typical E2F targets. (3) E2F responsive elements of atypical E2F targets specifically bind deregulated E2F and not physiologically activated E2F. (4) Unlike typical E2F targets, promoters of atypical E2F targets are not under repres‐ sion of RB and are specifically activated by deregulated E2F activity.

**4.** Regulatory mechanism of promoters is different between typical E2F targets and atypi‐ cal E2F targets. Promoters of typical E2F targets are repressed by E2F/RB complex. Growth stimulation inactivates RB and releases promoters from the repression by RB. Thus, so-called activation of typical E2F targets by physiological E2F is 'release from re‐ pression by RB'. In contrast, activation of atypical E2F targets by deregulated E2F is lit‐ erally 'activation'. Mutation of EREA, EREK or ERE73s in corresponding full-length promoter did not enhance basal promoter activities, indicating that these three promot‐ ers are not under repression through the E2F responsive elements. This is consistent with the observation that binding of physiological E2F to promoters of atypical E2F tar‐ gets was not observed in ChIP assay, including repressor type E2F4 (Figure 9).

It is generally accepted that the amount of free E2F is important for differential regula‐ tion of E2F targets, which have opposite roles in cell-fate determination, as proposed as threshold model [100]. According to this model, when the amount of free E2F (released from repression of RB) is below the threshold, E2F activates only growth-related target genes. When the amount of free E2F exceeds the threshold, E2F activates not only growth-related targets but also pro-apoptotic targets. However, molecular mechanism of how different amount of free E2F differentially regulates target genes is not yet elucidat‐ ed. The 'quantitative' difference of free E2F seems not sufficient to explain the four differ‐ ences between deregulated E2F and physiologically activated E2F. The above-mentioned differences strongly suggest the presence of qualitative difference between physiologically activated E2F by 'temporal release' from RB and deregulated E2F induced by 'dysfunc‐ tion' of the RB pathway [75-77] (Figure 9). The 'qualitative' difference between both of the E2Fs seems to be a useful cue to elucidate how cells discriminate oncogenic growth stim‐ ulation from physiological growth stimulation.

## **5. Conclusion and further research**

E2F/RB. The fact that deregulated E2F bind to atypical E2F targets, while physiological E2F does not bind to atypical E2F targets, suggest that there is difference in binding be‐

**Figure 9.** Differences in E2F regulation of target promoters between deregulated E2F and physiological E2F. (1) Se‐ quence of the atypical E2F responsive elements and surrounding regions are different from that of typical E2F targets. (2) E2F responsive elements of the atypical E2F targets locate far upstream from transcriptional start sites compared to that of typical E2F targets. (3) E2F responsive elements of atypical E2F targets specifically bind deregulated E2F and not physiologically activated E2F. (4) Unlike typical E2F targets, promoters of atypical E2F targets are not under repres‐

sion of RB and are specifically activated by deregulated E2F activity.

havior between deregulated E2F and physiologically activated E2F.

34 Future Aspects of Tumor Suppressor Gene

Accumulating evidence indicates that E2F plays essential roles in cell-fate determination, to grow, stop or die. Together with G1/S gatekeeper RB, E2F governs control of the restriction point, deciding whether to grow or not. Upon normal growth stimulation, physiologically acti‐ vated E2F facilitates cell proliferation. Upon dysfunction of the RB pathway, deregulated E2F suppresses cell growth by inducing p27Kip1 to restrain cells from aberrant cell growth. p21Cip1, in‐ duced by p53 through activation of the *ARF* gene, may also contribute to suppression of cell growth. When the arrest mechanism failed to stop the aberrant cell cycle progression, E2F indu‐ ces apoptosis through activation of p53 and TAp73 to protect cells from tumorigenesis. E2F seems to sense and discriminate between normal growth signals and abnormal growth signals originating from various oncogenic changes, asking cells whether to grow, stop or die.

Deregulated E2F activity specifically exists in cancer cell lines but not in normal growing fi‐ broblasts, suggesting that deregulated E2F activity may be a useful means to discriminate abnormally growing cancer cells from physiologically growing normal cells. Since the gen‐ eration of deregulated E2F activity is expected to be based on the mechanism of oncogene‐ sis, deregulated E2F activity could be a universal marker to discriminate cancer cells from normal growing cells. Analyses of atypical E2F targets suggest that deregulated E2F might be qualitatively different from physiologically activated E2F. One of the most intriguing is‐ sues in the future studies would be the molecular nature of deregulated E2F. By elucidating the molecular nature of deregulated E2F, we might be able to specifically approach cancer cells without affecting normal growing cells. For this purpose, qualitative difference be‐ tween deregulated E2F and physiological E2F is eager to be elucidated.

[7] Yamasaki L, Jacks T, Bronson R, Goillot E, Harlow E, Dyson NJ. Tumor induction

To Grow, Stop or Die? – Novel Tumor-Suppressive Mechanism Regulated by the Transcription Factor E2F

http://dx.doi.org/10.5772/54510

37

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## **Acknowledgements**

This work is supported by a Grant in-Aid for Science Research from the Ministry of Educa‐ tion, Culture, Sports, Science and Technology of Japan (21570180 to K. Ohtani and 22 2446 to E. Ozono)

## **Author details**

Eiko Ozono1,2, Shoji Yamaoka2 and Kiyoshi Ohtani1

1 Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, Japan

2 Department of Molecular Virology, Tokyo Medical and Dental University, Japan

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[87] Thomas DM, Johnson SA, Sims NA, Trivett MK, Slavin JL, Rubin BP, et al. Terminal osteoblast differentiation, mediated by runx2 and p27KIP1, is disrupted in osteosarco‐

[88] Binne UK, Classon MK, Dick FA, Wei W, Rape M, Kaelin WG Jr, et al. Retinoblasto‐ ma protein and anaphase-promoting complex physically interact and functionally

[89] Murray-Zmijewski F, Lane DP, Bourdon JC. p53/p63/p73 isoforms: an orchestra of isoforms to harmonise cell differentiation and response to stress. Cell Death Differ.

[90] Pietsch EC, Sykes SM, McMahon SB, Murphy ME. The p53 family and programmed

[91] Irwin MS, Kaelin WG. p53 family update: p73 and p63 develop their own identities.

cooperate during cell-cycle exit. Nat Cell Biol 2007;9(2) 225-232.

during specific phases of the cell cycle. Cell. 1989 ;58(6) 1097-1105.

1996;93(8) 3215-3220.

42 Future Aspects of Tumor Suppressor Gene

2009;9(11) 821-829.

663-673.

Growth Differ. 1994;5(5) 467-474.

product p19*ARF*. Cell. 1997;91(5) 649-659.

Sci U S A. 2003;100(26) 15930-15935.

senescence. Mol Cell Biol. 2001;21(11) 3616-3631.

trance Rb mutant. Mol Cell. 2004;16(1) 47-58.

cell death. Oncogene. 2008;27(50) 6507-6521.

Cell Growth Differ. 2001;12(7) 337-349.

ma. J Cell Biol. 2004;167(5) 925-934.

2006;13(6) 962-972.


**Chapter 3**

**MicroRNAs and lncRNAs as Tumour Suppressors**

Cancer is one of the most serious diseases around the world and it is the third leading cause of death, exceeded only by heart and infectious diseases [1]. There are five major steps for cancer development: initiation, promotion, malignant conversion, progression, and metastasis [2]. Cancer is result of process, where somatic cells mutate and escape the controlled balance of gene expression and cellular networks that maintain cellular homeostasis, which normally prevent unwanted expansion. Perturbations in these pathways results in cellular transforma‐ tion, where cancer cells differ from their normal counterparts in many characteristics, as is loss of differentiation, increased invasiveness, and decreased drug sensitivity [3-4]. There are six primary hallmarks of cancer: unlimited cell proliferation, autonomous growth without the need of external signals, resistance to growth inhibitory signals, escape from apoptosis the ability to recruit new vasculature and increased tissue invasion and metastasis [5]. The formation of cancer is therefore fundamentally genetic and epigenetic disease requiring accumulation of genomic alterations to inactivate tumour suppressor and activate protooncogenes [6]. These results in combined interaction of both tumour suppressors, that are not able to inhibit tumour development and protect cells against mutation that initiate transfor‐ mation, and cancer inducers, which promotes cancer development as initiators of cellular transformation. When cells exhibit abnormal growth and loss of apoptosis, it usually results

Genetic studies have revealed the mutational and epigenetic alterations of protein-coding genes that control DNA damage response, growth arrest, cell survival and apoptotic pathways [4]. Until recent years ago, the central dogma of molecular biology was that genetic information is stored in protein-coding genes with RNA as an intermediate between DNA sequence and its encoded protein [7]. Recent studies suggest that advanced stages of cancer are possessing more severe molecular perturbations and that this could be due to function of non-coding RNAs (ncRNAs), which were previously known only to have infrastructural functions (as

> © 2013 Boštjančič and Glavač; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

> © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2013 Boštjančič and Glavač; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Emanuela Boštjančič and Damjan Glavač

http://dx.doi.org/10.5772/54701

**1. Introduction**

in cancer formation [2].

Additional information is available at the end of the chapter

## **MicroRNAs and lncRNAs as Tumour Suppressors**

Emanuela Boštjančič and Damjan Glavač

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54701

## **1. Introduction**

Cancer is one of the most serious diseases around the world and it is the third leading cause of death, exceeded only by heart and infectious diseases [1]. There are five major steps for cancer development: initiation, promotion, malignant conversion, progression, and metastasis [2]. Cancer is result of process, where somatic cells mutate and escape the controlled balance of gene expression and cellular networks that maintain cellular homeostasis, which normally prevent unwanted expansion. Perturbations in these pathways results in cellular transforma‐ tion, where cancer cells differ from their normal counterparts in many characteristics, as is loss of differentiation, increased invasiveness, and decreased drug sensitivity [3-4]. There are six primary hallmarks of cancer: unlimited cell proliferation, autonomous growth without the need of external signals, resistance to growth inhibitory signals, escape from apoptosis the ability to recruit new vasculature and increased tissue invasion and metastasis [5]. The formation of cancer is therefore fundamentally genetic and epigenetic disease requiring accumulation of genomic alterations to inactivate tumour suppressor and activate protooncogenes [6]. These results in combined interaction of both tumour suppressors, that are not able to inhibit tumour development and protect cells against mutation that initiate transfor‐ mation, and cancer inducers, which promotes cancer development as initiators of cellular transformation. When cells exhibit abnormal growth and loss of apoptosis, it usually results in cancer formation [2].

Genetic studies have revealed the mutational and epigenetic alterations of protein-coding genes that control DNA damage response, growth arrest, cell survival and apoptotic pathways [4]. Until recent years ago, the central dogma of molecular biology was that genetic information is stored in protein-coding genes with RNA as an intermediate between DNA sequence and its encoded protein [7]. Recent studies suggest that advanced stages of cancer are possessing more severe molecular perturbations and that this could be due to function of non-coding RNAs (ncRNAs), which were previously known only to have infrastructural functions (as

© 2013 Boštjančič and Glavač; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Boštjančič and Glavač; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ribosomal RNA, transfer RNA, small nuclear and nucleolar RNA). Eukaryotic genomes are extensively transcribed into thousands of long and short ncRNAs, which are group of endogenous RNAs that also function as regulators of gene expression. They are involved in developmental, physiological as well as pathological processes [7,8].

**Functional role**

**RNA interference (RNAi)**

components of RNAi pathway [11].

**2.1. Brief introduction to miRNAs**

**Genomic organization**

miRNA [13,14].

The most widely studied and characterized of all the regulatory ncRNAs are miRNAs. The roles of regulatory ncRNAs, other than miRNAs, in the mediating transcriptional regulation, chromatin remodelling, post-transcriptional regulation, and other processes are less well understood. The contexts of gene regulation by ncRNAs in non-human systems provided insights into how these processes could function in human cells. Regulatory ncRNAs are involved in diverse cellular pathways, such as development and stem cell maintenance, response to stress and environmental stimuli, regulating chromatin structure and remodelling, chromosome architecture and genome integrity, transcription (positive or negative impact), and post-transcription processing (splicing, transport) and most commonly mRNA stability (translation, degradation) [8,9]. Some ncRNAs trigger different types of gene silencing that are

MicroRNAs and lncRNAs as Tumour Suppressors

http://dx.doi.org/10.5772/54701

47

RNAi is RNA-guided regulation of gene expression, historically known by other names, including post-transcriptional gene silencing. It is believed to be an evolutionary conserved mechanism in response to presence of foreign dsRNA in the cell. A key step in this silencing pathway is the processing of dsRNAs into short RNA duplexes of characteristic size and structure. The enzyme Dicer, which initiates the RNAi pathway, cleaves dsRNA to short double-stranded fragments of 20–25 base pairs (bp), named siRNAs. siRNAs usually possess perfect complementarity to the mRNA of target gene, thus causing its degradation. When the dsRNA is exogenous, coming from infection by a virus with RNA genome or laboratory manipulations, the RNA is imported directly into the cytoplasm where it is cleave by the Dicer. On other hand, the initiating dsRNA could be result of endogenously expressed RNA-coding genes from the genome. Some of small regulatory RNAs are processed in a similar way or with

miRNAs are endogenously expressed small (~22 nt), single-stranded ncRNAs. It is predicted that they constitute ~1-5 % of human genes [1,12] and in an update from August 2012, miRBase v19 was released with a list of 2019 unique mature human miRNAs. miRNAs are encoded as a single gene or gene clusters, with some of miRNA clusters being co-regulated and cotranscribed. Intergenic miRNAs are transcribed as an independent transcription unit, as a monocistronic, bicistronic or polycistronic primary transcripts [13]. Up to 60 % of currently known miRNAs are proposed to be from intronic sequences of either protein coding or noncoding transcription units and suggestion has been made that some miRNAs are also encoded in antisense DNA, which is not transcribed to the mRNA. Intronic miRNA are preferentially transcribed in the same orientation as the host gene and are together with their host transcripts co-regulated and co-transcribed from the same promoter. They are processed from introns, as are many snoRNA. Within the genome, there might be more than one copy of particular

collectively referred to as RNA silencing or RNA interference [11].

However, in this review, the following characteristics of ncRNA in human cancers will be summarized: (i) the current understanding of the critical role that lncRNAs and miRNAs may play in cancer as tumour suppressors; (ii) outline current knowledge about some specific lncRNA and miRNAs and their target genes in cancer; (iii) highlight their potential as bio‐ markers for patho-histological subtype classification; and (iv) highlight their potential as biomarkers and as circulating biomarkers and therapeutic targets in cancer.

Since the majority of research regarding regulatory ncRNAs as tumour suppressor was performed on miRNAs and in lesser extend on lncRNAs/lincRNAs, will this review further focused on these two groups of ncRNAs.

## **2. Brief overview of non-coding RNAs (ncRNAs)**

## **Classification**

Of transcribed eukaryotic genomes, only 1-2 % encode for proteins, whereas the vast majority are ncRNAs that are in more or less functional transcripts. The regulatory ncRNAs are important regulators of gene expression in many eukaryotes and are involved in a wide range of functions in eukaryotic biology [8,9].

Based on their function, ncRNAs can be divided into two groups. First is infrastructural group, with ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs). Second is regulatory group, with microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), small-interfering RNAs (siRNAs), long non-coding RNAs (lncRNAs), large intergenic non-coding RNAs (lincRNAs), promoter-associated small RNAs (PARs), repeat-associated short interfering RNAs (rasiRNAs) and enhancer RNAs (eRNAs) [8,9]. Recent findings suggest that some structural ncRNAs (e.g. snoRNAs) not only have infrastructural function but have regulatory as well [8].

Based on length, the regulatory ncRNAs can be divided in two groups: larger than 200 nucleotides (nt) are lncRNA, lincRNA, eRNA, whereas the others are smaller than 200 nt, with exception of PARs that are 16-30 nt long or up to 200 nt. Distinct classes of small RNAs are distinguished by their origins, and these are: snRNAs, snoRNAs, miRNAs, piRNAs, siRNAs, and rasiRNAs [8,9].

miRNAs and snoRNAs share similarities in processing pathways and protein interaction partners, genomic organization and location, as well as levels of conservation. However, similarities in sub-cellular localization have been also observed, since large proportion of human mature miRNAs have been detected in the nucleus as well as a subset of small RNAs derived from snoRNAs have been detected in the cytoplasm [10].

#### **Functional role**

ribosomal RNA, transfer RNA, small nuclear and nucleolar RNA). Eukaryotic genomes are extensively transcribed into thousands of long and short ncRNAs, which are group of endogenous RNAs that also function as regulators of gene expression. They are involved in

However, in this review, the following characteristics of ncRNA in human cancers will be summarized: (i) the current understanding of the critical role that lncRNAs and miRNAs may play in cancer as tumour suppressors; (ii) outline current knowledge about some specific lncRNA and miRNAs and their target genes in cancer; (iii) highlight their potential as bio‐ markers for patho-histological subtype classification; and (iv) highlight their potential as

Since the majority of research regarding regulatory ncRNAs as tumour suppressor was performed on miRNAs and in lesser extend on lncRNAs/lincRNAs, will this review further

Of transcribed eukaryotic genomes, only 1-2 % encode for proteins, whereas the vast majority are ncRNAs that are in more or less functional transcripts. The regulatory ncRNAs are important regulators of gene expression in many eukaryotes and are involved in a wide range

Based on their function, ncRNAs can be divided into two groups. First is infrastructural group, with ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs). Second is regulatory group, with microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), small-interfering RNAs (siRNAs), long non-coding RNAs (lncRNAs), large intergenic non-coding RNAs (lincRNAs), promoter-associated small RNAs (PARs), repeat-associated short interfering RNAs (rasiRNAs) and enhancer RNAs (eRNAs) [8,9]. Recent findings suggest that some structural ncRNAs (e.g. snoRNAs) not only have

Based on length, the regulatory ncRNAs can be divided in two groups: larger than 200 nucleotides (nt) are lncRNA, lincRNA, eRNA, whereas the others are smaller than 200 nt, with exception of PARs that are 16-30 nt long or up to 200 nt. Distinct classes of small RNAs are distinguished by their origins, and these are: snRNAs, snoRNAs, miRNAs, piRNAs, siRNAs,

miRNAs and snoRNAs share similarities in processing pathways and protein interaction partners, genomic organization and location, as well as levels of conservation. However, similarities in sub-cellular localization have been also observed, since large proportion of human mature miRNAs have been detected in the nucleus as well as a subset of small RNAs

developmental, physiological as well as pathological processes [7,8].

biomarkers and as circulating biomarkers and therapeutic targets in cancer.

