Mammary Adipocytes Research Articles

Lactate Metabolism in Breast Cancer Microenvironment: Contribution Focused on Associated Adipose Tissue and Obesity.

Metabolic reprogramming that favors high glycolytic flux with lactate production in normoxia is among cancer hallmarks. Lactate is an essential oncometabolite regulating cellular redox homeostasis, energy substrate partitioning, and intracellular signaling. Moreover, malignant phenotype’s chief characteristics are dependent on the interaction between cancer cells and their microenvironment. In breast cancer, mammary adipocytes represent an essential cellular component of the tumor milieu. We analyzed lactate concentration, lactate dehydrogenase (LDH) activity, and isozyme pattern, and LDHA/LDHB protein expression and tissue localization in paired biopsies of breast cancer tissue and cancer-associated adipose tissue in normal-weight and overweight/obese premenopausal women, compared to benign breast tumor tissue and adipose tissue in normal-weight and overweight/obese premenopausal women. We show that higher lactate concentration in cancer tissue is concomitant with a shift in isozyme pattern towards the “muscle-type” LDH and corresponding LDHA and LDHB protein expression changes. In contrast, significantly higher LDH activity in cancer-associated adipose tissue seems to be directed towards lactate oxidation. Moreover, localization patterns of LDH isoforms varied substantially across different areas of breast cancer tissue. Invasive front of the tumor showed cell-specific protein localization of LDHA in breast cancer cells and LDHB in cancer-associated adipocytes. The results suggest a specific, lactate-centric relationship between cancer tissue and cancer-associated adipose tissue and indicate how cancer-adipose tissue cross-talk may be influenced by obesity in premenopausal women.

Digital analysis of distant and cancer-associated mammary adipocytes

Adipocytes and cancer-associated adipocytes (CAAs) are poorly investigated cells in the tumor microenvironment. Different image analysis software exist for identifying and measuring these cells using scanned hematoxylin and eosin (H&E)-stained slides. It is however unclear which one is the most appropriate for breast cancer (BC) samples. Here, we compared three software (AdipoCount, Adiposoft, and HALO®). HALO® outperformed the other methods with regard to adipocyte identification, (> 96% sensitivity and specificity). All software performed equally good with regard to area and diameter measurement (concordance correlation coefficients > 0.97 and > 0.96, respectively). We then analyzed a series of 10 BCE samples (n = 51 H&E slides) with HALO®. Distant adipocytes were defined >2 mm away from cancer cells or fibrotic region, whereas CAAs as the first three lines of adipocytes close to the invasive front. Intra-mammary heterogeneity was limited, implying that measuring a single region of ∼500 adipocytes provides a reliable estimation of the distribution of their size features. CAAs had smaller areas (median fold-change: 2.62) and diameters (median fold-change: 1.64) as compared to distant adipocytes in the same breast (both p = 0.002). The size of CAAs and distant adipocytes was associated with the body mass index (BMI) of the patient (area: rho = 0.89, p = 0.001; rho = 0.71, p = 0.027, diameter: rho = 0.87 p = 0.002; rho = 0.65 p = 0.049, respectively). To conclude, we demonstrate that quantifying adipocytes in BC sections is feasible by digital pathology using H&E sections, setting the basis for a standardized analysis of mammary adiposity in larger series of patients.

Abstract 121: Mammary adipocytes drive breast tumor progression and angiogenesis via the VEGF/IL6/STAT3 signaling axis

Abstract Obesity is associated with a substantially increased risk (~50%) and a poor prognosis of breast cancer (BC) in postmenopausal (PM) women. The mechanism(s) underlying obesity-related BC are not clearly understood and, to date, most studies focus on the systemic effects of subcutaneous or visceral adipocytes on BC. We hypothesize that the increased local presence of ‘obese' mammary adipocytes within the breast microenvironment promotes the acquisition of an angiogenic and invasive breast tumor cell phenotype and thereby markedly accelerates tumor proliferation and progression. We first asked whether local interactions between mammary adipocytes and BC cells might promote tumor growth and if so, what the underlying mechanism(s) might be. Preadipocytes isolated from mammary adipose tissues of overweight/obese pre- (Pre-M; n=7) and post-menopausal (PM; n=9) women were differentiated into mature adipocytes using standard protocols. The effects of both normal (NA) and cancer-associated (CAA) mammary adipocyte secretome from Pre-M and PM obese women were tested on human and mouse BC cell lines respectively. BC cells (MDA-MB-436, MCF-7, M-Wnt), when treated with the secretome of mammary adipocytes from obese women (ObAd-CM), upregulated potent angiogenic factors, including VEGF-A, Angiopoietin-1, Jagged 1, HIF1-α and bFGF and significantly suppressed the angiogenesis inhibitor Tsp-1. ObAd-CM from both Pre-M and PM women significantly stimulated BC cell proliferation, migration and invasion. Importantly, compared to Pre-M patients, CAA Ad-CM from PM patients promoted higher levels of BC cell proliferation, migration and invasion. Pre-treatment with ObAd-CM resulted in increased endothelial cell (EC) recruitment by BC cells, an indispensable step in tumor angiogenesis. This effect was reversed by VEGF neutralization. In addition, ObAd-CM treatment significantly increased EC proliferation and capillary tube formation. Adipokines such as IL-6, IL-8, MCP-1, adiponectin and leptin were highly expressed in ObAd-CM. Of these, IL-6 neutralization alone inhibited the ObAd-CM-induced migratory and invasive phenotype of BC cells. Treatment with ObAd-CM also resulted in significant activation of pSTAT3, pAkt and pErk in BC cells. STAT3 inhibition reversed the ObAd-CM-mediated effects on BC cell proliferation and migration, suggesting that the pro-tumorigenic effects of Ob mammary adipocytes are mediated via IL-6/STAT3 signaling. Taken together, our results indicate that Ob mammary adipocytes promote migration, proliferation, invasion and the angiogenic phenotype in BC cells and suggest that targeting the VEGF/IL-6/STAT3 signaling axis may be a useful strategy in obesity-driven breast tumor growth and metastasis. [Supported by NIH RO1CA185530, the Karp Family Foundation and the Breast Cancer Research Foundation] Citation Format: Roopali Roy, Takaya Shimura, Lauren Merritt, Katherine Gonzalez, Emily Man, Margaret Lotz-Bousvaros, Susan Pories, Marsha A. Moses. Mammary adipocytes drive breast tumor progression and angiogenesis via the VEGF/IL6/STAT3 signaling axis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 121.

