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

Lipocalin2 Promotes Invasion, Tumorigenicity and Gemcitabine Resistance in Pancreatic Ductal Adenocarcinoma

<div><p>Lipocalin 2 (LCN2) is a small secreted protein and its elevated expression has been observed in pancreatic as well as other cancer types. LCN2 has been reported to promote resistance to drug-induced apoptosis, enhance invasion through its physical association with matrix metalloproteinase-9, and promote <em>in vivo</em> tumor growth. LCN2 was found to be commonly expressed in patient PDAC samples and its pattern of immunohistochemical staining intensified with increasing severity in high-grade precursor lesions. Downregulation of LCN2 in two pancreatic ductal adenocarcinoma cell lines (BxPC3 and HPAF-II) with high LCN2 expression significantly reduced attachment, invasion, and tumour growth <em>in vivo</em>, but not proliferation or motility. Downregulation of LCN2 in two pancreatic ductal adenocarcinoma cell lines (BxPC3 and HPAF-II) with high expression significantly reduced attachment, invasion, and tumour growth <em>in vivo</em>. In contrast, LCN2 overexpression in PANC1, with low endogenous expression, significantly increased invasion, attachment, and enhanced tumor growth. Suppression of LCN2 in BxPC3 and HPAF-II cells increased their sensitivity to gemcitabine <em>in vitro</em>, and <em>in vivo</em> when BxPC3 was tested. Furthermore, LCN2 promotes expression of VEGF and HIF1A which contribute to enhanced vascularity. These overall results demonstrate that LCN2 plays an important role in the malignant progression of pancreatic ductal carcinoma and is a potential therapeutic target for this disease.</p> </div

LCN2 promotes adhesion, invasion, and gemcitabine resistance in PDAC cells.

<p>Adhesion assays on the (A) H6c7 KrT, (B) BxPC3, HPAF-II, and PANC1 cell lines. Fold changes were calculated by comparing the KD to NS or LCN2 to EV (n = 3). The fold changes in invasive ability were calculated by comparing the effects of the shRNA constructs against the NS control, or LCN2 overexpression compared to the EV control. Invasive ability was assessed in (C) H6c7KrT cells (n = 3), (D) BxPC3, HPAF-II, and PANC1 cells (n = 6) were seeded onto Matrigel or collagen IV coated membranes. To assess MMP-9 activity gelatin zymography was performed on the conditioned media from (E) H6c7 KrT cells, (F) BxPC3, HPAF-II, and PANC1 cell lines (n = 3). (G) PI exclusion assays for cell death (n = 6) and (H) immunoblot analysis after 72 hours treatment by gemcitabine on the BxPC3, HPAF-II, and PANC1 cell lines (n = 3). (Gem., gemcitabine; C3, caspase 3; CC3, cleaved caspase 3; * denotes significant differences between the test and control samples student t-tests or one-way ANOVA and Bonferroni’s post hoc tests where appropriate.).</p

LCN2 promotes resistance to gemcitabine and angiogenesis.

<p>Effect of gemcitabine on the growth of tumors formed by (A) BxPC3-NS and –LCN2KD2 cell lines and (B) with PANC1-EV and –LCN2 cell lines [*denotes significance between vehicle treated cell lines (n = 10 per group, p<0.0001, mixed model multiple regression) †denotes significance between vehicle and gemcitabine treated mice injected with BxPC3 LCN2KD2 (n = 10 per group, p = 0.0003, mixed model multiple regression)]. (C) Protein lysates isolated from BxPC3 and PANC1 xenografts were assayed for caspase-3 cleavage after gemcitabine treatment (n = 10). (D) Representative histological images of xenografts formed by BxPC3 NS and –LCN2KD2, and PANC1-EV and –LCN2 cells after H&E, and immunostaining for LCN2, Ki67, and murine CD31. (E) Vascular density in five hot spots at high magnification in the BxPC3 NS and –LCN2KD2, and PANC1-EV and –LCN2 xenografts. The mRNA expression of (F) HIF1A and (G) VEGF in the BxPC3 and PANC1 xenografts. Gene expression was compared between KD and NS, or LCN2 and EV. [*denotes significance between KD and NS, or LCN2 and EV (n = 20; p<0.05, student t-test)].</p

LCN2 promotes tumor growth and invasion <i>in vivo</i>.

<p>Growth curves of tumors formed by (A) BxPC3 NS and –LCN2KD2, (B) HPAF-II NS and –LCN2KD2, and (C) PANC1 EV and –LCN2 cells implanted subcutaneously in SCID mice. Gelatin zymography was perform on protein lysates isolated from (D) BxPC3 NS and –LCN2KD2, (E) HPAF-II NS and –LCN2KD2, and (F) PANC1 EV and –LCN2 xenografts (*denotes significance between KD and NS, or LCN2 and EV, p<0.05, student t-tests, n = 5).</p

LCN2 promotes survival and adhesion.

<p>(A) LCN2 enhances the expression of anti-apoptotic genes and downregulated the pro-apoptotic genes. (B) LCN2 enhances adhesion and ECM. Target genes whose expression was up/downregulated by at least 1.5-fold in the control cell line and xenograft samples compared to the LCN2 downregulated cell line and xenograft samples. Red triangles denote increased expression and green triangles denote decreased expression. The mRNA expression of (C) AIFM, (D) BIRC2, (E) FAIM, (F) MCL-1, (G) LAMAC2, (H) MMP7, (I) CDH11, and (J) ITGA2 were assessed in BxPC3, HPAF-II, and PANC1 cell lines. (* denotes significant differences between the test and control samples (p<0.05, student t-tests, n = 3).</p

LCN2 expression in pancreatic neoplastic lesions and PDAC cell lines.

<p>(A) The LCN2 immunostaining pattern for normal (n = 31), PanIN1 (n = 22), PanIN-2 (n = 13), PanIN -3 and PDAC (n = 82). Mean scores and the SEM for LCN2 immunostaining are noted below the sections. (B) LCN2 gene expression was examined in 21 different PDAC cell lines. Relative expression was normalized using loading controls and then normalized to the H6c7 ratio. (C) Representative immunoblots of LCN2 and GAPDH protein expression in PDAC cell lines.</p