p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2

The kinase inhibitor p27Kip1 regulates the G1 cell cycle phase. Here, we present data indicating that the oncogenic kinase Src regulates p27 stability through phosphorylation of p27 at tyrosine 74 and tyrosine 88. Src inhibitors increase cellular p27 stability, and Src overexpression accelerates p27 proteolysis. Src-phosphorylated p27 is shown to inhibit cyclin E-Cdk2 poorly in vitro, and Src transfection reduces p27-cyclin E-Cdk2 complexes. Our data indicate that phosphorylation by Src impairs the Cdk2 inhibitory action of p27 and reduces its steady-state binding to cyclin E-Cdk2 to facilitate cyclin E-Cdk2-dependent p27 proteolysis. Furthermore, we find that Src-activated breast cancer lines show reduced p27 and observe a correlation between Src activation and reduced nuclear p27 in 482 primary human breast cancers. Importantly, we report that in tamoxifen-resistant breast cancer cell lines, Src inhibition can increase p27 levels and restore tamoxifen sensitivity. These data provide a new rationale for Src inhibitors in cancer therapy.     

Src Kinase Regulation in Progressively Invasive Cancer

Metastatic progression is a multistep process that involves tumor growth and survival, motility and invasion, and subsequent proliferation in an inappropriate environment. The Src protein tyrosine kinase has been implicated in many of the biochemical pathways that drive these behaviors. Although Src itself is only rarely mutated in human tumors, its aberrant activity has been noted in various cancers and suggested to serve as a barometer of metastatic potential. With these features in mind, we examined Src kinase regulation at the structural, enzymatic, and expression levels as a function of progressively invasive prostate cancer cell lines. Surprisingly, both total Src content and kinase activity decrease with increasing cell line aggressiveness, an observation that appears to be inconsistent with the well-documented role of Src in the signaling pathways that drive growth and invasion. However, we do observe a direct correlation between Src kinase specific activity (total Src kinase activity/total Src content) and metastatic aggressiveness, possibly suggesting that in highly aggressive cell lines, key signaling enzymes are globally recruited to drive the cancerous phenotype. In addition, although the expected enhanced phosphorylation of Src at Tyr-416 (activation site) is present in the most aggressive prostate cancer cell lines, unexpectedly high phosphorylation levels at the Tyr-527 inhibitory site are observed as well. The latter, rather than representative of inhibited enzyme, is more indicative of primed Src responsive to local phosphorylated binding partners.     

TRIM50 acts as a novel Src suppressor and inhibits ovarian cancer progression

Src is a known proto-oncogene and its aberrant activity is involved in a variety of cancers, including ovarian cancer, whereas the regulatory mechanism of Src has not been fully clarified. In this study, we identified tripartite motif-containing (TRIM) 50 as a novel negative regulator of Src protein. Our data showed that TRIM50 directly interacted with SH3 domain of Src via its B-box domain; and TRIM50 reduced Src stability by inducing RING domain-dependent K48-linked poly-ubiquitous modification. We further demonstrated that TRIM50 acted as a tumor suppressor in ovarian cancer cells by its negative regulation of Src protein. In vivo animal model verified that TRIM50 inhibited the xenograft tumor growth of ovarian cancer by suppressing Src protein. Clinical investigation showed that expression of TRIM50 in clinical specimens was inversely correlated with the clinical stages, pathology grades and lymph node metastatic status of the patients, which indicated the involvement of aberrant TRIM50 expression in disease progression. Further analysis verified the negative correlation between TRIM50 and Src expression in clinical specimens. Altogether, we identified TRIM50 as a novel suppressor of Src protein, and demonstrated that TRIM50 inhibited ovarian cancer progression by targeting Src and reducing its activity, which provided a novel therapeutic strategy for Src over-activated cancers by positive regulation of TRIM50.     

Src tyrosine kinase inhibits apoptosis through the Erk1/2- dependent degradation of the death accelerator Bik

Src, the canonical member of the non-receptor family of tyrosine kinases, is deregulated in numerous cancers, including colon and breast cancers. In addition to its effects on cell proliferation and motility, Src is often considered as an inhibitor of apoptosis, although this remains controversial. Thus, whether the ability of Src to generate malignancies relies on an intrinsic aptitude to inhibit apoptosis or requires preexistent resistance to apoptosis remains somewhat elusive. Here, using mouse fibroblasts transformed with v-Src as a model, we show that the observed Src-dependent resistance to cell death relies on Src ability to inhibit the mitochondrial pathway of apoptosis by specifically increasing the degradation rate of the BH3-only protein Bik. This effect relies on the activation of the Ras-Raf-Mek1/2-Erk1/2 pathway, and on the phosphorylation of Bik on Thr124, driving Bik ubiquitylation on Lys33 and subsequent degradation by the proteasome. Importantly, in a set of human cancer cells with Src-, Kras- or BRAF-dependent activation of Erk1/2, resistances to staurosporine or thapsigargin were also shown to depend on Bik degradation rate via a similar mechanism. These results suggest that Bik could be a rate-limiting factor for apoptosis induction of tumor cells exhibiting deregulated Erk1/2 signaling, which may provide new opportunities for cancer therapies.     

