Investigating AMPK signalling regulation of autophagy in a model of ovarian tumour dormancy

Investigating AMPK signalling regulation of autophagy in a model of ovarian tumour dormancy Jeremi Laski and Trevor G. Shepherd Background: Ovarian cancer is the most deadly gynecologic malignancy in women. A particular subset of this disease, epithelial ovarian cancer (EOC), is responsible for over 70% of all diagnosed ovarian cancer cases, yet the mechanisms governing disease progression are poorly understood. One of the unique hallmarks of EOC metastasis lies in the process of spheroid formation, whereby tumour cells aggregate into larger 3D structures. These EOC spheroids have been shown to be metabolically dormant, while concurrently up-regulating autophagy (cellular waste recycling) processes. This particular change in cellular activity seems to promote increased EOC chemotherapy resistance. My project examines the role of AMP-activated protein kinase (AMPK), a cellular metabolic sensor, and its role in regulating autophagy induction in EOC spheroids. Methods: Following AMPK knockdown using siRNA, protein markers for both AMPK and autophagy signalling will be assessed through immunoblotting, immunocytochemistry and fluorescence microscopy. Additional experiments will examine cellular viability of AMPK deficient EOC spheroids compared to their wild type counterparts following chemotherapy treatment. Expected Results: I expect that reducing the levels of cellular AMPK will disrupt autophagy induction, thereby sensitizing EOC spheroids to chemotherapy treatment. Discussion & Conclusion: Uncovering the mechanisms that govern autophagy induction in EOC spheroids will allow researchers to target a vulnerability in EOC to further develop therapeutics against this type of malignancy. Interdisciplinary Reflection: My research bridges molecular biology with clinical oncology, allowing for a more detailed understanding of the pathology of this deadly disease

Activated CAMKKβ-AMPK signaling promotes autophagy in a spheroid model of ovarian tumour metastasis

Background: A hallmark of epithelial ovarian cancer (EOC) metastasis is the process of spheroid formation, whereby tumour cells aggregate into 3D structures while in suspension in the peritoneal cavity. EOC spheroids are subjected to bioenergetic stress, thereby activating AMP-activated protein kinase (AMPK) signaling to enter a metabolically quiescent state, which can facilitate cell survival under nutrient-limiting conditions. Independently, we have also demonstrated that EOC spheroids induce autophagy, a process that degrades and recycles intracellular components to restore energy and metabolites. Herein, we sought to examine whether AMPK controls autophagy induction as a cell survival mechanism in EOC spheroids. Results: We observed a co-ordinate increase in phosphorylated AMPK and the autophagy marker LC3-II during EOC spheroid formation. Reduced AMPK expression by siRNA-mediated knockdown of PRKAA1 and PRKAA2 blocked autophagic flux in EOC spheroids as visualized by fluorescence microscopy using the mCherry-eGFP-LC3B reporter. A complementary approach using pharmacologic agents Compound C and CAMKKβ inhibitor STO-609 to inhibit AMPK activity both yielded a potent blockade of autophagic flux as well. However, direct activation of AMPK in EOC cells using oligomycin and metformin was insufficient to induce autophagy. STO-609 treatment of EOC spheroids resulted in reduced viability in 7 out of 9 cell lines, but with no observed effect in non-malignant FT190 cell spheroids. Conclusions: Our results support the premise that CAMKKβ-mediated AMPK activity is required, at least in part, to regulate autophagy induction in EOC spheroids and support cell viability in this in vitro model of EOC metastasis

AMPK Signalling As A Regulator Of Autophagy In A Model Of Ovarian Tumour Dormancy

One of the hallmarks of epithelial ovarian cancer (EOC) metastasis lies in the process of spheroid formation, whereby tumour cells aggregate into 3D structures. Previous literature suggests that as EOC cells form spheroids they undergo bioenergetic stress, activate AMP-activated protein kinase (AMPK) signaling, and thereby force cells to enter a metabolically quiescent state. We have previously shown that EOC spheroids up- regulate autophagy, a process that provides energy during starvation conditions. Herein, I examined the role of AMPK-mediated signaling regulation of autophagy in a model of ovarian tumour dormancy. Attenuation of AMPK signaling in EOC spheroids resulted in reduction of autophagic flux in all EOC lines although this reduction may occur in an LC3 and p62 independent manner. Interestingly, I identified that AMPK activation in spheroids is likely regulated by CAMKK-beta. The results of this study help further our understanding of the complex mechanisms driving autophagy in late-stage ovarian cancer

Investigating COVID-19 Vaccine Communication and Misinformation on TikTok: Cross-sectional Study

BackgroundThe COVID-19 pandemic has highlighted the need for reliable information, especially around vaccines. Vaccine hesitancy is a growing concern and a great threat to broader public health. The prevalence of social media within our daily lives emphasizes the importance of accurately analyzing how health information is being disseminated to the public. TikTok is of particular interest, as it is an emerging social media platform that young adults may be increasingly using to access health information. ObjectiveThe objective of this study was to examine and describe the content within the top 100 TikToks trending with the hashtag #covidvaccine. MethodsThe top 250 most viewed TikToks with the hashtag #covidvaccine were batch downloaded on July 1, 2021, with their respective metadata. Each TikTok was subsequently viewed and encoded by 2 independent reviewers. Coding continued until 100 TikToks could be included based on language and content. Descriptive features were recorded including health care professional (HCP) status of creator, verification of HCP status, genre, and misinformation addressed. Primary inclusion criteria were any TikToks in English with discussion of a COVID-19 vaccine. ResultsOf 102 videos included, the median number of plays was 1,700,000, with median shares of 9224 and 62,200 followers. Upon analysis, 14.7% (15/102) of TikToks included HCPs, of which 80% (12/102) could be verified via social media or regulatory body search; 100% (15/15) of HCP-created TikToks supported vaccine use, and overall, 81.3% (83/102) of all TikToks (created by either a layperson or an HCP) supported vaccine use. ConclusionsAs the pandemic continues, vaccine hesitancy poses a threat to lifting restrictions, and discovering reasons for this hesitancy is important to public health measures. This study summarizes the discourse around vaccine use on TikTok. Importantly, it opens a frank discussion about the necessity to incorporate new social media platforms into medical education, so we might ensure our trainees are ready to engage with patients on novel platforms