CCL2-Mediated Stromal Interactions Drive Macrophage Polarization to Increase Breast Tumorigenesis.

CCL2 is an inflammatory cytokine that regulates macrophage activity and is implicated in increased mammographic density and early breast tumorigenesis. The role of CCL2 in mediating stromal interactions that contribute to breast tumorigenesis has yet to be fully elucidated. THP-1-derived macrophages and mammary fibroblasts were co-cultured for 72 h. Fibroblasts and macrophages were analysed for phenotype, expression of inflammatory and ECM-regulatory genes and collagen production. Mice overexpressing CCL2 in the mammary glands were analysed for global gene expression by RNAseq at 12 weeks of age. These mice were cross-bred with PyMT mammary tumour mice to examine the role of CCL2 in tumorigenesis. The co-culture of macrophages with fibroblasts resulted in macrophage polarization towards an M2 phenotype, and upregulated expression of CCL2 and other genes associated with inflammation and ECM remodelling. CCL2 increased the production of insoluble collagen by fibroblasts. A global gene expression analysis of CCL2 overexpressing mice revealed that CCL2 upregulates cancer-associated gene pathways and downregulates fatty acid metabolism gene pathways. In the PyMT mammary tumour model, CCL2 overexpressing mice exhibited increased macrophage infiltration and early tumorigenesis. Interactions between macrophages and fibroblasts regulated by CCL2 can promote an environment that may increase breast cancer risk, leading to enhanced early tumorigenesis.

Attenuated TGFB signalling in macrophages decreases susceptibility to DMBA-induced mammary cancer in mice.

BACKGROUND: Transforming growth factor beta1 (TGFB1) is a multi-functional cytokine that regulates mammary gland development and cancer progression through endocrine, paracrine and autocrine mechanisms. TGFB1 also plays roles in tumour development and progression, and its increased expression is associated with an increased breast cancer risk. Macrophages are key target cells for TGFB1 action, also playing crucial roles in tumourigenesis. However, the precise role of TGFB-regulated macrophages in the mammary gland is unclear. This study investigated the effect of attenuated TGFB signalling in macrophages on mammary gland development and mammary cancer susceptibility in mice. METHODS: A transgenic mouse model was generated, wherein a dominant negative TGFB receptor is activated in macrophages, in turn attenuating the TGFB signalling pathway specifically in the macrophage population. The mammary glands were assessed for morphological changes through wholemount and H&E analysis, and the abundance and phenotype of macrophages were analysed through immunohistochemistry. Another cohort of mice received carcinogen 7,12-dimethylbenz(a)anthracene (DMBA), and tumour development was monitored weekly. Human non-neoplastic breast tissue was also immunohistochemically assessed for latent TGFB1 and macrophage marker CD68. RESULTS: Attenuation of TGFB signalling resulted in an increase in the percentage of alveolar epithelium in the mammary gland at dioestrus and an increase in macrophage abundance. The phenotype of macrophages was also altered, with inflammatory macrophage markers iNOS and CCR7 increased by 110% and 40%, respectively. A significant decrease in DMBA-induced mammary tumour incidence and prolonged tumour-free survival in mice with attenuated TGFB signalling were observed. In human non-neoplastic breast tissue, there was a significant inverse relationship between latent TGFB1 protein and CD68-positive macrophages. CONCLUSIONS: TGFB acts on macrophage populations in the mammary gland to reduce their abundance and dampen the inflammatory phenotype. TGFB signalling in macrophages increases mammary cancer susceptibility potentially through suppression of immune surveillance activities of macrophages.

Comparison of hormone-induced mRNA and protein biomarker expression changes in breast cancer cells.