The tumor suppressor DAPK is reciprocally regulated by tyrosine kinase Src and phosphatase LAR

Death-associated protein kinase (DAPK) is a calmodulin-regulated serine/threonine kinase and elicits tumor suppression function through inhibiting cell adhesion/migration and promoting apoptosis. Despite these biological functions, the signaling mechanisms through which DAPK is regulated remain largely elusive. Here, we show that the leukocyte common antigen-related (LAR) tyrosine phosphatase dephosphorylates DAPK at pY491/492 to stimulate the catalytic, proapoptotic, and antiadhesion/antimigration activities of DAPK. Conversely, Src phosphorylates DAPK at Y491/492, which induces DAPK intra-/intermolecular interaction and inactivation. Upon EGF stimulation, a rapid Src activation leads to subsequent LAR downregulation, and these two events act in synergism to inactivate DAPK, thereby facilitating tumor cell migration and invasion toward EGF. Finally, DAPK Y491/492 hyperphosphorylation is found in human cancers in which Src activity is aberrantly elevated. These results identify LAR and Src as a DAPK regulator through their reciprocal modification of DAPK Y491/492 residues and establish a functional link of this DAPK-regulatory circuit to tumor progression.     

Conformational basis for SH2-Tyr(P)527 binding in Src inactivation

Src protein-tyrosine kinase contains a myristoylation motif, a unique region, an Src homology (SH) 3 domain, an SH2 domain, a catalytic domain, and a C-terminal tail. The C-terminal tail contains a Tyr residue, Tyr527. Phosphorylation of Tyr527 triggers Src inactivation, caused by Tyr(P)527 binding to the SH2 domain. In this study, we demonstrated that a conformational contribution, not affinity, is the predominant force for the intramolecular SH2-Tyr(P)527 binding, and we characterized the structural basis for this conformational contribution. First, a phosphopeptide mimicking the C-terminal tail is an 80-fold weaker ligand than the optimal phosphopeptide, pYEEI, and similar to a phosphopeptide containing three Ala residues following Tyr(P) in binding to the Src SH2 domain. Second, the SH2-Tyr(P)527 binding is largely independent of the amino acid sequence surrounding Tyr(P)527, and only slightly decreased by an inactivating mutation in the SH2 domain. Furthermore, even the unphosphorylated C-terminal tail with the sequence of YEEI suppresses Src activity by binding to the SH2 domain. These experiments demonstrate that very weak affinity is sufficient for the SH2-Tyr(P)527 binding in Src inactivation. Third, the effective intramolecular SH2-Tyr(P)527 binding is attributed to a conformational contribution that requires residues Trp260 and Leu255. Although the SH3 domain is essential for Src inactivation by Tyr(P)527, it does not contribute to the SH2-Tyr(P)527 binding. These findings suggest a conformation-based Src inactivation model, which provides a unifying framework for understanding Src activation by a variety of mechanisms.     

Src Regulates the Activity of the ING1 Tumor Suppressor

The INhibitor of Growth 1 (ING1) is stoichiometric member of histone deacetylase (HDAC) complexes and functions as an epigenetic regulator and a type II tumor suppressor. It impacts cell growth, aging, apoptosis, and DNA repair, by affecting chromatin conformation and gene expression. Down regulation and mislocalization of ING1 have been reported in diverse tumor types and Ser/Thr phosphorylation has been implicated in both of these processes. Here we demonstrate that both in vitro and in vivo, the tyrosine kinase Src is able to physically associate with, and phosphorylate ING1, which results in a nuclear to cytoplasmic relocalization of ING1 in cells and a decrease of ING1 stability. Functionally, Src antagonizes the ability of ING1 to induce apoptosis, most likely through relocalization of ING1 and down regulation of ING1 levels. These effects were due to both kinase-dependent and kinase-independent properties of Src, and were most apparent at elevated levels of Src expression. These findings suggest that Src may play a major role in regulating ING1 levels during tumorigenesis in those cancers in which high levels of Src expression or activity are present. These data represent the first report of tyrosine kinase-mediated regulation of ING1 levels and suggest that kinase activation can impact chromatin structure through the ING1 epigenetic regulator.     

