Background
When activated, cell surface growth factor receptor tyrosine kinases (RTK) become phosphorylated at a number of tyrosine (Tyr) residues. Many of those located within the cytoplasmic tail of the receptor create binding sites for proteins containing Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains, which recognize phospho-Tyr residues within the context of specific adjacent amino acids. Proteins recruited to RTKs include enzymes, such as phospholipase Cγ (PLCγ) and phosphatidylinositol 3-kinase (PI3K); and adaptor proteins, including growth factor receptor-bound protein-2 (Grb2) and Src-homology collagen (Shc) proteins. These latter proteins contain multiple protein-protein interactions motifs. The resulting complex relays and amplifies an exquisitely fine-tuned regulation of multiple downstream signaling events, which depending on cellular context, mediate specific biological responses (reviewed in [
1]). This regulation is perturbed in cancers, including those of intestinal epithelial cell (IEC) origin such as colorectal cancer (CRC).
Deregulation of RTKs in CRC commonly involves the over-expression of the receptor and/or its ligand. As illustrated by the Met/hepatocyte growth factor (HGF) and epidermal growth factor receptor (EGFR) signaling axis, this dysregulation often takes place at the earliest stages of the disease and it is observed in virtually all metastatic CRC (reviewed in [
2‐
4]). Ligand or receptor deregulation may result in a lower threshold for growth factor stimulation, autocrine/paracrine ligand-receptor activation loops, and even ligand-independent constitutive receptor activation. Regardless of the mechanisms of RTK oncogenic activation, the outcome is the loss of the normally fine-tuned regulation of downstream signaling, which may ultimately contribute to the acquisition of cancer properties. Notably, its has been shown that the expression of constitutively activated forms of the Met receptor in non-transformed IECs promoted morphological transformation; enhanced proliferation rate; induced loss of both growth contact inhibition and anchorage-dependent growth; and increased
in vivo angiogenic, tumorigenic, and metastatic capacities [
5,
6].
Studies performed predominantly in fibroblast and breast cancer cell models have revealed that Grb2 and Shc adaptor proteins are among the signaling proteins that, upon recruitment by activated RTKs, mediate events directly linked to the initiation and progression of cancer [
7‐
12]. Many RTKs interact directly with Grb2, some rely on Shc family adaptors to recruit Grb2, and others do both [
1]. While direct Grb2/RTK interactions involve binding of the Grb2 SH2 domain to pYXNX motifs, Shc proteins interact with RTKs primarily through the binding of their N-terminal PTB domain to NPXpY motifs. The latter results in phosphorylation of Tyr residues within the Shc central collagen-homology domain 1 (CH1). These phosphorylated tyrosine residues constitute consensus-binding sites for the Grb2 SH2 domain, thus allowing Shc to engage Grb2-driven signaling pathways (reviewed in [
13]). The best-characterized role of the two adaptor proteins, Grb2 and Shc, is to link RTKs to the activation of the Ras/Raf/MEK/Erk mitogenic (Ras/MAPK) pathway. The constitutive association of the N-terminal Grb2 SH3 domain with the Ras guanine nucleotide exchange factor, Son of Sevenless (SOS) constitutes one component of this connection [
1]. Interaction of the C-terminal Grb2 SH3 domain with Grb2-associated binding (Gab) scaffold protein family members couples RTKs to the PI3K/Akt survival pathway and to the Ras/MAPK cascade by an alternate route [
14]. As such, the recruitment of Grb2 or Shc to RTKs has been shown to promote biologically redundant processes [
7,
8,
15,
16]. However, Shc proteins interact with diverse signaling molecules in addition to Grb2, thereby engage Grb2-independent pathways and biological functions [
9‐
13,
17‐
19].
Although the deregulation of RTKs is widely considered to be a major determinant in the progression of CRC, the specific contributions of the proximal signaling molecules engaged by these receptors in CRC remain virtually unexplored. Herein, we report the exploitation of well-characterized adaptor-specific RTK docking variants derived from the oncogenic Met receptor, Tpr-Met [
8,
9,
15,
16,
20], with shRNA and pharmacological interference approaches to define, for the first time, the cancer properties associated with early neoplastic transformation of IECs, induced upon oncogenic mediated activation of either Grb2 or Shc signaling.
Discussion
As normal intestinal epithelial cells become cancerous, they gain the ability to grow aberrantly by evading normal growth-inhibiting and death signals, as well as the ability to invade tissue [
38]. Experimental and clinical studies suggest that the deregulation of RTKs plays a critical role in the etiology and progression of human CRC [
2‐
4]. These studies highlight the ability of RTKs to induce biological characteristics linked with tumorigenesis and metastatic progression [
1,
38]. However, the proximal signaling molecules recruited by RTKs have not yet been assigned individual contributions to the neoplastic transformation of normal IECs. In this study, Met-derived docking-specific variants were used to define the cancer properties induced upon the RTK-mediated engagement of the Grb2 or Shc adaptor proteins in IECs. Our results show, for the first time in a non-transformed IEC model, that the sustained activation of signals downstream of either Grb2 or Shc alone is sufficient to promote morphological transformation, E-cadherin down-regulation, enhanced cell growth, loss of contact inhibition of growth, the acquisition of anchorage-independence of growth, and anoikis resistance (Figures
1,
2,
3). These oncogenic features are prerequisites for the progression of epithelial-derived cancers, favoring the survival and growth of cancerous cells in the matrix-poor, disorganized extracellular environments often found in primary tumors, and in systemic circulation, facilitating metastasis [
32,
38]. Thus, our results provide novel evidence for a causal role of RTK-linked Grb2 and Shc signaling pathways in important and common phenotypic features of neoplastic transformation of IECs and metastatic CRCs.
