The heterogeneity of cancer cell signaling is a significant obstacle for the effective development and clinical use of molecularly targeted therapies. As a contribution to a better understanding of the diversity of signaling activities in colorectal cancers (CRCs), we have analyzed the activity of Src family kinases (SFKs), which are implicated in human cancer development, in 64 CRC cell lines. A striking diversity of SFK activity was observed within this panel.Importantly, all CRC lines tested depend on SFK activity for their growth. In addition, SFK activity levels strongly correlated with global levels of tyrosine-phosphorylated (pTyr) proteins in CRC lines. SFK inhibition substantially reduced these pTyr levels, suggesting that SFKs may function as signal integration points and master controllers for the pTyr protein status in CRC lines. The majority of analyzed CRC lines with high-SFK activity express activated c-Met (pYpY1234/1235), a receptor tyrosine kinase contributing to the regulation of cell proliferation, migration, and invasion. Inhibition of SFKs reduced c-Met phosphorylation in most cases, indicating a reversed signal flow from SFK to c-Met. We conclude that SFK activity is important for the growth of CRC lines, although only low activity levels are required. If this also is true for CRC patients, tumors with low-SFK activity may be particularly sensitive to SFK inhibitors, and such patients should be targeted in clinical trials testing SFK inhibitors.colon ͉ therapy ͉ kinase inhibition ͉ molecular heterogeneity

The p53 regulatory network is critically involved in preventing the initiation of cancer. In unstressed cells, p53 is maintained at low levels and is largely inactive, mainly through the action of its two essential negative regulators, HDM2 and HDMX. p53 abundance and activity are up-regulated in response to various stresses, including DNA damage and oncogene activation. Active p53 initiates transcriptional and transcription-independent programs that result in cell cycle arrest, cellular senescence, or apoptosis. p53 also activates transcription of HDM2, which initially leads to the degradation of HDMX, creating a positive feedback loop to obtain maximal activation of p53. Subsequently, when stress-induced post-translational modifications start to decline, HDM2 becomes effective in targeting p53 for degradation, thus attenuating the p53 response. To date, no clear function for HDMX in this critical attenuation phase has been demonstrated experimentally. Like HDM2, the HDMX gene contains a promoter (P2) in its first intron that is potentially inducible by p53. We show that p53 activation in response to a plethora of p53-activating agents induces the transcription of a novel HDMX mRNA transcript from the HDMX-P2 promoter. This mRNA is more efficiently translated than that expressed from the constitutive HDMX-P1 promoter, and it encodes a long form of HDMX protein, HDMX-L. Importantly, we demonstrate that HDMX-L cooperates with HDM2 to promote the ubiquitination of p53 and that p53-induced HDMX transcription from the P2 promoter can play a key role in the attenuation phase of the p53 response, to effectively diminish p53 abundance as cells recover from stress.The tumor suppressor protein p53 functions primarily as a stress-inducible transcriptional activator of genes that promote cell cycle arrest and apoptosis (1). Stress-induced p53 activation can form a rate-limiting barrier to tumorigenesis (2, 3), and the manipulation of p53 function is key to the mechanism of action of many cancer chemotherapeutic strategies (4, 5). In unstressed cells, p53 is maintained at low levels and inactive, largely through the action of several p53-inducible negative feedback pathways, the most extensively studied of which involves the oncoproteins HDM2 and HDMX (also called MDM4) (MDM2 and MDMX/MDM4 in mice) (6, 7). Considerable research effort has been applied to understanding the mechanisms whereby these two proteins regulate p53 function. HDM2 and HDMX both contain an N-terminal pocket that binds to the primary transactivation domain of p53; they can, therefore, function independently of each other to repress p53-dependent transcription (8 -10). HDM2 also forms both HDM2-HDM2 homodimers and HDM2-HDMX heterodimers. These function as E3 ubiquitin ligases for p53; monoubiquitination of p53 by HDM2 inhibits p53 activity by both inhibiting acetylation and promoting nuclear export, whereas polyubiquitination promotes proteasome-mediated p53 degradation and is largely responsible for the rapid turnover of p53 protein that occurs in proli...

BackgroundSrc family kinases (SFK) are implicated in the development of some colorectal cancers (CRC). One SFK member, Lck, is not detectable in normal colonic epithelium, but becomes aberrantly expressed in a subset of CRCs. Although SFK have been extensively studied in fibroblasts and different types of immune cells, their physical and functional targets in many epithelial cancers remain poorly characterised.Results64 CRC cell lines were tested for expression of Lck. SW620 CRC cells, which express high levels of Lck and also contain high basal levels of tyrosine phosphorylated (pY) proteins, were then analysed to identify novel SFK targets. Since SH2 domains of SFK are known to often bind substrates after phosphorylation by the kinase domain, the LckSH2 was compared with 14 other SH2s for suitability as affinity chromatography reagent. Mass spectrometric analyses of LckSH2-purified pY proteins subsequently identified several proteins readily known as SFK kinase substrates, including cortactin, Tom1L1 (SRCASM), GIT1, vimentin and AFAP1L2 (XB130). Additional proteins previously reported as substrates of other tyrosine kinase were also detected, including the EGF and PDGF receptor target Odin. Odin was further analysed and found to contain substantially less pY upon inhibition of SFK activity in SW620 cells, indicating that it is a formerly unknown SFK target in CRC cells.ConclusionRapid identification of known and novel SFK targets in CRC cells is feasible with SH2 domain affinity chromatography. The elucidation of new SFK targets like Odin in epithelial cancer cells is expected to lead to novel insight into cancer cell signalling mechanisms and may also serve to indicate new biomarkers for monitoring tumor cell responses to drug treatments.