Potentiation of Epidermal Growth Factor-Mediated Oncogenic Transformation by Sialidase NEU3 Leading to Src Activation

We previously demonstrated that sialidase NEU3, a key glycosidase for ganglioside degradation, is up-regulated in various human cancers, leading to increased cell invasion, motility and survival of cancer cells possibly through activation of EGF signaling. Its up-regulation is also important for promotion of the stage of colorectal carcinogenesis in vivo in human NEU3 transgenic mice treated with azoxymethane for the induction of aberrant crypt foci in the colon mucosa, accompanied by enhanced phosphorylation of EGF receptor (EGFR). To address whether the activation of EGF signaling by the sialidase is associated with oncogenic transformation, we here analyzed the effects of overexpression of NEU3 and EGFR in NIH-3T3 cells. When NEU3 was stably transfected with or without EGFR, it was associated with significant increases in clonogenic growth, clonogenicity on soft agar and in vivo tumor growth in nude mice either with or without the receptor overexpression in the presence of EGF, compared with the levels in their vector controls. Despite the fact that the endogenous level of EGFR is known to be extremely low in these cells, NEU3 significantly enhanced the phosphorylation of Akt and ERK, as well as that of the receptor. The NEU3-mediated activation was largely abrogated by the EGFR inhibitor AG1478 or PD153035, but significant clonogenic growth still remained. NEU3 was then found to activate Src kinase, and the clonogenicity was completely suppressed by an Src inhibitor, PP2. The activity-null mutants failed to activate Src and EGFR, indicating that ganglioside modulation by NEU3 may be necessary for the activation. NEU3 and Src were co-immunoprecipitated with EGFR in NEU3- and EGFR- transfected cells. These findings identify NEU3 as an essential participant in tumorigenesis through the EGFR/Src signaling pathway and a potential target for inhibiting EGFR-mediated tumor progression.     

A redox-silent analogue of tocotrienol inhibits hypoxic adaptation of lung cancer cells

We have previously reported that a redox-silent analogue of α-tocotrienol (T3), 6-O-carboxypropyl-α-tocotrienol (T3E) shows more potential anti-carcinogenic property than T3 in a lung cancer cell (A549 cell). However, the mechanisms by which T3E exerts its potential anti-carcinogenic effect is still unclear. As tumor malignancy is associated with hypoxia adaptation, in this study, we examined whether T3E could suppress survival and invasion in A549 cells under hypoxia. Hypoxia treatment drastically-induced activation of the protein tyrosine kinase, Src, and its regulated signaling required for hypoxia adaptation of A549 tumor cells. The survival and invasion capacity of the tumor cells under hypoxia was suppressed by T3E via the inactivation of Src. More specifically, T3E-dependent inhibition of Src-induced Akt activation contributed to suppression of cell survival under hypoxia, and the reduction of fibrinolytic factors such as plasminogen activator-1(PAI-1) via the decrease of hypoxia-inducible factor-2α by T3E led to inhibition of hypoxic invasion. Overall these results suggest that T3E suppresses hypoxia adaptation of A549 cells by the inhibition in hypoxia-induced activation of Src signaling.     

Targeting Androgen Receptor/Src Complex Impairs the Aggressive Phenotype of Human Fibrosarcoma Cells

Background

Hormones and growth factors influence the proliferation and invasiveness of human mesenchymal tumors. The highly aggressive human fibrosarcoma HT1080 cell line harbors classical androgen receptor (AR) that responds to androgens triggering cell migration in the absence of significant mitogenesis. As occurs in many human cancer cells, HT1080 cells also express epidermal growth factor receptor (EGFR).

Experimental

Findings: We report that the pure anti-androgen Casodex inhibits the growth of HT1080 cell xenografts in immune-depressed mice, revealing a novel role of AR in fibrosarcoma progression. In HT1080 cultured cells EGF, but not androgens, robustly increases DNA synthesis. Casodex abolishes the EGF mitogenic effect, implying a crosstalk between EGFR and AR. The mechanism underlying this crosstalk has been analyzed using an AR-derived small peptide, S1, which prevents AR/Src tyrosine kinase association and androgen-dependent Src activation. Present findings show that in HT1080 cells EGF induces AR/Src Association, and the S1 peptide abolishes both the assembly of this complex and Src activation. The S1 peptide inhibits EGF-stimulated DNA synthesis, cell matrix metalloproteinase-9 (MMP-9) secretion and invasiveness of HT1080 cells. Both Casodex and S1 peptide also prevent DNA synthesis and migration triggered by EGF in various human cancer-derived cells (prostate, breast, colon and pancreas) that express AR.

