**miRNAs as Essential Mediators of the Actions of Retinoic Acid in Neuroblastoma Cells**

[152] Yu AL, Gilman AL, Ozkaynak MF, London WB, Kreissman SG, Chen HX, et al. Anti-GD2 antibody with GM-CSF, interleukin-2, and isotretinoin for neuroblastoma. N

[153] Sabzevari H, Gillies SD, Mueller BM, Pancook JD, Reisfeld RA. A recombinant anti‐ body-interleukin 2 fusion protein suppresses growth of hepatic human neuroblasto‐ ma metastases in severe combined immunodeficiency mice. Proc Natl Acad Sci U S

[154] Becker JC, Varki N, Gillies SD, Furukawa K, Reisfeld RA. Long-lived and transfera‐ ble tumor immunity in mice after targeted interleukin-2 therapy. J Clin Invest. 1996

[155] Osenga KL, Hank JA, Albertini MR, Gan J, Sternberg AG, Eickhoff J, et al. A phase I clinical trial of the hu14.18-IL2 (EMD 273063) as a treatment for children with refrac‐ tory or recurrent neuroblastoma and melanoma: a study of the Children's Oncology

[156] Shusterman S, London WB, Gillies SD, Hank JA, Voss SD, Seeger RC, et al. Antitu‐ mor activity of hu14.18-IL2 in patients with relapsed/refractory neuroblastoma: a Children's Oncology Group (COG) phase II study. J Clin Oncol. 2010 Nov 20;28(33):

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4969-75.

246 Neuroblastoma

Salvador Meseguer, Juan-Manuel Escamilla and Domingo Barettino

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55444

#### **1. Introduction**

The discovery of microRNAs (miRNAs, miRs) led to a profound change on our vision about the regulation of gene expression in eukaryotes. MicroRNAs are an emerging class of small noncoding endogenous RNAs that participate on the fine tuning of gene expression at the posttranscriptional level. First discovered at the early 90s in the nematode *C. elegans* [1], microRNAs have been involved in multiple important biological processes both in animal as in plant cells. These regulatory RNAs are transcribed as primary longer transcripts, which are then processed into 19-23-nt mature miRNAs. One strand of the mature miRNA is then incorporated into the RNA-induced silencing complex (RISC) to regulate gene expression by targeting the 3'-untrans‐ lated region (3'UTR) of mRNAs with consequent translational repression and/or target mRNA degradation. This mode of action demonstrates the great regulatory potential of miRNAs, since a unique mRNA can be targeted by diverse miRNAs and conversely each miRNA may have hundreds of different target mRNAs. In recent years miRNAs have been established as impor‐ tant regulators of tumor development, progression and metastasis, and have demonstrated to be useful for tumor diagnosis and classification. Moreover, miRNA regulation might represent a new avenue for cancer treatment in a near future.

Neuroblastoma is the most common extracranial solid tumor in childhood and the most common tumor in infants, which originates from aberrant development of primordial neural crest cells. Several lines of evidence support the idea that microRNA deregulation could contribute to neuroblastoma pathogenesis and progression [2, 3], and the usefulness of miRNA profiles for neuroblastoma diagnostics, classification and prognosis has been recently reported [4]. Neuroblastoma cell lines can be induced to differentiate *in vitro* by several agents, including Retinoic Acid (RA) [5, 6], the biologically active form of vitamin A. RA treatments lead to

© 2013 Meseguer 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 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.

proliferative arrest and neuronal differentiation [5, 7] and to a reduction of the biological aggressiveness of neuroblastoma cells, by reducing their migratory and invasive abilities [8-10]. As a consequence of this, RA and its derivatives have been introduced into therapeutic protocols for neuroblastoma patients [11-13].

*seed* region, located at nucleotides 2-7 of the guide strand. The lack of stringency results in a many-to-many relationship between miRNAs and mRNA targets, with the consequence that a high percentage of the genome may be regulated post-transcriptionally by a comparatively small set of miRNAs. A consequence of that is also that bioinformatic prediction of miRNA target mRNAs becomes relatively inaccurate. The guide strand binds to its complementary region in the 3′UTR of its target mRNA through Watson–Crick base pairing of the seed residues. Several alternative seed binding arrangements have been observed that involved

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different number of residues and therefore could have different binding affinity [24].

**Figure 1. miRNA Biogenesis.** The scheme depicts the different steps in the biogenesis of miRNAs, the enzymes in‐

**2.3. Suppression of mRNA translation and/or mRNA degradation mediated by miRNAs**

The binding of miRNA-RISC complex to its cognate mRNA target leads to mRNA silencing through suppression of mRNA translation and/or mRNA decay. [25, for review] Several mechanisms involving different protein complexes have been proposed. mRNA translation could be blocked at initiation step as well as post-initiation stages. The miRNA-RISC complex inhibits translation initiation by interfering with eIF4F-cap recognition and 40S small riboso‐ mal subunit recruitment or by antagonizing 60S subunit joining and preventing 80S ribosomal

volved and the intermediate miRNA forms.

In this article we want to review the evidences supporting the contribution of miRNA regulation to RA-induced differentiation of neuroblastoma cells. We will show that miRNA contribute to the gene-expression changes associated with neuroblastoma cell differentiation and that specific RA-induced miRNAs target the expression of relevant genes in the context of neural differentiation. In addition RA-regulated miRNAs contribute to the reduction in the biological aggressiveness elicited by RA *in vitro*. We put forward the idea that miRNA regulation is part of the RA signaling pathway, and that miRNAs are essential mediators of the actions of RA in neuroblastoma cells.

