The Cellular Form Of The Src Gene Biology Essay

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Src has been one of the oldest and most studied proto-oncogenes in scientific literature. The cellular Src holds a critical role in several human malignancies and has emerged as a key factor that promotes tumor progression during the multistep process of colorectal cancer (CRC) pathogenesis. The robust activation of Src in CRC tumors of aggressive phenotype and poor prognosis seems to be a subsequent event of a strong link between its deregulated activity and the tumor's cell adhesion properties, invasiveness and metastatic potential. The rarely detected genetic defects drive interest in signaling networks that control Src kinase activity and integrate the association of Src with receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR). Therefore, a dynamic crosstalk is being formed with oncogenic capacity and therapeutic applications, since Src inhibition seems to sensitize previously unresponsive cancer cells to chemotherapy and anti-EGFR inhibitors. The present review explores the molecular basis behind Src inhibition in colorectal carcinomas. Furthermore, preclinical studies and clinical trials of Src inhibitors and combination regimens are discussed, providing new insights for further investigation and new therapeutic strategies.

Introduction

The cellular form of the src gene (c-src), which was the first proto-oncogene that was discovered in the vertebrate genome, remains the cornerstone of understanding how signaling networks contribute to cancer biology. The skepticism about Rous' findings ninety years ago, regarding filterable agents that induce solid tumors, turned into a major breakthrough several decades later culminating at the 1970's with the identification of the viral src gene (v-src) and its cellular counterpart c-src. Although the investigation of c-src has been a diachronic research field linked with the development and progression of cancer, only recently there has been a renewed interest in the encoded c-src protein as a molecular target for several types of human malignancies [1].

The c-src is a non-receptor protein tyrosine kinase with a 60-kD molecular weight. It is a member of a 9-gene family named the Src family kinases (SFKs), including Src, Blk, Fgr, Fyn, Hcy, Lck, Lyn, Yes and Yrk. Src, Fyn and Yes are ubiquitously expressed, whereas the other six proteins have been primarily detected in hematopoietic cells [2]. Aberrant c-src activation has been described in many types of cancer, among others in colorectal cancer (CRC), in breast, pancreatic, gastric, lung and prostate tumors. Exploring CRC tumorigenesis, c-src has been recognized as a dominant molecule at the crossroad of several signal transduction pathways, mostly those driven by receptor tyrosine kinases (RTKs) and especially epidermal growth factor receptor (EGFR)-induced signaling. Therefore, SFK inhibitors have been developed recently entering clinical trials either in single-agent therapies or, more prominently, in combination regimens [3].

The present review explores the molecular rationale behind Src inhibition in CRC pathobiology, focusing in Src and RTKs/EGFR emerging interplay. Additionally, the efforts regarding the application of the respective signaling pathways crosstalk into the clinic are being discussed, providing new insights for further investigation in favor of pharmaceutical treatment.

Src structure and function

The c-src protein is composed of a unique amino (N)-terminal domain, four Src homology (SH) domains and a negative regulatory carboxyl (C)-terminal segment. The N-terminal domain is myristoylated in order for the protein to be associated with the cell membrane and the following SH3 modular domain has high affinity for structures that are rich in proline forming the polyproline type II (PPII) helix. The SH2 modular domain consists of two recognition pockets, the first one for phosphotyrosine residues and the second one for isoleucine residues. The SH1 kinase domain is a bilobal protein kinase fold that owns the autophosphorylation site at Tyr419, whereas the C-terminal tail owns the negative-regulatory tyrosine residue at Tyr530, a segment missing from the constitutively active v-src [4] (Figure 1).

