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UBC Theses and Dissertations

Caveolin-1 mediates mitochondrial potential and ROS through ROCK/AMPK regulation of mitophagy Timmins, Logan Rayne


Cancer cells often exhibit altered metabolisms that support aerobic glycolysis and reduced mitochondrial function to promote cell survival. Caveolin-1 (Cav1) is an integral membrane protein and the main structural component of caveolae. Caveolae formation requires the adaptor protein Cavin1 and without it, Cav1 forms scaffolds at the plasma membrane. Cav1 can be phosphorylated at tyrosine-14 (pCav1) and is involved in signal transduction, cell migration, and mitochondrial function. Cav1 plays contrasting roles in cancer progression and its precise role in metabolism and, specifically, mitochondrial function remains poorly defined. Using a potential- dependent mitochondrial reporter, MitoView 633, and a mitochondrial reactive oxygen species (ROS) reporter, MitoSOX Red, we report that CRISPR/Cas9 knockout of Cav1 in MDA-MB- 231 triple-negative breast cancer cells increases mitochondrial potential and reduces ROS. These effects are reversed upon reintroduction of Cav1 by stably expressing Cav1 in KO cells. Further, introduction of the Cav1 Y14D phosphomimetic mutant decreased mitochondrial potential in Cav1 KO cells while the Y14F non-phosphorylatable mutant did not. pCav1 is a known effector of Rho-associated kinase (ROCK) signaling. Treatment of MDA-MB-231 cells with Y-27632, a ROCK1/2 inhibitor, or with ROCK1/2 siRNA increased mitochondrial potential and decreased ROS in Cav1-expressing cells. Similar results were observed in PC3 prostate cancer cells that lack caveolae suggesting this is a caveolae-independent effect. Use of the mito-Keima mitophagy probe reported increased basal mitophagy in Cav1 KO MDA-MB-231 and PC3 cells and induction of mitophagy by ROCK inhibition in Cav1-expressing cells. Knockdown of the autophagy protein ATG5 and mitophagy regulator PINK1 in Cav1 KO MDA-MB-231 cells reduced potential and increased ROS while Cav1-expressing cells were not significantly affected, demonstrating that mitophagy promotes mitochondrial health and reduces ROS in Cav1 KO cells. Finally, we show that Cav1 KO MDA-MB-231 cells have more active AMP-activated protein kinase (AMPK), a major mitochondrial homeostasis protein whose activity is inhibited by ROCK. Knockdown of AMPK resulted in decreased mitochondrial potential, increased ROS, and decreased mitophagy in Cav1 KO cells while the Cav1-expressing cells were not affected. Overall, our results suggest that Cav1 expression in cancer cells reduces mitochondrial potential and increases ROS via ROCK/AMPK regulated mitophagy.

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