We have recently identified a metastasis suppressor gene for colorectal cancer: AES/Aes, which encodes an endogenous inhibitor of NOTCH signaling. When Aes is knocked out in the adenomatous epithelium of intestinal polyposis mice, their tumors become malignant, showing marked submucosal invasion and intravasation. Here, we show that one of the genes induced by NOTCH signaling in colorectal cancer is DAB1/Dab1. Genetic depletion of DAB1 suppresses cancer invasion and metastasis in the NOTCH signaling–activated mice. DAB1 is phosphorylated by ABL tyrosine kinase, which activates ABL reciprocally. Consistently, inhibition of ABL suppresses cancer invasion in mice. Furthermore, we show that one of the targets of ABL is the RAC/RHOGEF protein TRIO, and that phosphorylation at its Tyr residue 2681 (pY2681) causes RHO activation in colorectal cancer cells. Its unphosphorylatable mutation TRIO Y2681F reduces RHOGEF activity and inhibits invasion of colorectal cancer cells. Importantly, TRIO pY2681 correlates with significantly poorer prognosis of patients with colorectal cancer after surgery. Significance: These results indicate that TRIO pY2681 is one of the downstream effectors of NOTCH signaling activation in colorectal cancer, and can be a prognostic marker, helping to determine the therapeutic modality of patients with colorectal cancer. Cancer Discov; 5(2); 198–211. ©2014 AACR. See related commentary by Kranenburg, p. 115 This article is highlighted in the In This Issue feature, p. 97

Caudal-related homeoprotein CDX2 is expressed in intestinal epithelial cells, in which it is essential for their development and differentiation. A tumor suppressor function is suggested by evidence that CDX2 levels are decreased in human colon cancer specimens and that an inactivating mutation of Cdx2 in Apc^Δ716 mice markedly increases the incidence of colonic polyps. In this study, we investigated roles for transcriptional and nontranscriptional functions of CDX2 in suppression of colonic tumorigenesis. Mutagenic analysis of CDX2 revealed that loss of function stabilizes CDK inhibitor p27^Kip1 by a nontranscriptional but homeodomain-dependent mechanism that inhibits cyclin E-CDK2 activity and blocks G0/G1-S progression in colon cancer cells. p27^Kip1 stabilization was mediated by an inhibition of ubiquitylation-dependent proteolysis associated with decreased phosphorylation of Thr187 in p27^Kip1. siRNA-mediated knockdown of p27^Kip1 relieved the decrease in cyclin E-CDK2 activity and S-phase cell fraction elicited by CDX2 expression. Together, these results implicate a nontranscriptional function of CDX2 in tumor suppression mediated by p27^Kip1 stabilization. Up to approximately 75% of low-CDX2 human colon cancer lesions show reduced levels of p27^Kip1, whereas approximately 68% of high-CDX2 lesions retain expression of p27^Kip1. These results show that low levels of CDX2 accelerate colon tumorigenesis by reducing p27^Kip1 levels. Cancer Res; 71(2); 593–602. ©2011 AACR.

Recent advances allowed culturing and examination of patient-derived colorectal cancer (PD-CRC) cells as organoids or spheroids. To be applied to practical personalized medicine, however, current methods still need to be strengthened for higher efficiency. Here we report an improved method to propagate PD-CRC tumor initiating cells (TICs) in spheroid culture. We established > 100 cancer spheroid lines derived from independent colorectal cancer patients employing a serum-containing medium with additional inhibitors, Y27632 and SB431542. Because colorectal cancer spheroids showed wide-range growth rates depending on the patient tumors, we searched for supplementary factors that accelerated proliferation of slow-growing CRC-TIC spheroids. To this end, we introduced a convenient growth-monitoring method using a luciferase reporter. We found that epidermal growth factor (EGF) and/or basic fibroblast growth factor (bFGF) were critical for steady propagation of a subset of CRC-TIC spheroids carrying the wild-type RAS and RAF genes. We also identified 5'-(N-ethyl-carboxamido)-adenosine (NECA), an adenosine receptor agonist, as an essential supplement for another subset of spheroids. Based on these results, we propose to optimize culture conditions for CRC-TIC spheroids by adjusting to the respective tumor samples. Our method provides a versatile tool that can be applied to personalized chemotherapy evaluation in prospective clinical studies.