**2. Brief overview of non-coding RNAs (ncRNAs)**

infrastructural function but have regulatory as well [8].

derived from snoRNAs have been detected in the cytoplasm [10].

focused on these two groups of ncRNAs.

46 Future Aspects of Tumor Suppressor Gene

of functions in eukaryotic biology [8,9].

**Classification**

and rasiRNAs [8,9].

The most widely studied and characterized of all the regulatory ncRNAs are miRNAs. The roles of regulatory ncRNAs, other than miRNAs, in the mediating transcriptional regulation, chromatin remodelling, post-transcriptional regulation, and other processes are less well understood. The contexts of gene regulation by ncRNAs in non-human systems provided insights into how these processes could function in human cells. Regulatory ncRNAs are involved in diverse cellular pathways, such as development and stem cell maintenance, response to stress and environmental stimuli, regulating chromatin structure and remodelling, chromosome architecture and genome integrity, transcription (positive or negative impact), and post-transcription processing (splicing, transport) and most commonly mRNA stability (translation, degradation) [8,9]. Some ncRNAs trigger different types of gene silencing that are collectively referred to as RNA silencing or RNA interference [11].

#### **RNA interference (RNAi)**

RNAi is RNA-guided regulation of gene expression, historically known by other names, including post-transcriptional gene silencing. It is believed to be an evolutionary conserved mechanism in response to presence of foreign dsRNA in the cell. A key step in this silencing pathway is the processing of dsRNAs into short RNA duplexes of characteristic size and structure. The enzyme Dicer, which initiates the RNAi pathway, cleaves dsRNA to short double-stranded fragments of 20–25 base pairs (bp), named siRNAs. siRNAs usually possess perfect complementarity to the mRNA of target gene, thus causing its degradation. When the dsRNA is exogenous, coming from infection by a virus with RNA genome or laboratory manipulations, the RNA is imported directly into the cytoplasm where it is cleave by the Dicer. On other hand, the initiating dsRNA could be result of endogenously expressed RNA-coding genes from the genome. Some of small regulatory RNAs are processed in a similar way or with components of RNAi pathway [11].

#### **2.1. Brief introduction to miRNAs**

#### **Genomic organization**

miRNAs are endogenously expressed small (~22 nt), single-stranded ncRNAs. It is predicted that they constitute ~1-5 % of human genes [1,12] and in an update from August 2012, miRBase v19 was released with a list of 2019 unique mature human miRNAs. miRNAs are encoded as a single gene or gene clusters, with some of miRNA clusters being co-regulated and cotranscribed. Intergenic miRNAs are transcribed as an independent transcription unit, as a monocistronic, bicistronic or polycistronic primary transcripts [13]. Up to 60 % of currently known miRNAs are proposed to be from intronic sequences of either protein coding or noncoding transcription units and suggestion has been made that some miRNAs are also encoded in antisense DNA, which is not transcribed to the mRNA. Intronic miRNA are preferentially transcribed in the same orientation as the host gene and are together with their host transcripts co-regulated and co-transcribed from the same promoter. They are processed from introns, as are many snoRNA. Within the genome, there might be more than one copy of particular miRNA [13,14].

#### **Biogenesis**

miRNAs expression is determined by intrinsic cellular factors and diverse environmental variables [1]. As for protein-coding genes it is known, that regulation of miRNA tran‐ scription and expression depends on transcription factors and epigenetic mechanisms (e.g. p53, Myc, and myogenin). In general, from genes encoding miRNAs is transcription guided by RNA-polymerase II (Pol II). Resulting primary transcript (several hundred bas‐ es to several kilobases), named *pri-miRNA*, forms distinctive hairpin-shaped stem-loop secondary structure and contains poly-A tail and a cap, similarly to protein-coding mRNA. *pri-miRNA* is processed in the nucleus by Drosha, an RNase III enzyme. The re‐ sulting 70-nt stem-loop structure called *pre-miRNA* with a 5' phosphate and 3' 2-nt over‐ hang is imported into the cytoplasm by a transporter protein, Exportin 5. The doublestranded RNA portion of *pre-miRNA* is bound and cleaved by Dicer, another RNase III enzyme, which produces a miRNA:miRNA\* duplex (a transient intermediate in miRNA biogenesis, 20–25 nt). One of the two strands of each fragment is together with proteins argonaute (Ago), incorporated into a complex called the miRNA-containing ribonucleo‐ protein complex (miRNP). It is believed that the *guide strand* is determined on the basis of the less energetically stable 5' end. The resulting complex base-pair with complementa‐ ry 3'-UTR mRNA sequences. The other strand, miRNA\* is presumably degraded, al‐ though there are increasing evidence that either or both strands may be functional [2,9]. The schematic overview of canonical miRNA biosynthesis pathway has been represented elsewhere [15].

miRNAs. Proteins or mRNA secondary structures could restrict miRNP accessibility to the

MicroRNAs and lncRNAs as Tumour Suppressors

http://dx.doi.org/10.5772/54701

49

There is the prospect that some miRNA might specify more than just post-transcriptional repression [9,13]. miRNAs may also target promoter to regulate transcription through epigenetic mechanism. miRNAs have been paradoxically also shown to up-regulate gene

Translational repression, as major mechanism of miRNAs, may in normal cell conditions occur in different ways: as switch off the targets, that is for mRNAs that should not be expressed in a particular cell type, the protein production is reduced to inconsequential levels; as fine-tuners of target expression, that is when miRNAs can adjust protein output for customized expression in different cell types; as neutralizers of target expression, that is when miRNAs act as bystanders, where down-regulation by miRNAs is tolerated or reversed by feedback processes [13]. Role of miRNA can be further divided in three paradigms: combinatorial control (defined as cooperativity), cell-to-cell variation, specific (tissue-specific and/or cell-type specific) and

Despite the large number of identified miRNAs, the scope of their roles in regulating cellular gene expression is not fully understood [11]. It is believed that miRNAs through negative gene regulation influence at least 50 % of genes within the human genome [9]. Expression profiling of many miRNAs in various normal and diseased tissues have demonstrated unique spatial and temporal expression patterns. Many miRNAs are important at distinct stages of develop‐ ment and have been found to regulate a variety of physiological and pathological processes [11]. miRNAs are involved in a numerous biological processes, such as stem cell division and developmental timing, proper organ formation, embryonic pattering and body growth, proliferation and differentiation, apoptosis, epithelial-mesenchymal-transition (EMT), cholesterol metabolism and regulation of insulin secretion, resistance to viral infection and oxidative stress, immune response etc. [2,11]. All these effects may occur by regulating or being regulated by the expression of signalling molecules, such as cytokines, growth factors, transcription factors, pro-apoptotic and anti-apoptotic genes [21]. With all different genes and expression patterns, it is reasonable to propose that every cell type at each developmental stage

Up to date, over 2000 human miRNAs have been identified and this number is still growing. All annotated miRNAs are collected in miRBase [22]. The first step in miRNA target identification is usually defining reciprocally regulated miRNA-mRNA or miRNAprotein. Since miRNAs target mRNA mainly by incomplete base-pairing, many computa‐ tional methods have been recently developed for further identifying potential miRNA targets [23]. Most of these methods search for three criteria in predicting miRNA target genes: first, multiple conserved regions of miRNA complementarities within 3'-UTR of target mRNA (evolutionary conservation); second, interaction between seven consecutive nucleotides in the target mRNAs 3'-UTR and the 1-8 nt ("seed sequence") at the 5' miR‐

UTRs, or may facilitate recognition of the authentic mRNA targets [12,13,18,19].

expression by enhancing translation under specific conditions [9].

**Biological function**

housekeeping functions [20].

might have a distinct miRNA expression profile.

**Defining miRNA targets and databases**

Numerous alternative pathways differing from canonical miRNA biogenesis pathway have been described recently and subset of several diverse longer non-coding RNAs can serve as precursors for miRNAs [10,16]. As an example, intronic miRNAs presumably by‐ pass Drosha cleavage, since through *pre-mRNA* splicing/debranching machinery is pro‐ duced an approx. 60-nt hairpin precursor miRNA (*pre-miRNA*) that enter biogenesis pathway at the step of Exportin 5 [14]. However, some of the post-transcriptional mecha‐ nism include miRNA editing, which is mechanism mediated by adenine deaminase of al‐ teration of adenines to inosines, and not yet thoroughly studied regulations of miRNA, such as export step from nucleus or miRNAs turnover rate [17].

#### **miRNAs mechanism**

The functional role of miRNA varies, but the primary mechanism of miRNA action in mammals is believed to be base-pairing to 3'-UTR of target mRNA followed by inhibition of mRNA translation (when base pairing between these two molecules is incomplete) or deade‐ nylation and degradation (perfect complementarity of miRNA:mRNA binding) [9]. Especially in animals, the primary mechanism of miRNA action is reducing mRNA translation and each miRNA can inhibit the translation of as many as 200 target genes. In addition, mRNA can be regulated by more than one miRNA. The cooperative action of multiple identical (multiplicity) or different miRNPs (cooperativity) appears to provide the most efficient translational inhibition. Additional mechanism to increase the specificity of miRNAs is combinatorial control of gene expression, which may be also provided by a set of co-ordinately expressed miRNAs. Proteins or mRNA secondary structures could restrict miRNP accessibility to the UTRs, or may facilitate recognition of the authentic mRNA targets [12,13,18,19].

There is the prospect that some miRNA might specify more than just post-transcriptional repression [9,13]. miRNAs may also target promoter to regulate transcription through epigenetic mechanism. miRNAs have been paradoxically also shown to up-regulate gene expression by enhancing translation under specific conditions [9].

## **Biological function**

**Biogenesis**

48 Future Aspects of Tumor Suppressor Gene

elsewhere [15].

**miRNAs mechanism**

miRNAs expression is determined by intrinsic cellular factors and diverse environmental variables [1]. As for protein-coding genes it is known, that regulation of miRNA tran‐ scription and expression depends on transcription factors and epigenetic mechanisms (e.g. p53, Myc, and myogenin). In general, from genes encoding miRNAs is transcription guided by RNA-polymerase II (Pol II). Resulting primary transcript (several hundred bas‐ es to several kilobases), named *pri-miRNA*, forms distinctive hairpin-shaped stem-loop secondary structure and contains poly-A tail and a cap, similarly to protein-coding mRNA. *pri-miRNA* is processed in the nucleus by Drosha, an RNase III enzyme. The re‐ sulting 70-nt stem-loop structure called *pre-miRNA* with a 5' phosphate and 3' 2-nt over‐ hang is imported into the cytoplasm by a transporter protein, Exportin 5. The doublestranded RNA portion of *pre-miRNA* is bound and cleaved by Dicer, another RNase III enzyme, which produces a miRNA:miRNA\* duplex (a transient intermediate in miRNA biogenesis, 20–25 nt). One of the two strands of each fragment is together with proteins argonaute (Ago), incorporated into a complex called the miRNA-containing ribonucleo‐ protein complex (miRNP). It is believed that the *guide strand* is determined on the basis of the less energetically stable 5' end. The resulting complex base-pair with complementa‐ ry 3'-UTR mRNA sequences. The other strand, miRNA\* is presumably degraded, al‐ though there are increasing evidence that either or both strands may be functional [2,9]. The schematic overview of canonical miRNA biosynthesis pathway has been represented

Numerous alternative pathways differing from canonical miRNA biogenesis pathway have been described recently and subset of several diverse longer non-coding RNAs can serve as precursors for miRNAs [10,16]. As an example, intronic miRNAs presumably by‐ pass Drosha cleavage, since through *pre-mRNA* splicing/debranching machinery is pro‐ duced an approx. 60-nt hairpin precursor miRNA (*pre-miRNA*) that enter biogenesis pathway at the step of Exportin 5 [14]. However, some of the post-transcriptional mecha‐ nism include miRNA editing, which is mechanism mediated by adenine deaminase of al‐ teration of adenines to inosines, and not yet thoroughly studied regulations of miRNA,

The functional role of miRNA varies, but the primary mechanism of miRNA action in mammals is believed to be base-pairing to 3'-UTR of target mRNA followed by inhibition of mRNA translation (when base pairing between these two molecules is incomplete) or deade‐ nylation and degradation (perfect complementarity of miRNA:mRNA binding) [9]. Especially in animals, the primary mechanism of miRNA action is reducing mRNA translation and each miRNA can inhibit the translation of as many as 200 target genes. In addition, mRNA can be regulated by more than one miRNA. The cooperative action of multiple identical (multiplicity) or different miRNPs (cooperativity) appears to provide the most efficient translational inhibition. Additional mechanism to increase the specificity of miRNAs is combinatorial control of gene expression, which may be also provided by a set of co-ordinately expressed

such as export step from nucleus or miRNAs turnover rate [17].

Translational repression, as major mechanism of miRNAs, may in normal cell conditions occur in different ways: as switch off the targets, that is for mRNAs that should not be expressed in a particular cell type, the protein production is reduced to inconsequential levels; as fine-tuners of target expression, that is when miRNAs can adjust protein output for customized expression in different cell types; as neutralizers of target expression, that is when miRNAs act as bystanders, where down-regulation by miRNAs is tolerated or reversed by feedback processes [13]. Role of miRNA can be further divided in three paradigms: combinatorial control (defined as cooperativity), cell-to-cell variation, specific (tissue-specific and/or cell-type specific) and housekeeping functions [20].

Despite the large number of identified miRNAs, the scope of their roles in regulating cellular gene expression is not fully understood [11]. It is believed that miRNAs through negative gene regulation influence at least 50 % of genes within the human genome [9]. Expression profiling of many miRNAs in various normal and diseased tissues have demonstrated unique spatial and temporal expression patterns. Many miRNAs are important at distinct stages of develop‐ ment and have been found to regulate a variety of physiological and pathological processes [11]. miRNAs are involved in a numerous biological processes, such as stem cell division and developmental timing, proper organ formation, embryonic pattering and body growth, proliferation and differentiation, apoptosis, epithelial-mesenchymal-transition (EMT), cholesterol metabolism and regulation of insulin secretion, resistance to viral infection and oxidative stress, immune response etc. [2,11]. All these effects may occur by regulating or being regulated by the expression of signalling molecules, such as cytokines, growth factors, transcription factors, pro-apoptotic and anti-apoptotic genes [21]. With all different genes and expression patterns, it is reasonable to propose that every cell type at each developmental stage might have a distinct miRNA expression profile.

#### **Defining miRNA targets and databases**

Up to date, over 2000 human miRNAs have been identified and this number is still growing. All annotated miRNAs are collected in miRBase [22]. The first step in miRNA target identification is usually defining reciprocally regulated miRNA-mRNA or miRNAprotein. Since miRNAs target mRNA mainly by incomplete base-pairing, many computa‐ tional methods have been recently developed for further identifying potential miRNA targets [23]. Most of these methods search for three criteria in predicting miRNA target genes: first, multiple conserved regions of miRNA complementarities within 3'-UTR of target mRNA (evolutionary conservation); second, interaction between seven consecutive nucleotides in the target mRNAs 3'-UTR and the 1-8 nt ("seed sequence") at the 5' miR‐ NA end; third, stability of base pairing and predicted binding energy. Further complicat‐ ing target site prediction in mammals is the fact that not all 3'-UTR sites with perfect complementarities to the miRNA seed nucleotides are functional. Moreover, mRNAs sites with imperfect seed complementarities can themselves be very good miRNA targets [24,25]. Bioinformatics is therefore much noisier and more prone to false positive and false negative predictions. Among many available programs for predicting mRNA targets for specific miRNA, none of these programs can be used as an independently approach for validating the targets, and all predicted targets must be validated *in vitro* and/or *in vivo.* Thus the gold standard for miRNA target identification is the experimental demon‐ stration that a luciferase reporter fused to the 3'-UTR of the predicted target is repressed by over-expression of the miRNA and that this repression is abrogated by point muta‐ tion in the target sequences in 3'-UTR [26,27]. Finaly, expression profiling in human dis‐ ease gives the starting point for target verification/validation and association to disease prognosis and pathogenesis. All identified disease related miRNAs are listed in The hu‐ man microRNA disease database (HMDD), where you can search for specific miRNA, for tissue expression of annotated miRNAs, and for disease related miRNAs [28].

**Function**

**Database**

lncRNA [29].

within nucleus [31,32].

**3. Involvement of ncRNA in cancer**

**3.1. Mutations, SNPs and epigenetics of ncRNAs**

lncRNAs may act through diverse molecular mechanisms, and play regulatory as well as struc‐ tural roles in different biological processes [3]. Many of the identified lncRNAs show spatialand temporal-specific patterns of expression. Almost every step in the life cycle of genes – transcription, mRNAs splicing, RNA decay, and translation – can be influenced by lncRNAs. Generally lncRNAs have been implicated in gene-regulatory roles, such as chromatin dosagecompensation, imprinting, epigenetic regulation, cell cycle control, nuclear and cytoplasmic trafficking, cell differentiation etc. [7]. A number of studies suggest that lncRNAs are key com‐ ponents of the epigenetic regulatory network [4]. Two general modes of lncRNAs regulation seem to be important: interaction with chromatin remodelling complexes that promote silenc‐ ing of specific genes; and modulation of splicing factors. Chromatin remodelling guided by ncRNAs contributes to the establishment of chromatin structure and to the maintenance of epi‐ genetic memory. Various ncRNAs have been identified as regulators of chromatin structure

MicroRNAs and lncRNAs as Tumour Suppressors

http://dx.doi.org/10.5772/54701

51

and gene expression [30]. Additional mechanisms of action are yet to be revealed [3].

