Key Points Question Is there an increased risk for metastasis of breast cancers that are diagnosed in young women post partum that extends beyond 5 years from the last childbirth, and what association do standard clinical prognostic factors have with metastatic risk in these young women when categorized by parity? Findings In a cohort study of 701 women 45 years or younger with breast cancer, those with stage I or II cancer diagnosed up to 10 years post partum had an increased risk for distant metastasis, with both estrogen receptor–positive and estrogen receptor–negative disease significantly affected. Meaning Postpartum status may be a prognostic indicator in young women with breast cancer and should be routinely identified, as up to 45% of women 45 years or younger with breast cancer fall into this category and could be at increased risk for metastasis.

Brain metastases (BM) are a devastating consequence of breast cancer. BM occur more frequently in patients with estrogen receptor-negative (ER−) breast cancer subtypes; HER2 overexpressing (HER2+) tumors and triple-negative (TN) (ER−, progesterone receptor-negative (PR−) and normal HER2) tumors. Young age is an independent risk factor for development of BM, thus we speculated that higher circulating estrogens in young, pre-menopausal women could exert paracrine effects through the highly estrogen-responsive brain microenvironment. Using a TN experimental metastases model, we demonstrate that ovariectomy decreased the frequency of MRI detectable lesions by 56% as compared to estrogen supplementation, and that the combination of ovariectomy and letrozole further reduced the frequency of large lesions to 14.4% of the estrogen control. Human BM expressed 4.2-48.4% ER+ stromal area, particularly ER+ astrocytes. In vitro, E2-treated astrocytes increased proliferation, migration and invasion of 231BR-EGFP cells in an ER-dependent manner. E2 upregulated EGFR ligands Egf, Ereg, and Tgfa mRNA and protein levels in astrocytes, and activated EGFR in brain metastatic cells. Co-culture of 231BR-EGFP cells with E2-treated astrocytes led to upregulation of the metastatic mediator S100 Calcium-binding protein A4 (S100A4) (1.78-fold, P<0.05). Exogenous EGF increased S100A4 mRNA levels in 231BR-EGFP cells (1.40±0.02 fold, P<0.01 compared to vehicle-control) and an EGFR/HER2 inhibitor blocked this effect, suggesting that S100A4 is a downstream effector of EGFR activation. ShRNA-mediated S100A4 silencing in 231BR-EGFP cells decreased their migration and invasion in response to E2-CM, abolished their increased proliferation in co-cultures with E2-treated astrocytes, and decreased brain metastatic colonization. Thus, S100A4 is one effector of the paracrine action of E2 in brain metastatic cells. These studies provide a novel mechanism by which estrogens, acting through ER+ astrocytes in the brain microenvironment, can promote BM of TN breast cancers, and suggests existing endocrine agents may provide some clinical benefit towards reducing and managing BM.

BackgroundObesity and type II diabetes are linked to increased breast cancer risk in postmenopausal women. Patients treated with the antidiabetic drug metformin for diabetes or metabolic syndrome have reduced breast cancer risk, a greater pathologic complete response to neoadjuvant therapy, and improved breast cancer survival. We hypothesized that metformin may be especially effective when targeted to the menopausal transition, as this is a lifecycle window when weight gain and metabolic syndrome increase, and is also when the risk for obesity-related breast cancer increases.MethodsHere, we used an 1-methyl-1-nitrosourea (MNU)-induced mammary tumor rat model of estrogen receptor (ER)-positive postmenopausal breast cancer to evaluate the long-term effects of metformin administration on metabolic and tumor endpoints. In this model, ovariectomy (OVX) induces rapid weight gain, and an impaired whole-body response to excess calories contributes to increased tumor glucose uptake and increased tumor proliferation. Metformin treatment was initiated in tumor-bearing animals immediately prior to OVX and maintained for the duration of the study.ResultsMetformin decreased the size of existing mammary tumors and inhibited new tumor formation without changing body weight or adiposity. Decreased lipid accumulation in the livers of metformin-treated animals supports the ability of metformin to improve overall metabolic health. We also found a decrease in the number of aromatase-positive, CD68-positive macrophages within the tumor microenvironment, suggesting that metformin targets the immune microenvironment in addition to improving whole-body metabolism.ConclusionsThese findings suggest that peri-menopause/menopause represents a unique window of time during which metformin may be highly effective in women with established, or at high risk for developing, breast cancer.

Triple positive breast cancers overexpress both the human epidermal growth factor receptor 2 (HER2) oncogene and the hormonal receptors (HR) to estrogen and progesterone. These cancers represent a unique therapeutic challenge because of a bidirectional cross-talk between the estrogen receptor alpha (ERα) and HER2 pathways leading to tumor progression and resistance to targeted therapy. Attempts to combine standard of care HER2-targeted drugs with antihormonal agents for the treatment of HR+/HER2+ breast cancer yielded encouraging results in preclinical experiments but did improve overall survival in clinical trial. In this review, we dissect multiple mechanisms of therapeutic resistance typical of HR+/HER2+ breast cancer, summarize prior clinical trials of targeted agents, and describe novel rational drug combinations that include antihormonal agents, HER2-targeted drugs, and CDK4/6 inhibitors for treatment of the HR+/HER2+ breast cancer subtype.