PURPOSE: Protein biomarkers estrogen receptor (ER), progesterone receptor (PR), and marker of proliferation (Ki67) are routinely assessed by immunohistochemistry to guide treatment decisions for breast cancer. Now, quantification of mRNA encoding these proteins is being adopted in the clinic. However, mRNA and protein biomarkers may be differentially regulated by fluctuations in estrogen and progesterone that occur across the menstrual cycle in premenopausal breast cancer patients. This study aimed to compare how estrogen and progesterone affect mRNA and protein biomarker expression in hormone-responsive breast cancer cells. METHODS: Hormone-responsive ZR-75-1 and T-47D human breast cancer cell lines were xenografted into the mammary fat pad of BALB/c nude mice supplemented with estrogen. Progesterone or vehicle was administered prior to dissection of tumors. Protein expression of ER, PR and Ki67 was quantified by immunohistochemistry, and mRNA encoding these proteins, ESR1, PGR and KI67, respectively, was quantified by real-time PCR. mRNA expression was also quantified in breast cancer cell lines treated with estrogen and progesterone in vitro. RESULTS: In T-47D-xenografted tumors, estrogen and progesterone treatment reduced PGR and KI67 mRNA expression, and reduced PR and Ki67 protein positivity, compared to estrogen treatment alone. In ZR-75-1 xenografted tumors, no significant differences in protein or mRNA biomarker expression were observed. In vitro, estrogen and progesterone co-treatment significantly reduced ESR1 and PGR mRNA expression in both T-47D and ZR-75-1 cell lines. CONCLUSIONS: Estrogen and progesterone similarly affect mRNA and protein biomarker expression in hormone-responsive breast cancer xenografts. Further research is needed to investigate concordance between protein and mRNA biomarkers in premenopausal breast cancer.

Ovarian cycle stage critically affects 21-gene recurrence scores in Mmtv-Pymt mouse mammary tumours.

BACKGROUND: The Oncotype DX 21-gene Recurrence Score is predictive of adjuvant chemotherapy benefit for women with early-stage, estrogen receptor (ER)-positive, HER2-negative breast cancer. In premenopausal women, fluctuations in estrogen and progesterone during the menstrual cycle impact gene expression in hormone-responsive cancers. However, the extent to which menstrual cycling affects the Oncotype DX 21-gene signature remains unclear. Here, we investigate the impact of ovarian cycle stage on the 21-gene signature using a naturally cycling mouse model of breast cancer. METHODS: ER-positive mammary tumours were dissected from naturally cycling Mmtv-Pymt mice at either the estrus or diestrus phase of the ovarian cycle. The Oncotype DX 21-gene signature was assessed through quantitative real time-PCR, and a 21-gene experimental recurrence score analogous to the Oncotype DX Recurrence Score was calculated. RESULTS: Tumours collected at diestrus exhibited significant differences in expression of 6 Oncotype DX signature genes (Ki67, Ccnb1, Esr1, Erbb2, Grb7, Bag1; p ≤ 0.05) and a significant increase in 21-gene recurrence score (21.8 ± 2.4; mean ± SEM) compared to tumours dissected at estrus (15.5 ± 1.9; p = 0.03). Clustering analysis revealed a subgroup of tumours collected at diestrus characterised by increased expression of proliferation- (p < 0.001) and invasion-group (p = 0.01) genes, and increased 21-gene recurrence score (p = 0.01). No correlation between ER, PR, HER2, and KI67 protein abundance measured by Western blot and abundance of mRNA for the corresponding gene was observed, suggesting that gene expression is more susceptible to hormone-induced fluctuation compared to protein expression. CONCLUSIONS: Ovarian cycle stage at the time of tissue collection critically affects the 21-gene signature in Mmtv-Pymt murine mammary tumours. Further studies are required to determine whether Oncotype DX Recurrence Scores in women are similarly affected by menstrual cycle stage.

Discordance in 21-gene recurrence scores between paired breast cancer samples is inversely associated with patient age.

