Oxygen-Dependent Phase Separation of PHD3 Links the Hypoxic Microenvironment to Mitochondrial Metabolic Reprogramming in ccRCC

Abstract

Clear cell renal cell carcinoma (ccRCC) is characterized by profound metabolic dysregulation, with both PHD3 and pyruvate carboxylase (PC) independently implicated in disease progression. While each influences patient outcomes, a direct mechanistic interplay between these two regulators has remained elusive. Here, we uncover a novel regulatory axis involving PHD3 and PC by identifying an unexpected subcellular behavior of PHD3, namely its dual localization to the cytosol and the mitochondrial matrix. We show that mitochondrial import of PHD3 is driven by liquid–liquid phase separation (LLPS), a biophysical process modulated by PHD3 hydroxylase activity and oxygen levels. Once in the matrix, PHD3 directly hydroxylates PC, suppressing its enzymatic activity. In ccRCCs with elevated PHD3 expression, this modification restricts anaplerotic flux into the tricarboxylic acid (TCA) cycle, leading to impaired proliferation, reduced metastasis, and enhanced apoptosis. Together, our findings provide a new framework for targeting cancer metabolism by establishing a previously unrecognized mechanistic link between PHD3- mediated oxygen sensing within the tumor microenvironment (TME) and the regulation of ccRCC mitochondrial metabolism through LLPS-driven translocation.