Abstract Introduction: Exposure of non-transformed breast cells and breast microstructures to the medium-chain (MC) fatty acid (FA) octanoic acid (OA) induces a metabolic shift toward the serine, one-carbon, glycine and methionine pathways (SOG/methionine), enhancing epigenetic plasticity, increasing reactive oxygen species (ROS), promoting cell survival and disrupting cell-cell communication. Similarly, the aged mammary gland is characterized by disrupted cell-cell communication, epigenetic plasticity and increased ROS. We hypothesize that FA-induced metabolic reprogramming leads to biological aging of the mammary gland, contributing to pro-tumorigenic alterations observed during chronologic aging. Methods: MCF-10A cells were exposed to OA for proteomics. Breast microstructures exposed to ± OA were analyzed by scRNAseq. Breast microstructures and 3D mammary spheres derived from primary cells were embedded in Matrigel, exposed to ± OA for 7 days, stained for luminal and basal markers, F-actin, and nuclei, and imaged by confocal microscopy to assess migration/invasion. Migratory cell populations enriched in OA-containing media were identified with scRNAseq. Raman spectroscopy (RS) was used to characterize the lipid content in normal breast tissue. Results: OA treatment induced changes previously reported in aging and tumorigenic contexts, including: (1) upregulation (p < 0.01) of aging-related genes (GDF15, MDK, PLIN2), and downregulation (p < 0.01) of lineage markers and MMP7, a gene whose downregulation promotes mammary epithelial aging; (2) upregulation (p < 0.01) of Senescence-Associated Secretory Phenotype (SASP) genes, including AREG (reprogramming) and ANGPTL4 (migration); (3) increased secreted signaling via AREG, GDF15, and MDK; and (4) reduced extracelular matrix (ECM)-receptor and cell-cell interactions. Ex vivo, OA altered tissue architecture disrupting the basal barrier and promoting cellular migration. BMYO1, LASP1, and LHS1 epithelial subtypes were among the migratory cells in OA media and expressed SASP, cancer (MYC, EGFR, SREBF1), migration (S100A4, NCAM1), aging and SOG/methionine genes. FB1 fibroblasts dominated in vehicle media, but OA favored ECM-disassembling FB2 cells. RS analysis demonstrates the presence of both saturated and unsaturated FAs and revealed the presence of MCFAs, such as OA, with higher intensities observed in the postmenopausal tissue supporting the in vivo plausibility of our in vitro/ex vivo findings. Conclusions: Our data supports a model suggesting that chronological and biological aging processes increase the release of free FAs, due to elevated GDF15-induced lipolysis. The rise in FAs drives mammary gland remodeling and accelerates aging of the gland. Chronological and biological aging increase vulnerability to breast cancer. This model suggests potential preventive strategies such as targeting GDF15 and SOG/methionine. Citation Format: Mariana Bustamante Eduardo, Abul B.M.M.K. Islam, Curtis W. McCloskey, Maria Paula Zappia, Ashok Z. Samuel, Maxim V. Frolov, Rama Khokha, Rohit Bhargava, Elizaveta V. Benevolenskaya, Seema A. Khan, Susan E. Clare. Fatty acid exposure promotes age-related mammary tissue alterations with pro-tumorigenic potential [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 6019.

Epidemiologic and preclinical studies suggest a potential protective effect of breast implants against breast cancer, but underlying mechanisms remain unclear. The authors hypothesize that local inflammation after breast implant placement induces immunosurveillence. The authors of this study investigated whether serum from implant-exposed (IE) women reduced breast cancer cell viability compared with implant-naive (IN) women, and whether antibodies mediated this effect. Serum from IE and IN women with high or low antibody levels against estrogen receptor-alpha, mucin-1, or mammaglobin-A was incubated with cancer cell lines overexpressing each antigen (MCF7, T47D, and SKBR3, respectively). 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assays measured cell viability. Rescue experiments utilized recombinant antigen to neutralize antibodies. Thirty-six patients were enrolled, and 12 samples were used to assess viability for each cell line. Serum with high antibody concentrations reduced cell viability compared with serum with low antibody concentrations across all cell lines. IE serum reduced cell viability compared with IN serum across all cell lines. Rescue experiments reversed differences between high and low antibody serum, as well as between IE and IN serum, implicating antibodies as the mediators of reduced viability. Peripheral serum from IE women decreased breast cancer cell viability compared with IN women, even when matched for the same antibody levels. Antibody neutralization reversed differences. This suggests implant exposure altered antibody function or other serum components. These findings support the hypothesis that the inflammatory response to breast implants may increase breast cancer immunosurveillance, although significantly more work is needed to confirm a mechanism. Level of Evidence: 3 (Therapeutic).

