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The oncoprotein v-Src and its cellular homologue (c-Src) are tyrosine kinases that modulate the actin cytoskeleton and cell adhesions. Through the concerted action of their protein-interaction and kinase domains, they are targeted to cell–matrix integrin adhesions or cadherin-dependent junctions between epithelial cells, where they phosphorylate substrates that induce adhesion turnover and actin re-modelling. Recent experiments have defined some of the key targets and effector pathways that mediate the pleiotropic oncogenic effects of v-Src. Key Points v-Src was the first identified tyrosine kinase and the first known oncoprotein. It is located in adhesion sites at the plasma membrane, from where it induces pleiotropic effects on cells that result in the transformed phenotype. The intracellular targeting of v-Src, and c-Src, in mesenchymal cells is dependent on actin stress fibres and peripheral actin re-modelling that is, in turn, controlled by the Rho family of small GTPases. The non-catalytic domains of Src — SH3 (modulated also by SH2) domains — are essential for proper intracellular targeting, and are mediated by interaction with protein partners, such as PI3K. In addition, the adaptor functions of Src might also have a role in the assembly of adhesion signalling complexes after integrin engagement. The result of the unrestrained catalytic activity of v-Src at adhesion sites is to induce adhesion turnover and disorganization of the associated actin cytoskeleton. When this is unregulated in primary cells, the result is complete loss of cell-substratum adhesion and detachment. In established cell lines, the result is enhanced adhesion turnover that facilitates rapid cell migration. This is in keeping with the evidence that the Src family kinases have a key role in integrin-dependent cell motility. v-Src-induced activation of the calcium-dependent protease, calpain, and tyrosine phosphorylation of focal adhesion kinase (FAK), lead to FAK proteolysis, events that are associated with adhesion disruption in transformed cells. v-Src also induces actin re-modelling, and specifically induces formation of actin-rich podosomes that contain several regulators of actin assembly, including cortactin, that is a v-Src substrate. In addition, v-Src induces tyrosine phosphorylation of p190 Rho-GAP that might mediate stress-fibre disorganization by antagonizing the actin assembly function of RhoA. v-Src activity at the cell periphery also triggers intracellular signalling that promotes cell-cycle progression, and specifically induces rapid transit through the pRb checkpoint at the G1/S boundary. Surprisingly, calpain activity is required also for the cell–cycle changes that are induced by the oncprotein. The catalytic activity of v-Src, and c-Src in epithelial cells results in disruption of cadherin-mediated cell-cell adhesions. Although this is not yet well understood in molecular terms, new potential Src effectors at adherens junctions are being identified. Weakened cadherin-dependent adhesions might be an important consequence of elevated Src activity in epithelial cancers.

Pancreatic ductal adenocarcinoma (PDAC) cells usually overexpress the epidermal growth factor receptor (EGFR); however, most are resistant to the anti-EGFR monoclonal antibody, cetuximab. In this study, we report that the molecular mechanism of resistance to cetuximab in PDAC cells is mediated by the overexpression of active integrin β1 with downstream Src-Akt activation; this triggers an EGFR ligand-independent proliferation signaling, bypassing EGFR-blocking effect. Knockdown of integrin β1 or inhibition of Src or Akt sensitized cetuximab-resistant (Ctx R ) PDAC cells to cetuximab. We found that neuropilin-1 (NRP1) physically interacts with active integrin β1, but not inactive one, on the cell surface. To inhibit active integrin β1-driven signaling by targeting NRP1, while suppressing EGFR signaling, we generated an EGFR and NRP1 dual targeting antibody, Ctx-TPP11, by genetic fusion of the NRP1-targeting peptide, TPP11, to the C terminus of the cetuximab heavy chain (Ctx-TPP11). We demonstrate that Ctx-TPP11 efficiently inhibited the growth of Ctx R PDAC cells, in vitro and in vivo . The sensitization mechanism involved downregulating active integrin β1 levels through NRP1-coupled internalization mediated by the TPP11 moiety, leading to the inhibition of active integrin β1-driven bypass signaling. Our findings identify aberrant active integrin β1-driven Src-Akt hyperactivation as a primary resistance mechanism to cetuximab in PDAC cells and offer an effective therapeutic strategy to overcome this resistance using an EGFR and NRP1 dual targeting antibody.

