A large collaborative analysis of data from 47 epidemiological studies concluded that longer duration of breastfeeding reduces the risk of developing breast cancer. Despite the strong epidemiological ...evidence, the molecular mechanisms linking prolonged breastfeeding to decreased risk of breast cancer remain poorly understood.
We modeled two types of breastfeeding behaviors in wild type FVB/N mice: (1) normal or gradual involution of breast tissue following prolonged breastfeeding and (2) forced or abrupt involution following short-term breastfeeding. To accomplish this, pups were gradually weaned between 28 and 31 days (gradual involution) or abruptly at 7 days postpartum (abrupt involution). Mammary glands were examined for histological changes, proliferation, and inflammatory markers by immunohistochemistry. Fluorescence-activated cell sorting was used to quantify mammary epithelial subpopulations. Gene set enrichment analysis was used to analyze gene expression data from mouse mammary luminal progenitor cells. Similar analysis was done using gene expression data generated from human breast samples obtained from parous women enrolled on a tissue collection study, OSU-2011C0094, and were undergoing reduction mammoplasty without history of breast cancer.
Mammary glands from mice that underwent abrupt involution exhibited denser stroma, altered collagen composition, higher inflammation and proliferation, increased estrogen receptor α and progesterone receptor expression compared to those that underwent gradual involution. Importantly, when aged to 4 months postpartum, mice that were in the abrupt involution cohort developed ductal hyperplasia and squamous metaplasia. Abrupt involution also resulted in a significant expansion of the luminal progenitor cell compartment associated with enrichment of Notch and estrogen signaling pathway genes. Breast tissues obtained from healthy women who breastfed for < 6 months vs ≥ 6 months showed significant enrichment of Notch signaling pathway genes, along with a trend for enrichment for luminal progenitor gene signature similar to what is observed in BRCA1 mutation carriers and basal-like breast tumors.
We report here for the first time that forced or abrupt involution of the mammary glands following pregnancy and lack of breastfeeding results in expansion of luminal progenitor cells, higher inflammation, proliferation, and ductal hyperplasia, a known risk factor for developing breast cancer.
Orchestrating cell-cycle-dependent mRNA oscillations is critical to cell proliferation in multicellular organisms. Even though our understanding of cell-cycle-regulated transcription has improved ...significantly over the last three decades, the mechanisms remain untested in vivo. Unbiased transcriptomic profiling of G0, G1-S, and S-G2-M sorted cells from FUCCI mouse embryos suggested a central role for E2Fs in the control of cell-cycle-dependent gene expression. The analysis of gene expression and E2F-tagged knockin mice with tissue imaging and deep-learning tools suggested that post-transcriptional mechanisms universally coordinate the nuclear accumulation of E2F activators (E2F3A) and canonical (E2F4) and atypical (E2F8) repressors during the cell cycle in vivo. In summary, we mapped the spatiotemporal expression of sentinel E2F activators and canonical and atypical repressors at the single-cell level in vivo and propose that two distinct E2F modules relay the control of gene expression in cells actively cycling (E2F3A-8-4) and exiting the cycle (E2F3A-4) during mammalian development.
Display omitted
•E2F expression during cell division, differentiation, and quiescence is measured in vivo•E2F3A, E2F8, and E2F4 accumulate sequentially in the nucleus of cycling cells•E2F3A-4 nuclear accumulation controls gene expression during cell-cycle exit•Deep learning tools are applied to nuclear segmentation of complex mammalian tissues
The study of E2Fs in vivo has been challenging. Cuitiño et al. reconstruct the spatiotemporal expression of E2F activators (E2F3A) and canonical (E2F4) and atypical (E2F8) repressors during the mammalian cell cycle and propose that orchestrated accumulation of different E2F combinations control gene expression in proliferating (E2F3A-8-4) and differentiating (E2F3A-4) cells.
