Genome-wide CRISPR/Cas9 knockout library screening identified PTPMT1 for cardiolipin synthesis as a crucial metabolic regulator for hypoxic survival in hepatocellular carcinoma

Abstract

Hypoxia, low oxygen (O2) tension, due to deficiency of blood supply and dysregulated cancer cell proliferation, is a universal feature for all solid tumours, including hepatocellular carcinoma (HCC). Hypoxia induces metabolic rewiring that culminates in refractory and aggressive phenotypes in cancer. Novel targetable vulnerabilities are urgently warranted to combat the continuous dismal clinical outcomes of hypoxic HCC.

We employed genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 knockout library screening to unbiasedly identify candidates that confer hypoxic survival advantages to HCC cells. HIF-1α and HIF-1β, which dimerize to form HIF-1 complex, the well-studied master regulator for oxygen sensing and hypoxic adaptation, ranked as the first and second most important candidates in our screening. The prioritization of these positive control genes highlights the validity and reliability of our screening. In contrast to the conventional view that mitochondria were insignificant hypoxic condition, we, from the screening, identified protein tyrosine phosphatase mitochondrial 1 (PTPMT1) as the third most significant gene for the survival of HCC cells under hypoxia, right after the HIF-1 complex. PTPMT1 is a pivotal enzyme for the synthesis of cardiolipin (CL), which is exclusively found in the mitochondrial with the greatest abundance at the mitochondrial inner membrane (MIM). CL physically interacts with and stabilizes the assembly of electron transport chain (ETC) complexes located at MIM to optimize electron transfer and oxidative phosphorylation (OXPHOS) to produce ATP. We envisioned that the loss of PTPMT1 prevented CL maturation, leading to the disruption of ETC. Electron leakage further resulted in excessive reactive oxygen species (ROS) accumulation and extensive cell death in hypoxic HCC cells where O2, the final electron accepter, is depleted. PTPMT1 knockout substantially disrupted mitochondrial cristae structure, reduced mitochondrial capacity and activity, increased cellular ROS level and induced apoptotic cell death of hypoxic HCC cells. Lipidomics and targeted mass spectrometry (MS) analyses confirmed the impairment of CL synthesis pathway and decrease of CL in PTPMT1 KO HCC cells. In vivo, loss of PTPMT1 greatly reduced HCC growth and tumor aggressiveness in orthotopic implantation model and liver-specific Ptpmt1 knockout model. Intriguingly, HCC cells treated with a specific PTPMT1 inhibitor, alexidine dihydrochloride (AD), recapitulated the phenotypes of PTPMT1 knockout cells. AD greatly sensitized HCC cells under hypoxia by inducing extensive cell death. Importantly, we showed that AD consistently induced oxidative stress in different cancer types in vitro and effectively suppressed development of ovarian cancer and colorectal cancer in vivo. Clinically, PTPMT1 was frequently upregulated in HCC patients and its overexpression was associated with poor survival and prognosis.

Taken together, we unprecedented leveraged CRISPR/Cas9 library screening to identify PTPMT1, an important metabolic regulator for hypoxic survival by maintaining mitochondrial integrity in cancer cells. PTPMT1 inhibitor, AD, also represents a novel therapeutic regimen to combat not only HCC, but also other hypoxic solid tumors.