**6. Cancer stem cells and apoptosis**

132 Apoptosis and Medicine

**5.2. Restoration of tumour suppressor miRNAs** 

different treatments needs to be improved.

breast cancer MCF7 cells [195]

**5.3. miRNAs and drug resistance** 

The restoration of tumour suppressor miRNAs, as a therapeutic strategy, includes viral delivery or synthetic miRNA mimics. Elevation of the expression levels of miRNAs can restore tumour inhibitory functions in cancer cells. Adeno-associated virus delivery of miRNAs or miRNA antagonists has the advantage of being efficient and because the virus does not integrate into the genome, non-mutagenic. In Myc-induced liver tumours, intravenous injection of adeno-associated virus 8 (AAV8)-expressing miR-26 resulted in the suppression of tumourigenicity by inducing tumour-apoptosis, without signs of toxicity [191]. These findings indicate a possibility strategy for the treatment of liver cancer, however, before this approach achieve widespread clinical use, the delivery and safety of

Another strategy to increase the expression of a tumour-suppressor miRNA in cancer could be overcome by miRNA mimics, which are small, chemically modified double-stranded RNA molecules designed to mimic endogenous mature miRNAs [192]. Introduction of synthetic miRNA mimics with tumour-suppressor function in cancer cells have been implicated to induce cell death and block proliferation in several studies [30,147,178,184,186]. In prostate and AMl cell lines mimics of miR-15a and miR-29 respectively, induced apoptosis by repression of anti-apoptotic genes Mcl-1 and Bcl-2 [184].

Multiple miRNAs have been found to inhibit the apoptotic pathway following their over expression during cancer development. Reduced expression of miR-15, miR-16, and let-7 has been observed in different types of cancers and as one consequence anti-apoptotic genes and apoptotic signalling pathways have been activated in these cancer cells [30,187,193]. As well, transfection of anti-miR-24 oligonucleotides has been proved to induce apoptosis in several cell lines [194]. It has been reported that miR-195, miR-24-2 and miR-365-2 act as negative regulators of the anti-apoptotic proto-oncogene Bcl2. The overexpression of these miRNAs caused an increase in apoptosis and also augmented the apoptotic effect of etoposide in

Several miRNA, some of them related to apoptosis, have been associated with drug resistance. Deregulation of miR-214 is a recurrent event in human ovarian cancer and it has been shown that miR-214 induces cell survival and cisplatin resistance primarily through targeting the PTEN/Akt pathway [196]. Also, is known that the let-7 family of miRs plays a role in a host of cellular functions such as modulation of drug sensitivity. The miRNA let-7a which directly targeting caspase-3 is over-expressed in some human cancers and has been shown to induce resistance to a variety of drugs caspase-3-dependent apoptosis, including doxorubicin, paclitaxel and interferon-gamma. Let-7e was up-regulated in some ovarian cancer cell lines with increased resistance to doxorubicin. On other hand, it has been reported that let-7i is down-regulated in chemotherapy-resistant ovarian cancer, and reintroduction of let-7i can sensitize resistant ovarian cancer cell lines to platinum-based Some cancers are originated in cells with intrinsic self-renewal activity or in differentiated cells in which self-renewal is activated by oncogenic mechanisms; hence, the study of normal self-renewal is important to improve our understanding of these mechanisms. Cancers express a spectrum of aberrantly differentiated cells, ranging from those that appear well differentiated to those that appear undifferentiated, and these phenotypes are commonly evident in the same tumour. This suggests that the transformation process can induce defects throughout the multistep differentiation process. Recent data suggest that cancers arise from rare self-renewing stem cells that are biologically distinct from their more numerous differentiated progeny. A small number of cells identied as cancer stem cells (CSCs) from solid tumours usually express organ-specic markers, contribute to chemotherapy resistance and are able to generate a new tumour in immunodecient mice. Moreover, there is growing evidence that pathways regulating normal stem cell self-renewal and differentiation are also present in cancer cells and CSCs [197].

