**Abstract**

Neuroblastoma, the most common extra-cranial pediatric solid tumor, is responsible for 9–15% of all pediatric cancer deaths. Its intrinsic heterogeneity makes it difficult to successfully treat, resulting in overall survival of 50% for half of the patients. Here we analyze the role in neuroblastoma of the adaptor protein p140Cap, encoded by the *SRCIN1* gene. RNA-Seq profiles of a large cohort of neuroblastoma patients show that *SRCIN1* mRNA levels are an independent risk factor inversely correlated to disease aggressiveness. In high-risk patients, *SRCIN1* was frequently altered by hemizygous deletion, copy-neutral loss of heterozygosity, or disruption. Functional assays demonstrated that p140Cap is causal in dampening both Src and Jak2 kinase activation and STAT3 phosphorylation. Moreover, p140Cap expression decreases *in vitro* migration and anchorage-independent cell growth, and impairs *in vivo* tumor progression, in terms of tumor volume and number of spontaneous lung metastasis. p140Cap also contributes to an increased sensitivity of neuroblastoma cells to chemotherapy drugs and to the combined usage of doxorubicin and etoposide with Src inhibitors. Overall, we provide the first evidence that *SRCIN1*/p140Cap is a new independent prognostic marker for patient outcome and treatment, with a causal role in curbing the aggressiveness of neuroblastoma. We highlight the potential clinical impact of *SRCIN1*/p140Cap expression in neuroblastoma tumors, in terms of reducing cytotoxic effects of chemotherapy, one of the main issues for pediatric tumor treatment.

**Keywords:** p140Cap, *SRCIN1* gene, Src kinase, Signal transducer and activator of transcription 3, chemotherapy, neuroblastoma, Src inhibitors

### **1. Introduction**

Neuroblastoma (NB) is the most frequent embryonic malignancy among children particularly before 5 years of age [1]. It originates from primitive sympathetic neural precursor cells of the peripheral nervous system [2]. The majority of these tumors develop in the adrenal medulla; however, NB can arise anywhere along the

sympathetic nervous system (neck, chest, abdomen or pelvis). Primary tumors in the neck or upper chest can cause Horner's syndrome (ptosis, miosis, and anhidrosis). Tumors arising along the spinal column can expand through the intraforaminal spaces and cause cord compression, with resulting paralysis [3].

NB is a complex disease with different outcomes, going from metastasis to one or more distant sites [4] to spontaneous regression or differentiation, even in the absence of any specific treatment [5]. Given the high heterogeneous features of NB, the International Neuroblastoma Staging System (INSS), considers a plethora of criteria to rank patients. Namely, the degree of surgical excision of primary tumor, lymph node involvement, dissemination to distant organs, degree of bone marrow involvement and the age of infant [6]. Accordingly, stages 1, 2A and 2B include patients with localized tumor, without propagation to lymph nodes. Stage 3 and stage 4 comprehends patients with metastatic disease. Stage 4S specifies a metastatic disease in children under the age of one year, which may undergo spontaneous regression, usually associated with 90% survival rate at 5 years [7].

The genetic etiology of NB includes some established markers such as the presence of segmental chromosome abnormalities (chromosomes 1p, 3p, 4p, 11q loss and of 1q, 2p, 17q gains) [8] and DNA ploidy [9]. At the molecular level, the Anaplastic Lymphoma Kinase (*ALK*) oncogene is the most frequently mutated gene in hereditary familial NB, where it is amplified or constitutively activated in its tyrosine kinase domain [10, 11]. Amplification of the *N-MYC* oncogene (*MYCN*) occurs in 20% of NB, representing a poor prognostic factor for this embryonic malignancy [12, 13]. Tropomyosin receptor kinase B (*TrkB)* and Brain-Derived Neurotrophic Factor (*BDNF)* are both expressed in aggressive NB with *MYCN* amplification [14]. In addition, driver mutations in the lin-28 homolog B (*LIN28B*) [15] and in the Paired-like Homeobox 2b (*PHOX2B*) [16] genes have been reported.

NB therapeutic standard of care worldwide is based on multi-modality therapy including chemotherapy, surgery, radiation therapy, myeloablative therapy with stem cell transplant, immunotherapy and differentiation therapy [17–19]. However, a more accurate stratification of patients based on newly identified prognostic markers would allow the development of additional therapeutic strategies with increased effectiveness and reduced toxicity.

p140Cap (Cas-associated protein), also known as SNIP (Snap25-interacting protein) [20], is a scaffold protein codified by the gene *SRCIN1*. It is highly expressed in the brain, testis and epithelial rich tissue [21]. In human cancer patients, p140Cap/*SRCIN1* is a new favorable prognostic marker in HER2-related breast cancer, where p140Cap expression is associated with good prognosis [22]. At the molecular level, p140Cap impairs breast cancer growth and metastatic progression, interfering with both Src kinase [23] and Rac1 GTPases [22] activation.

More recently we have investigated p140Cap/*SRCIN1* relevance in NB. This chapter aims to present data supporting p140Cap/*SRCIN1* as a key biological determinant of NB outcome, representing a new independent prognostic marker for patient outcome and treatment. We highlight the potential clinical impact of *SRCIN1*/p140Cap expression in NB tumors in terms of reducing cytotoxic effects of chemotherapy, one of the main issues for pediatric tumor treatment.

