Abstract Triple-negative breast cancer (TNBC) remains one of the most aggressive and therapeutically challenging breast cancer subtypes, characterized by high heterogeneity, intrinsic resistance, and poor immune infiltration. There is a critical unmet need for less toxic approaches that can both suppress tumor growth and remodel the tumor microenvironment (TME). EZH2, a histone methyltransferase, is frequently overexpressed in TNBC, associated with immunosuppression, and disease progression. We previously reported combining GSK126 (an EZH2 inhibitor) with A77636 (a dopamine D1 receptor [DRD1] agonist) produced superior antitumor effects compared to monotherapies. We hypothesized synergistic targeting of EZH2 and DRD1 would convert the immunologically “cold” TNBC microenvironment into a “hot,” immune-permissive state, thereby enhancing therapeutic efficacy and overcoming resistance mechanisms. Female NSG mice (4-6 weeks old) were orthotopically implanted with MDA-MB-231 cells, randomized into four treatment groups (n=8): vehicle, GSK126 (2 mg/kg), A77636 (50 mg/kg), or combination. Treatments were administered intraperitoneally weekly for four weeks. Tumor volume was measured weekly and at endpoint. Tumors and immune tissues were analyzed by flow cytometry. Combination therapy significantly reduced tumor weight (mean difference = 0.278g, 95% CI: 0.109-0.446, p=0.0018) and volume (mean difference = 101 mm3, 95% CI: 51.7-151, p<0.0001) compared with vehicle and single agents. Notably, the combination markedly decreased monocyte populations in both blood and tumor tissue and downregulated EZH2 expression in tumor-associated monocytes and neutrophils. Kinetic profiling revealed a biphasic monocyte response-initial Ly6C^hi recruitment followed by Ly6C^lo transition. The combination suppressed Ly6C^hi infiltration (0.32 vs. 0.92; 65% decrease, p=0.0138) while promoting Ly6C^lo accumulation (2.5 vs. 1.5; 1.67-fold increase, p=0.5126). By using a combinational treatment strategy to redirect monocyte phenotypes, we were able to suppress the pro-inflammatory IL-1β environment while simultaneously enhancing anti-inflammatory IL-10 signaling-ultimately creating a tumor-suppressive immune milieu. In summary, dual modulation of EZH2 and DRD1 effectively halts TNBC progression by reshaping the immunological landscape. These findings uncover a distinctive therapeutic avenue that integrates epigenetic regulation with dopaminergic activation to restore immune responsiveness in TNBC. (This work was supported by DOD W81XWH2010065 to Eswar Shankar). Citation Format: Rajni Kant Shukla, Kate Ormiston, Gautam Sarathy, Shivani Dhekne, Dionisia Marie Quiroga, Sanjay Gupta, Daniel G. Stover, Pierre Giglio, Christian Rolfo, Eswar Shankar. A novel dual-target strategy against TNBC: Combining EZH2 inhibition and dopamine D1 receptor activation to restore immune balance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7473.

In the published publication [...]

Epidemiological data link a lack of breastfeeding with an increased risk of breast cancer. Breast tissue remodels after pregnancy through involution. Long-term breastfeeding results in gradual involution (GI), and a lack of breastfeeding leads to abrupt involution (AI). AI causes increased mammary gland estrogen signaling, causing adipocyte redifferentiation through neutrophil infiltration. Adipocyte differences and metabolic implications of involution have not been explored between AI and GI. As breast cancer is characterized as highly metabolic, we explored how adipocyte differences and metabolism during involution may support breast cancer risk. FVB/n was randomized to AI/GI and standardized to 6 pups on day 0/birth. AI mice had pups removed on day 7. GI mice had 3 pups removed on days 28 and 31. Mammary glands were harvested at 28, 56, and 120 days. A subset of AI mice were given tamoxifen for 21 days. Day 28 AI glands had upregulation of estrogen signaling, neutrophil degranulation, and glucose metabolism and downregulation of adipogenesis and glycolysis compared to Day 56 GI. Day 120 AI glands had downregulation of oxidative phosphorylation and upregulation of mitochondrial dysfunction similar to estrogen receptor-negative (ER-) pregnancy-associated breast cancer (PABC). AI with tamoxifen resulted in a similar metabolic phenotype to GI. Early metabolic phenotypes in AI and GI glands may be related to estrogen signaling. AI long-term transcriptional metabolic effects were similar to breast cancer.

