Abstract 1385: Oncogenic regulation of autophagy and DNA emission
Bibliographic record
Abstract
Abstract Background: Cancer cells release higher levels of cell-free DNA (cfDNA) into the circulatory system than normal cells. However, the molecular mechanisms underlying the process of programmed DNA elimination remain poorly understood. Emerging evidence has indicated that oncogenes such as HRAS can be released through extracellular vesicles (EVs). Moreover, studies have shown an active mechanism of DNA emission through autophagy. Autophagy is an evolutionarily conserved process that degrades and recycles cytoplasmic materials such as organelles and proteins in the lysosomes. Interestingly, Microtubule-associated protein 1 light chain-3 (LC3B) is known to degrade nuclear lamina components in HRAS-induced cells, potentially producing a pool of cytoplasmic chromatin. Cytoplasmic chromatin is ultimately degraded in lysosomes or re-routed outside of the cells. The aim of our study was to evaluate the role of autophagy proteins in circulating tumor (ctDNA) and EV-associated DNA emission in cancer cell lines harboring oncogenes (RAS and BRAF) as this aspect of the process is relatively understudied. Methods: We have assessed protein and RNA expression of autophagy genes (LC3B, ATG5 and ATG7) in IEC-18 cells that have been transduced with the HRAS oncogene (RAS3 cells) and human colorectal cancer cell lines SW620 and HCT116 using RT-qPCR and western blot (WB). All conditions were compared to their respective control cells (IEC-18, CCD-841 CoN and CCD18-Co cells. To determine the role of LC3 in nuclear membrane degradation, we employed transmission electron microscopy (TEM). We have further validated the role of autophagy genes in EV-DNA emission by shRNA knockdown using shRNA against highly expressed autophagy genes, such as LC3B in RAS3 cells. EV-DNA was measured using droplet digital PCR (ddPCR). Results: To determine whether autophagy expels oncogenic DNA via EVs in KRAS-mutant CRC cells, we focused on the role of ATG5, LC3B and ATG7, as this aspect of the process is understudied. Our data shows higher LC3B and ATG7 expression in transformed RAS3 cells, compared to control cell lines (IEC18). Moreover, ATG5 is overexpressed in SW620 and HT29 colorectal cancer cells compared to control cell lines. LC3 is known to degrade the nuclear membrane in HRAS-activated cells. Indeed, we observed degradation of the nuclear membrane in RAS3 cells leading to the accumulation of cytosolic DNA shown by TEM. Furthermore, overexpression of LC3B in IEC18 cells using GFP-tagged LC3B resulted in increased EV emission. These findings suggest that the autophagy axis may contribute to oncogenic DNA emission via EVs. Lastly, shRNA knockdown of LC3 resulted in reduction of EV-DNA emission (ddPCR). Impact: EV biogenesis and autophagy pathways play pivotal and interconnected roles during DNA processing and extracellular emission. Therefore, understanding the detailed mechanisms is critical for biomarker discovery and potential therapeutic strategies targeting this process. Citation Format: Thupten Tsering, Kyle Dickinson, Laura Montermini, Janusz Rak, Julia Burnier. Oncogenic regulation of autophagy and DNA emission [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1385.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.003 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".