#### **2. The molecular bases of miRNA action**

#### **2.1. Biogenesis of miRNAs**

miRNAs use complementary base pairing and the RNA induced silencing complex (RISC) to bind and either block translation and/or promote degradation of their target mRNAs. miRNAs are 19-22 nt-long RNA molecules transcribed mainly from non-coding regions of the genome, although some are embedded within genes, primarily as part of intronic sequences [14]. In addition, clusters of miRNAs were also found in the genome [15]. miRNAs are transcribed as large hairpin-containing molecules, called pri-miRNA, that are cleaved in the nucleus by the microprocessor complex, involving Drosha and Pasha/DGCR8 proteins [16, 17]. The result of this cleavage is a shorter precursor hairpin (approx. 70 nt), called pre-miRNA. Pre-miRNAs are exported through RAN GTPase and exportin-5 to the cytoplasm [18] where undergo further cleavage by Dicer to yield a transient intermediate imperfect duplex of approx 19-22 bp miRNA [19]. Subsequently, the duplex unwinds and miRNA strand is loaded into RISC complex together with proteins of the Argonaute (Ago) family [20]. The miRNA strand in RISC acts as a guide strand to find the complementary site in mRNA, and thereby suppressing the translational activity of the target mRNA. The complementary strand (known as miRNA\* or as passenger strand) is degraded when the duplex is unwound, although recent evidences show that in some cases miRNA\* accumulated at physiological levels and support the idea of a role for miRNA\* on gene regulation [21]. (see Figure 1)

#### **2.2. miRNA target binding**

miRNAs interact primarily with the 3'-untranslated (3'UTR) region of their target mRNAs, although recent evidences show that miRNAs can also associate with sites located within the coding region of target genes [22]. In fact, complex arrays of multiple binding sites for either the same or different miRNAs located both in the 3'UTR as well as in the coding region of the target genes have been reported [23]. The base pairing of miRNA and mRNA in vertebrates requires only partial homology, with a preference for contiguous pairing occurring only at the *seed* region, located at nucleotides 2-7 of the guide strand. The lack of stringency results in a many-to-many relationship between miRNAs and mRNA targets, with the consequence that a high percentage of the genome may be regulated post-transcriptionally by a comparatively small set of miRNAs. A consequence of that is also that bioinformatic prediction of miRNA target mRNAs becomes relatively inaccurate. The guide strand binds to its complementary region in the 3′UTR of its target mRNA through Watson–Crick base pairing of the seed residues. Several alternative seed binding arrangements have been observed that involved different number of residues and therefore could have different binding affinity [24].

proliferative arrest and neuronal differentiation [5, 7] and to a reduction of the biological aggressiveness of neuroblastoma cells, by reducing their migratory and invasive abilities [8-10]. As a consequence of this, RA and its derivatives have been introduced into therapeutic

In this article we want to review the evidences supporting the contribution of miRNA regulation to RA-induced differentiation of neuroblastoma cells. We will show that miRNA contribute to the gene-expression changes associated with neuroblastoma cell differentiation and that specific RA-induced miRNAs target the expression of relevant genes in the context of neural differentiation. In addition RA-regulated miRNAs contribute to the reduction in the biological aggressiveness elicited by RA *in vitro*. We put forward the idea that miRNA regulation is part of the RA signaling pathway, and that miRNAs are essential mediators of

miRNAs use complementary base pairing and the RNA induced silencing complex (RISC) to bind and either block translation and/or promote degradation of their target mRNAs. miRNAs are 19-22 nt-long RNA molecules transcribed mainly from non-coding regions of the genome, although some are embedded within genes, primarily as part of intronic sequences [14]. In addition, clusters of miRNAs were also found in the genome [15]. miRNAs are transcribed as large hairpin-containing molecules, called pri-miRNA, that are cleaved in the nucleus by the microprocessor complex, involving Drosha and Pasha/DGCR8 proteins [16, 17]. The result of this cleavage is a shorter precursor hairpin (approx. 70 nt), called pre-miRNA. Pre-miRNAs are exported through RAN GTPase and exportin-5 to the cytoplasm [18] where undergo further cleavage by Dicer to yield a transient intermediate imperfect duplex of approx 19-22 bp miRNA [19]. Subsequently, the duplex unwinds and miRNA strand is loaded into RISC complex together with proteins of the Argonaute (Ago) family [20]. The miRNA strand in RISC acts as a guide strand to find the complementary site in mRNA, and thereby suppressing the translational activity of the target mRNA. The complementary strand (known as miRNA\* or as passenger strand) is degraded when the duplex is unwound, although recent evidences show that in some cases miRNA\* accumulated at physiological levels and support the idea of

miRNAs interact primarily with the 3'-untranslated (3'UTR) region of their target mRNAs, although recent evidences show that miRNAs can also associate with sites located within the coding region of target genes [22]. In fact, complex arrays of multiple binding sites for either the same or different miRNAs located both in the 3'UTR as well as in the coding region of the target genes have been reported [23]. The base pairing of miRNA and mRNA in vertebrates requires only partial homology, with a preference for contiguous pairing occurring only at the

protocols for neuroblastoma patients [11-13].

the actions of RA in neuroblastoma cells.

**2.1. Biogenesis of miRNAs**

248 Neuroblastoma

**2.2. miRNA target binding**

**2. The molecular bases of miRNA action**

a role for miRNA\* on gene regulation [21]. (see Figure 1)

**Figure 1. miRNA Biogenesis.** The scheme depicts the different steps in the biogenesis of miRNAs, the enzymes in‐ volved and the intermediate miRNA forms.

#### **2.3. Suppression of mRNA translation and/or mRNA degradation mediated by miRNAs**

The binding of miRNA-RISC complex to its cognate mRNA target leads to mRNA silencing through suppression of mRNA translation and/or mRNA decay. [25, for review] Several mechanisms involving different protein complexes have been proposed. mRNA translation could be blocked at initiation step as well as post-initiation stages. The miRNA-RISC complex inhibits translation initiation by interfering with eIF4F-cap recognition and 40S small riboso‐ mal subunit recruitment or by antagonizing 60S subunit joining and preventing 80S ribosomal complex formation. The interaction of the GW182 protein with the poly(A)-binding protein (PABP) might interfere with the closed-loop formation mediated by the eIF4G-PABP interac‐ tion and thus contribute to the repression of translation initiation. The miRNA-RISC might inhibit also translation at post-initiation steps by inhibiting ribosome elongation, inducing ribosome drop-off, or promoting proteolysis of nascent polypeptides. To promote mRNA degradation, the miRNA-RISC complex interacts with the CCR4-NOT1 deadenylase complex to facilitate deadenylation of the mRNA poly(A) tail. Deadenylation requires the direct interaction of the GW182 protein with PABP. Following deadenylation, the 5′-terminal cap (m7 G) is removed by the DCP1-DCP2 decapping complex. Although miRNA-mediated deadenylation followed by mRNA degradation appear to be widespread events, not all miRNA-targeted mRNAs are destabilized. miRNA-targeted translationally repressed mRNAs can accumulate in discrete cytoplasmic foci, such as P or GW bodies, or stress granules. A fraction of GW bodies co-localizes with multivesicular bodies (MVBs), membrane structures that play a role in miRNA-mediated repression. Compelling evidences support a role for miRNAs at the nucleus, acting on transcriptional regulation via chromatin remodeling and epigenetic mechanisms [26].

outgrowth was impaired in cells with experimentally reduced levels of miR-10a or -10b, and

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the expression of several neural differentiation markers like Tyrosine Kinase receptors *NTRK2*

**Figure 2. miRNA expression profiling in differentiating SH-SY5Y cells.** Relative expression values detected in Taq‐ Man microRNA Low Density Arrays for microRNAs with FDR<0.05 at least in two of the three treated versus non-treat‐ ed comparisons, for upregulated **(A)** and downregulated miRNAs **(B)**. The values for 24 (empty bars), 48 (grey bars)

and 96 (black bars) h of RA treatment are represented.