Abstract Introduction: Estrogen receptor-negative (ER-neg) breast cancer (BC) disproportionatelyaffects younger women, women of African descent, and underserved populations. Nopreventive agents are approved for this subtype. ER-neg BCs, including triple-negative (TNBC)and HER2+ tumors, often metastasize to the brain early, yet remain undetected until neurologicsymptoms emerge, when prognosis is poor. Current therapies, including ER-neg adjuvantagents, offer limited brain efficacy due to poor blood-brain barrier penetration. Radio-surgicalinterventions for brain metastases have severe side effects with no survival benefit. There is acritical need for safe, risk-reducing therapies that prevent both ER-neg BC and braindissemination.We identified L024, a first-in-class agent with efficacy against ER-neg BC. Mechanistically, L024inhibits SREBP1-driven lipogenesis, suppresses the PI3K-AKT axis, and downregulates NF-κB-mediated inflammation, key pathways in ER-neg progression. We have demonstrated that L024suppresses tumor growth and we hypothesize that it will intercept early brain metastasis, offering a novel preventive and interceptive approach. Methods: We assessed L024’s antiproliferative activity in six ER-neg BC lines: MDA-MB-231,HCC-1937, HCC-3153, 4T1, 4T1-BM2, and MDA-MB-231-Br using IncuCyte live cell imaging. Xenografts were developed using MDA-MB-231 cells in nude mice; L024 (80 mg/kg/day) was administered when tumors reached threshold size. Tumor growth was monitored over 28 days;RNA-seq was performed on excised tumors. Transwell assays with/without matrix assessedinvasion/migration of brain-tropic cells after a sublethal L024 dose (2.5-5 µM). Western blotswere performed after 24 h L024 (5 µM) exposure to assess downstream effectors. ADMEproperties were evaluated in silico (ACD Lab). PK was analyzed in female CD-1 mice dosed with L024 (50 mg/kg), with drug levels in plasma, mammary, and brain measured with LC-MS/MS, and modeled using SAAM II. Results: L024 significantly inhibited TNBC proliferation in a dose-dependent manner, with sustained effects six days post-treatment. It reduced tumor growth and epithelial-to-mesenchymal transition in the xenografts. Sublethal doses impaired invasion and migration ofbrain-tropic cells. Western blots showed reduced PI3K, AKT, and pAKT (Ser473). In silicoprofiling predicted CNS permeability: molecular weight <400 Da, LogP = 3.9, TPSA = 66.76 Å2,and no P-gp/BCRP efflux. In vivo PK confirmed high brain exposure (Kp >1 between 1-8 h),cleared by 24 h, supporting prevention/therapy potential. Conclusions: L024 reprograms metabolic pathways essential for ER-neg BC progression,specifically targeting multiple steps of the metastatic cascade. CNS PK supports furtherevaluation in immunocompetent TNBC models with brain metastatic potential. Citation Format: Atieh Hajirahimkhan, Alexander D. Eremin, Elizabeth Bartom, Saktimayee M. Roy, Daniel M. Watterson, Susan E. Clare, Seema A. Khan. Prevention and interception of triple-negative breast cancer and its metastasis to the brain with brain-permeant L024 [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 967.

