Concomitant Loss of Smad4 and Activation of Wnt Signaling Triggers Enterocyte De-differentiation and Adenoma Formation

In the current work, we add to the understanding of differentiated-cell-derived tumorigenesis by demonstrating that simultaneous loss of SMAD4 and activation of the WNT pathway triggers stem cell properties and adenoma formation in the differentiated epithelium. Under normal conditions, SMAD4 loss does not immediately affect the normal tissue homeostasis in the intestine. However, after approximately 6 months, adenomas will develop and feature elevated WNT signaling, suggesting that SMAD4 loss predisposes to WNT-driven tumors. Interestingly, ectopic elevation of WNT in the context of a SMAD4 mutant background triggers stem cell activity and adenoma formation in the differentiated epithelium. Thus Smad4 functions to suppress villus cells from re-entering the cell cycle and functioning as stem cells upon exposure to high levels of WNT. Thus, we report a new mechanism through which differentiated cells can contribute to tumor formation. Overall design: RNAs were extracted from crypt jejunal epithelia of the indicated genotypes (Smad4f/f;Villin-CreER(T2) and Smad4f/f; Ex3f/+;Villin-CreER(T2)) in triplicates, one day following 4 consecutive days of tamoxifen injection. Uninjected mice served as control. After flushing the freshly harvested jejunum with cold PBS, the epithelia was dissociated from underlying mesenchyme by incubating with 3 mM EDTA/PBS at 4°C as described previously (Perekatt, 2014), and filtered through 70 micron filter to isolate the crypts. The crypts were washed with PBS, and pelleted to remove excess PBS prior to addition of TRIzol for RNA extraction according to manufacturer's protocols.

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Perekatt AO, Shah PP, Cheung S, Jariwala N, Wu A, Gandhi V, Kumar N, Feng Q, Patel N, Chen L, Joshi S, Zhou A, Taketo MM, Xing J, White E, Gao N, Gatza ML, Verzi MP