A risk-associated Active transcriptome phenotype expressed by histologically normal human breast tissue and linked to a pro-tumorigenic adipocyte population

BackgroundPrevious studies have identified and validated a risk-associated Active transcriptome phenotype commonly expressed in the cancer-adjacent and histologically normal epithelium, stroma, and adipose containing peritumor microenvironment of clinically established invasive breast cancers, conferring a 2.5- to 3-fold later risk of dying from recurrent breast cancer. Expression of this Active transcriptome phenotype has not yet been evaluated in normal breast tissue samples unassociated with any benign or malignant lesions; however, it has been associated with increased peritumor adipocyte composition.MethodsDetailed histologic and transcriptomic (RNAseq) analyses were performed on normal breast biopsy samples from 151 healthy, parous, non-obese (mean BMI = 29.60 ± 7.92) women, ages 27–66 who donated core breast biopsy samples to the Komen Tissue Bank, and whose average breast cancer risk estimate (Gail score) at the time of biopsy (1.27 ± 1.34) would not qualify them for endocrine prevention therapy.ResultsFull genome RNA sequencing (RNAseq) identified 52% (78/151) of these normal breast samples as expressing the Active breast phenotype. While Active signature genes were found to be most variably expressed in mammary adipocytes, donors with the Active phenotype had no difference in BMI but significantly higher Gail scores (1.46 vs. 1.18; p = 0.007). Active breast samples possessed 1.6-fold more (~ 80%) adipocyte nuclei, larger cross-sectional adipocyte areas (p < 0.01), and 0.5-fold fewer stromal and epithelial cell nuclei (p < 1e−6). Infrequent low-level expression of cancer gene hotspot mutations was detected but not enriched in the Active breast samples. Active samples were enriched in gene sets associated with adipogenesis and fat metabolism (FDR q ≤ 10%), higher signature scores for cAMP-dependent lipolysis known to drive breast cancer progression, white adipose tissue browning (Wilcoxon p < 0.01), and genes associated with adipocyte activation (leptin, adiponectin) and remodeling (CAV1, BNIP3), adipokine growth factors (IGF-1, FGF2), and pro-inflammatory fat signaling (IKBKG, CCL13).ConclusionsThe risk-associated Active transcriptome phenotype first identified in cancer-adjacent breast tissues also occurs commonly in healthy women without breast disease who do not qualify for breast cancer chemoprevention, and independently of breast expressed cancer-associated mutations. The risk-associated Active phenotype appears driven by a pro-tumorigenic adipocyte microenvironment that can predate breast cancer development.

Creatine Mediated Crosstalk between Adipocytes and Cancer Cells Regulates Obesity-Driven Breast Cancer

Obesity is a major risk factor for adverse outcomes in breast cancer; however, the underlying molecular mechanisms have not been elucidated. To investigate the role of crosstalk between mammary adipocytes and neoplastic cells in the tumor microenvironment, we performed transcriptomic analysis of cancer cells and adjacent adipose tissue in a murine model of obesity-accelerated breast cancer and identified glycine amidinotransferase (Gatm) in adipocytes and Acsbg1 in cancer cells as required for obesity-driven tumor progression. Gatm is the rate-limiting enzyme in creatine biosynthesis, and deletion in adipocytes specifically attenuated obesity-driven tumor growth. Similarly, inhibition of creatine import into cancer cells by knockdown of the creatine transporter (Slc6a8) reduced tumor growth in obesity. In parallel, breast cancer cells in obese animals markedly upregulated the fatty acyl-CoA synthetase, Acsbg1, driving creatine-dependent tumor progression. Analyses of human breast tumors revealed that increased Slc6a8 and Acsbg1 expression were significantly associated with worse tumor grade and that increased Slc6a8 expression was associated with reduced survival in triple negative breast cancer.These findings reveal key nodes in the crosstalk between adipocytes and cancer cells in the tumor microenvironment necessary for obesity-dependent breast cancer progression.