## **3. Profiling miRNA expression during retinoic-acid-induced neuroblastoma cell differentiation**

#### **3.1. Profiling miRNA expression during retinoic-acid induced neuroblastoma cell differentiation**

Several studies have addressed the changes in the expression of miRNAs upon RA-dependent induction of differentiation of neuroblastoma cells, with somewhat different results depending on the cell line, treatment duration, analysis platform used, etc. [2, 27-30]. To analyze the contribution of microRNA regulation to RA-induced differentiation of neuroblastoma cells, we have studied the changes in the pattern of expression of 667 different human miRNAs upon RA treatment of SH-SY5Y neuroblastoma cells. We used miRNA profiling with TaqMan RT-PCR Low Density Arrays, and we found that 452 miRNAs were expressed above detection level. From them, 42 specific miRNAs change significatively their expression levels (26 upregulated and 16 downregulated) during RA-induced differentiation (Figure 2). This suggests miRNAs as an additional post-transcriptional regulatory layer under RA control [30].

#### **3.2. A role for miRNAs-10a and -10b in RA-dependent regulation of neuroblastoma differentiation**

We have focused our study on the closely related miR-10a and -10b, that showed the most prominent expression changes in SH-SY5Y cell line. Similar results have been reported for other neuroblastoma cell lines, like LA-N-1, LAN5 and SK-N-BE [29, 30].

Loss of function experiments with anti-sense anti-miRs antagonists could show that miR-10a and -10b contribute to the regulation of RA-induced differentiation. RA-induced neurite outgrowth was impaired in cells with experimentally reduced levels of miR-10a or -10b, and the expression of several neural differentiation markers like Tyrosine Kinase receptors *NTRK2*

complex formation. The interaction of the GW182 protein with the poly(A)-binding protein (PABP) might interfere with the closed-loop formation mediated by the eIF4G-PABP interac‐ tion and thus contribute to the repression of translation initiation. The miRNA-RISC might inhibit also translation at post-initiation steps by inhibiting ribosome elongation, inducing ribosome drop-off, or promoting proteolysis of nascent polypeptides. To promote mRNA degradation, the miRNA-RISC complex interacts with the CCR4-NOT1 deadenylase complex to facilitate deadenylation of the mRNA poly(A) tail. Deadenylation requires the direct interaction of the GW182 protein with PABP. Following deadenylation, the 5′-terminal cap

G) is removed by the DCP1-DCP2 decapping complex. Although miRNA-mediated deadenylation followed by mRNA degradation appear to be widespread events, not all miRNA-targeted mRNAs are destabilized. miRNA-targeted translationally repressed mRNAs can accumulate in discrete cytoplasmic foci, such as P or GW bodies, or stress granules. A fraction of GW bodies co-localizes with multivesicular bodies (MVBs), membrane structures that play a role in miRNA-mediated repression. Compelling evidences support a role for miRNAs at the nucleus, acting on transcriptional regulation via chromatin remodeling and

**3. Profiling miRNA expression during retinoic-acid-induced**

**3.1. Profiling miRNA expression during retinoic-acid induced neuroblastoma cell**

**3.2. A role for miRNAs-10a and -10b in RA-dependent regulation of neuroblastoma**

other neuroblastoma cell lines, like LA-N-1, LAN5 and SK-N-BE [29, 30].

We have focused our study on the closely related miR-10a and -10b, that showed the most prominent expression changes in SH-SY5Y cell line. Similar results have been reported for

Loss of function experiments with anti-sense anti-miRs antagonists could show that miR-10a and -10b contribute to the regulation of RA-induced differentiation. RA-induced neurite

Several studies have addressed the changes in the expression of miRNAs upon RA-dependent induction of differentiation of neuroblastoma cells, with somewhat different results depending on the cell line, treatment duration, analysis platform used, etc. [2, 27-30]. To analyze the contribution of microRNA regulation to RA-induced differentiation of neuroblastoma cells, we have studied the changes in the pattern of expression of 667 different human miRNAs upon RA treatment of SH-SY5Y neuroblastoma cells. We used miRNA profiling with TaqMan RT-PCR Low Density Arrays, and we found that 452 miRNAs were expressed above detection level. From them, 42 specific miRNAs change significatively their expression levels (26 upregulated and 16 downregulated) during RA-induced differentiation (Figure 2). This suggests miRNAs as an additional post-transcriptional regulatory layer under RA control [30].

(m7

250 Neuroblastoma

epigenetic mechanisms [26].

**differentiation**

**differentiation**

**neuroblastoma cell differentiation**

**Figure 2. miRNA expression profiling in differentiating SH-SY5Y cells.** Relative expression values detected in Taq‐ Man microRNA Low Density Arrays for microRNAs with FDR<0.05 at least in two of the three treated versus non-treat‐ ed comparisons, for upregulated **(A)** and downregulated miRNAs **(B)**. The values for 24 (empty bars), 48 (grey bars) and 96 (black bars) h of RA treatment are represented.

