Secretory Mitophagy Exports p53: A new pro-tumor survival mechanism.

Mitochondrial dysfunction is associated with many life-threatening illnesses, from Parkinson’s disease to malignant cancers. Cells remove damaged, aged, or stressed mitochondria through a process called mitophagy. Our team is investigating a potential pro-tumor survival mechanism cancer cells enact with the help of mitophagy initiation and subsequent export. Mitophagy initiation is sensed by the molecule PINK1, triggering the isolation and packaging of the damaged mitochondrial segment for degradation through the lysosome. Cancer cell mitophagy is triggered by elevated oxidative stress and mitochondrial DNA damage caused by hypoxia, chemotherapy, radiotherapy, and immunotherapy. In our recent study, we discovered a secretory form of mitophagy in which damaged mitochondrial segments are packaged and exported outside of the cell within PINK1+ extracellular vesicles (EV). Additionally, we found that these PINK1+ EVs export tumor suppressors (TS) phospho-P53 and Merlin along with mitochondrial segments outside the tumor cell. It has been discovered that p53 is phosphorylated by PINK1, ultimately enhancing mitophagy. We also found that Merlin, an unappreciated TS associated with neurofibromatosis, interacts with p53 through the MDM2 mediator. We hypothesize that secretory mitophagy export of TS and other mitophagy-related proteins is an adaptive mechanism of cancer cells to withstand oxidative stress and avoid apoptosis. The export of TS p53 and Merlin were analyzed in 4T1 & IOMM-Lee cells. We isolated EVs via differential ultracentrifugation followed by immunoprecipitation of purified EV samples with PINK1 and phospho-p53 to identify protein-protein interactions. Western blotting and mass spectrometry were performed to validate immunoprecipitation results and PINK1 and p53 interaction. Understanding the role of EV-mediated export of TS in secretory mitophagy can reveal novel mechanisms aiding cancer cells’ survival under oxidative stress caused by chemotherapy or other treatments, offering potential therapeutic targets for various cancers.

Mitochondrial dysfunction is associated with many life-threatening illnesses, from Parkinson’s disease to malignant cancers. Cells remove damaged, aged, or stressed mitochondria through a process called mitophagy. Mitochondria are known as the powerhouse of the cell for a reason: they help produce the majority of the energy needed to power our cells. In Parkinson’s disease, scientists have found that the process of mitophagy is impaired, leading to an accumulation of dysfunctional mitochondria within cells. On the other hand, in cancer cells, mitophagy plays a more complex role and may even be enhanced. As evidenced by my research, our lab at the Center for Applied Proteomics and Molecular Medicine found that the process may serve as a kind of survival mechanism used by cancer cells in the face of oxidative stress caused by chemotherapy or radiation.
Mitophagy initiation is sensed by the molecule PINK-1, triggering the isolation and packaging of the damaged mitochondrial segment for degradation through the lysosome. Our team discovered a secretory form of mitophagy in which mitochondrial segments are packaged and exported outside of the cell within extracellular vesicles (EVs) derived from the interstitial fluid of breast cancer tumors. Moreover, it has been discovered that the tumor suppressor molecule p53 interacts and becomes phosphorylated by PINK-1, ultimately enhancing mitophagy and carcinogenesis. Pancreatic cancer (PC) p53 mutations are associated with tumor aggressiveness. Decreased levels of intercellular p53 leads to increased genetic instability, higher tumor growth rate, and survival. Pancreatic cancer is the 3rd most fatal cancer in the U.S. due to high rates of metastasis and late diagnoses. Understanding and targeting this imbalance may result in new forms of personalized medicine for PC patients.
In terms of materials and methods, I cultured PANC-1 and BXPC-3 cell lines and incubated them for 5 days, then applied a drug called CCCP to each flask. CCCP induces oxidative stress to mimic the conditions experienced by cancer cells undergoing treatment like chemotherapy or radiation. Afterwards, I confirmed that I had protein in my sample, and used various stages of ultracentrifugation to isolate the extracellular vesicles produced by these cells. The vesicles came in 3 sizes: 2K (the largest), 10K, and 100K (the smallest). To analyze the proteins within my samples, I used Western Blotting and mass spectrometry, and used the ExoView R200 to examine and categorize the EVs used in the experiment.

Here you can see some images of me working in the lab: doing cell culture, running Western Blots, and observing my pancreatic cancer cells.

My project produced some very interesting results. I compared the relative concentrations of p53, the tumor suppressor protein, and PINK-1, the mitophagy-associated signalling molecule, and found that there is a very high and positive correlation between the export of PINK-1 p-p53 via EVs when oxidative stress is induced, indicating that p53 is degraded and exported alongside PINK-1 in EVs.Exported p53 may aid tumor progression and constitute a novel diagnostic method of non-invasively determining the mitochondrial health and p53 status within PC. PC EVs positive for phospho-p53 represent a novel diagnostic biomarker indicative of tumor stress. Targeting EV pathways in combination with oxidative stress could be a novel method of treating PC. Our lab is currently investigating if secretory mitophagy & EV export of tumor suppressors is common among other kinds of cancer, as well.

We recently published a paper on the topic of secretory mitophagy, but again, we hope to connect secretory mitophagy to the export of other tumor suppressors in future studies.

I wanted to thank my mentors and colleagues at the Center for Applied Proteomics and Molecular Medicine for their continued guidance and support, including the following people: Purva Gade, my direct mentor, Dr. Lance Liotta, Dr. Marissa Howard, Sofie Strompf, Angela Rojas, and Thomas Philipson.

I would also like to thank the GMU OSCAR URSP program and Dr. Karen Lee, as I received funding and guidance from OSCAR throughout the past semester.

Thank you very much for listening to my presentation!