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

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

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

of the endothelium compared to control.

highly aggressive tumors.

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

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

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

The molecular mechanisms underpinning the tumor-suppressive properties of p140Cap in NB may rely on the modulation of specific intracellular signaling

**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

antimiR-373 in NB cell proliferation, migration and invasion.

pathways that will be dissected in the next sections.

**82**

cells from apoptosis, and dual inhibition of these kinases may be important when designing therapeutic interventions for this tumor [69]. Immunohistochemical staining showed FAK to be present in 73% of human NB specimens examined. In addition, p125FAK staining was significantly increased in stage IV tumors with amplification of the *MYCN*. Src expression in NB patients has been associated with poor outcomes [70, 71]. Indeed, Src family kinases promote cell survival/ proliferation and reduce cell aggregation of NBs. Conversely, its inhibition results in decreased proliferation and enhanced apoptosis in NB cells [72, 73], suggesting that Src family kinase inhibitors may be good candidates for molecular targeted therapy [74]. Of note, dasatinib, a well-known Src kinase inhibitor, is a potent inhibitor of NB cell viability with an IC(50) in the submicromolar range. As a consequence, dasatinib decreased anchorage-independent growth, affecting senescence and apoptosis. Interestingly, in the HTLA-230 NB model, dasatinib decreased c-Kit and Src activation together with a strong MAPK and Akt impairment. Dasatinib was also tested *in vivo* in a murine orthotopic model, where NB cells were injected directly in the adrenal gland in a microenvironment that closely mimics the human tumors conditions. HTLA-230 tumors were reduced in size and cellularity, with proliferation disease. Drug treatment in the orthotopic model utilizing HTLA-230 cells produced a significant reduction of tumor burden. Nevertheless, dasatinib activity *in vivo* was also significantly inhibited, but complete tumor eradication was not achieved [75]. Recently, the scaffold protein PAG1 was involved in the regulation of Src family kinase (SFK) signaling in NB. The NB cell line expressing PAG1™− lacks the membrane-spanning domain of PAG1 and is located in the cytoplasm. PAG1™− cells exhibited higher amounts of active SFKs and increased growth rate. Under differentiation conditions, PAG1™− cells continued to proliferate and did not undergo differentiation. Activated FYN was sequestered in PAG1™− cells, suggesting that disruption of FYN localization led to the observed defects in differentiation. Overall, PAG1 is an additional example of how a scaffold protein may control SFK intracellular localization, impacting on their activity and signaling that induces differentiation events, that may be crucial in the control of NB aggressiveness [76].

IL-6-dependent activation of STAT3 [77] has already been reported in NB, where STAT3 is critical in mediating increased survival and drug resistance [78–80]. Interestingly, very recently, the antiapoptotic and prometastatic JAK-STAT3 pathway was activated in chemoresistant tumors, generated in the Th-*MYCN* CPM32 model. This model derives from a multicycle treatment with cyclophosphamide of the Th-*MYCN* genetically engineered mice which develop rapidly progressive chemosensitive NB, but lack clinically relevant metastases. Copy number aberrations in these tumors reflect the genomic alterations typical of human *MYCN*-amplified NB, e.g. copy number gains at mouse chromosome 11, syntenic with gains on human chromosome 17q. The Th-*MYCN* CPM32 model is characterized by chemoresistance and progression in spontaneous bone marrow metastatic events. NB tumors show a huge remodeling of the immune microenvironment, with augmented tumor-associated fibroblasts and stroma. Treatment with the JAK1/JAK2 inhibitor CYT387 reduced progression of chemoresistant tumors and increased survival, highlighting that under treatment conditions that mimic chemotherapy in human patients, Th-*MYCN* CPM32 mice develop genomic, microenvironmental, and clinical features reminiscent of human chemorefractory disease, with dysregulation of signaling pathways such as JAK-STAT3 that could be targeted to improve treatment of aggressive disease [81].

Our recent data show that p140Cap expression in NB cells is sufficient to down-modulate the tyrosine phosphorylation of Src Tyr 416 (p-Src), a marker of active Src, as well as of p130Cas, a well-known Src substrate [82] (**Figure 4A, B**).

