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Effects Of Obesity On Adipose Stromal Cell Biology: Contributions To Breast Cancer Risk
Mahsa Taskindoust1, Xiaoshuang Guo, MD2, Tingjun Xie, MD2, Valery M. Nelson, MS1, Scott T. Hollenbeck, MD1, Robin E. Bachelder, PhD1.
1Duke University School of Medicine, Durham, NC, USA, 2Peking Union Medical College, Beijing, China.

Purpose: Obesity in premenopausal women increases their lifetime risk of developing an aggressive subtype of breast cancer (triple-negative breast cancer; TNBC). Senescent cells are associated with multiple disease states, partially due to their secretion of inflammatory cytokines. We previously demonstrated that BRCA1 mutations, which are associated with increased TNBC risk, directly impact adipose stromal cell (ASC) biology, resulting in cellular senescence and the secretion of inflammatory cytokines. In the current studies, we investigated the hypothesis that ASCs from obese premenopausal women are senescent, and secrete inflammatory cytokines that may contribute to TNBC initiation by acting in a paracrine manner on the breast epithelium.
Methods: ASCs were isolated from breast adipose tissue obtained from obese (BMI>30) or lean (BMI<27) women undergoing breast reductions. ASC marker expression (CD44, CD90, CD105) was confirmed by flow cytometry. ASCs from women with differing BMIs were also obtained from Zenbio. Senescence was measured in ASCs by beta galactosidase staining. Cytokines were measured by ELISA in ASC conditioned media. The ability of an IL6 receptor-neutralizing antibody to reverse senescence in obese ASCs was tested. We also tested if recombinant IL6 promotes Stat3 signaling and senescence in ASCs from low BMI patients. Effects of conditioned media from obese ASCs on breast epithelial cell growth were tested.
Results: ASCs from obese premenopausal women were senescent and supported autocrine IL6/Stat3 signaling. Treatment of ASCs from lean women with IL6 also promoted Stat3 signaling and senescence. An anti-IL6 receptor antibody blocked autocrine IL6 signaling in ASCs from obese women. We will discuss how this obesity-altered ASC biology may contribute to TNBC initiation.
Conclusions: ASCs from obese premenopausal women are senescent, and secrete senescence-associated cytokines that can act in a paracrine fashion on breast epithelial cells to promote cell behaviors associated with breast cancer initiation. Ultimately, determining how obesity impacts ASC biology, and how this biology may drive TNBC initiation, will provide an important foundation for developing ASC-targeting therapies that reduce mortalities associated with TNBC.
Figure 1: ASCs from high BMI women are senescent, and produce senescence-associated cytokines that activate Stat3 signaling. A. ASCs from premenopausal women with a BMI of 26.5 or 38 (passage 7) were stained using the Senescence Beta-galactosidase Staining Kit (Cell Signaling Technology). Note increased beta gal staining in BMI 38 ASCs compared to that in BMI 26.5 ASCs. B and C. Conditioned media (72 h) were collected from ASCs from women with the indicated BMIs (p7; triplicate wells). IL6 (B) and IL8 (C) were quantified by ELISA (R&D Systems; pg/ml/cell (+/- SD) **, p<0.001). SFM= serum-free media. D. ASCs (BMI 26.5) were pulsed for 10 min with IL6 [rIL6 (R&D Systems;10 pg/mL) +/- soluble IL6R [sIL6R (R&D Systems; 150 pg/mL)]. Equivalent amounts of nuclear protein were immunoblotted with phospho-Stat3 (Tyr705) or GAPDH antibodies (Cell Signaling Technology). Addition of both IL6 and sIL6R increased phospho-Stat3 levels, indicating that these ASCs support trans-IL6 signaling.



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