BACKGROUND: The Oncotype DX 21-gene Recurrence Score is a genomic-based algorithm that guides adjuvant chemotherapy treatment decisions for women with early-stage, oestrogen receptor (ER)-positive breast cancer. However, there are age-related differences in chemotherapy benefit for women with intermediate Oncotype DX Recurrence Scores that are not well understood. Menstrual cycling in younger women is associated with hormonal fluctuations that might affect the expression of genomic predictive biomarkers and alter Recurrence Scores. Here, we use paired human breast cancer samples to demonstrate that the clinically employed Oncotype DX algorithm is critically affected by patient age. METHODS: RNA was extracted from 25 pairs of formalin-fixed paraffin-embedded, invasive ER-positive breast cancer samples that had been collected approximately 2 weeks apart. A 21-gene signature analogous to the Oncotype DX platform was assessed through quantitative real-time PCR, and experimental recurrence scores were calculated using the Oncotype DX algorithm. RESULTS: There was a significant inverse association between patient age and discordance in the recurrence score. For every 1-year decrease in age, discordance in recurrence scores between paired samples increased by 0.08 units (95% CI - 0.14, - 0.01; p = 0.017). Discordance in recurrence scores for women under the age of 50 was driven primarily by proliferation- and HER2-associated genes. CONCLUSION: The Oncotype DX 21-gene Recurrence Score algorithm is critically affected by patient age. These findings emphasise the need for the consideration of patient age, particularly for women younger than 50, in the development and application of genomic-based algorithms for breast cancer care.

Altered liver metabolism post-wean abolishes efficacy of vitamin D for breast cancer prevention in a mouse model.

Young women have increased risk of vitamin D deficiency, which may increase breast cancer incidence. Here, we assessed the anti-cancer efficacy of vitamin D in mouse models of young-onset breast cancer. In never-pregnant mice, vitamin D supplementation increased serum 25(OH)D and hepatic 1,25(OH)2D3, reduced tumor size, and associated with anti-tumor immunity. These anti-tumor effects were not replicated in a mouse model of postpartum breast cancer, where hepatic metabolism of vitamin D was suppressed post-wean, which resulted in deficient serum 25(OH)D and reduced hepatic 1,25(OH)2D3. Treatment with active 1,25(OH)2D3 induced hypercalcemia exclusively in post-wean mice, highlighting metabolic imbalance post-wean. RNAseq revealed suppressed CYP450 expression postpartum. In sum, we provide evidence that vitamin D anti-tumor activity is mediated through immunomodulatory mechanisms and is ineffective in the post-wean window due to altered hepatic metabolism. These findings have implications for suppressed xenobiotic metabolism in postpartum women beyond vitamin D.

Postpartum Breast Cancer and Survival in Women With Germline BRCA Pathogenic Variants.

Importance: In young-onset breast cancer (YOBC), a diagnosis within 5 to 10 years of childbirth is associated with increased mortality. Women with germline BRCA1/2 pathogenic variants (PVs) are more likely to be diagnosed with BC at younger ages, but the impact of childbirth on mortality is unknown. Objective: To determine whether time between most recent childbirth and BC diagnosis is associated with mortality among patients with YOBC and germline BRCA1/2 PVs. Design, Setting, and Participants: This prospective cohort study included women with germline BRCA1/2 PVs diagnosed with stage I to III BC at age 45 years or younger between 1950 and 2021 in the United Kingdom, who were followed up until November 2021. Data were analyzed from December 3, 2021, to November 29, 2023. Exposure: Time between most recent childbirth and subsequent BC diagnosis, with recent childbirth defined as 0 to less than 10 years, further delineated to 0 to less than 5 years and 5 to less than 10 years. Main Outcomes and Measures: The primary outcome was all-cause mortality, censored at 20 years after YOBC diagnosis. Mortality of nulliparous women was compared with the recent post partum groups and the 10 or more years post partum group. Cox proportional hazards regression analyses were adjusted for age, tumor stage, and further stratified by tumor estrogen receptor (ER) and BRCA gene status. Results: Among 903 women with BRCA PVs (mean [SD] age at diagnosis, 34.7 [6.1] years; mean [SD] follow-up, 10.8 [9.8] years), 419 received a BC diagnosis 0 to less than 10 years after childbirth, including 228 women diagnosed less than 5 years after childbirth and 191 women diagnosed 5 to less than 10 years after childbirth. Increased all-cause mortality was observed in women diagnosed within 5 to less than 10 years post partum (hazard ratio [HR], 1.56 [95% CI, 1.05-2.30]) compared with nulliparous women and women diagnosed 10 or more years after childbirth, suggesting a transient duration of postpartum risk. Risk of mortality was greater for women with ER-positive BC in the less than 5 years post partum group (HR, 2.35 [95% CI, 1.02-5.42]) and ER-negative BC in the 5 to less than 10 years post partum group (HR, 3.12 [95% CI, 1.22-7.97]) compared with the nulliparous group. Delineated by BRCA1 or BRCA2, mortality in the 5 to less than 10 years post partum group was significantly increased, but only for BRCA1 carriers (HR, 2.03 [95% CI, 1.15-3.58]). Conclusions and Relevance: These findings suggest that YOBC with germline BRCA PVs was associated with increased risk for all-cause mortality if diagnosed within 10 years after last childbirth, with risk highest for ER-positive BC diagnosed less than 5 years post partum, and for ER-negative BC diagnosed 5 to less than 10 years post partum. BRCA1 carriers were at highest risk for poor prognosis when diagnosed at 5 to less than 10 years post partum. No such associations were observed for BRCA2 carriers. These results should inform genetic counseling, prevention, and treatment strategies for BRCA PV carriers.

