UK SPORE in Gastrointestinal Cancer

The incidence of HCC (HCC) is rapidly increasing in the U.S., particulariy among African American and Hispanic males. Strong circumstantial evidence suggests that this is due to an increased prevalence of hepatitis C virus (HCV). Antiviral therapies may prevent progression to HCC, but often fail. We believe that a better understanding of the mechanisms underiying HCV-related liver cancer will offer opportunities for improved preventive, diagnostic and therapeutic approaches to control of HCC. This project thus seeks to determine the clinical relevance and potential role in hepatic carcinogenesis of interactions between host cell and virally-encoded proteins that have been demonstrated in recent cell culture-based experiments. The retinoblastoma susceptibility protein (pRb), p5S, and the DEAD-box RNA helicases, DDXS and DDX5, contribute to normal regulation of the cell cycle as well as DNA damage responses. We have shown that the HCV RNA-dependent RNA polymerase, NS5B, forms a complex with pRb, targeting it for E6AP-dependent ubiquitination and degradation via the proteasome. This results in reduced pRb abundance, activation of E2Fresponsive promoters, and proliferation of cultured cells. NS5B also binds DDXS, causing its redistribution from the nucleus to the cytoplasm, and the viral core protein binds DDXS, altering its abundance and cytoplasmic localization. Other evidence suggests that two other viral proteins, NSS and NS5A, bind pSS and disrupt its function. In addition, HCV proteins directly cause ER and mitochondrial oxidative stress. Our central hypothesis is that these viral-host interactions promote proliferation of infected hepatocytes and also impair the ability of infected cells to respond to DNA damage, thereby contributing to the cancer-promoting activity of HCV. Our long-term goal is to determine whether HCV infection alters expression of these tumor suppressors in vivo and promotes proliferation of hepatocytes, and whether interferon therapy reduces this enhanced cell proliferation and thereby lessens the risk of HCC. To test this hypothesis, we propose a series of experiments with the following Specific Aims: Aim 1. We will (i) develop and validate sensitive methods for detecting HCV proteins (core, NSS and NSSB) in cultured cells and paraffin-embedded or frozen tissues using fluorescent semiconductor quantum dot (Qdot) probes, and (ii) assess the impact of HCV proteins on expression of tumor suppressor proteins (pRb, pSS, DDXS and DDXS) in vitro and in vivo in both normal and HCV transgenic mice. Aim 2. Applying the imaging methods developed in Aim 1 to human liver tissue, we will determine whether the abundance and cellular localization of pRb, pSS, DDXS, and DDXS are altered in HCV-infected human hepatocytes in vivo. Aim 3. To test the hypothesis that eariy events in carcinogenesis occur preferentially in HCV-infected cells, we will determine whether HCV protein expression is associated with increased expression of cell proliferation markers, activation of E2F-responsive promoters, and formation of oxidative protein adducts and DNA modifications in vivo, and whether the proportion of hepatocytes displaying these cellular abnormalities is reduced following interferon therapy.