Epidemiological data links lack of breastfeeding with increased risk of breast cancer. Breast tissue undergoes remodeling to pre-pregnancy state after birth through involution. Long-term breastfeeding leads to gradual involution (GI). Lack of breastfeeding leads to abrupt involution (AI). While estrogen impacts repopulation of adipocytes, AI causes several precancerous changes in the mouse mammary gland. The impact of AI on adipocyte repopulation and metabolism is yet to be elucidated. Objectives:To investigate effects of AI on mammary gland metabolism and its potential link to breast cancer. Methods:At partum (day 0), FVB/n dams were randomized to AI or GI and standardized to 6 pups. AI mice had pups removed on day 7 postpartum to mimic short-term breastfeeding. GI mice had 3 pups each were removed on day 28 and 31 postpartum to mimic gradual weaning. Mammary glands were harvested on day 28, 56, and 120 postpartum. Subset of AI mice had long-term sustained release tamoxifen placed subscapular on day 8 postpartum. Metabolic changes were assessed using: 1) transcriptional; 2) functional; 3) oxidative stress; and 4) metabolites analysis. Results:Day 28 GI glands sustains/continues milk synthesis pathways impacting metabolic comparison with day28 AI glands. Day 28 AI when compared to day 56 GI showed upregulation of estrogen signaling, neutrophil degranulation, glucose metabolism, RNA synthesis, and down regulation of adipogenesis and glycolysis. At day 120, AI glands had downregulation of oxidative phosphorylation and upregulation of mitochondria dysfunction similar to pregnancy associated estrogen receptor negative breast cancer. Tamoxifen treatment of AI dams showed metabolic pathways and estrogen signaling similar to that of GI glands on day 28. Conclusion:Early metabolic phenotypes in AI and GI glands may be caused by differences in adipocyte repopulation related to estrogen signaling. Long-term metabolic effects of AI lead to similar metabolic effects found in breast cancer.

Epidemiological studies associate an increase in breast cancer risk, particularly triple-negative breast cancer (TNBC), with lack of breastfeeding. This is more prevalent in African American women, with significantly lower rate of breastfeeding compared to Caucasian women. Prolonged breastfeeding leads to gradual involution (GI), whereas short-term or lack of breastfeeding leads to abrupt involution (AI) of the breast. Our previous study utilizing a murine model demonstrated precancerous changes, specifically hyperplasia, a non-obligate precursor of breast cancer in the mammary glands of AI mice. Here we investigated mechanisms during early events of AI that prompts precancerous changes in mouse mammary glands. Uniparous FVB/N mice were randomized to AI and GI on postpartum day 7 when all pups were removed from AI dams. GI dams were allowed to nurse the pups till day 31. Cell death kinetics and gene expression were assessed by TUNEL assay and qPCR respectively. Immune cell changes were investigated by flow cytometry, cytokine array and multiplex immunofluorescence. 3D-organoid cultures were used for in vitro assay of luminal progenitor cells. AI results in rapid cell death, DNA repair response, and immunosuppressive myeloid cells infiltration, leading to a chronically inflamed microenvironment. GI elicits a more controlled immune response and extended cell death. At the peak of cell death, AI glands harbored more immunosuppressive myeloid-derived suppressor cells (MDSCs) and CD206 + M2-like macrophages, known to promote oncogenic events, compared to GI glands. AI glands exhibit an enrichment of CCL9-producing MDSCs and CD206 + M2-like macrophages that promote expansion of ELF5 + /ERα- luminal cells, both in vitro and in vivo. Multiplex imaging of AI glands demonstrated an increase in ELF5 + /WNT5a + luminal cells alongside a reduction in the ELF5 + /ERα + population when involution appeared histologically complete. A significantly higher number of CD206 + cells in post involution AI gland attests to a chronically inflamed state induced by AI. Our findings reveal significant disparities between AI and GI gland dynamics at the early phase of involution. CCL9, secreted by immune cells at the peak of cell death promotes expansion of Elf5 + /ERα- luminal progenitor cells, the putative precursors of TNBC connecting early events of AI with increased breast cancer risk.

