**8. p140Cap increases NB cell sensitivity to Src kinase inhibitors**

As already said above, Src family kinases are proto-oncogene tyrosine-protein kinases which are involved in tumor progression in several cancer types and are considered as a target for a low toxic anti-tumor treatment. High Src levels are generally associated with a poor prognosis and play an important role in the differentiation, cell-adhesion and survival of NB cells. Indeed, the inhibition of such kinase is an effective approach for NB treatment and several Src- inhibitors have been developed, holding a promising antiproliferative effect, cell cycle arrest, apoptosis induction and decreased adhesion/invasiveness [69, 72, 73].

Since active Src was significantly down-regulated in p140Cap tumors over mock tumors and p140Cap overexpressing cells showed lower levels of active Src (**Figure 3F** and **4A**), we hypothesized that Src activity may be involved in NB cell viability [89]. In mock cells, Src activity was highly sensitive to two well-known Src inhibitors, saracatinib (which also inhibits the Abl kinase [90] at 100 nM), and sugen (used in preclinical NB models [91] at 1 μM). At 72 h, in mock cells both inhibitors decreased cell viability of 20–25%. Interestingly, the same treatment in p140Cap cells leads to a reduction in viability of nearly 40%. Moreover, viability to Src inhibitors was increased in cells silenced for p140Cap compared to p140Cap overexpressing cells. However, the partially silenced cells were still more sensitive than mock cells, indicating that there is a direct correlation between p140Cap expression and the augmented sensitivity to Src inhibitors.

We observed a decreased viability in mock cells upon treating them with Src inhibitors coupled with drugs that induce a DNA damage (in particular, doxorubicin or etoposide have been used at a concentration of 10 nM and 100 nM in association with saracatinib and sugen).

The decreased viability of mock cells (approximately at 50%) in these conditions indicates that may Src inhibitors concur in increasing chemotherapy cytotoxic effect in those cells which do not express p140Cap.

In addition, the use of both genotoxic drugs and Src inhibitors in the same treatment confers to p140Cap overexpressing cells a lower viability, in particular in cells treated with doxorubicin. Taken together, our data suggest that a combined treatment with Src inhibitors could increase NB cells sensitivity to etoposide and doxorubicin.

Upon a treatment with augmented doses of etoposide and doxorubicin (in a range of 1 nM-1 mM) used alone or in association with the same concentrations of Src inhibitors, we observed that the combined experimental setting was synergistic in both the cell lines (mock and p140Cap overexpressing cells).

Indeed, the Combination Index (CI) values computed for the different combinations of drugs were < 1 in all the experimental settings [92]. The p140Cap overexpressing cells still showed an increased sensitivity to the Src inhibitors in the combined treatment, with a shift of the sensitivity to lower doses (**Figure 6**). Therefore, our data show that chemotherapy and Src inhibitors combination synergistically decreases NB cell viability and this effect can be further increased by p140Cap expression [43].

**89**

**Figure 7.**

*good outcome in NB patients.*

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

This chapter highlights the original involvement of *SRCIN1*/p140Cap in NB, providing evidence that *SRCIN1* gene expression may be exploited as a marker of good outcomes in NB (**Figure 7**). *SRCIN1* mRNA levels are clinically relevant in NB

*Overview of* SRCIN1 *involvement in NB. The data reported here indicate a key causal role of* SRCIN1*/ p140Cap in dampening cell signaling, tumor growth, metastasis and drug sensitivity in NB cells, leading to a* 

*Synergistic analysis of combined treatments with chemotherapy drugs and Src inhibitors. The CI (combination index to reduce viable cells to 50%) and the DRI50 (the dose-reduction necessary to decrease viable cells to 50%, were calculated using the CalcuSyn software (www.biosoft.com/w/calcusyn.htm); r: Linear regression* 

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

**9. Conclusions**

**Figure 6.**

*coefficient.*

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


#### **Figure 6.**

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

sensitivity to drug-dependent DNA damage [43].

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

**8. p140Cap increases NB cell sensitivity to Src kinase inhibitors**

induction and decreased adhesion/invasiveness [69, 72, 73].

expression and the augmented sensitivity to Src inhibitors.

in both the cell lines (mock and p140Cap overexpressing cells).

effect in those cells which do not express p140Cap.

tion with saracatinib and sugen).

As already said above, Src family kinases are proto-oncogene tyrosine-protein kinases which are involved in tumor progression in several cancer types and are considered as a target for a low toxic anti-tumor treatment. High Src levels are generally associated with a poor prognosis and play an important role in the differentiation, cell-adhesion and survival of NB cells. Indeed, the inhibition of such kinase is an effective approach for NB treatment and several Src- inhibitors have been developed, holding a promising antiproliferative effect, cell cycle arrest, apoptosis

Since active Src was significantly down-regulated in p140Cap tumors over mock tumors and p140Cap overexpressing cells showed lower levels of active Src (**Figure 3F** and **4A**), we hypothesized that Src activity may be involved in NB cell viability [89]. In mock cells, Src activity was highly sensitive to two well-known Src inhibitors, saracatinib (which also inhibits the Abl kinase [90] at 100 nM), and sugen (used in preclinical NB models [91] at 1 μM). At 72 h, in mock cells both inhibitors decreased cell viability of 20–25%. Interestingly, the same treatment in p140Cap cells leads to a reduction in viability of nearly 40%. Moreover, viability to Src inhibitors was increased in cells silenced for p140Cap compared to p140Cap overexpressing cells. However, the partially silenced cells were still more sensitive than mock cells, indicating that there is a direct correlation between p140Cap

We observed a decreased viability in mock cells upon treating them with Src inhibitors coupled with drugs that induce a DNA damage (in particular, doxorubicin or etoposide have been used at a concentration of 10 nM and 100 nM in associa-

The decreased viability of mock cells (approximately at 50%) in these conditions indicates that may Src inhibitors concur in increasing chemotherapy cytotoxic

In addition, the use of both genotoxic drugs and Src inhibitors in the same treatment confers to p140Cap overexpressing cells a lower viability, in particular in cells treated with doxorubicin. Taken together, our data suggest that a combined treatment with Src inhibitors could increase NB cells sensitivity to etoposide and

Upon a treatment with augmented doses of etoposide and doxorubicin (in a range of 1 nM-1 mM) used alone or in association with the same concentrations of Src inhibitors, we observed that the combined experimental setting was synergistic

Indeed, the Combination Index (CI) values computed for the different combinations of drugs were < 1 in all the experimental settings [92]. The p140Cap overexpressing cells still showed an increased sensitivity to the Src inhibitors in the combined treatment, with a shift of the sensitivity to lower doses (**Figure 6**). Therefore, our data show that chemotherapy and Src inhibitors combination

synergistically decreases NB cell viability and this effect can be further increased by

**88**

doxorubicin.

p140Cap expression [43].

*Synergistic analysis of combined treatments with chemotherapy drugs and Src inhibitors. The CI (combination index to reduce viable cells to 50%) and the DRI50 (the dose-reduction necessary to decrease viable cells to 50%, were calculated using the CalcuSyn software (www.biosoft.com/w/calcusyn.htm); r: Linear regression coefficient.*
