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Aim: To quantitatively assess complex cancer cell phenotypes after gene inhibition by RNAi interference (RNAi), and assess the effect of these distinct morphologies on cell viability and cell motility using semi-automated image-based high-throughput screening. Method: GOhTRT cells were seeded and treated with the siRNA Human Druggable Genome Library (Dharmacon) by reverse transfection. Cells immunostained for DNA, tubulin and actin were imaged with the InCell Analyzer 1000 and processed using the InCell Analyzer software, CellHTS2 and RNAither. Statistical z-score analysis was performed on the combined A-T metric (F-actin area-α-tubulin area). The effect of RNAi knockdown on cell viability and cell motility were assessed using MTT cell proliferation assay and scratch wound assay. Results:127 high confidence hits (Z-factor>2) was refined to six genes (RRM2, ITGB8, GPS1, SPRY1, NOL1, MYO9B) on the basis of distinct morphologies, reproducible metrics, and functional pathway analysis. In siRRM2 cells nuclear displacement (ND) and A-T area was 1.598±0.076; p

Background & Aims During colorectal cancer pathogenesis, mutations and epigenetic events cause neoplastic behavior in epithelial cells by deregulating the Wnt, Ras-Raf-extracellular signal-regulated kinase (ERK), and transforming growth factor (TGF)-β-signaling pathways, among others. The TGF-β-signaling pathway is often inactivated in colon cancer cells by mutations in the gene encoding the TGF-β receptor TGFBR2 . The RAS-RAF-ERK pathway is frequently up-regulated in colon cancer via mutational activation of KRAS or BRAF . We assessed how these pathways interact in vivo and affect formation of colorectal tumors. Methods We analyzed intestinal tumors that arose in mice that express an oncogenic (active) form of Kras and that have Tgfbr2 inactivations—2 common molecular events observed in human colorectal tumors. LSL-KrasG12D mice were crossed with Villin-Cre;Tgfbr2E2flx/E2flx mice, which do not express Tgfbr2 in the intestinal epithelium. Results Neither inactivation of Tgfbr2 nor expression of oncogenic Kras alone was sufficient to induce formation of intestinal neoplasms. Histologic abnormalities arose in mice that expressed Kras , but only the combination of Tgfbr2 inactivation and Kras activation led to intestinal neoplasms and metastases. The cancers arose via a β-catenin-independent mechanism; the epidermal growth factor-signaling pathway was also activated. Cells in the resulting tumors proliferated at higher rates, expressed decreased levels of p15, and expressed increased levels of cyclin D1 and cdk4, compared with control cells. Conclusions A combination of inactivation of the TGF-β-signaling pathway and expression of oncogenic Kras leads to formation of invasive intestinal neoplasms through a β-catenin-independent pathway; these adenocarcinomas have the capacity to metastasize.