Background: EZH2 component of the polycomb repressive complex 2 (PRC2) is a histone methyltransferase whose function is to methylate lysine 27 of histone 3. EZH2 is overexpressed in 49% of breast cancers and is associated with worse outcomes especially in TNBC. EZH2 is overrepresented in African American and Hispanic women, suggesting that clinical targeting of this protein may particularly help improve the disproportionately poor outcomes in these populations. In addition to its role in metastasis, EZH2 regulates tumor immune microenvironment (TIME) by inhibiting T cell activation via suppression of MHC-1 antigen presentation pathway, upregulating PD-L1 expression causing suppression of an antitumor immune response. Current EZH2 inhibitors only targets the catalytic activity of EZH2, leaving its function as a gene activator unaffected. Despite the advancement in the discovery of inhibitors for EZH2 that attenuate its catalytic activity, resistance to these small molecules limits their use in solid tumors. The neurotransmitter dopamine via its D1 receptor activation in TNBC cell lines induces apoptosis and autophagy, as well as inhibits the invasion and regress in mammary tumors in vivo. In addition, dopamine D1 receptor signaling has been reported to attenuate the immunosuppressive effects of myeloid-derived suppressor cells (MDSCs) on T cell proliferation and IFN-g production. We hypothesize that combined treatment of dopamine D1 receptor agonist (A77636) and EZH2 inhibitors (GSK126) inhibits tumor growth and metastasis of TNBC cells both in vitro and in vivo. Methods: To test the efficacy of the combination inhibiting metastasis we employed an in vivo model system to confirm the results we had obtained from our invitro 3D culture system and 3D organ-on-chip-based microphysiological (MPS) platform (SynTumor). 4-6 weeks old female NSG mice were injected with MDA-MB-231 cells on the breast fat pads. When the tumors became palpable, they were randomized into four groups, Vehicle, GSK126 (2mg/kg BW), A77637 (50mg/kg BW) and the combination. The drugs individually or in combination were administered intraperitoneally five days for 4 weeks. Tumor measurements were also done during the time and at the end of 4 weeks the animals were euthanized. The tissues, tumor, bone, blood, and spleen were harvested and processed for flow cytometer analysis. Tumor weight and volume were also calculated. Results: The combination of GSK126 and A77636 demonstrated a synergistic effect inhibiting the tumor weight and tumor volume when compared to the individual treatments or the vehicle treated animals. These results matched with our in-vitro data, where the combination synergistically inhibited the growth of spheroids. in the microfluidic SynTumor model, the combination reduced circulating tumor cell numbers by half. The combination significantly decreased the monocyte population in the blood and tumor. Also, the EZH2 expression in the monocytes and neutrophils were significantly decreased by the combination. Conclusion: Our data indicate that the combinatorial effect of DRD1 agonist and EZH2 inhibitor efficiently attenuates the EZH2-mediated tumor growth and innate immune environment in TNBC (This work is supported by DOD: W81XWH2010065, for Eswar Shankar). Dedicated in memory of Dr. Bhuvaneswari Ramaswamy. Citation Format: Eswar Shankar, Rajni Kant Shukla, Gautam Sarathy, Kate Ormiston, Xilal Rima, Chunyu Hu, Divya S Patel, Radha vaddavalli, Deborah Ramsey, Gwen Fewell, Bhuvaneswari Ramaswamy, Eduardo Reátegui. Enhancer of zeste homologue 2 (EZH2) inhibition in Triple Negative Breast Cancer (TNBC) attenuates tumor growth in vitro and in vivo altering the tumor immune microenvironment [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-09-20.