Currently, there are two theories on the origin of cancer: the classic clonal evolution theory or stochastic model, by which malignant transformation results from random mutations and subsequent clonal selection of cancer cells with similar potential to regenerate the tumour growth [198,199]; and the CSC hypothesis, which considers the tumour to be formed by a small population of cells with stem cell-like properties. The features in common with stem cells are: indefinite self-replication, asymmetric cell division, and resistance to toxic agents, owing, in part, to elevated expression of ABC transporters [199,200,201]. In addition, they are also characterized by genetic instability (chromosomal and microsatellite), changes of chromatin, transcription and epigenetics, mobilization of cellular resources, and modified microenvironment interactions (tumour cells, stromal cells, extracellular, endothelium) [202]. Both paradigms of tumour propagation are likely to exist in human cancer but only the CSC model is hierarchical. It is important to note that the two models are not mutually exclusive, as CSCs undergo clonal evolution, as shown for leukaemia stem cells [203].

The theory of cancer stem cells is not new, having started wonderings in the 19th century when comparing cancerous and embryonic tissue in the microscope and certain similarities were observed, annotating the idea that tumours arising from embryonic-like cells. This theory continued to evolve, and the isolation of four different tumour subpopulations from a single breast cancer in a mouse was reported in the decade of the 80s[204]. Tumour heterogeneity is reflected in different phenotypic aspects such as cell morphology, gene expression, metabolism, motility and proliferation, immunogenic, angiogenic and metastatic potential [202].

Apoptosis as a Therapeutic Target in Cancer and Cancer Stem Cells: Novel Strategies and Futures Perspectives 135

Molecular characterization of CSCs is necessary to develop a targeted therapy (Table 6). They have been isolated from several tumour types including haematological malignancies (the first evidence) [205], breast, brain, colon, lung, head and neck, prostate, pancreas and liver cancers and melanoma. The subpopulation of CSCs self renews, differentiates, and regenerates a phenocopy of the original tumour when injected into immunodeficient mice

Tumour stem cells may display multidrug resistance that is conferred by ABC transporters. These ABC transporters have been reported as CSCs markers in melanoma and osteosarcoma, among others. These transporters are up-regulated in CSCs, and the lowstaining fraction of cells with the ability to efux Hoechst 33342 dye is commonly known as the side population (SP) [33]. Targeted inactivation of ABC transporters could reinstate drug sensitivity in CSCs, resulting in their death. Moreover, pathways that regulate self-renewal of normal stem cells, such as Wnt, Notch and Hedgehog, tumor suppressor genes, such as PTEN and p53, have been implicated in the control of CSCs self-renewal. These pathways are believed to be deregulated in CSCs, leading to uncontrolled self-renewal and generation of tumours that are resistant to conventional therapies [220]. The above mentioned characteristics and the ability of CSCs to evade cell death signals contribute to the failure of existing therapies to eradicate malignant tumours, causing resistance to treatment and an increased morbidity and mortality [201,221]. Apoptosis is one of the most critical and wellstudied mechanisms, governing tissue development and homeostasis through a complex network of molecules that mediate death and survival signals. Escape from death program is a prerequisite for any tumour-initiating cell and may support the survival of CSCs in response to chemo- or radio-therapy. Thus, manipulating the apoptotic machinery to

eradicate tumour-initiating cells holds enormous therapeutic potential [206].

or intrinsic pathways to treat cancer.

**6.1. TRAIL** 

Several studies have focused on apoptosis induction in CSCs by intervening in the extrinsic

TRAIL has been demonstrated to induce apoptosis in a wide range of cancer types both in vitro and also in various mouse models of human cancers [106]. In certain types of tumours a correlation has been established between the expression of DR and CSCs. Chemotherapyresistant colon cancer SP cells express high levels of DR4 [222]**;** in some bladder cancer cell lines there is an increased expression of DR5 [223], and, in glioblastoma and lung CSCs express DRs [206]. More importantly, several clinical trials have explored the response to the use of DRs as a treatment. Studies concluded that SP cells of colon cancer displayed higher sensitivity to TRAIL compared to the non-SP cells [222]. In bladder cancer treatment sensitivity was greater in those showing increased expression of DR5 [223]. Moreover, isolated neurospheres from glioblastomas with characteristics of stem cells showed differences in apoptosis after treatment with TRAIL and it was effective in those who keep the route intact for caspase 8. Genomic heterogeneity in glioblastomas suggests the presence of multiple mechanisms in TRAIL resistance in both CSCs and non-stem cells. Future

[206].

