Background: Mitochondrial dynamics underlies malignant transformation, cancer progression, and response to treatment. Current research presents conflicting evidence for functions of mitochondrial fission and fusion in tumor progression. Here, we investigated how mitochondrial fission and fusion states regulate underlying processes of cancer progression and metastasis in triple-negative breast cancer (TNBC). Methods: We enforced mitochondrial fission and fusion states through chemical or genetic approaches and measured migration and invasion of TNBC cells in 2D and 3D in vitro models. We also utilized kinase translocation reporters (KTRs) to identify single cell effects of mitochondrial state on signaling cascades, PI3K/Akt/mTOR and Ras/ Raf/MEK/ERK, commonly activated in TNBC. Furthermore, we determined effects of fission and fusion states on metastasis, bone destruction, and signaling in mouse models of breast cancer. Results: Enforcing mitochondrial fission through chemical or genetic approaches inhibited migration, invasion, and metastasis in TNBC. Breast cancer cells with predominantly fissioned mitochondria exhibited reduced activation of Akt and ERK both in vitro and in mouse models of breast cancer. Treatment with leflunomide, a potent activator of mitochondrial fusion proteins, overcame inhibitory effects of fission on migration, signaling, and metastasis. Mining existing datasets for breast cancer revealed that increased expression of genes associated with mitochondrial fission correlated with improved survival in human breast cancer. Conclusions: In TNBC, mitochondrial fission inhibits cellular processes and signaling pathways associated with cancer progression and metastasis. These data suggest that therapies driving mitochondrial fission may benefit patients with breast cancer.

Migration and invasion of cancer cells constitute fundamental processes in tumor progression and metastasis. Migratory cancer cells commonly upregulate expression of plasminogen activator inhibitor 1 (PAI1), and PAI1 correlates with poor prognosis in breast cancer. However, mechanisms by which PAI1 promotes migration of cancer cells remain incompletely defined. Here we show that increased PAI1 drives rearrangement of the actin cytoskeleton, mitochondrial fragmentation, and glycolytic metabolism in triple-negative breast cancer (TNBC) cells. In two-dimensional environments, both stable expression of PAI1 and treatment with recombinant PAI1 increased migration, which could be blocked with the specific inhibitor tiplaxtinin. PAI1 also promoted invasion into the extracellular matrix from coculture spheroids with human mammary fibroblasts in fibrin gels. Elevated cellular PAI1 enhanced cytoskeletal features associated with migration, actin-rich migratory structures, and reduced actin stress fibers. In orthotopic tumor xenografts, we discovered that TNBC cells with elevated PAI1 show collagen fibers aligned perpendicular to the tumor margin, an established marker of invasive breast tumors. Further studies revealed that PAI1 activates ERK signaling, a central regulator of motility, and promotes mitochondrial fragmentation. Consistent with known effects of mitochondrial fragmentation on metabolism, fluorescence lifetime imaging microscopy of endogenous NADH showed that PAI1 promotes glycolysis in cell-based assays, orthotopic tumor xenografts, and lung metastases. Together, these data demonstrate for the first time that PAI1 regulates cancer cell metabolism and suggest targeting metabolism to block motility and tumor progression.Implications: We identified a novel mechanism through which cancer cells alter their metabolism to promote tumor progression.

The chemokine receptor CXCR4 regulates fundamental processes in development, normal physiology, and diseases, including cancer. Small subpopulations of CXCR4-positive cells drive the local invasion and dissemination of malignant cells during metastasis, emphasizing the need to understand the mechanisms controlling responses at the single-cell level to receptor activation by the chemokine ligand CXCL12. Using single-cell imaging, we discovered that short-term cellular memory of changes in environmental conditions tuned CXCR4 signaling to Akt and ERK, two kinases activated by this receptor. Conditioning cells with growth stimuli before CXCL12 exposure increased the number of cells that initiated CXCR4 signaling and the amplitude of Akt and ERK activation. Data-driven, single-cell computational modeling revealed that growth factor conditioning modulated CXCR4-dependent activation of Akt and ERK by decreasing extrinsic noise (preexisting cell-to-cell differences in kinase activity) in PI3K and mTORC1. Modeling established mTORC1 as critical for tuning single-cell responses to CXCL12-CXCR4 signaling. Our single-cell model predicted how combinations of extrinsic noise in PI3K, Ras, and mTORC1 superimposed on different driver mutations in the ERK and/or Akt pathways to bias CXCR4 signaling. Computational experiments correctly predicted that selected kinase inhibitors used for cancer therapy shifted subsets of cells to states that were more permissive to CXCR4 activation, suggesting that such drugs may inadvertently potentiate pro-metastatic CXCR4 signaling. Our work establishes how changing environmental inputs modulate CXCR4 signaling in single cells and provides a framework to optimize the development and use of drugs targeting this signaling pathway.

Chemokine CXCL12 gradients drive chemotaxis in a CXCR4-dependent mechanism and have been implicated in cancer metastasis. While CXCL12 gradients are typically studied in organized, defined environments, the tumor microenvironment is disorganized. In vivo, CXCL12 gradients depend on many factors: the number and arrangement of cells secreting and degrading CXCL12, isoform-dependent binding to the extracellular matrix, diffusion, and circadian fluctuations. We developed a computational model of the tumor microenvironment to simulate CXCL12 gradient dynamics in disorganized tissue. There are four major findings from the model. First, CXCL12-β and -γ form higher magnitude (steeper) gradients compared to CXCL12-α. Second, endothelial CXCR7+ cells regulate CXCL12 gradient direction by controlling concentrations near but not far from the vasculature. Third, the magnitude and direction of CXCL12 gradients are dependent on the local composition of secreting and scavenging cells within the tumor. We theorize that “micro-regions” of cellular heterogeneity within the tumor are responsible for forming strong gradients directed into the blood. Fourth, CXCL12 circadian fluctuations influence gradient magnitude but not direction. Our simulations provide predictions for future experiments in animal models. Understanding the generation of CXCL12 gradients is crucial to inhibiting cancer metastasis.