EZH2, a histone methyltransferase linked to poor outcomes in cancer, is overexpressed in triple-negative breast cancer (TNBC), disproportionately affecting African-American and Hispanic women. EZH2 regulates the tumor immune microenvironment (TIME) by inhibiting MHC-I antigen presentation, upregulating PD-L1, leading to reduced antitumor immune responses. Current EZH2 inhibitors target its catalytic activity; however, the development of resistance mechanisms in cancer cells frequently undermines their effectiveness. The neurotransmitter dopamine, through activation of its D1 receptor, promotes apoptosis and autophagy while suppressing cancer cell invasion. Dopamine D1 receptor signaling mitigates the immunosuppressive effects of myeloid-derived suppressor cells (MDSCs) on T cell proliferation and IFN-γ production. Using in silico approaches such as cryptic pocket identification, docking, and molecular dynamics (MD) simulations, initially we assessed the binding affinity of the DRD1 agonist to EZH2. Our in-silico analyses uncovered stable binding profiles, indicating novel interactions at previously uncharacterized sites within the CXC domain and the EED-binding region for both GSK126 and A77636. Next, we performed in vivo experiments combining GSK126 and A77636 to evaluate their effect on tumor growth and the TIME using an NSG mouse model. MDA-MB-231 cells were injected into the breast fat pads of 4-6-week-old female NSG mice. Once tumors were palpated, the mice were randomized into four groups: vehicle treatment, GSK126 (2 mg/kg), A77636 (50 mg/kg), and the combination of both, with treatments administered intraperitoneally for four weeks. Tumor growth was monitored throughout the treatment period, and at the termination, blood, tumor, and spleen were collected and processed into single-cell suspensions for high-dimensional flow cytometry analysis. GSK126 and A77636 treatment in combination significantly reduced tumor volume and decreased EZH2 expression in monocytes and neutrophils within the blood and tumor microenvironment and lowering the levels of pro-inflammatory cytokine IL-1β. In summary, this approach unveils new possibilities for treating TNBC by concurrently targeting EZH2 and DRD1 through a combinatorial strategy. (This work is supported by DOD: W81XWH2010065, for Eswar Shankar). Rajni Kant Shukla, Kate Ormiston, Gautam Sarathy, Shivani Dhekne, Sanjay Gupta, Daniel Stover, Christian Rolfo, Eswar Shankar. Synergistic inhibition of TNBC via enhancer of zeste homologue 2 (EZH2) inhibition and D1 receptor agonism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 509.