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

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 invivo. 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 invivo. These findings help explain why postmenopausal ER+ breast cancer increases with obesity, and offer new strategies for prevention and therapy.

Abstract P6-15-07: Impact of increased mammary adiposity on DCIS progression

Abstract Background: The incidence of ductal carcinoma in situ (DCIS) has greatly increased since the introduction of mammographic screening. However, so far, it is still not possible to predict which patient with DCIS will further develop invasive breast cancer. Adipocyte hypertrophy and adipocyte inflammation has been associated with increased risk of developing breast cancer in postmenopausal women and with worse prognosis in breast cancer patients. In this study, we aimed 1) at evaluating the association between mammary adiposity, as assessed by digital pathology, with DCIS progression using a unique case-control series with long-term follow up and 2) study the interaction between DCIS cells and the surrounding adipocytes. Patients and methods: Mammary adipocyte size was measured in surgical specimens of 279 women with primary DCIS. These mammary adipocyte measurements were compared in 109 women with primary DCIS who subsequently developed ipsilateral invasive breast cancer (iIBC) (cases) and 170 women who did not (controls). Patients were matched for age and follow-up duration. Median follow-up time was 12.8 years. Post-menopausal patients were defined as &amp;gt;50 years at diagnosis and represented 220/279 (78,9%) of patients. All patients were treated with breast cancer-conserving surgery only. Intact adipocytes distant from the DCIS lesions were measured (largest diameter and area) using HALO™ image analysis software (Indica Labs, Corrales, NM) on H&amp;E-stained whole slide images of primary DCIS. Given the specific interest in the larger adipocytes, we systematically considered the 75th percentile as unique value for each patient. An average of 4259 adipocytes (min:70, max:21107) was measured per patient. Tissue segmentation was used to measure the adipose area. Adipose triglyceride lipase (ATGL clone 2138, Cell Signaling) immunohistochemistry (IHC) was applied to study ATGL expression in DCIS cells. Her2, ER and COX-2 IHC and RNAseq of microdissected pure DCIS was already available for this series (LL. Visser et al. Clin Can Res 24 (15) 2018). Conditional logistic regression was applied to investigate differences in adipocyte hypertrophy between groups of patients (e.g., cases and controls). Results: Adipocytes were larger and the proportion of adipose tissue was higher in post-menopausal patients (both p&amp;lt;0.001). Mean adipocyte maximum diameter and mean adipocyte area of the 75th percentile ranged from 46.9-115.9 µm and 2016-11336 µm2 respectively. For every 1000 µm2 increase in mean adipocyte area a DCIS patient had a 1.18 increased risk for iIBC (95% CI 1.02-1.36, p= 0.028). DCIS patients with large adipocytes (above the 75th percentile) had a 2.1 increased risk for developing iIBC (95% CI: 1.18-3.72, p=0.011). This observation remained when restricting the analysis to post-menopausal patients (OR 1.19 per 1000 µm2 95% CI 1.01-1.40, p=0.038 and adipocytes above 75th percentile OR 2.2, 95% CI 1.22-3.99, p= 0.009). To further explore the potential interactions between the epithelial cells from DCIS with the adjacent adipocytes, we compared the size of the DCIS-adjacent adipocytes to those away from DCIS lesions. DCIS-adjacent adipocytes showed a reduction in size, suggesting delipidation. Adipose triglyceride lipase (ATGL) is a rate limiting lipase that can release stored free fatty acids (FFA) in DCIS cells for fatty acid ß-oxidation. In a pilot series strong ATGL expression was observed in a subset of DCIS lesions (4 of 25). Conclusion and perspective: This study is the first to demonstrate that mammary adipocyte hypertrophy is associated with an increased risk of progression for patients with DCIS. Our findings might help to distinguish potentially hazardous from harmless DCIS, enabling overtreatment of indolent DCIS. Adipocyte size will be correlated to immunohistochemical expression of ATGL, Her2, ER and COX2 in DCIS, and RNAseq data of pure microdissected DCIS. Citation Format: Mathilde Matthea Machteld Almekinders, Michael Schaapveld, Bram Thijssen, Ingrid Hofland, Marjolijn Mertz, Dennis Peters, Annegien Broeks, Lodewijk Wessels, Wilbert Zwart, Jos Jonkers, Esther Lips, Christine Desmedt, Jelle Wesseling, on behalf of the PRECISION Team. Impact of increased mammary adiposity on DCIS progression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-15-07.