Conclusion

This study shows that targeting the AR domain involved in AR/Src association impairs EGF signaling in human fibrosarcoma HT1080 cells. The EGF-elicited processes inhibited by the peptide (DNA synthesis, MMP-9 secretion and invasiveness) cooperate in increasing the aggressive phenotype of HT1080 cells. Therefore, AR represents a new potential therapeutic target in human fibrosarcoma, as supported by Casodex inhibition of HT1080 cell xenografts. The extension of these findings in various human cancer-derived cell lines highlights the conservation of this process across divergent cancer cells and identifies new potential targets in the therapeutic approach to human cancers.     

Cigarette smoke extract stimulates epithelial-mesenchymal transition through Src activation

Epithelial-mesenchymal transition (EMT) is implicated in the pathogenesis of lung fibrosis and cancer metastasis, two conditions associated with cigarette smoke (CS). CS has been reported to promote the EMT process. CS is the major cause of lung cancer and nearly half of lung cancer patients are active smokers. Nonetheless, the mechanism whereby CS induces EMT remains largely unknown. In this study we investigated the induction of EMT by CS and explored the underlying mechanisms in the human non-small-cell lung carcinoma (H358) cell line. We demonstrate that exposure to an extract of CS (CSE) decreases E-cadherin and increases N-cadherin and vimentin, markers of EMT, in H358 cells cultured in RPMI 1640 medium with 1% fetal bovine serum. Pretreatment with N-acetylcysteine (NAC), a potent antioxidant and precursor of glutathione, abrogated changes in these EMT markers. In addition, CSE activated Src kinase (shown as increased phosphorylation of Src at Tyr418), and the Src kinase inhibitor PP2 inhibited CS-stimulated EMT changes, suggesting that Src is critical in CSE-stimulated EMT induction. Furthermore, NAC treatment abrogated CSE-stimulated Src activation. However, co-incubation with catalase had no effect on CSE-mediated Src activation. Finally, acrolein, an unsaturated aldehyde present in CSE, caused Src activation. Taken together, these data suggest that CSE initiates EMT through Src, which is activated by CS through redox modification.     

Protein-tyrosine phosphatase PTPL1/FAP-1 triggers apoptosis in human breast cancer cells

Studies in Jurkat leukemia cells have suggested that protein-tyrosine phosphatase PTPL1/FAP-1 rescues Fas-induced cell death. However, we have previously shown that this enzyme triggers 4-hydroxytamoxifen-induced growth inhibition in human breast cancer cells. The present study addresses the role of PTPL1/FAP-1 in antiestrogen-regulated apoptotic effect and insulin-like growth factor-I survival action in MCF7 cells and further identifies the impacted signaling pathway. By terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and cytoplasmic nucleosome enzyme-linked immunosorbent assay, we demonstrated that 4-hydroxytamoxifen-induced apoptosis was totally lost in PTPL1/FAP-1 antisense transfectants in which enzyme expression was abrogated, revealing the crucial role of this phosphatase in the apoptotic process in human breast cancer cells. Time-dependent expression of PTPL1/FAP-1 in MCF7 cells completely abolished the survival action of insulin-like growth factor-I. This effect occurred through a highly significant reduction in phosphatidylinositol 3-kinase/Akt pathway activation (80% reduction in phosphatidylinositol 3-kinase activity, 55% inhibition of Akt activation) accompanied by a 65% decrease in insulin receptor substrate-1 growth factor-induced tyrosine phosphorylation. These results provide the first evidence that PTPL1/FAP-1 has a key role in the apoptotic process in human breast cancer cells independent of Fas but associated with an early inhibition of the insulin receptor substrate-1/phosphatidylinositol 3-kinase pathway. Our data therefore suggest new therapeutic routes and strengthen the importance of identifying endogenous regulators and substrates of this phosphatase in breast tumors.     

