The Major Pre- and Postmenopausal Estrogens Play Opposing Roles in Obesity-Driven Mammary Inflammation and Breast Cancer Development

•Menopause, obesity, and cancer increase pro-inflammatory cytokines in human breast fat•Estrone stimulates and estradiol relieves the inflammation of obesity in vivo•Estrone cooperates with NFκB to induce inflammatory mediators, but estradiol does not•HSD17B14 increases intracellular estrone to drive inflammation and ER+ CSC expansion Many inflammation-associated diseases, including cancers, increase in women after menopause and with obesity. In contrast to anti-inflammatory actions of 17β-estradiol, we find estrone, which dominates after menopause, is pro-inflammatory. In human mammary adipocytes, cytokine expression increases with obesity, menopause, and cancer. Adipocyte:cancer cell interaction stimulates estrone- and NFκB-dependent pro-inflammatory cytokine upregulation. Estrone- and 17β-estradiol-driven transcriptomes differ. Estrone:ERα stimulates NFκB-mediated cytokine gene induction; 17β-estradiol opposes this. In obese mice, estrone increases and 17β-estradiol relieves inflammation. Estrone drives more rapid ER+ breast cancer growth in vivo. HSD17B14, which converts 17β-estradiol to estrone, associates with poor ER+ breast cancer outcome. Estrone and HSD17B14 upregulate inflammation, ALDH1 activity, and tumorspheres, while 17β-estradiol and HSD17B14 knockdown oppose these. Finally, a high intratumor estrone:17β-estradiol ratio increases tumor-initiating stem cells and ER+ cancer growth in vivo. These findings help explain why postmenopausal ER+ breast cancer increases with obesity, and offer new strategies for prevention and therapy. Many inflammation-associated diseases, including cancers, increase in women after menopause and with obesity. In contrast to anti-inflammatory actions of 17β-estradiol, we find estrone, which dominates after menopause, is pro-inflammatory. In human mammary adipocytes, cytokine expression increases with obesity, menopause, and cancer. Adipocyte:cancer cell interaction stimulates estrone- and NFκB-dependent pro-inflammatory cytokine upregulation. Estrone- and 17β-estradiol-driven transcriptomes differ. Estrone:ERα stimulates NFκB-mediated cytokine gene induction; 17β-estradiol opposes this. In obese mice, estrone increases and 17β-estradiol relieves inflammation. Estrone drives more rapid ER+ breast cancer growth in vivo. HSD17B14, which converts 17β-estradiol to estrone, associates with poor ER+ breast cancer outcome. Estrone and HSD17B14 upregulate inflammation, ALDH1 activity, and tumorspheres, while 17β-estradiol and HSD17B14 knockdown oppose these. Finally, a high intratumor estrone:17β-estradiol ratio increases tumor-initiating stem cells and ER+ cancer growth in vivo. These findings help explain why postmenopausal ER+ breast cancer increases with obesity, and offer new strategies for prevention and therapy. Estrogen receptor-positive (ER+) breast cancer risk increases with obesity after menopause, but not before. A new clue to this paradox lies in the opposing actions of pre- and postmenopausal estrogens on inflammation. After menopause, 17β-estradiol production falls and estrone dominates. Here, researchers from the University of Miami and their colleagues show that while premenopausal 17β-estradiol is anti-inflammatory, estrone promotes inflammation in diet-induced obesity. Estrone activates pro-inflammatory genes associated with poor ER+ breast cancer outcome. A high estrone:17β-estradiol ratio, as exists after menopause and increases with obesity, drives inflammation and stimulates hormone-sensitive breast cancer initiation and tumor growth. This work sheds new light on the increase in inflammatory diseases, including cancer, in women after menopause. Recent decades have seen an alarming rise in the prevalence of obesity worldwide. It has been estimated that by 2030, up to 51% of the population will be obese (Finkelstein et al., 2012Finkelstein E.A. Khavjou O.A. Thompson H. Trogdon J.G. Pan L. Sherry B. Dietz W. Obesity and severe obesity forecasts through 2030.Am. J. Prev. Med. 2012; 42: 563-570Abstract Full Text Full Text PDF PubMed Scopus (722) Google Scholar, Ward et al., 2019Ward Z.J. Bleich S.N. Cradock A.L. Barrett J.L. Giles C.M. Flax C. Long M.W. Gortmaker S.L. Projected U.S. state-level prevalence of adult obesity and severe obesity.N. Engl. J. Med. 2019; 381: 2440-2450Crossref PubMed Scopus (258) Google Scholar). In addition to known associations with heart disease and diabetes, obesity is associated with increased risk and worse outcome for several cancers, including breast cancer (Renehan et al., 2015Renehan A.G. Zwahlen M. Egger M. Adiposity and cancer risk: new mechanistic insights from epidemiology.Nat. Rev. Cancer. 2015; 15: 484-498Crossref PubMed Scopus (287) Google Scholar). 