Epidemiological data links higher parity and lack of breastfeeding with increased risk of breast cancer, specifically triple negative breast cancer (TNBC). TNBC is the aggressive hormone receptor and HER2 negative subtype associated with higher mortality rate1. Following pregnancy and lactation, breast remodels to near pre-pregnancy stage through apoptotic cell death and adipocyte repopulation process2. 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 place2. Our studies comparing GI vs. AI in a mouse model have shown several precancerous changes, such as increased collagen deposition, inflammation, and hyperplasia in the mammary gland of mice after AI2. While our preliminary data indicates metabolic shifts in the AI glands, the impact of AI on mammary gland metabolism and how this increases risk of breast cancer is yet to be elucidated. Objectives: Determine the impact of AI on metabolic changes within the mammary gland and decipher the underlying mechanism that could link AI to increased breast cancer risk. Methods: Eight-week-old FVB/n mice were paired for breeding. At partum (day 0), dams were randomized to AI or GI cohort, standardized to 6 pups per dam and housed individually. 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 31 ppm to mimic gradual weaning. Mammary glands were harvested on day 28, 56, and 120 ppm to assess short-term, intermediate, and long-term effects of AI vs. GI. Total mammary gland RNA was subjected to global gene expression analysis using Affymetrix and analyzed Ingenuity pathway analysis software. Differentially expressed genes were validated by qPCR and western blot. Oxidative stress was measured via MitoSox and H2CDFDA using Flow Cytometry. Whole mammary glands were subjected to untargeted metabolomics and lipidomics. Results: On day 28 ppm, AI glands had marked upregulation of oxidative phosphorylation, ATP synthesis, and mitochondrial fatty acid b-oxidation compared to GI glands. AI glands had significantly higher levels of mitochondrial oxidative stress and enrichment of oxidized glycerophospholipids. On day 56 ppm, AI glands were metabolically comparable to GI glands. However, AI glands had an upregulation of genes related to fatty acid synthesis (PPARg, ACLY, Chrebp, GLUT4 and SLC25A1) and mitochondrial biogenesis (PGC1a). Interestingly, on day 120 ppm, AI glands showed significant downregulation of oxidative phosphorylation, ATP synthesis, glucose metabolism, and marked upregulation of mitochondria dysfunction. Amyloid precursor protein (APP) associated with mitochondria dysfunction and downregulation of energy metabolism, was found to be elevated in AI vs. GI glands on day 120 ppm. Conclusion: AI of the mammary gland leads to metabolic changes over time that disrupt mitochondrial function. Multiple studies have associated high levels of APP in human breast tumors and breast cancer cell lines with disruption of mitochondria function, enhanced cell proliferation, metastasis and invasion. Increased expression of APP in AI mammary glands suggests a key role of this protein in mitochondrial and metabolic dysfunction induced by AI. Further investigation is underway to decipher the role of APP in AI induced changes. Significance: For the first time, this study demonstrates a metabolic shift in the mammary gland caused by AI. Targeting one or more key players in this metabolic deregulation could provide options for lowering breast cancer risk in women who are unable to breast feed. *Funding NCI RO1 – CA237185 PI-Ramaswamy/Ganju Citation Format: Kate Ormiston, Neelam Shinde, Gautam Sarathy, Allen Zhang, Morgan Bauer, Rajni Kant Shukla, Sara Alsammerai, Annapurna Gupta, Djawed Bennouna, Xiaoli Zhang, Rachel Kopec, Eswar Shankar, Ramesh Ganju, Kristin I. Stanford, Sarmila Majumder, Bhuvaneswari Ramaswamy. Abrupt Involution Leads to Long-Term Mitochondrial Dysfunction and Metabolic Shift – Increasing Risk of Breast Cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-03-06.

Chemotherapy agents in breast cancer are associated with chemotherapy-related cognitive impairments (CRCI). Mechanisms are not fully clear, but alterations of glucose and lipid metabolism, neuroinflammation and neurodegeneration may contribute to CRCI. The aim of this study was to investigate the combined effects of a high fat (HF) diet combined with doxorubicin-based chemotherapy on glucose and lipid metabolism, neuroinflammation, and neurodegeneration in mice. Additionally, we examined the therapeutic potential of dietary eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to attenuate these effects. Female C57Bl/6 mice (n = 42) were fed HF, HFn-3 (2 % kcals as EPA + DHA) or Low Fat (LF) diets for seven weeks, with and without chemotherapy. In this study, two chemotherapy injections led to weight and body fat loss associated with a decrease in insulin resistance measured by HOMA-IR. HOMA-IR was significantly greater in HF versus LF groups; but HOMA-IR in HFn-3 group did not significantly differ from either HF or LF groups. Chemotherapy resulted in higher brain concentrations of the inflammatory chemokine KC/GRO. Compared to LF diet plus chemotherapy, HF diet plus chemotherapy upregulated multiple genes involved in neuroinflammation and neurodegeneration pathways. HFn-3 diet plus chemotherapy attenuated gene expression by downregulating multiple genes involved in neuroinflammation and blood brain barrier regulation, including Mapkapk2, Aqp4, and s100b, and upregulating Kcnb1 and Atxn3, genes involved in reduction of oxidative stress and anxiety, respectively. Overall, a HF diet combined with chemotherapy is associated with neuroinflammatory and neurodegenerative gene expression changes in this mouse model; dietary enrichment of EPA and DHA attenuated these effects. Further studies are needed to understand how diet impacts behavioral outcomes of CRCI.

