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Hyperactivation of SRC-family protein kinases (SFKs) contributes to the initiation and progression of human colorectal cancer (CRC). Since oncogenic mutations of SFK genes are rare in human CRC, we investigated if SFK hyperactivation is linked to dysregulation of their upstream inhibitors, C-terminal SRC kinase (CSK) and its homolog CSK-homologous kinase (CHK/MATK). We demonstrate that expression of CHK/MATK but not CSK was significantly downregulated in CRC cell lines and primary tumours compared to normal colonic tissue. Investigation of the mechanism by which CHK/MATK expression is down-regulated in CRC cells uncovered hypermethylation of the CHK/MATK promoter in CRC cell lines and primary tumours. Promoter methylation of CHK/MATK was also observed in several other tumour types. Consistent with epigenetic silencing of CHK/MATK, genetic deletion or pharmacological inhibition of DNA methyltransferases increased CHK/MATK mRNA expression in CHK/MATK-methylated colon cancer cell lines. SFKs were hyperactivated in CHK/MATK-methylated CRC cells despite expressing enzymatically active CSK, suggesting loss of CHK/MATK contributes to SFK hyperactivation. Re-expression of CHK/MATK in CRC cell lines led to reduction in SFK activity via a non-catalytic mechanism, a reduction in anchorage-independent growth, cell proliferation and migration in vitro, and a reduction in tumour growth and metastasis in a zebrafish embryo xenotransplantation model in vivo, collectively identifying CHK/MATK as a novel putative tumour suppressor gene in CRC. Furthermore, our discovery that CHK/MATK hypermethylation occurs in the majority of tumours warrants its further investigation as a diagnostic marker of CRC.

RNA interference has been used to dissect the importance of individual gene products in various human disease processes, including cancer. Small-interfering RNA, or siRNA, is one of the tools utilized in this regard, but specially-designed delivery agents are required to allow the siRNA to gain optimal access to the cell interior. Our laboratory has utilized two different siRNA-binding delivery peptides containing a polyarginine core, and modified by myristoylation and targeting motifs (iRGD or Lyp-1). A third peptide was designed to assist with endosomal release. Various ratios of the peptides and siRNA were combined and assayed for the ability to form stable complexes, and optimized ratios were determined. The complexes were found to form particles, with the majority having a diameter of 100-300 nm, as visualized by electron microscopy. These siRNA complexes have enhanced protection from nucleases present in serum, as compared to "naked" unprotected siRNA. The particles were internalized by the cells and could be detected in the cell cytoplasm by confocal fluorescence microscopy. In functional assays, peptide/siRNA complexes were shown to cause the knock down of corresponding targeted proteins. The peptide with the LyP-1 targeting motif was more effective at knockdown in MDA-MB-231 breast cancer cells than the peptide with the iRGD motif. Inclusion of the endosomal release peptide in the complexes greatly enhanced the peptide/siRNA effects. Peptide/siRNA complexes simultaneously targeting Stat3 and c-Myc caused a marked reduction in anchorage-independent growth, a property correlated with tumorigenicity. This study demonstrates the ability of a peptide-based siRNA-delivery system to deliver siRNA into breast cancer cells and cause both protein knockdown and suppression of the malignant phenotype. Such peptide complexes are likely to become highly useful siRNA-delivery vehicles for the characterization, and potentially for the treatment, of human cancer.

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.

Excitotoxicity resulting from overstimulation of glutamate receptors is a major cause of neuronal death in cerebral ischemic stroke. The overstimulated ionotropic glutamate receptors exert their neurotoxic effects in part by overactivation of calpains, which induce neuronal death by catalyzing limited proteolysis of specific cellular proteins. Here, we report that in cultured cortical neurons and in vivo in a rat model of focal ischemic stroke, the tyrosine kinase Src is cleaved by calpains at a site in the N-terminal unique domain. This generates a truncated Src fragment of ~52 kDa, which we localized predominantly to the cytosol. A cell membrane-permeable fusion peptide derived from the unique domain of Src prevents calpain from cleaving Src in neurons and protects against excitotoxic neuronal death. To explore the role of the truncated Src fragment in neuronal death, we expressed a recombinant truncated Src fragment in cultured neurons and examined how it affects neuronal survival. Expression of this fragment, which lacks the myristoylation motif and unique domain, was sufficient to induce neuronal death. Furthermore, inactivation of the prosurvival kinase Akt is a key step in its neurotoxic signaling pathway. Because Src maintains neuronal survival, our results implicate calpain cleavage as a molecular switch converting Src from a promoter of cell survival to a mediator of neuronal death in excitotoxicity. Besides unveiling a new pathological action of Src, our discovery of the neurotoxic action of the truncated Src fragment suggests new therapeutic strategies with the potential to minimize brain damage in ischemic stroke.