Isogenic Mammary Models of Intraductal Carcinoma Reveal Progression to Invasiveness in the Absence of a Non-Obligatory In Situ Stage.

In breast cancer, progression to invasive ductal carcinoma (IDC) involves interactions between immune, myoepithelial, and tumor cells. Development of IDC can proceed through ductal carcinoma in situ (DCIS), a non-obligate, non-invasive stage, or IDC can develop without evidence of DCIS and these cases associate with poorer prognosis. Tractable, immune-competent mouse models are needed to help delineate distinct mechanisms of local tumor cell invasion and prognostic implications. To address these gaps, we delivered murine mammary carcinoma cell lines directly into the main mammary lactiferous duct of immune-competent mice. Using two strains of immune-competent mice (BALB/c, C57BL/6), one immune-compromised (severe combined immunodeficiency; SCID) C57BL/6 strain, and six different murine mammary cancer cell lines (D2.OR, D2A1, 4T1, EMT6, EO771, Py230), we found early loss of ductal myoepithelial cell differentiation markers p63, α-smooth muscle actin, and calponin, and rapid formation of IDC in the absence of DCIS. Rapid IDC formation also occurred in the absence of adaptive immunity. Combined, these studies demonstrate that loss of myoepithelial barrier function does not require an intact immune system, and suggest that these isogenic murine models may prove a useful tool to study IDC in the absence of a non-obligatory DCIS stage-an under-investigated subset of poor prognostic human breast cancer.

Vitamin D as a Potential Preventive Agent For Young Women's Breast Cancer.

Clinical studies backed by research in animal models suggest that vitamin D may protect against the development of breast cancer, implicating vitamin D as a promising candidate for breast cancer prevention. However, despite clear preclinical evidence showing protective roles for vitamin D, broadly targeted clinical trials of vitamin D supplementation have yielded conflicting findings, highlighting the complexity of translating preclinical data to efficacy in humans. While vitamin D supplementation targeted to high-risk populations is a strategy anticipated to increase prevention efficacy, a complimentary approach is to target transient, developmental windows of elevated breast cancer risk. Postpartum mammary gland involution represents a developmental window of increased breast cancer promotion that may be poised for vitamin D supplementation. Targeting the window of involution with short-term vitamin D intervention may offer a simple, cost-effective approach for the prevention of breast cancers that develop postpartum. In this review, we highlight epidemiologic and preclinical studies linking vitamin D deficiency with breast cancer development. We discuss the underlying mechanisms through which vitamin D deficiency contributes to cancer development, with an emphasis on the anti-inflammatory activity of vitamin D. We also discuss current evidence for vitamin D as an immunotherapeutic agent and the potential for vitamin D as a preventative strategy for young woman's breast cancer.

Modern, exogenous exposures associated with altered mammary gland development: A systematic review.