#### **2. The p140Cap adaptor protein**

The human *SRCIN1* gene, located on chromosome 17q12, includes 27 exons, and it is highly conserved in vertebrates and mammals [24]. The genomic region immediately bordering *SRCIN1* contains several genes involved in breast cancer onset and progression such as *ERBB2* (17q12), *BRCA1* (17q21), retinoic acid receptor-α

**77**

**for NB**

*The Scaffold Protein p140Cap as a Molecular Hub for Limiting Cancer Progression…*

(*RAR*A; 17q21) and signal transducer and activator of transcription 3 (*STAT3*; 17q21). These genes often undergo a gain of function role in human tumors [25]. Moreover, the 17q gain occurs in 50–70% of all high stage NB and is associated with

*The structure of the adaptor protein p140Cap. p140Cap protein analysis reveals the presence of a putative N-terminal myristoylation site, a tyrosine-rich region (Tyr-rich), an actin-binding domain (ABD), a proline rich domain (Pro1), a coil-coiled region (C1-C2), two domains rich in charged amino acids (CH1, CH2) and a C-terminal proline-rich domain (Pro2). Tyrosine phosphorylation (PY) EPLYA and EGLYA are shown. The interactors Tiam1, Csk,* β*-catenin, Vinexin, EB3, Cortactin, p130Cas and Src are associated with specific domains.*

p140Cap shares different Intrinsically Disordered Regions (IDRs) that classify p140Cap as "Intrinsic Disorder Protein" (IDP) [30, 31]. The IDP features of p140Cap could allow the interaction with several partners and promote protein–protein interactions that are the elected functions for a scaffold protein. The p140Cap protein can interact with multiple partners [30] (**Figure 1**). In particular, p140Cap associates with the tyrosine kinases Csk and Src. This macromolecular complex triggers Csk activity to phosphorylate Src on its inhibitory tyrosine, resulting in Src inactivation and in the suppression of downstream pathways regulating motility and invasion of cancer cells [23]. Indeed, at the structural level, p140Cap contains a tyrosine rich domain, important for the interaction with Csk [32], two coiled coil regions, that can mediate the binding with beta-catenin [10] and two different proline rich domains responsible for the association with the microtubule

The physiologic role of p140Cap has been mainly investigated in the brain [35], where it is expressed in neurons both in the presynapse [10, 36] and in the postsynapse [10, 37–41]. In differentiated neurons, it controls synaptic plasticity [33, 40], and regulates GABAergic synaptogenesis and development of hippocampal inhibitory circuits [36]. In particular, p140Cap enters and accumulates in the dendritic spine (DS) through EB3 binding [33]. In this compartment p140Cap acts as hub interacting with Cortactin, a protein that regulates actin branching and new filament polymerization [42] and with Citron-N [40] resulting in mature DS stabilization. In both the pre- and post-synaptic regions, p140Cap is involved in a network of protein–protein interactions as confirmed by its interactome in synaptosomes. p140Cap interactors converge on key synaptic processes, including transmission across chemical synapses, actin cytoskeleton remodeling and cell–cell junction organization [41].

**3.** *SRCIN1* **mRNA expression is an independent prognostic marker** 

To address the involvement of p140Cap in NB patients, we first investigated the relationship between *SRCIN1* mRNA levels and patient outcomes, by using the R2 genomics analysis and visualization platform (R2: Genomics analysis and

poor prognosis as an independent marker of adverse outcome [26–29].

associated protein EB3 [33], Cortactin [34] and Vimentin [35].

*DOI: http://dx.doi.org/10.5772/intechopen.96383*

**Figure 1.**

*The Scaffold Protein p140Cap as a Molecular Hub for Limiting Cancer Progression… DOI: http://dx.doi.org/10.5772/intechopen.96383*

#### **Figure 1.**

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

increased effectiveness and reduced toxicity.

**2. The p140Cap adaptor protein**

sympathetic nervous system (neck, chest, abdomen or pelvis). Primary tumors in the neck or upper chest can cause Horner's syndrome (ptosis, miosis, and anhidrosis). Tumors arising along the spinal column can expand through the intraforaminal

NB is a complex disease with different outcomes, going from metastasis to one or more distant sites [4] to spontaneous regression or differentiation, even in the absence of any specific treatment [5]. Given the high heterogeneous features of NB, the International Neuroblastoma Staging System (INSS), considers a plethora of criteria to rank patients. Namely, the degree of surgical excision of primary tumor, lymph node involvement, dissemination to distant organs, degree of bone marrow involvement and the age of infant [6]. Accordingly, stages 1, 2A and 2B include patients with localized tumor, without propagation to lymph nodes. Stage 3 and stage 4 comprehends patients with metastatic disease. Stage 4S specifies a metastatic disease in children under the age of one year, which may undergo spontaneous

spaces and cause cord compression, with resulting paralysis [3].

regression, usually associated with 90% survival rate at 5 years [7].

The genetic etiology of NB includes some established markers such as the presence of segmental chromosome abnormalities (chromosomes 1p, 3p, 4p, 11q loss and of 1q, 2p, 17q gains) [8] and DNA ploidy [9]. At the molecular level, the Anaplastic Lymphoma Kinase (*ALK*) oncogene is the most frequently mutated gene in hereditary familial NB, where it is amplified or constitutively activated in its tyrosine kinase domain [10, 11]. Amplification of the *N-MYC* oncogene (*MYCN*) occurs in 20% of NB, representing a poor prognostic factor for this embryonic malignancy [12, 13]. Tropomyosin receptor kinase B (*TrkB)* and Brain-Derived Neurotrophic Factor (*BDNF)* are both expressed in aggressive NB with *MYCN* amplification [14]. In addition, driver mutations in the lin-28 homolog B (*LIN28B*) [15] and in the Paired-like Homeobox 2b (*PHOX2B*) [16] genes have been reported. NB therapeutic standard of care worldwide is based on multi-modality therapy including chemotherapy, surgery, radiation therapy, myeloablative therapy with stem cell transplant, immunotherapy and differentiation therapy [17–19]. However, a more accurate stratification of patients based on newly identified prognostic markers would allow the development of additional therapeutic strategies with

p140Cap (Cas-associated protein), also known as SNIP (Snap25-interacting protein) [20], is a scaffold protein codified by the gene *SRCIN1*. It is highly expressed in the brain, testis and epithelial rich tissue [21]. In human cancer patients, p140Cap/*SRCIN1* is a new favorable prognostic marker in HER2-related breast cancer, where p140Cap expression is associated with good prognosis [22]. At the molecular level, p140Cap impairs breast cancer growth and metastatic progression,

interfering with both Src kinase [23] and Rac1 GTPases [22] activation.

chemotherapy, one of the main issues for pediatric tumor treatment.