Inhibitors of PI3-K/Akt are currently being assessed clinically in patients with advanced RCC. Identification of therapeutic strategies that might enhance the efficacy of PI3-K/Akt inhibitors is therefore of great interest. As PI3-K inhibition would be expected to have many pro-apoptotic effects, we hypothesized that there may be unique synergy between PI3-K inhibitors and BH3-mimetics. Towards this end, we assessed the combination of the PI3K inhibitor LY 294002 and the Bcl-2 family inhibitor ABT-737 in RCC cell lines. We found that the combinatorial treatment with these agents led to a significant increase in PARP cleavage and cell death in all RCC cell lines. The synergized cell death was correlated with decreased levels of Mcl-1 and XIAP, and increased levels in Bim, and appears critically dependent upon the activation of caspase 3 and 8. The enhanced lethality observed with the combination also appears dependent upon the regulation of XIAP, Mcl-1 and Bim levels. Our results suggest that the combination of PI3-K inhibitors with BH3-mimetics may be a viable therapeutic strategy in RCC., (© 2013 The Authors. Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Objective: Src is a protein tyrosine kinase that plays important roles in cancer development, and Src kinase activity has been found to be elevated in several types of cancers. However, the cause of the elevation of Src kinase activity in the majority of human colon carcinomas is still largely unknown. We aim at finding the cause of elevated Src kinase activity in human colon carcinomas., Methods: We employed normal colon epithelial FHC cells and examined Src activation in human colon carcinoma specimens from 8 patients. Protein expression levels were determined by Western blotting, and the activity of Src kinase by kinase assay., Results: Actin levels were different between tumor and normal tissues, demonstrating the complexities and inhomogeneities of the tissue samples. Src kinase activities were increased in the majority of the colon carcinomas as compared with normal colon epithelial cells (range 13-29). Src protein levels were reduced in the colon carcinomas. Src Y530 phosphorylation levels were reduced to a higher extent than protein levels in the carcinomas., Conclusion: The results suggest that Src specific activities were highly increased in human colon carcinomas; phosphorylation at Src Y530 was reduced, contributing to the highly elevated Src specific activity and Src kinase activity.

Background: Src and signaling molecules downstream of Src, including signal transducer and activator of transcription 3 (Stat3) and cMyc, have been implicated in the development, maintenance and/or progression of several types of human cancers, including breast cancer. Here we report the ability of siRNA-mediated Src knock-down alone, and simultaneous knock-down of Src and Stat3 and/or cMyc to inhibit the neoplastic phenotype of a highly metastatic human model breast cancer cell line, MDA-MB-435S, a widely used model for breast cancer research., Methodology/results: Src and its downstream signaling partners were specifically targeted and knocked-down using siRNA. Changes in the growth properties of the cultured cancer cells/tumors were documented using assays that included anchorage-dependent and -independent (in soft agar) cell growth, apoptosis, and both primary and metastatic tumor growth in the mouse tumor model. siRNA-mediated Src knock-down alone, and simultaneous knock-down of Src and Stat3 and/or cMyc inhibited the neoplastic phenotype of a highly metastatic human model breast cancer cell line, MDA-MB-435S. This knock-down resulted in reduced growth in monolayer and soft agar cultures, and a reduced ability to form primary tumors in NOD/SCID mice. In addition, direct intra-tumoral injection of siRNAs targeting these signaling molecules resulted in a substantial inhibition of tumor metastases as well as of primary tumor growth. Simultaneous knock-down of Src and Stat3, and/or Myc exhibited the greatest effects resulting in substantial inhibition of primary tumor growth and metastasis., Conclusions/significance: These findings demonstrate the effectiveness of simultaneous targeting of Src and the downstream signaling partners Stat3 and/or cMyc to inhibit the growth and oncogenic properties of a human cancer cell line. This knowledge may be very useful in the development of future therapeutic approaches involving targeting of specific genes products involved in tumor growth and metastasis.

The von Hippel-Lindau tumor suppressor protein (VHL), when mutated and inactivated, has been associated with renal and CNS cancer development. VHL normally plays an important role in targeting for degradation of the HIF-1α (hypoxia inducible factor-1α) transcription factor, a primary positive regulator of vascular endothelial growth factor (VEGF) production. In this report we demonstrate that VHL destabilization can be induced by Src kinase and may be involved in other cancers, including breast cancer. We have found that elevated Src can trigger a drastic reduction in VHL stability even under normoxic conditions, through phosphorylation of VHL tyrosine residue 185, leading to ubiquitination and proteasome-mediated degradation of VHL. The Src-induced degradation of VHL protein leads to increased HIF-1α levels and transcriptional activity and increased VEGF production. In this manner, Src regulation of VHL protein stability may play an important role in promoting VEGF expression, tumor angiogenesis, and cancer progression.

