Obesity and adverse breast cancer risk and outcome: Mechanistic insights and strategies for intervention.

Answer questions and earn CME/CNE Recent decades have seen an unprecedented rise in obesity, and the health impact thereof is increasingly evident. In 2014, worldwide, more than 1.9 billion adults were overweight (body mass index [BMI], 25-29.9 kg/m2 ), and of these, over 600 million were obese (BMI ≥30 kg/m2 ). Although the association between obesity and the risk of diabetes and coronary artery disease is widely known, the impact of obesity on cancer incidence, morbidity, and mortality is not fully appreciated. Obesity is associated both with a higher risk of developing breast cancer, particularly in postmenopausal women, and with worse disease outcome for women of all ages. The first part of this review summarizes the relationships between obesity and breast cancer development and outcomes in premenopausal and postmenopausal women and in those with hormone receptor-positive and -negative disease. The second part of this review addresses hypothesized molecular mechanistic insights that may underlie the effects of obesity to increase local and circulating proinflammatory cytokines, promote tumor angiogenesis and stimulate the most malignant cancer stem cell population to drive cancer growth, invasion, and metastasis. Finally, a review of observational studies demonstrates that increased physical activity is associated with lower breast cancer risk and better outcomes. The effects of recent lifestyle interventions to decrease sex steroids, insulin/insulin-like growth factor-1 pathway activation, and inflammatory biomarkers associated with worse breast cancer outcomes in obesity also are discussed. Although many observational studies indicate that exercise with weight loss is associated with improved breast cancer outcome, further prospective studies are needed to determine whether weight reduction will lead to improved patient outcomes. It is hoped that several ongoing lifestyle intervention trials, which are reviewed herein, will support the systematic incorporation of weight loss intervention strategies into care for patients with breast cancer. CA Cancer J Clin 2017;67:378-397. © 2017 American Cancer Society.

Estradiol and Estrone Have Different Biological Functions to Induce NF-κB-Driven Inflammation, EMT and Stemness in ER+ Cancer Cells.

In general, the risk of being diagnosed with cancer increases with age; however, the development of estrogen-receptor-positive (ER+) cancer types in women are more closely related to menopausal status than age. In fact, the general risk factors for cancer development, such as obesity-induced inflammation, show differences in their association with ER+ cancer risk in pre- and postmenopausal women. Here, we tested the role of the principal estrogens in the bloodstream before and after menopause, estradiol (E2) and estrone (E1), respectively, on inflammation, epithelial-to-mesenchymal transition (EMT) and cancer stem cell enrichment in the human ER+ cervical cancer cell line HeLa. Our results demonstrate that E1, contrary to E2, is pro-inflammatory, increases embryonic stem-transcription factors (ES-TFs) expression and induces EMT in ER+ HeLa cells. Moreover, we observed that high intratumoural expression levels of 17ß-Hydroxysteroid dehydrogenase (HSD17B) isoforms involved in E1 synthesis is a poor prognosis factor, while overexpression of E2-synthetizing HSD17B isoforms is associated with a better outcome, for patients diagnosed with ER+ ovarian and uterine corpus carcinomas. This work demonstrates that E1 and E2 have different biological functions in ER+ gynaecologic cancers. These results open a new line of research in the study of ER+ cancer subtypes, highlighting the potential key oncogenic role of E1 and HSD17B E1-synthesizing enzymes in the development and progression of these diseases.

Dual Role of Fibroblasts Educated by Tumour in Cancer Behavior and Therapeutic Perspectives.