More recently we have investigated p140Cap/*SRCIN1* relevance in NB. This chapter aims to present data supporting p140Cap/*SRCIN1* as a key biological determinant of NB outcome, representing a new independent prognostic marker for patient outcome and treatment. We highlight the potential clinical impact of *SRCIN1*/p140Cap expression in NB tumors in terms of reducing cytotoxic effects of

The human *SRCIN1* gene, located on chromosome 17q12, includes 27 exons, and it is highly conserved in vertebrates and mammals [24]. The genomic region immediately bordering *SRCIN1* contains several genes involved in breast cancer onset and progression such as *ERBB2* (17q12), *BRCA1* (17q21), retinoic acid receptor-α

**76**

*The structure of the adaptor protein p140Cap. p140Cap protein analysis reveals the presence of a putative N-terminal myristoylation site, a tyrosine-rich region (Tyr-rich), an actin-binding domain (ABD), a proline rich domain (Pro1), a coil-coiled region (C1-C2), two domains rich in charged amino acids (CH1, CH2) and a C-terminal proline-rich domain (Pro2). Tyrosine phosphorylation (PY) EPLYA and EGLYA are shown. The interactors Tiam1, Csk,* β*-catenin, Vinexin, EB3, Cortactin, p130Cas and Src are associated with specific domains.*

(*RAR*A; 17q21) and signal transducer and activator of transcription 3 (*STAT3*; 17q21). These genes often undergo a gain of function role in human tumors [25]. Moreover, the 17q gain occurs in 50–70% of all high stage NB and is associated with poor prognosis as an independent marker of adverse outcome [26–29].

p140Cap shares different Intrinsically Disordered Regions (IDRs) that classify p140Cap as "Intrinsic Disorder Protein" (IDP) [30, 31]. The IDP features of p140Cap could allow the interaction with several partners and promote protein–protein interactions that are the elected functions for a scaffold protein. The p140Cap protein can interact with multiple partners [30] (**Figure 1**). In particular, p140Cap associates with the tyrosine kinases Csk and Src. This macromolecular complex triggers Csk activity to phosphorylate Src on its inhibitory tyrosine, resulting in Src inactivation and in the suppression of downstream pathways regulating motility and invasion of cancer cells [23]. Indeed, at the structural level, p140Cap contains a tyrosine rich domain, important for the interaction with Csk [32], two coiled coil regions, that can mediate the binding with beta-catenin [10] and two different proline rich domains responsible for the association with the microtubule associated protein EB3 [33], Cortactin [34] and Vimentin [35].

The physiologic role of p140Cap has been mainly investigated in the brain [35], where it is expressed in neurons both in the presynapse [10, 36] and in the postsynapse [10, 37–41]. In differentiated neurons, it controls synaptic plasticity [33, 40], and regulates GABAergic synaptogenesis and development of hippocampal inhibitory circuits [36]. In particular, p140Cap enters and accumulates in the dendritic spine (DS) through EB3 binding [33]. In this compartment p140Cap acts as hub interacting with Cortactin, a protein that regulates actin branching and new filament polymerization [42] and with Citron-N [40] resulting in mature DS stabilization. In both the pre- and post-synaptic regions, p140Cap is involved in a network of protein–protein interactions as confirmed by its interactome in synaptosomes. p140Cap interactors converge on key synaptic processes, including transmission across chemical synapses, actin cytoskeleton remodeling and cell–cell junction organization [41].

### **3.** *SRCIN1* **mRNA expression is an independent prognostic marker for NB**

To address the involvement of p140Cap in NB patients, we first investigated the relationship between *SRCIN1* mRNA levels and patient outcomes, by using the R2 genomics analysis and visualization platform (R2: Genomics analysis and Visualization Platform (http://r2.amc.nl)). The bioinformatics analysis performed on a dataset containing clinical and gene expression data of 498 NB patients revealed that *SRCIN1* positively impacts on patients' outcome. In fact, the Kaplan– Meier analysis showed that high expression of *SRCIN1* is associated with good prognosis in 403 patients, whereas low expression is observed in 95 poor prognosis patients (**Figure 2A**). Furthermore, high *SRCIN1* expression was significantly