**Figure 7. A.** The clonal evolution model of cancer is based on the fact that accumulation of a series of mutations (inhibition of apoptosis, angiogenesis,…) in any somatic cell can cause a tumour. **B.** The cancer stem cell model is based on the principle that a progenitor cell capable of self-renewal and proliferation (stem cell characteristics) is the cause of formation of a tumour.


**Table 6.** Phenotypic markers of CSCs in various tumours.

Molecular characterization of CSCs is necessary to develop a targeted therapy (Table 6). They have been isolated from several tumour types including haematological malignancies (the first evidence) [205], breast, brain, colon, lung, head and neck, prostate, pancreas and liver cancers and melanoma. The subpopulation of CSCs self renews, differentiates, and regenerates a phenocopy of the original tumour when injected into immunodeficient mice [206].

Tumour stem cells may display multidrug resistance that is conferred by ABC transporters. These ABC transporters have been reported as CSCs markers in melanoma and osteosarcoma, among others. These transporters are up-regulated in CSCs, and the lowstaining fraction of cells with the ability to efux Hoechst 33342 dye is commonly known as the side population (SP) [33]. Targeted inactivation of ABC transporters could reinstate drug sensitivity in CSCs, resulting in their death. Moreover, pathways that regulate self-renewal of normal stem cells, such as Wnt, Notch and Hedgehog, tumor suppressor genes, such as PTEN and p53, have been implicated in the control of CSCs self-renewal. These pathways are believed to be deregulated in CSCs, leading to uncontrolled self-renewal and generation of tumours that are resistant to conventional therapies [220]. The above mentioned characteristics and the ability of CSCs to evade cell death signals contribute to the failure of existing therapies to eradicate malignant tumours, causing resistance to treatment and an increased morbidity and mortality [201,221]. Apoptosis is one of the most critical and wellstudied mechanisms, governing tissue development and homeostasis through a complex network of molecules that mediate death and survival signals. Escape from death program is a prerequisite for any tumour-initiating cell and may support the survival of CSCs in response to chemo- or radio-therapy. Thus, manipulating the apoptotic machinery to eradicate tumour-initiating cells holds enormous therapeutic potential [206].

Several studies have focused on apoptosis induction in CSCs by intervening in the extrinsic or intrinsic pathways to treat cancer.

## **6.1. TRAIL**

134 Apoptosis and Medicine

potential [202].

heterogeneity is reflected in different phenotypic aspects such as cell morphology, gene expression, metabolism, motility and proliferation, immunogenic, angiogenic and metastatic

**Figure 7. A.** The clonal evolution model of cancer is based on the fact that accumulation of a series of mutations (inhibition of apoptosis, angiogenesis,…) in any somatic cell can cause a tumour. **B.** The cancer stem cell model is based on the principle that a progenitor cell capable of self-renewal and

[209,210]

**TUMOUR TYPE MARKERS Reference Hematological malignancies** CD34+ / CD38- [207] **CNS** CD133+ [208]

CD166+

**Breast** CD24-/low /CD44+ [211] **Lung** CD133+ [212] **Pancreas** CD44+ / CD24+ / EpCAM+/ CD133+ [213] **Liver** CD90+ [214] **Prostate** CD44+ / CD133 / α2β1hi [215] **Bladder** CD44+ / CK5+/ CD20- [216] **Ovaries** CD44+ / CD117+ [217] **Head and neck** CD44+ [218] **Melanoma** ABCB5+ [219]

proliferation (stem cell characteristics) is the cause of formation of a tumour.