Background Extracellular vesicles (EVs) are small membrane particles that contribute to cancer progression and metastases by transporting biologically significant proteins and nucleic acids. They may also serve as biomarkers of various disease states or important therapeutic targets. Breast cancer EVs have the potential to change the behavior of other cells in their microenvironment. However, the proteomic content of EVs isolated from young women’s breast cancer patients and the mechanisms underlying the influence of EVs on tumor cell behavior have not yet been reported. Methods In our current translational studies, we compared the proteomic content of EVs isolated from invasive breast cancer cell lines and plasma samples from young women’s breast cancer (YWBC) patients and age-matched healthy donors using mass spectrometry. We analyzed the functionality of EVs in two dimensional tumor cell invasion assays and the gene expression changes in tumor cells after incubation with EVs. Results We found that treatment with EVs from both invasive breast cancer cell lines and plasma of YWBC patients altered the invasive properties of non-invasive breast cancer cells. Proteomics identified differences between EVs from YWBC patients and healthy donors that correlated with their altered function. Further, we identified gene expression changes in non-invasive breast cancer cells after treatment with EVs that implicate the Focal Adhesion Kinase (FAK) signaling pathway as a potential targetable pathway affected by breast cancer-derived EVs. Conclusions Our results suggest that the proteome of EVs from breast cancer patients reflects their functionality in tumor motility assays and may help elucidate the role of EVs in breast cancer progression.

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Background: Young women's breast cancer[YWBC] affects 27,000 US women age ≤45 annually. Half of these cancers occur within 5-10 years of a prior childbirth, a postpartum breast cancer[PPBC], incurring a 3 fold increased risk for metastasis and death. Recently, extracellular vesicles[EV] have been identified in human circulation, released from cancer cells, that have paracrine and autocrine effects, alter the tumor microenvironment and establish metastatic niches. EVs isolated from breast cancer lines increase proliferation and invasion of other breast cancer cells in vitro. However, the impact of EVs isolated from primary breast cancer patients on tumor invasion, metastasis and their role in tumor immune suppression is largely unknown. We hypothesized that EVs from YWBC/PPBC patients may contain unique pro-metastatic cargo, influence aggressive breast cancer cell behavior and may demonstrate the ability to alter immune cell function. Method: We isolated EVs using size-exclusion chromatography [SEC] from the plasma of 10 unaffected young women and 20 YWBC patients balanced for parity, age, subtype and stage. We compared the breast cancer-specific EV proteins within various clinical groups of YWBC and PPBC to identify significant proteomic differences by parity, sub-type, stage, and disease recurrence. We determined the functional impact of these EVs on tumor cell motility and proliferation, and analyzed the effect of breast cancer derived EVs on immune cell phenotype, function, and T cell proliferation assays. Results: Of the 582 proteins, 22 proteins are significantly increased in the EVs of YWBC compared to unaffected donors. The protein set includes breast cancer antigens [MUC 1, 2, 5b], transcriptional regulators [Myc target protein], enzymes [catalase, MMP inhibitor 1], and signaling molecules [Annexin 1, latent TGFb binding protein 1], among others. Several identified proteins specifically track with those YWBC cases with subsequent metastases. Furthermore, 8 unique proteins track with PPBC, including cartilage oligomeric matrix protein, a novel breast cancer biomarker that correlates with increased invasiveness, and decreased recurrence-free survival. EVs isolated from the plasma of newly diagnosed YWBC increase breast cancer invasion and EVs derived from breast cancer are engulfed by the majority of monocytic immune cells, including dendritic cells, classical and activated monocytes, but not by lymphocytes. Specifically, CD14+ monocytic myeloid derived suppressor cells engulfed the EVs while the CD15+ granulocytic subset did not. Once engulfed, phenotypic changes occurr in the EV containing monocytes and a significantly reduction in T cell stimulation in standard mixed-lymphocyte reactions is observed. Conclusion: EVs isolated from YWBC & PPBC cases have unique protein content and increase breast cancer invasiveness, which suggests potential mechanistic roles for EVs as increasing metastatic risk and provides novel candidate biomarkers. We identified an immunomodulatory effect of breast cancer EVs on human monocytes that may contribute to immunosuppression in breast cancer and a role for EVs as directly modulating the host and tumor microenvironment. Citation Format: Borges VF, Jordan KR, Hall JK, Schedin T, Hansen K, Schedin P. Extracellular vesicles from young women's and postpartum breast cancer display unique proteomic content, alter breast cancer aggressive behavior, and influence immune cell function [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-12-05.