COBRE Pilot Project for Tianyan Gao: Center of Research in Obesity and Cardiovascular Disease

Cancer cells are rapidly dividing cells that have increased demands for energy and macromolecules. To cope with these elevated requirements cancer cells undergo major metabolic modifications, a process known as metabolic reprogramming. Evidence starts to emerge suggesting that the primary functions of activated oncogenes and inactivated tumor suppressors are to rewire cellular metabolic pathways in order to drive tumorigenesis. Among the metabolic alterations, increased de novo lipid biosynthesis has been recognized as one of the hallmarks associated with cancer cells. Since active FA synthesis occurs in normal tissues such as adipocytes and hormone-sensitive cells, we hypothesize that cancer cells hijack the normal lipogenic pathway to fuel membrane biogenesis for cell proliferation and to improve the overall fitness of cancer cells in order to promote tumor progression and resistance to chemotherapy drugs. However, relatively few studies have rigorously examined whether increased lipogenesis promotes tumor progression in colorectal cancer (CRC) and how lipogenic pathways are regulated. Our lab has been focused on understanding the role of a novel family of protein phosphatases, PHLPP, in inhibiting CRC initiation and progression. Following our initial discovery of PHLPP as a negative regulator of Akt, over the last five years we have: i) discovered that loss of PHLPP expression is frequently found in CRC patients, and re-expression of PHLPP in CRC cells inhibits cell proliferation in vitro and tumorigenesis in vivo1; ii) demonstrated that PHLPP expression is downregulated by hypoxia and decreased PHLPP expression contributes to hypoxia-induced chemoresistance in CRC cells2; iii) identified PHLPP as a novel inhibitor of mTOR-mediated protein translation by directly inactivating S6K3; and iv) demonstrated that PHLPP-loss induces EMT by upregulating RAS/RAF signaling in CRC cells; and genetic deletion of Phlpp1 promotes the development of invasive intestinal adenocarcinoma and decreases survival in ApcMin mice4. Collectively, our studies provide strong evidence supporting the tumor suppressor role of PHLPP in CRC. Given the close link between PHLPP and PI3K/Akt/mTOR pathway, we have begun to investigate the effect of PHLPP-loss on cellular metabolism in CRC. In exciting recent findings, we demonstrate that the expression of nuclear SREBP1, a key activator of lipid biosynthesis, is increased in PHLPP knockdown cells suggesting a potential upregulation of lipogenesis. Consistently, MEF cells isolated from PHLPP knockout mice have enhanced lipogenesis during differentiation into adipocytes. In addition, silencing PHLPP expression leads to increased glucose uptake, lactate production, and Krebs cycle activity in CRC cells indicating altered metabolism. Therefore, the central hypothesis driving this proposed study is that PHLPP plays an essential role in inhibiting lipogenesis by negatively regulating the PI3K/Akt/mTOR pathway, and loss of PHLPP expression promotes CRC progression as the result of metabolic reprogramming (Fig. 1). We have designed experiments with the following Specific Aims: Aim 1. To delineate the molecular mechanism by which PHLPP regulates lipogenesis in CRC cells. We will test the hypothesis that PHLPP regulates lipogenesis by controlling SREBP1 activation downstream of Akt and S6K. The effect of PHLPP-loss on promoting lipogenesis will be determined. In addition, we will examine how PHLPP-loss affects lipid metabolism in CRC cells in PHLPP knockdown CRC cells using the Stable Isotope-Resolved Metabolomics (SIRM) analysis. Aim 2. To determine the functional importance of PHLPP-mediated regulation of lipogenesis in CRC. We will determine if increased lipogenesis protect PHLPP deficient CRC cells from energy stress. We have previously shown that PHLPP depletion promotes chemo-resistance in CRC cells. Here, we will test the hypothesis that PHLPP-loss renders CRC cells more resistant to chemotherapy drugs as the result of alteration in lipogensis. By collaborating with Dr. Evers, we have an ongoing collection of fresh CRC tumor tissues from patients who under surgery at the Markey Cancer Center. Primary CRC cells will be generated from patient samples. The effect of PHLPP-mediated regulation of lipogenesis on drug sensitivity will be determined in a panel of CRC cell lines, as well as primary CRC cells. In summary, our proposed study stems from a novel hypothesis that PHLPP-loss plays a pivotal role in driving CRC progression by controlling lipid metabolism. Results from our study on determine how PHLPPmediated effect on lipogenesis affects chemo-sensitivity is clinically important as they provide the mechanistic insight needed to guide better application of cancer therapies. Fig. 1. PHLPP inhibits lipogenesis in cancer cells by negatively regulating the PI3K/AKT/mTOR pathway