(*trkB*) and *RET*, *GAP43,* Neuron-specific Enolase (*ENO2*), medium-size neurofilament protein NEFM and the enzyme Tyrosine Hydroxylase (TH) was abrogated or severely impaired after suppression of miR-10a or -10b (Figure 3).

tiation itself and although the mRNA levels of *RET*, *NTRK2*, *GAP43* and *ENO2* or the protein levels of NEFM and TH were slightly enhanced by transfection of pre-miR-10a and -10b, the attained expression levels for all the markers analyzed were far below those obtained by RA treatment. Similarly, ectopic expression of miR-10a and -10b led to certain increase in neurite outgrowth, but lower to that obtained for RA treatment [30]. Therefore, miR-10a and-10b appeared to be necessary but not sufficient for full neural differentiation, and consequently

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**3.3. miRNAs-10a and -10b contribute to the reduction on the biological aggressiveness of**

It has been reported that RA treatment of neuroblastoma cells results in a reduction in their biological aggressiveness, by decreasing their migratory and invasive abilities [8-10]. We wanted to analyze whether RA-induced expression of miR-10a and -10b could be related to the reduction in migratory and invasive potential of neuroblastoma cells. To test the migratory potential of SH-SY-5Y cells we used a modified, light-opaque Boyden chamber assay (Falcon HTS FluoroBlok, 8 μm pore size). Cells were transfected with anti-miR-10a or -10b or the corresponding Negative Control anti-miR, treated with 1 μM RA or vehicle in culture medium during 96 h, and labeled in the plate with Calcein AM. Labeled cells were counted and added to the upper chamber of the Boyden chamber, and allow to migrate towards de lower chamber, filled with medium containing 10% FBS as chemoattractant. The results show that indeed RAtreatment reduced the migration of neuroblastoma cells. However suppressing miR-10a or -10b expression not only abolished that reduction but increased migration over basal levels, supporting a contribution of RA-induced miR-10a and 10b to the reduction of migratory

**Figure 4. Involvement of miR-10a and -10b on the effects of RA in migratory and invasive potential of neuro‐ blastoma cells.** Mock-transfected cells and cells transfected with Negative Control (NC) anti-miR, anti-miR-10a or antimiR-10b were treated with 1 μM RA or vehicle for 96 h and used in migration **(A)** or matrigel invasion **(B)** transwell assays. The graph shows a representative experiment performed in triplicate (mean ± SD). Statistical significance was analyzed by comparing samples transfected with anti-miR-10a and -10b with those transfected with NC-anti-miR.

For invasion assays we used a similar assay, with the difference that the porous membrane separating the upper and lower chambers of the Boyden chamber was covered with BD *Matrigel*

in serum-free medium). The lower chamber contained 10% FBS as chemoattrac‐

additional actions of RA must contribute to differentiation.

**neuroblastoma cells induced by RA**

activity produced by RA [30]. (Figure 4A)

matrix (5 μg/cm2

**Figure 3. Knock-down of miR-10a and -10b impaired RA-induced differentiation.** Blocking the action of miR-10a and -10b by transfection of their cognate anti-miRs diminished neurite outgrowth (A) and reduced the expression of neuronal differentiation markers *NTRK2* (B), *RET* (C), *GAP43* (D) and *ENO2* (E), as shown by quantitative RT-PCR. (Statis‐ tical signification in the Figures: \*: p<0.05; \*\*: p<0.01; \*\*\*: p<0,001; ns: non significative)

Conversely, the downregulation of the members of the ID gene family, *ID1, ID2* and *ID3* was abolished in RA-treated cells transfected with anti-miR-10a and anti-miR-10b. However, miR-10a and -10b did not appear to play a relevant role in RA-induced proliferation arrest, because the strong reduction of the incorporation of 3 H-Thymidine (to approximately 30% of the control values) and the decrease in the percentage of cells in S- phase (to 50% of the control) induced by RA treatment, was equivalent in neuroblastoma cells transfected with anti-miR-10a and anti-miR-10b [30]. However, a reduction in the cell growth in SK-N-BE neuroblastoma cells when transfected with pre-miR-10a and -10b has been reported [29]. Overexpression of miR-10 and -10b by transfecting synthetic precursor pre-miRs could not trigger full differen‐ tiation itself and although the mRNA levels of *RET*, *NTRK2*, *GAP43* and *ENO2* or the protein levels of NEFM and TH were slightly enhanced by transfection of pre-miR-10a and -10b, the attained expression levels for all the markers analyzed were far below those obtained by RA treatment. Similarly, ectopic expression of miR-10a and -10b led to certain increase in neurite outgrowth, but lower to that obtained for RA treatment [30]. Therefore, miR-10a and-10b appeared to be necessary but not sufficient for full neural differentiation, and consequently additional actions of RA must contribute to differentiation.

(*trkB*) and *RET*, *GAP43,* Neuron-specific Enolase (*ENO2*), medium-size neurofilament protein NEFM and the enzyme Tyrosine Hydroxylase (TH) was abrogated or severely impaired after

**Figure 3. Knock-down of miR-10a and -10b impaired RA-induced differentiation.** Blocking the action of miR-10a and -10b by transfection of their cognate anti-miRs diminished neurite outgrowth (A) and reduced the expression of neuronal differentiation markers *NTRK2* (B), *RET* (C), *GAP43* (D) and *ENO2* (E), as shown by quantitative RT-PCR. (Statis‐

Conversely, the downregulation of the members of the ID gene family, *ID1, ID2* and *ID3* was abolished in RA-treated cells transfected with anti-miR-10a and anti-miR-10b. However, miR-10a and -10b did not appear to play a relevant role in RA-induced proliferation arrest,

the control values) and the decrease in the percentage of cells in S- phase (to 50% of the control) induced by RA treatment, was equivalent in neuroblastoma cells transfected with anti-miR-10a and anti-miR-10b [30]. However, a reduction in the cell growth in SK-N-BE neuroblastoma cells when transfected with pre-miR-10a and -10b has been reported [29]. Overexpression of miR-10 and -10b by transfecting synthetic precursor pre-miRs could not trigger full differen‐

H-Thymidine (to approximately 30% of

tical signification in the Figures: \*: p<0.05; \*\*: p<0.01; \*\*\*: p<0,001; ns: non significative)

because the strong reduction of the incorporation of 3

suppression of miR-10a or -10b (Figure 3).

252 Neuroblastoma

#### **3.3. miRNAs-10a and -10b contribute to the reduction on the biological aggressiveness of neuroblastoma cells induced by RA**

It has been reported that RA treatment of neuroblastoma cells results in a reduction in their biological aggressiveness, by decreasing their migratory and invasive abilities [8-10]. We wanted to analyze whether RA-induced expression of miR-10a and -10b could be related to the reduction in migratory and invasive potential of neuroblastoma cells. To test the migratory potential of SH-SY-5Y cells we used a modified, light-opaque Boyden chamber assay (Falcon HTS FluoroBlok, 8 μm pore size). Cells were transfected with anti-miR-10a or -10b or the corresponding Negative Control anti-miR, treated with 1 μM RA or vehicle in culture medium during 96 h, and labeled in the plate with Calcein AM. Labeled cells were counted and added to the upper chamber of the Boyden chamber, and allow to migrate towards de lower chamber, filled with medium containing 10% FBS as chemoattractant. The results show that indeed RAtreatment reduced the migration of neuroblastoma cells. However suppressing miR-10a or -10b expression not only abolished that reduction but increased migration over basal levels, supporting a contribution of RA-induced miR-10a and 10b to the reduction of migratory activity produced by RA [30]. (Figure 4A)

**Figure 4. Involvement of miR-10a and -10b on the effects of RA in migratory and invasive potential of neuro‐ blastoma cells.** Mock-transfected cells and cells transfected with Negative Control (NC) anti-miR, anti-miR-10a or antimiR-10b were treated with 1 μM RA or vehicle for 96 h and used in migration **(A)** or matrigel invasion **(B)** transwell assays. The graph shows a representative experiment performed in triplicate (mean ± SD). Statistical significance was analyzed by comparing samples transfected with anti-miR-10a and -10b with those transfected with NC-anti-miR.