**85**

*protein.*

**Figure 4.**

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

*p140Cap affects signaling pathways in NB cells. (A) Src activation was evaluated on mock and p140 ACN cells by WB analysis of Tyr 416 phosphorylation (Y416) and Src protein level as loading control. Antibodies to GAPDH (as loading control) were used. Quantification on the right is the ratio between phosphorylated Src and total Src protein; (B) p130Cas phosphorylation was evaluated with antibodies to phosphorylated p130Cas at Tyr 410, p130Cas and GAPDH antibodies for loading control. Quantification on the right is the ratio between phosphorylated p130Cas and total p130Cas; (C) STAT3 phosphorylation was evaluated with antibodies to phosphorylated STAT3 at Tyr705 and STAT3 antibodies for loading control. Quantification on the right is the ratio between phosphorylated STAT3 and total STAT3 protein from three independent experiment; (D) Jak2 activation was evaluated with antibodies to phosphorylated Jak2 at Tyr1007/1008 and Jak2 antibodies for loading control. Quantification on the right is the ratio between phosphorylated Jak2 and total Jak2 protein; in E-G p140Cap silenced cells were tested for p140Cap WB, and GAPDH as loading control (E), for Src activation at Tyr 416 phosphorylation (Y416) and Src protein level as loading control (F), and for phosphorylated STAT3 at Tyr 705 and STAT3 protein level as loading control (G). Quantification is shown on the left as the ratio between phosphorylated Src/STAT3 and total Src/STAT3* 

*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 4.**

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

Under differentiation conditions, PAG1™−

cells from apoptosis, and dual inhibition of these kinases may be important when designing therapeutic interventions for this tumor [69]. Immunohistochemical staining showed FAK to be present in 73% of human NB specimens examined. In addition, p125FAK staining was significantly increased in stage IV tumors with amplification of the *MYCN*. Src expression in NB patients has been associated with poor outcomes [70, 71]. Indeed, Src family kinases promote cell survival/ proliferation and reduce cell aggregation of NBs. Conversely, its inhibition results in decreased proliferation and enhanced apoptosis in NB cells [72, 73], suggesting that Src family kinase inhibitors may be good candidates for molecular targeted therapy [74]. Of note, dasatinib, a well-known Src kinase inhibitor, is a potent inhibitor of NB cell viability with an IC(50) in the submicromolar range. As a consequence, dasatinib decreased anchorage-independent growth, affecting senescence and apoptosis. Interestingly, in the HTLA-230 NB model, dasatinib decreased c-Kit and Src activation together with a strong MAPK and Akt impairment. Dasatinib was also tested *in vivo* in a murine orthotopic model, where NB cells were injected directly in the adrenal gland in a microenvironment that closely mimics the human tumors conditions. HTLA-230 tumors were reduced in size and cellularity, with proliferation disease. Drug treatment in the orthotopic model utilizing HTLA-230 cells produced a significant reduction of tumor burden. Nevertheless, dasatinib activity *in vivo* was also significantly inhibited, but complete tumor eradication was not achieved [75]. Recently, the scaffold protein PAG1 was involved in the regulation of Src family kinase (SFK) signaling in NB. The NB cell line expressing PAG1™− lacks the membrane-spanning domain of PAG1 and is located in the cytoplasm.

cells exhibited higher amounts of active SFKs and increased growth rate.