The lncRNA database provides sequence, structural, and conservation evidence for multispecies lncRNAs, together with a list of lncRNAs that are experimentally known to interact with coding mRNAs, harbouring other short ncRNAs and other characteristics of specific

Three major mechanisms are known to give rise to deregulated ncRNAs function, genetic alterations, epigenetic alterations, and in case of miRNAs, an aberrant miRNA biogenesis machinery. Since brief overview of first two mechanisms is described below, will be here mentioned only aberrant machinery of miRNA processing. Proteins involved in miRNA biogenesis (Drosha, Dicer, Ago) are deregulated in several cancers. Co-factors involved in miRNA biogenesis can be mutated causing consequently deregulation of Dicer; Exportin 5, mediating pre-miRNA nuclear export, is often mutated and truncated, leaving pre-miRNAs

Cancer cells have different genetic and epigenetic changes from their normal counterparts and the role of ncRNAs in mediating these differences is beginning to emerge. Specific genetic polymorphisms are associated with the risk of developing several types of cancer [7-9]. Multiple studies have identified small-scale and large-scale mutations and genomic alterations affecting also noncoding regions of the genome. Some of these mutations are structural alterations, rearrangements and chromosomal translocation, amplification, loss of heterozi‐ gocity and copy-number variation, nucleotide expansion, and single-nucleotide polymor‐ phisms (SNPs), and they are linking distinct types of mutations in ncRNA genes with diverse

## **2.2. Brief introduction to lncRNA**

#### **Genomic organization**

LncRNAs are those longer than 200 nt, and many of them can also act as primary tran‐ scripts for the production of short RNAs [9]. It is estimated that total number of lncRNA transcripts, including new unexplored, is approx. 15000. Thousands of protein-coding genes in humans harbour natural antisense transcripts (approx. 61 % of transcribed re‐ gions show antisense transcription) belonging to the lncRNA, and majority of known lncRNAs in some way overlap protein-coding loci. All these data are giving the impor‐ tance to lncRNA annotation [29].

#### **Classification**

LncRNAs can be classified according to their proximity to protein coding genes. There are five categories of lncRNAs: sense, antisense, bidirectional, intronic, intergenic. Just to mention a few of them, lincRNAs, a class of ncRNAs, exhibit a high conservation be‐ tween different species; they both up- and down- regulate hundreds of gene expression and participate in the establishment of cell type-specific epigenetic states [9]. Further, ncRNAs were found expressed at enhancer regions, suggesting that some enhancer RNA is also transcribed with an average size of 800 nt; these transcripts are termed eRNAs. Studies propose a possible role for eRNAs as transcriptional activators, however, ques‐ tion remains whether such eRNAs are in fact a subset of the activating lncRNAs. Similar to eRNA, a novel diverse class of ncRNAs has been linked to the promoters, called PARs, ranging from 16-36 nt to 200 nt. It is suggested that they participate in the tran‐ scriptional regulation [9]. Most lncRNAs are characterized by low expression levels, low level of sequence conservation, by composition of poly-A tail and without poly-A tail as well as by spliced and un-spliced forms. They are believed to have nuclear localization, but can also accumulate in cytoplasm of cells [3].

#### **Function**

NA end; third, stability of base pairing and predicted binding energy. Further complicat‐ ing target site prediction in mammals is the fact that not all 3'-UTR sites with perfect complementarities to the miRNA seed nucleotides are functional. Moreover, mRNAs sites with imperfect seed complementarities can themselves be very good miRNA targets [24,25]. Bioinformatics is therefore much noisier and more prone to false positive and false negative predictions. Among many available programs for predicting mRNA targets for specific miRNA, none of these programs can be used as an independently approach for validating the targets, and all predicted targets must be validated *in vitro* and/or *in vivo.* Thus the gold standard for miRNA target identification is the experimental demon‐ stration that a luciferase reporter fused to the 3'-UTR of the predicted target is repressed by over-expression of the miRNA and that this repression is abrogated by point muta‐ tion in the target sequences in 3'-UTR [26,27]. Finaly, expression profiling in human dis‐ ease gives the starting point for target verification/validation and association to disease prognosis and pathogenesis. All identified disease related miRNAs are listed in The hu‐ man microRNA disease database (HMDD), where you can search for specific miRNA, for

tissue expression of annotated miRNAs, and for disease related miRNAs [28].

LncRNAs are those longer than 200 nt, and many of them can also act as primary tran‐ scripts for the production of short RNAs [9]. It is estimated that total number of lncRNA transcripts, including new unexplored, is approx. 15000. Thousands of protein-coding genes in humans harbour natural antisense transcripts (approx. 61 % of transcribed re‐ gions show antisense transcription) belonging to the lncRNA, and majority of known lncRNAs in some way overlap protein-coding loci. All these data are giving the impor‐

LncRNAs can be classified according to their proximity to protein coding genes. There are five categories of lncRNAs: sense, antisense, bidirectional, intronic, intergenic. Just to mention a few of them, lincRNAs, a class of ncRNAs, exhibit a high conservation be‐ tween different species; they both up- and down- regulate hundreds of gene expression and participate in the establishment of cell type-specific epigenetic states [9]. Further, ncRNAs were found expressed at enhancer regions, suggesting that some enhancer RNA is also transcribed with an average size of 800 nt; these transcripts are termed eRNAs. Studies propose a possible role for eRNAs as transcriptional activators, however, ques‐ tion remains whether such eRNAs are in fact a subset of the activating lncRNAs. Similar to eRNA, a novel diverse class of ncRNAs has been linked to the promoters, called PARs, ranging from 16-36 nt to 200 nt. It is suggested that they participate in the tran‐ scriptional regulation [9]. Most lncRNAs are characterized by low expression levels, low level of sequence conservation, by composition of poly-A tail and without poly-A tail as well as by spliced and un-spliced forms. They are believed to have nuclear localization,

**2.2. Brief introduction to lncRNA**

50 Future Aspects of Tumor Suppressor Gene

tance to lncRNA annotation [29].

but can also accumulate in cytoplasm of cells [3].

**Genomic organization**

**Classification**

lncRNAs may act through diverse molecular mechanisms, and play regulatory as well as struc‐ tural roles in different biological processes [3]. Many of the identified lncRNAs show spatialand temporal-specific patterns of expression. Almost every step in the life cycle of genes – transcription, mRNAs splicing, RNA decay, and translation – can be influenced by lncRNAs. Generally lncRNAs have been implicated in gene-regulatory roles, such as chromatin dosagecompensation, imprinting, epigenetic regulation, cell cycle control, nuclear and cytoplasmic trafficking, cell differentiation etc. [7]. A number of studies suggest that lncRNAs are key com‐ ponents of the epigenetic regulatory network [4]. Two general modes of lncRNAs regulation seem to be important: interaction with chromatin remodelling complexes that promote silenc‐ ing of specific genes; and modulation of splicing factors. Chromatin remodelling guided by ncRNAs contributes to the establishment of chromatin structure and to the maintenance of epi‐ genetic memory. Various ncRNAs have been identified as regulators of chromatin structure and gene expression [30]. Additional mechanisms of action are yet to be revealed [3].

#### **Database**

The lncRNA database provides sequence, structural, and conservation evidence for multispecies lncRNAs, together with a list of lncRNAs that are experimentally known to interact with coding mRNAs, harbouring other short ncRNAs and other characteristics of specific lncRNA [29].

## **3. Involvement of ncRNA in cancer**

Three major mechanisms are known to give rise to deregulated ncRNAs function, genetic alterations, epigenetic alterations, and in case of miRNAs, an aberrant miRNA biogenesis machinery. Since brief overview of first two mechanisms is described below, will be here mentioned only aberrant machinery of miRNA processing. Proteins involved in miRNA biogenesis (Drosha, Dicer, Ago) are deregulated in several cancers. Co-factors involved in miRNA biogenesis can be mutated causing consequently deregulation of Dicer; Exportin 5, mediating pre-miRNA nuclear export, is often mutated and truncated, leaving pre-miRNAs within nucleus [31,32].

#### **3.1. Mutations, SNPs and epigenetics of ncRNAs**

Cancer cells have different genetic and epigenetic changes from their normal counterparts and the role of ncRNAs in mediating these differences is beginning to emerge. Specific genetic polymorphisms are associated with the risk of developing several types of cancer [7-9]. Multiple studies have identified small-scale and large-scale mutations and genomic alterations affecting also noncoding regions of the genome. Some of these mutations are structural alterations, rearrangements and chromosomal translocation, amplification, loss of heterozi‐ gocity and copy-number variation, nucleotide expansion, and single-nucleotide polymor‐ phisms (SNPs), and they are linking distinct types of mutations in ncRNA genes with diverse diseases [7]. First, lncRNA have already been implicated in human diseases such as cancer and neurodegeneration [33]. Second, approx. half of miRNA genes are encoded in genomic region prone to cancer-associated rearrangements or in fragile chromosomal sites (amplified, deleted or rearranged) that are often associated with cancer, such as ovarian and breast carcinomas, and melanomas [8,11]. Third, presence of SNPs in miRNAs, where disruption of miRNA target interaction either in the miRNA gene or its target site (3′-UTR mRNA) can lead to complete gain or loss of the miRNA function or target gene thus causing disease [34,35]. In contrast to the miRNA target sites in mRNA transcripts, where the potential of variation is huge, variants identified in miRNA precursor sequences tend to be rarer [36]. The presence of SNPs in *primiRNA* or *pre-miRNA* can in addition affect the processing of miRNAs, their expression and/or binding to target mRNA [27,37]. Forth, recent advances in miRNA research have provided evidence of a miRNA association with epigenetic mechanisms activated in diseased human tissues [38]. Heritable changes in gene expression that do not involve coding sequence modification are referred as epigenetics. Gene regulation by ncRNAs was considered as an epigenetic mechanism, but ncRNAs can be regulated by the same mechanism in which they participate [39]. DNA methylation, one of the two major epigenetic mechanisms, leads to gene silencing, and serves as an alternative mechanism of gene inactivation. The aberrant DNA methylation of gene promoters has been shown to result in the inactivation of tumour suppressor genes [40]. For an example, *miR-34* family is a family of tumour suppressors' genes, with *miR-34a* being deregulated by DNA methylation in both epithelial and haematological cancers. *miR-34* is an important component of the p53 tumour suppressor network, and p53 is a predicted target for members of the *miR-34* family. *miR-34a* reinforces the tumour suppressor function of p53, transactivation of *miR-34a* by p53 was also shown to promote apoptosis [11,41-44]. Another example is that *miR-29s* could target two enzymes of methylation process, DNMT3A and DNMT3B [39]. Antisense ncRNAs have been recently showed to be implicated in the silencing of tumour suppressor genes through epigenetic remodelling events [30]. All these miRNAs abnormalities suggest that they play a broad role in cancer pathogenesis.

NAs in preneoplasia or their usefulness to predict progression from preneoplasia to cancer. Several lines of evidence suggest the potential usefulness of ncRNAs, particularly miRNAs: first, as signature of early events in carcinogenesis and as biomarkers in early cancer detec‐ tion, second, as differential indicators of benign tumours, preneoplasia, and neoplasia, and third, that miRNAs and perhaps other ncRNAs might be useful in determining which pre‐ neoplastic lesions are likely to progress to cancer. Distinguishing benign diseases and cer‐ tain non-precancerous lesions from precancerous lesions and metastatic tumours would improve patient outcomes, survival, and reduce patient discomfort [45]. Characterization of ncRNAs involved in the development or maintenance of oncogenic states may therefore de‐ fine ncRNAs as early biomarkers for the emergence of cancer, and could have have an im‐

MicroRNAs and lncRNAs as Tumour Suppressors

http://dx.doi.org/10.5772/54701

53

miRNAs are believed to be promising potential biomarkers for cancer diagnosis, prognosis and targets for therapy. As potential markers for diagnosis are better classification factors than mRNAs. miRNAs seems to be evolutionarily selected gene regulatory molecules, their expression profiles might therefore be rich in gene regulatory information. Only small per‐ centage of the 16000 genes on the mRNA-expression arrays are regulatory molecules. This difference may be responsible for more efficient microRNA expression arrays in classifying cancer than mRNA-expression arrays [21,45,46]. Some of the key features of miRNAs that make them useful as potential biomarkers can be briefly summarized. First, expression pat‐ terns of miRNAs in human cancers appear to be tissue specific. Second, miRNA profiles ap‐ pear to reflect developmental lineage and differentiation state of the tumours. Third, miRNAs can successfully classify poorly differentiated tumours with high accuracy (~70 %). In contrast, mRNA profiles in the same set of specimens had an accuracy of only 6 %. There‐ fore, a combination of both miRNA and mRNA profiling data has the potential of enhancing accuracy of tumour classification. Forth, miRNAs can also be profiled and quantitatively measured in formalin-fixed paraffin-embedded tissues. And last, miRNAs are stable in hu‐ man body fluids of plasma and serum and can be quantitatively measured in microliter

Highly stable cell-free circulating nucleic acid (cfCNA), both RNA and DNA, has been dis‐ covered in the blood, plasma, and urine in humans. Since there is good correlation between tumours and genetic, epigenetic and/or transcriptomic changes and alterations in cfCNA levels, it gives a usefulness of cfCNA as biomarkers for clinical applications. Release of cfCNA in body fluids is probably related to apoptosis and necrosis. Circulating RNAs are stable in serum and plasma in spite of high amounts of RNAase in blood of cancer patients [48]. They are packed in microparticles, of which the most analyzed are in recent years exo‐ somes [3,49,50]. Tumour derived exosomes are small membrane vesicles of endocytic origin released by the tumour and found in peripheral circulation. Several recent reports showed that exosomes could be an important resource of cf-lncRNA/cf-miRNA in serum or plasma [51]. Small size, relative stability and resistance to RNAase degradation make the miRNAs more superior molecular markers than mRNAs [1]. Using non-invasive diagnostic proce‐ dures, the extraction and reliable determination of cf-miRNAs, circulating in body fluids

like plasma, serum, and others, could serve as circulating tumour biomarkers [52,53].

pact on the development of tools for disease diagnosis and treatment [30].

quantities of human sera or plasma using qPCR. [45-47].

#### **3.2. Promising role of ncRNAs in cancer: As cancer-subtype classifiers and detection in body fluids**

ncRNAs have been recognized as gene-specific regulators. They are similar in activity to a large number of protein transcription factors that are known to be critical in the transformation of cells to a malignant state. Majority of research has been involved in defining the role of miRNAs in cancer; however, lincRNAs have been shown to play role in tumour development by promoting the expression of genes involved in metastasis and angiogenesis [9]. Genomewide analyses have shown that ncRNAs have distinct signatures specific for a certain cancer type. Importance of combining ncRNAs with other biomarkers for cancer detection and prognosis would improve cancer risk assessment, detection, and prognosis. Thus, there is a need to combine genomic mutations with ncRNA markers to develop marker panels for more accurate risk assessment and early diagnosis [7-9].

Most cancers are diagnosed in advance stages, leading to poor outcome. Intense investiga‐ tion is going on seeking specific molecular changes that are able to identify patients with early cancer or precursor lesions [1]. Genome-wide expression profiling has examined miR‐ NAs in preneoplasia or their usefulness to predict progression from preneoplasia to cancer. Several lines of evidence suggest the potential usefulness of ncRNAs, particularly miRNAs: first, as signature of early events in carcinogenesis and as biomarkers in early cancer detec‐ tion, second, as differential indicators of benign tumours, preneoplasia, and neoplasia, and third, that miRNAs and perhaps other ncRNAs might be useful in determining which pre‐ neoplastic lesions are likely to progress to cancer. Distinguishing benign diseases and cer‐ tain non-precancerous lesions from precancerous lesions and metastatic tumours would improve patient outcomes, survival, and reduce patient discomfort [45]. Characterization of ncRNAs involved in the development or maintenance of oncogenic states may therefore de‐ fine ncRNAs as early biomarkers for the emergence of cancer, and could have have an im‐ pact on the development of tools for disease diagnosis and treatment [30].

diseases [7]. First, lncRNA have already been implicated in human diseases such as cancer and neurodegeneration [33]. Second, approx. half of miRNA genes are encoded in genomic region prone to cancer-associated rearrangements or in fragile chromosomal sites (amplified, deleted or rearranged) that are often associated with cancer, such as ovarian and breast carcinomas, and melanomas [8,11]. Third, presence of SNPs in miRNAs, where disruption of miRNA target interaction either in the miRNA gene or its target site (3′-UTR mRNA) can lead to complete gain or loss of the miRNA function or target gene thus causing disease [34,35]. In contrast to the miRNA target sites in mRNA transcripts, where the potential of variation is huge, variants identified in miRNA precursor sequences tend to be rarer [36]. The presence of SNPs in *primiRNA* or *pre-miRNA* can in addition affect the processing of miRNAs, their expression and/or binding to target mRNA [27,37]. Forth, recent advances in miRNA research have provided evidence of a miRNA association with epigenetic mechanisms activated in diseased human tissues [38]. Heritable changes in gene expression that do not involve coding sequence modification are referred as epigenetics. Gene regulation by ncRNAs was considered as an epigenetic mechanism, but ncRNAs can be regulated by the same mechanism in which they participate [39]. DNA methylation, one of the two major epigenetic mechanisms, leads to gene silencing, and serves as an alternative mechanism of gene inactivation. The aberrant DNA methylation of gene promoters has been shown to result in the inactivation of tumour suppressor genes [40]. For an example, *miR-34* family is a family of tumour suppressors' genes, with *miR-34a* being deregulated by DNA methylation in both epithelial and haematological cancers. *miR-34* is an important component of the p53 tumour suppressor network, and p53 is a predicted target for members of the *miR-34* family. *miR-34a* reinforces the tumour suppressor function of p53, transactivation of *miR-34a* by p53 was also shown to promote apoptosis [11,41-44]. Another example is that *miR-29s* could target two enzymes of methylation process, DNMT3A and DNMT3B [39]. Antisense ncRNAs have been recently showed to be implicated in the silencing of tumour suppressor genes through epigenetic remodelling events [30]. All these miRNAs abnormalities suggest that they play a broad role in cancer pathogenesis.

**3.2. Promising role of ncRNAs in cancer: As cancer-subtype classifiers and detection in body**

ncRNAs have been recognized as gene-specific regulators. They are similar in activity to a large number of protein transcription factors that are known to be critical in the transformation of cells to a malignant state. Majority of research has been involved in defining the role of miRNAs in cancer; however, lincRNAs have been shown to play role in tumour development by promoting the expression of genes involved in metastasis and angiogenesis [9]. Genomewide analyses have shown that ncRNAs have distinct signatures specific for a certain cancer type. Importance of combining ncRNAs with other biomarkers for cancer detection and prognosis would improve cancer risk assessment, detection, and prognosis. Thus, there is a need to combine genomic mutations with ncRNA markers to develop marker panels for more

Most cancers are diagnosed in advance stages, leading to poor outcome. Intense investiga‐ tion is going on seeking specific molecular changes that are able to identify patients with early cancer or precursor lesions [1]. Genome-wide expression profiling has examined miR‐

accurate risk assessment and early diagnosis [7-9].