BACKGROUND: Many women report low milk supply as the reason for premature breastfeeding cessation. Altered mammary gland development may impact a woman's lactation ability. OBJECTIVE: This review identifies modern exogenous exposures which alter mammary gland development during embryonic life, puberty and pregnancy. METHODS: A systematic review was undertaken whereby Medline, CINAHL and Embase articles published from January 1, 2005 to November 20, 2020 were searched using the keywords puberty or embry* or fetal or foetal or foetus or fetus or pregnan* or gestation* AND "mammary gland development" or "breast development" or "mammary development" or "mammary gland function" or "mammary function" or "insufficient glandular tissue" or "mammary hypoplasia" or "breast hypoplasia" or "mammary gland hypoplasia" or "tubular breast*" or "tuberous breast*" or "glandular tissue" or "breast composition" or "mammary composition" or "mammary gland composition". After initial screening of 1207 records, 60 full texts were assessed for eligibility; 6 were excluded due to lack of information about exposure or outcome, leaving 54 studies. RESULTS: The review included results from 52 animal (rats and mice, monkeys, rabbits, sheep, goats pigs and cows) and 2 human studies. Various endocrine disrupting chemicals and an obesogenic diet were found to be associated with altered mammary gland morphology during key development stages. CONCLUSIONS: To improve lactation outcomes, future studies need to focus on lactation as the endpoint and be conducted in a standardised manner to allow for a more significant contribution to the literature that allows for better comparison across studies.

Timing of breast cancer surgery during the menstrual cycle-is there an optimal time of the month?

An intriguing relationship between menstrual cycle phase at the time of breast cancer surgery and clinical outcomes was first proposed in the late 1980s. Despite a number of clinical studies conducted to address this, as well as meta-analyses and systematic reviews, there remains significant controversy surrounding the effect of menstrual cycle phase at time of surgery on the prognosis of premenopausal breast cancer. While some studies have suggested that surgery performed during the luteal phase results in the most favourable outcome, other studies report the follicular phase is more favourable, and others show no association. Given the conflicting results, there remains insufficient evidence to determine whether there is an optimal time of the month to perform surgery. This issue has dogged breast cancer surgery for decades; knowledge of an optimal time of the month to conduct surgery would be a simple approach to improving patient outcomes. This review explores the potential biological mechanisms through which the hormonal milieu might contribute to differences in prognosis, and why clinical findings are so variable. It is concluded that a significant problem with current clinical research is the lack of insight from mechanistic studies. While there are a number of plausible biological mechanisms that could lead to altered survival, supporting evidence is limited. There are also variable approaches to defining the menstrual cycle phase and hormone receptor status of the tumour and few studies controlled for prognostic factors such as tumour size and stage, or addressed the impact of adjuvant treatments. Elucidation of the specific confounding factors, as well as biological mechanistic pathways that could explain the potential relationship between timing of surgery and survival, will greatly assist in designing robust well-controlled prospective clinical studies to evaluate this paradigm.

Hormonal Modulation of Breast Cancer Gene Expression: Implications for Intrinsic Subtyping in Premenopausal Women.

Clinics are increasingly adopting gene-expression profiling to diagnose breast cancer subtype, providing an intrinsic, molecular portrait of the tumor. For example, the PAM50-based Prosigna test quantifies expression of 50 key genes to classify breast cancer subtype, and this method of classification has been demonstrated to be superior over traditional immunohistochemical methods that detect proteins, to predict risk of disease recurrence. However, these tests were largely developed and validated using breast cancer samples from postmenopausal women. Thus, the accuracy of such tests has not been explored in the context of the hormonal fluctuations in estrogen and progesterone that occur during the menstrual cycle in premenopausal women. Concordance between traditional methods of subtyping and the new tests in premenopausal women is likely to depend on the stage of the menstrual cycle at which the tissue sample is taken and the relative effect of hormones on expression of genes versus proteins. The lack of knowledge around the effect of fluctuating estrogen and progesterone on gene expression in breast cancer patients raises serious concerns for intrinsic subtyping in premenopausal women, which comprise about 25% of breast cancer diagnoses. Further research on the impact of the menstrual cycle on intrinsic breast cancer profiling is required if premenopausal women are to benefit from the new technology of intrinsic subtyping.