Abstract P3-08-20: The normal breast Active transcriptome associated with future breast cancer risk is driven by a dysregulated adipocyte microenvironment

Abstract Background: Previous studies of breast samples from cosmetic surgeries, benign biopsies associated with abnormal mammograms or cancer-adjacent tissue have identified at least two different transcriptional phenotypes of “normal” human breast tissue (Troester, 2009; Haakensen, 2011), including an “Active” phenotype linked to increased risk of later breast cancer mortality (Roman-Perez, 2012; Troester, 2016). This study compares breast histology and transcriptional phenotypes from healthy parous women with no prior history of breast disease who donated breast core biopsies for research and supplied reproductive histories enabling breast cancer risk calculation. Since the Active transcriptome phenotype was recently associated with increased mammary adipocyte content, we focused on the possibility that adipocyte activation contributes to the Active transcriptome and drives breast cancer risk. Methods: RNA from paraffin-embedded tissue sections sufficient for RNAseq analysis (~100ng) was extracted from 151/200 core biopsies donated to the Komen Tissue Bank by healthy, parous white women (age range: 27-66, median = 45) with no history of breast cancer. Questionnaire data enabled breast cancer risk (Gail) score calculation; and digitized H&amp;E images were used for histologic analyses. A previously validated classifying signature was used in unsupervised hierarchical clustering to identify samples with Active (78/151) vs. Inactive (73/151) transcriptome phenotypes for comparison with donor risk factors, breast tissue composition, and expression of candidate genes and gene signatures. Results: Mean (+/-SD) BMI and Gail score values were 29.60 (+/-7.92) and 1.27 (+/- 1.34), respectively; BMI scores were not significantly different by phenotype, but Gail scores were significantly higher for donors with an Active phenotype (1.46 vs. 1.18; p=0.007, Wilcoxon rank-sum). Active normal breast tissue samples possessed significantly more (%) adipocyte nuclei (p=3.9e-11) and greater adipocyte size (p&amp;lt;0.01), with fewer (%) stromal (p=4.3e-7) and (%) epithelial (p=1.2e-10) nuclei. Differentially expressed genes between the Active and Inactive phenotypes were significantly enriched for gene sets (GSEA) involved in fat differentiation and metabolism (14% at FDR q≤10%). Signature scores for cAMP-dependent lipolysis (known to drive breast cancer progression), as well as white adipose tissue “browning,” were significantly higher (Wilcoxon p&amp;lt;0.01) for the Active samples, as were specific genes reflecting adipocyte activation (leptin, adiponectin), remodeling (CAV1, BNIP3), adipokine growth factors (IGF-1, FGF2), and pro-inflammatory fat signaling (IKBKG, CCL13). Conclusion: In this cohort of predominantly non-obese women without breast disease, ~50% express an Active transcriptome phenotype previously linked to later-life breast cancer mortality. While &amp;gt;80% of this donor cohort would not qualify for breast cancer chemoprevention, those with Active transcriptomes had significantly higher Gail scores supporting their increased future risk for breast cancer development. The Active breast transcriptome is strongly associated with increased adipocyte content, size, and overexpression of signatures and genes (including those previously linked to breast cancer progression) indicating a differentially activated adipocyte population. This dysregulated mammary adipocyte microenvironment not only appears to underlie the Active transcriptome phenotype but also precedes and potentially predicts the future histologic development of breast cancer. Citation Format: Christopher C Benz, Taekyu Kang, Christina Yau, Chris Wong, Yulia Newton, Charlie Vaske, Stephen C Benz, Gregor Krings, Roman Camarda, Jill E Henry, Josh Stuart, Mark Powell. The normal breast Active transcriptome associated with future breast cancer risk is driven by a dysregulated adipocyte microenvironment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-20.

STAT3 Activation-Induced Fatty Acid Oxidation in CD8+ T Effector Cells Is Critical for Obesity-Promoted Breast Tumor Growth

Although obesity is known to be critical for cancer development, how obesity negatively impacts antitumor immune responses remains largely unknown. Here, we show that increased fatty acid oxidation (FAO) driven by activated STAT3 in CD8+ T effector cells is critical for obesity-associated breast tumor progression. Ablating Tcell Stat3 or treatment with an FAO inhibitor in obese mice spontaneously developing breast tumor reduces FAO, increases glycolysis and CD8+ T effector cell functions, leading to inhibition of breast tumor development. Moreover, PD-1 ligation in CD8+ Tcells activates STAT3 to increase FAO, inhibiting CD8+ T effector cell glycolysis and functions. Finally, leptin enriched in mammary adipocytes and fat tissues downregulates CD8+ Tcell effector functions through activating STAT3-FAO and inhibiting glycolysis. We identify a critical role of increased oxidation of fatty acids driven by leptin and PD-1 through STAT3 in inhibiting CD8+ T effector cell glycolysis and in promoting obesity-associated breast tumorigenesis.

Low-dose Bisphenol-A regulates inflammatory cytokines through GPR30 in mammary adipose cells.