**fluids**

52 Future Aspects of Tumor Suppressor Gene

miRNAs are believed to be promising potential biomarkers for cancer diagnosis, prognosis and targets for therapy. As potential markers for diagnosis are better classification factors than mRNAs. miRNAs seems to be evolutionarily selected gene regulatory molecules, their expression profiles might therefore be rich in gene regulatory information. Only small per‐ centage of the 16000 genes on the mRNA-expression arrays are regulatory molecules. This difference may be responsible for more efficient microRNA expression arrays in classifying cancer than mRNA-expression arrays [21,45,46]. Some of the key features of miRNAs that make them useful as potential biomarkers can be briefly summarized. First, expression pat‐ terns of miRNAs in human cancers appear to be tissue specific. Second, miRNA profiles ap‐ pear to reflect developmental lineage and differentiation state of the tumours. Third, miRNAs can successfully classify poorly differentiated tumours with high accuracy (~70 %). In contrast, mRNA profiles in the same set of specimens had an accuracy of only 6 %. There‐ fore, a combination of both miRNA and mRNA profiling data has the potential of enhancing accuracy of tumour classification. Forth, miRNAs can also be profiled and quantitatively measured in formalin-fixed paraffin-embedded tissues. And last, miRNAs are stable in hu‐ man body fluids of plasma and serum and can be quantitatively measured in microliter quantities of human sera or plasma using qPCR. [45-47].

Highly stable cell-free circulating nucleic acid (cfCNA), both RNA and DNA, has been dis‐ covered in the blood, plasma, and urine in humans. Since there is good correlation between tumours and genetic, epigenetic and/or transcriptomic changes and alterations in cfCNA levels, it gives a usefulness of cfCNA as biomarkers for clinical applications. Release of cfCNA in body fluids is probably related to apoptosis and necrosis. Circulating RNAs are stable in serum and plasma in spite of high amounts of RNAase in blood of cancer patients [48]. They are packed in microparticles, of which the most analyzed are in recent years exo‐ somes [3,49,50]. Tumour derived exosomes are small membrane vesicles of endocytic origin released by the tumour and found in peripheral circulation. Several recent reports showed that exosomes could be an important resource of cf-lncRNA/cf-miRNA in serum or plasma [51]. Small size, relative stability and resistance to RNAase degradation make the miRNAs more superior molecular markers than mRNAs [1]. Using non-invasive diagnostic proce‐ dures, the extraction and reliable determination of cf-miRNAs, circulating in body fluids like plasma, serum, and others, could serve as circulating tumour biomarkers [52,53].

LncRNAs show greater tissue specificity compared to protein-coding mRNAs, making them attractive in the search of novel diagnostics and/or prognostics cancer biomarkers in body fluid samples. For an example, lncRNA PCA3 was initially identified as over-expressed in prostate tumours relative to benign prostate hyperplasia and normal epithelium. It was latter showed that is very specific prostate cancer gene, whose mechanism is not yet identified, but it can be detected in urine samples and has been shown to improve diagnosis of prostate cancer [3].

#### **3.3. ncRNAs can act as both tumour suppressor genes and oncogenes**

There are different ways in which miRNAs appear to be involved in cancer: as tumour suppressors, as oncogenes, or as agents involved in affecting genome stability. Below is discussed role of miRNAs acting both, as tumours suppressor and as oncogenes, since are much more investigated in this field than are lncRNAs. Care must be taken in assigning oncogenic or tumour suppressor activity to a miRNA, since miRNA expression patterns are highly specific for cell-type and cellular differentiation status. The same miRNA can function as tumour suppressor in one cell type and as potential oncogene in other cell type. Some of the aberrant miRNA expression observed in tumours may also be a secon‐ dary consequence of the loss of normal cellular function that accompanies malignant transformation. Up- or down-regulation of a miRNA in a given tumour type is not obvi‐ ous a causative role in tumorigenesis [6].

The increased expression of oncogenic miRNAs appears to act in a manner analogous to an oncogene. Over-expression of oncogenic miRNAs are presumed to function by downregulating the levels of protein product of target tumour suppressor gene or by reduction of tumour suppressor processes, such as apoptosis [2,6,20]. A loss of expression of tumour suppressor miRNA may lead to elevated levels of the protein products of target oncogenes [6], activation of an oncogenic processes, such as proliferation [2,20]. MicroRNAs with antiproliferative and pro-apoptotic activity are likely to function as tumour suppressors and thus may be under-expressed in cancer cells. Figure 1 represents schematic overview of miRNAs acting as tumour suppressors or oncogenes in comparison to non-cancerous cells.

There should be at least four type of evidence before assigning tumour suppressor function to ncRNAs: (i) data about widespread deregulation in diverse cancer, (ii) gain or loss of function in tumours owing to deletion, amplification or mutation, (iii) direct documentation of tumour suppressing activity using cell line or animal models, (iv) the identification and verification of cancer relevant targets that define mechanisms through which miRNAs participate in onco‐ genesis [6].

## **4. ncRNAs as potential therapeutic targets in cancer**

#### **4.1. RNAi in therapeutic applications**

Using RNAi approaches, ncRNAs may in future serve as therapeutic targets. For ncRNA that is under-expressed and possess tumour suppressor function, re-introduction of the mature

**Figure 1.** Schematic overview of miRNAs acting as tumour suppressors or oncogenes.

MicroRNAs and lncRNAs as Tumour Suppressors

http://dx.doi.org/10.5772/54701

55

MicroRNAs and lncRNAs as Tumour Suppressors http://dx.doi.org/10.5772/54701 55

LncRNAs show greater tissue specificity compared to protein-coding mRNAs, making them attractive in the search of novel diagnostics and/or prognostics cancer biomarkers in body fluid samples. For an example, lncRNA PCA3 was initially identified as over-expressed in prostate tumours relative to benign prostate hyperplasia and normal epithelium. It was latter showed that is very specific prostate cancer gene, whose mechanism is not yet identified, but it can be detected in urine samples and has been shown to improve diagnosis of prostate cancer [3].

There are different ways in which miRNAs appear to be involved in cancer: as tumour suppressors, as oncogenes, or as agents involved in affecting genome stability. Below is discussed role of miRNAs acting both, as tumours suppressor and as oncogenes, since are much more investigated in this field than are lncRNAs. Care must be taken in assigning oncogenic or tumour suppressor activity to a miRNA, since miRNA expression patterns are highly specific for cell-type and cellular differentiation status. The same miRNA can function as tumour suppressor in one cell type and as potential oncogene in other cell type. Some of the aberrant miRNA expression observed in tumours may also be a secon‐ dary consequence of the loss of normal cellular function that accompanies malignant transformation. Up- or down-regulation of a miRNA in a given tumour type is not obvi‐

The increased expression of oncogenic miRNAs appears to act in a manner analogous to an oncogene. Over-expression of oncogenic miRNAs are presumed to function by downregulating the levels of protein product of target tumour suppressor gene or by reduction of tumour suppressor processes, such as apoptosis [2,6,20]. A loss of expression of tumour suppressor miRNA may lead to elevated levels of the protein products of target oncogenes [6], activation of an oncogenic processes, such as proliferation [2,20]. MicroRNAs with antiproliferative and pro-apoptotic activity are likely to function as tumour suppressors and thus may be under-expressed in cancer cells. Figure 1 represents schematic overview of miRNAs

There should be at least four type of evidence before assigning tumour suppressor function to ncRNAs: (i) data about widespread deregulation in diverse cancer, (ii) gain or loss of function in tumours owing to deletion, amplification or mutation, (iii) direct documentation of tumour suppressing activity using cell line or animal models, (iv) the identification and verification of cancer relevant targets that define mechanisms through which miRNAs participate in onco‐

Using RNAi approaches, ncRNAs may in future serve as therapeutic targets. For ncRNA that is under-expressed and possess tumour suppressor function, re-introduction of the mature

acting as tumour suppressors or oncogenes in comparison to non-cancerous cells.

**4. ncRNAs as potential therapeutic targets in cancer**

**4.1. RNAi in therapeutic applications**

**3.3. ncRNAs can act as both tumour suppressor genes and oncogenes**

ous a causative role in tumorigenesis [6].

54 Future Aspects of Tumor Suppressor Gene

genesis [6].

**Figure 1.** Schematic overview of miRNAs acting as tumour suppressors or oncogenes.

ncRNA into the affected tissue would restore the regulation of the target gene. By contrast, over-expressed ncRNA with oncogenic function could be down-regulated by reducing mature ncRNA level by its direct targeting [54].

by influencing drug resistance or enhancing responsiveness to therapy. Current limitation is need for improvement of efficiency of delivery to target tissue, for systemic drug ad‐ ministration, potential inhibition of non-target genes ("off-target effect"), redundancy among miRNAs efficacy, potential toxicity and immunogeneic responses. However, stud‐ ies introducing miRNAs strategies to inhibit cancer propagation in animal models are

MicroRNAs and lncRNAs as Tumour Suppressors

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57

Therapeutic delivery to animal models was demonstrated using miRNA-mimics of the tumour suppressor miRNAs, *miR-34a* and *let-7a*, both of which are often down-regulated or lost in lung cancer. It has been shown that re-introduction of *let-7* directly represses cancer growth in the lung [58] and that development of chemically synthesized therapeutic *miR-34a* and lipid-based delivery vehicle block tumour growth in mouse models of non-small-cell lung cancer (NSCLC) [59]. Systemic treatment of these mice led to significant decrease in tumour burden. Mice treated with *miR-34a* displayed a 60 % reduction in tumour area compared to mice treated with

Successful inhibition of lncRNAs seems to be more difficult than inhibition of miRNAs. Our growing knowledge of other ncRNAs might exploit in future to develop new therapeutic strategies not only against cancer, but also for other diseased states. The findings regarding lncRNA and Alzheimer disease are attracting the attention of pharmaceutical and biotechnol‐ ogy industries [8]. Therapy using small RNAs that targets ncRNA transcripts, such as eRNAs or PARs, may represent a new way to treat disease conditions caused by epigenetic changes [9].

Another possible approach for manipulation of ncRNAs level may also be by altering DNA methylation. As mentioned above, DNA methylation is a crucial mechanism associated with epigenetic regulation. It has been shown that in cancer cells treated with DNA demethylating agent reactivation of certain miRNAs occurs [40]. ncRNAs mediated therapy may also be useful in combination with DNA methyltransferase inhibitors that are other way toxic [39].

In the following section, down-regulated miRNAs will be describe and miRNAs with sug‐ gested tumours suppressive roles in different types of cancer. However, down-regulation does

*Leukaemia.* Chronic lymphocytic leukaemia (CLL) is characterized by overexpression of the protein Bcl-2 in B cells and represents the most common human leukaemia. In less than 5 %

**5. ncRNAs as tumour suppressor in different types of cancers**

a miRNA control. Similar results were obtained with the *let-7* mimic [60].

showing promising results [32].

**Examples for miRNA**

**Targeting lncRNAs**

**Targeting both, lncRNAs and miRNAs**

**5.1. miRNAs as tumour suppressors**

**Hematological cancers**

not ncessary mean that miRNA is tumours uppressor.

Due to the interferon response it is difficult to introduce long dsRNAs into mammalian cells, however, the use of RNAi as a therapeutic approach has been successfully used. Among the first applications to reach clinical trials were in the treatment of macular degeneration and respiratory syncytical virus infection, reversal of induced liver failure in mouse models, antiviral therapies, neurodegenerative diseases, and cancer. Cancer was treated by silencing up-regulated genes in tumour cells or genes involved in cell division. A key area of research in the use of RNAi for clinical applications is the development of a safe delivery method, which to date has involved mainly viral (lentivirus, adenovirus, adeno-associated virus) and nonviral (nanoparticles, aptamers, stable nucleic-acid-lipid particle, e.g.) vector systems similar to those suggested for gene therapy [55].

## **4.2. ncRNAs with tumour suppressor function as therapeutic targets**

#### **Replenishing small RNAs/miRNAs**

Pharmacological manipulation of miRNAs is still in its infancy; however, the correlation between the expression of miRNAs and their effects on target oncogenes, on tumorigene‐ sis, and on the proliferation of cancer cells has gained experimental support. miRNAs are small molecules, making their *in vivo* delivery feasible. It has been shown that miRNAs can be delivered systematically, and can reduce invasion, proliferation and growth as well as induce radio-sensitivity and resistance. miRNAs may therefore serve as therapeu‐ tic targets in the future.

For miRNA that is under-expressed, re-introduction of the mature miRNA into the affected tissue would restore regulation of the target gene. For this purpose, artificial miRNA (miRNAmimic) have been developed to enhance the expression of beneficial miRNAs or the introduc‐ tion of short hairpin duplex, similar to *pre-miRNA*, into the cell. This suggests that individual miRNAs are potential therapeutic agents, provided that their expression or delivery can be targeted to appropriate tissue. Most of the developed protocols have used local administration in easily accessible tissue; systemic delivery has also give some promising results; the major challenge remains tissue and cell-type specific targeting [56].

miRNA mimic can only last a couple of days and the long term biological effects were not observed very effectively. To overcome this, the cells were infected with a lentivirus that expressed mature miRNAs. This generated stable cell expressing miRNAs. miRNA mimics and lentiviral miRNAs showed great potential in restoring tumour suppressor miRNAs. However, viral and non-viral delivery systems have been developed. Viral vector-directed methods show high gene transfer efficiency, but have some limitations. However, non-viral gene transfer vectors have been also developed: cationic liposome mediated gene transfer system, lipoplexes, neutral lipid emulsion, etc. [57].

Expression of miRNA-mimic would simultaneously suppress many gene targets. miR‐ NAs-mimic would be useful in conjunction with standard chemotherapy or radiotherapy, by influencing drug resistance or enhancing responsiveness to therapy. Current limitation is need for improvement of efficiency of delivery to target tissue, for systemic drug ad‐ ministration, potential inhibition of non-target genes ("off-target effect"), redundancy among miRNAs efficacy, potential toxicity and immunogeneic responses. However, stud‐ ies introducing miRNAs strategies to inhibit cancer propagation in animal models are showing promising results [32].

#### **Examples for miRNA**

ncRNA into the affected tissue would restore the regulation of the target gene. By contrast, over-expressed ncRNA with oncogenic function could be down-regulated by reducing mature

Due to the interferon response it is difficult to introduce long dsRNAs into mammalian cells, however, the use of RNAi as a therapeutic approach has been successfully used. Among the first applications to reach clinical trials were in the treatment of macular degeneration and respiratory syncytical virus infection, reversal of induced liver failure in mouse models, antiviral therapies, neurodegenerative diseases, and cancer. Cancer was treated by silencing up-regulated genes in tumour cells or genes involved in cell division. A key area of research in the use of RNAi for clinical applications is the development of a safe delivery method, which to date has involved mainly viral (lentivirus, adenovirus, adeno-associated virus) and nonviral (nanoparticles, aptamers, stable nucleic-acid-lipid particle, e.g.) vector systems similar to

Pharmacological manipulation of miRNAs is still in its infancy; however, the correlation between the expression of miRNAs and their effects on target oncogenes, on tumorigene‐ sis, and on the proliferation of cancer cells has gained experimental support. miRNAs are small molecules, making their *in vivo* delivery feasible. It has been shown that miRNAs can be delivered systematically, and can reduce invasion, proliferation and growth as well as induce radio-sensitivity and resistance. miRNAs may therefore serve as therapeu‐

For miRNA that is under-expressed, re-introduction of the mature miRNA into the affected tissue would restore regulation of the target gene. For this purpose, artificial miRNA (miRNAmimic) have been developed to enhance the expression of beneficial miRNAs or the introduc‐ tion of short hairpin duplex, similar to *pre-miRNA*, into the cell. This suggests that individual miRNAs are potential therapeutic agents, provided that their expression or delivery can be targeted to appropriate tissue. Most of the developed protocols have used local administration in easily accessible tissue; systemic delivery has also give some promising results; the major

miRNA mimic can only last a couple of days and the long term biological effects were not observed very effectively. To overcome this, the cells were infected with a lentivirus that expressed mature miRNAs. This generated stable cell expressing miRNAs. miRNA mimics and lentiviral miRNAs showed great potential in restoring tumour suppressor miRNAs. However, viral and non-viral delivery systems have been developed. Viral vector-directed methods show high gene transfer efficiency, but have some limitations. However, non-viral gene transfer vectors have been also developed: cationic liposome mediated gene transfer

Expression of miRNA-mimic would simultaneously suppress many gene targets. miR‐ NAs-mimic would be useful in conjunction with standard chemotherapy or radiotherapy,

ncRNA level by its direct targeting [54].

56 Future Aspects of Tumor Suppressor Gene

those suggested for gene therapy [55].

**Replenishing small RNAs/miRNAs**

tic targets in the future.

**4.2. ncRNAs with tumour suppressor function as therapeutic targets**

challenge remains tissue and cell-type specific targeting [56].

system, lipoplexes, neutral lipid emulsion, etc. [57].

Therapeutic delivery to animal models was demonstrated using miRNA-mimics of the tumour suppressor miRNAs, *miR-34a* and *let-7a*, both of which are often down-regulated or lost in lung cancer. It has been shown that re-introduction of *let-7* directly represses cancer growth in the lung [58] and that development of chemically synthesized therapeutic *miR-34a* and lipid-based delivery vehicle block tumour growth in mouse models of non-small-cell lung cancer (NSCLC) [59]. Systemic treatment of these mice led to significant decrease in tumour burden. Mice treated with *miR-34a* displayed a 60 % reduction in tumour area compared to mice treated with a miRNA control. Similar results were obtained with the *let-7* mimic [60].

#### **Targeting lncRNAs**

Successful inhibition of lncRNAs seems to be more difficult than inhibition of miRNAs. Our growing knowledge of other ncRNAs might exploit in future to develop new therapeutic strategies not only against cancer, but also for other diseased states. The findings regarding lncRNA and Alzheimer disease are attracting the attention of pharmaceutical and biotechnol‐ ogy industries [8]. Therapy using small RNAs that targets ncRNA transcripts, such as eRNAs or PARs, may represent a new way to treat disease conditions caused by epigenetic changes [9].