#### **Figure 2.**

*Stratification by SRCIN1 mRNA expression in primary NB patients and SRCIN1 gene status. A) Kaplan– Meier curves for overall (upper panel) and event-free (bottom panel) survival stratified by SRCIN1 expression in a cohort of 498 NB patients. Cut off for high or low SRCIN1 expression was chosen by Kaplan–Meier scan method. Survival curves were compared by log-rank test. P-values were corrected for multiple hypotheses testing by Bonferroni method. Each plot reports the corrected P-value (P). Corrected P-values lower than 0.05 were considered statistically significant. The number of patients with high or low expression of SRCIN1 mRNA is reported in every curve. B) Box and whisker plot for the expression of SRCIN1 mRNA in the two risk groups defined by INSS stages (st1, st2, st3, st4s vs. st4). The significance of the mean expression was measured by unpaired student t-test. A P-value lower than 0.05 was considered significant. C) Multivariate cox regression analysis for overall survival (OS) and event-free survival (EFS). The prognostic value of SRCIN1 mRNA expression (high and low) was tested in the context of known risk factors: Age at diagnosis (>12 months vs. <12 months) MYCN amplification (normal vs. amplified) INSS stages (st1, st2, st3, st4s vs. st4). Cut off for high or low SRCIN1 expression was chosen by Kaplan–Meier scan method. Cox regression coefficient (coefficient), hazard ratio (HR), 95% of confidence interval (95% CI) and P-value are shown for each variable in the OS and EFS panel. Significant P-values are lower than 0.05 (OS: HR 0.34 95% CI 0.2–0.5 P < 0.0001; EFS: HR 0.27 95% CI 0.1–0.4, P < 0.0001).*

**79**

**Table 1.**

*The Scaffold Protein p140Cap as a Molecular Hub for Limiting Cancer Progression…*

Case 1 disrupted in the breakpoint Chr17: 36696338–81029941

Case 2 disrupted in the breakpoint Chr17: 36694901–80943345

Case 3 loss Chr17: 25311574–36777884

Case 4 disrupted in the breakpoint Chr17: 36696338–80969424

Case 5 disrupted in the breakpoint Chr17: 36696279–81029941

Case 6 copy neutral LOH Chr17: 25569094–42949451

Case 7 copy neutral LOH Chr17: 29149425–45297941

Case 8 copy neutral LOH Chr17: 31571877–40588363

Case 9 loss Chr17: 25278114–37876263

Case 10 loss Chr17: 25278114–68301170

Case 11 disrupted in the breakpoint Chr17: 36696279–81029941

Case 12 disrupted in the breakpoint Chr17: 36696338–81029941

Case 13 disrupted in the breakpoint Chr17: 36740844–80943189

Case 14 disrupted in the breakpoint Chr17: 36740903–80993001

Case 15 loss Chr17: 25278114–81029941

Case 16 disrupted in the breakpoint Chr17: 36672992–77470237

Case 17 disrupted in the breakpoint Chr17: 36694044–81099040

*SRCIN1 loss/cn-LOH or disruption in the breakpoint on 17 NB patients.*

**NB patients** *SRCIN1* **gene status CHROMOSOMAL COORDINATES**

Cytoband: 17q12-q25.3 Size: 44.33 Mb

Cytoband: 17q12-q25.3 Size: 44.24 Mb

Cytoband: 17q11.1-q12 Size: 11.46 Mb

Cytoband: 17q12-q25.3 Size: 44.27 Mb

Cytoband: 17q12-q25.3 Size: 44.33 Mb

Cytoband: 17q11.1-q21.31 Size: 17.38 Mb

Cytoband: 17q11.1-q21.31 Size: 16.14 Mb

Cytoband: 17q11.2-q21.2 Size: 9.01 Mb

Cytoband: 17q11.1-q12 Size: 12.59 Mb

Cytoband: 17q11.1-q24.3 Size: 43.02 Mb

Cytoband: 17q12-q25.3 Size: 44.33 Mb

Cytoband: 17q12-q25.3 Size: 44.33 Mb

Cytoband: 17q12-q25.3 Size: 44.20 Mb

Cytoband: 17q12-q25.3 Size: 44.25 Mb

Cytoband: 17q11.1-q25.3 Size: 55.75 Mb

Cytoband: 17q12-q25.3 Size: 40.79 Mb

Cytoband: 17q12-q25.3 Size: 44.40 Mb

*DOI: http://dx.doi.org/10.5772/intechopen.96383*


*The Scaffold Protein p140Cap as a Molecular Hub for Limiting Cancer Progression… DOI: http://dx.doi.org/10.5772/intechopen.96383*

#### **Table 1.**

*SRCIN1 loss/cn-LOH or disruption in the breakpoint on 17 NB patients.*

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

Visualization Platform (http://r2.amc.nl)). The bioinformatics analysis performed on a dataset containing clinical and gene expression data of 498 NB patients revealed that *SRCIN1* positively impacts on patients' outcome. In fact, the Kaplan– Meier analysis showed that high expression of *SRCIN1* is associated with good prognosis in 403 patients, whereas low expression is observed in 95 poor prognosis patients (**Figure 2A**). Furthermore, high *SRCIN1* expression was significantly

*Stratification by SRCIN1 mRNA expression in primary NB patients and SRCIN1 gene status. A) Kaplan– Meier curves for overall (upper panel) and event-free (bottom panel) survival stratified by SRCIN1 expression in a cohort of 498 NB patients. Cut off for high or low SRCIN1 expression was chosen by Kaplan–Meier scan method. Survival curves were compared by log-rank test. P-values were corrected for multiple hypotheses testing by Bonferroni method. Each plot reports the corrected P-value (P). Corrected P-values lower than 0.05 were considered statistically significant. The number of patients with high or low expression of SRCIN1 mRNA is reported in every curve. B) Box and whisker plot for the expression of SRCIN1 mRNA in the two risk groups defined by INSS stages (st1, st2, st3, st4s vs. st4). The significance of the mean expression was measured by unpaired student t-test. A P-value lower than 0.05 was considered significant. C) Multivariate cox regression analysis for overall survival (OS) and event-free survival (EFS). The prognostic value of SRCIN1 mRNA expression (high and low) was tested in the context of known risk factors: Age at diagnosis (>12 months vs. <12 months) MYCN amplification (normal vs. amplified) INSS stages (st1, st2, st3, st4s vs. st4). Cut off for high or low SRCIN1 expression was chosen by Kaplan–Meier scan method. Cox regression coefficient (coefficient), hazard ratio (HR), 95% of confidence interval (95% CI) and P-value are shown for each variable in the OS and EFS panel. Significant P-values are lower than 0.05 (OS: HR 0.34 95% CI 0.2–0.5* 

**78**

*P < 0.0001; EFS: HR 0.27 95% CI 0.1–0.4, P < 0.0001).*

**Figure 2.**

associated with event-free survival (EFS) (321 patients) whereas a low expression was significantly associated with reduced metastatic recurrence (177 patients) (**Figure 2B**). *SRCIN1* mRNA expression was a favorable prognostic factor, both in terms of overall survival (OS) and EFS, regardless of the other known risk factors, including *MYCN* amplification, INSS stage, and age at diagnosis (**Figure 2C**).

