c-MET and KRAS: Signalling and Clinical Implications in Colorectal Cancer

Colorectal cancer (CRC) is the third leading cause of death from cancer in North America. The KRAS gene is mutated in approximately 40-50% of all CRC, and this mutation precludes treatment with promising targeted therapeutics. c-MET is a receptor tyrosine kinase that is overexpressed in ~70% of CRCs, and expression is correlated with disease progression. We hypothesized that high c-MET plus mutant KRAS would result poor survival of CRC patients, by activating unique signalling pathways that may be targeted for therapeutic purposes. To this end, we used phosphoproteomics in a KRAS mutant cell line, and identified proteins phosphorylated on tyrosine in response to HGF stimulation, including a subset of those that contain SRC family kinase consensus motifs. Small molecule inhibitors of either SRC or c-MET reduced tyrosine phosphorylation of both proteins, indicating reciprocal signalling. We chose the c-MET target p190RhoGAP for future study, as it is often ubiquitously bound to p120RasGAP via phosphorylated tyrosine. We found that RasGAP expression is mediated in part by KRAS signalling, and that expression of RasGAP could partly rescue tumourigenicity of a CRC cell line where the mutant KRAS allele has been inactivated, indicating the requirement of both mutant KRAS and RasGAP expression in this model. We then conclude by looking at CRC patient samples to determine the role of KRAS mutation in the progression and survival of CRC. We found that both KRAS and c-MET copy number are correlated to KRAS mutation status, and that c-MET polysomy plus KRAS mutation leads to worse overall survival than KRAS mutation alone. Overall, we identified novel targets of c-MET and KRAS oncogenic signaling, and identify a population which may derive the most benefit from treatments targeting both of these lesions.Ph

TRIM14 is a Putative Tumor Suppressor and Regulator of Innate Immune Response in Non-Small Cell Lung Cancer

Non-small-cell lung carcinoma (NSCLC) accounts for 85% of malignant lung tumors and is the leading cause of cancer deaths. Our group previously identified Tripartite Motif 14 (TRIM14) as a component of a prognostic multigene expression signature for NSCLC. Little is known about the function of TRIM14 protein in normal or disease states. We investigated the functional and prognostic role of TRIM14 in NSCLC using in vitro and in vivo perturbation model systems. Firstly, a pooled RNAi screen identified TRIM14 to effect cell proliferation/survival in NSCLC cells. Secondly, silencing of TRIM14 expression significantly enhanced tumor growth in NSCLC xenograft mouse models, while exogenous TRIM14 expression attenuated tumorigenesis. In addition, differences in apoptotic activity between TRIM14-deficient and control tumors suggests that TRIM14 tumor suppressor activity may depend on cell death signaling pathways. TRIM14-deficient cell lines showed both resistance to hypoxia-induced cell death and attenuation of interferon response via STAT1 signaling. Consistent with these phenotypes, multivariate analyses on published mRNA expression datasets of over 600 primary NSCLCs demonstrated that low TRIM14 mRNA levels are significantly associated with poorer prognosis in early stage NSCLC patients. Our functional data therefore establish a novel tumor suppressive role for TRIM14 in NSCLC progression

p120RasGAP is a mediator of rho pathway activation and tumorigenicity in the DLD1 colorectal cancer cell line.

KRAS is mutated in ∼40% of colorectal cancer (CRC), and there are limited effective treatments for advanced KRAS mutant CRC. Therefore, it is crucial that downstream mediators of oncogenic KRAS continue to be studied. We identified p190RhoGAP as being phosphorylated in the DLD1 CRC cell line, which expresses a heterozygous KRAS G13D allele, and not in DKO4 in which the mutant allele has been deleted by somatic recombination. We found that a ubiquitous binding partner of p190RhoGAP, p120RasGAP (RasGAP), is expressed in much lower levels in DKO4 cells compared to DLD1, and this expression is regulated by KRAS. Rescue of RasGAP expression in DKO4 rescued Rho pathway activation and partially rescued tumorigenicity in DKO4 cells, indicating that the combination of mutant KRAS and RasGAP expression is crucial to these phenotypes. We conclude that RasGAP is an important effector of mutant KRAS in CRC

Assay interference and off-target liabilities of reported histone acetyltransferase inhibitors

A substantial obstacle in basic research is the use of poorly validated tool compounds with purported useful biological functions. Here, the authors systematically profile widely used histone acetyltransferase inhibitors and find that the majority are nonselective interference compounds

RhoGAP phosphorylation and RasGAP expression in DLD1 and DKO4 cell lines.

