Abstract 5006: SOX2 inhibits Wnt driven colon cancer cell proliferation.
Authors: Debasish Boral, Man-Tzu Wang, Daotai Nie
Journal: Cancer Research (2013)

Abstract APC mutation/s resulting in aberrant Wnt signaling, initiates intestinal polyposis and drives adenoma to carcinoma progression in human colon. This represents a target for specific therapeutic intervention and has generated extreme interest among the scientific community. However, it is becoming increasingly apparent that though Wnt signaling may cause robust proliferation, it is the quiescent cell population, defined by stem-ness related gene signatures, that evades chemo-radio therapy targeted at rapidly dividing cells, and perpetuates self-renewal ultimately causing relapse at local site and/or recurrence as distant metastases. We report that SOX2, an embryonic stem cell transcription factor, counteracts Wnt driven tumor cell proliferation and maintains quiescence in colo-rectal cancers (CRC). SOX2 expression increases with progressive staging of colon cancer patients. Using colon cancer cell lines transduced with Wnt reporter constructs, we identified distinct sub-populations with varying degrees of Wnt activity and found that SOX2 expression and Wnt activity are mutually exclusive in SW480 primary CRC cell line, but co-expressed in the isogenic SW620 metastatic CRC cell line. Knockdown of SOX2 in SW620 cell increased anchorage independence in vitro and tumor growth in xenograft models. This was accompanied by an increase of cells in S-phase and increased Ki67 index in IHC of xenograft tissue. Over-expression of SOX2 in SW480 cells resulted in decreased colony formation in soft agar and primary tumor growth in mice. Over-expression also caused increased fraction of cells in G0-G1 phase and decreased Ki67 staining in tumor tissue. In SW620 cell line, SOX2 knockdown not only increased basal Wnt activity, but also caused acquisition of Wnt activity in hitherto Wnt negative cells. Intriguingly, over-expression of SOX2 in SW480 cells, reversed the enhanced Wnt activity gained by loss-of-function APC mutation. Experiments conducted with both SOX2 silenced and over-expressed cell lines revealed that SOX2 inhibits phosphorylation of β-catenin at Serine 552 and 675 thereby decreasing its nuclear sequestration and activation. Screening with protein kinase and phosphatase inhibitors identified PHLPP and PHLPPL as candidate genes that are controlled by SOX2 and are responsible for its effects on β-catenin. Western Blot and qPCR analyses showed that SOX2 enhances PHLPP/L expression. Reporter assays demonstrated that SOX2 controls PHLPP/L promoter activity while ChIP assays confirmed binding of SOX2 to various regions of the respective promoters. IHC of SOX2 silenced xenograft tissue showed decreased PHLPP/L and increased pβ-catenin staining while SOX2 over-expression showed reciprocal changes. Our findings delineate the mechanism by which CRC cells suppress Wnt activity thereby maintaining a pool of quiescent cancer cells, and lay the basis for further research to identify and target these cells. Citation Format: Debasish Boral, Man-Tzu Wang, Daotai Nie. SOX2 inhibits Wnt driven colon cancer cell proliferation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5006. doi:10.1158/1538-7445.AM2013-5006