Tumours are complex systems with dynamic interactions between tumour cells, non-tumour cells, and extracellular components that comprise the tumour microenvironment (TME). The majority of TME's cells are cancer-associated fibroblasts (CAFs), which are crucial in extracellular matrix (ECM) construction, tumour metabolism, immunology, adaptive chemoresistance, and tumour cell motility. CAF subtypes have been identified based on the expression of protein markers. CAFs may act as promoters or suppressors in tumour cells depending on a variety of factors, including cancer stage. Indeed, CAFs have been shown to promote tumour growth, survival and spread, and secretome changes, but they can also slow tumourigenesis at an early stage through mechanisms that are still poorly understood. Stromal-cancer interactions are governed by a variety of soluble factors that determine the outcome of the tumourigenic process. Cancer cells release factors that enhance the ability of fibroblasts to secrete multiple tumour-promoting chemokines, acting on malignant cells to promote proliferation, migration, and invasion. This crosstalk between CAFs and tumour cells has given new prominence to the stromal cells, from being considered as mere physical support to becoming key players in the tumour process. Here, we focus on the concept of cancer as a non-healing wound and the relevance of chronic inflammation to tumour initiation. In addition, we review CAFs heterogeneous origins and markers together with the potential therapeutic implications of CAFs "re-education" and/or targeting tumour progression inhibition.

Cardiomyogenic differentiation potential of human endothelial progenitor cells isolated from patients with myocardial infarction.

BACKGROUND AIMS: Endothelial progenitor cells (EPCs) are known to play a beneficial role by promoting postnatal vasculogenesis in pathological events, such as ischemic heart disease and peripheral artery disease. However, little is known about the potential of EPCs to restore heart damage tissue. We compared the cardiac differentiation capacity of EPCs isolated from peripheral blood of patients with acute myocardial infarction (AMI) with EPCs obtained from umbilical cord blood (UCB). METHODS: EPCs from both origins were isolated by density gradient centrifugation and characterized through the use of endothelial markers (UEA-1lectin, CD133 and KDR) and endothelial cell colony-forming unit assay. Cardiac differentiation capacity of EPCs was assessed by immunofluorescence and reverse transcriptase-polymerase chain reaction after 5-azacytidine (5-aza) induction. RESULTS: No significant differences were observed between the number of endothelial cell colony-forming units in peripheral blood of patients with AMI and samples from UCB. Moreover, 5-aza induced the appearance of myotube-like structures and the positive expression of sarcomeric α-actinin, cardiac troponin I and T and desmin in a similar pattern for both cell sources, which indicates a comparable acquisition of a cardiac-like phenotype. CONCLUSIONS: For the first time, we have compared, in vitro, the cardiomyogenic potential of EPCs derived from patients with AMI with UCB-derived EPCs. Our data indicate that EPCs obtained from both origins have similar plasticity and functions and suggest a potential therapeutic efficacy in cardiac cell therapy.

Estrone, the major postmenopausal estrogen, binds ERa to induce SNAI2, epithelial-to-mesenchymal transition, and ER+ breast cancer metastasis.

Recent work showed that the dominant post-menopausal estrogen, estrone, cooperates with nuclear factor κB (NF-κB) to stimulate inflammation, while pre-menopausal 17ß-estradiol opposes NF-κB. Here, we show that post-menopausal estrone, but not 17ß-estradiol, activates epithelial-to-mesenchymal transition (EMT) genes to stimulate breast cancer metastasis. HSD17B14, which converts 17ß-estradiol to estrone, is higher in cancer than normal breast tissue and in metastatic than primary cancers and associates with earlier metastasis. Treatment with estrone, but not 17ß-estradiol, and HSD17B14 overexpression both stimulate an EMT, matrigel invasion, and lung, bone, and liver metastasis in estrogen-receptor-positive (ER+) breast cancer models, while HSD17B14 knockdown reverses the EMT. Estrone:ERα recruits CBP/p300 to the SNAI2 promoter to induce SNAI2 and stimulate an EMT, while 17ß-estradiol:ERα recruits co-repressors HDAC1 and NCOR1 to this site. Present work reveals novel differences in gene regulation by these estrogens and the importance of estrone to ER+ breast cancer progression. Upon loss of 17ß-estradiol at menopause, estrone-liganded ERα would promote ER+ breast cancer invasion and metastasis.