#### **Targeting both, lncRNAs and miRNAs**

Another possible approach for manipulation of ncRNAs level may also be by altering DNA methylation. As mentioned above, DNA methylation is a crucial mechanism associated with epigenetic regulation. It has been shown that in cancer cells treated with DNA demethylating agent reactivation of certain miRNAs occurs [40]. ncRNAs mediated therapy may also be useful in combination with DNA methyltransferase inhibitors that are other way toxic [39].

## **5. ncRNAs as tumour suppressor in different types of cancers**

#### **5.1. miRNAs as tumour suppressors**

In the following section, down-regulated miRNAs will be describe and miRNAs with sug‐ gested tumours suppressive roles in different types of cancer. However, down-regulation does not ncessary mean that miRNA is tumours uppressor.

#### **Hematological cancers**

*Leukaemia.* Chronic lymphocytic leukaemia (CLL) is characterized by overexpression of the protein Bcl-2 in B cells and represents the most common human leukaemia. In less than 5 % of cases, over-expression of Bcl-2 is due to a translocation of the Bcl-2 gene, whereas for the majority of CLL cases no explanation for the deregulation of Bcl-2 has been reported. It has been demonstrated that mutations in genomic regions containing miRNAs were associated with disease progression in a number of CLL patients. In this type of cancer, *miR-15* and *miR-16* expression is often reduced and indeed, one of the first associations between miRNAs and cancer development was observed for *miR-15* and *miR-16* in CLL [46]. Both miRNAs are located in a 30 kb region on chromosome 13 that had been found deleted in more than half of B cell CLL (chromosome 13q14 deletion) [8,46], and *miR-15a* and *miR-16-1* have been shown to be deleted or translocated in approx. 65 % of CLL patients [2,11]. Several papers indicate that miRNA regulates cell growth and apoptosis. Indeed, over-expression of *miR-15* and *miR-16* directly inhibit anti-apoptotic Bcl-2, a key player in many types of human cancers, and thus activate apoptotic processes [2,11]. However, it was further demonstrated that other mutations in miRNA genes are frequent in CLL; many mutations were located in the flanking sequence of *pre-miRNA*, thus cell culture assay indicated that a point mutation of the *miR-16-1* precursor abolishes expression of mature *miR-16* [21]. Few other miRNAs were also recognized as tumour suppressors in CLL. *miR-29a* and *miR-29b* are associated with fragile site FRA7H that is not associated with any known tumour suppressor gene. Over-expression of *miR-29b* may target TCL1 and reduces anti-apoptotic Mcl-1 protein in CLL patients [11]. Another wellknown tumour suppressor was analysed. Low expression of *miR-34a* in CLL was found to be associated with p53 inactivation, impaired DNA damage response, apoptosis resistance and chemotherapy-refractory disease irrespective to p53 mutation (cases with CLL with p53 mutation are resistant to chemotherapy). It was latter showed that *miR-34a* is induced by p53. In another type of leukaemia, particularly acute myeloid leukaemia, an inverse correlation between *miR-34b* and CREB expression has been observed. After restoring expression of *miR-34b*, cell cycle abnormalities, reduces growth and altered CREB expression has been observed, suggesting tumour suppressor potential of this miRNA [47].

cancer cells, and enhanced expression decreased tumour cell proliferation [11]. Cyclin D1 has been identified as a direct target for *miR-17/20* that functions to suppress proliferation of breast cancer cells. Additional miRNAs have also been shown to be down-regulated and have tumour suppressor function in other types of cancer: *let-7, miR-145, miR-34a, miR-214,* and *miR-205*. MicroRNAs, *miR-31, miR-126, miR-146a/b, miR-206* and *miR-335* have been shown as anti-

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59

Colorectal cancer is the third most commonly diagnosed cancer in the world, but it is more common in developed countries. Also in colorectal neoplasia miRNAs expression is associated to the tumour formation. Reduced expression of *miR-143* and *miR-145* have been shown to be a frequent feature of colorectal tumours (adenomatous and cancer stage) when compared to normal mucosa [2,6,64]. A tumour suppressive role of *miR-143* has been elucidated in the epigenetic aberration of CRC with DNMT3A as a target. Restoration of *miR-143* expression in CRC decreases tumour cell growth and down-regulates DNMT3A expression [47]. *let-7* has been implicated in development of colon cancers and progression of colorectal cancers; together with *miR-143* and *miR-18a* was observed to be down-regulated and target KRAS [11, 64]. *miR-145* has been proposed as a tumour suppressor and it has been shown that target IRS-1, and when over-expressed it dramatically inhibits the growth of colon cancer cells. A ubiquitous loss of *miR-126* expression in colon cancer lines was observed and its reconstitution resulted in a significant growth reduction. Also, in a panel of matched normal colon and primary colon tumours, each of the tumours demonstrated *miR-126* down-regulation [64]. Down-regulation of *miR-200* family is a hallmark of EMT as well as up-regulation of ZEB1 transcription factor, and it was shown that in colorectal cells ZEB1 directly suppress tran‐ scription of *miR-141* and *miR-200c* [57]. It was found that *miR-192* and *miR-215* was downregulated in CRC, and their anti-proliferative effect was identified in CRC cell lines. It was further defined that both are regulated by p53 and that their targets are a number of transcripts that regulate cell cycle checkpoints [57]. An inverse correlation between COX-2 and *miR-101* was reported in CRC cell lines, and this was further confirmed in colon cancer tissue and liver metastases derived from CRC patients. *miR-16*, *miR-125b*, *miR-31*, *miR-133b*, and *miR-96* were along with already mentioned miRNAs showed to be down-regulated in colorectal cancer [1].

Aberrant DNA methylation may further induce silencing of specific miRNAs in CRC. While methylation of *miR-129* and *miR-137* CpG islands is frequently observed in CRC, is methylation of *miR-9-1* associated with the presence of lymph node metastasis and expression of *miR-9* in CRC inversely correlated with the methylation of its promoter regions [47]. In human colon cancer cell lines, *miR-34a* was showed to participate in the apoptotic program triggered by p53 activation and loss of *miR-34a* expression occurs frequently in cancer cells. p53 directly binds to the genomic region defined as the *miR-34a* promoter with consequent *miR-34a* targeting genes of cell cycle, DNA repair, mitotic checkpoint, DNA integrity checkpoint, cell prolifera‐ tion, and angiogenesis. Among the down-regulated targets of *miR-34* family were wellcharacterized p53 targets, such as CDK4/6, cyclin E2, E2F5, BIRC3 and Bcl-2. These effects were nearly identical irrespective of whether *miR-34a*, *miR-34b* or *miR-34c* was introduced into cell lines. Another target was identified for *miR-34a*, SIRT1, negative regulator of apoptosis.

metastatic miRNAs [62,63].

**Colorectal Cancer (CRC)**

*Lymphoma.miR-142* gene was found at the junction of the t(8;17) translocation, which may contribute to the progression of an indolent lymphoma into aggressive B-cell leukaemia [21].

#### **Breast cancer**

Breast cancer is one of the most important cancers in adult females. *miR-125b, miR-145*, *miR-21* and *miR-155* were significantly reduced in breast cancer tissue and this expression was correlated with specific breast cancer pathologic features, such as tumour stage, proliferation, oestrogen and progesterone receptor expression, and vascular invasion. Some of these miRNAs act as oncogenes (e.g. *miR-21*) in many cancer types, so it is suggested that some miRNAs act as tumour suppressors in one cancer type and as oncogenes in another [2]. *miR-125b-1* is located on a fragile site on chromosome 11q24, which is deleted in a subset of patients with breast cancer [61]. Down-regulation of *mir-221* in breast cancers was detected, whereas germ line mutation in mature *miR-125a* is highly associated with breast cancer tumorigenesis, suggesting its tumour suppressor role. *miR-125a* is also down-regulated in human breast cancer and when over-expressed post-trancriptionally regulates CYP24 result‐ ing in an anti-proliferative effect. Ectopic expression of *miR-30e* suppresses cell growth in breast cancer, probably through targeting Ubc9 [47]. *miR-17-5p* was down-regulated in breast cancer cells, and enhanced expression decreased tumour cell proliferation [11]. Cyclin D1 has been identified as a direct target for *miR-17/20* that functions to suppress proliferation of breast cancer cells. Additional miRNAs have also been shown to be down-regulated and have tumour suppressor function in other types of cancer: *let-7, miR-145, miR-34a, miR-214,* and *miR-205*. MicroRNAs, *miR-31, miR-126, miR-146a/b, miR-206* and *miR-335* have been shown as antimetastatic miRNAs [62,63].

#### **Colorectal Cancer (CRC)**

of cases, over-expression of Bcl-2 is due to a translocation of the Bcl-2 gene, whereas for the majority of CLL cases no explanation for the deregulation of Bcl-2 has been reported. It has been demonstrated that mutations in genomic regions containing miRNAs were associated with disease progression in a number of CLL patients. In this type of cancer, *miR-15* and *miR-16* expression is often reduced and indeed, one of the first associations between miRNAs and cancer development was observed for *miR-15* and *miR-16* in CLL [46]. Both miRNAs are located in a 30 kb region on chromosome 13 that had been found deleted in more than half of B cell CLL (chromosome 13q14 deletion) [8,46], and *miR-15a* and *miR-16-1* have been shown to be deleted or translocated in approx. 65 % of CLL patients [2,11]. Several papers indicate that miRNA regulates cell growth and apoptosis. Indeed, over-expression of *miR-15* and *miR-16* directly inhibit anti-apoptotic Bcl-2, a key player in many types of human cancers, and thus activate apoptotic processes [2,11]. However, it was further demonstrated that other mutations in miRNA genes are frequent in CLL; many mutations were located in the flanking sequence of *pre-miRNA*, thus cell culture assay indicated that a point mutation of the *miR-16-1* precursor abolishes expression of mature *miR-16* [21]. Few other miRNAs were also recognized as tumour suppressors in CLL. *miR-29a* and *miR-29b* are associated with fragile site FRA7H that is not associated with any known tumour suppressor gene. Over-expression of *miR-29b* may target TCL1 and reduces anti-apoptotic Mcl-1 protein in CLL patients [11]. Another wellknown tumour suppressor was analysed. Low expression of *miR-34a* in CLL was found to be associated with p53 inactivation, impaired DNA damage response, apoptosis resistance and chemotherapy-refractory disease irrespective to p53 mutation (cases with CLL with p53 mutation are resistant to chemotherapy). It was latter showed that *miR-34a* is induced by p53. In another type of leukaemia, particularly acute myeloid leukaemia, an inverse correlation between *miR-34b* and CREB expression has been observed. After restoring expression of *miR-34b*, cell cycle abnormalities, reduces growth and altered CREB expression has been

observed, suggesting tumour suppressor potential of this miRNA [47].

**Breast cancer**

58 Future Aspects of Tumor Suppressor Gene

*Lymphoma.miR-142* gene was found at the junction of the t(8;17) translocation, which may contribute to the progression of an indolent lymphoma into aggressive B-cell leukaemia [21].

Breast cancer is one of the most important cancers in adult females. *miR-125b, miR-145*, *miR-21* and *miR-155* were significantly reduced in breast cancer tissue and this expression was correlated with specific breast cancer pathologic features, such as tumour stage, proliferation, oestrogen and progesterone receptor expression, and vascular invasion. Some of these miRNAs act as oncogenes (e.g. *miR-21*) in many cancer types, so it is suggested that some miRNAs act as tumour suppressors in one cancer type and as oncogenes in another [2]. *miR-125b-1* is located on a fragile site on chromosome 11q24, which is deleted in a subset of patients with breast cancer [61]. Down-regulation of *mir-221* in breast cancers was detected, whereas germ line mutation in mature *miR-125a* is highly associated with breast cancer tumorigenesis, suggesting its tumour suppressor role. *miR-125a* is also down-regulated in human breast cancer and when over-expressed post-trancriptionally regulates CYP24 result‐ ing in an anti-proliferative effect. Ectopic expression of *miR-30e* suppresses cell growth in breast cancer, probably through targeting Ubc9 [47]. *miR-17-5p* was down-regulated in breast

Colorectal cancer is the third most commonly diagnosed cancer in the world, but it is more common in developed countries. Also in colorectal neoplasia miRNAs expression is associated to the tumour formation. Reduced expression of *miR-143* and *miR-145* have been shown to be a frequent feature of colorectal tumours (adenomatous and cancer stage) when compared to normal mucosa [2,6,64]. A tumour suppressive role of *miR-143* has been elucidated in the epigenetic aberration of CRC with DNMT3A as a target. Restoration of *miR-143* expression in CRC decreases tumour cell growth and down-regulates DNMT3A expression [47]. *let-7* has been implicated in development of colon cancers and progression of colorectal cancers; together with *miR-143* and *miR-18a* was observed to be down-regulated and target KRAS [11, 64]. *miR-145* has been proposed as a tumour suppressor and it has been shown that target IRS-1, and when over-expressed it dramatically inhibits the growth of colon cancer cells. A ubiquitous loss of *miR-126* expression in colon cancer lines was observed and its reconstitution resulted in a significant growth reduction. Also, in a panel of matched normal colon and primary colon tumours, each of the tumours demonstrated *miR-126* down-regulation [64]. Down-regulation of *miR-200* family is a hallmark of EMT as well as up-regulation of ZEB1 transcription factor, and it was shown that in colorectal cells ZEB1 directly suppress tran‐ scription of *miR-141* and *miR-200c* [57]. It was found that *miR-192* and *miR-215* was downregulated in CRC, and their anti-proliferative effect was identified in CRC cell lines. It was further defined that both are regulated by p53 and that their targets are a number of transcripts that regulate cell cycle checkpoints [57]. An inverse correlation between COX-2 and *miR-101* was reported in CRC cell lines, and this was further confirmed in colon cancer tissue and liver metastases derived from CRC patients. *miR-16*, *miR-125b*, *miR-31*, *miR-133b*, and *miR-96* were along with already mentioned miRNAs showed to be down-regulated in colorectal cancer [1].

Aberrant DNA methylation may further induce silencing of specific miRNAs in CRC. While methylation of *miR-129* and *miR-137* CpG islands is frequently observed in CRC, is methylation of *miR-9-1* associated with the presence of lymph node metastasis and expression of *miR-9* in CRC inversely correlated with the methylation of its promoter regions [47]. In human colon cancer cell lines, *miR-34a* was showed to participate in the apoptotic program triggered by p53 activation and loss of *miR-34a* expression occurs frequently in cancer cells. p53 directly binds to the genomic region defined as the *miR-34a* promoter with consequent *miR-34a* targeting genes of cell cycle, DNA repair, mitotic checkpoint, DNA integrity checkpoint, cell prolifera‐ tion, and angiogenesis. Among the down-regulated targets of *miR-34* family were wellcharacterized p53 targets, such as CDK4/6, cyclin E2, E2F5, BIRC3 and Bcl-2. These effects were nearly identical irrespective of whether *miR-34a*, *miR-34b* or *miR-34c* was introduced into cell lines. Another target was identified for *miR-34a*, SIRT1, negative regulator of apoptosis. *miR-34a* promoter hyper-methylation was observed in 3 of 23 cases of colon cancer, *miR-34b/c* were found to be epigenetically silenced in 9 of 9 cell lines and in 101 of 111 primary CRC tumours [42,64].

tumour suppressor miRNAs involved in pancreatic cancer are: *miR-100, miR-181a*, and

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61

*miR-200* family, potential tumour suppressors, is up-regulated in pancreatic cancer. Low expression of *miR-200* family genes and higher expression of their target is common in different cancers. However, most pancreatic cancer cell investigations showed hypo-methylation of *miR-200a/b* and its over-expression, in contrary its targets are hyper-methylated, suggesting

Primary liver cancer mainly refers to HCC, which is one of the most common malignant tumours in liver and accounts for 85-90 % of primary liver cancers. Cyclins D2 and E2 were validated as direct targets for *miR-26a*, which exhibit reduced expression in HCC [68]. In another early study on animal models, down-regulation of number of miRNAs has been detected, including known tumour suppressor miRNAs, such as: *miR-15/16*, *miR-34a, miR-150* and *miR-195* [69]. *miR-122*, which represents 70 % of all liver miRNAs, was found to be frequently down-regulated in HCCs. Loss of *miR-122* expression in tumour cells segregates with specific gene expression profiles linking to HCC progression. *miR-122* is specifically repressed in a subset of primary HCCs that are characterized by poor prognosis and is therefore suggested as tumour suppressor miRNA [47,68]. As one of *miR-122* targets, cyclin G1 was identified, through its regulation *miR-122* influences p53 protein stability and transcriptional activity. Two other *miR-122* targets, which promote tumorigenesis, are SRF and IGF1R. The cellular mRNAs and protein levels of Bcl-w were also repressed by *miR-122*. Other proapoptotic functions were assigned for *let-7* through targeting Bcl-xL, for *miR-101* through targeting Mcl-1, and for *miR-29* through targeting Mcl-1 and Bcl2. *miR-195* was significantly reduced in HCC tissues and cell lines, it suppress tumorigenicity through targeting cyclin D1, CDK6, and E2F3. CDK6 was showed to be also target for *miR-124*, which is silenced through CpG methylation in HCC; *miR-124* in addition targets vimentin, SET, and MYND. *Let-7g* inhibits the proliferation of HCC by down-regulating c-Myc. Methylation of *miR-1* in HCC results in enhanced tumour cell growth, probably through release of its oncogenic targets c-Met, FoxP1, HDAC4. *miR-223* targets Stahmin 1 and is down-regulated in HCC whereas *miR-375* inhibits the proliferation and invasion of HCC cells by targeting Hippo-signalling effectors YAP [68,70]. Research on expression profiling in HCC and adjunct non-tumour tissue defined that *miR-199a\*, miR-195, miR-199a, miR-200a*, and *miR-125a* were also under-expressed in HCC tissue [2]. Anti-metastatic functions were showed for *miR-122* by targeting desintegrin and metalloprotease, and *let-7g* by targeting type I collagen A2. c-Met is target for *miR-1, miR-34a, miR-23b* and *miR-199a-3p*, and all of these miRNAs are down-regulated in HCC

Lung cancer is one of the most common cancers of adults and is also leading cause of cancerrelated deaths in many economically developed countries [2]. *Let-7* family is a family of miRNAs, whose genes map to different chromosome regions that are frequently deleted in lung cancer [71]. Significantly worse survival was observed among patients with low expres‐

that this pathway is not involved in metastases in most pancreatic cancer [67]

*miR-15b*, but are as well as *miR-200* family up-regulated [67].