To date, p140Cap expression by immunohistochemistry (IHC) on NB samples has not been studied owing to the lack of available cancer tissues, but *SRCIN1* mRNA expression correlates with a good outcome and is an independent prognostic marker for NB.

The *SRCIN1* gene is located on chromosome 17q12, a genomic region frequently involved in genetic abnormalities in NB. Therefore, a large cohort of 225 NB patients of all stages with 17q gain with poor prognosis, was analyzed by high-resolution oligonucleotide array-Comparative Genomic Hybridization (a-CGH) and Single Nucleotide Polymorphism - array (SNP-array). *SRCIN1* was hemizygously deleted in four NB tumors and it was subjected to copy-neutral Loss Of Heterozigosity (cn-LOH) in three specimens. Moreover, ten tumors displayed *SRCIN1* loss due to a breakpoint involved in the generation of 17q gain [43] (**Table 1**). However, because of the limited number of analyzed cases, survival differences between patients harboring these alterations did not reach statistical significance. Similar results come out in NB cell lines, as shown in SK-N-SH cells, where cn-LOH (8.72 Mb) that included *SRCIN1* gene, correlates with weak protein expression, suggesting an effective partial knockout of gene expression, originally proving that in NB patients the *SRCIN1* gene status may affect p140Cap expression, affecting prognosis.

#### **4. p140Cap negatively affects tumorigenic features**

The data obtained in NB patients support the hypothesis that p140Cap may curb the intrinsic biological aggressiveness of NB tumors. NB originates from the developing sympathetic nervous system, with a preferential localization in sympathetic ganglia and adrenal glands. Interestingly, we found that p140Cap is expressed in the main site of origin of NB tumors, in the medulla of normal human neonatal adrenal glands (**Figure 3A**). p140Cap is also expressed in a board panel of human NB cell lines which represent valid surrogate models for NB research [44]. Among these cell lines, p140Cap level was highly detected in HTLA-230, IMR-5, IMR-32, LAN-1 and SH-SY-5Y cell lines, weakly in SK-N-SH cells and undetectable in ACN cell line, a neuroblast-like cell line derived from bone marrow metastasis [45] (**Figure 3B**). According to the protein level analysis, the genomic profiling revealed a wide spectrum of *SRCIN1* gene abnormalities. Indeed, the *SRCIN1* gene was lost in ACN, while a single copy was found in SH-SY-5Y, IMR-32, and HTLA-230 cell lines. Moreover, a genomic gain was observed in the LAN-1 cell line, whereas SK-N-SH cells displayed a cn-LOH.

The absence of p140Cap protein renders the ACN cell line a suitable tool for the generation of a p140Cap-expressing NB cell line via retroviral infection that might be leveraged for further functional investigations (**Figure 3C**). It is well established that p140Cap inhibits breast cancer cell features such as migration and proliferation [22]. Consistently, p140Cap-overexpressing ACN (p140Cap-ACN) cells exhibited decreased migration properties in a Wound Healing assay, and impaired anchorageindependent growth of NB cells, one of the main hallmarks of cancer. Cancer cells are known to avoid apoptosis by increasing or decreasing the expression of apoptotic and anti-apoptotic genes, respectively [46]. A specific type of apoptotic process, called anoikis, occurs in cells in response to loss of adhesion to the extracellular matrix.

**81**

**Figure 3.**

*tumor growth. Mock and p140 cells (2 × 105*

*The Scaffold Protein p140Cap as a Molecular Hub for Limiting Cancer Progression…*

Upon anoikis, p140Cap-ACN cells showed both a lower upregulation of the antiapoptotic protein Bcl-2 compared to mock cells, and a significantly higher percentage of apoptotic cells detected by annexin V labeling. Overall, p140Cap can limit anchorage-independent growth, migration and apoptosis of NB cells, suggesting a

*In vitro expression and in vivo role of p140Cap. (A) p140Cap staining is visible in the chromaffin cells of the adrenal medulla (upper panel), as confirmed by the chromogranin a staining (lower panel). Scale bar: 50 μM; (B) p140Cap expression in NB cell lines, by western blot of equal amounts of proteins from the indicated cell lines; (C) p140Cap expression in a pool of clones of ACN cells upon viral infection; (D) p140Cap limits in vivo* 

*region of male NSG mice. Average tumor volume. The size of the tumors was evaluated twice a week using digital calipers in blind experiments and significance was quantified by unpaired t-test (\*\*P < 0.01); (E) WB analysis of p140Cap expression on explanted tumors by SDS-PAGE. Antibodies to p140Cap and tubulin (as loading control) were used; (F) Src kinase activation in tumor extracts. Tyr 416 phosphorylation (Y416) and Src protein level is shown. Quantification on the right is the ratio between phosphorylated Src and total Src* 

 *cells in 0.2 ml of PBS) were subcutaneously injected into the dorsal* 

To date, the *in vivo* models commonly used for NB research and drug efficacy studies ranges from genetically engineered mouse models to xenograft murine systems [47] and from syngeneic mice to zebrafish and chick embryo chorioallantoic membrane [48, 49]. Each animal model has its own strengths and limitations, and

causal involvement of this protein in curbing NB cancer cell properties.