Obtaining Human Breast Adipose Cells for Breast Cancer Cell Co-culture Studies.

Primary human breast cancers invade surrounding fat and contact adipocytes, inflammatory infiltrates, and fibrous stroma. This tissue niche influences breast tumor progression. Here, we present a protocol to enable the in vitro study of the complex interactions that occur between breast cancer cells and adipose cells. We describe how to obtain different adipose cell populations, including adipose-derived stem cells, immature adipocytes, and mature adipocytes, from human breast fat tissue and detail the application for co-culture assays with breast cancer cells. For complete details on the use and execution of this protocol, please refer to Picon-Ruiz et al. (2016) and Qureshi et al. (2020).

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 17ß-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 17ß-estradiol-driven transcriptomes differ. Estrone:ERα stimulates NFκB-mediated cytokine gene induction; 17ß-estradiol opposes this. In obese mice, estrone increases and 17ß-estradiol relieves inflammation. Estrone drives more rapid ER+ breast cancer growth in vivo. HSD17B14, which converts 17ß-estradiol to estrone, associates with poor ER+ breast cancer outcome. Estrone and HSD17B14 upregulate inflammation, ALDH1 activity, and tumorspheres, while 17ß-estradiol and HSD17B14 knockdown oppose these. Finally, a high intratumor estrone:17ß-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.

Low adherent cancer cell subpopulations are enriched in tumorigenic and metastatic epithelial-to-mesenchymal transition-induced cancer stem-like cells.

Cancer stem cells are responsible for tumor progression, metastasis, therapy resistance and cancer recurrence, doing their identification and isolation of special relevance. Here we show that low adherent breast and colon cancer cells subpopulations have stem-like properties. Our results demonstrate that trypsin-sensitive (TS) breast and colon cancer cells subpopulations show increased ALDH activity, higher ability to exclude Hoechst 33342, enlarged proportion of cells with a cancer stem-like cell phenotype and are enriched in sphere- and colony-forming cells in vitro. Further studies in MDA-MB-231 breast cancer cells reveal that TS subpopulation expresses higher levels of SLUG, SNAIL, VIMENTIN and N-CADHERIN while show a lack of expression of E-CADHERIN and CLAUDIN, being this profile characteristic of the epithelial-to-mesenchymal transition (EMT). The TS subpopulation shows CXCL10, BMI-1 and OCT4 upregulation, differing also in the expression of several miRNAs involved in EMT and/or cell self-renewal such as miR-34a-5p, miR-34c-5p, miR-21-5p, miR-93-5p and miR-100-5p. Furthermore, in vivo studies in immunocompromised mice demonstrate that MDA-MB-231 TS cells form more and bigger xenograft tumors with shorter latency and have higher metastatic potential. In conclusion, this work presents a new, non-aggressive, easy, inexpensive and reproducible methodology to isolate prospectively cancer stem-like cells for subsequent biological and preclinical studies.

Interactions between Adipocytes and Breast Cancer Cells Stimulate Cytokine Production and Drive Src/Sox2/miR-302b-Mediated Malignant Progression.

Consequences of the obesity epidemic on cancer morbidity and mortality are not fully appreciated. Obesity is a risk factor for many cancers, but the mechanisms by which it contributes to cancer development and patient outcome have yet to be fully elucidated. Here, we examined the effects of coculturing human-derived adipocytes with established and primary breast cancer cells on tumorigenic potential. We found that the interaction between adipocytes and cancer cells increased the secretion of proinflammatory cytokines. Prolonged culture of cancer cells with adipocytes or cytokines increased the proportion of mammosphere-forming cells and of cells expressing stem-like markers in vitro. Furthermore, contact with immature adipocytes increased the abundance of cancer cells with tumor-forming and metastatic potential in vivo. Mechanistic investigations demonstrated that cancer cells cultured with immature adipocytes or cytokines activated Src, thus promoting Sox2, c-Myc, and Nanog upregulation. Moreover, Sox2-dependent induction of miR-302b further stimulated cMYC and SOX2 expression and potentiated the cytokine-induced cancer stem cell-like properties. Finally, we found that Src inhibitors decreased cytokine production after coculture, indicating that Src is not only activated by adipocyte or cytokine exposures, but is also required to sustain cytokine induction. These data support a model in which cancer cell invasion into local fat would establish feed-forward loops to activate Src, maintain proinflammatory cytokine production, and increase tumor-initiating cell abundance and metastatic progression. Collectively, our findings reveal new insights underlying increased breast cancer mortality in obese individuals and provide a novel preclinical rationale to test the efficacy of Src inhibitors for breast cancer treatment.