The dramatic rise in obesity and metabolic syndrome can be related, at least in part, to environmental chemical factors such as Bisphenol-A (BPA). In this study, we aimed to understand the effects of low-dose Bisphenol-A on the human mature adipocytes and stromal vascular fraction (SVF) cells, obtained from subcutaneous mammary adipose tissue of overweight female patients, undergoing surgical mammary reduction. 24 and/or 48-h exposure to BPA 0.1 nM elicited significant increase of the inflammatory molecules interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemo-attractant protein 1α (MCP1α) and induced G protein-coupled estrogen receptor 30 (GPR30) levels more than two-fold both in mature adipocytes and SVF cells. These effects were similar to that obtained in the presence of GPR30-specific agonist G1 (100 nM) and were reverted by G15 (1 µM), a GPR30-selective antagonist. As a result of BPA-GPR30 signaling activation, fatty acid synthase (FAS) and leptin mRNA levels were significantly higher upon BPA exposure (P < 0.05) in mature adipocytes, with an opposite effect on adiponectin (ADIPOQ). In addition, an increase in SVF cell proliferation and ERK1/2 phosphorylation, was observed, compared to untreated cells. G15 reverted all of these effects. Interestingly, the action of BPA on SVF cell growth was mimicked by IL-8 treatment and was reverted by incubation with anti-IL8 antibodies. All these data suggest that BPA at 0.1 nM, a ten times lower concentration than environmental exposure, increases the expression of pro-inflammatory cytokines via GPR30 both in mature mammary adipocytes and in SVF cells with a possible involvement of IL-8.

Remodeling of Murine Mammary Adipose Tissue during Pregnancy, Lactation, and Involution.

White adipocytes in the mammary gland stroma comprise the majority of the mammary gland mass. White adipocytes regulate numerous hormonal and metabolic processes and exhibit compositional and phenotypic plasticity. This plasticity is exemplified by the ability of mammary adipocytes to regress during lactation, when mammary epithelial cells expand to establish sufficient milk-producing alveoli. Upon weaning, the process reverses through mammary involution, during which adipocytes extensively regenerate, and alveolar epithelial cells disappear through cell death, returning the mammary gland to the non-lactating state. Despite intensive studies on the development and involution of the mammary alveolar epithelium, the fate of mammary adipocytes during pregnancy and lactation, and the origins of mammary adipocytes regenerated during mammary involution, is poorly understood. Here, we discuss the recent discoveries of the fate of mammary adipocytes during pregnancy and lactation in a number of different mouse models, and the lineage origin of mammary adipocytes regenerated during involution.

Abstract 3314: Normal breast tissue at risk for cancer development: A breast cancer initiating role for mammary adipocytes

Abstract Adipocytes are the predominant cell population in the normal breast and while recent attention has pointed to adipocyte-tumor cell crosstalk as a driver of breast cancer biology there have been few reports on the potential role of adipocytes in driving breast cancer initiation. Because normal breast tissue studies have invariably used reduction mammoplasty, benign biopsy or cancer-adjacent tissues, we studied random breast core biopsy samples donated by 145 healthy, parous, non-obese, white women (median age = 45, range 27-66 y) without any history of breast cancer. Using questionnaire data to calculate future breast cancer risk (Gail scores), we compared digitized microscopic breast tissue (H&amp;E) images with whole genome transcriptome profiling (RNAseq) from FFPE-extracted RNA. We used unsupervised hierarchical clustering of 1487 genes (normalized, median centered, log2-scaled RSEM values) to identify 32% of normal samples with an “Active” (vs. 68% “Inactive”) transcriptome phenotype previously associated with later-life risk of death from breast cancer. Despite slightly lower BMI values, donors with the Active transcriptome phenotype showed significantly higher Gail scores as well as higher mammary adipocyte nuclei counts (median 80% vs. 60%, p=2.3e-6). Tissue resident leukocytes were uncommon but Active transcriptome tissues expressed significantly altered immune modules enriched in TGFβ, interferon and macrophage gene signatures (including single gene increase in CD68) and depleted (relative to Inactive samples) of CD8+ T-cell and serum response/inflammation/wound healing signatures. Active samples were not enriched in cell senescence, SASP or DNA damage response gene signatures but were enriched in an autophagy-to-senescence-transition (AST) signature with increased CAV1 (caveolin-1, p=2.7e-12) and BNIP3 (Bcl2 interacting protein-3, p=4.7e-05) expression, genes that also regulate lipoprotein digestion/mobilization and adipocyte remodeling. Strongest among significant associations linking Active with adipocyte-enriched normal breast samples were increases in two adipokine growth factors, IGF-1 (p=2.2e-16) and FGF2 (p=3.0e-11), the adipokine (resistin) receptor CAP1 (p=0.04) recently linked to poor breast cancer outcomes, and a cAMP-dependent pro-lipolytic signature (p=0.01) known to drive breast cancer progression which, in these samples, correlated positively with average adipocyte area values. Altogether, the collective histologic and molecular features characterizing the normal breast tissue of &amp;gt;30% of healthy parous and non-obese women with increased predicted breast cancer risk seem to implicate a dysregulated mammary adipocyte microenvironment similar to but distinct from that associated with established breast tumors, that precedes microscopic and clinical evidence of breast tumorigenesis. Citation Format: Taekyu Kang, Christina Yau, Stephen Benz, Gregor Krings, Roman Camarda, Jill E. Henry, Mark Powell, Christopher C. Benz. Normal breast tissue at risk for cancer development: A breast cancer initiating role for mammary adipocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3314.

