Karla Andrade de Oliveira

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 (pPresentation: Thursday, June 15, 2023

Endocrine therapy can lead to nutrient deprivation by reducing glucose and glutamine uptake and total cellular ATP production. The ability of cells to bypass this metabolic stress is fundamental to how they regulate BC growth and acquire endocrine resistance. Sirtuins (SIRTs) are NAD+-dependent deacylases and regulate a widerange of intracellular processes including metabolism. SIRT3, 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 metabolic pathways, 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. We used Differential Dependency Network analysis to compare the wiring of the SIRTs and key metabolism-related genes in matched antiestrogen- sensitive vs. resistant breast cancer (BC) cells, which identified several novel signaling hubs including CDKN2A (p16), linking SIRT3 to cell proliferation. 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. Using endocrine sensitive (LCC1) and resistant (LCC9) BC cells, we confirmed the higher expression of SIRT3 in LCC9, compared to LCC1 cells. Upregulation of SIRT3 expression within 24 hrs of Fulvestrant (ICI 1 µM) treatment (p=0.0176) in LCC1 was shown, but no effect was observed in LCC9 cells. Conversely, treatment with 17β-estradiol (E2- 10 nM) did not significantly affect SIRT3 expression in either LCC1 or LCC9 cells. Inhibition of SIRT3 acitivity, using the LC0296 inhibitor (15 µM), reduced growth rate in LCC1 and SIRT3-silenced LCC9 cells under treatment with ICI (1 nM and 1 µM, respectively) (p< 0.0001, p< 0.001 respectively). Treatment with SIRTUIN 3 inhibitor and ICI induced apoptosis in LCC1 and SIRT3-silenced LCC9 cells compared to the untreated control and ICI only treated cells (p< 0.001). The combination SIRT3 inhibitor (15 µM) plus ICI (0.5 µM) increased ROS production in LCC9-SIRT3 silenced cells in 24 hours of treatment compared to the untreated control and ICI only treated cells (p< 0.001). SIRT3 inhibitor (15 µM) decreased LCC1 cell spheroid formation (p< 0.05), however it didn’t show additive effect to the effect of ICI (0.5 µM) in the same conditions. The combination of SIRT3 inhibitor (15 µM) and ICI (1 µM) reduced the growth of LCC9 SIRT3-silenced cells in detached condition compared to untreated control and ICI only treated cells (p< 0.001). The results show that SIRT3 may have a role in Fulvestrant response in BC cells; the mechanism is under investigation in our laboratory. Citation Format: Karla Andrade de Oliveira, Surojeet Sengupta, Lu Jin, Fabia de Oliveira Andrade, Melike Ozgu-Onal, Anil Yadav, Robert Clarke. Inhibition of Sirtuin 3 Sensitizes Breast Cancer Cells to Fulvestrant [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-08.

Objective: To determine the prevalence of metabolic syndrome (MS) and its components among Brazilian adolescents. Data source: Databases, such as LILACS, MEDLINE, and SciELO, were searched for original cross-sectional studies published between 2010 and 2021. The inclusion criteria were determined based on the mnemonic CoCoPop — Condition, Context, and Population: studies determining the prevalence of MS and its components (condition) in the general population of Brazilian adolescents, female and male (population), enrolled in public or private schools in rural or urban areas (context). Reviews, editorials, and articles that did not directly relate to the prevalence of MS or that included non-adolescent age groups or groups with specific health conditions (obesity/overweight and others) were excluded. Data synthesis: A total of 15 studies, including 43,227 adolescents, were identified. MS prevalence (95% confidence interval [95%CI]) was 2.9% (2.65–3.18) and 2.4% (1.90–2.90) (p<0.001) in males and females, respectively, by using the International Diabetes Federation (IDF) criteria. There was a significant difference in MS prevalence among Brazilian regions (Q=24.7; p<0.001). The lowest MS prevalence (95%CI) was determined for North Region of Brazil, 1.8% (1.52–2.13), and the highest for Northeast Region of Brazil, 2.9% (2.62–3.23). Regarding MS components, a higher prevalence (95%CI) was found for low high-density lipoprotein (HDL), 22.1% (12.49–36.17), followed by abdominal obesity, 11.0% (8.05–14.94), and arterial hypertension, 10.3% (7.84–13.48). Conclusions: This study allowed the determination of the prevalence of MS and the MS components in Brazilian adolescents, highlighting relevant aspects to be addressed on public health management.

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.