*protein in 5 tumors per group, as mean ± SEM (right) (unpaired t-test \*\*P < 0.01).*

*DOI: http://dx.doi.org/10.5772/intechopen.96383*

*The Scaffold Protein p140Cap as a Molecular Hub for Limiting Cancer Progression… DOI: http://dx.doi.org/10.5772/intechopen.96383*

#### **Figure 3.**

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

marker for NB.

affecting prognosis.

cells displayed a cn-LOH.

associated with event-free survival (EFS) (321 patients) whereas a low expression was significantly associated with reduced metastatic recurrence (177 patients) (**Figure 2B**). *SRCIN1* mRNA expression was a favorable prognostic factor, both in terms of overall survival (OS) and EFS, regardless of the other known risk factors, including *MYCN* amplification, INSS stage, and age at diagnosis (**Figure 2C**).

To date, p140Cap expression by immunohistochemistry (IHC) on NB samples

has not been studied owing to the lack of available cancer tissues, but *SRCIN1* mRNA expression correlates with a good outcome and is an independent prognostic

The *SRCIN1* gene is located on chromosome 17q12, a genomic region frequently involved in genetic abnormalities in NB. Therefore, a large cohort of 225 NB patients of all stages with 17q gain with poor prognosis, was analyzed by high-resolution oligonucleotide array-Comparative Genomic Hybridization (a-CGH) and Single Nucleotide Polymorphism - array (SNP-array). *SRCIN1* was hemizygously deleted in four NB tumors and it was subjected to copy-neutral Loss Of Heterozigosity (cn-LOH) in three specimens. Moreover, ten tumors displayed *SRCIN1* loss due to a breakpoint involved in the generation of 17q gain [43] (**Table 1**). However, because of the limited number of analyzed cases, survival differences between patients harboring these alterations did not reach statistical significance. Similar results come out in NB cell lines, as shown in SK-N-SH cells, where cn-LOH (8.72 Mb) that included *SRCIN1* gene, correlates with weak protein expression, suggesting an effective partial knockout of gene expression, originally proving that in NB patients the *SRCIN1* gene status may affect p140Cap expression,

The data obtained in NB patients support the hypothesis that p140Cap may curb the intrinsic biological aggressiveness of NB tumors. NB originates from the developing sympathetic nervous system, with a preferential localization in sympathetic ganglia and adrenal glands. Interestingly, we found that p140Cap is expressed in the main site of origin of NB tumors, in the medulla of normal human neonatal adrenal glands (**Figure 3A**). p140Cap is also expressed in a board panel of human NB cell lines which represent valid surrogate models for NB research [44]. Among these cell lines, p140Cap level was highly detected in HTLA-230, IMR-5, IMR-32, LAN-1 and SH-SY-5Y cell lines, weakly in SK-N-SH cells and undetectable in ACN cell line, a neuroblast-like cell line derived from bone marrow metastasis [45] (**Figure 3B**). According to the protein level analysis, the genomic profiling revealed a wide spectrum of *SRCIN1* gene abnormalities. Indeed, the *SRCIN1* gene was lost in ACN, while a single copy was found in SH-SY-5Y, IMR-32, and HTLA-230 cell lines. Moreover, a genomic gain was observed in the LAN-1 cell line, whereas SK-N-SH

The absence of p140Cap protein renders the ACN cell line a suitable tool for the generation of a p140Cap-expressing NB cell line via retroviral infection that might be leveraged for further functional investigations (**Figure 3C**). It is well established that p140Cap inhibits breast cancer cell features such as migration and proliferation [22]. Consistently, p140Cap-overexpressing ACN (p140Cap-ACN) cells exhibited decreased migration properties in a Wound Healing assay, and impaired anchorageindependent growth of NB cells, one of the main hallmarks of cancer. Cancer cells are known to avoid apoptosis by increasing or decreasing the expression of apoptotic and anti-apoptotic genes, respectively [46]. A specific type of apoptotic process, called anoikis, occurs in cells in response to loss of adhesion to the extracellular matrix.

**4. p140Cap negatively affects tumorigenic features**

**80**

*In vitro expression and in vivo role of p140Cap. (A) p140Cap staining is visible in the chromaffin cells of the adrenal medulla (upper panel), as confirmed by the chromogranin a staining (lower panel). Scale bar: 50 μM; (B) p140Cap expression in NB cell lines, by western blot of equal amounts of proteins from the indicated cell lines; (C) p140Cap expression in a pool of clones of ACN cells upon viral infection; (D) p140Cap limits in vivo tumor growth. Mock and p140 cells (2 × 105 cells in 0.2 ml of PBS) were subcutaneously injected into the dorsal region of male NSG mice. Average tumor volume. The size of the tumors was evaluated twice a week using digital calipers in blind experiments and significance was quantified by unpaired t-test (\*\*P < 0.01); (E) WB analysis of p140Cap expression on explanted tumors by SDS-PAGE. Antibodies to p140Cap and tubulin (as loading control) were used; (F) Src kinase activation in tumor extracts. Tyr 416 phosphorylation (Y416) and Src protein level is shown. Quantification on the right is the ratio between phosphorylated Src and total Src protein in 5 tumors per group, as mean ± SEM (right) (unpaired t-test \*\*P < 0.01).*

Upon anoikis, p140Cap-ACN cells showed both a lower upregulation of the antiapoptotic protein Bcl-2 compared to mock cells, and a significantly higher percentage of apoptotic cells detected by annexin V labeling. Overall, p140Cap can limit anchorage-independent growth, migration and apoptosis of NB cells, suggesting a causal involvement of this protein in curbing NB cancer cell properties.