Src-specific activity has been reported to be elevated in a high percentage of colon cancer cell lines and tumors, but the underlying mechanisms are largely unknown. In this study, we report that, in the seven cancer cell lines tested, Src-specific activity was elevated (5.2- to 18.7-fold) relative to normal colon cells (FHC). This activation of Src correlated with reduced phosphorylation at Y530 of Src, whereas there was no significant change in the level of phosphorylation at Y419. The membrane tyrosine phosphatase activity for a Src family-specific phosphopeptide substrate FCP (Fyn COOH-terminal peptide phosphorylated by Csk) was greatly increased in the cancer cells and was attributed to PTP1B in most of the cell lines. Membrane PTP1B protein levels were also greatly increased. Overexpression of PTP1B increased Src specific activity in colon cancer cells by reducing phosphorylation at Y530 of Src. It also increased anchorage-independent cell growth and this increase was blocked by the Src inhibitor PP2 and Src small interfering RNA (siRNA). Down-regulating PTP1B activity by PTP1B inhibitor CinnGEL 2Me or knocking down PTP1B using siRNA also reduced Src kinase activity and colony formation ability of colon cancer cells. PTP1B siRNA reduced tumor growth in nonobese diabetic/severe combined immunodeficient mice. This study suggests that (a) PTP1B can act as an important activator of Src in colon cancer cells via dephosphorylation at Y530 of Src and (b) elevated levels of PTP1B can increase tumorigenicity of colon cancer cells by activating Src.

The Src family of protein kinases (SFKs) mediates mitogenic signal transduction, and constitutive SFK activation is associated with tumorigenesis. To prevent constitutive SFK activation, the catalytic activity of SFKs in normal mammalian cells is suppressed mainly by two inhibitors called C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK), which inactivate SFKs by phosphorylating a consensus tyrosine near the C terminus of SFKs (Y(T)). The phosphorylated Y(T) intramolecularly binds to the SH2 domain of SFKs. This interaction, known as pY(T)/SH2 interaction, together with binding between the SH2 kinase linker and the SH3 domain of SFKs (linker/SH3 interaction) stabilizes SFKs in a "closed" inactive conformation. We previously discovered an alternative mechanism CHK employs to inhibit SFKs. This mechanism, referred to as the non-catalytic inhibitory mechanism, involves tight binding of CHK to SFKs; the binding alone is sufficient to inhibit SFKs. Herein, we constructed multiple active conformations of an SFK member, Hck, by systematically disrupting the two inhibitory interactions. We found that CHK employs the non-catalytic mechanism to inactivate these active conformations of Hck. However, CHK does not bind Hck when it adopts the inactive conformation in which both inhibitory interactions are intact. These data indicate that binding of CHK to SFKs via the non-catalytic mechanism is governed by the conformations of SFKs. Although CSK is also an inhibitor of SFKs, it does not inhibit SFKs by a similar non-catalytic mechanism. Thus, the non-catalytic inhibitory mechanism is a unique property of CHK that allows it to down-regulate multiple active conformations of SFKs.

Although C-terminal Src kinase (CSK)-homologous kinase (CHK) is generally believed to inactivate Src-family tyrosine kinases (SFKs) by phosphorylating their consensus C-terminal regulatory tyrosine (Tyr(T)), exactly how CHK inactivates SFKs is not fully understood. Herein, we report that in addition to phosphorylating Tyr(T), CHK can inhibit SFKs by a novel non-catalytic mechanism. First, CHK directly binds to the SFK members Hck, Lyn, and Src to form stable protein complexes. The complex formation is mediated by a non-catalytic Tyr(T)-independent mechanism because it occurs even in the absence of ATP or when Tyr(T) of Hck is replaced by phenylalanine. Second, the non-catalytic CHK-SFK interaction alone is sufficient to inactivate SFKs by inhibiting the catalytic activity of autophosphorylated SFKs. Third, CHK and Src co-localize to specific plasma membrane microdomains of rat brain cells, suggesting that CHK is in close proximity to Src such that it can effectively inactivate Src in vivo. Fourth, native CHK.Src complex exists in rat brain, and recombinant CHK.Hck complex exists in transfected HEK293T cells, implying that CHK forms stable complexes with SFKs in vivo. Taken together, our findings suggest that CHK inactivates SFKs (i) by phosphorylating their Tyr(T) and (ii) by this novel Tyr(T)-independent mechanism involving direct binding of CHK to SFKs. It has been documented that autophosphorylated SFKs can still be active, in some cases even when their Tyr(T) is phosphorylated. Thus, the ability of the Tyr(T)-independent mechanism to suppress the activity of both non-phosphorylated and autophosphorylated SFKs represents a fail-safe measure employed by CHK to down-regulate SFK signaling under all circumstances.