Abstract 2145: Untargeted sequencing of human breast cancer reveals induction of pro-apoptotic and anti-proliferative transcriptional signatures in response to mitochondrial uncoupling
Authors: Zunica Elizabeth R. , Axelrod Christopher L. , Cho Eunhan , Spielmann Guillaume , Yang Shengping , Gilmore L Anne , Kirwan John P. 
Published: July 2021

Background: Targeting mitochondrial function has regained traction as a viable anti-cancer strategy. Here, we characterized the phenotypic and transcriptomic landscape of human triple negative breast cancer (TNBC) cells following exposure to the mitochondrial uncoupler BAM15.

Methods: Cell viability and IC50 were determined by MTS assay after treatment (24-hours) with doxorubicin, cyclophosphamide, or BAM15 (0-100 µM). Mitochondrial membrane potential (ΔΨm) was measured by TMRM staining in live (Annexin V negative) cells using flow cytometry after acute (15-minutes) and chronic (16-hours) exposure to BAM15. Untargeted RNA sequencing was conducted after chronic (16-hours) exposure to BAM15 (1, 10, and 20 µM). Differentially expressed genes were determined by using DESeq2 package in R with the following cutoffs: false discovery rate &lt;0.05 and fold change &gt;1.5. Pathway enrichment analysis was then used to identify canonical signaling networks and upstream regulators and specific gene targets were validated by real-time qPCR.

Results: BAM15 reduced cell viability with an IC50 similar to doxorubicin and superior to cyclophosphamide. Acute exposure to BAM15 reduced ΔΨm by ~60% which saturated at 1 µM. After 16-hour exposure to BAM15, ΔΨm recovered at 1 µM, but was reduced by ~40% at 10 and 20 µM conditions. Through untargeted sequencing, we identified 2108 transcripts that were differentially regulated by BAM15 (20 µM vs. vehicle), 968 of which were upregulated and 1140 downregulated. To contextualize these findings, we performed pathway enrichment analysis on genes differentially expressed by BAM15. Canonical signaling pathways related cell survival, proliferation, energy production, and DNA damage were differentially regulated by BAM15. A number of known transcriptional factors required for cell division, replication, and survival, including CCNE2, E2F1, CDC6, CDC20, MCM3, MCM6, AURKA, UBEC2C, and S100A4, were inhibited by BAM15. Notably, genes and pathways essential to ATP production, including both glycolysis and oxidative phosphorylation, were dramatically reduced by BAM15, including LDHA and MT-CO1.

Conclusions: Taken together, these findings reveal transcriptomic signatures and canonical signaling pathways that mediate the pro-apoptotic and anti-proliferative effects of sustained mitochondrial uncoupling.

Citation Format: Elizabeth R. Zunica, Christopher L. Axelrod, Eunhan Cho, Guillaume Spielmann, Shengping Yang, L Anne Gilmore, John P. Kirwan. Untargeted sequencing of human breast cancer reveals induction of pro-apoptotic and anti-proliferative transcriptional signatures in response to mitochondrial uncoupling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2145.