**Hepatocellular Carcinoma (HCC)**

[68,70].

**Lung cancer**

## **Gastric cancer**

Gastric cancer is the fourth most common cancer and the second leading cause of cancer death in the world. It was reported that loss of Ago2, which leads to premature stopping of miRNAs biogenesis and general deregulation of miRNAs expression, was observed in 40 % of human gastric cancer patients with high microsatellite instability. A number of miRNAs were reported to be down-regulated. Among these, *miR-141* was significantly low expressed in 80 % of primary gastric carcinoma compared to non-cancer adjacent tissue. It targets FGFR2 and its down-regulation means proliferative potential and poor differentiation of gastric cancer cells [65]. It appears that down-regulation of *miR-451* is related to the worse prognosis of the gastric cancer patients. Over-expression of *miR-451* in gastric cancer cells regulates the oncogene MIF production, reduces cell proliferation and increases sensitivity to radiotherapy [47]. *miR-101* was down-regulated in gastric cancer cells, its targets are: EZH2, Cox-2, Mcl-1, and Fos [65]. Other potential tumour suppressor miRNAs in gastric cancer are: *miR-181b/c* and *miR-432AS*, and for *miR-181* it was proposed that modulate expression of Bcl-2. Low level or loss of expression in gastric cancer also showed *let-7a, miR-486*, and *miR-449*. However, a proposed role for *miR-107* and *miR-126* is controversial in gastric cancer, either tumour-suppressive or oncogenic [66].

An epigenetic silencing of *miR-512-5p* was observed in gastric cancer cells. As its target is was shown anti-apoptotic protein Mcl-1 and after epigenetic treatment (demethylation), it results in apoptosis of gastric cancer cells [65].

The association between genetic polymorphism of *miR-196a-2* and risk of gastric cancer has been identified; it was found that the variant homozygous genotype of *miR-196a-2* was associated with significantly increased risk of gastric cancer [65].

#### **Pancreatic cancer**

Pancreatic cancer is the eighth most common cause of cancer-related deaths worldwide and it has a poor prognosis for all stages. It is usually diagnosed at advent stages, therefore it is an urgent need to find some specific biomarkers and key components of carcinogenesis. Several miRNAs were reported to suppress metastasis. In pancreatic cancer cell lines, *miR-146a* was decreased compared to normal ductal epithelial cell line, and its ectopic expression inhibited invasive capacity of pancreatic cancer cell lines. *miR-96* is believed to be a potential tumour suppressor through targeting KRAS. It is significantly down-regulated in pancreatic cancer, its ectopic expression induces apoptosis, inhibits cell proliferation, migration and invasion. In human clinical samples there is observed inverse correlation between *miR-96* and KRAS. Further, ectopic expression of *miR-520h* has inhibitory effect on pancreatic cancer cell migration and invasion, *miR-20a*, with metastasis-suppressing effect, is reduced in pancreatic cancer and its cell lines. [67]. In pancreatic ductal carcinoma, *miR-345*, *miR-139*, and *miR-142-p* were the most down-regulated miRNAs in tumour tissue compared to normal tissue [1]. Other potential tumour suppressor miRNAs involved in pancreatic cancer are: *miR-100, miR-181a*, and *miR-15b*, but are as well as *miR-200* family up-regulated [67].

*miR-200* family, potential tumour suppressors, is up-regulated in pancreatic cancer. Low expression of *miR-200* family genes and higher expression of their target is common in different cancers. However, most pancreatic cancer cell investigations showed hypo-methylation of *miR-200a/b* and its over-expression, in contrary its targets are hyper-methylated, suggesting that this pathway is not involved in metastases in most pancreatic cancer [67]

#### **Hepatocellular Carcinoma (HCC)**

*miR-34a* promoter hyper-methylation was observed in 3 of 23 cases of colon cancer, *miR-34b/c* were found to be epigenetically silenced in 9 of 9 cell lines and in 101 of 111 primary CRC

Gastric cancer is the fourth most common cancer and the second leading cause of cancer death in the world. It was reported that loss of Ago2, which leads to premature stopping of miRNAs biogenesis and general deregulation of miRNAs expression, was observed in 40 % of human gastric cancer patients with high microsatellite instability. A number of miRNAs were reported to be down-regulated. Among these, *miR-141* was significantly low expressed in 80 % of primary gastric carcinoma compared to non-cancer adjacent tissue. It targets FGFR2 and its down-regulation means proliferative potential and poor differentiation of gastric cancer cells [65]. It appears that down-regulation of *miR-451* is related to the worse prognosis of the gastric cancer patients. Over-expression of *miR-451* in gastric cancer cells regulates the oncogene MIF production, reduces cell proliferation and increases sensitivity to radiotherapy [47]. *miR-101* was down-regulated in gastric cancer cells, its targets are: EZH2, Cox-2, Mcl-1, and Fos [65]. Other potential tumour suppressor miRNAs in gastric cancer are: *miR-181b/c* and *miR-432AS*, and for *miR-181* it was proposed that modulate expression of Bcl-2. Low level or loss of expression in gastric cancer also showed *let-7a, miR-486*, and *miR-449*. However, a proposed role for *miR-107* and *miR-126* is controversial in gastric cancer, either tumour-suppressive or

An epigenetic silencing of *miR-512-5p* was observed in gastric cancer cells. As its target is was shown anti-apoptotic protein Mcl-1 and after epigenetic treatment (demethylation), it results

The association between genetic polymorphism of *miR-196a-2* and risk of gastric cancer has been identified; it was found that the variant homozygous genotype of *miR-196a-2*

Pancreatic cancer is the eighth most common cause of cancer-related deaths worldwide and it has a poor prognosis for all stages. It is usually diagnosed at advent stages, therefore it is an urgent need to find some specific biomarkers and key components of carcinogenesis. Several miRNAs were reported to suppress metastasis. In pancreatic cancer cell lines, *miR-146a* was decreased compared to normal ductal epithelial cell line, and its ectopic expression inhibited invasive capacity of pancreatic cancer cell lines. *miR-96* is believed to be a potential tumour suppressor through targeting KRAS. It is significantly down-regulated in pancreatic cancer, its ectopic expression induces apoptosis, inhibits cell proliferation, migration and invasion. In human clinical samples there is observed inverse correlation between *miR-96* and KRAS. Further, ectopic expression of *miR-520h* has inhibitory effect on pancreatic cancer cell migration and invasion, *miR-20a*, with metastasis-suppressing effect, is reduced in pancreatic cancer and its cell lines. [67]. In pancreatic ductal carcinoma, *miR-345*, *miR-139*, and *miR-142-p* were the most down-regulated miRNAs in tumour tissue compared to normal tissue [1]. Other potential

was associated with significantly increased risk of gastric cancer [65].

tumours [42,64].

60 Future Aspects of Tumor Suppressor Gene

**Gastric cancer**

oncogenic [66].

**Pancreatic cancer**

in apoptosis of gastric cancer cells [65].

Primary liver cancer mainly refers to HCC, which is one of the most common malignant tumours in liver and accounts for 85-90 % of primary liver cancers. Cyclins D2 and E2 were validated as direct targets for *miR-26a*, which exhibit reduced expression in HCC [68]. In another early study on animal models, down-regulation of number of miRNAs has been detected, including known tumour suppressor miRNAs, such as: *miR-15/16*, *miR-34a, miR-150* and *miR-195* [69]. *miR-122*, which represents 70 % of all liver miRNAs, was found to be frequently down-regulated in HCCs. Loss of *miR-122* expression in tumour cells segregates with specific gene expression profiles linking to HCC progression. *miR-122* is specifically repressed in a subset of primary HCCs that are characterized by poor prognosis and is therefore suggested as tumour suppressor miRNA [47,68]. As one of *miR-122* targets, cyclin G1 was identified, through its regulation *miR-122* influences p53 protein stability and transcriptional activity. Two other *miR-122* targets, which promote tumorigenesis, are SRF and IGF1R. The cellular mRNAs and protein levels of Bcl-w were also repressed by *miR-122*. Other proapoptotic functions were assigned for *let-7* through targeting Bcl-xL, for *miR-101* through targeting Mcl-1, and for *miR-29* through targeting Mcl-1 and Bcl2. *miR-195* was significantly reduced in HCC tissues and cell lines, it suppress tumorigenicity through targeting cyclin D1, CDK6, and E2F3. CDK6 was showed to be also target for *miR-124*, which is silenced through CpG methylation in HCC; *miR-124* in addition targets vimentin, SET, and MYND. *Let-7g* inhibits the proliferation of HCC by down-regulating c-Myc. Methylation of *miR-1* in HCC results in enhanced tumour cell growth, probably through release of its oncogenic targets c-Met, FoxP1, HDAC4. *miR-223* targets Stahmin 1 and is down-regulated in HCC whereas *miR-375* inhibits the proliferation and invasion of HCC cells by targeting Hippo-signalling effectors YAP [68,70]. Research on expression profiling in HCC and adjunct non-tumour tissue defined that *miR-199a\*, miR-195, miR-199a, miR-200a*, and *miR-125a* were also under-expressed in HCC tissue [2]. Anti-metastatic functions were showed for *miR-122* by targeting desintegrin and metalloprotease, and *let-7g* by targeting type I collagen A2. c-Met is target for *miR-1, miR-34a, miR-23b* and *miR-199a-3p*, and all of these miRNAs are down-regulated in HCC [68,70].

#### **Lung cancer**

Lung cancer is one of the most common cancers of adults and is also leading cause of cancerrelated deaths in many economically developed countries [2]. *Let-7* family is a family of miRNAs, whose genes map to different chromosome regions that are frequently deleted in lung cancer [71]. Significantly worse survival was observed among patients with low expres‐ sion of *let-7a-2* compared to those with opposite expression pattern, independent of disease stage [2]. Over-expression of *let-7* in lung adenocarcinoma cell lines inhibited cancer cell growth and reduced cell cycle progression. These findings reflect that *let-7* mediates tumour suppressive function [2,45]. It has been shown that *let-7* regulates the expression of several oncogenes, RAS, MYC, HMGA2, and cell-cycle progression regulators, CDC25, CDK6, cyclin D2. *let-7* is down regulated in lung tumours and its expression anti-correlated with that of RAS relative to the normal lung tissue [46,71]. In animal models, ectopic *let-7g* expression reduces tumour burden and intranasal administration repress lung adenocarcinoma. A SNP in *let-7* complementary site 6 in 3'-UTR of its target KRAS is significantly associated with increased risk for NSCLC among moderate smokers [71]. The *let-7* family was subsequently found to be deregulated in a large number of tumour types [46].

Notch-2 and CDK6. Ectopic up-regulation of *miR-124* was shown to inhibit cell proliferation, and it was demonstrated that *miR-124* target and regulates CDK6. *miR-125b*, *miR-324-5p*, and *miR-326* over-expression inhibit medulloblastoma cell growth by targeting Hedgehog signal‐ ling pathway. *miR-128* also inhibits growth by targeting Bmi-1 oncogene. Another miRNA,

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In contrary to medulloblastomas, are gliomas the most common and deadly primary human brain tumours, and its subtype glioblastomas are highly invasive, very aggressive, and one of the most incurable [2]. *miR-181a, miR-181b* and *miR-181c* were originally identified as downregulated in glioblastoma cells and tumours when compared to normal brain controls. *miR-181a* and to a greater extent *miR-181b* were subsequently described as tumour suppressors that inhibit growth and induce apoptosis of glioma cells. *miR-181a* over-expression downregulates Bcl-2. Several other miRNAs have been implicated in glioma malignancy as tumour suppressors. *miR-15b* was suggested to target CCNE1, the gene encoding cyclin E1, however, a direct link between CCNE1 down-regulation by *miR-15* and cell cycle regulation was not demonstrated. *miR-146b* was shown to inhibit glioma cell migration and invasion, and was identified as one of miRNAs that is significantly deregulated in human glioblastoma tissue. *miR-146b* over-expression or knock-down did not affect the growth of human glioblastoma cell line, while it significantly reduced the migration and invasion of one glioblastoma cell line. MMP16 was identified as one of the downstream targets of *miR-146b*. *miR-125b* was shown to induce cell cycle arrest and inhibits CDK6 and CDC25A expression in glioma cell lines. However, another study suggested oncogene function for *miR-125b*. *miR-153* decrease cell proliferation and increased apoptosis (pro-apoptotic miRNA), it inhibited Bcl-2 and Mcl-1. *miR-17* and *miR-184* were identified as two miRNAs with reduced expression in higher grades of glioblastomas. Their over-expression inhibited viability, proliferation, invasion and decreased expression of AKT 2 and several other genes. *let-7* over-expression effect was investigated in glioma cells, its transfection reduced expression of RAS oncogenes, prolifera‐

tion in vitro, migration of the cells, and reduced the size of tumours generated [73].

Head and neck tumours are a heterogenous group with different behaviour at the various sites arising from anatomical factors, cell-type variation, and differences in exposure to risk factors including tobacco, alchocol, and viruses [74]. Head and neck squamous cell carcinoma is represented by epithelial cancers of the oral cavity, pharynx, nasal cavity, paranasal sinuses, salivary glands and larynx [75]. Studies were made in expression profiling regarding different sites of head and neck tumours, tongue, tonsil, larynx, hypopharynx, nasopharynx, saliva, oral cavity, salivary gland and animal models. Several miRNAs were identified as down-regulated,

Low expression of *miR-205* is significantly associated with local-regional recurrence inde‐ pendent of disease severity at diagnosis and treatment. Combined low expression of *let-7d* and *miR-205* is significantly associated with poor survival. In nasopharengyal carcinoma downregulation of *miR-34* family, *miR-145* and *miR-143* was also observed [47]. Tumour suppressive role in HNSCC has been suggested for *let-7* family, *miR-125a/b*, *miR-200*, *miR-133a/b*, and *miR-100* [75]. General down-regulation of *miR-1*, *miR-133a* and *let-7b* was also observed [74].

**Head and Neck Squamous Cell Carcinoma (HNSCC)**

and for some of their target genes were validated [74].

*miR-199b-5p* negatively regulates proliferation and cell growth [72].

Mutations in EGFR gene are more frequent in NSCLC patients who never smoked tobacco; a significant down-regulation of *miR-145* has been demonstrated in the cancer tissues of these patients. Restoration of this miRNA can inhibit cancer cell growth in EGFR mutant lung AD [47,71]. *miR-128b* has been also showed as direct regulator of EGFR, whereas *miR-128b* loss-ofheterozigocity is frequently found in NSCLC. *miR-7* is frequently down-regulated in lung cancer, it suppress EGFR and Raf1, it attenuates activation of Akt and ERK, suggesting that is negative regulator of EGFR pathway [71].

It was proposed that *miR-140* regulates PDGF in lung cancer development, but this has not yet been thoroughly investigated. *miR-29* is down-regulated in lung cancers, its targets are Mcl-1, DNMT3A and DNMT3B, suggesting that it is pro-apoptotic and that it has role in regulating epigenetic DNA methylation. Further, in lung cancer cells induction of *miR-34* results in apoptosis; miRNA profiling revealed that *miR-34a/b/c* are directly correlated with expression of p53. Decreased expression of *miR-126* and increased expression of VEGFA was found in various lung cancer cell lines. Introduction of *miR-126* down-regulates VEGFA, inhibits growth, and reduces average tumour weight. Mouse model of lung adenocarcinoma showed that *miR-200* family possess anti-metastatic abilities [5]. *miR-125b-1* is located on a fragile site on chromosome 11q24 which is deleted in a subset of patients with lung cancer [61].

#### **Human brain cancer**

The phrase "brain tumours" describes an inhomogeneous collection of various tumours of the brain, which represents primary tumours of nervous central system or metastases. Glioblas‐ tomas (belongs to family of gliomas) are the most frequent occurrence and malignant form of primary brain tumors in contrary to medulloblastomas, which have a better prognosis [2,11]. Several articles have described the effects of ectopic miRNA modulation on medulloblastoma cell proliferation and growth.

Rescued expression of *miR-9* and *miR-125a* were shown to promote medulloblastoma cell growth arrest and apoptosis by targeting TrkC, whereas *miR-29* has been shown to be downregulated in neuroblastoma and brain tumour [47,72]. Further, *miR-34a* induces apoptosis in neuroblastoma cells, possibly by targeting the transcription factor E2F3 [11]. Transient transfection in medullablastoma cells with *miR-34a* strongly inhibited cell proliferation, cell cycle progression, cell survival and cell invasion. *miR-34a* was shown to inhibit c-Met, Notch-1, Notch-2 and CDK6. Ectopic up-regulation of *miR-124* was shown to inhibit cell proliferation, and it was demonstrated that *miR-124* target and regulates CDK6. *miR-125b*, *miR-324-5p*, and *miR-326* over-expression inhibit medulloblastoma cell growth by targeting Hedgehog signal‐ ling pathway. *miR-128* also inhibits growth by targeting Bmi-1 oncogene. Another miRNA, *miR-199b-5p* negatively regulates proliferation and cell growth [72].

In contrary to medulloblastomas, are gliomas the most common and deadly primary human brain tumours, and its subtype glioblastomas are highly invasive, very aggressive, and one of the most incurable [2]. *miR-181a, miR-181b* and *miR-181c* were originally identified as downregulated in glioblastoma cells and tumours when compared to normal brain controls. *miR-181a* and to a greater extent *miR-181b* were subsequently described as tumour suppressors that inhibit growth and induce apoptosis of glioma cells. *miR-181a* over-expression downregulates Bcl-2. Several other miRNAs have been implicated in glioma malignancy as tumour suppressors. *miR-15b* was suggested to target CCNE1, the gene encoding cyclin E1, however, a direct link between CCNE1 down-regulation by *miR-15* and cell cycle regulation was not demonstrated. *miR-146b* was shown to inhibit glioma cell migration and invasion, and was identified as one of miRNAs that is significantly deregulated in human glioblastoma tissue. *miR-146b* over-expression or knock-down did not affect the growth of human glioblastoma cell line, while it significantly reduced the migration and invasion of one glioblastoma cell line. MMP16 was identified as one of the downstream targets of *miR-146b*. *miR-125b* was shown to induce cell cycle arrest and inhibits CDK6 and CDC25A expression in glioma cell lines. However, another study suggested oncogene function for *miR-125b*. *miR-153* decrease cell proliferation and increased apoptosis (pro-apoptotic miRNA), it inhibited Bcl-2 and Mcl-1. *miR-17* and *miR-184* were identified as two miRNAs with reduced expression in higher grades of glioblastomas. Their over-expression inhibited viability, proliferation, invasion and decreased expression of AKT 2 and several other genes. *let-7* over-expression effect was investigated in glioma cells, its transfection reduced expression of RAS oncogenes, prolifera‐ tion in vitro, migration of the cells, and reduced the size of tumours generated [73].