PURPOSE:Glucose dysregulation may occur during chemotherapy treatment of patients with breast cancer. Absent appropriate management, patients may experience worsened morbidity and mortality. Mechanisms regarding glucose dysregulation are well-studied, but the role of chemotherapy is not. Previous studies of glucose dysregulation among patients undergoing chemotherapy have yielded inconsistent results. METHODS:We evaluate changes in blood glucose in women with breast cancer undergoing chemotherapy and explore effects of covariates. Records of 981 patients with stage I-IV breast cancer were evaluated, including blood glucose levels and other clinical and histopathological data. Patients were treated at The Ohio State University, an NCI-designated Comprehensive Cancer Center, from 2016 to 2018. Subgroups reflecting populations studied by previous reports were analyzed. RESULTS:Overall, blood glucose decreased slightly after chemotherapy versus baseline (131.3 mg/dL vs. 134.8 mg/dL, P = .048). Among patients with baseline random glucose levels lower than 125 mg/dL, levels increased versus baseline (113 vs 99 mg/dL, P < .001). Among patients younger than 70 and without diabetes mellitus, there was no statistically significant change (120 vs 122 mg/dL, P = .47). Adjusting for baseline variables including glucose levels, estimated change from baseline is higher among patients exposed to docetaxel/cyclophosphamide versus other regimens assessed (P < .001). CONCLUSIONS:Random blood glucose decreased slightly during chemotherapy. Differences in results in previous reports are attributable to differing study populations. Baseline glucose levels, age, and chemotherapy regimen are predictors of change in glucose level.

Chemotherapy-related cognitive impairment (CRCI) and affective symptoms negatively impact quality of life in breast cancer survivors. The aim of this study was to determine the efficacy of high eicosapentaenoic acid + docosahexaenoic acid (EPA+DHA) and low sucrose diets to alleviate these symptoms in a mouse model of chemotherapy. Potential mechanisms involving insulin resistance were explored. We hypothesized that diets enriched in EPA+DHA and low amounts of sucrose would protect against the impact of chemotherapy on measures of CRCI. Female C57Bl/6 mice were randomized to 1 of 4 diets (2% kcal eicosapentaenoic acid + docosahexaenoic acid [EPA+DHA]/high or low sucrose, low omega-3/high or low sucrose) for 6 weeks and treated with two injections of doxorubicin-based chemotherapy or vehicle during week 2 and 4. Behavioral tests were performed 7 days after second injection. Chemotherapy increased serum insulin and decreased body weight, locomotion and exploratory behavior (all p < .05). Low sucrose consumption resulted in better long-term memory regardless of chemotherapy or vehicle injection (p < .05). 2% EPA+DHA consumption lessened insulin resistance (p < .05); however, controlling for body weight attenuated this effect (p = .08). There were no significant differences by diet or injection on liver lipid content; however, liver lipid content was positively correlated with insulin resistance scores (p < .05). Low sucrose diets may protect long-term memory during chemotherapy. The effect of EPA+DHA on insulin resistance and affective side effects during chemotherapy requires further investigation.