Adipocytes promote breast cancer resistance to chemotherapy, a process amplified by obesity: role of the major vault protein\xa0(MVP)

IntroductionClinical studies suggest that obesity, in addition to promoting breast cancer aggressiveness, is associated with a decrease in chemotherapy efficacy, although the mechanisms involved remain elusive. As chemotherapy is one of the main treatments for aggressive or metastatic breast cancer, we investigated whether adipocytes can mediate resistance to doxorubicin (DOX), one of the main drugs used to treat breast cancer, and the mechanisms associated.MethodsWe used a coculture system to grow breast cancer cells with in vitro differentiated adipocytes as well as primary mammary adipocytes isolated from lean and obese patients. Drug cellular accumulation, distribution, and efflux were studied by immunofluorescence, flow cytometry, and analysis of extracellular vesicles. Results were validated by immunohistochemistry in a series of lean and obese patients with cancer.ResultsAdipocytes differentiated in vitro promote DOX resistance (with cross-resistance to paclitaxel and 5-fluorouracil) in a large panel of human and murine breast cancer cell lines independently of their subtype. Subcellular distribution of DOX was altered in cocultivated cells with decreased nuclear accumulation of the drug associated with a localized accumulation in cytoplasmic vesicles, which then are expelled into the extracellular medium. The transport-associated major vault protein (MVP), whose expression was upregulated by adipocytes, mediated both processes. Coculture with human mammary adipocytes also induced chemoresistance in breast cancer cells (as well as the related MVP-induced DOX efflux) and their effect was amplified by obesity. Finally, in a series of human breast tumors, we observed a gradient of MVP expression, which was higher at the invasive front, where tumor cells are at close proximity to adipocytes, than in the tumor center, highlighting the clinical relevance of our results. High expression of MVP in these tumor cells is of particular interest since they are more likely to disseminate to give rise to chemoresistant metastases.ConclusionsCollectively, our study shows that adipocytes induce an MVP-related multidrug-resistant phenotype in breast cancer cells, which could contribute to obesity-related chemoresistance.

Cyclical Dedifferentiation and Redifferentiation of Mammary Adipocytes

Adipocytes are generally thought to be terminally differentiated cells; however, recent evidence suggests a subset may have greater plasticity in certain contexts. In this issue of Cell Metabolism, Wang etal. (2018) demonstrate a novel capacity for mammary adipocytes to dedifferentiate into preadipocyte-like precursors during lactation and redifferentiate upon weaning.

Abstract 4501: Mammary adipocytes mediate cytokine production and malignant progression of ER-positive breast cancer through NF-kB activation

Abstract Obesity increases the adverse prognosis of all breast cancer subtypes at any age, and is also associated with an increased risk of developing estrogen receptor-positive (ER+) breast cancers after menopause. However, mechanisms thereof are not fully known. We have identified one mechanism whereby obesity contributes to tumor progression. Cancers contain a subset of self-renewing stem cells that mediate treatment resistance and metastasis. We showed prolonged co-culture of in vitro differentiated adipocytes together with breast cancer lines or cultured primary dissociated human breast cancer cells increases secretion of pro-inflammatory cytokines IL6, IL8, CCL2, CCL5 and IP10. Prolonged exposure to these fat cells or to each cytokine increased the proportion of both ER+ and ER- breast cancer cells that form mammospheres and express ALDH1 activity in vitro and that can initiate primary tumors and metastasis in vivo. Differentiated adipocyte or cytokine exposures activate Src, and Src family kinase activity induced Sox2, cMyc and Nanog upregulation and miR302b induction. miR302b upregulation is Sox2-dependent, promotes cytokine-driven sphere formation, and in turn, stimulates cMYC and SOX2 expression. Finally Src was not only activated by differentiated adipocyte or cytokine exposures, it was also required to sustain cytokine induction, since Src inhibitors decreased cytokine production after co-culture. Thus, cancer cell invasion into local fat would establish feed-forward loops to activate Src, maintain pro-inflammatory cytokine production and increase tumor initiating cell abundance, tumor growth and metastasis. It is well known that obesity mediates a chronic inflammatory state through NF-κB pathway driven pro-inflammatory cytokine expression. Our ongoing work investigates the role of NF-kB pathway in the inflammatory tumor promoting effects of obesity. We have observed that cytokine induction in breast cancer cell exposed to isolated mammary breast adipocytes is estrogen:ER dependent, since this effect in ER+ breast cancer cell is much greater than that in ER- cancer cells; and since the aromatase inhibitor, letrozole, reduced co-culture induced cytokine induction. We found NF-kB inhibitors prevent co-culture mediated cytokine induction, and that adipocyte co-culture increased RelA (p65) levels and its nuclear localization. Following invasion into local obese fat, high aromatase levels would increase local estrogen to drive cytokine induction upon mature adipocyte:cancer contact, and increase ER+ cancer stem cells and metastasis. Citation Format: Manuel Picon Ruiz, Cynthia Morata Tarifa, Rehana Qureshi, Hyunho Yoon, Miyoung Shin, Hetakshi Kurani, Joyce M. Slingerland. Mammary adipocytes mediate cytokine production and malignant progression of ER-positive breast cancer through NF-kB activation [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 4501.