To date, the *in vivo* models commonly used for NB research and drug efficacy studies ranges from genetically engineered mouse models to xenograft murine systems [47] and from syngeneic mice to zebrafish and chick embryo chorioallantoic membrane [48, 49]. Each animal model has its own strengths and limitations, and

provides insights to specific biological questions. Immunodeficient mouse models such as the NOD Scid Gamma (NSG) mice represent a valuable tool for the study of engrafted human cell lines and PDX tumors [50]. In particular, NSG mice exhibit a complete deficiency in the adaptive immunity and a severe deficiency in the innate immunity as a consequence of mutations in the IL2-receptor common gamma chain, the *Prkdc* gene, which determines the so-called "scid" mutation, and the Rag1 or Rag2 null mutation [50]. Therefore, the NSG preclinical model was a suitable candidate to investigate the *in vivo* tumor-suppressing role of p140Cap. Upon subcutaneous injection into the dorsal region of the NSG mice, p140Cap cells gave origin to smaller tumors, compared to mock cells. p140Cap tumors were also extensively poorly proliferative, in terms of proliferation marker KI67 (**Figure 3D, E**).

In NB, angiogenesis has a prominent role in determining tumor phenotype. A study published by Meitar D *et al.* demonstrated that higher vascularity in NB correlates with metastasis, unfavorable histology, and poor outcome [51]. p140Cap tumors showed a slight but significant lower number of vessels positive for CD31 and CD105 endothelial cell markers compared to control. Histological sections were also stained for AML and NG2 markers of mature or young pericytes, respectively, in order to evaluate the pericyte coverage of vessels. In line with the idea that p140Cap limits the angiogenic activity of cancer cells leading to the formation of larger and more stable vessels, p140Cap tumors exhibited higher pericyte coverage of the endothelium compared to control.

As already mentioned, p140Cap has been widely demonstrated to limit breast cancer cells growth and metastasis formation [22, 23]. The ability of p140Cap to inhibit cancer cell adhesion, migration and proliferation may contribute to the overall reduced occurrence of metastatic events. p140Cap tumors gave rise to a significantly reduced number of lung metastases compared to control. Overall, p140Cap impairs NB tumor growth and spontaneous metastasis *in vivo*, with a significant decrease in proliferation markers and an increase in tumor vessel pericyte coverage. These results are in line with those obtained in HER2 positive breast cancer patients and preclinical models [22], where p140Cap dampens the aggressiveness of these highly aggressive tumors.

Further evidence supporting the biological relevance of p140Cap in curbing NB aggressiveness was provided by the recent work of Yuan XL *et al*. [52]. Yuan XL *et al*. demonstrated that *SRCIN1* is a direct target of the microRNA-373 (miR-373) and that their expression has a negative correlation in both NB human samples and cell lines. miR-373 functions as an oncomiRNA promoting proliferation, migration and invasion of NB cells. Inhibition of miR-373 by using a specific anti-miRNA in SK-N-BE(2) cells led to a significant decrease of tumor growth in a mouse xenograft model that was paralleled with increased p140Cap mRNA and protein levels in the resected tumors. Silencing of *SRCIN1* partially abrogated the inhibitory effect of antimiR-373 in NB cell proliferation, migration and invasion.

The molecular mechanisms underpinning the tumor-suppressive properties of p140Cap in NB may rely on the modulation of specific intracellular signaling pathways that will be dissected in the next sections.

#### **5. Molecular mechanisms and therapeutic targets in neuroblastoma**

Over the last years, genomic analysis, exome and whole-genome sequencing, genome-wide association studies, transcriptomics and drug screenings have shed light on NB biology [53]. The ongoing phase relies on translating NB biology and genetics into improved prognostic stratification and precision medicine. New druggable targets could come out from the identification of predictors for response and

**83**

**pathways**

*The Scaffold Protein p140Cap as a Molecular Hub for Limiting Cancer Progression…*

outcome as well as from the discovery of molecular aberrations in the tumors (for a recent review see [53]). Of the genetic aberrations described in NB only *MYCN* overexpression and activating mutations of the tyrosine kinase receptor (RTK) *ALK* have been proven to be de novo oncogenic drivers as mutation or overexpression of these molecules give rise to NB in genetically engineered mouse models [54, 55]. The oncogenic transcription factor *MYCN* is a hallmark of poor prognosis in NB patients [56]. However, the compounds that can interfere with *MYCN* interaction with its partner MAX as a way to block its transcriptional action, were not efficient *in vivo* [57], dampening their development in clinical testing. Strategies to exploit *MYCN* as a tumor-associated antigen for immunotherapy deserve further functional validation [58]. The *ALK* gene is altered by gain-of-function point mutations in around 14% of high-risk NB and represents an ideal therapeutic target given its low or absent expression in healthy tissue postnatally [59]. ALK signaling can be blocked in ALK-mutant NB cell lines and mouse models by different means, including RNA interference and small-molecule inhibitors. Moreover, the STAT3, PI3K/AKT and Ras/MAPK are the main pathways involved in full-length ALK signaling [60]. In particular, Mass Spectrometry-based phosphotyrosine profiling of signaling events associated with the full-length ALK receptor, showed robust activation of STAT3 on Tyr705 in a number of independent NB cell lines. STAT3 silencing reduces *MYCN* protein levels downstream of ALK signaling, together with inhibition of NB cell growth in the presence of STAT3 inhibitors. Overall these data suggest that activation of STAT3 is important for ALK signaling activity in NB [61]. On the other hand, ERK5 can mediate ALK-induced transcription of *MYCN* and proliferation of NB, suggesting that targeting both ERK5 and ALK may be benefi-