The importance of the tumor-associated stroma in cancer progression is clear. However, it remains uncertain whether early events in the stroma are capable of initiating breast tumorigenesis. Here, we ...show that in the mammary glands of non-tumor bearing mice, stromal-specific phosphatase and tensin homolog (Pten) deletion invokes radiation-induced genomic instability in neighboring epithelium. In these animals, a single dose of whole-body radiation causes focal mammary lobuloalveolar hyperplasia through paracrine epidermal growth factor receptor (EGFR) activation, and EGFR inhibition abrogates these cellular changes. By analyzing human tissue, we discover that stromal PTEN is lost in a subset of normal breast samples obtained from reduction mammoplasty, and is predictive of recurrence in breast cancer patients. Combined, these data indicate that diagnostic or therapeutic chest radiation may predispose patients with decreased stromal PTEN expression to secondary breast cancer, and that prophylactic EGFR inhibition may reduce this risk.
Lithium ferrites have attracted considerable attention because they have been used as replacements for garnets due to their low cost. A series of polycrystalline ferrite samples were prepared with ...the composition of LiXZn(0.6−2X)Cu0.4Fe2O4(X=0.05, 0.1, 0.15, 0.2, 0.25, 0.3) at chemical reaction temperature 150°C by sintering with microwave assisted combustion method. The characterization shows the formation of single phase cubic structure when carried out by using the X-rays technique and I–R technique. Magnetization parameters such as saturation magnetization, coercivity, magnetic moment were calculated by using the Hysteresis graph. The Curie temperature obtained using the susceptibility data are found to be in the range 350–700°C. Anhysteric remanent magnetization is used for estimating the grain size and domain structure of the composition. An attempt has been made to synthesis the nano-particles at lower reaction temperature by using non-conventional microwave sintering method. The advantage of this method is its lower sintering temperature and time compared to the conventional ceramic technique and direct formation of nano-ferrites without ball-milling.
•Single spinel phases of LiZnCu ferrite are studied.•Different sized particles, ranging 43–63nm were formed by using microwave sintering method.•The substitution of Zn2+ ions alters the domain structure from SD to MD and then to SP.•SD particle have higher ARM intensities per unit mass compared to MD particles.•A combination of lower chemical reaction temperature with microwave sintering could be useful for obtaining nano-ferrites.
Ni 0.5 Cu x/2 Zn (0.5-x/2) Fe 2 O 4 (where x\(= 0.3\) , 0.4, 0.5, and 0.6) ferrites were prepared at different chemical reaction temperatures (100 °C, 125 °C, and 150 °C) and then were further ...sintered using domestic microwave oven. X-ray diffraction revealed the ferrite phase formation and proved that chemical reaction temperature affects the structural properties. Magnetic properties, such as saturation magnetization, coercivity, Curie temperature, and normalized susceptibility were found to be dependent not only on composition, but also on chemical reaction temperature. Grain size was found to be in range of 41.866-200.909 nm.
Invasive lobular carcinoma (ILC) accounts for 10% to 15% of breast cancers in the United States, 80% of which are estrogen receptor (ER)‐positive, with an unusual metastatic pattern of spread to ...sites such as the serosa, meninges, and ovaries, among others. Lobular cancer presents significant challenges in detection and clinical management given its multifocality and multicentricity at presentation. Despite the unique features of ILC, it is often lumped with hormone receptor‐positive invasive ductal cancers (IDC); consequently, ILC screening, treatment, and follow‐up strategies are largely based on data from IDC. Despite both being treated as ER‐positive breast cancer, querying the Cancer Genome Atlas database shows distinctive molecular aberrations in ILC compared with IDC, such as E‐cadherin loss (66% vs. 3%), FOXA1 mutations (7% vs. 2%), and GATA3 mutations (5% vs. 20%). Moreover, compared with patients with IDC, patients with ILC are less likely to undergo breast‐conserving surgery, with lower rates of complete response following therapy as these tumors are less chemosensitive. Taken together, this suggests that ILC is biologically distinct, which may influence tumorigenesis and therapeutic strategies. Long‐term survival and clinical outcomes in patients with ILC are worse than in stage‐ and grade‐matched patients with IDC; therefore, nuanced criteria are needed to better define treatment goals and protocols tailored to ILC's unique biology. This comprehensive review highlights the histologic and clinicopathologic features that distinguish ILC from IDC, with an in‐depth discussion of ILC's molecular alterations and biomarkers, clinical trials and treatment strategies, and future targets for therapy.
