Determining the Impact of Metabolic Adaptations on the Immune-Tumor Microenvironment in Metastatic Breast Cancer

The tumor microenvironment (TME) of TNBC is extremely heterogenous, which plays critical roles in shaping tumor aggressiveness and response to therapies. Within the TME, tumor cells adapt to the selective pressures of limited nutrient and oxygen supplies, evade immune surveillance, and emerge as aggressive metastatic variants. We recently uncovered that the metabolic enzyme PFKFB4 is a potential driver of aggressive breast cancer. PFKFB4 is one of the rare bifunctional enzymes containing both a kinase and a phosphatase domain and controls the flow of glucose flux towards anabolic pathways. We discovered a non-canonical function of PFKFB4 acting as a protein kinase that phosphorylates the transcriptional coregulator SRC-3 to regulate gene activation. Our preliminary findings indicate that increased expression of PFKFB4 is significantly associated with poor survival in TNBC patients, and elevated levels were enriched in the metastatic lesions. Orthotopic implantation of human TNBC cells expressing inducible shRNA-PFKFB4 constructs significantly attenuated metastasis in xenograft models, whereas in syngeneic mouse TNBC models, depletion of PFKFB4 significantly increased CD8+ T cell infiltration and activation, which effectively cleared tumors. Photoacoustic imaging in live animals confirmed that hypoxia within mouse tumors robustly activated PFKFB4 expression. RNAseq analysis identified a pro-metastatic gene signature in the integrin αvβ3 pathway that was selectively activated by PFKFB4. We hypothesize that bifunctional activation of PFKFB4 in response to TME stressors accentuate TNBC progression to metastasis through intrinsic mechanisms that regulate metabolic and genetic plasticity, and extrinsic effects on the TME that enhance immune suppression. In Aim 1, we will investigate the bifunctional mechanisms of the PFKFB4 kinase and phosphatase activities regulating metabolic and genetic plasticity in the hypoxic TME and identify the mechanisms that promote selection of clones with metastatic competence. Aim 2 will interrogate the impact of PFKFB4-dependent tumor metabolic adaptations on the immune-TME and identify mechanisms of immunosuppression and trafficking that impair CD8+ T cell infiltration and/or their effector function leading to tumor-immune escape. Our proposed research will unmask the novel mechanisms in the hostile TME that accentuates the development and progression of metastatic TNBC progression by increasing tumor-immune escape.