In addition to ALK, signaling through the EGFR and ERBB2 RTK, both found to be non-mutational activated in subsets of NB, converge at MAPK, with increased MAPK signaling. Further, MAPK/ERK kinase (MEK) inhibitors have been shown to inhibit the growth of NB cells *in vitro* [63] and *in vivo*, alone or in synergy with the CDK4/6 inhibitor, ribociclib to suppress tumor growth in a panel of murine xenograft models of NB [64]. However, a recent preclinical study advises against trametinib as monotherapy in ALK-addicted NB due to increased feedback activation of other signaling pathways including PI3K/AKT in both cell lines and mice xenografts [65]. Interestingly, high-risk NBs without *MYCN* amplification may deregulate *MYC* and other oncogenic genes via altered beta-catenin signaling providing a potential candidate pathway for therapeutic inhibition [66]. XAV939, a tankyrase 1 inhibitor, promotes cell apoptosis in NB cell lines by inhibiting Wnt/beta-catenin signaling pathway, by reducing the expression of anti-apoptotic markers and decreasing colony formation *in vitro* [67]. O6-methylguanine-DNA methyltransferase (MGMT) is commonly overexpressed in cancers and is implicated in the development of chemoresistance. A significant correlation between Wnt signaling and MGMT expression was found in several cancers, including NB. Further, immunofluorescence analysis on human tumor tissues showed co-localization of nuclear beta-catenin and MGMT in subtypes of NB. Pharmacological or genetic inhibition of Wnt activity downregulates MGMT expression and restores chemosensitivity of DNA-alkylating drugs [68].

**6. p140Cap impairs the Src/p130Cas and the STAT3/Jak2 signaling** 

Focal adhesion kinase (FAK) and Src are two non-receptor intracellular kinases highly expressed in a number of human tumors including NB, and together regulate both cellular adhesion and survival. Both FAK and Src play a role in protecting NB

*DOI: http://dx.doi.org/10.5772/intechopen.96383*

cial in NB patients [62].

#### *The Scaffold Protein p140Cap as a Molecular Hub for Limiting Cancer Progression… DOI: http://dx.doi.org/10.5772/intechopen.96383*

outcome as well as from the discovery of molecular aberrations in the tumors (for a recent review see [53]). Of the genetic aberrations described in NB only *MYCN* overexpression and activating mutations of the tyrosine kinase receptor (RTK) *ALK* have been proven to be de novo oncogenic drivers as mutation or overexpression of these molecules give rise to NB in genetically engineered mouse models [54, 55]. The oncogenic transcription factor *MYCN* is a hallmark of poor prognosis in NB patients [56]. However, the compounds that can interfere with *MYCN* interaction with its partner MAX as a way to block its transcriptional action, were not efficient *in vivo* [57], dampening their development in clinical testing. Strategies to exploit *MYCN* as a tumor-associated antigen for immunotherapy deserve further functional validation [58]. The *ALK* gene is altered by gain-of-function point mutations in around 14% of high-risk NB and represents an ideal therapeutic target given its low or absent expression in healthy tissue postnatally [59]. ALK signaling can be blocked in ALK-mutant NB cell lines and mouse models by different means, including RNA interference and small-molecule inhibitors. Moreover, the STAT3, PI3K/AKT and Ras/MAPK are the main pathways involved in full-length ALK signaling [60]. In particular, Mass Spectrometry-based phosphotyrosine profiling of signaling events associated with the full-length ALK receptor, showed robust activation of STAT3 on Tyr705 in a number of independent NB cell lines. STAT3 silencing reduces *MYCN* protein levels downstream of ALK signaling, together with inhibition of NB cell growth in the presence of STAT3 inhibitors. Overall these data suggest that activation of STAT3 is important for ALK signaling activity in NB [61]. On the other hand, ERK5 can mediate ALK-induced transcription of *MYCN* and proliferation of NB, suggesting that targeting both ERK5 and ALK may be beneficial in NB patients [62].

In addition to ALK, signaling through the EGFR and ERBB2 RTK, both found to be non-mutational activated in subsets of NB, converge at MAPK, with increased MAPK signaling. Further, MAPK/ERK kinase (MEK) inhibitors have been shown to inhibit the growth of NB cells *in vitro* [63] and *in vivo*, alone or in synergy with the CDK4/6 inhibitor, ribociclib to suppress tumor growth in a panel of murine xenograft models of NB [64]. However, a recent preclinical study advises against trametinib as monotherapy in ALK-addicted NB due to increased feedback activation of other signaling pathways including PI3K/AKT in both cell lines and mice xenografts [65].

Interestingly, high-risk NBs without *MYCN* amplification may deregulate *MYC* and other oncogenic genes via altered beta-catenin signaling providing a potential candidate pathway for therapeutic inhibition [66]. XAV939, a tankyrase 1 inhibitor, promotes cell apoptosis in NB cell lines by inhibiting Wnt/beta-catenin signaling pathway, by reducing the expression of anti-apoptotic markers and decreasing colony formation *in vitro* [67]. O6-methylguanine-DNA methyltransferase (MGMT) is commonly overexpressed in cancers and is implicated in the development of chemoresistance. A significant correlation between Wnt signaling and MGMT expression was found in several cancers, including NB. Further, immunofluorescence analysis on human tumor tissues showed co-localization of nuclear beta-catenin and MGMT in subtypes of NB. Pharmacological or genetic inhibition of Wnt activity downregulates MGMT expression and restores chemosensitivity of DNA-alkylating drugs [68].