Abstract Introduction: A meta-analysis of 47 global epidemiological studies highlights a higher breast cancer risk in women who did not breastfeed or breastfed for a short time. Further studies showed this is especially true for triple-negative breast cancer (TNBC) patients. Premenopausal AA women (AAW) have a lower prevalence of breastfeeding and a higher incidence of TNBC and mortality. Our previous study compares short-term breastfeeding, abrupt involution (AI) with prolonged breastfeeding called gradual involution (GI), revealing that AI alone induces ductal hyperplasia four months postpartum. Our current investigation delves into early events during AI versus GI, employing a comprehensive approach encompassing histology, gene expression, and myeloid cell involvement. Methods: Utilizing FVB female mice, we conducted a comparative analysis of AI and GI. AI involved early pup removal, while GI was achieved through staggered weaning. The evaluation included analysis of histomorphology, gene/protein expression, and myeloid cell infiltration. Sequential mammary gland (MG) changes were monitored through H&E staining, TUNEL assay, and DNA damage analysis. 3D-organoid cultures of luminal progenitors (LPs) were employed to assess the impact of AI versus GI. qRT-PCR, IHC, Western blot, and flow cytometry/multiplex imaging were employed for the differential expression analysis of molecular and cellular factors associated with AI/GI. Results: Our research showed that AI had early adipocyte repopulation, rapid cell death, DNA repair, and myeloid cell infiltration, resulting in a chronically inflamed microenvironment. In contrast, the GI triggers a controlled immune response and prolonged cell death, facilitating comprehensive remodeling of the MG. Our flow cytometric or multiplexing imaging analyses revealed that AI-affected glands exhibit an enrichment of CCL9-producing CD206+ M2-like macrophages and CD11b+Gr1+ myeloid-derived suppressor cells. Moreover, exogenous CCL9 treatment on LPs in 3D-organoid culture results in disorganized acinar-type organoids, mirroring morphological differences observed in LPs from AI mammary glands on day56 PPM. Further analysis of CCL9 treated organoids revealed the expansion of Esr1- LPs population in ex-vivo organoid culture which might indicate an increase in the putative cells of origin of TNBC. Conclusion: Our studies comparing AI and GI demonstrate that AI is producing a pro-tumorigenic environment in the breast. It is important to note that prolonged breastfeeding protects the breast, although this cannot be a singular risk factor for TNBC. Therefore, understanding the mechanism will lead to prevention strategies to improve outcomes for all women but, has the potential to have a significant benefit in AAW. Citation Format: Sanjay Mishra, Neelam Shinde, Maria Cuitino, Morgan Bauer, Dinesh Ahirwar, Vijaya Bharti, Kate Ormiston, Resham Mawalkar, Sara Alsammerai, Gautam Sarathy, Xiaoli Zhang, Anna Vilgelm, Ramesh Ganju, Sarmila Majumder, Bhuvaneswari Ramaswamy. Understanding the link between breastfeeding and the risk of breast cancer through comparative analysis of the murine-based model of mammary gland involution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2138.