ing that disruption of FYN localization led to the observed defects in differentiation. Overall, PAG1 is an additional example of how a scaffold protein may control SFK intracellular localization, impacting on their activity and signaling that induces differentiation events, that may be crucial in the control of NB aggressiveness [76]. IL-6-dependent activation of STAT3 [77] has already been reported in NB, where STAT3 is critical in mediating increased survival and drug resistance [78–80]. Interestingly, very recently, the antiapoptotic and prometastatic JAK-STAT3 pathway was activated in chemoresistant tumors, generated in the Th-*MYCN* CPM32 model. This model derives from a multicycle treatment with cyclophosphamide of the Th-*MYCN* genetically engineered mice which develop rapidly progressive chemosensitive NB, but lack clinically relevant metastases. Copy number aberrations in these tumors reflect the genomic alterations typical of human *MYCN*-amplified NB, e.g. copy number gains at mouse chromosome 11, syntenic with gains on human chromosome 17q. The Th-*MYCN* CPM32 model is characterized by chemoresistance and progression in spontaneous bone marrow metastatic events. NB tumors show a huge remodeling of the immune microenvironment, with augmented tumor-associated fibroblasts and stroma. Treatment with the JAK1/JAK2 inhibitor CYT387 reduced progression of chemoresistant tumors and increased survival, highlighting that under treatment conditions that mimic chemotherapy in human patients, Th-*MYCN* CPM32 mice develop genomic, microenvironmental, and clinical features reminiscent of human chemorefractory disease, with dysregulation of signaling pathways such as JAK-STAT3 that could be targeted to improve treatment of aggressive disease [81]. Our recent data show that p140Cap expression in NB cells is sufficient to down-modulate the tyrosine phosphorylation of Src Tyr 416 (p-Src), a marker of active Src, as well as of p130Cas, a well-known Src substrate [82] (**Figure 4A, B**).

undergo differentiation. Activated FYN was sequestered in PAG1™−

cells continued to proliferate and did not

cells, suggest-

**84**

PAG1™−

*p140Cap affects signaling pathways in NB cells. (A) Src activation was evaluated on mock and p140 ACN cells by WB analysis of Tyr 416 phosphorylation (Y416) and Src protein level as loading control. Antibodies to GAPDH (as loading control) were used. Quantification on the right is the ratio between phosphorylated Src and total Src protein; (B) p130Cas phosphorylation was evaluated with antibodies to phosphorylated p130Cas at Tyr 410, p130Cas and GAPDH antibodies for loading control. Quantification on the right is the ratio between phosphorylated p130Cas and total p130Cas; (C) STAT3 phosphorylation was evaluated with antibodies to phosphorylated STAT3 at Tyr705 and STAT3 antibodies for loading control. Quantification on the right is the ratio between phosphorylated STAT3 and total STAT3 protein from three independent experiment; (D) Jak2 activation was evaluated with antibodies to phosphorylated Jak2 at Tyr1007/1008 and Jak2 antibodies for loading control. Quantification on the right is the ratio between phosphorylated Jak2 and total Jak2 protein; in E-G p140Cap silenced cells were tested for p140Cap WB, and GAPDH as loading control (E), for Src activation at Tyr 416 phosphorylation (Y416) and Src protein level as loading control (F), and for phosphorylated STAT3 at Tyr 705 and STAT3 protein level as loading control (G). Quantification is shown on the left as the ratio between phosphorylated Src/STAT3 and total Src/STAT3 protein.*

Moreover, we showed that STAT3 Tyr 705 (pSTAT3) is less phosphorylated, and JAK2 kinase is less active in p140Cap cells (**Figure 4C, D**). Consistent with these data, silencing of the endogenous p140Cap in SH-SY-5Y cells RNA [22] caused increased Src activation of STAT3 phosphorylation, confirming that p140Cap can regulate these two signaling pathways (**Figure 4E**-**G**). Overall, p140Cap ability to influence the Src/p130Cas and the JAK2/STAT3 pathways could be causal for the impairment of NB progression observed in patients [70, 72, 78–80, 83]. p140Cap also impairs Src kinase activity in breast cancer cells upon integrin-mediated adhesion or growth factor treatment stimulation [23, 84]. Overall, p140Cap may negatively regulate Src activity at least two tumor types, as a key event in dampening their migratory and invasive phenotype.

Based on the pro-survival role of STAT3 in NB, we also performed anoikis assays, showing that p140Cap-expressing cells were characterized by a significant decrease in the level of pSTAT3. Only the forced expression of the constitutive active STAT3C mutant is able to decrease p140Cap sensitivity to anoikis-dependent death. Overall, our data indicate that in NB cells, p140Cap expression may affect cell death, by impairing the pro apoptotic signaling sustained by the JAK2/STAT3- Bcl2 survival pathway.