Implications for Practice
The majority of invasive lobular breast cancers (ILCs) are hormone receptor (HR)‐positive and low grade. Clinically, ILC is treated similar to HR‐positive invasive ductal cancer (IDC). However, ILC differs distinctly from IDC in its clinicopathologic characteristics and molecular alterations. ILC also differs in response to systemic therapy, with studies showing ILC as less sensitive to chemotherapy. Patients with ILC have worse clinical outcomes with late recurrences. Despite these differences, clinical trials treat HR‐positive breast cancers as a single disease, and there is an unmet need for studies addressing the unique challenges faced by patients diagnosed with ILC.
Invasive lobular carcinoma is the second most common subtype of invasive breast cancer and presents unique challenges in detection and treatment. This review reports on the distinct morphological, molecular, and clinical features of invasive lobular carcinoma and highlights the challenges in the management of this cancer.
Abstract only
1538
Background: Epidemiological studies have shown that prolonged breastfeeding is associated with a reduced risk of developing triple negative/basal-like breast cancer (TN/BLBC). We ...have modeled abrupt involution (AI) following short breastfeeding and gradual involution (GI) of the mammary gland that occurs over time upon prolonged breastfeeding in wild-type FVB/N mice and discovered prominent histological and molecular changes in the AI glands over time. Further, we demonstrated that breast tissue from healthy women who breastfed <6 months showed enrichment in stem-cell and cell renewal pathways. Here, we corroborate those studies using normal human breast tissue obtained from Susan G. Komen for the Cure Tissue Bank (KTB). Methods: FFPE breast tissue sections obtained from KTB (Protocol #2017CO184). Donors were parous women, aged 18 to 45, without history of breast cancer and for whom breastfeeding history was available. H&E sections and TDLU, the primary anatomical source of most breast cancers, of women who breastfed for ≥6 months (GI, n=49) vs. those who breastfed for ≤3 months (AI, n=20) were evaluated by a blinded pathologist. Masson Trichrome stain was used to measure collagen deposition. Ki67 immunohistochemistry was utilized to determine proliferation. Statistical significance was measured using Fisher’s exact t-test and two-sample t-test with a p-value of <0.05. Results: H&E analysis revealed that breast tissue obtained from women in the AI cohort exhibited histological features of inflammation (p-value= 0.025). Using Ki67 IHC (AI, n=15; GI, n=32) and Masson Trichome stain (AI, n=3; GI, n=4), sections in the AI cohort showed 2-fold increase in proliferation of lobular epithelium (p-value= 0.048) and 1.4-fold increase in periductal collagen deposition (p-value= 0.027) when compared to GI cohort. Age, race, and BMI were not statistically different between AI and GI cohorts. Conclusions: Breast tissue from parous women who breastfed ≤3 months is histologically different than tissue of women with ≥6 months history of breastfeeding. We are currently staining more breast tissue samples obtained from KTB. Experiments are underway to assess the long-term effect of breastfeeding on breast epithelial cell hierarchy and biomarkers of inflammation. Understanding this mechanistic link will help in developing prevention strategies, particularly for African-American women who have lower prevalence of breastfeeding and higher incidence of TN/BLBC.
