Identification of a new tumor suppressor pathway modulating rapamycin sensitivity in colorectal cancer

Abstract

Both genetic and epigenetic defects causing alterations to gene expression are implicated in cancer development. Epigenetic repression of gene transcription through DNA methylation is one of the fundamental mechanisms for inactivation of tumor suppressor genes in many cancers. Thus, identification of these silencing tumor suppressor genes could provide insight into the biological processes and pathways underlying tumorigenesis. In this thesis, we provide a comprehensive approach that integrates gene expression and ChIP-seq data for identification of DNA methylation silencing tumor suppressors and their-associated signaling pathways in colorectal cancer. A total of 203 colon cancer methylation silencing (CMS) genes have been identified and further characterized. Among the 203 CMS genes, PPP2R2B, one of the regulatory B subunits of protein phosphatase 2A (PP2A), was selected for further functional study. Tumor suppressor PP2A complex is a major serine/threonine phosphatase that serves as a critical cellular regulator of cell growth, proliferation, and survival. However, how its change in human cancer confers growth advantage is largely unknown. This study shows that PPP2R2B, encoding the B55? regulatory subunit of PP2A complex, is epigenetically inactivated by DNA hypermethylation in most of human colorectal cancer patients. Functional studies show that PPP2R2B re-expression in colorectal cancer (CRC) cells resulted in senescence, decreased cell proliferation, and xenograft tumor growth inhibition. In addition, PPP2R2B knockdown promotes cellular transformation in immortalized human epithelial cells. Therefore, gain- and loss-of-function data suggest that the loss of PPP2R2B facilitates oncogenic transformation. Mechanistically, we have demonstrated that PPP2R2B forms a functional PP2A complex targeting and inhibiting p70S6K and Myc phosphorylation. Taken together, our data show that PPP2R2B-specific PP2A complex functions as a tumor suppressor and its loss contributes to the deregulated S6K and Myc signaling, leading to growth advantage of CRC. Furthermore, we show that PPP2R2B-regulated tumor suppressor pathway has a role in modulating mTOR inhibitor sensitivity. The mTOR signaling pathway plays a central role in tumor development, making this pathway as attractive target for cancer therapy. Small molecule drugs targeting mTOR, such as rapamycin, have been shown to be promising for cancer therapy. However, the clinical responses to the rapamycin as mTOR-targeted therapy are frequently confounded by acquired resistance. In colon cancer, loss of PPP2R2B leads to induction of PDK1-dependent Myc phosphorylation in response to rapamycin. Conversely, re-expression of PPP2R2B blocks the PDK1-Myc signaling, leading to re-sensitization to rapamycin. We also show that genetic ablation or pharmacologic inhibition of PDK1 abrogates rapamycin-induced Myc phosphorylation, leading to rapamycin sensitization. Thus, our data demonstrate a new mechanism underlying rapamycin resistance in CRC, which is independent of PI3K-AKT and MAPK negative feedback loops. Together, these results identified PPP2R2B as a new biomarker to predict the rapamycin response and also provided a new therapeutic strategy to overcome the rapamycin resistance in cancer therapy.