Abstract P2-02-05: Targeting PFKFB3 enzyme induces cell death via reactive oxygen species-mediated toxicity in endocrine therapy-resistant breast cancers
Authors: Sengupta S , Sevigny CM , Jones BC , Jin L , Pohlmann PR , Calrke R 
Published: May 2019

Reprogrammed glucose metabolism is one of the key hallmarks of cancer. High-energy demand in cancer cells leads to increased glycolysis to maintain anabolic processes that are often driven by altered enzyme levels. 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) is an enzyme that converts fructose-6-phosphate (F6P) to fructose-2,6-bisphosphate (F2,6BP). F2,6BP is an allosteric activator of phosphofructokinase-1 (PFK1), a critical rate-limiting control within glycolysis. PFKFB3 expression and activity are upregulated in many cancers including breast cancer, facilitating the increased glycolytic activity associated with neoplasia. In ER+ breast cancer cells, PFKFB3 is further upregulated by estrogen, but whether this affects responsiveness to endocrine therapies is largely unknown.

Expression of PFKFB3 mRNA and protein was measured in estrogen-responsive MCF7 cells and in endocrine therapy-resistant LCC9 and MCF7:5C cells (estrogen-independent; 4-hydroxytamoxifen (4OHT) and fulvestrant (Fulv) crossresistant). Both MCF7:5C and LCC9 cells expressed higher levels of PFKFB3 compared to MCF7 cells. Cells with higher PFKFB3 levels also showed increased basal glucose uptake and lactate secretion, consistent with the enhanced aerobic glycolysis often referred to as the “Warburg Effect.”

To evaluate the potential translational relevance of these observations, the association of PFKFB3 mRNA expression with clinical outcomes in ER+, node-negative breast cancer patients treated with an endocrine therapy was studied in two publicly available data bases. High expression of PFKFB3 was strongly associated with adverse recurrence-free survival (hazard ratio = 4.12 and p= 5.5x10-5).

Next, the effect of pharmacological inhibition of PFKFB3 was investigated on the growth of endocrine-resistant breast cancer cells using PFK158, an inhibitor of PFKFB3 that is currently being evaluated in clinical trials. PFK158 treatment suppressed the basal glucose uptake in LCC9 and MCF7:5C cells. PFK158 treatment alone was effective in inducing apoptotic cell death in both cell models. However, combining a lower, sub-optimal concentration of PFK158 with either 4OHT or Fulv significantly enhanced cell death in endocrine-resistant LCC9 and MCF7:5C cells. Notably, the cytotoxic effects of PFK158 alone or in combination with 4OHT or Fulv were markedly diminished by blocking reactive oxygen species (ROS) using either of the ROS scavengers N-acetyl cysteine (NAC) or Tempol.Overall, this study reveals that (1) PFKFB3 expression drives high glycolytic activity in endocrine-therapy resistant breast cancer cells; (2) PFKFB3 mRNA expression in ER+ LN- breast cancer is a prognostic factor; and (3) targeting PFKFB3 in combination with endocrine-based therapy induces enhanced cell death mediated by high ROS levels. Based on this intriguing data, combination of the PFKFB3 inhibitor PFK158 with an antiestrogen should therefore be regarded as a potential therapeutic intervention for patients with endocrine therapy-resistant breast cancer.

Citation Format: Sengupta S, Sevigny CM, Jones BC, Jin L, Pohlmann PR, Calrke R. Targeting PFKFB3 enzyme induces cell death via reactive oxygen species-mediated toxicity in endocrine therapy-resistant breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-02-05.