#### **7. p140Cap increases NB cell sensitivity to chemotherapeutic treatment**

Despite advances in the molecular exploration of pediatric cancers, approximately 50% of children with high-risk NB lack effective treatment [85]. NB treatments are designed on the basis of a risk classification, which takes into account a subset of prognostic factors associated with a patient's outcome. Clinical features (for instance, the tumor stage or patient's age at diagnosis) and biological tumor properties (such as histology, genetic alteration and molecular markers) can be used as prognostic factors [9, 19] to classify NB patients in low risk, intermediate risk (IR) or high risk (HR) groups [19].

Non-high-risk represent slightly more than half of newly diagnosed patients. Outcomes are generally excellent for these children, with variable treatment strategies including observation alone, surgical resection, or moderate doses of chemotherapy [86, 87]. On the other hand, high-risk NB are very difficult to treat and require multi-modal therapy. Intensification of therapy has vastly improved survival rates, and research is focused on novel treatments to further improve survival rates [88].

Children with an intermediate or high risk often receive chemotherapy, namely carboplatin, cyclophosphamide, doxorubicin, etoposide, busulfan, ifosfamide or vincristine [9]. However, the side effects of chemotherapy and the outcome depend on the individual and the dose used. In this context, we demonstrated that p140Cap correlates with an increased sensitivity to chemotherapy. Namely, we tested five chemotherapeutic drugs commonly used in NB patients (ciclophosphamide, carboplatin, doxorubicin, etoposide, and vincristine) in dose viability assays. NB cell lines overexpressing p140Cap showed significantly increased sensitivity to low doses (10 nM, 100 nM) of cyclophosphamide, vincristine, doxorubicin and etoposide (**Figure 5A**-**C**). Consistently, in SH-SY-5Y cells, p140Cap silencing resulted in increased viability to both doxorubicin and etoposide [43] (**Figure 5D**).

Both etoposide and doxorubicin prevent ligation of the DNA strands, stopping the process of replication. The number of foci/cells of phosphorylated histone H2AX (gamma H2AX), an established marker of DNA damage [89], was counted

**87**

*gamma-H2AX and vinculin.*

**Figure 5.**

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

*p140Cap regulates cell viability to chemotherapeutic drugs. (A) Dose dependence viability to chemotherapy drugs. Mock and p140 cells were treated with the four indicated doses and cell viability was quantified at 72 h of treatment; (B-C) time dependent viability. Mock and p140 cells were processed as in (a); (D) cell viability in SH-5YSY cells silenced for p140Cap. Cells were transfected with appropriate siRNA and after 24 h treated with 1 μM etoposide or doxorubicin. Cell viability was quantified at 48 h; (E) visualization of nuclear foci for gamma H2AX histone as a marker of DNA damage. Mock and p140 cells on glass slides were treated for 6 h with 1 μM etoposide and doxorubicin. Green: Gamma H2AX foci; blue: DAPI for nuclear staining. Scale bar: 10 μm; (F) quantification of gamma H2AX foci/cell. Mock: Red; p140: Green. 50 nuclei were evaluated for each experiment; (G) gamma H2AX levels upon chemotherapy treatment. Mock and p140 cells were acutely treated with 1* μ*M etoposide or doxorubicin for 6 h. extracts were analyzed by western blot with antibodies to gamma-H2AX, H2AX and vinculin for loading controls. Quantification on the right is the ratio between* 

*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 5.**

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

ing their migratory and invasive phenotype.

Bcl2 survival pathway.

(IR) or high risk (HR) groups [19].

survival rates [88].

Moreover, we showed that STAT3 Tyr 705 (pSTAT3) is less phosphorylated, and JAK2 kinase is less active in p140Cap cells (**Figure 4C, D**). Consistent with these data, silencing of the endogenous p140Cap in SH-SY-5Y cells RNA [22] caused increased Src activation of STAT3 phosphorylation, confirming that p140Cap can regulate these two signaling pathways (**Figure 4E**-**G**). Overall, p140Cap ability to influence the Src/p130Cas and the JAK2/STAT3 pathways could be causal for the impairment of NB progression observed in patients [70, 72, 78–80, 83]. p140Cap also impairs Src kinase activity in breast cancer cells upon integrin-mediated adhesion or growth factor treatment stimulation [23, 84]. Overall, p140Cap may negatively regulate Src activity at least two tumor types, as a key event in dampen-