For invasion assays we used a similar assay, with the difference that the porous membrane separating the upper and lower chambers of the Boyden chamber was covered with BD *Matrigel* matrix (5 μg/cm2 in serum-free medium). The lower chamber contained 10% FBS as chemoattrac‐ tant to promote cell invasion. In this case RA treatment results in increased invasive potential, whereas in cells transfected with anti-miR-10a or 10b the same treatment the increase in inva‐ sion induced by RA treatment is even larger, supporting the idea that the expression of miR-10a and -10b contributes to a reduction in the invasive potential [30]. (Figure 4B)

targets in the literature or in databases as TarBase [41]. A validated target for miR-10b in breast cancer cells is the homeobox gene *HOXD10* [34, 42]. However, we could not find regulation of *HOXD10* in SH-SY5Y neuroblastoma cells, when treated with RA or when the levels of miR-10a

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A lot of effort has been made to generate computational miRNA target prediction tools [reviewed in 43], mainly based on the search for complementary sequences in the genome.

**Figure 5. Chorioallantoic Membrane Metastasis Assay.** (A) Schematic representation of the experiment. Cells from the different treatment groups were transferred to the upper chorioallantoic membrane of 10-day-old chicken em‐ bryos and the number of metastatized cells into the lungs evaluated 7 days later. (B) Cells transfected with Negative Control (NC) anti-miR or anti-miR-10a were treated with 1 μM RA or vehicle for 96 h as indicated in the figure. The graph represents the values obtained from six parallel assays (mean ± SD). Statistical significance was analyzed by comparing samples transfected with anti-miR-10a with those transfected with NC-anti-miR. In addition samples trans‐

fected with NC-anti-miR treated with vehicle were compared to those treated with RA.

and -10b were experimentally altered [30].

To analyze the effects of RA treatment on the metastatic potential of neuroblastoma cells we used the chicken embryo chorioallantoic membrane assays (also known as CAM assay; [31]). This assay is useful to study intravasation and metastasis *in vivo*, since it recapitulates all the steps of the metastatic process. In the CAM assay the cells to be tested are inoculated on the chorioallantoic membrane of 10-day-old chicken embryos. After a week, the egg is opened, the embryo is obtained and secondary organs like the lungs were dissected. The presence of human cells in the chicken organ is evaluated and quantified after obtaining their genomic DNA, by detecting the presence of human-specific *Alu*-sequences by Real-Time PCR (Figure 3). As this is a complex technique that requires a higher number of replicate experiments we have simplified the study to analyze only the effects of miR-10a suppression. Neuroblastoma cells could be detected in the chicken lungs after 7 days incubation. Suppression of miR-10a expression with its cognate anti-miR resulted in an increase of the metastatic cells. As expected, RA treatment led to a reduction of the number of neuroblastoma cells reaching the lungs. However this inhibitory effect of RA was abolished in cells having a reduced amount of miR-10a by transfecting the corresponding anti-miR-10a [30].

In good agreement with our results, it has been reported that miR-10a and -10b reduces the ability of neuroblastoma cells to form colonies in soft agar [29], a phenotype that is character‐ istic of malignant cells. All these results support the idea that miR-10a and -10b expression contribute to reduction of migratory, invasive and metastatic activities induced by RA. In a recent report it has been shown that a protein involved in cell migration, Tiam1, is targeted by miR-10b in mammary tumor cells. Overexpression of miR-10b suppresses the ability of breast carcinoma cells to migrate and invade [32]. Consistent to that, it has been reported an associ‐ ation between lower miR-10a expression and lower overall survival for a subclass of neuro‐ blastoma tumors (11q- tumor cohort) [29]. However other reports seem to involve the members of the miR-10 family as promoters of migration and metastasis in different tumors [33-40]. This apparent controversy may suggest that the role of the microRNAs from the miR-10 family in tumorigenesis and metastasis would depend on their molecular targets and therefore would depend on the cellular context.

## **4. Molecular targets of miR-10a and -10b in the differentiation of neuroblastoma cells**

#### **4.1. The search for the molecular targets for miRNAs**

The identification of molecular targets for miRNAs is a crucial step towards the understanding of miRNA function. Because an ever growing number of experimentally validated targets for miRNAs are being reported, a simple way to identify miRNA targets is to search for validated targets in the literature or in databases as TarBase [41]. A validated target for miR-10b in breast cancer cells is the homeobox gene *HOXD10* [34, 42]. However, we could not find regulation of *HOXD10* in SH-SY5Y neuroblastoma cells, when treated with RA or when the levels of miR-10a and -10b were experimentally altered [30].

tant to promote cell invasion. In this case RA treatment results in increased invasive potential, whereas in cells transfected with anti-miR-10a or 10b the same treatment the increase in inva‐ sion induced by RA treatment is even larger, supporting the idea that the expression of miR-10a

To analyze the effects of RA treatment on the metastatic potential of neuroblastoma cells we used the chicken embryo chorioallantoic membrane assays (also known as CAM assay; [31]). This assay is useful to study intravasation and metastasis *in vivo*, since it recapitulates all the steps of the metastatic process. In the CAM assay the cells to be tested are inoculated on the chorioallantoic membrane of 10-day-old chicken embryos. After a week, the egg is opened, the embryo is obtained and secondary organs like the lungs were dissected. The presence of human cells in the chicken organ is evaluated and quantified after obtaining their genomic DNA, by detecting the presence of human-specific *Alu*-sequences by Real-Time PCR (Figure 3). As this is a complex technique that requires a higher number of replicate experiments we have simplified the study to analyze only the effects of miR-10a suppression. Neuroblastoma cells could be detected in the chicken lungs after 7 days incubation. Suppression of miR-10a expression with its cognate anti-miR resulted in an increase of the metastatic cells. As expected, RA treatment led to a reduction of the number of neuroblastoma cells reaching the lungs. However this inhibitory effect of RA was abolished in cells having a reduced amount of