The c-src protein can be found both in an active and in an inactive state. Crystallographic studies have shown that the SH2 domain binds the C-terminal segment when phosphorylated at Tyr530, whereas the SH3 domain forms interactions with the linker between the SH2 domain and the SH1 kinase domain. These two conformational alterations set c-src protein in a closed configuration which "hides" the SH1 kinase domain and reduces the potential for substrate interactions [1]. Mutations, C-terminal tail dephosphorylation and displacement of the inhibitory protein interactions disrupt either SH2-tail or SH3-linker associations and are sufficient for c-src activation. However, phosphorylation of the SH1 kinase domain at Tyr419 emerges as the dominant effect for c-src full activation [5]. The structure domains described, which are prominent for protein-protein interactions, along with c-src downstream kinase activity highlight c-src as an important molecule in cell proliferation, migration, differentiation, survival, adhesion, morphology and motility processes. Its function is regulated by RTKs, integrin receptors, antigen and G-protein coupled receptors (GPCRs), cytokine receptors and other factors, thus forming cellular crosstalks and complex regulatory networks [6].

Src in CRC

Src phenotype in CRC

In CRC cell lines and primary tumors both c-src kinase activity and protein expression levels have been found elevated in various studies compared either to normal colonic mucosal cells and fibroblasts or normal adjacent tissue, respectively [7-9]. In particular, it is suggested that c-src participates both in early stages of carcinogenesis and in tumor invasion and metastatic processes. Increased specific activity and expression levels have been detected in colonic polyps of high malignant potential and severe dysplasia [9, 10]. C-src, though, seems to have a robust increase regarding expression and kinase activity in liver metastases originating from CRC compared to those derived from other tumors types [8, 9, 11]. In corroboration, high activity emerges also in extrahepatic CRC metastases with site-specific activity differences [11]. As anticipated, c-src high activity was proved to be correlated with poor overall survival in all stages and reduced disease-free survival, emerging as an independent aggravating prognostic factor [12].

Animal models bring about ambiguities regarding scientific conclusions. In nude mice that were injected with KM12C and SW480 cells, stably transfected in order to overexpress c-src, only primary tumor growth was increased, whereas metastatic potential in vivo and cell proliferation in vitro remained unaffected [13]. In addition, immortalized rat colon epithelial cells overexpressing c-src were detected to be poorly transforming but conferred invasion capacity and anchorage-independent growth [14]. Equivocal conclusions render since in HCT116 and SW480 cells that inducibly express c-src, the elevated kinase activity and expression levels promote cell proliferation neither in vitro nor in vivo. Moreover, inducible overexpression of the mutant c-src Y527F seems to inhibit even tumor growth itself [15]. Evaluating src transformation capacity, a "Src transformation fingerprint" has been identified in rat 3Y1 fibroblasts. In comparison between rat cDNA array and orthologous genes extracted from the normal colon and 50 staged tumors, the upregulation of 73 genes in both datasets was revealed with 40% of the genes having correlated expression patterns which encoded for transcription factors (TFs), heat shock proteins, proteins of cell growth, proliferation and other important cellular components [16].

Genetic and epigenetic regulation

A truncating mutation of c-src has been identified at codon 531 in 12% of advanced CRC cases. The mutation was associated with elevated kinase activity and proved to be tumorigenic, to promote metastases and to be adequately transforming as it was shown after the transfection of 531 mutants into rat 3Y1 and NIH3T3 fibroblasts [17]. However, various studies that followed in colo-rectal advanced cancers identified no mutations at the same codon among different populations, indicating that such alteration may be rare, ethnic-specific and certainly not one of the primary tumorigenic mechanisms in CRC [18-21]. This notion was enhanced by a study that patterned protein kinase mutations among 210 human cancers, including CRC tumors and cell lines. Mutations were detected in SFK members Hck, Lyn and Fyn, but c-src was not included among the genes that were probable of carrying at least one driver mutation [22].

