SRCIN1 loss/cn-LOH or disruption in the breakpoint on 17 NB patients.
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.
- SRCIN1 gene
- Src kinase
- Signal transducer and activator of transcription 3
- Src inhibitors
Neuroblastoma (NB) is the most frequent embryonic malignancy among children particularly before 5 years of age . It originates from primitive sympathetic neural precursor cells of the peripheral nervous system . 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 .
NB is a complex disease with different outcomes, going from metastasis to one or more distant sites  to spontaneous regression or differentiation, even in the absence of any specific treatment . 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 . 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 .
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)  and DNA ploidy . At the molecular level, the Anaplastic Lymphoma Kinase (
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, 18, 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) , is a scaffold protein codified by the gene
More recently we have investigated p140Cap/
2. The p140Cap adaptor protein
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  (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 . Indeed, at the structural level, p140Cap contains a tyrosine rich domain, important for the interaction with Csk , two coiled coil regions, that can mediate the binding with beta-catenin  and two different proline rich domains responsible for the association with the microtubule associated protein EB3 , Cortactin  and Vimentin .
The physiologic role of p140Cap has been mainly investigated in the brain , where it is expressed in neurons both in the presynapse [10, 36] and in the postsynapse [10, 37, 38, 39, 40, 41]. In differentiated neurons, it controls synaptic plasticity [33, 40], and regulates GABAergic synaptogenesis and development of hippocampal inhibitory circuits . In particular, p140Cap enters and accumulates in the dendritic spine (DS) through EB3 binding . In this compartment p140Cap acts as hub interacting with Cortactin, a protein that regulates actin branching and new filament polymerization  and with Citron-N  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 .
SRCIN1mRNA expression is an independent prognostic marker for NB
To address the involvement of p140Cap in NB patients, we first investigated the relationship between
To date, p140Cap expression by immunohistochemistry (IHC) on NB samples has not been studied owing to the lack of available cancer tissues, but
|NB patients||CHROMOSOMAL COORDINATES|
|Case 1||disrupted in the breakpoint||Chr17: 36696338–81029941|
Size: 44.33 Mb
|Case 2||disrupted in the breakpoint||Chr17: 36694901–80943345|
Size: 44.24 Mb
|Case 3||loss||Chr17: 25311574–36777884|
Size: 11.46 Mb
|Case 4||disrupted in the breakpoint||Chr17: 36696338–80969424|
Size: 44.27 Mb
|Case 5||disrupted in the breakpoint||Chr17: 36696279–81029941|
Size: 44.33 Mb
|Case 6||copy neutral LOH||Chr17: 25569094–42949451|
Size: 17.38 Mb
|Case 7||copy neutral LOH||Chr17: 29149425–45297941|
Size: 16.14 Mb
|Case 8||copy neutral LOH||Chr17: 31571877–40588363|
Size: 9.01 Mb
|Case 9||loss||Chr17: 25278114–37876263|
Size: 12.59 Mb
|Case 10||loss||Chr17: 25278114–68301170|
Size: 43.02 Mb
|Case 11||disrupted in the breakpoint||Chr17: 36696279–81029941|
Size: 44.33 Mb
|Case 12||disrupted in the breakpoint||Chr17: 36696338–81029941|
Size: 44.33 Mb
|Case 13||disrupted in the breakpoint||Chr17: 36740844–80943189|
Size: 44.20 Mb
|Case 14||disrupted in the breakpoint||Chr17: 36740903–80993001|
Size: 44.25 Mb
|Case 15||loss||Chr17: 25278114–81029941|
Size: 55.75 Mb
|Case 16||disrupted in the breakpoint||Chr17: 36672992–77470237|
Size: 40.79 Mb
|Case 17||disrupted in the breakpoint||Chr17: 36694044–81099040|
Size: 44.40 Mb
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 . 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  (Figure 3B). According to the protein level analysis, the genomic profiling revealed a wide spectrum of
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 . Consistently, p140Cap-overexpressing ACN (p140Cap-ACN) cells exhibited decreased migration properties in a Wound Healing assay, and impaired anchorage-independent 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 . A specific type of apoptotic process, called anoikis, occurs in cells in response to loss of adhesion to the extracellular matrix. Upon anoikis, p140Cap-ACN cells showed both a lower upregulation of the anti-apoptotic 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 NB, angiogenesis has a prominent role in determining tumor phenotype. A study published by Meitar D
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
Further evidence supporting the biological relevance of p140Cap in curbing NB aggressiveness was provided by the recent work of Yuan XL
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 . 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 outcome as well as from the discovery of molecular aberrations in the tumors (for a recent review see ). Of the genetic aberrations described in NB only
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
Interestingly, high-risk NBs without
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 cells from apoptosis, and dual inhibition of these kinases may be important when designing therapeutic interventions for this tumor . 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
IL-6-dependent activation of STAT3  has already been reported in NB, where STAT3 is critical in mediating increased survival and drug resistance [78, 79, 80]. Interestingly, very recently, the antiapoptotic and prometastatic JAK-STAT3 pathway was activated in chemoresistant tumors, generated in the Th-
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  (Figure 4A,B). 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  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, 79, 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 . 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 .
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 .