In good agreement with our results, it has been reported that miR-10a and -10b reduces the ability of neuroblastoma cells to form colonies in soft agar [29], a phenotype that is character‐ istic of malignant cells. All these results support the idea that miR-10a and -10b expression contribute to reduction of migratory, invasive and metastatic activities induced by RA. In a recent report it has been shown that a protein involved in cell migration, Tiam1, is targeted by miR-10b in mammary tumor cells. Overexpression of miR-10b suppresses the ability of breast carcinoma cells to migrate and invade [32]. Consistent to that, it has been reported an associ‐ ation between lower miR-10a expression and lower overall survival for a subclass of neuro‐ blastoma tumors (11q- tumor cohort) [29]. However other reports seem to involve the members of the miR-10 family as promoters of migration and metastasis in different tumors [33-40]. This apparent controversy may suggest that the role of the microRNAs from the miR-10 family in tumorigenesis and metastasis would depend on their molecular targets and therefore would

**4. Molecular targets of miR-10a and -10b in the differentiation of**

The identification of molecular targets for miRNAs is a crucial step towards the understanding of miRNA function. Because an ever growing number of experimentally validated targets for miRNAs are being reported, a simple way to identify miRNA targets is to search for validated

and -10b contributes to a reduction in the invasive potential [30]. (Figure 4B)

miR-10a by transfecting the corresponding anti-miR-10a [30].

depend on the cellular context.

254 Neuroblastoma

**neuroblastoma cells**

**4.1. The search for the molecular targets for miRNAs**

A lot of effort has been made to generate computational miRNA target prediction tools [reviewed in 43], mainly based on the search for complementary sequences in the genome.

**Figure 5. Chorioallantoic Membrane Metastasis Assay.** (A) Schematic representation of the experiment. Cells from the different treatment groups were transferred to the upper chorioallantoic membrane of 10-day-old chicken em‐ bryos and the number of metastatized cells into the lungs evaluated 7 days later. (B) Cells transfected with Negative Control (NC) anti-miR or anti-miR-10a were treated with 1 μM RA or vehicle for 96 h as indicated in the figure. The graph represents the values obtained from six parallel assays (mean ± SD). Statistical significance was analyzed by comparing samples transfected with anti-miR-10a with those transfected with NC-anti-miR. In addition samples trans‐ fected with NC-anti-miR treated with vehicle were compared to those treated with RA.

However, that is not an easy task, because short sequences are problematic for the algorithms usually developed for complementarity analysis. As indicated in 2.2, the base pairing of miRNA and mRNA in vertebrates requires only partial homology, with a preference for contiguous pairing occurring only at the "seed" region, located at nucleotides 2-7 of the guide strand, and this makes even more difficult to find the right target sequence in the genome. Several authors have approached this problem from different startpoints, using mainly complementarity analysis of the complete miRNA sequence, complementarity analysis of the seed sequence, or adding thermodynamic stability analysis of duplex sequences or 3'UTR sequence conservation to the complementarity analysis. Nowadays a set of miRNA target prediction resources are available, mainly as web-based tools. However it becomes striking to the new users of these tools how different results can be obtained when using the same sequence with different prediction tools. In addition, prediction tools generate lists of hun‐ dreds of genes for each of the miRNAs, and the fact of having sequence diversity at the 3'UTR by alternative polyadenylation sites could also complicate the analysis [for discussion, see 44]. **4.2. Regulation of** *SFRS1* **(SF2/ASF) by miR-10a and -10b**

The regulation of *SFRS1* (SF2/ASF) by miR-10a and-10b was experimentally validated at mRNA and protein levels in HeLa and SH-SY5Y cells (Figure 6). In addition regulation by miR-10a and -10b was shown in transfection experiments with reporter plasmids containing *SFRS1* 3'UTR sequences linked to the Luciferase gene. miR-10a and -10b are new players in the complex regulation of *SFRS1* protein through a mechanism involving enhanced mRNA cleavage. In addition, we showed how changes in miR-10a and -10b expression levels may influence some molecular activities in which the product of *SFRS1* is involved, such as translation enhancement of certain mRNAs and alternative splicing, that could have impor‐ tance in the neural differentiation process [30] (Figure 7). We have reported that the activation of signaling pathways by RA treatment results in rapid changes in the phosphorylation pattern of SR proteins, including SFRS1 and subsequently, changes in alternative splicing selection and an increase of the translation of mRNAs containing SFRS1 binding sites take place [48]. In this context, the reduction in *SFRS1* levels through miR-10a and -10b regulation could be interpreted as the closing of the feedback regulatory loop of RA on the activities of *SFRS1*.

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257

**Figure 7.** Experimental alteration of miR-10a and -10b levels resulted in an impairment of SFRS1 functions in the regu‐ lation of alternative splicing. Alternative splicing of *tau* protein exon 10 is altered by transfection of anti-miR-10a and -10b. RT-PCR was performed on RNA extracted from anti-miR-10a, -10b or negative control (NC) anti-miR transfected SH-SY5Y cells. *tau* Exon 10 flanking primers were used in RT-PCR reaction according to [49]. Quantification of the per‐ centage of exon 10 inclusion. The graph shows the average from 3 independent experiments. Statistical analysis was made by comparing the values from cells transfected with pre-miR-10a or -10b to those from cells transfected with

NC-pre-miR.

To find relevant targets for miR-10a and-10b in neuroblastoma cells we choose to combine bioinformatic prediction tools together with experimental analysis. We created a list of potential miR-10a and -10b targets by including the common predicted genes using three different prediction resources: miRbase targets [45], TargetScan [46] and PicTar [47]. Only mRNAs that contained evolutionarily conserved miRNA binding sequences on their 3'UTR were considered. This list was crossed with the dataset of an Affymetrix microarray experiment containing the genes downregulated after 48 h RA treatment. In the resulting list, two members of the Arginine/serine-rich splicing factors, *SFRS1* (SF2/ASF) and *SFRS10* (TRA2B), as well as the nuclear receptor co-repressor *NCOR2* (SMRT) were on top [30].