Expression of the cell adhesion molecule, E-cadherin, is typically depleted from cell-cell contacts in epithelial cancer cells, or even shut down altogether [
31]. Cellular loss of E-cadherin leads to dissolution of adherens junctions and to a reduction in cell-cell contacts, facilitating migration and invasion, both of which are key processes for metastatic dissemination of epithelial tumor cells. Notably, an inverse correlation exists between E-cadherin levels in human CRC specimens and cancer grade, invasiveness of tumor phenotype, metastatic disease progression, and poor patient prognosis [
39]. Multiple mechanisms have been identified that promote E-cadherin down-regulation in epithelial cells, in response to different stimuli and/or in different cell types. These include transcriptional silencing via deregulation of transcription factors (Snail, Twist, and Zeb) or promoter hyper-methylation, and internalization followed by subsequent lysosomal degradation mediated by post-translational modifications [
31,
40]. E-cadherin protein levels were further reduced in IEC-6 cells expressing the Shc docking-specific oncoproteins than those transformed by TM-Grb2 or Tpr-Met (Figure
1). Differential expression levels of these oncoproteins cannot fully account for this response, therefore suggesting that Grb2 and Shc might mediate E-cadherin down-regulation by distinct mechanisms. However, E-cadherin levels and an epithelial non-transformed typical morphology were restored upon pharmacological inhibition of MEK1/2 but not of PI3K, activities in IECs transformed by these oncoproteins (Figure
5 and Additional file
3). These findings suggest that the engagement of Grb2 and Shc, like Met, promotes these oncogenic features through shared signaling pathways. Furthermore, E-cadherin repression induced by oncogenic Met signaling pathways in IECs, like that driven by Grb2 and Shc, was associated with an up-regulation of the repressors of E-cadherin transcription
Snail2,
Twist1, or
Twist2, but not of
Snail1 or
Zeb1 (Additional file
2). Future studies will be needed to define the shared and distinct mechanisms that result in E-cadherin down-regulation in IECs downstream of Grb2 or Shc. Nonetheless, our results suggest that these adaptor proteins are important integrators of signals leading to the neoplastic transformation and E-cadherin dysfunction in CRC harboring the deregulated Met receptor, and most likely other RTKs.
A role for deregulated RTKs in conferring anoikis resistance to IECs through cross-talk with cell adhesion receptors, is well-established [
32]. While transforming growth factor-α (TGF-α) stimulation protects IECs from anoikis [
41], CRC cells that express high levels of EGF/TGF-α evade anoikis through autocrine stimulation of the EGFR [
33]. However, the importance of Grb2 or Shc functions in anoikis resistance in IECs has not been addressed. Furthermore, of the very few investigations of the HGF/Met receptor axis in anoikis control and cancer, a single study has reported a key role for the Met receptor in CRC cells [
42]. Activation of the Met receptor has been reported to prevent anoikis in human colon, ovarian, pancreatic, and head and neck carcinoma cell lines by mechanisms dependent on PI3K, but with varied requirement for MEK1/2 [
42‐
45]. Herein, we have shown that while individual inhibition of PI3K activity, but not of MEK1/2, partially restored anoikis sensitivity in Tpr-Met-IEC-6 cells, concurrent inhibition of these pathways exerted a synergistic effect (Figure
6). Notably, anoikis resistance driven by the oncogenic Met receptor in IECs is partly dependent on Grb2 functions, whereas Shc functions appear dispensable (Figure
4). Our findings suggest a model whereby deregulation of Met might promote anoikis resistance in CRC cells, through the integration of both MEK and PI3K signaling pathways, and likely involving the engagement of Grb2. Collectively, our results provide novel evidence that signaling pathways engaged by deregulated RTKs in CRC, including those reliant on Grb2 or Shc, may represent important regulators of anoikis resistance in IECs, a process of outmost relevance in cancer metastasis.