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We recently showed that interaction between breast cancer cells and immature adipocytes upregulates cytokines IL6, IL8, CCL5, and CCL2, and activates Src to increase cancer stem-like cells, tumor initiation, and metastasis (Picon-Ruiz et al., 2016Picon-Ruiz M. Pan C. Drews-Elger K. Jang K. Besser A.H. Zhao D. Morata-Tarifa C. Kim M. Ince T.A. Azzam D.J. et al.Interactions between adipocytes and breast cancer cells stimulate cytokine production and drive Src/Sox2/miR-302b-mediated malignant progression.Cancer Res. 2016; 76: 491-504Crossref PubMed Scopus (91) Google Scholar). While both cell types upregulated inflammatory cytokines, production by adipocytes was considerably greater than that of the cancer cells; thus, cancer-associated adipocytes may be major drivers of inflammation. Each cytokine contributed to cancer stem cell (CSC) expansion, which would facilitate tumor progression following local invasion into breast fat (Picon-Ruiz et al., 2016Picon-Ruiz M. Pan C. Drews-Elger K. Jang K. Besser A.H. Zhao D. Morata-Tarifa C. Kim M. Ince T.A. Azzam D.J. et al.Interactions between adipocytes and breast cancer cells stimulate cytokine production and drive Src/Sox2/miR-302b-mediated malignant progression.Cancer Res. 2016; 76: 491-504Crossref PubMed Scopus (91) Google Scholar). The present work investigated how the dominant estrogens before and after menopause interact with NFκB to affect inflammation and breast cancer development. It is paradoxical that ER+ breast cancer increases after menopause and that the excess risk of ER+ breast cancer with obesity is limited to the postmenopausal period, when total estrogen levels decrease. Here, we investigated the hypothesis that the dominant postmenopausal estrogen, E1, might have an opposite effect to E2, and activate NFκB-mediated inflammation to promote ER+ tumor emergence after menopause. Present data indicate that, in contrast to E2, which has anti-inflammatory effects, E1 cooperates with NFκB to mediate inflammation. E1 increases inflammation accompanying high-fat diet (HFD)-induced obesity in vivo, while E2 opposes it. In the context of TNFα activation, E1 and E2 stimulate different global gene expression patterns, with E1 further upregulating genes of poor prognostic significance in ER+ breast cancer. While E2 opposes coactivator CBP recruitment to κB sites on cytokine gene promoters, E1 stimulates its recruitment and chromatin H3K27 acetylation to drive cytokine expression and increase CSCs and tumorigenesis. Present work may help explain the rise in ER+ breast cancer incidence after menopause and inform the pathophysiology of chronic inflammation-related diseases of higher male prevalence that increase after menopause in women. While obese adipose tissue is a site of chronic inflammation (Tornatore et al., 2012Tornatore L. Thotakura A.K. Bennett J. Moretti M. Franzoso G. The nuclear factor kappa B signaling pathway: integrating metabolism with inflammation.Trends Cell Biol. 2012; 22: 557-566Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar), how estrogens regulate production of inflammatory mediators in mammary fat is not fully characterized. Circulating E2, of ovarian origin, falls from 30–500 pg/mL before to <10 pg/mL after menopause. In contrast, pre- and postmenopausal serum E1 levels are similar (25–30 pg/mL), but more than double in obese women to 60–80 pg/mL (Eliassen et al., 2006Eliassen A.H. Missmer S.A. Tworoger S.S. Spiegelman D. Barbieri R.L. Dowsett M. Hankinson S.E. Endogenous steroid hormone concentrations and risk of breast cancer among premenopausal women.J. Natl. Cancer Inst. 2006; 98: 1406-1415Crossref PubMed Scopus (286) Google Scholar, Kaaks et al., 2005Kaaks R. Rinaldi S. Key T.J. Berrino F. Peeters P.H. Biessy C. Dossus L. Lukanova A. Bingham S. 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American Physiologic Society, 1973: 615-629Google Scholar) and pro-inflammatory cytokine production (Picon-Ruiz et al., 2016Picon-Ruiz M. Pan C. Drews-Elger K. Jang K. Besser A.H. Zhao D. Morata-Tarifa C. Kim M. Ince T.A. Azzam D.J. et al.Interactions between adipocytes and breast cancer cells stimulate cytokine production and drive Src/Sox2/miR-302b-mediated malignant progression.Cancer Res. 2016; 76: 491-504Crossref PubMed Scopus (91) Google Scholar), we assayed cytokine secretion from mature human adipocytes from fat tissues. Of over 45 cytokines assayed, CCL2, IL6, and IL8 were most abundant (Figures S1A and S1B). Their expression was then compared in mammary adipocytes from women of different body weight and menopausal status. Expression of CCL2, IL6, and IL8 was higher in mammary adipocytes from obese (n = 11) than from non-obese premenopausal women (n = 11; Figure 1A). Postmenopausal obese donors had higher mammary adipocyte CCL2 and IL8 expression than obese premenopausal donors (trend to significance for CCL2, p < 0.01 for IL8; Figure 1B). In women undergoing bilateral mastectomy for breast cancer, peritumoral breast fat expressed higher levels of CCL2, IL6, and IL8 than adipocytes from the contralateral unaffected breast (Figure 1C). Thus, mammary adipocyte inflammation appears to increase with obesity, when ovarian E2 falls after menopause and in cancer-associated breast tissue. Breast cancer cells invading beyond ductal basement membranes encounter peritumoral adipocytes, the most abundant mammary stromal component. We previously showed breast cancer cell interaction with adipocytes stimulates pro-inflammatory cytokine production (Picon-Ruiz et al., 2016Picon-Ruiz M. Pan C. Drews-Elger K. Jang K. Besser A.H. Zhao D. Morata-Tarifa C. Kim M. Ince T.A. Azzam D.J. et al.Interactions between adipocytes and breast cancer cells stimulate cytokine production and drive Src/Sox2/miR-302b-mediated malignant progression.Cancer Res. 2016; 76: 491-504Crossref PubMed Scopus (91) Google Scholar). Here, we evaluated how estrogens affect this cytokine upregulation. Mammary adipocytes from obese postmenopausal women not only expressed higher CCL2 and IL8 than those from premenopausal donors (Figure 1B), but they also stimulated greater cytokine induction by MCF7 ER+ breast cancer cells following co-culture (Figure 1D). Furthermore, co-culture of mature adipocytes with ER+ or ER− breast cancer cells showed CCL2, IL6, and IL8 induction was greater in ER+ than in ER− breast cancer lines