**Figure 6. miR-10a/-10b knockdown leads to increased SFRS1 protein and mRNA levels in SH-SY5Y cells. (Left panel)** Western blot of SFRS1 protein expression after anti-miR-10a, -10b and negative control NC-anti-miR transfec‐ tion of SH-SY5Y cells followed by 1μM RA treatment. The blot was reprobed with actin beta antibodies as loading control. **(Right panel).** RT-qPCR analysis of SFRS1 mRNA levels in same conditions. The graph shows expression levels relative to that of RA untreated, NC-anti-miR transfected cells (mean ± SD of a triplicate experiment). Statistical analy‐ sis for right panel was made by comparing the values from cells transfected with anti-miR-10a or -10b to those from cells transfected with NC-anti-miR; ns= non significative.

#### **4.2. Regulation of** *SFRS1* **(SF2/ASF) by miR-10a and -10b**

However, that is not an easy task, because short sequences are problematic for the algorithms usually developed for complementarity analysis. As indicated in 2.2, the base pairing of miRNA and mRNA in vertebrates requires only partial homology, with a preference for contiguous pairing occurring only at the "seed" region, located at nucleotides 2-7 of the guide strand, and this makes even more difficult to find the right target sequence in the genome. Several authors have approached this problem from different startpoints, using mainly complementarity analysis of the complete miRNA sequence, complementarity analysis of the seed sequence, or adding thermodynamic stability analysis of duplex sequences or 3'UTR sequence conservation to the complementarity analysis. Nowadays a set of miRNA target prediction resources are available, mainly as web-based tools. However it becomes striking to the new users of these tools how different results can be obtained when using the same sequence with different prediction tools. In addition, prediction tools generate lists of hun‐ dreds of genes for each of the miRNAs, and the fact of having sequence diversity at the 3'UTR by alternative polyadenylation sites could also complicate the analysis [for discussion, see 44].

256 Neuroblastoma

To find relevant targets for miR-10a and-10b in neuroblastoma cells we choose to combine bioinformatic prediction tools together with experimental analysis. We created a list of potential miR-10a and -10b targets by including the common predicted genes using three different prediction resources: miRbase targets [45], TargetScan [46] and PicTar [47]. Only mRNAs that contained evolutionarily conserved miRNA binding sequences on their 3'UTR were considered. This list was crossed with the dataset of an Affymetrix microarray experiment containing the genes downregulated after 48 h RA treatment. In the resulting list, two members of the Arginine/serine-rich splicing factors, *SFRS1* (SF2/ASF) and *SFRS10* (TRA2B), as well as

**Figure 6. miR-10a/-10b knockdown leads to increased SFRS1 protein and mRNA levels in SH-SY5Y cells. (Left panel)** Western blot of SFRS1 protein expression after anti-miR-10a, -10b and negative control NC-anti-miR transfec‐ tion of SH-SY5Y cells followed by 1μM RA treatment. The blot was reprobed with actin beta antibodies as loading control. **(Right panel).** RT-qPCR analysis of SFRS1 mRNA levels in same conditions. The graph shows expression levels relative to that of RA untreated, NC-anti-miR transfected cells (mean ± SD of a triplicate experiment). Statistical analy‐ sis for right panel was made by comparing the values from cells transfected with anti-miR-10a or -10b to those from

the nuclear receptor co-repressor *NCOR2* (SMRT) were on top [30].

cells transfected with NC-anti-miR; ns= non significative.

The regulation of *SFRS1* (SF2/ASF) by miR-10a and-10b was experimentally validated at mRNA and protein levels in HeLa and SH-SY5Y cells (Figure 6). In addition regulation by miR-10a and -10b was shown in transfection experiments with reporter plasmids containing *SFRS1* 3'UTR sequences linked to the Luciferase gene. miR-10a and -10b are new players in the complex regulation of *SFRS1* protein through a mechanism involving enhanced mRNA cleavage. In addition, we showed how changes in miR-10a and -10b expression levels may influence some molecular activities in which the product of *SFRS1* is involved, such as translation enhancement of certain mRNAs and alternative splicing, that could have impor‐ tance in the neural differentiation process [30] (Figure 7). We have reported that the activation of signaling pathways by RA treatment results in rapid changes in the phosphorylation pattern of SR proteins, including SFRS1 and subsequently, changes in alternative splicing selection and an increase of the translation of mRNAs containing SFRS1 binding sites take place [48]. In this context, the reduction in *SFRS1* levels through miR-10a and -10b regulation could be interpreted as the closing of the feedback regulatory loop of RA on the activities of *SFRS1*.

**Figure 7.** Experimental alteration of miR-10a and -10b levels resulted in an impairment of SFRS1 functions in the regu‐ lation of alternative splicing. Alternative splicing of *tau* protein exon 10 is altered by transfection of anti-miR-10a and -10b. RT-PCR was performed on RNA extracted from anti-miR-10a, -10b or negative control (NC) anti-miR transfected SH-SY5Y cells. *tau* Exon 10 flanking primers were used in RT-PCR reaction according to [49]. Quantification of the per‐ centage of exon 10 inclusion. The graph shows the average from 3 independent experiments. Statistical analysis was made by comparing the values from cells transfected with pre-miR-10a or -10b to those from cells transfected with NC-pre-miR.

#### **4.3. Regulation of** *NCOR2* **(SMRT) by miR-10a and -10b**

The regulation of *NCOR2* by miR-10a and-10b was experimentally validated at mRNA and protein levels in SK-N-BE neuroblastoma cells. Moreover, a luciferase reporter construct containing the *NCOR2* 3'UTR showed a significant decrease in luciferase activity when cotransfected with mature miR-10a, -10b or 10a/10b mimics in SK-N-BE cells. This decrease in luciferase activity was completely abolished when the putative miR-10a and -10b target site was mutated in its seed sequence. Knock-down of *NCOR2* expression through transfection of siRNAs to SK-N-BE cells recapitulates most of the changes induced by RA, like neurite outgrowth, proliferative arrest, expression of neural markers, downregulation of *MYCN* and expression of miR-10a [29]. *NCOR2* acts as co-repressor in the regulation of many genes, especially as co-regulator of nuclear receptor-regulated genes. Bound to the unliganded receptor, *NCOR2* maintains the promoters of nuclear receptor-regulated genes in a repressed state, and its release from the complex with the receptor upon ligand binding allows tran‐ scriptional activation [50]. It has been reported that *NCOR2* represses expression of the jumonji-domain containing gene *JMJD3*, a direct retinoic-acid-receptor target that functions as a histone H3 trimethyl K27 demethylase and which is capable of activating specific compo‐ nents of the neurogenic program [51]. Therefore, downregulation of *NCOR2* by miR-10a and -10b would potentiate the actions of RA through RARs and RXRs and could contribute to some of the changes in gene expression associated with neural differentiation.