Breast cancer (BC) risk-reducing drugs have minimal impact due to their adverse effects and low acceptance. Novel non-endocrine agents with reduced toxicity and efficacy beyond HR+ BC are needed. We recently showed that in high-risk women’s breast treated with licochalcone A (LicA) ex vivo, changes in metabolism flux led to reduced proliferation, NF-kB-dependent inflammation, and SREBP1-dependent lipogenesis. Additionally, LicA suppressed the proliferation of pre-malignant and malignant breast cell lines (HR+ and HR-) and their xenografts in mice. Now we present confirmatory evidence that LicA prevents BC by reducing SREBP1-dependent metabolism and inflammation. We also show the promising pharmacokinetic (PK) profiles of our novel oral formulations. We performed NanoString metabolism panel assay in MDA-MB-231 (HR-) and MCF-7 (HR+) cells treated with LicA (10 µM, 24 h) and analyzed data by ROSALIND. We also conducted Proteome Integral Solubility Alteration (PISA) proteomics and Enrichr analysis to confirm transcriptomic data and the main targets of LicA. We validated the findings with western blots. In addition, we evaluated spatiotemporal changes in cholesterol concentrations in the inner leaflet of the plasma membrane. Five proprietary novel formulations of LicA were developed and administered orally and intravenously to female BALB/c mice and Sprague-Dawley rats followed by LC-MS/MS analysis of the plasma and mammary tissue, and PK modeling using SAAM II. We observed significant (adj P < 0.05) upregulation of anti-inflammatory signals (up to 9-fold) such as HMOX1, and downregulation (up to 6-fold) of NF-kB-dependent inflammatory pathways such as prostaglandin E2 synthesis at the level of genes and proteins. We also observed significant downregulation of SREBP1-dependent lipogenesis signals such as ACAT2, FASN, SCD, and proliferation markers such as MKI67. Proteomics results further revealed a reduction in neddylation responsible for stabilizing SREBP1, and a metabolic shift from lipogenesis to the degradation of fatty acids and branched amino acids, and an enhanced TCA cycle. Western blots revealed significant suppression of SREBP1 (4-fold), and the reduced phosphorylation of PI3K (5-fold) and AKT (6-fold, at Ser 473). These results were confirmed by cholesterol depletion in the inner leaflet of the plasma membrane in HR- (8-fold) and HR+ (4-fold) cells. These results demonstrate that LicA reduces cell proliferation by suppressing SREBP1-dependent lipogenesis and inflammation. Our novel oral formulation of LicA, L13 showed promising PK (Cmax ≈ 6 µM, T1/2 = 26 h) suitable for BC prevention efficacy. Our data suggests that reprogramming SREBP1-dependent metabolism and inflammation by LicA prevents BC regardless of HR status. We will test L13 in immunocompetent models of BC to further establish its preventive efficacy. Atieh Hajirahimkhan, Elizabeth T. Bartom, xingyu Guo, Kyli Berkley, Ruohui Chen, Wonhwa Cho, Michael Avram, Susan E. Clare, Seema A. Khan. Reprogramming SREBP1-dependent metabolism and inflammation in high-risk breast with licochalcone A for the prevention of breast cancer [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 6339.