Resveratrol inhibits obesity-associated adipose tissue dysfunction and tumor growth in a mouse model of postmenopausal claudin-low breast cancer.

Adipose tissue dysregulation, a hallmark of obesity, contributes to a chronic state of low-grade inflammation and is associated with increased risk and progression of several breast cancer subtypes, including claudin-low breast tumors. Unfortunately, mechanistic targets for breaking the links between obesity-associated adipose tissue dysfunction, inflammation, and claudin-low breast cancer growth have not been elucidated. Ovariectomized female C57BL/6 mice were randomized (n = 15/group) to receive a control diet, a diet-induced obesity (DIO) diet, or a DIO + resveratrol (0.5% wt/wt) diet. Mice consumed these diets ad libitum throughout study and after 6 weeks were orthotopically injected with M-Wnt murine mammary tumor cells, a model of estrogen receptor (ER)-negative claudin-low breast cancer. Compared with controls, DIO mice displayed adipose dysregulation and metabolic perturbations including increased mammary adipocyte size, cyclooxygenase-2 (COX-2) expression, inflammatory eicosanoid levels, macrophage infiltration, and prevalence of crown-like structures (CLS). DIO mice (relative to controls) also had increased systemic inflammatory cytokines and decreased adipocyte expression of peroxisome proliferator-activated receptor gamma (PPARγ) and other adipogenesis-regulating genes. Supplementing the DIO diet with resveratrol prevented obesity-associated increases in mammary tumor growth, mammary adipocyte hypertrophy, COX-2 expression, macrophage infiltration, CLS prevalence, and serum cytokines. Resveratrol also offset the obesity-associated downregulation of adipocyte PPARγ and other adipogenesis genes in DIO mice. Our findings suggest that resveratrol may inhibit obesity-associated inflammation and claudin-low breast cancer growth by inhibiting adipocyte hypertrophy and associated adipose tissue dysregulation that typically accompanies obesity.

Mammary adipocytes stimulate breast cancer invasion through metabolic remodeling of tumor cells.

In breast cancer, a key feature of peritumoral adipocytes is their loss of lipid content observed both in vitro and in human tumors. The free fatty acids (FFAs), released by adipocytes after lipolysis induced by tumor secretions, are transferred and stored in tumor cells as triglycerides in lipid droplets. In tumor cell lines, we demonstrate that FFAs can be released over time from lipid droplets through an adipose triglyceride lipase-dependent (ATGL-dependent) lipolytic pathway. In vivo, ATGL is expressed in human tumors where its expression correlates with tumor aggressiveness and is upregulated by contact with adipocytes. The released FFAs are then used for fatty acid β-oxidation (FAO), an active process in cancer but not normal breast epithelial cells, and regulated by coculture with adipocytes. However, in cocultivated cells, FAO is uncoupled from ATP production, leading to AMPK/acetyl-CoA carboxylase activation, a circle that maintains this state of metabolic remodeling. The increased invasive capacities of tumor cells induced by coculture are completely abrogated by inhibition of the coupled ATGL-dependent lipolysis/FAO pathways. These results show a complex metabolic symbiosis between tumor-surrounding adipocytes and cancer cells that stimulate their invasiveness, highlighting ATGL as a potential therapeutic target to impede breast cancer progression.

Abstract LB-316: Social isolation alters abdominal and mammary adipocytes to potentially increase mammary tumorigenesis in mice