Abstract
Background: Epidemiological studies indicate that prolonged breast feeding reduces the risk of triple negative breast cancer (TNBC), which carries the worst prognosis. Prolonged ...breastfeeding allows gradual involution (GI) of the breast while lack of or short-term breast feeding leads to abrupt involution (AI). We developed a novel murine model mimicking AI and GI of breast, and found that GI offers better protection to mammary glands from tissue remodeling associated injuries. Our data showed that AI leads to the development of pro-tumorigenic microenvironment and ductal hyperplasia1. Tissue remodeling involves orchestrated cell death and repopulation, and is closely associated with metabolic reprogramming from mitochondrial oxidative phosphorylation (OXPHOS) to glycolysis. Such metabolic alterations can contribute to cellular changes aiding malignant transformation2. We used our murine model to evaluate whether AI affects cellular metabolism differentially when compared to GI. Methods: Wild-type mice of FVB background were used in all our experiments. Twelve to fourteen week old uniparous mice were allowed to nurse (6 pups/dam) for 7 days postpartum. All pups were removed on day7 postpartum from the dams in AI cohort and three each on day28 and day31 from dams in GI cohort. Whole mammary glands and sorted luminal progenitor (LP) cells harvested on postpartum day28 were subjected to Affymetrix microarray analysis. Gene Set Enrichment Analysis (GSEA) was used to compute pathway enrichments in AI vs. GI glands. Differentially expressed genes were validated using qRT-PCR. Mammary glands harvested on day28 postpartum were subjected to mass spectrometry based untargeted metabolic profiling using Agilent QTOF. Raw data were analyzed using XCMS to assess key metabolic networks altered in AI vs GI glands. Targeted analysis for lactate, pyruvate, succinate and palmitic acid were performed using C13 labelled internal standards to compare OXPHOS vs glycolysis reliance in the AI and GI glands. Results: We observed enrichment of mitochondrial OXPHOS pathway, fatty acid metabolism and Myc target genes in both whole mammary gland and LP cells of AI vs. GI mice. Adipogenesis and hypoxia related genes were enriched in AI-glands. We observed significant upregulation of genes involved in glucose transport and fatty acid synthesis in AI glands, namely, Glut-5, Cidea, Acss2, Acsm3, Acly, Atp6v0d2, Acot11, and Elvol3. Several factors indicating a higher reliance on OXPHOS vs glycolysis, such as, Ppar-γ, Pgc1α, Cpt-2, Srebp1c and Chrebp were upregulated in AI glands. Upregulation of Cpt-2 and Srebp1c in the AI glands indicate higher flux through fatty acid oxidation and reliance on cholesterol synthesis. Metabolomic profiling revealed significant alteration in L-carnitine, GMP and XMP in AI glands which reflect mitochondrial fatty acid transport and nucleotide biosynthesis via guanine-guanosine salvage pathway. Pyruvate and lactate associated with glycolysis were increased in GI vs. AI glands. Conclusion: We show for the first time that in the abruptly involuting (AI) mammary glands following short-term breast feeding, there is a significant shift in the metabolic pathways towards mitochondrial OXPHOS and fatty acid oxidation compared to GI glands. Studies are underway to determine the effect of this metabolic shift on cellular transformation and tumorigenesis and the potential to target these pathways to reverse the detrimental effects of AI. 1. Basree MM, Shinde N, Koivisto C, et al. Breast Cancer Res. 2019; 21(1):80. 2. Ward PS, Thompson CB. Cancer Cell. 2012; 21(3):297.
Citation Format: Neelam Shinde, Kirti Kaul, Allen Zhang, Saba Mehra, Resham Mawalkar, Hee Kyung Kim, Ramesh Ganju, Sarmila Majumder, Bhuvaneswari Ramaswamy. Abrupt involution of lactating mammary gland induces metabolic reprogramming conducive to pro-tumorigenic changes abstract. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-28.
