Vinculin Deficiency Disrupts the Metabolic Phenotype in Breast Cancer Cells

Introduction:: Cytoskeletal remodeling and ATP dephosphorylation fuel cancer cell migration. Actin dynamics are coupled to cell metabolism, and actin cytoskeleton assembly is correlated with increased levels of regulatory enzymes in glycolysis [1-2]. However, our understanding of cellular bioenergetics in response to cytoskeleton disruption, and how this may affect downstream events such as cell migration and proliferation remains poorly understood. Adhesion to the extracellular matrix (ECM) facilitates migration through integrin signaling and mechanical anchorage. Clusters of integrins, focal adhesions (FAs), transmit adhesive and traction forces between the cytoskeleton and the ECM which is pivotal for cell migration. Our preliminary work shows that knockdown of vinculin (Vcl), a FA protein anchoring the actin cytoskeleton to integrins, in metastatic MDA-MB-231 (MDA) breast cancer cells results in a greater ATP:ADP ratio of the cells, suggesting increased energy utilization. We therefore hypothesize that with reduced Vcl, cells may alter bioenergetics in response to their loss in ability to transfer force from the cytoskeleton to the ECM.


Materials and Methods:: A Vcl knockout (KO) cell line was made using electroporation to transfect MDA cells with CRISPR/Cas9 targeting Vcl. Immunofluorescent staining was used to observe Vcl and actin organization in the cells. Corrected total cell fluorescence (CTCF) was measured by manually outlining cells in Fiji. The XFe24 Seahorse analyzer from Agilent was used to measure extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Oligomycin was used to inhibit mitochondrial ATP synthesis, while antimycin A and rotenone allow for complete inhibition of mitochondrial respiration. Cell migration was tracked using time-lapse microscopy for 6 hours, and centroid displacement was used to determine cell speed in Fiji. Cell proliferation was measured using the percentage EdU incorporation in thymidine bases during S-phase.

Results, Conclusions, and Discussions:: Results: Since Vcl anchors actin to integrins, we investigated the effect of Vcl depletion on cytoskeletal structure. Using immunofluorescence, MDAs depleted of Vcl lacked polarized actin bundle formation (Fig. 1A), confirming that Vcl KO alters actin organization. Since cytoskeletal dynamics are energetically dependent processes, we then probed glucose uptake and mitochondrial membrane potential (MMP) using 2-NBDG and TMRM, respectively (Fig. 1B). To further assess metabolism, we utilized an ATP rate assay to measure total ATP production rate. Vcl KO cells exhibited overall greater ATP production from glycolytic and mitochondrial ATP than parental MDAs, measured by ECAR and OCR, respectively (Fig. 1C). Together, higher glucose uptake, mitochondrial activity, and ATP production suggests that Vcl KOs exhibit increased energetics. To determine how higher energetics influences Vcl KO cell behavior, migration and proliferation, energetically demanding processes, were assessed. Interestingly, Vcl KO and control cells showed no differences in migration speed or proliferation rates (Fig. 1D-E). Taken together, this suggests that the higher energy in Vcl KOs may not be efficiently transmitted to affect cellular processes.

Discussion: Together with prior work indicating that cytoskeletal dynamics are tied to bioenergetics, the altered glucose uptake, MMP, and metabolic pathway utilization in Vcl KO cells suggests that Vcl may mediate metabolism. Notably, however, two energetically demanding processes, migration speed and proliferation, were not significantly altered with Vcl KO despite increased energetics. Together, these findings suggest that changes in ATP are not translating to changes in cell behaviors which raises the question of how cells are expending this excess energy. Since Vcl plays a key role in connecting the cytoskeleton to the ECM, the unchanged migration speed with Vcl KO could be due to cellular inability to efficiently transfer force between the ECM.

Conclusions: Here we showed that Vcl-deficient cells exhibit increased energy production without affecting migration speed or proliferation, suggesting that Vcl deficiency disrupts force transfer between the cytoskeleton and matrix. Since altered bioenergetics are a hallmark of cancer, understanding the link between Vcl and the energetic consequences that Vcl KO imposes from cytoskeletal disruption may elucidate therapeutic avenues for minimizing breast cancer metastases.


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References (Optional): : [1] Liu et al. Eur J Cell Biol. 2022 [2] Park et al. Nature, 2020.