Abstract Social isolation, characterized as withdrawal from interactions with others, is an equally powerful predictor of early all-cause mortality as smoking and high cholesterol. Social isolation has been consistently linked to increased breast cancer risk and mortality in animal studies and humans. Recent animal studies in our laboratory show that social isolation potentiates the effects of high fat (HF) diet on carcinogen- induced mammary cancer. Social isolation coupled with HF diet also magnified adverse metabolic effects associated with obesity, including insulin resistance. To investigate how these metabolic effects manifest, we measured and compared metabolic endpoints in abdominal and mammary gland adipose cells in 7- and 23-week-old female mice kept in social isolation for 4 or 20 weeks, respectively. Four groups of C56BL/6 mice were studied: control diet fed group housed mice (C-G), HF diet fed group housed mice (HF-G), control diet fed socially isolated mice (C-ISO) and HF diet fed socially isolated mice (HF-ISO). At 7 weeks of age, abdominal adipocytes of HF diet fed HF-G mice were significantly larger than in C-G control mice (C-G) (p = 0.012), indicating adipocyte hypertrophy, a hallmark of obesity. Adipocytes in HF-ISO were also enlarged, but not significantly. At 23 weeks of age, however, mammary adipocytes of HF-ISO mice were significantly reduced compared with C-G controls (p = 0.046) and HF-G group (p = 0.009). Abdominal adipocytes were reduced both in C-ISO (p&amp;lt;0.001) and HF-ISO (p = 0.002) compared with HF-G. Histological analysis indicated more pockets or areas of multi-locular adipocytes in the mammary adipocytes of socially isolated mice (both HF-ISO and C-ISO), suggesting the presence of beige adipocytes. Beige adipocytes are specialized white adipocytes that have an appearance and function similar to brown adipocytes that dissipate energy. An increase in beige adipocytes has been proposed to prevent and reverse weight gain and metabolic diseases, but it is not clear whether these cells affect the development of breast and other cancers. Immunohistochemical analysis for beige specific adipocyte markers in abdominal and mammary adipose tissue showed higher intensity staining for Ucp1 and Pgc1 in HF-G, HF-ISO and C-ISO, compared with C-G controls. In 7 week old mice, western blot analysis of mammary gland samples of these groups indicated more Ucp1 expression, albeit with no statistical significance. However, Bmp4, an activator of beige adipocytes, was downregulated in C-ISO (p = 0.015), HF-G (p = 0.020), and HF-ISO (p = 0.057) groups with C-G group, suggesting that if present, the beige adipocytes may not be active in socially isolated mice. Our findings suggest that social isolation may alter phenotype and function of abdominal and mammary adipocytes, and these changes could contribute to the increased breast cancer risk and mortality in socially isolated women and animal models. Citation Format: Madisa Macon, Allison Sumis, Wei Yu, Leena Hilakivi-Clarke. Social isolation alters abdominal and mammary adipocytes to potentially increase mammary tumorigenesis in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-316.

Mammary Adipose Tissue-Derived Lysophospholipids Promote Estrogen Receptor–Negative Mammary Epithelial Cell Proliferation

Lysophosphatidic acid (LPA), acting in an autocrine or paracrine fashion through G protein-coupled receptors, has been implicated in many physiologic and pathologic processes, including cancer. LPA is converted from lysophosphatidylcholine (LPC) by the secreted phospholipase autotaxin (ATX). Although various cell types can produce ATX, adipocyte-derived ATX is believed to be the major source of circulating ATX and also to be the major regulator of plasma LPA levels. In addition to ATX, adipocytes secrete numerous other factors (adipokines); although several adipokines have been implicated in breast cancer biology, the contribution of mammary adipose tissue-derived LPC/ATX/LPA (LPA axis) signaling to breast cancer is poorly understood. Using murine mammary fat-conditioned medium, we investigated the contribution of LPA signaling to mammary epithelial cancer cell biology and identified LPA signaling as a significant contributor to the oncogenic effects of the mammary adipose tissue secretome. To interrogate the role of mammary fat in the LPA axis during breast cancer progression, we exposed mammary adipose tissue to secreted factors from estrogen receptor-negative mammary epithelial cell lines and monitored changes in the mammary fat pad LPA axis. Our data indicate that bidirectional interactions between mammary cancer cells and mammary adipocytes alter the local LPA axis and increase ATX expression in the mammary fat pad during breast cancer progression. Thus, the LPC/ATX/LPA axis may be a useful target for prevention in patients at risk of ER-negative breast cancer. Cancer Prev Res; 9(5); 367-78. ©2016 AACR.

Mammary epithelial tubes elongate through MAPK-dependent coordination of cell migration

Mammary branching morphogenesis is regulated by receptor tyrosine kinases (RTKs). We sought to determine how these RTK signals alter proliferation and migration to accomplish tube elongation in mouse. Both behaviors occur but it has been difficult to determine their relative contribution to elongation in vivo, as mammary adipocytes scatter light and limit the depth of optical imaging. Accordingly, we utilized 3D culture to study elongation in an experimentally accessible setting. We first used antibodies to localize RTK signals and discovered that phosphorylated ERK1/2 (pERK) was spatially enriched in cells near the front of elongating ducts, whereas phosphorylated AKT was ubiquitous. We next observed a gradient of cell migration speeds from rear to front of elongating ducts, with the front characterized by both high pERK and the fastest cells. Furthermore, cells within elongating ducts oriented both their protrusions and their migration in the direction of tube elongation. By contrast, cells within the organoid body were isotropically protrusive. We next tested the requirement for proliferation and migration. Early inhibition of proliferation blocked the creation of migratory cells, whereas late inhibition of proliferation did not block continued duct elongation. By contrast, pharmacological inhibition of either MEK or Rac1 signaling acutely blocked both cell migration and duct elongation. Finally, conditional induction of MEK activity was sufficient to induce collective cell migration and ductal elongation. Our data suggest a model for ductal elongation in which RTK-dependent proliferation creates motile cells with high pERK, the collective migration of which acutely requires both MEK and Rac1 signaling.