Abstract
Objective: Epidemiological studies indicate a direct relation between length of breast feeding and protection against risk of triple negative breast cancer (TNBC), an aggressive subtype. ...While prolonged breastfeeding allows gradual involution (GI) of the breast, short-term or no breast feeding leads to abrupt involution (AI). We modelled AI and GI of breast in mice, and showed that mice subjected to GI have better protection from tissue remodeling associated injuries in the mammary gland. Our data revealed late development of ductal hyperplasia aided by pro-tumorigenic microenvironment in the AI glands1. Detailed mechanism of mammary gland involution immediately following cessation of lactation has been studied in the past, but only in the AI setting, that is after abrupt removal of pups at the peak of lactation2,3. The goal of this study is to conduct stepwise comparison of the mechanism of GI vs. AI early on to understand the protective effect of GI against breast cancer risk.Methods: Wild-type FVB mice were used in all our studies. Females were mated at 8 weeks of age and uniparous mice were allowed to nurse (6 pups/dam) for 7 days. Females were then assigned randomly to AI or GI cohort. All pups were removed from the AI dams on postnatal day7 (PND7). Three pups each were removed from GI dams on day28 and 31. Mammary glands were harvested intermittently between PND7 and PND35. H&E stained sections were used for histological studies. Unstained FFPE sections were used for immunohistochemistry and TUNEL assay. Total RNA and protein from whole mammary gland was used for qPCR and western blot respectively. Results: GI glands transitioned from fully active lactating to near involuted glands over a period of 8 days (PND17- PND25), while for AI glands it took < 4 days (PND8.5-PND12). The shrinkage and flattening of tall epithelia and loss of acini was gradual in GI glands as opposed to rapid breakdown of acini and adipocyte repopulation in AI gland. Apoptotic cell count peaked on PND11 (5%) in AI vs. PND25 (3%) in GI glands. The pStat3Y705+ cells were highest on PND8.5 (25%) in AI vs. on PND25 (11%) in GI glands. Macrophage infiltration (F4/80+) peaked on PND11 (35%) and remained elevated at ~24% till PND25, while in GI glands increase was gradual from PND17 through PND25 (27%). Expression of key genes identified in AI mice2,3 have markedly different expression pattern in GI mice (Table). While some peaked at a later time point in GI vs. AI coinciding with maximum cell death, expression of some are significantly low or undetectable in GI glands at both RNA and protein level. Conclusions: We show for the first time that kinetics of cell death, adipocyte repopulation, immune cell infiltration and inflammatory state of glands undergoing abrupt vs. gradual involution are markedly different. Several genes known to play a key role during AI are either not expressed or barely detectable in the GI glands at any time point during involution. These data suggests that not only the kinetics, but mechanism of GI and AI are not identical. We conclude that orchestrated cell death in GI protects from drastic lysosomal, and immune cell activities that predisposes mammary glands to higher risk of neoplastic changes.Significance: Epidemiological data highlights the benefits of prolonged breastfeeding in protecting against breast cancer, particularly, TNBC, an aggressive subtype prevalent in the African American women. Our study highlights the mechanism underlying the benefits of gradual involution of breast.
GeneFold Change compared to PND7 (GI vs. AI) Peaked on (GI vs. AI)Stat34.0 vs. 5.9PND25 vs. PND8.5Ctsb2.8 vs. 3.4PND25 vs. PND8.5CD143.4 vs. 20PND25 vs. PND8.5Orm11.7 vs. 10PND25 vs. PND12Lrg130 vs. 216PND25 vs. PND12MMP215.8 vs. 27PND25 vs. PND12Chi3L11350 vs. 572, 859PND28 vs. PND11, 28Cebpδnone vs. 4.4None vs. PND8.5CtsLnone vs. 5.7None vs. PND8.5Orm2none vs. 370None vs. PND12Slpinone vs. 92None vs. PND8.5
Citation Format: Bhuvaneswari Ramaswamy, Neelam Shinde, Morgan Bauer, Maria Cuitino, Saba Mehra, Resham Mawalkar, Mustafa Basree, Allen Zhang, Kirti Kaul, Xiaoli Zhang, Ramesh Ganju, Sarmila Majumder. Mechanistic differences between abrupt and gradual involution of mouse mammary gland abstract. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-08.