**Colon** CD133+/ EpCAMhi / CD44+ /

**Table 6.** Phenotypic markers of CSCs in various tumours.

TRAIL has been demonstrated to induce apoptosis in a wide range of cancer types both in vitro and also in various mouse models of human cancers [106]. In certain types of tumours a correlation has been established between the expression of DR and CSCs. Chemotherapyresistant colon cancer SP cells express high levels of DR4 [222]**;** in some bladder cancer cell lines there is an increased expression of DR5 [223], and, in glioblastoma and lung CSCs express DRs [206]. More importantly, several clinical trials have explored the response to the use of DRs as a treatment. Studies concluded that SP cells of colon cancer displayed higher sensitivity to TRAIL compared to the non-SP cells [222]. In bladder cancer treatment sensitivity was greater in those showing increased expression of DR5 [223]. Moreover, isolated neurospheres from glioblastomas with characteristics of stem cells showed differences in apoptosis after treatment with TRAIL and it was effective in those who keep the route intact for caspase 8. Genomic heterogeneity in glioblastomas suggests the presence of multiple mechanisms in TRAIL resistance in both CSCs and non-stem cells. Future clinical trials of TRAIL apoptotic pathway targeted therapies may consider genomic analysis of tumour tissue to identify the genomic status of TRAIL apoptotic genes such as *caspase 8* and use it as a genomic marker to predict tumour resistance to TRAIL apoptotic pathwaytargeted therapies [224]. In addition, recent studies are focused on combining mesenchymal stem cells (MSCs) expressing TRAIL and chemotherapy. These MSCs migrated to tumours and reduced the growth of primary cancers and metastases by induction of apoptosis, death and reduced colony formation of the SP and were synergistic when combined with traditional chemotherapy in apoptosis induction [225].

Apoptosis as a Therapeutic Target in Cancer and Cancer Stem Cells: Novel Strategies and Futures Perspectives 137

expression levels of the anti-apoptoticc Bcl-2 protein, Mcl-1, were associated with resistance to treatment with Bcl-2 inhibitor ABT-737 in glioma stem cells [233]. In hematopoietic malignances, it has been showed that despite the over-expression of Bcl-2 this is not the critical point for the generation, selection and maintenance of leukemia stem cells [234].

The transcription factor NF-κB has been connected to multiple aspects of oncogenesis, including inhibition of apoptosis by increasing the expression of survival factor. In fact, aberrant regulation of NF-κB has been observed in many cancers, including both solid and hematopoietic tumours [235]. A fairly representative example is found in pancreatic cancer, where there is a clear correlation between the basal activity of NF-kB and the ability to generate angiogenesis and metastasis of pancreatic tumour cells [236] and, more recently it has been found that not only in this way, but also in the non-canonical NF-kB is also activated and functional [237]. A very interesting study by [238] showed that in CD44+ breast CSCs the expression of CD24 potentiated DNA-induced apoptosis by suppressing anti-apoptotic NF-κB signaling. Several therapies are being developed to inhibit this factor because there are many tumours which relate the decrease in the activity of NF-kB with a

A classic mechanism involved in the induction of apoptosis is in response to DNA damage by p53 action. This gene is mutated in most human cancers and inactivated in about 50% of cancers [244]. p53 was found to repress the CSC marker gene CD44 in an experimental breast tumour model and the over-expression of CD44 blocked p53-dependent apoptosis, leading to expansion of tumour-initiating cells [245]. Moreover, glioma CSCs resist radiation through preferential activation of the retainer DNA damage response and an increase in DNA repair capacity. In addition, the radioresistance of CD133+ glioma stem cells could be reversed with a specific inhibitor of Chk1 and Chk2 checkpoint kinases [208]. Another report reinforces the tumour-promoting effect of DNA damage response activation in leukemia stem cells by demonstrating that cell-cycle inhibitor p21 was indispensable for

Recently, miRNAs have emerged as key regulators of "stemness", collaborating in the maintenance of pluripotency, control of self-renewal, and differentiation of stem cells. Moreover, certain miRNAs involved in apoptosis appear to influence the CSC fate by controlling self-renewal. It has been shown that restoration of miR-34 modulates selfrenewal in pancreatic CSCs by directly regulating down-stream target gene Notch and Bcl-2 [247]. Also the restoration of miR-34, inhibit p53-mutant gastric cancer tumourspheres growth in vitro and tumour formation in vivo, which is reported to be correlated to the selfrenewal of CSCs [248]. As miR-34 is a significant tumour suppressor of CSCs by regulation

decrease in the size and tumor growth [239,240,241,242,243].

**6.5. NF-kB** 

**6.6. DNA repair capacity** 

maintaining self-renewal of these CSCs [246].

**6.7. miRNAs, apoptosis and CSCs** 