#### **Head and Neck Squamous Cell Carcinoma (HNSCC)**

sion of *let-7a-2* compared to those with opposite expression pattern, independent of disease stage [2]. Over-expression of *let-7* in lung adenocarcinoma cell lines inhibited cancer cell growth and reduced cell cycle progression. These findings reflect that *let-7* mediates tumour suppressive function [2,45]. It has been shown that *let-7* regulates the expression of several oncogenes, RAS, MYC, HMGA2, and cell-cycle progression regulators, CDC25, CDK6, cyclin D2. *let-7* is down regulated in lung tumours and its expression anti-correlated with that of RAS relative to the normal lung tissue [46,71]. In animal models, ectopic *let-7g* expression reduces tumour burden and intranasal administration repress lung adenocarcinoma. A SNP in *let-7* complementary site 6 in 3'-UTR of its target KRAS is significantly associated with increased risk for NSCLC among moderate smokers [71]. The *let-7* family was subsequently found to be

Mutations in EGFR gene are more frequent in NSCLC patients who never smoked tobacco; a significant down-regulation of *miR-145* has been demonstrated in the cancer tissues of these patients. Restoration of this miRNA can inhibit cancer cell growth in EGFR mutant lung AD [47,71]. *miR-128b* has been also showed as direct regulator of EGFR, whereas *miR-128b* loss-ofheterozigocity is frequently found in NSCLC. *miR-7* is frequently down-regulated in lung cancer, it suppress EGFR and Raf1, it attenuates activation of Akt and ERK, suggesting that is

It was proposed that *miR-140* regulates PDGF in lung cancer development, but this has not yet been thoroughly investigated. *miR-29* is down-regulated in lung cancers, its targets are Mcl-1, DNMT3A and DNMT3B, suggesting that it is pro-apoptotic and that it has role in regulating epigenetic DNA methylation. Further, in lung cancer cells induction of *miR-34* results in apoptosis; miRNA profiling revealed that *miR-34a/b/c* are directly correlated with expression of p53. Decreased expression of *miR-126* and increased expression of VEGFA was found in various lung cancer cell lines. Introduction of *miR-126* down-regulates VEGFA, inhibits growth, and reduces average tumour weight. Mouse model of lung adenocarcinoma showed that *miR-200* family possess anti-metastatic abilities [5]. *miR-125b-1* is located on a fragile site

on chromosome 11q24 which is deleted in a subset of patients with lung cancer [61].

The phrase "brain tumours" describes an inhomogeneous collection of various tumours of the brain, which represents primary tumours of nervous central system or metastases. Glioblas‐ tomas (belongs to family of gliomas) are the most frequent occurrence and malignant form of primary brain tumors in contrary to medulloblastomas, which have a better prognosis [2,11]. Several articles have described the effects of ectopic miRNA modulation on medulloblastoma

Rescued expression of *miR-9* and *miR-125a* were shown to promote medulloblastoma cell growth arrest and apoptosis by targeting TrkC, whereas *miR-29* has been shown to be downregulated in neuroblastoma and brain tumour [47,72]. Further, *miR-34a* induces apoptosis in neuroblastoma cells, possibly by targeting the transcription factor E2F3 [11]. Transient transfection in medullablastoma cells with *miR-34a* strongly inhibited cell proliferation, cell cycle progression, cell survival and cell invasion. *miR-34a* was shown to inhibit c-Met, Notch-1,

deregulated in a large number of tumour types [46].

negative regulator of EGFR pathway [71].

62 Future Aspects of Tumor Suppressor Gene

**Human brain cancer**

cell proliferation and growth.

Head and neck tumours are a heterogenous group with different behaviour at the various sites arising from anatomical factors, cell-type variation, and differences in exposure to risk factors including tobacco, alchocol, and viruses [74]. Head and neck squamous cell carcinoma is represented by epithelial cancers of the oral cavity, pharynx, nasal cavity, paranasal sinuses, salivary glands and larynx [75]. Studies were made in expression profiling regarding different sites of head and neck tumours, tongue, tonsil, larynx, hypopharynx, nasopharynx, saliva, oral cavity, salivary gland and animal models. Several miRNAs were identified as down-regulated, and for some of their target genes were validated [74].

Low expression of *miR-205* is significantly associated with local-regional recurrence inde‐ pendent of disease severity at diagnosis and treatment. Combined low expression of *let-7d* and *miR-205* is significantly associated with poor survival. In nasopharengyal carcinoma downregulation of *miR-34* family, *miR-145* and *miR-143* was also observed [47]. Tumour suppressive role in HNSCC has been suggested for *let-7* family, *miR-125a/b*, *miR-200*, *miR-133a/b*, and *miR-100* [75]. General down-regulation of *miR-1*, *miR-133a* and *let-7b* was also observed [74]. Reduced expression for majority of members of the *let-7* family (except *let-7i*) was observed in HNSCC. KRAS and HMGA2 have been characterized as targets for *let-7* [75]. Notable among down-regulated was also *miR-98*. However, another group identified *miR-98* as another regulator of an oncogene HMGA2 [74]. A possible molecular mechanism of *miR-125a/b* downregulation might be through targeting ERBB2, since its higher level of expression was observed in oral SCC. Other target were also suggested for *miR-125*, namely KLF13, CXCL11 and FOXA1 [74,75]. Down-regulation of *miR-133a/b* in primary HNSCC may further contribute to increased cell proliferation and decreased apoptosis. PKM2 has been validated as cellular target for both, *miR-133a* and *miR-133b*, and increased expression of PKM2 has been associated with cancer progression. Finally, *miR-100* has been observed at suppressed levels in primary HNSCC and derived cell lines. A few of its targets are known, namely FGFR1, MMP13, ID1, FGFR3, EGR2. The exact role has to be investigated yet, but suggestion has been made that down-regulation of *miR-100* means higher rate of cell proliferations.

inhibition and induces cell death. *mir-129* is shown to target GALNT1 and SOX4. Trans‐ fection of *miR-30-3p*, *miR-133a* and *miR-199a\** results in decrease of tumour cell growth in bladder cancer. *miR-101* inhibits cell proliferation in bladder transitional cell carcinoma by targeting EZH2 and altering global chromatin structure. Down-regulation of *miR-145* in

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*Prostate cancer.* A major signal transduction pathway in prostate cancer is PI3K/Akt signalling pathway that is hyper-activated in approx. 30-50 % of prostate cancer. Many of the predicted miRNAs that are predicted to target proteins of this pathway are differentially regulated in prostate cancer. Second pathway is androgen receptor (AR) pathway. *miR-125b* is an androgensensitive miRNA, which has been shown to regulate apoptosis through inhibition of BAK1. *miR-101* has been shown to be up-regulated in human prostate cancer and it seems that through inhibition of EZH2 reduce invasion and induce morphological changes in prostate cancer cells [76]. *miR-221* is found to be progressively down-regulated in aggressive forms of prostate cancer. Down-regulation of *miR-221* is linked to cancer progression and recurrence in a high risk prostate cohort. Progressive miR-221 down-regulation also hallmarks metastasis [47,76]. *miR-499a* has been shown to be down-regulated in prostate cancer tissue. Its introduction into prostate cancer cells results in cell cycle arrest and apoptosis, where it regulates cell growth and viability in part by repressing the expression of HDAC1 [47]. *miR-15a-miR-16-1* cluster,

located at chromosome 13q14, is deleted in most cases of prostate cancer [21].

Dicer and Ago, are over-expressed in melanoma [32].

related to melanoma progression.

Melanoma is the most aggressive type of skin cancer, and it is resistant to therapy in its advanced stages [77]. Abnormalities in several signal transduction pathways, which are important for normal melanocyte development, only partly explain molecular mechanism directly linking UV radiation to the development of melanoma. miRNAs are emerging as important causal factors to melanoma initiation and progression [78]. In 45 primary cul‐ tured melanoma cell lines, there was observed that many genomic loci containing miR‐ NAs are frequently affected (85.9 %) by copy number abnormalities. For an example, copy number losses of the region containing *miR-218-1* and SLIT2 were shown in 33 % of all investigated melanoma lines [79]. Proteins involved in miRNA biogenesis, Drosha,

However, deregulation of miRNAs expression is not always explained. *let-7* plays a role in melanoma development and progression, its targets are many cancer-promoting mole‐ cules, such as NRAS, Raf, c-myc, cyclin D1/D3, and CDK4 [77]. Analyzing 10 melanocytic nevi and 10 primary melanomas, it was revealed that five members of the *let-7* family were significantly down-regulated in melanoma [80]. Over-expression of *let-7b* leads to in‐ hibition of cell cycle progression and inhibition of its targets (cyclin D1/D3/A and CDK4) [77]. A direct interaction of *let-7b* with the cyclin D1 3'-UTR was showed. *Let-7a* was thus demonstrated to regulate expression of integrin beta3 and RAS oncogene, which is highly

Another family, *miR-34*, has tumour suppressive role. Expression of *miR-34a* is silenced due to an aberrant CpG methylation in 43.2 % of melanoma cell lines and in 62.5 % of primary

bladder cancer has been also observed [47].

**Melanomas**

Deregulation of miRNAs in cancer can occur through epigenetic changes (promoter CpG island hyper-methylation in the case of *miR-200* family) [8]. Suppressed *miR-200a* was detected in primary oral SCC. Members of *miR-200* family inhibit EMT by directly targeting ZEB1/ZEB2, suppressed levels of *miR-200a* may promote EMT. At last, it was observed that *miR-137* and *miR-193a* could be also silenced via hyper-methylation, CDK6 and E2F6 has been suggested as their major targets. It was also shown that *miR-137* hyper-methylation is associated with poorer average survival [74,75].

#### **Urological tumours**

*Renal cell carcinoma (RCC).* The VHL tumour suppressor signalling pathway is the most important deregulated pathway in clear cell RCC, the dominant subtype of kidney can‐ cers. The VHL gene can be spontaneously deleted or hyper-methylated. The regulation of the VHL pathway by miRNAs has not been well studied in RCC. The interactions have been proposed but a direct relation between miRNAs and VHL or HIF1A were not pro‐ ven. A subset of miRNAs has been identified as regulated by VHL pathway, 3 miRNAs were up-regulated and 6 were down-regulated. The second commonly deregulated path‐ way is VEGF signalling pathway, which is transcriptionally regulated by HIF (after hypo‐ xia) or due to loss of VHL. Interaction between miRNAs and VEGF has not been well studied in RC, and only *miR-29b* has been shown to indirectly regulate VEGF. However, strong inverse correlation between *miR-200* family and VEGFA has been observed with suggestion that VEGFA is direct target of these miRNAs [76]. Down-regulation of *miR-141* was found in malignant compared to matched non-malignant tissue samples [47].

*Bladder cancer.* Mutation or over-expression of the FGFR3 gene occurs in approx. 80 % of all patients with low-grade non-invasive urothelial carcinomas. Some well-established de‐ regulated miRNAs in bladder cancer are predicted to target FGFR3, and regulation of FGFR3 by *miR-99* and *miR-100* of four predicted miRNAs has been experimentally vali‐ dated. Family *miR-200* is associated with an epithelial phenotype; its ectopic expression in bladder cancer cell lines induces up-regulation of epithelial and down-regulation of mes‐ enchymal markers. Up-regulation of *miR-143* is accompanied by down-regulation of RAS [76]. Transfection of bladder cancer cell lines with *pre-miR-129* exerts significant growth inhibition and induces cell death. *mir-129* is shown to target GALNT1 and SOX4. Trans‐ fection of *miR-30-3p*, *miR-133a* and *miR-199a\** results in decrease of tumour cell growth in bladder cancer. *miR-101* inhibits cell proliferation in bladder transitional cell carcinoma by targeting EZH2 and altering global chromatin structure. Down-regulation of *miR-145* in bladder cancer has been also observed [47].

*Prostate cancer.* A major signal transduction pathway in prostate cancer is PI3K/Akt signalling pathway that is hyper-activated in approx. 30-50 % of prostate cancer. Many of the predicted miRNAs that are predicted to target proteins of this pathway are differentially regulated in prostate cancer. Second pathway is androgen receptor (AR) pathway. *miR-125b* is an androgensensitive miRNA, which has been shown to regulate apoptosis through inhibition of BAK1. *miR-101* has been shown to be up-regulated in human prostate cancer and it seems that through inhibition of EZH2 reduce invasion and induce morphological changes in prostate cancer cells [76]. *miR-221* is found to be progressively down-regulated in aggressive forms of prostate cancer. Down-regulation of *miR-221* is linked to cancer progression and recurrence in a high risk prostate cohort. Progressive miR-221 down-regulation also hallmarks metastasis [47,76]. *miR-499a* has been shown to be down-regulated in prostate cancer tissue. Its introduction into prostate cancer cells results in cell cycle arrest and apoptosis, where it regulates cell growth and viability in part by repressing the expression of HDAC1 [47]. *miR-15a-miR-16-1* cluster, located at chromosome 13q14, is deleted in most cases of prostate cancer [21].

#### **Melanomas**

Reduced expression for majority of members of the *let-7* family (except *let-7i*) was observed in HNSCC. KRAS and HMGA2 have been characterized as targets for *let-7* [75]. Notable among down-regulated was also *miR-98*. However, another group identified *miR-98* as another regulator of an oncogene HMGA2 [74]. A possible molecular mechanism of *miR-125a/b* downregulation might be through targeting ERBB2, since its higher level of expression was observed in oral SCC. Other target were also suggested for *miR-125*, namely KLF13, CXCL11 and FOXA1 [74,75]. Down-regulation of *miR-133a/b* in primary HNSCC may further contribute to increased cell proliferation and decreased apoptosis. PKM2 has been validated as cellular target for both, *miR-133a* and *miR-133b*, and increased expression of PKM2 has been associated with cancer progression. Finally, *miR-100* has been observed at suppressed levels in primary HNSCC and derived cell lines. A few of its targets are known, namely FGFR1, MMP13, ID1, FGFR3, EGR2. The exact role has to be investigated yet, but suggestion has been made that down-regulation

Deregulation of miRNAs in cancer can occur through epigenetic changes (promoter CpG island hyper-methylation in the case of *miR-200* family) [8]. Suppressed *miR-200a* was detected in primary oral SCC. Members of *miR-200* family inhibit EMT by directly targeting ZEB1/ZEB2, suppressed levels of *miR-200a* may promote EMT. At last, it was observed that *miR-137* and *miR-193a* could be also silenced via hyper-methylation, CDK6 and E2F6 has been suggested as their major targets. It was also shown that *miR-137* hyper-methylation is associated with

*Renal cell carcinoma (RCC).* The VHL tumour suppressor signalling pathway is the most important deregulated pathway in clear cell RCC, the dominant subtype of kidney can‐ cers. The VHL gene can be spontaneously deleted or hyper-methylated. The regulation of the VHL pathway by miRNAs has not been well studied in RCC. The interactions have been proposed but a direct relation between miRNAs and VHL or HIF1A were not pro‐ ven. A subset of miRNAs has been identified as regulated by VHL pathway, 3 miRNAs were up-regulated and 6 were down-regulated. The second commonly deregulated path‐ way is VEGF signalling pathway, which is transcriptionally regulated by HIF (after hypo‐ xia) or due to loss of VHL. Interaction between miRNAs and VEGF has not been well studied in RC, and only *miR-29b* has been shown to indirectly regulate VEGF. However, strong inverse correlation between *miR-200* family and VEGFA has been observed with suggestion that VEGFA is direct target of these miRNAs [76]. Down-regulation of *miR-141*

was found in malignant compared to matched non-malignant tissue samples [47].

*Bladder cancer.* Mutation or over-expression of the FGFR3 gene occurs in approx. 80 % of all patients with low-grade non-invasive urothelial carcinomas. Some well-established de‐ regulated miRNAs in bladder cancer are predicted to target FGFR3, and regulation of FGFR3 by *miR-99* and *miR-100* of four predicted miRNAs has been experimentally vali‐ dated. Family *miR-200* is associated with an epithelial phenotype; its ectopic expression in bladder cancer cell lines induces up-regulation of epithelial and down-regulation of mes‐ enchymal markers. Up-regulation of *miR-143* is accompanied by down-regulation of RAS [76]. Transfection of bladder cancer cell lines with *pre-miR-129* exerts significant growth

of *miR-100* means higher rate of cell proliferations.

poorer average survival [74,75].

64 Future Aspects of Tumor Suppressor Gene

**Urological tumours**

Melanoma is the most aggressive type of skin cancer, and it is resistant to therapy in its advanced stages [77]. Abnormalities in several signal transduction pathways, which are important for normal melanocyte development, only partly explain molecular mechanism directly linking UV radiation to the development of melanoma. miRNAs are emerging as important causal factors to melanoma initiation and progression [78]. In 45 primary cul‐ tured melanoma cell lines, there was observed that many genomic loci containing miR‐ NAs are frequently affected (85.9 %) by copy number abnormalities. For an example, copy number losses of the region containing *miR-218-1* and SLIT2 were shown in 33 % of all investigated melanoma lines [79]. Proteins involved in miRNA biogenesis, Drosha, Dicer and Ago, are over-expressed in melanoma [32].