Children with an intermediate or high risk often receive chemotherapy, namely carboplatin, cyclophosphamide, doxorubicin, etoposide, busulfan, ifosfamide or vincristine . 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  (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 , was counted 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 .
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 . In mock cells, Src activity was highly sensitive to two well-known Src inhibitors, saracatinib (which also inhibits the Abl kinase  at 100 nM), and sugen (used in preclinical NB models  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 . 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 .
This chapter highlights the original involvement of
Overall, the protein p140Cap acts as a tumor suppressor gene in NB tumors, dampening tumor volume and decreasing progression towards distant metastasis. This might occur because of an increased ability to undergo apoptosis and a decreased capability of p140Cap NB cells to proliferate
An urgent need in NB is to increase the five-year OS rate of high-risk NB patients, which is still less than 40% . Despite emerging new therapies, the impact of treatments is very heavy for affected children, which can have serious consequences for years to come . The data showing that p140Cap expressing NB have significantly increased sensitivity to low doses (10 nM concentration) of doxorubicin and etoposide, two drugs used in first line NB treatment, open new perspectives. Further, the fact that a combo treatment with Src inhibitors and low doses doxorubicin or etoposide, sensitize mock cells, reducing cell viability to that of p140Cap cells treated with chemotherapy alone, is an encouraging result. Therefore, it would be interesting to set combinatorial approaches with low doses of both chemotherapy drugs and specific inhibitors, including also the Jak2 pathway, to quantify the additional/synergistic effects. Further, to increase the understanding of the mechanism of action of p140Cap on the sensitization to specific drugs, it would be very useful to identify the vital molecular signaling mechanisms involved. To achieve these results, both automated platforms for cell viability and genome-wide CRISPR-CAS9 technology are largely available. In conclusion, we believe that these data demonstrate the potential clinical impact of SRCIN1/p140Cap expression and of p140Cap-regulated pathways in NB tumors. These results pave the way to include
The figures and Tables have been adapted from ref.  Grasso et al., 2020. We give here appropriate acknowledgment to the original authors of this publication (Silvia Grasso, Melissa Alzona, Alessia Lamolinara, Andrea Saglietto, Federico Tommaso Bianchi, Sara Cabodi, Iris Chiara Salaroglio, Federica Fusella, Marzia Ognibene, Manuela Iezzi, Annalisa Pezzolo, Valeria Poli, Ferdinando Di Cunto, Alessandra Eva, Chiara Riganti, Luigi Varesio). This work was supported by AIRC (Associazione Italiana Ricerca Cancro) to PD (IG- 20107), Compagnia San Paolo, Torino, Progetto DEFLECT to PD; Fondazione CRT 2020.1798 to PD.
Conflict of interest
There is no competing of interest to declare.
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|Abl||Abelson Tyrosine-Protein Kinase|
|a-CGH||array - Comparative Genomic Hybridization|
|AKT||AKT serine/threonine kinase 1|
|ALK||Anaplastic Lymphoma Kinase|
|AML||Acute myeloid leukemia|
|Bcl-2||B-cell lymphoma 2|
|Bcl-2||B-cell lymphoma gene 2|
|BDNF||Brain-Derived Neurotrophic Factor|
|BRCA1||BRCA1 DNA repair associated|
|CD31||platelet and endothelial cell adhesion molecule 1|
|CDK4/6||cyclin D-cyclin-dependent kinase 4/6|
|cn-LOH||copy-neutral Loss Of Heterozygosity|
|Csk||C-terminal Src kinase|
|EB3||microtubule associated protein RP/EB family member 3|
|EFS||event free survival|
|ERBB2||erb-b2 receptor tyrosine kinase 2|
|ERK||extracellular signal-regulated kinase|
|ERK5||Extracellular signal-regulated kinase 5|
|FAK||focal adhesion kinase|
|FYN||FYN oncogene related to SRC, FGR, YES|
|H2AX||H2A histone family member X|
|IC50||the half maximal inhibitory concentration|
|IDP||Intrinsic Disorder Protein|
|IDR||Intrinsically Disordered Regions|
|INSS||International Neuroblastoma Staging System|
|Ki67||marker of proliferation Ki-67|
|LIN28B||lin-28 homolog B|
|MAPK||mitogen-activated protein kinase|
|MAX||myc-associated factor X|
|NG2||Neural/glial antigen 2|
|NSG||NOD Scid Gamma mice|
|PAG1TM-||PAG1 fragment that lacks the membrane spanning domain|
|PDX||patient derived xenograft|
|PHOX2B||Paired-like Homeobox 2b|
|Prkdc||protein kinase, DNA-activated, catalytic subunit|
|Rag-1 and 2||Recombinant activating 1,2|
|RARA||retinoic acid receptor-α|
|Ras||Ras-related C3 botulinum toxin substrate|
|RTK||tyrosine kinase receptor|
|SFK||Src family kinase|
|SNIP||SNAP25 Interacting Protein|
|SNP-array||Single Nucleotide Polymorphism – array|
|Src||proto-oncogene tyrosine-protein kinase Src|
|SRCIN1||SRC kinase signaling inhibitor 1|
|STAT3||Signal transducer and activator of transcription 3|
|TrkB||Tropomyosin receptor kinase B|
|Wnt||Wingless-related integration site|