Abstract
BackgroundStabilization of RAS is a key event for the hyper-activation of Wnt/β-catenin signaling and activation of cancer stem cell (CSC) in colorectal cancer (CRC). WD Repeat protein 76 (WDR76) mediates the polyubiquitination-dependent degradation of RAS in hepatocellular carcinoma (HCC). We investigated whether WDR76 destabilizes RAS and acts as a tumor suppressor inhibiting CSC activation in CRC.MethodsWe generated mice with deletion of Wdr76 (Wdr76−/−) and crosses of Wdr76−/− with ApcMin/+ (Wdr76−/−; ApcMin/+) and compared them with wildtype mice (Wdr76+/+) and ApcMin/+ mice (Wdr76+/+; ApcMin/+), respectively. Intestinal crypt lengthening, tumorigenesis and CSC activation were analyzed by histology, immunohistochemistry, and immunoblotting. CRC cell line was engineered to stably express or knockdown WDR76 or control vector and was analyzed after spheroid culture.ResultsWdr76−/− mice, with increased Ras level, displayed crypt elongation and hyper-proliferation. Wdr76−/−; ApcMin/+ mice developed more tumors with bigger sizes than ApcMin/+ mice and their tumors showed increased proliferation and CSC activation with elevated RAS and β-catenin levels. In CRC cells, overexpression or knockdown of WDR76 decreased or increased the numbers and sizes of CRC spheroids with inhibition or activation of CSC markers, respectively. In human CRC, lower level of WDR76 was associated with poor patient survival.ConclusionsIn analyses of mice with deletion of Wdr76 and CRC spheroids, we found that RAS stability plays important roles in tumorigenesis by affecting proliferation and CSC activation. Our results suggest that destabilization of RAS by WDR76 is a potential strategy for targeting malignant CRC involving CSC activation.Graphic abstract
Highlights
Stabilization of RAS is a key event for the hyper-activation of Wnt/β-catenin signaling and activation of cancer stem cell (CSC) in colorectal cancer (CRC)
Based on our observation of increased RAS protein levels in CSC-like population with high expression of CSC markers compared to low CSC counterpart sorted from a human CRC cell line, we investigated the role of WD Repeat protein 76 (WDR76) as a modulator of RAS abundance and CSC activation contributing to tumorigenesis in CRC
We found that pan-Ras was stabilized in the intestinal crypt base, where Lgr5+ intestinal stem cell (ISC) reside in the murine small intestine (Additional file 1: Figure S1A, B)
Summary
Stabilization of RAS is a key event for the hyper-activation of Wnt/β-catenin signaling and activation of cancer stem cell (CSC) in colorectal cancer (CRC). We investigated whether WDR76 destabilizes RAS and acts as a tumor suppressor inhibiting CSC activation in CRC. Results: Wdr76−/− mice, with increased Ras level, displayed crypt elongation and hyper-proliferation. Wdr76−/−; ApcMin/+ mice developed more tumors with bigger sizes than ApcMin/+ mice and their tumors showed increased proliferation and CSC activation with elevated RAS and β-catenin levels. In CRC cells, overexpression or knockdown of WDR76 decreased or increased the numbers and sizes of CRC spheroids with inhibition or activation of CSC markers, respectively. The intestinal crypt stem cell niche provides Wnt and epidermal growth factor (EGF) signals that maintain resident stem cells and can instruct progenitor cells to revert to a stem cell state when the original stem cells are lost. When CSCs are eradicated, transit amplifying (TA) cells and differentiated cells can be reprogrammed into CSCs by the niche through plasticity.
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