Drp1 and Opa1 reciprocally structure the inner mitochondrial membrane and electron transport chain in lung adenocarcinoma

Author: ORCID icon orcid.org/0000-0002-9754-2791
Sessions, Dane, Microbiology - School of Medicine, University of Virginia
Kashatus, David, MD-MICR Microbiology, University of Virginia

Cancer is the number two cause of US mortality after cardiovascular disease and lung cancer is the highest mortality form of cancer. Lung adenocarcinoma (LUAD) represents almost half of all lung cancer cases and has a current five-year survival rate of 24%. The high mortality is significantly driven by the fact that many LUAD cases are diagnosed at late stages after primary tumor cells have already metastasized to distant sites such as the brain. The standard of care for LUAD includes surgical resection, if possible, combined with traditional platinum-based chemotherapies (i.e. cisplatin), immunotherapy, and targeted molecular therapy, when possible (i.e. anti-EGFR antibodies). About half of LUAD tumors demonstrate activating driver mutations in genes of the mitogen-activated protein kinase (MAPK) pathway, such as EGFR (12%), KRAS (30%), and BRAF (7%), that normally transmits proliferation, survival, and growth signals from the cell surface to the nucleus. Activating mutations in MAPK components confer metabolic changes to tumor cells compared to their normal tissue counterparts. In particular, KRAS mutations upregulate glycolytic metabolism to promote rapid cytoplasmic ATP synthesis and to generate biosynthetic precursor molecules required for nucleotide and lipid synthesis. Mitochondria are organelles that are the primary orchestrators of cellular metabolism and regulate intrinsic apoptosis, signaling molecule generation like reactive oxygen species, and buffer ions like calcium. Mitochondria are not static within the cell, but rather demonstrate complex fusion-fission dynamics regulated by large dynamin-related GTPases: Mfn1/2 and Opa1 are the direct effectors of fusion of the mitochondrial outer and inner membranes, respectively, and are functionally opposed by Drp1 that directly executes mitochondrial fission and reorganizes cristae during apoptosis. Opa1 is localized to themitochondrial inner membrane and intermembrane space and also functions to promote cristae morphology and oxidative phosphorylation capacity. Oncogenic signaling regulates mitochondrial dynamics. In particular, our lab has demonstrated that oncogenic KRas promotes mitochondrial fission through Erk2- mediated phosphorylation of Drp1. Our group also established that Drp1 is required for KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) and that depletion of Drp1 in this system inhibits tumor metabolism and development in vitro and in vivo. The goals of this dissertation are twofold: 1) to elucidate the requirements for mitochondrial dynamics machinery in KRAS-mutant LUAD, as tumors of different tissue origin are known to demonstrate differential metabolic signatures and dependencies, and 2) to understand how fusion and fission effectors interact with each other to promote mitochondrial function and drive tumorigenesis. To approach these questions, we established in vitro and in vivo systems of mitochondrial dynamics perturbation in LUAD that inactivate mitochondrial fission through Drp1 depletion, fusion through Opa1 depletion, or both simultaneously. We find that contrary to PDAC, KRAS-mutant LUAD growth and development are insensitive to inactivation of mitochondrial fission. We also demonstrate that LUAD requires Opa1 to maintain electron transport chain (ETC) function to regenerate the electron carrying molecule NAD+ required for oxidative biosynthesis and that Opa1 is only required when Drp1 is expressed and enzymatically active. We find that Opa1 depletion destroys ETC complex I assembly and function required to regenerate NAD+, and that co-depletion with Drp1 rescues this phenotype. We propose a model in which steady-state Drp1-mediated fission reorganizes cristae in a manner deleterious to ETC function, and that Opa1 opposes fission by reestablishing cristae morphology to restore ETC assembly and function.

PHD (Doctor of Philosophy)
mitochondrial dynamics, lung cancer, electron transport chain
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