Introduction: We have investigated the breast microenvironment to identify factors that promote Estrogen Receptor Negative Breast Cancer (ERneg BC) and that may be disrupted for prevention. To that end, we have identified a lipid metabolism gene signature associated with the risk of ERneg BC. To better understand lipid metabolism in the breast, we studied the effect of fatty acids (FA) on non-transformed breast epithelial cells and tissues. FA exposure alters histone methylation, affecting gene expression and increase flux through serine, one-carbon, glycine (SOG) and methionine pathways. The association of the serine pathway and ERneg BC was first observed over a decade ago. A SOG pathway gene signature is significantly correlated with ERneg status. We hypothesized that the metabolism of FA results in a metabolic shift toward the de novo serine synthesis pathway (SSP), which ultimately increases S-adenosylmethionine (SAM), altering histone methylation, profoundly changing gene expression and fostering ERneg oncogenesis. Methods: Non-transformed MCF-10A cells were used for in vitro metabolic and epigenomic analyses. Cells exposed to the medium-chain FA octanoic acid (OA) were utilized for proteomics and U13C-glucose tracing. SAM, glutathione (GSH) and 2-hydroxyglutarate (2-HG) concentrations were measured following treatment with OA ± blockade of the serine pathway. Reactive Oxygen Species (ROS)-induced redox changes were monitored live cells. Comet assay was performed to detect DNA damage. CUT&RUN was performed for H3K4me3. Human breast tissue derived microstructures were utilized for genomic analysis. Single-cell RNA-seq (scRNAseq) was performed in microstructures exposed to ± OA. Metabolic flux analyses was performed using Compass. Results: 13C flux analysis revealed that OA led to increased flux to methylation. OA significantly increased the main methyl donor SAM, the antioxidant GSH via the transsulfuration pathway and the oncometabolite 2-HG after 15 min exposure. Blocking the first and rate limiting enzyme in the SSP, PHGDH, prevented these increases. Proteomics revealed the overexpression of PHGDH following OA exposure. Upon exposure to OA, scRNAseq analysis revealed increased expression of the SSP transcription factor (TF) ATF3 and the SSP genes PHGDH and PSAT1 in epithelial and non-epithelial clusters. Upon OA the proportion of three subtypes within the epithelial compartment increased: basal BSL1, Hormone sensing HS1 and luminal progenitor LP3. Compass, an algorithm to characterize cellular metabolic states, revealed flux greatly increased through the three enzymes of the SSP: PHGDH, PSAT1 and PSPH secondary to OA exposure in BSL1, LP3 and HS1 cells. H3K4me3 CUT&RUN revealed 661 differential peaks (FDR < 0.05) comparing OA to control. Motif analysis revealed an overrepresentation of binding sites for SSP TFs ATF3/4 (p < 0.05). After 5 min OA exposure, mitochondrial and nuclear ROS increased significantly (p < 0.01), peaking at 15 min. OA exposure triggered DNA damage likely due to ROS increase in the nucleus. Compass predicted an increase in GSH metabolism and ROS detoxification in BSL1. Conclusions: Protein levels of PHGDH are elevated in 70% of ERneg BCs. This cannot be explained by gene amplification alone as PHGDH gene amplification is observed in only approximately 6% of all breast cancers. This suggests that there are mechanisms other than gene amplification that contribute to PHGDH dysregulation. One of those mechanisms may be the lipid induced metabolic shift toward the SOG and methionine pathways that we have identified. The increased SAM and 2-HG foster epigenetic phenotypic plasticity via altered histone methylation. ROS increase shortly after OA exposure and are controlled by antioxidant defenses (e.g. GSH), which favors the survival of specific cell subtypes with acquired DNA damage which likely facilitates malignant transformation. Citation Format: Mariana Bustamante Eduardo, Curtis W. McCloskey, Gannon Cottone, Shiyu Liu, Flavio R. Palma, Maria Paula Zappia, Abul B.M.M.K. Islam, Jason Locasale, Marcelo G. Bonini, Maxim V. Frolov, Elizaveta V. Benevolenskaya, Rama Khokha, Navdeep S. Chandel, Seema A. Khan, Susan E. Clare. Medium chain fatty acids shift metabolism towards the de novo serine pathway fostering epigenetic plasticity and oxidative DNA damage [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 P1-05-02.