In conjunction with potential genetic regulation of Src, an epigenetic interplay has emerged as an additional factor of CRC tumorigenesis and impairment of c-src function [23]. Downregulation and reduction of c-src transcripts has been found after treatment of CRC cell lines with histone deacetylase (HDAC) inhibitors, a finding that reverses the widespread notion of HDACs functioning only as transcriptional repressors [24, 25]. HDACs have been suggested also to downregulate Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (Cbp/PAG1), a transmembrane adaptor protein that promotes c-terminal src kinase (Csk)-dependent src inactivation, via the mitogen-activated protein kinase/phosphoinisitide 3-kinase (MAPK/PI3K) pathway. Cbp/PAG1 expression was restored after silencing HDACs in HT-29 cells [26]. Micro-RNAs (miR) also entered the game mediating a positive feedback loop of c-src activation. MiR-542-3p represents a potential mediator of c-src oncogenic signaling since c-src upregulation is correlated with miR-542-3p downregulation. An upregulation of integrin-linked kinase (ILK) follows, which promotes further c-src and focal adhesion kinase (FAK) activation along with tumorigenic and invasive potential [27]. Finally, classic epigenetic silencing through DNA hypermethylation has been detected on reversion-induced LIM (RIL), a c-src inhibitor that functions via protein tyrosine phosphatase L1 (PTPL1)-dependent phosphorylation [28].

Regulation of kinase activity

C-src kinase activity is balanced by the orchestrated function of protein kinases and phosphatases targeting mainly Tyr530 at the C-terminal tail. Csk operates as a c-src negative regulator that phosphorylates c-src at the C-terminal segment [29] (Figure 1). Deregulation of this specific interaction constitutes a potential mechanism of c-src activation in CRC pathogenesis. Csk downregulated levels and kinase activity have been found to be strongly correlated inversely with upregulated c-src levels and kinase activity [30]. In vivo models further enhance Csk suppressing role. Csk overexrpession in highly metastatic mouse NL-17 CRC cells reduces src kinase activity in vitro, suppresses lung metastases and tumor invasiveness in vivo [31]. In an azoxymethane-rat model of CRC, Csk downregulation in preneoplastic colonic mucosa has been proved to provoke increased proliferation of CRC cells, implicating Csk deregulation in early CRC oncogenesis [32]. In addition, human CRC tumors and cell lines were detected with the presence of Csk autoantibodies in patients with early-stage tumors and precancerous lesions, although in the same study Csk was overexpressed and had no correlation with src kinase activity, implying that additional mechanisms contribute to c-src aberrant function in CRC [33]. These increased Csk expression levels described have been also detected in further studies strongly suggesting the alternative mechanisms that take place in Csk-dependent c-src regulation. It seems that Csk membrane delocalization is a key-event that allows SFKs dependent invasiveness in CRC cells and is also associated with Cbp/PAG downregulation [34].

On the other hand, several cytoplasmic and transmembrane protein tyrosine phosphatases (PTPs) have been found to dephosphorylate c-src at Tyr530 regulating its kinase activity [35]. The downregulation of the transmembrane PTPα has been proven to inhibit src kinase activity and to induce apoptosis in CRC cells [36]. Moreover, splice mutants of receptor-like PTPα (RPTPα) have been detected in 30% of colon, breast and liver tumors. In particular, expression of the transforming RPTPα245 mutant in tumors facilitates the elevation of src dephosphorylation/activation via RPTPα/endogenous RPTPα binding [37]. RPTPα was also found to regulate the recognition of extracellular physical stimuli in SW480 cells. RPTPα co-localizes with paxillin and β1-integrins inducing the formation of novel adhesion sites and regulating invasive and adhesive properties in vivo [38]. In addition, PTP1β increased expression provokes higher levels of src kinase activity by reducing Tyr530 phosphorylation. The subsequent inhibition of PTP1β suppresses src activity and colony formation as well as tumor growth in immunodeficient mice [39]. Finally, re-expression of the lost RIL/LIM and recognition of the active src in CRC cells facilitates PTPL1-mediated inactivation of src, along with RIL's dissociation and the initiation of a new src inactivation cycle [28].

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