Posttranslational modifications mediated by ubiquitin-like proteins regulate a variety of cellular pathways. Although small ubiquitin-like modifier (SUMO) is a new member of this family, it has attracted a great deal of attention recently because of its novel and distinct functions. The sumoylation cycle is a multistep process, involving maturation, activation, conjugation and ligation;it is catalyzed by multiple enzymes, including E1, E2 and E3 enzymes. Ubiquitin-conjugating enzyme 9 (UBC9) is the sole E2 conjugating enzyme and is the best characterized enzyme in the sumoylation cycle. UBC9 transfers the activated SUMO to the target protein and is believed to play an important role in regulating the substrate specificity and enhancing the efficiency of sumoylation in vivo. Genotoxic stress induces sumoylation of a broad number of proteins involved in nuclear function as well as proteins involved in important signaling pathways, such as the nuclear factor kappa B (NF- :B). UBC9 and SUMO are highly expressed in human premalignant conditions in response to low-grade, long- term genotoxic stress, implying that upregulation of sumoylation may be an adaptive process to genotoxic stress. Furthermore, UBC9 is overexpressed in several malignancies, such as lung adenocarcinoma, ovarian carcinoma, and melanoma. Antagonizing UBC9 function in MCF-7 breast cancer cells transplanted in nude mice inhibited cell growth and increased apoptosis via Bcl-2 dependent mechanisms. Inactivating mutations of UBC9's SUMO conjugating activity enhances sensitivity to DNA damaging agents. UBC9 may be fundamental for tumorigenesis and tumor progression by preventing activation of apoptotic pathways and by minimizing the acute cellular stress response associated with the accumulating DNA damage of tumor progression. Whether UBC9 is deregulated in hepatocellular carcinoma (HCC) is unknown. How UBC9 protein stability is regulated post-translationally is also unknown. Finally, whether UBC9 expression is altered in response to alcohol is also unknown. S-adenosylmethionine (SAMe) is the principle biological methyl donor that is made in all mammalian cells as the first product of methionine metabolism, catalyzed by methionine adenosylmethionine (MAT). Besides being a methyl donor, accumulating evidence show SAMe regulates many critical cellular pathways that control growth and apoptosis. In mammals two MAT genes, MAT1A and MAT2A, encode for two catalytic subunits of MAT, 11 and 12, respectively. Patients with chronic liver disease including alcohol have impaired hepatic SAMe biosynthesis due to decreased MAT1A mRNA levels and post-translational inhibition of the MAT1A-encoded isoenzymes. Chronic hepatic SAMe deficiency occurs in MAT1A knockout (KO) mice, which exhibit increased propensity to choline-deficient diet induced fatty liver, higher level of lipid peroxidation, spontaneous development of steatohepatitis and HCC. We have recently shown that there is increased genotoxic stress in this model as early as at one month of age. Consistently, we found UBC9 expression is increased in the MAT1A KO livers and importantly administration of SAMe lowered UBC9 expression at the protein level. Treatment of HepG2 and HuH-7 cells, two human hepatoma cell lines, with SAMe also lowered the UBC9 protein level. This is a surprise finding as we reported recently that SAMe inhibits proteosomal activity. Since alcohol feeding lowers hepatic SAMe level, we also examined whether UBC9 expression is altered in this model and found that like the MAT1A KO livers, hepatic UBC9 protein level is increased in response to alcohol feeding. In this proposal we are testing several novel hypotheses, 1) UBC9 protein half-life is affected by phosphorylation so that phosphorylation at critical site(s) protects against degradation, 2) SAMe and its metabolite methylthioadenosine (MTA) can lower the UBC9 protein half-life by lowering its phosphorylation at these critical sites, 3) SAMe and MTA, known to be pro-apoptotic in liver cancer cells, cause apoptosis by lowering UBC9 expression, 4) UBC9 expression and hence sumoylation are increased during the development of alcoholic liver injury and they may contribute to many of the abnormalities seen.
Three aims are proposed in this application to test these hypotheses and elucidate how SAMe affects signaling pathways that are important in hepatocarcinogenesis and development of alcoholic liver injury.
S-adenosylmethionine (SAMe) is an important biological molecule that controls growth, death and anti-oxidant response in cells. SAMe is synthesized by methionine adenosyltransferase (MAT) proteins. SAMe levels fall during alcoholic liver injury and cancer. This project aims to understand how SAMe controls UBC9, an important protein that is induced in cancer development. Given that liver cancer is a leading cause of cancer death worldwide, if the proposed studies are successfully accomplished, they may uncover very novel insights into the development and therapy of liver cancer and alcoholic liver disease and as such they have very high public health relevance.