Alterations in estrogen-mediated cellular signaling have largely been implicated in the pathogenesis of breast cancer. Here, we investigated the signaling regulation of a splice variant of the estrogen receptor, namely estrogen receptor (ERα-36), associated with a poor prognosis in breast cancers. Coupling in vitro and in vivo approaches we determined the precise sequential molecular events of a new estrogen signaling network in an ERα-negative cell line and in an original patient-derived xenograft. After estrogen treatment, ERα-36 rapidly associates with Src at the level of the plasma membrane, initiating downstream cascades, including MEK1/ERK activation and paxillin phosphorylation on S126, which in turn triggers a higher expression of cyclin D1. Of note, the direct binding of ERα-36 to ERK2 prevents its dephosphorylation by MKP3 and enhances the downstream signaling. These findings improve our understanding of the regulation of non-genomic estrogen signaling and open new avenues for personalized therapeutic approaches targeting Src or MEK in ERα-36-positive patients.

Hypertrophic scar (HS) is a serious fibrotic skin condition with currently no satisfactory therapy due to undefined molecular mechanism. FAK and Src are two important non-receptor tyrosine kinases that have been indicated in HS pathogenesis. Here we found both FAK and Src were activated in HS vs. normal skin (NS), NS fibroblasts treated with TGF-β1 also exhibited FAK/Src activation. Co-immunoprecipitation and dual-labelled immunofluorescence revealed an enhanced FAK-Src association and co-localization in HS vs. NS. To examine effects of FAK/Src activation and their interplay on HS pathogenesis, site-directed mutagenesis followed by gene overexpression was conducted. Results showed only simultaneous overexpression of non-phosphorylatable mutant FAK Y407F and phosphomimetic mutant Src Y529E remarkably down-regulated the expression of Col I, Col III and α-SMA in cultured HS fibroblasts, alleviated extracellular matrix deposition and made collagen fibers more orderly in HS tissue vs. the effect from single transfection with wild-type or mutational FAK/Src. Glabridin, a chemical found to block FAK-Src complex formation in cancers, exhibited therapeutic effects on HS pathology probably through co-deactivation of FAK/Src which further resulted in FAK-Src de-association. This study suggests FAK-Src complex could serve as a potential molecular target and FAK/Src double deactivation might be a novel strategy for HS therapy.

Cofilin is a major regulator of actin dynamics involved in the regulation of cell spreading and migration through its actin depolymerizing and severing activities. v-Src is an activated Src tyrosine kinase and a potent oncogene known to phosphorylate a variety of cellular proteins in cell transformation process including altered cell adhesion, spreading and migration. Recently, it has been suggested that cofilin is a potential substrate of v-Src (Rush et al. , 2005). Here, we show direct tyrosine phosphorylation of cofilin by v-Src and identify Y68 as the major phosphorylation site. Cofilin phosphorylation at Y68 did not change its activity per se , but induced increased ubiquitination of cofilin and its degradation through the proteosome pathway. Furthermore, the negative effect of cofilin on cellular F-actin contents was inhibited by coexpression of v-Src, whereas that of cofilin mutant Y68F (Y68 mutated to F) was not affected, suggesting that v-Src-mediated cofilin phosphorylation at Y68 is required for the degradation of cofilin in vivo . Lastly, inhibition of cell spreading by v-Src was rescued partially by coexpression of cofilin, and to a greater extent by the Y68F mutant, which is not subjected to v-Src-induced degradation through phosphorylation, suggesting that v-Src-mediated changes in cell spreading is, at least in part, through inhibiting the function of cofilin through phosphorylating it at Y68. Together, these results suggest a novel mechanism by which cofilin is regulated by v-Src through tyrosine phosphorylation at Y68 that triggers the degradation of cofilin through ubiquitination–proteosome pathway and consequently inhibits cofilin activity in reducing cellular F-actin contents and cell spreading.

