Expression and localization of HIF prolyl 4‐hydroxylases in rat hepatocytes and JM1 tumor cells

Abstract

Hepatocellular carcinoma (HCC) is the is the 5th most common solid tumor worldwide and the 4th leading cause of cancer-related death. Due to its highly vascular nature, HCC has a strong propensity for invasion. Angiogenic signals such as VEGF are upregulated by hypoxia-inducible factors (HIFs), transcription factors that induce hypoxia-responsive genes. Developing anti-angiogenic therapies that target the HIF pathway may provide effective chemotherapy. HIFs are post-translationally regulated by a family of recently identified oxygen-dependent HIF prolyl 4-hydroxylases (PHD). There are currently four known PHD enzymes, whose functions are non-redundant in other cell types. PHDs are highly expressed in normal liver, an organ which is itself unique due to its physiologic O2 gradient, and they may be dysregulated in tumors; however, a detailed mechanism for HIF over-expression in HCC has not yet been described. We hypothesize that alterations in PHD expression, localization, and/or activity can contribute to excessive HIF induction and hypervascularity in HCC. In this study we analyze the regulation of PHD1-4 expression and localization in normal rat liver and JM1 hepatoma tissues and cells. In culture, the PHDs exhibit a reversible nuclear-to-cytoplasmic translocation in response to hypoxia, with sequestration localized to peroxisomes. In normal liver, peri-venous localization of PHD2-4 is observed, consistent with areas of low pO2. Surprisingly, increased PHD1-3 are observed in JM1 tumors versus normal adjacent liver. To our knowledge, this is the first detailed report of these novel but critical enzymes in the liver. Understanding these mechanisms at the molecular level will help advance our knowledge of HCC pathology and treatment. (Supported by PHS1T32EB001026, CA35373, & CA103958)