Mitochondrial dynamics in Ovarian Cancer: Role of alternative splice variants of fission protein Drp1
Restricted (Penn State Only)
- Author:
- Javed, Zaineb
- Graduate Program:
- Biomedical Sciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 29, 2024
- Committee Members:
- Vonn Walter, Outside Field Member
Shengyu Yang, Major Field Member
Lisa Shantz, Outside Unit Member
Lisa Shantz, Program Head/Chair
Hong-Gang Wang, Chair & Dissertation Advisor
Nadine Hempel, Special Member
Mohamed Trebak, Special Member - Keywords:
- Drp1
DNM1L splice variants
mitochondrial dynamics
ovarian cancer - Abstract:
- Mitochondrial dynamics, particularly the balance between fission and fusion, are crucial for maintaining cellular homeostasis, energy production, and metabolism. Dynamin-related protein 1 (Drp1), encoded by the DNM1L gene, is a key regulator of mitochondrial fission, with dysregulation implicated in various pathologies, including cancer. Drp1's activity is tightly controlled by post-translational modifications, interactions with adapter proteins, and alternative splicing, producing variants with distinct functions and tissue distributions. In epithelial ovarian cancer (EOC), the deadliest gynecologic malignancy, Drp1 is often overexpressed, correlating with poor patient outcomes. However, the specific roles of Drp1 splice variants in EOC have not been previously elucidated. This thesis addresses this gap by exploring the expression and functional impact of Drp1 splice variants in EOC. We present the first comprehensive description of Drp1/DNM1L transcript variants in ovarian cancer, emphasizing their potential roles in disease progression and therapeutic resistance. Analysis of The Cancer Genome Atlas (TCGA) RNA sequencing data revealed that transcripts resulting from exon 16 splicing are highly expressed in ovarian cancer cells, with their relative expression to full-length Drp1 mRNA correlating with poor patient outcomes. Additionally, we discovered a novel C-terminal truncation variant, ΔC-Drp1, defective in fission. Clinical validation using patient ascites and matched tumor samples confirmed the pathophysiological relevance of Drp1 splice variants in EOC. Functional assays revealed that the Drp1 variant lacking exon 16 localizes with microtubules, resulting in reduced fission and a more interconnected mitochondrial network with increased cristae density. This morphological change leads to enhanced mitochondrial respiration and metabolic flexibility, evidenced by increased oxygen consumption rates, ATP production, and elevated levels of glycolytic and TCA cycle metabolites. In vitro and in vivo experiments demonstrated that Drp1(-/17) expression enhances cell proliferation, migration, and metastatic potential. Moreover, the lack of exon 16 in Drp1 confers resistance to chemotherapeutic agents, such as cisplatin and paclitaxel, by decreasing apoptosis and increasing cell survival, underscoring the importance of Drp1 splicing in cancer therapy resistance. Using splice variant-specific siRNA, the study validated that the pro-tumorigenic effects of Drp1(-/17) are not artifacts of overexpression. Manipulating the endogenous ratios of Drp1 splice variants confirmed that increased Drp1(-/17) or decreased Drp1(16/17) expression leads to more fused mitochondrial networks, enhanced respiratory function, increased proliferation, migration, and chemoresistance. Furthermore, we explored the unique transcriptional and protein interaction landscapes of Drp1 variants. Utilizing RNA sequencing, we discovered that the Drp1(-/17) variant significantly upregulates genes such as AQP3, GDF15, IDO1, and FOS, linked to cancer progression. Gene set enrichment analysis revealed that Drp1(-/17) expression is associated with metabolic and cancer regulatory signaling pathways. To delineate protein interactions, we employed APEX2-mediated proximity labeling. Notably, Drp1(-/17) interacted with the Tubulin beta chain, confirming its microtubule association. Under fission stimulus (FCCP treatment), Drp1(16/17) exhibited a more robust interaction profile, with 55 interactors compared to 26 for Drp1(-/17). Gene Ontology and Reactome pathway analyses highlighted that Drp1(-/17) is enriched in processes related to cell fate determination, mitotic cell cycle, and chromatin binding, whereas Drp1(16/17) showed strong interactions with mitochondrial fission processes. These findings underscore the multifaceted roles of Drp1 variants in cellular processes beyond mitochondrial fission. In conclusion, this research highlights the distinct functions of Drp1 splice variants in regulating mitochondrial dynamics and their consequences on ovarian cancer progression. These findings underscore the pathophysiological significance of Drp1 alternative splicing in ovarian cancer and emphasize the need to consider Drp1 alternative splicing in future cancer research.