Based on the pro-survival role of STAT3 in NB, we also performed anoikis assays, showing that p140Cap-expressing cells were characterized by a significant decrease in the level of pSTAT3. Only the forced expression of the constitutive active STAT3C mutant is able to decrease p140Cap sensitivity to anoikis-dependent death. Overall, our data indicate that in NB cells, p140Cap expression may affect cell death, by impairing the pro apoptotic signaling sustained by the JAK2/STAT3-

**7. p140Cap increases NB cell sensitivity to chemotherapeutic treatment**

Despite advances in the molecular exploration of pediatric cancers, approximately 50% of children with high-risk NB lack effective treatment [85]. NB treatments are designed on the basis of a risk classification, which takes into account a subset of prognostic factors associated with a patient's outcome. Clinical features (for instance, the tumor stage or patient's age at diagnosis) and biological tumor properties (such as histology, genetic alteration and molecular markers) can be used as prognostic factors [9, 19] to classify NB patients in low risk, intermediate risk

Non-high-risk represent slightly more than half of newly diagnosed patients.

Children with an intermediate or high risk often receive chemotherapy, namely carboplatin, cyclophosphamide, doxorubicin, etoposide, busulfan, ifosfamide or vincristine [9]. However, the side effects of chemotherapy and the outcome depend on the individual and the dose used. In this context, we demonstrated that p140Cap correlates with an increased sensitivity to chemotherapy. Namely, we tested five chemotherapeutic drugs commonly used in NB patients (ciclophosphamide, carboplatin, doxorubicin, etoposide, and vincristine) in dose viability assays. NB cell lines overexpressing p140Cap showed significantly increased sensitivity to low doses (10 nM, 100 nM) of cyclophosphamide, vincristine, doxorubicin and etoposide (**Figure 5A**-**C**). Consistently, in SH-SY-5Y cells, p140Cap silencing resulted in

Both etoposide and doxorubicin prevent ligation of the DNA strands, stopping the process of replication. The number of foci/cells of phosphorylated histone H2AX (gamma H2AX), an established marker of DNA damage [89], was counted

Outcomes are generally excellent for these children, with variable treatment strategies including observation alone, surgical resection, or moderate doses of chemotherapy [86, 87]. On the other hand, high-risk NB are very difficult to treat and require multi-modal therapy. Intensification of therapy has vastly improved survival rates, and research is focused on novel treatments to further improve

increased viability to both doxorubicin and etoposide [43] (**Figure 5D**).

**86**

*p140Cap regulates cell viability to chemotherapeutic drugs. (A) Dose dependence viability to chemotherapy drugs. Mock and p140 cells were treated with the four indicated doses and cell viability was quantified at 72 h of treatment; (B-C) time dependent viability. Mock and p140 cells were processed as in (a); (D) cell viability in SH-5YSY cells silenced for p140Cap. Cells were transfected with appropriate siRNA and after 24 h treated with 1 μM etoposide or doxorubicin. Cell viability was quantified at 48 h; (E) visualization of nuclear foci for gamma H2AX histone as a marker of DNA damage. Mock and p140 cells on glass slides were treated for 6 h with 1 μM etoposide and doxorubicin. Green: Gamma H2AX foci; blue: DAPI for nuclear staining. Scale bar: 10 μm; (F) quantification of gamma H2AX foci/cell. Mock: Red; p140: Green. 50 nuclei were evaluated for each experiment; (G) gamma H2AX levels upon chemotherapy treatment. Mock and p140 cells were acutely treated with 1* μ*M etoposide or doxorubicin for 6 h. extracts were analyzed by western blot with antibodies to gamma-H2AX, H2AX and vinculin for loading controls. Quantification on the right is the ratio between gamma-H2AX and vinculin.*

after an acute 6 h treatment with 1 μM etoposide and doxorubicin. p140Cap cells showed a significant increase in this marker over mock cells, indicating that the increased sensitivity of p140Cap cells to these drugs was associated with increased DNA lesions (**Figure 5E**-**G**). Overall, our study indicates that p140Cap NB cells display a significant decrease in cell viability upon drug treatment, with an increased sensitivity to drug-dependent DNA damage [43].