However, deregulation of miRNAs expression is not always explained. *let-7* plays a role in melanoma development and progression, its targets are many cancer-promoting mole‐ cules, such as NRAS, Raf, c-myc, cyclin D1/D3, and CDK4 [77]. Analyzing 10 melanocytic nevi and 10 primary melanomas, it was revealed that five members of the *let-7* family were significantly down-regulated in melanoma [80]. Over-expression of *let-7b* leads to in‐ hibition of cell cycle progression and inhibition of its targets (cyclin D1/D3/A and CDK4) [77]. A direct interaction of *let-7b* with the cyclin D1 3'-UTR was showed. *Let-7a* was thus demonstrated to regulate expression of integrin beta3 and RAS oncogene, which is highly related to melanoma progression.

Another family, *miR-34*, has tumour suppressive role. Expression of *miR-34a* is silenced due to an aberrant CpG methylation in 43.2 % of melanoma cell lines and in 62.5 % of primary melanoma samples. The tumour suppressive function of *miR-34a* has not yet been investigated, however, there was shown a reduced expression of *miR-34b/c* and *miR-199a*\* and it was proposed that their target is MET oncogene [80]. Another miRNA with potentially tumour suppressor role is embedded in CpG island and epigenetically regulated in melanoma, this is *miR-370* [79].

**5.2. lncRNAs as tumour suppressors**

**MEG3**

**GAS5**

**LincRNA-21**

proposed tumour suppressor function in cancer.

types of cancer, as well as their gene copy number loss [9].

major role in silencing the *MEG3* gene in tumours [84].

of a critical subset of genes with tumour suppressive consequences [4].

lncRNAs are known to mediate epigenetic modifications of DNA by recruiting chromatin complexes to specific loci [8]. Only a handful of lncRNAs have been characterized, and their involvement in control of gene expression [3]. We therefore presented four lncRNAs with

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67

*MEG3*, located in chromosome 14q32, is maternally expressed imprinted gene, which repre‐ sents lncRNA, but also hosted miRNAs and snoRNAs. It plays role in cell proliferation, and its expression is under epigenetic control. *MEG3* and its hosted miRNAs and snoRNA could represent a tumour suppressor gene, since aberrant CpG methylation (promoter hypermethylation, and hyper-methylation of the intergenic region) has been observed in several

*MEG3* ncRNA might modulate binding of p53 on the promoter of its target genes [9]. It was later verified that *MEG3* was associated with p53 and that this association was required for p53 activation, further suggesting tumour suppressor role for *MEG3*. It was demonstrated that *MEG3* expression is markedly decreased in glioma tissues compared to adjunct normal tissues. Ectopic expression of *MEG3* inhibited cell proliferation and promoted cell apoptosis in glioma cell lines [83]. Growth inhibition is partially due to apoptosis induced by *MEG3*, which induces accumulation of p53, stimulates transcription from p53-dependent promoter and regulates p53 target gene expression. Loss or significantly reduction of *MEG3* expression has been further found in other cancer cell lines examined, bladder, bone marrow, breast, cervix, colon, liver, lung, meninges, and prostate, as well as in other primary tumours, neuroblastoma, hepato‐ cellular carcinoma, and meningioma. It has been suggested that DNA methylation plays a

LncRNA *GAS5* is highly expressed in cells that have arrested growth and can sensitize a cell to apoptosis by regulating activity of glucocorticoids in response to nutrient starvation. It has been linked with breast cancer. *GAS5* transcript levels are significantly reduced compared to un-affected normal breast epithelia, suggesting that could act as tumour suppressor. *GAS5* maintain sufficient caspase activity to activate appropriate apoptotic response in diseased cells. Chromosomal translocation affecting 1q25 locus that contains the *GAS5* gene has been detected in melanoma, B-cell lymphoma, prostate and breast cancer [7,85]. *GAS5* regulates expression

*LincRNA-p21* is required for the global repression of genes that interfere with p53 function regulating cellular apoptosis; it physically interacts with a protein hnRNP-K, allows it localization to promoters of genes that need to be repressed in a p53-dependent manner [4]. In response to DNA damage, lncRNAs are induced by the p53 tumour suppressor pathway. *lincRNA-p21* plays an important role in cellular response to apoptotic signal, it is induced by p53 and act as an inhibitor of the p53-dependent transcriptional response by repressing the

*miR-203* has also an important tumours suppressor role in a lot of cancers, and it is often lost due to deletion or due to promoter CpG hyper-methylation. *miR-203* target p63, and their relationship might be relevant also in melanoma, since it is known that *miR-203* functions as switch between epidermal proliferation and differentiation [81].

#### **Gynaecological tumours**

*miR-125b-1* is located on a fragile site on chromosome 11q24 which is deleted in a subset of patients with ovarian and cervical cancer [61].

*Cervical cancer.* Cervical cancer aetiology is strongly linked to HPV infection, and involvement of virus protein E6 and E7 in pathogenesis is well established. The exact pathway from infection to tumorigenesis has not been elucidated yet. However, down-regulation was observed for: *let-7b/c, miR-23b, miR-196b*, *miR-143*, and *miR-145. miR-143* and *miR-145* were equally downregulated in all cell lines of cervical cancer (HPV infected and HPV not infected), whereas miR-*218* was the unique miRNA down regulated only in HPV-16 and HPV-18 positive cell lines. Down-regulation of *miR-214* is related to the ability of this miRNA to inhibit HeLa cells proliferation through targeting MEK3 and JNK1 transcripts. HPV protein E6 induces destabi‐ lization of p53, down-regulation of *miR-34a* and increased proliferation of pre-malignant HPV infected cervical cancer cell lines [82].

*Endometrial cancer*. Reciprocal association between down-regulation of *miR-192-2* and SOX4 expression was determined; it was further established that restoration of *miR-192-2* induced a decrease in SOX4 expression and this resulted in diminished cell proliferation. Decreased expression for *miR-152* and *miR-101* was found to consist of an independent risk factor for disease free survival. Restoration of those miRNAs by transfection in cell lines lead to dimin‐ ished cell proliferation. Down-regulation of *miR-101* was correlated with strong positive immunoreactivity of COX2, which was previously shown to be associated with worse prognosis. To date, no data are available for relationship between miRNAs and oestrogen response in endometrial cancer [82].

*Ovarian cancer.* Inconsistencies are observed between results in ovarian cancer studies for wellknown tumour suppressors. These could be due to the differences in study populations and methodologies used, due to the choice of control group and type of control. For instance, the number of studies used as control cell lines and another number of studies used whole normal ovaries. The existence of significant discrepancies in expression profiles of certain miRNAs indicate the need of further and more in-depth research that would establish those results [82].

#### **5.2. lncRNAs as tumour suppressors**

lncRNAs are known to mediate epigenetic modifications of DNA by recruiting chromatin complexes to specific loci [8]. Only a handful of lncRNAs have been characterized, and their involvement in control of gene expression [3]. We therefore presented four lncRNAs with proposed tumour suppressor function in cancer.

## **MEG3**

melanoma samples. The tumour suppressive function of *miR-34a* has not yet been investigated, however, there was shown a reduced expression of *miR-34b/c* and *miR-199a*\* and it was proposed that their target is MET oncogene [80]. Another miRNA with potentially tumour suppressor role is embedded in CpG island and epigenetically regulated in melanoma, this is

*miR-203* has also an important tumours suppressor role in a lot of cancers, and it is often lost due to deletion or due to promoter CpG hyper-methylation. *miR-203* target p63, and their relationship might be relevant also in melanoma, since it is known that *miR-203* functions as

*miR-125b-1* is located on a fragile site on chromosome 11q24 which is deleted in a subset of

*Cervical cancer.* Cervical cancer aetiology is strongly linked to HPV infection, and involvement of virus protein E6 and E7 in pathogenesis is well established. The exact pathway from infection to tumorigenesis has not been elucidated yet. However, down-regulation was observed for: *let-7b/c, miR-23b, miR-196b*, *miR-143*, and *miR-145. miR-143* and *miR-145* were equally downregulated in all cell lines of cervical cancer (HPV infected and HPV not infected), whereas miR-*218* was the unique miRNA down regulated only in HPV-16 and HPV-18 positive cell lines. Down-regulation of *miR-214* is related to the ability of this miRNA to inhibit HeLa cells proliferation through targeting MEK3 and JNK1 transcripts. HPV protein E6 induces destabi‐ lization of p53, down-regulation of *miR-34a* and increased proliferation of pre-malignant HPV

*Endometrial cancer*. Reciprocal association between down-regulation of *miR-192-2* and SOX4 expression was determined; it was further established that restoration of *miR-192-2* induced a decrease in SOX4 expression and this resulted in diminished cell proliferation. Decreased expression for *miR-152* and *miR-101* was found to consist of an independent risk factor for disease free survival. Restoration of those miRNAs by transfection in cell lines lead to dimin‐ ished cell proliferation. Down-regulation of *miR-101* was correlated with strong positive immunoreactivity of COX2, which was previously shown to be associated with worse prognosis. To date, no data are available for relationship between miRNAs and oestrogen

*Ovarian cancer.* Inconsistencies are observed between results in ovarian cancer studies for wellknown tumour suppressors. These could be due to the differences in study populations and methodologies used, due to the choice of control group and type of control. For instance, the number of studies used as control cell lines and another number of studies used whole normal ovaries. The existence of significant discrepancies in expression profiles of certain miRNAs indicate the need of further and more in-depth research that would establish those results [82].

switch between epidermal proliferation and differentiation [81].

patients with ovarian and cervical cancer [61].

infected cervical cancer cell lines [82].

response in endometrial cancer [82].

*miR-370* [79].

**Gynaecological tumours**

66 Future Aspects of Tumor Suppressor Gene

*MEG3*, located in chromosome 14q32, is maternally expressed imprinted gene, which repre‐ sents lncRNA, but also hosted miRNAs and snoRNAs. It plays role in cell proliferation, and its expression is under epigenetic control. *MEG3* and its hosted miRNAs and snoRNA could represent a tumour suppressor gene, since aberrant CpG methylation (promoter hypermethylation, and hyper-methylation of the intergenic region) has been observed in several types of cancer, as well as their gene copy number loss [9].

*MEG3* ncRNA might modulate binding of p53 on the promoter of its target genes [9]. It was later verified that *MEG3* was associated with p53 and that this association was required for p53 activation, further suggesting tumour suppressor role for *MEG3*. It was demonstrated that *MEG3* expression is markedly decreased in glioma tissues compared to adjunct normal tissues. Ectopic expression of *MEG3* inhibited cell proliferation and promoted cell apoptosis in glioma cell lines [83]. Growth inhibition is partially due to apoptosis induced by *MEG3*, which induces accumulation of p53, stimulates transcription from p53-dependent promoter and regulates p53 target gene expression. Loss or significantly reduction of *MEG3* expression has been further found in other cancer cell lines examined, bladder, bone marrow, breast, cervix, colon, liver, lung, meninges, and prostate, as well as in other primary tumours, neuroblastoma, hepato‐ cellular carcinoma, and meningioma. It has been suggested that DNA methylation plays a major role in silencing the *MEG3* gene in tumours [84].

## **GAS5**

LncRNA *GAS5* is highly expressed in cells that have arrested growth and can sensitize a cell to apoptosis by regulating activity of glucocorticoids in response to nutrient starvation. It has been linked with breast cancer. *GAS5* transcript levels are significantly reduced compared to un-affected normal breast epithelia, suggesting that could act as tumour suppressor. *GAS5* maintain sufficient caspase activity to activate appropriate apoptotic response in diseased cells. Chromosomal translocation affecting 1q25 locus that contains the *GAS5* gene has been detected in melanoma, B-cell lymphoma, prostate and breast cancer [7,85]. *GAS5* regulates expression of a critical subset of genes with tumour suppressive consequences [4].

#### **LincRNA-21**

*LincRNA-p21* is required for the global repression of genes that interfere with p53 function regulating cellular apoptosis; it physically interacts with a protein hnRNP-K, allows it localization to promoters of genes that need to be repressed in a p53-dependent manner [4]. In response to DNA damage, lncRNAs are induced by the p53 tumour suppressor pathway. *lincRNA-p21* plays an important role in cellular response to apoptotic signal, it is induced by p53 and act as an inhibitor of the p53-dependent transcriptional response by repressing the transcription of genes that interfere with apoptosis (guidance of hnRNP-K to the promoters of genes repressed by p53). *LincRNA-p21* has not been directly associated with disease yet, but loss of function of *lincRNA-p21* might be involved in cancer initiation since functions to trigger cell death through the induction of apoptosis program [7,9,85].

[2] Zhang B, Pan X, Cobb GP, Anderson TA. microRNAs as oncogenes and tumor

MicroRNAs and lncRNAs as Tumour Suppressors

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## **CCND1**

It is involved in the regulation of Cyclin D1 gene expression. Cyclin D1 is a cell cycle regulator often mutated, amplified and over-expressed in various types of cancer. After binding of this lncRNA on RNA-binding protein, consequently inhibition of enzymatic activities of the histone acetyltransferases occurs, leading to silencing of cyclin D1 gene. These studies suggest that this lncRNA is a tumour suppressor RNA, which can be rapidly induced by cellular stress to regulate it sense gene expression [85].

## **6. Conclusion**

The rest of ncRNAs, other than miRNAs, in regulation biological functions are more or less unexplored, and this should be further investigated in future research. Regarding therapeutic approaches, we still need more knowledge concerning which miRNAs to target, how to produce and stabilize them, how to direct them to the target tissue. The specificity of drug-like oligonucleotides is important, because of the off-target effect. The off-target effect is also a significant challenge, especially considering that miRNA-mediated repression often requires a homology of only six to seven nucleotides in the seed region of the miRNA and mRNA target site. Toxicity due to chemical modifications, which is used to facilitate cellular uptake and prevent degradation, should be take into account. However, only recently was described the possibility of using exososmes and exosomal tumour-suppressive miRNAs as novel cancer therapy [86].

## **Author details**

Emanuela Boštjančič and Damjan Glavač

Department of Molecular Genetics, Institute of Pathology, Faculty of Medicine Ljubljana, University of Ljubljana, Slovenia

## **References**

[1] Paranjape T, Slack FJ, Weidhaas JB. MicroRNAs: tools for cancer diagnostics. Gut 2009;58(11): 1546-54.

[2] Zhang B, Pan X, Cobb GP, Anderson TA. microRNAs as oncogenes and tumor suppressors. Dev Biol 2007;302: 1-12.

transcription of genes that interfere with apoptosis (guidance of hnRNP-K to the promoters of genes repressed by p53). *LincRNA-p21* has not been directly associated with disease yet, but loss of function of *lincRNA-p21* might be involved in cancer initiation since functions to trigger

It is involved in the regulation of Cyclin D1 gene expression. Cyclin D1 is a cell cycle regulator often mutated, amplified and over-expressed in various types of cancer. After binding of this lncRNA on RNA-binding protein, consequently inhibition of enzymatic activities of the histone acetyltransferases occurs, leading to silencing of cyclin D1 gene. These studies suggest that this lncRNA is a tumour suppressor RNA, which can be rapidly induced by cellular stress

The rest of ncRNAs, other than miRNAs, in regulation biological functions are more or less unexplored, and this should be further investigated in future research. Regarding therapeutic approaches, we still need more knowledge concerning which miRNAs to target, how to produce and stabilize them, how to direct them to the target tissue. The specificity of drug-like oligonucleotides is important, because of the off-target effect. The off-target effect is also a significant challenge, especially considering that miRNA-mediated repression often requires a homology of only six to seven nucleotides in the seed region of the miRNA and mRNA target site. Toxicity due to chemical modifications, which is used to facilitate cellular uptake and prevent degradation, should be take into account. However, only recently was described the possibility of using exososmes and exosomal tumour-suppressive miRNAs as novel cancer

Department of Molecular Genetics, Institute of Pathology, Faculty of Medicine Ljubljana,

[1] Paranjape T, Slack FJ, Weidhaas JB. MicroRNAs: tools for cancer diagnostics. Gut

cell death through the induction of apoptosis program [7,9,85].

to regulate it sense gene expression [85].

68 Future Aspects of Tumor Suppressor Gene

**CCND1**

**6. Conclusion**

therapy [86].

**Author details**

**References**

Emanuela Boštjančič and Damjan Glavač

University of Ljubljana, Slovenia

2009;58(11): 1546-54.


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**Chapter 4**

**Roles of Tumor Suppressor**

**Signaling on Reprogramming and**

Arthur Kwok Leung Cheung, Yee Peng Phoon,

Additional information is available at the end of the chapter

The pioneering landmark, established by Takahashi and Yamanaka (Takahashi et al., 2007; Takahashi and Yamanaka, 2006) in reprogramming somatic cells into induced pluripotent stem (iPS) cells using the four transcriptional factors of Oct4, Sox2, Klf4, and c-Myc, represents one of the most important paradigm shifts in current stem cell biology. This unprecedented discovery could potentially revolutionize regenerative medicine, cell-based therapy and personalized medicine. Despite recent great advancement in cell reprogramming, there are still considerable technical challenges to circumvent restrictions of applications of reprogram‐ ming technology (Kawamura et al., 2009; Saha and Jaenisch, 2009). The utilization of overexpressed transcriptional factors, which of many play oncogenic roles, during somatic reprogramming posts the risk of malignant transformation, thus, limiting its clinical applica‐ tions. Moreover, the reprogramming process using these factors is still inefficient in some of cell types, and is not always successful in other kinds of cells (Kawamura et al., 2009; Marion et al., 2009; Menendez et al., 2012). Therefore, the underlying mechanisms for signaling control

Somatic cell reprogramming is a complicated cellular process that is controlled by many signaling networks. Accumulated evidence indicated that stemness transition can be detected in some tumor cells following the introduction of relevant signal stimulation, and cancer cells or differentiated cells can be changed into stem cell-like cells that go through less-differentiated stages (Chen et al., 2008; Fodde and Brabletz, 2007; Huang et al., 2009; Liu et al., 2009a).

and reproduction in any medium, provided the original work is properly cited.

© 2013 Cheung et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Cheung et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Hong Lok Lung, Josephine Mun Yee Ko,

of these factors still need to be further explored.

Yue Cheng and Maria Li Lung

http://dx.doi.org/10.5772/55712

**1. Introduction**

**Stemness Transition in Somatic Cells**

[86] Kosaka N, Takeshita F, Yoshioka Y, Hagiwara K, Katsuda T, Ono M, Ochiya T. Exosomal tumor-suppressive microRNAs as novel cancer therapy: "Exocure" is another choice for cancer treatment. Adv Drug Deliv Rev 2012

## **Chapter 4**