HER2-signaling pathway, JNK and ERKs kinases, and cancer stem-like cells are targets of Bozepinib small compound.

Identification of novel anticancer drugs presenting more than one molecular target and efficacy against cancer stem-like cells (CSCs) subpopulations represents a therapeutic need to combat the resistance and the high risk of relapse in patients. In the present work we show how Bozepinib [(RS)-2,6-dichloro-9-[1-(p-nitrobenzenesulfonyl)-1,2,3,5-tetrahydro-4,1-benzoxazepin-3-yl]-9H-purine], a small anti-tumor compound, demonstrated selectivity on cancer cells and showed an inhibitory effect over kinases involved in carcinogenesis, proliferation and angiogenesis. The cytotoxic effects of Bozepinib were observed in both breast and colon cancer cells expressing different receptor patterns. Bozepinib inhibited HER-2 signaling pathway and JNK and ERKs kinases. In addition, Bozepinib has an inhibitory effect on AKT and VEGF together with anti-angiogenic and anti-migratory activities. Moreover, the modulation of pathways involved in tumorigenesis by Bozepinib was also evident in microarrays analysis. Interestingly, Bozepinib inhibited both mamo- and colono-spheres formation and eliminated ALDH+ CSCs subpopulations at a low micromolar range similar to Salinomycin. Bozepinib induced the down-regulation of c-MYC, ß-CATENIN and SOX2 proteins and the up-regulation of the GLI-3 hedgehog-signaling repressor. Finally, Bozepinib shows in vivo anti-tumor and anti-metastatic efficacy in xenotransplanted nude mice without presenting sub-acute toxicity. These findings support further studies on the therapeutic potential of Bozepinib in cancer patients.

Triple negative breast cancer initiating cell subsets differ in functional and molecular characteristics and in γ-secretase inhibitor drug responses.

Increasing evidence suggests that stem-like cells mediate cancer therapy resistance and metastasis. Breast tumour-initiating stem cells (T-ISC) are known to be enriched in CD44(+) CD24(neg/low) cells. Here, we identify two T-ISC subsets within this population in triple negative breast cancer (TNBC) lines and dissociated primary breast cancer cultures: CD44(+) CD24(low+) subpopulation generates CD44(+) CD24(neg) progeny with reduced sphere formation and tumourigenicity. CD44(+) CD24(low+) populations contain subsets of ALDH1(+) and ESA(+) cells, yield more frequent spheres and/or T-ISC in limiting dilution assays, preferentially express metastatic gene signatures and show greater motility, invasion and, in the MDA-MB-231 model, metastatic potential. CD44(+) CD24(low+) but not CD44(+) CD24(neg) express activated Notch1 intracellular domain (N1-ICD) and Notch target genes. We show N1-ICD transactivates SOX2 to increase sphere formation, ALDH1+ and CD44(+) CD24(low+) cells. Gamma secretase inhibitors (GSI) reduced sphere formation and xenograft growth from CD44(+) CD24(low+) cells, but CD44(+) CD24(neg) were resistant. While GSI hold promise for targeting T-ISC, stem cell heterogeneity as observed herein, could limit GSI efficacy. These data suggest a breast T-ISC hierarchy in which distinct pathways drive developmentally related subpopulations with different anti-cancer drug responsiveness.