We show that oncogenic Met receptor-dependent signals, like those activated downstream of Grb2 and Shc, trigger negative feedback upon the Ras/MAPK and PI3K/Akt pathways in IECs, restricting Erk and Akt activation (Figure
5 and Additional file
3). Although somewhat controversial, some studies suggest that Erk hyperactivation may only occur in a small subset of CRC tumors, and that Erk activity is more often elevated in adjacent normal tissue [
46‐
48]. Also, Erk activity in human CRC tumors appears to be a poor predictor of activating
K-RAS mutation status and of the effectiveness of MEK inhibition [
48‐
50]. We report the inhibition of the IEC transformation and E-cadherin down-regulation induced by each of our oncoproteins by inhibitors of MEK activity, but not of PI3K activity (Figure
5 and Additional file
3). Cell growth and anoikis resistance evoked by Tpr-Met, on the other hand, was blocked by concomitant treatment with MEK and PI3K inhibitors (Figure
6). Thus, our findings suggest that while growth factor stimulation is linked to the activation of the Ras/MAPK and PI3K/Akt pathways, in part through Grb2 and Shc, Erk or Akt activity levels in CRC may not reliably predict the extent of RTK deregulation, nor the sensitivity to therapies targeting them.
The Tpr-Met and the Grb2- and Shc-specific docking oncoproteins are all predicted to promote cancer features in IECs by engaging similar signaling pathways. Indeed, they share the ability to complex with the Gab1 scaffolding protein. While binding to TM-Grb2 and TM-Shc oncoproteins by Grb2-dependent mechanisms, Gab1 also interacts directly with the Met receptor [
8,
20,
51]. Notably, Gab1 has been shown to be required for Erk and Akt activation, and many oncogenic functions downstream of Met, and the Grb2- and Shc-docking oncoproteins in fibroblast, MDCK epithelial, and
Xenopus cell models [
20,
28]. Thus, it may be that Gab1 provides a platform for the integration of Ras/MAPK and PI3K/Akt positive and negative signals downstream of these oncoproteins and relevant to their oncogenic functions in IECs. However, Tpr-Met-IEC-6 cells were observed to display stronger transformed phenotypes than cells expressing the Grb2 or Shc-binding variants oncoproteins, for example in focus-formation and growth in soft agar (Figures
2 and
3). This suggests that Tpr-Met may activate pathways not engaged by the Tpr-Met Shc or Tpr-Met Grb2 oncoproteins. Furthermore, it is now acknowledged that Shc, by interacting with proteins other than Grb2 such as IQGAP1, Crk and Sgk269, can promote Grb2-independent pathways and functions [
13,
17‐
19], underscoring the complexity of the cellular networks that these adaptor proteins can engage downstream of RTKs. It is therefore anticipated that the Grb2- and Shc-specific docking oncoproteins, and Tpr-Met may prove, upon further analyses, to mediate distinct signaling pathways, and therefore specific cancer processes in IECs.
Therapies targeting RTKs are recognized as a promising avenue for the treatment of cancer, but the clinical benefits observed with these agents have so far been modest. As typified by EGFR-targeted therapies for metastatic CRC (e.g.: panitumumab and cetuximab), this modest response is attributed to the innate and acquired proficiency of cancer cells to escape EGFR inhibition by engaging alternative oncogenic signals [
2,
3,
52]. Multiple mechanisms of resistance have been proposed, including the manifest heterogeneity of RTKs being deregulated in CRC cells [
52,
53]. Notably, activation of the Met/HGF receptor axis is emerging as an important mechanism of resistance to drugs targeting oncogenic kinases in human cancers, including CRC, while concurrent inhibition of multiple RTKs in CRC cells seems to offer better therapeutic effects than targeting a specific RTK [
53‐
60]. An alternative way to achieve similar outcomes might be offered by targeting RTK-proximal signaling effectors engaged by all, or at least several RTKs, particularly those regulating biological processes critical for the initiation and/or progression of CRCs. In this regard, we show that although oncogenic engagement of Grb2 or Shc triggers redundant cancer properties in IECs (Figures
1,
2,
3), these adaptor proteins were proven, through analysis of the impact of their silencing in Tpr-Met-transformed IECs, to be necessary for non-overlapping functions (Figure
4). The silencing of Shc in Tpr-Met-IEC-6 cells was demonstrated to partly reduce cell growth without impacting anoikis resistance, but slightly increasing transformation and E-cadherin down-regulation. These results indicate that the Met receptor has the intrinsic capacity to circumvent the loss of Shc functions by engaging alternative oncogenic signals, likely involving the adaptor proteins Grb2, Gab1, or others effectors. Conversely, inhibition of Grb2 functions restored normal non-transformed epithelial morphology, E-cadherin expression, and anoikis sensitivity in these same Met-transformed IECs. Incidentally, Grb2 SH2 domain-binding antagonists were shown
in vitro to block HGF-induced migration and invasion in MDCK epithelial cells
, metastasis formation of melanoma and prostate cancer cells
in vivo, and the motility of human SW620 CRC cells in wound-healing
in vitro assays [
61‐
63]. Considering these observations, with our current findings, we suggest the targeting of Grb2 signaling in CRC, particularly in the context of deregulated Met, as a potentially effective therapeutic strategy to reduce CRC metastasis.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
VP contributed to the conception of the study, performed cell and molecular studies, and drafted the manuscript. ML carried out part of the anoikis and pharmacological studies. JB generated and carried out the initial characterization of the IEC populations expressing the Tpr-Met and docking-specific variants. PHV participated in drafting the manuscript. CS coordinated all aspects of the study and participated in the writing of the manuscript. All authors read and approved the final manuscript.