Inhibition of PFKFB4 and Glycolysis to Suppress HER2-Positive Breast Cancer Growth

Primary and metastatic breast tumors are driven by oncogenic mutations to rely on glucose metabolism to support their rapid proliferation. Of these genetic alterations, the increased activity of the human epidermal growth factor receptor 2 (HER2/ErbB2) is observed in a third of all breast tumors. HER2 has been shown to activate downstream effectors (e.g. Ras, HIF-1α) that stimulate glycolysis - in part by activation of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase family of enzymes (PFKFB1-4). The PFKFBs produce fructose-2,6-bisphosphate (F26BP) which activates a key rate-limiting glycolytic enzyme, 6-phosphofructo-1-kinase (PFK1) and therefore play a critical regulatory role in glycolysis. In preliminary studies, we determined that the PFKFB4 family member was highly expressed in a series of patient - derived HER2+ breast tumors relative to normal breast tissue with markedly higher expression in matched metastatic lymph nodes. We next found that decreasing PFKFB4 expression markedly reduced glycolysis and cell survival in HER2+ breast cancer cells and inhibited established tumor growth in vivo. Through virtual screening, we discovered a first-in-class small molecule inhibitor of PFKFB4, termed 5MPN, that selectively inhibits PFKFB4 activity with a resultant decrease in glycolysis and cancer cell growth and have now identified a potent analog of this compound, termed 5MPN-a, with significantly greater activity. PFKFB4 inhibition also caused a significant decrease in the invasion and migration of HER2+ breast cancer cells in vitro, importantly at concentrations well below levels that affect cell viability, and marked suppression in the growth of transgenic HER2+ mammary tumors without signs of systemic toxicity. In additional studies, we have determined that a frequently co-expressed and related enzyme (PFKFB3) is also expressed in HER2+ breast tumors. Previous studies from our group found that the PFKFB4 enzyme localized to the cytoplasm (where PFK1 and the glycolytic pathway function) indicating a possible dominant role for PFKFB4 in glycolytic regulation. PFKFB3 however largely localized to the nucleus where it serves an important function in proliferation via activation of cyclin-dependent kinases. Based on the different roles played by these enzymes in cancer cells, we evaluated the effects of simultaneous PFKFB4 ± PFKFB3 inhibition on survival and proliferation in HER2+ breast cancer and found that the combination synergistically decreased the viability and growth of several HER2+ breast cancer cell lines. Our data indicate that targeting PFKFB4 may be a viable therapeutic option against breast cancer and strongly support the further evaluation of co-targeting the PFKFB4 and PFKFB3 enzymes as a potential strategy for the successful treatment of HER2+ breast cancer. These studies have been supported by the Brown Cancer Center, University of Louisville, the Dept. of Defense CDMRP Breast Cancer Research Program, NIH/REACH ExCITE Program at the University of Louisville and the Brinkley Endowed Professorship.