FIP1L1-PDGFRA is a constitutively activated kinase described in chronic eosinophilic leukemia (CEL) and hypereosinophilic syndrome (HES). Imatinib is clinically active in FIP1L1-PDGFRA-positive diseases. Using in vitro screening to identify imatinib-resistant mutations, we frequently detected a Phe to Ser exchange at position 604 (F604S) of FIP1L1-PDGFRA alone or in combination with other exchanges. Surprisingly, FIP1L1-PDGFRA/F604S did not increase the biochemical or cellular IC50 value of imatinib when compared with unmutated FIP1L1-PDGFRA. However, FIP1L1-PDGFRA/F604S more efficiently induced growth factor independence in cell lines and primary mouse bone marrow cells. Pulse chase analysis revealed that the F604S exchange strongly stabilized FIP1L1-PDGFRA/F604S. The F604S mutation creates a binding site for the phosphatase domain of SHP-2, leading to lower autophosphorylation of FIP1L1-PDGFRA/F604S. This is associated with a reduced activation of SRC and CBL by FIP1L1-PDGFRA/F604S compared with the unmutated oncogene. As SRC inhibition and knockdown resulted in FIP1L1-PDGFRA stabilization, this explains the extended half-life of FIP1L1-PDGFRA/F604S. Interestingly, FIP1L1-PDGFRA/L629P, a recently identified mutation in an imatinib-resistant CEL patient, also showed protein stabilization similar to that observed with FIP1L1-PDGFRA/F604S. Therefore, resistance mutations in FIP1L1-PDGFRA that do not interfere with drug binding but rather increase target protein stability seem to be one of the drug-resistance mechanisms in FIP1L1-PDGFRA-positive disease.

Kruppel-like factor 8 (KLF8) is a member of the family of KLF transcription factors. Several KLF members have been shown to play a role in oncogenesis. We have previously demonstrated that KLF8 mediates cell cycle progression downstream of focal adhesion kinase (FAK) by upregulating cyclin D1. FAK plays a critical role in transformation and tumorigenesis and is aberrantly upregulated in many types of human cancer. Little is known about the function of KLF8 in these regards. Here we provide evidence suggesting a novel role of KLF8 in oncogenic transformation. We show that KLF8 expression is elevated in several types of human cancer cells and primary tumor tissues. Induced expression of ectopic KLF8 causes serum-independent growth and morphological transformation in NIH3T3 cells and enhances anchorage-independent growth of v-Src-transformed cells. In contrast, expression of a dominant-negative mutant of KLF8 dramatically suppresses the transformed phenotypes induced by v-Src. In addition, the KLF8-enhanced transformation in the v-Src cells was prevented by ablating cyclin D1 expression. Overall, these results indicate that KLF8 is required for v-Src-induced transformation and may play a role in tumor progression of human cancer.

Among androgen-regulated genes, soluble guanylyl cyclase α1 (sGCα1) is significant in promoting the survival and growth of prostate cancer cells and does so independent of nitric oxide (NO) signaling. Peptides were designed targeting sGCα1 to block its pro-cancer functions and one peptide is discussed here. Peptide B-8R killed both androgen-dependent and androgen-independent prostate cancer cells that expressed sGCα1, but not cells that do not express this gene. Peptide B-8R induced apoptosis of prostate cancer cells. Importantly, Peptide B-8R does not affect nor its cytotoxicity depend on NO signaling, despite the fact that it associates with sGCα1, which dimerizes with sGCβ1 to form the sGC enzyme. Just as with a previously studied Peptide A-8R, Peptide B-8R induced elevated levels of reactive oxygen species (ROS) in prostate cancer cells, but using a ROS-sequestering agent showed that ROS was not responsible the cytotoxic activity of Peptide B-8R. Interestingly, Peptide B-8R induced elevated levels of p53 and phosphorylated p38, but neither of these changes is the cause of the peptide's cytotoxicity. Additional drugs were used to alter levels of iron levels in cells and these studies showed that Peptide B-8R activity does not depend on Ferroptosis. Thus, future work will be directed at defining the mechanism of cytotoxic action of Peptide B-8R against prostate cancer cells.

Stats1 and 3 (signal transducers and activators of transcription) can be activated simultaneously, although not necessarily to the same degree or duration, by the interaction of cells with the same polypeptide ligand (EGF, PDGF, or high concentrations of IL-6, for example). However, these two Stat proteins can mediate opposing effects on cell growth and survival. Stat1 activation slows growth and promotes apoptosis. In contrast, activated Stat3 can protect cells from apoptosis. Furthermore, a constitutively active form of Stat3, Stat3-C (bridged by S-S linkages between cysteines instead of phosphotyrosines) can induce cellular transformation of fibroblasts. We have determined that fibroblasts transformed by Stat3-C are more resistant to proapoptotic stimuli than nontransformed cells. Also, to examine the potential opposing roles in apoptosis of Stat1 and Stat3, we studied the cervical carcinoma-derived cell line, Me 180, which undergoes Stat1-dependent, IFNγ-induced apoptosis. Me 180 cells that express Stat3-C are protected against IFNγ-mediated apoptosis.