Although multifactorial in origin, one of the most impactful consequences of social isolation is an increase in breast cancer mortality. How this happens is unknown, but many studies have shown that social isolation increases circulating inflammatory cytokines and impairs mitochondrial metabolism. Using a preclinical Sprague Dawley rat model of estrogen receptor-positive breast cancer, we investigated whether social isolation impairs the response to tamoxifen therapy and increases the risk of tumors emerging from dormancy, and thus their recurrence. We also studied which signaling pathways in the mammary glands may be affected by social isolation in tamoxifen treated rats, and whether an anti-inflammatory herbal mixture blocks the effects of social isolation. Social isolation increased the risk of dormant mammary tumor recurrence after tamoxifen therapy. The elevated recurrence risk was associated with changes in multiple signaling pathways including an upregulation of IL6/JAK/STAT3 signaling in the mammary glands and tumors and suppression of the mitochondrial oxidative phosphorylation (OXPHOS) pathway. In addition, social isolation increased the expression of receptor for advanced glycation end-products (RAGE), consistent with impaired insulin sensitivity and weight gain linked to social isolation. In socially isolated animals, the herbal product inhibited IL6/JAK/STAT3 signaling, upregulated OXPHOS signaling, suppressed the expression of RAGE ligands S100a8 and S100a9, and prevented the increase in recurrence of dormant mammary tumors. Increased breast cancer mortality among socially isolated survivors may be most effectively prevented by focusing on the period following the completion of hormone therapy using interventions that simultaneously target several different pathways including inflammatory and mitochondrial metabolism pathways.

Reprogrammed glucose metabolism is recognized as one of the hallmarks of cancer. High energy demand in cancer cells leads to increased glycolysis to maintain anabolic processes that may be driven by altered enzyme levels. The second committed step in the glycolysis pathway is catalyzed by phosphofructokinase1 (PFK1), which converts fructose-6 phosphate (F6P) to fructose 1,6-bisphosphate (F1,6BP). Another enzyme, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), converts F6P to fructose 2,6-bisphosphate (F2,6BP) that then functions as an allosteric activator of PFK1 and drives glycolysis. PFKFB3 enzyme is upregulated in many cancers including breast cancers. PFKFB3 is regulated by estrogen in ER-positive breast cancer cells but its role in endocrine-therapy resistant breast cancer cells is largely unknown. We investigated the expression of PFKFB3 mRNA and protein in estrogen-responsive MCF7 cells and compared it with LCC9 cells that are estrogen-independent and resistant to 4-hydroxytamoxifen (4OHT) and fulvestrant (Fulv). Twofold higher PFKFB3 mRNA and protein levels were expressed in LCC9 cells compared to MCF7 cells. In addition, the estrogen-mediated stimulation of PFKFB3 that was evident in MCF7 cells was not observed in LCC9 cells. We further studied the effect of pharmacological inhibition of PFKFB3, on the growth of therapy-resistant breast cancer cells using a compound PFK158, a potent inhibitor of the PFKFB3 enzyme. Treatment with PFK158 inhibited the growth of LCC9 cells as well as another cell line, MCF7:5C that is also resistant to 4OHT and partially resistant to Fulv. PFK158 alone was effective in suppressing the growth of both the cell lines at a concentration of 5 µM or higher. Notably, combining PFK158 with either 4OHT or Fulv significantly potentiated the effect of PFK158 treatment in blocking the growth of the cells. In MCF7:5C cells the combination treatment of 2.5 µM PFK158 and Fulv (500nM) or 4OHT(500nM) inhibited cell growth by ~70% as compared to ~30% inhibition in the presence of PFK 158 alone. In LCC9 cells >70% of growth was inhibited in the combination treatment as compared to no inhibition when PFK158, 2.5 µM or 4OHT or Fulv alone was used. In publicly available datasets of ER+, node-negative breast cancer patients, high expression of PFKFB3 was associated with adverse recurrence-free survival (hazard ratio = 4.12 and p= 5.5x10^-5). Our study shows an increased basal expression of PFKFB3 mRNA and protein in estrogen-independent, endocrine-therapy resistant breast cancer cells as compared to estrogen-responsive breast cancer cells. Targeting PFKFB3 in combination with anti-estrogens should be explored for potential therapeutic intervention in endocrine therapy-resistant breast cancers. Citation Format: Surojeet Sengupta, Catherine M. Sevigny, Xiaoyi Liu, Lu Jin, Paula R. Pohlmann, Robert Clarke. Targeting glycolysis enzyme, PFKFB3, in endocrine therapy resistant breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 907.

