Colorectal cancer (CRC) initiation and growth is often attributed to stem cells, yet little is known about the regulation of these cells. We show here that a subpopulation of Prox1-transcription-factor-expressing cells have stem cell activity in intestinal adenomas, but not in the normal intestine. Using in vivo models and 3D ex vivo organoid cultures of mouse adenomas and human CRC, we found that Prox1 deletion reduced the number of stem cells and cell proliferation and decreased intestinal tumor growth via induction of annexin A1 and reduction of the actin-binding protein filamin A, which has been implicated as a prognostic marker in CRC. Loss of Prox1 also decreased autophagy and the survival of hypoxic tumor cells in tumor transplants. Thus, Prox1 is essential for the expansion of the stem cell pool in intestinal adenomas and CRC without being critical for the normal functions of the gut.

Estrogen receptor-α (ERα) and transforming growth factor (TGF)-β signaling pathways are major regulators during mammary gland development, function and tumorigenesis. Predominantly, they have opposing roles in proliferation and apoptosis. While ERα signaling supports growth and differentiation and is antiapoptotic, mammary gland epithelia cells are very sensitive to TGF-β-induced cell cycle arrest and apoptosis. Their regulatory pathways intersect, and ERα blocks TGF-β pathway by multiple means, including direct interactions of its signaling components, Smads. However, relatively little is known of the dysfunction of their interactions in cancer. A better understanding would help to develop new strategies for breast cancer treatment.

Membrane fusion requires the formation of a complex between a vesicle protein (v-SNARE) and the target membrane proteins (t-SNAREs). Syntaxin 4 is a t-SNARE that, according to previous overexpression studies, is predominantly localized at the plasma membrane. In the present study endogenous syntaxin 4 was found in intracellular vesicular structures in addition to regions of the plasma membrane. In these vesicular structures syntaxin 4 colocalized with rab11, a marker of recycling endosomes. Furthermore, syntaxin 4 colocalized with actin at the dynamic regions of the plasma membrane. Treatment with N-ethylmaleimide, the membrane transport inhibitor, caused an increased accumulation of syntaxin 4/rab11 positive vesicles in actin ¢lament-like structures. Finally, puri¢ed recombinant syntaxin 4 but not syntaxin 2 or 3 cosedimented with actin ¢laments in vitro, suggesting direct interaction between these two proteins. Taken together, these data suggest that syntaxin 4 regulates secretion at the actin-rich areas of the plasma membrane and may be recycled through rab11 positive intracellular membranes. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

In the majority of microsatellite-stable colorectal cancers (CRCs), an initiating mutation occurs in the adenomatous polyposis coli (APC) or β-catenin gene, activating the β-catenin/TCF pathway. The progression of resulting adenomas is associated with oncogenic activation of KRas and inactivation of the p53 and TGF-β/Smad functions. Most established CRC cell lines contain mutations in the TGF-β/Smad pathway, but little is known about the function of TGF-β in the early phases of intestinal tumorigenesis. We used mouse and human ex vivo 3D intestinal organoid cultures and in vivo mouse models to study the effect of TGF-β on the Lgr5 + intestinal stem cells and their progeny in intestinal adenomas. We found that the TGF-β-induced apoptosis in Apc-mutant organoids, including the Lgr5 + stem cells, was mediated by up-regulation of the BH3-only proapoptotic protein Bcl-2-like protein 11 (Bim). BH3-mimetic compounds recapitulated the effect of Bim not only in the adenomas but also in human CRC organoids that had lost responsiveness to TGF-β-induced apoptosis. However, wild-type intestinal crypts were markedly less sensitive to TGF-β than Apcmutant adenomas, whereas the KRas oncogene increased resistance to TGF-β via the activation of the Erk1/2 kinase pathway, leading to Bim down-regulation. Our studies identify Bim as a critical mediator of TGF-β-induced apoptosis in intestinal adenomas and show that the common progression mutations modify Bim levels and sensitivity to TGF-β during intestinal adenoma development.A ctivation of the β-catenin/TCF transcription factor (Wnt) pathway is the initiating event in the majority of human colorectal cancers (CRCs) and one of the key regulators of CRC pathogenesis (1, 2). The β-catenin level in cells is controlled through a multiprotein complex that contains the adenomatous polyposis coli (APC) protein. In 70-80% of individuals suffering from the sporadic version of CRC, both APC alleles are mutated and thus inactivated, resulting in elevated nuclear β-catenin levels (1). Recently, the intestinal stem cells have been identified as the cells of origin of CRC (3, 4).According to the model presented by Fearon and Vogelstein (5), the initiating APC or CTNNB1 mutation is followed by oncogenic activation of the KRAS gene and inactivation of the TGF-β signal transduction pathway and the TP53 tumor suppressor. Recent genome-wide analysis by the Cancer Genome Atlas Network has demonstrated the high frequency of mutations in the β-catenin/TCF, TGF-β, phosphoinositide 3-kinase (PI3K) and p53 pathways in CRC (6). Full understanding of the roles of the driver mutations in colorectal tumors has been hampered by lack of appropriate ex vivo model systems for studies of CRC progression, and thus it has been difficult to study the effects of TGF-β in intestinal adenoma cells at the early stages of tumor development. Although TGF-β receptors have been deleted in Apc-mutant mouse models of CRC (7,8), these studies do not reveal the mechanism of TGF-β action in intestinal adenomas or give further i...