**Author details**

Valencia, Spain

**References**

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Salvador Meseguer, Juan-Manuel Escamilla and Domingo Barettino

Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas,

miRNAs as Essential Mediators of the Actions of Retinoic Acid in Neuroblastoma Cells

http://dx.doi.org/10.5772/55444

259

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#### **5. Conclusion**

MicroRNAs are essential players in the process on neural differentiation of neuroblastoma cells, and contribute to the transduction of Retinoic Acid signaling. In addition, miRNAs have been reported to participate in the pathogenesis and progression of human neuroblastoma tumors [2, 3], and miRNA profiles have been recently proven to be useful for classification and prognosis [4]. Finally miRNAs open new avenues for the treatment of neuroblastoma cells, and proof of concept experiments showing a therapeutic action of miRNA-based treatments in animal models of neuroblastoma [52-54] and other tumors [33] have been reported. Therefore, we have to expect in the next years an increased interest in the study of microRNAs among the neuroblastoma researchers community.

#### **Acknowledgements**

The research work described in this article was financed through grants of the Spanish National Plan for Research, Development and Innovation (SAF2007-60780, SAF2010-15032 and SAF2011-23869), Generalitat Valenciana (ACOMP 09/212) and Genoma España to D. Barettino. S. Meseguer was the recipient of an EACR training and travel fellowship award and a CSIC I3P predoctoral fellowship/contract.

#### **Author details**

**4.3. Regulation of** *NCOR2* **(SMRT) by miR-10a and -10b**

of the changes in gene expression associated with neural differentiation.

among the neuroblastoma researchers community.

MicroRNAs are essential players in the process on neural differentiation of neuroblastoma cells, and contribute to the transduction of Retinoic Acid signaling. In addition, miRNAs have been reported to participate in the pathogenesis and progression of human neuroblastoma tumors [2, 3], and miRNA profiles have been recently proven to be useful for classification and prognosis [4]. Finally miRNAs open new avenues for the treatment of neuroblastoma cells, and proof of concept experiments showing a therapeutic action of miRNA-based treatments in animal models of neuroblastoma [52-54] and other tumors [33] have been reported. Therefore, we have to expect in the next years an increased interest in the study of microRNAs

The research work described in this article was financed through grants of the Spanish National Plan for Research, Development and Innovation (SAF2007-60780, SAF2010-15032 and SAF2011-23869), Generalitat Valenciana (ACOMP 09/212) and Genoma España to D. Barettino. S. Meseguer was the recipient of an EACR training and travel fellowship award and a CSIC

**5. Conclusion**

258 Neuroblastoma

**Acknowledgements**

I3P predoctoral fellowship/contract.

The regulation of *NCOR2* by miR-10a and-10b was experimentally validated at mRNA and protein levels in SK-N-BE neuroblastoma cells. Moreover, a luciferase reporter construct containing the *NCOR2* 3'UTR showed a significant decrease in luciferase activity when cotransfected with mature miR-10a, -10b or 10a/10b mimics in SK-N-BE cells. This decrease in luciferase activity was completely abolished when the putative miR-10a and -10b target site was mutated in its seed sequence. Knock-down of *NCOR2* expression through transfection of siRNAs to SK-N-BE cells recapitulates most of the changes induced by RA, like neurite outgrowth, proliferative arrest, expression of neural markers, downregulation of *MYCN* and expression of miR-10a [29]. *NCOR2* acts as co-repressor in the regulation of many genes, especially as co-regulator of nuclear receptor-regulated genes. Bound to the unliganded receptor, *NCOR2* maintains the promoters of nuclear receptor-regulated genes in a repressed state, and its release from the complex with the receptor upon ligand binding allows tran‐ scriptional activation [50]. It has been reported that *NCOR2* represses expression of the jumonji-domain containing gene *JMJD3*, a direct retinoic-acid-receptor target that functions as a histone H3 trimethyl K27 demethylase and which is capable of activating specific compo‐ nents of the neurogenic program [51]. Therefore, downregulation of *NCOR2* by miR-10a and -10b would potentiate the actions of RA through RARs and RXRs and could contribute to some

Salvador Meseguer, Juan-Manuel Escamilla and Domingo Barettino

Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas, Valencia, Spain

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**Chapter 12**

**Copper as a Target for Treatment of Neuroblastoma:**

Copper is a trace metal essential to the catalysis of a wide range of enzymatic activities, including those involved in the process of energy production (cytochrome c oxidase), the cell response to oxidant injuries (Cu,Zn-superoxide dismutase), the catecholamine (dopamine βmonooxygenase) and melanin (tyrosinase) production, the remodelling of extracellular matrix (lysyl oxidase), blood clotting processes (Factors V and VIII) and iron metabolism (ceruloplas‐ min and hephaestin) [1]. The catalytic properties of copper are linked to its ability to easily

trigger severe cell alterations through the generation of hydroxyl radicals in Fenton-like reactions [2,3]. When the cytosolic copper concentration is above the optimal level, the newly formed reactive oxygen species (ROS) rapidly bind to DNA, thus inducing the breaking of the nucleic acid strands and initiating a series of cascade events that can lead to significant damage

Considerable intrinsic oxidative stress and enhanced serum and tissue copper levels depict a disease condition that often accompanies the progression of several tumour forms, in turn resulting from a perturbed energy metabolism, mitochondrial dysfunction, release of cyto‐ kines and inflammation [5]. Copper is intimately involved in all these cell functions, thus targeting the elevated copper levels would be an ideal therapeutic strategy to effectively

This issue is anyway highly debated. In fact, the topical delivery of copper complexes to tumour tissues has been demonstrated to kill the cancer cells through a "therapeutic" induction of oxidative stress [6]. At the same time, especially in the case of solid tumours, as *neuroblas‐*

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

© 2013 Urso and Maffia; 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,

) states, but just the metal reactive behaviour can

**Molecular and Cellular Mechanisms**

Additional information is available at the end of the chapter

**1.1. Copper and carcinogenesis, a double-edged sword**

assume the oxidized (Cu2+) and reduced (Cu+

to cell structures and function [4].

counteract the tumour development [5].

Emanuela Urso and Michele Maffia

http://dx.doi.org/10.5772/55423

**1. Introduction**