Disclosure: K.A. de Oliveira: None. F.D. Andrade: None. M. Ozgul-Onal: None. A. Yadav: None. T. Onal: None. L. Jin: None. S. Sengupta: None. R. Clarke: None. Endocrine therapy can lead to cellular nutrient deprivation by reducing glucose and glutamine uptake and total cellular ATP production in breast cancer cells. The ability to bypass this metabolic stress is fundamental to how breast cancer cells regulate growth and acquire endocrine resistance. SIRT3, which can be activated by calorie restriction, modulates mitochondrial adaptation to low energy input. It has a key role in mitochondrial integrity and function, regulating cell survival, death, and metabolism, regulating the shift to amino-acid and fatty-acid catabolism. SIRT3 can maintain ROS levels at the appropriate levels for sustaining a proliferative phenotype, preventing apoptosis and promoting carcinogenesis. A role of SIRT3 in epigenetic regulation has also been reported. The ability of SIRTs to sense and respond to changes in energy, coupled with their intrinsic deacetylase/deacylase functions, could provide a mechanism for the cell to rewire signaling and maintain a newly acquired drug-resistant phenotype. Using antiestrogen sensitive (LCC1) and cross-resistant (LCC9) breast cancer cells, we confirmed higher expression of SIRT3 in LCC9, compared with LCC1 cells. Upregulation of SIRT3 expression within 24 hrs of Fulvestrant (ICI 1 µM) treatment (p=0.0176) in LCC1 was shown, whereas no effect was observed in LCC9 cells. Inhibition of SIRT3 activity, using the inhibitor LC0296 (15 µM), reduced growth rate in both LCC1 and SIRT3-silenced LCC9 cells under treatment with ICI (p<0.0001, p<0.001 respectively). Treatment with SIRTUIN 3 inhibitor and ICI induced caspase-independent cell death in LCC1 and SIRT3-silenced LCC9 cells compared to the untreated control and ICI only treated cells (p<0.001). The combination of SIRT3 inhibition (LC0296, 15 µM) and ICI treatment (0.5 µM) increased ROS production in LCC9-SIRT3 silenced cells in 24 hrs compared with the untreated control and ICI only treated cells (p<0.001). The combination of LC0296 and ICI induced a decrease in mitochondrial membrane potential and ATP production, and a decrease in glycolytic capacity and glycolytic reserve in both LCC1 and SIRT3-silenced LCC9 cells, compared with the untreated control (p<0.001). Moreover, ICI significantly reduced ATP production in the mitochondria of the SIRT3-silenced LCC9 cells (p<0.001) but not in the non-silenced (control) LCC9 cells. Taken together, these results show that SIRT3 may have a key role in determining the response to fulvestrant response in ER+ breast cancer cells; a more detailed understanding of this role for SIRT3 is under investigation in our laboratory. Presentation: Thursday, June 15, 2023

Estrogen receptor positive (ER+) breast cancers (BC) that do not respond to endocrine therapies exhibit lower response rates to chemotherapy than other subtypes, implying that endocrine resistance may confer cross-resistance to cytotoxic drugs and contribute to the poor responses to chemotherapy in recurrent ER+ disease. Endocrine therapies can reduce glucose (GLC) and glutamine uptake and total cellular ATP production in BC cells, and the ability of cells to bypass this metabolic stress is fundamental to how endocrine therapies regulate BC growth in a way that leads to acquired endocrine resistance and cross-resistance to other drugs. Sirtuins are NAD+-dependent deacylases. SIRT1 deacetylates histones and various transcription factors, and regulates GLC metabolism through TORC2, PGC1α, FOXO1. SIRT3, activated by caloric restriction, modulates mitochondrial adaptation to low energy input. SIRT3 destabilizes HIF-1α, and then regulates glycoytic gene expression. The role of SIRT3 in epigenetic regulation has been reported. Using Differential Dependency Network analysis to compare the wiring of the SIRTs and key metabolism-related genes in matched antiestrogen- sensitive vs. resistant BC cells, guided by gene related to the nicotinate and nicotinamide metabolism, and associated with clinical outcome in ER+ patients treated with an endocrine therapy, we identifyed several novel signaling hubs including: CDKN2A (p16) Hub, linking SIRT3 to cell proliferation; TP53 Hub, linking SIRT5 to clinical resistance to aromatase inhibitors, cell survival and proliferation; ATM Hub that comprises ATM/SIRT2/SIRT4/HIF1A and may functionally link endocrine resistance to chemoresistance, consistent with the relatively poor responses of ER+ BC to cytotoxic drugs. Hence, the ability of SIRTs to sense and respond to changes in energy, coupled with their deacetylase/deacylase functions, could provide a mechanism for the cell to rewire signaling and maintain a newly acquired drug-resistant phenotype enabled by epigenetically maintained changes in genes that control metabolism. Using endocrine sensitive (LCC1) and resistant (LCC9) BC cells, we confirmed the higher expression of SIRT1 and 3 in LCC9, compared to LCC1 cells. We further showed an upregulation of SIRT3 expression within 24 hrs of Fulvestrant (1 µM) treament (p=0.0176) in LCC1, but no effect was observed in LCC9 cells. Conversely, treatment with 17β-estradiol (10 nM) did not significantly affect SIRT3 expression in either LCC1 or LCC9 cells. SIRT 1 expression in LCC1 cells was not regulated by ICI within 72 hrs of treatment. In ER+ BC patients, lower expression of SIRT3 is associated with poor relapse free survival (RFS) (HR=0.57; CI=0.43-0.76); p= 7.7x 10^-5) whereas higher expression of SIRT5 is associated with poor RFS (HR=2.5; CI=0.99-6.59; p=4.4x10^-2). We will use transfection assays for knockdown/overexpression of SIRTs to investigate their roles in therapy resistance. Citation Format: Karla A. De Oliveira, Surojeet Sengupta, Lu Jin, Robert Clarke. Fulvestrant regulation of SIRT1 and SIRT3 expression in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2339.