Global tyrosine kinome profiling of human thyroid tumors identifies Src as a promising target for invasive cancers

Background: Novel therapies are needed for the treatment of invasive thyroid cancers. Aberrant activation of tyrosine kinases plays an important role in thyroid oncogenesis. Because current targeted therapies are biased toward a small subset of tyrosine kinases, we conducted a study to reveal novel therapeutic targets for thyroid cancer using a bead-based, high-throughput system.Methods: Thyroid tumors and matched normal tissues were harvested from twenty-six patients in the operating room. Protein lysates were analyzed using the Luminex immunosandwich, a bead-based kinase phosphorylation assay. Data was analyzed using GenePattern 3.0 software and clustered according to histology, demographic factors, and tumor status regarding capsular invasion, size, lymphovascular invasion, and extrathyroidal extension. Survival and invasion assays were performed to determine the effect of Src inhibition in papillary thyroid cancer (PTC) cells.Results: Tyrosine kinome profiling demonstrated upregulation of nine tyrosine kinases in tumors relative to matched normal thyroid tissue: EGFR, PTK6, BTK, HCK, ABL1, TNK1, GRB2, ERK, and SRC. Supervised clustering of well-differentiated tumors by histology, gender, age, or size did not reveal significant differences in tyrosine kinase activity. However, supervised clustering by the presence of invasive disease showed increased Src activity in invasive tumors relative to non-invasive tumors (60% v. 0%, p < 0.05). In vitro, we found that Src inhibition in PTC cells decreased cell invasion and proliferation.Conclusion: Global kinome analysis enables the discovery of novel targets for thyroid cancer therapy. Further investigation of Src targeted therapy for advanced thyroid cancer is warranted.     

To C or not to C: Direct and indirect redox regulation of Src protein tyrosine kinase

Src protein tyrosine kinase is a master regulator of cell proliferation by modulating cell metabolism, division, survival and migration, thus the mechanisms that regulate Src function are of great interest to cancer research. One emerging mode of Src regulation is its response to reactive oxygen species (ROS). ROS have historically been viewed as damaging agents in cells under oxidative stress, but recent studies establish H2O2 as a secondary messenger to growth signals. A large number of cellular events respond to ROS, and many responses require the activity of Src, suggesting that Src may be a primary target of ROS. How Src kinase responds to ROS has not been established, as conflicting reports of Src activation or inactivation in response to increased concentration of ROS in the cells have been published. To determine how Src directly responds to oxidation, we investigated the effect of the redox environment on purified Src enzyme in vitro. The study reveals that Src is active in the reducing environment, and retains only 8-25% of activity in the absence of reducing agents. The inactivation is mediated by oxidation of Cys277, which leads to Src homodimers linked by a disulfide bond between the Cys277 residues of two Src monomers. A similar inactivation mechanism appears to be conserved in eight of more than 90 PTKs, including three Src family kinases and all four members of the FGFR family. The finding contradicts the view that Src is activated by oxidation, and suggests a complex response by Src to redox regulation. In this Extra View, we examine the conflicting observations in the context of complex mechanisms of Src regulation.     

The PDZ-ligand and Src-homology type 3 domains of epidemic avian influenza virus NS1 protein modulate human Src kinase activity during viral infection

The Non-structural 1 (NS1) protein of avian influenza (AI) viruses is important for pathogenicity. Here, we identify a previously unrecognized tandem PDZ-ligand (TPL) domain in the extreme carboxy terminus of NS1 proteins from a subset of globally circulating AI viruses. By using protein arrays we have identified several human PDZ-cellular ligands of this novel domain, one of which is the RIL protein, a known regulator of the cellular tyrosine kinase Src. We found that the AI NS1 proteins bind and stimulate human Src tyrosine kinase, through their carboxy terminal Src homology type 3-binding (SHB) domain. The physical interaction between NS1 and Src and the ability of AI viruses to modulate the phosphorylation status of Src during the infection, were found to be influenced by the TPL arrangement. These results indicate the potential for novel host-pathogen interactions mediated by the TPL and SHB domains of AI NS1 protein.     

c‐Src drives intestinal regeneration and transformation

The non‐receptor tyrosine kinase c‐Src, hereafter referred to as Src, is overexpressed or activated in multiple human malignancies. There has been much speculation about the functional role of Src in colorectal cancer (CRC), with Src amplification and potential activating mutations in up to 20% of the human tumours, although this has never been addressed due to multiple redundant family members. Here, we have used the adult Drosophila and mouse intestinal epithelium as paradigms to define a role for Src during tissue homeostasis, damage‐induced regeneration and hyperplasia. Through genetic gain and loss of function experiments, we demonstrate that Src is necessary and sufficient to drive intestinal stem cell (ISC) proliferation during tissue self‐renewal, regeneration and tumourigenesis. Surprisingly, Src plays a non‐redundant role in the mouse intestine, which cannot be substituted by the other family kinases Fyn and Yes. Mechanistically, we show that Src drives ISC proliferation through upregulation of EGFR and activation of Ras/MAPK and Stat3 signalling. Therefore, we demonstrate a novel essential role for Src in intestinal stem/progenitor cell proliferation and tumourigenesis initiation in vivo.