ABS 024: Detachment from the extracellular matrix induces loss of the mitochondrial phosphatase PGAM5 in cancer cells

Konrad R. Czyzewski ¹ ,Matthew J. Fink ¹ , Michael Douglas ¹ , Connor McCloskey ¹ , Frances Ubogu ¹ , and Zachary T. Schafer ¹

¹ Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556

Van Wickle (2025) Volume 1, ABS 024

Introduction: Cancer cells often experience altered mitochondrial metabolism due to loss of integrin-mediated attachment to the extracellular matrix (ECM). Relatedly, work from our lab has revealed that ECM-detached cells undergo mitophagy, which subsequently can activate cell death pathways. Cancer cells, however, can circumvent mitophagy induction during ECM-detachment to promote their viability1. However, the mechanisms underlying mitophagy regulation during ECM- detachment remain poorly understood. The mitochondrial phosphatase PGAM5 is part of a proteinaceous complex initiating mitophagy during ECM-detachment, but its regulation in this context has not been extensively studied. Interestingly, we find that certain cancer cell lines have an innate ability to reduce PGAM5 protein levels when cultured under ECM-detached conditions. We find this phenomenon is primarily due to alterations in PGAM5 transcription, rather than alterations in protein stability. Given that ECM-detachment has been previously shown to impair proliferation, we analyzed PGAM5 expression in ECM-attached cells treated with CDK4/6 inhibitors. Our data suggest expression of PGAM5 is indeed linked to proliferation, as arresting cells in G1 results in decreased PGAM5 expression. We also examined the consequences of reduced PGAM5 on mitochondrial dynamics. Previous research has discovered that PGAM5 dephosphorylates Drp1, a key regulator of mitochondrial fission. Indeed, we observe that decreased expression of PGAM5 (during ECM-detachment and after CDK4/6 inhibitor treatment) results in elevated phosphorylation of Drp1, suggesting increased mitochondrial fusion in detached and G1 arrested cells are likely. These data collectively support the conclusion that ECM-detachment causes changes in mitochondrial dynamics as a consequence of impaired proliferation and subsequent loss of PGAM.

Methods: To investigate the regulation of PGAM5 during extracellular matrix (ECM) detachment, we cultured multiple cancer cell lines under both ECM-attached and detached conditions. PGAM5 expression was quantified via RT-qPCR and Western blotting across different time points. To determine whether PGAM5 downregulation was linked to cell cycle arrest, cells were treated with CDK4/6 inhibitors—Palbociclib and Abemaciclib—and PGAM5 expression was again measured. Flow cytometry using propidium iodide staining confirmed G1 arrest. Additionally, mitochondrial dynamics were assessed by measuring the phosphorylation status of Drp1 at serine 637 (p-Drp1(S637)) via Western blot, since PGAM5 is known to dephosphorylate Drp1. Finally, protein stability assays were conducted to determine whether PGAM5 loss was due to decreased transcription or enhanced degradation. These experiments were designed to clarify how ECM detachment and impaired proliferation affect mitochondrial regulation in cancer cells.

Results: We have demonstrated that ECM detachment leads to significant downregulation of PGAM5 at the mRNA and protein level. This effect is not due to altered protein stability, but rather to transcriptional regulation. Furthermore, arresting cells in G1 via CDK4/6 inhibition also decreases PGAM5 expression, implicating proliferation as a key modulator. Reduced PGAM5 is accompanied by elevated phosphorylation of Drp1, indicating a shift toward mitochondrial fusion. These findings collectively suggest that both ECM-detachment and G1 arrest drive mitochondrial remodeling by repressing PGAM5.

Discussion: Our findings identify ECM-detachment and G1 cell cycle arrest as key triggers for transcriptional downregulation of PGAM5 in cancer cells, resulting in altered mitochondrial dynamics through increased Drp1 phosphorylation. This supports a model where decreased PGAM5 promotes mitochondrial fusion during non-proliferative states. These results enhance our understanding of how cancer cells adapt to ECM-detachment by evading mitophagy and remodeling mitochondria. Future studies will aim to identify the transcription factors responsible for PGAM5 regulation and explore whether targeting PGAM5-low cancer cells may expose vulnerabilities that could be exploited for therapeutic intervention.