Abstract Epidemiological data links higher parity and lack of breastfeeding with increased risk of breast cancer, specifically aggressive triple negative breast cancer (TNBC), and higher mortality ...rate. Following pregnancy and lactation, breast remodels to near pre-pregnancy stage through apoptotic cell death and adipocyte repopulation process. Long-term breastfeeding and gradual weaning of an infant leads to gradual involution (GI) of the breast, while lack of or abrupt discontinuation of breastfeeding after birth leads to abrupt involution (AI), when rapid and massive cell death takes place. Our studies have shown several precancerous changes, such as increased collagen deposition, inflammation, and hyperplasia in the mammary gland of mice after AI. However, the systemic impact of AI and how this increases risk of breast cancer is yet to be elucidated. Objectives: Our objective is to evaluate the systemic effects that are prompted by the AI mammary gland. We hypothesize that AI leads to marked alteration in lipid metabolism and systemic inflammation that enhances risk for breast cancer. Methods: FVB/n mice (8week old) were paired for breeding. At partum (day 0), dams were randomized to AI or GI cohort and standardized to 6 pups. AI mice had pups removed on day 7 postpartum (ppm) to mimic short-term breastfeeding. For GI mice 3 pups each were removed on day 28 and 31ppm to mimic gradual weaning. Tissues harvested on day 28, 56, and 120 postpartum. Body composition was measured by echo MRI. Glucose tolerance test (GTT) was performed after a 6 hour fast using a 2g/kg glucose intraperitoneal injection. Blood glucose was measured by glucometer. Serum insulin was analyzed by ELISA. HOMA-IR was calculated using blood glucose and serum insulin results. Serum was analyzed using multiplex ELISA by MesoScale Diagnostics. Mammary glands were subjected to untargeted lipidomics. Results: There were no significant differences in body weight, percent body fat or lean mass between AI and GI groups at any time point. However, at day 120 ppm (4 months after partum), we have observed significantly larger amount (1.29-fold increase) of perigonadal adipose tissue (visceral adipose) in AI mice than GI mice (p=0.0112; n=24-38/group). There were no significant differences in blood glucose, serum insulin, HOMA-IR, or GTT results between AI and GI groups at day 120. AI mice had significantly higher levels of cytokines IL-1β (3.1-fold increase, p=0.0417) and KC/GRO (1.5-fold increase, p=0.0196) than GI mice at day 120. At day 28, AI mammary glands had significantly higher amounts of level 3 identified oxidized ceramide containing sphingolipids that were linked to insulin resistance and diabetes. At day 56, GI mammary glands had significantly higher amounts of level 2 and 3 identified metabolites linked to cellular signaling and lipid metabolism. On day 120, there were no significant differences in lipid metabolites between groups. Conclusion: Although histologically GI and AI mammary glands return to near pre-pregnancy state within a month after partum, our data shows specific lipid changes in the AI mammary gland similar to what has been shown in women with TNBC. Furthermore, AI of the mammary gland leads to systemic effects on adiposity and inflammation that could be key to increased breast cancer risk. Further studies along these lines are in progress to understand the whole-body effects of AI and stratify preventive measures for women who cannot breast feed. Significance: Lack of breastfeeding is more prevalent in African American (AA) women and linked to higher risk of developing aggressive TNBC1. Our novel animal models of AI and GI help to link the impact of AI and systemic changes that may enhance breast cancer risk. In particular, we see an increase in visceral adiposity with AI. Understanding this mechanism will help identify strategies to reduce risk in women who are unable or choose not to breastfeed and ultimately help to reduce TNBC and TNBC-related mortality in AA women. Citation Format: Kate Ormiston, Kirti Kaul, Neelam Shinde, Djawed Bennouna, Rachel Kopec, Ramesh Ganju, Sarmila Majumder, Bhuvaneswari Ramaswamy. Abrupt involution of mouse mammary gland leads to inflammatory systemic changes along with mammary specific metabolic shifts that may enhance risk of breast cancer abstract. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-09-11.
PDF