Abstract Epidemiological data links higher parity and lack of breastfeeding with increased risk of breast cancer, specifically aggressive triple negative breast cancer, and associated higher mortality rate. 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 show several precancerous changes, such as increased collagen deposition, inflammation, and hyperplasia in the mammary gland (MG) of mice after AI1. Recent studies indicate peroxisome proliferator-activated receptor-gamma coactivator 1-apha (PGC1α) as a regulator of involution and energy metabolism. As metabolic reprogramming is a hallmark of cancer, metabolic changes in the MG related to involution warrant investigation. Objective: Our objective was to evaluate the metabolic effects of AI in MG. We hypothesized that AI leads to marked alteration in mammary lipid metabolism, mitochondrial biogenesis, and oxidative stress through elevations of PGC1α, which cause long-term metabolic reprogramming and genomic instability. Methods: FVB/n mice were paired for breeding. At partum, dams were randomized to AI or GI cohort and standardized to 6 pups per dam. AI mice had pups removed on day 7 postpartum (PPM). For GI mice 3 pups each were removed on day 28 and 31ppm. Tissues were harvested on day 28, 56, and 120 PPM. MG were subjected to Affymetrix, Gene Set Enrichment Analysis (GSEA), Seahorse Analysis, and lipidomics. Results were validated by qPCR and Western Blot. Superoxide species were detected by flow cytometry. DNA damage was analyzed via 8-hydroxy-2’-deoxygnuanosine (8-OHdG) ELISA. Results: Day 28 AI glands had significantly higher PGC1α expression than GI glands (p=0.006). Affymetrix and GSEA data showed day 28 AI glands to have enriched pathways related to fatty acid metabolism (p=0.004) and oxidative phosphorylation (p<0.001). Lipidomics showed elevated levels of oxidized sphingolipids and production of prostaglandin J2 (PGJ2) in day 28 AI glands (all p<0.02). Day 56 AI glands had higher levels of mitochondria superoxide species (p<0.0001) and higher PGJ2 synthesis (p=0.01). Day 120 AI MG had upregulation of an oxidized lipid (p=0.0476). Day 120 AI glands had significantly higher 8-OHdG levels (p=0.02), higher reliance of fatty acid substrates for energy (p=0.0185), and elevated extracellular acidification rates (p=0.0194). Conclusion: Although histologically both GI and AI MG return to near pre-pregnancy state within a month, our data shows long-term metabolic reprogramming in the AI MG similar to what is shown in breast cancer cells. These metabolic changes link to early elevations in PGC1α. Addressing the metabolic changes by targeting PGC1α provide a potential option to reduce the risk of developing breast cancer if a woman is unable to breastfeed. PMID6637535 *Fellowship T32CA229114 Citation Format: Kate S Ormiston, Kirti Kaul, Neelam Shinde, Gautam Sarathy, Morgan Bauer, Djawed Bennouna, Rachel Kopec, Ramesh Ganju, Sarmila Majumder, Bhuvaneswari Ramaswamy. Elevated PGC1α during abrupt mammary gland involution leads to long-term metabolic reprogramming and genomic instability; hallmarks of breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A045.

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.

Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer with higher rates of recurrence and distant metastasis, as well as decreased 5-year survival rates. Racial disparities are evident in the incidence and mortality rates of triple negative breast cancer particularly increased in young African American women. Concurrently, young African American women have multiple risk factors for TNBC including higher rates of premenopausal abdominal obesity (higher waist-hip ratio) and lower rates of breastfeeding with higher parity, implicating these factors as potentially contributors to poor outcomes. By understanding the mechanisms of how premenopausal obesity and lack of breastfeeding may be associated with increased risk of triple negative breast cancer, we can determine the best strategies for intervention and awareness to improve outcomes in TNBC.

The intestinal microbial population is recognized for its impact on cancer treatment outcomes. Little research has reported microbiome changes during cancer progression or the interplay of disease progression, dietary sugar/fat intake, and the microbiome through surgery and chemotherapy. In this study, the murine gut microbiome was used as a model system, and changes in microbiome diversity, richness, and evenness over the progression of the cancer and treatment were analyzed. Mice were categorized into four diet cohorts, combinations of either high or low sucrose and high or low omega-3 fatty acids, and two treatment cohorts, saline vehicle or chemotherapy, for a total of eight groups. Fecal samples were collected at specific timepoints to assess changes due to diet implementation, onset of cancer, lumpectomy, and chemotherapy. Akkermansia muciniphila abundance was very high in some samples and negatively correlated with overall Amplicon Sequence Variant (ASV) richness (r(64) = −0.55, p = 3 × 10−8). Throughout the disease progression, ASV richness significantly decreased and was impacted by diet and treatment. Alpha-diversity and differential microbial abundance were significantly affected by disease progression, diet, treatment, and their interactions. These findings help establish a baseline for understanding how cancer progression, dietary macronutrients, and specific treatments impact the murine microbiome, which may influence outcomes.