<p>A) RhoGAP and RasGAP were immunoprecipitated from DLD1 and DKO4 cells. Immunoprecipitates were subjected to Western blotting and probed for total phosphotyrosine, RasGAP and RhoGAP. Western Blotting of whole cell lysates (A) and rt-QPCR (C) were used to determine total protein and mRNA levels respectively in these cell lines.</p

RasGAP expression is mediated in part by KRAS.

<p>Wild-type (WT) or mutant KRAS was overexpressed in DKO4 cells. mRNA was extracted from cells and quantified using rt-qPCR to measure KRAS (A) or RASA1 (B). C) Western blotting showing levels of these proteins, along with activation status of KRAS. Correlation of mRNA (D) and protein expression using densitometry analysis of Western blotting (E) of KRAS and RASA1 after knockdown of KRAS using 11 different shRNAs. For protein correlation, outliers over 3 standard deviations from the mean were excluded. All quantification is relative to empty vector. Statistical analysis of expression using unpaired t-test, ***p<0.001, *p<0.05.</p

An overview of the c-MET signaling pathway

c-MET is a receptor tyrosine kinase that, after binding with its ligand, hepatocyte growth factor, activates a wide range of different cellular signaling pathways, including those involved in proliferation, motility, migration and invasion. Although c-MET is important in the control of tissue homeostasis under normal physiological conditions, it has also been found to be aberrantly activated in human cancers via mutation, amplification or protein overexpression. This paper provides an overview of the c-MET signaling pathway, including its role in the development of cancers, and provides a rationale for targeting the pathway as a possible treatment option

Overexpression of RasGAP expression in DLD1 cells rescues RhoGAP phosphorylation and overall GAP activity.

<p>A) mRNA expression of RasGAP after overexpression in DKO4 cells compared to the GFP vector control. B) Real-time NMR analysis of RasGAP activity, showing mean rate of GTP hydrolysis (B) and GAP activity over time (C). Each curve in (C) is derived from a single representative experiment. Error bars in (B) denote standard error of the mean (SEM). D) RAS activity assay showing levels of active KRAS after RasGAP overexpression. Numbers denote densitometry values from this blot, which is representative of three biological replicates. E) RhoGAP was immunoprecipitated from cell lines, subjected to Western blotting, then probed for total phosphotyrosine, RasGAP and RhoGAP.</p

Summary of findings and proposed mechanism.

<p>In DLD1 cells, active KRAS stabilizes RasGAP expression, which in turn binds to and stabilizes RhoGAP phosphorylation. This complex then activates Rho pathway activation, either by sequestration of RhoGAP away from Rho, or by increasing Rho turnover. In DKO4 cells, RasGAP is not expressed, due in part to a truncating mutation and in part to lack of expression downstream of active KRAS. In this situation, RhoGAP is not phosphorylated, and so Rho pathway phenotypes are inactive. When RasGAP is overexpressed in DKO4, RhoGAP is once again phosphorylated and Rho pathway is active- however, lack of stabilization and/or contributing signaling pathways from active RAS means that tumorigenicity does not attain the same level as DLD1. The bottom of the figure summarizes the main characteristics of RhoGAP and RasGAP in these cell lines.</p

Identification of a novel truncating mutation in RASA1.

<p>A) Chromatogram showing relative intensities of each base pair after Sanger sequencing in both the genomic and cDNA derived from the cell lines. B) Illustration of location of the mutation at the RasGAP protein level. C) RASA1 expression data derived from all cell lines in the Broad Institute Cancer Cell Line Encyclopedia. Bars represent mean.</p