Anti-estrogens have had a limited impact on breast cancer (BC) prevention. Novel agents with better tolerability, and efficacy beyond estrogen receptor (ER) positive BC are needed. We studied licochalcone A (LicA) for ER-agnostic BC prevention. We demonstrated that LicA significantly reduced proliferation in seven human breast cell lines and suppressed ER+ and ER- xenograft tumors in mice. We confirmed these observations ex vivo in the contralateral unaffected breast (CUB) of women with unilateral sporadic BC, and BC cell lines using RNA sequencing, metabolism flux modeling, confirmatory NanoString nCounter metabolic pathway panel analysis in independent sets of specimens, proteomics, and western blots. We found that LicA targets sterol regulatory element binding protein 1 (SREBP1) with subsequent metabolic-inflammatory changes, lowering spatiotemporally resolved cholesterol levels inside malignant cells to the levels in normal mammary cells. Mechanistically, in CUBs we observed that LicA downregulated PI3K-AKT-SREBP1-dependent lipogenesis, NF-kB-dependent inflammation, and de novo nucleotide biosynthesis, stalling proliferation. Studies in cell lines showed suppression of PI3K and AKT phosphorylation, SREBP1 protein expression, and the SREBP1-dependent enzymes such as ACAT2, ACLY, FASN, SCD, consistent with reduced NEDD8 required for SREBP1 stabilization. We found a significant reduction in NF-kB expression, its nuclear translocation mediator karyopherin β1, and prostaglandin E2 synthesis. We demonstrated a reduction in PRPS1-catalyzed de novo nucleotide biosynthesis, and downregulation of proliferative markers MKI67, RRM2, and the survival marker BCL2. LicA reduces pro-tumorigenic aberrations in lipid homeostasis and inflammation through SREBP1. It is a promising non-endocrine candidate for BC prevention. Future studies in immunocompetent BC prevention models are warranted.

Epigenetic changes, particularly DNA methylation, are crucial to breast cancer development. Tumor-adjacent normal (AN) tissue frequently serves as a reference for characterizing genomic alterations but is reported to share some characteristics with tumors. However, it is unclear whether AN’s epigenetic profiles reflect a predisposition to cancer or a response to the presence of the tumor. We address this gap by systematically comparing methylation profiles of tumor, AN, and matched-benign tissues from both breasts, as well as to healthy donated breast tissue. We studied four different sample categories from 69 cancer cases: tumor (TU), AN, ipsilateral opposite quadrant (OQ), and contralateral unaffected breast (CUB); and healthy donated breast (HDB) tissue from 182 cancer-unaffected donors. These constitute a “tumor proximity axis” (TPxA): HDB→CUB→OQ→AN→TU. Methylation profiles were assayed using Illumina’s Infinium Methylation EPICv1.0 BeadChip. Differential methylation (DM) analysis was conducted, and the significantly DM CpGs were analyzed for enrichment of transcription factor binding sites (TFBS) and other features. Following data processing and quality control, there were 69 TU, 60 AN, 67 OQ, 68 CUB, and 182 HDB samples for analysis. DM analysis showed distinct methylation profiles of TU relative to benign tissues, whereas case-benign tissues were similar to each other but distinct from HDB. Hypomethylated sites in case-benign versus HDB were enriched for TF binding sites of TP63, GATA3, ESR1, PR, AR, NR3C1, and GREB1. TU hypermethylation events were enriched for Polycomb-repressive complex 2 (PRC2) binding, including EZH2, SUZ12, and JARID2, with hypermethylation enrichment for PRC2-related binding motifs in both ER + and ER- tumors. TU methylation profiles were otherwise highly distinct by ER status: TFBS enrichment of hypomethylation events for hormone receptor-related pathways in ER + tumors and for hematopoiesis/immune-related pathways in ER- tumors. We found no differential methylation between benign tissues from patients with ER + vs. ER- tumors. DNA methylation profiles differ profoundly at two points: tumor to case-benign and case-benign to HDB, with clear distinction between ER + and ER- tumors. Case-benign tissues are not epigenetically “normal”, are similar across both breasts, and do not differ by ER status of paired tumors.

Introduction: Epigenetic changes play a crucial role in cancer development. Among these, DNA methylation is one of the most significant due to its impact on gene expression and genome stability. Hyper-methylation generally suppresses transcription and hypo-methylation promotes it. In breast cancer research, tumor-adjacent tissue is often used as a reference for characterizing epigenetic alterations, but numerous studies suggest that the tissue adjacent to a tumor shares genomic characteristics with the tumor itself. The extent to which epigenetic profiles overlap between tumor, histologically normal tissues from the involved and uninvolved breast, and benign tissues from cancer-free individuals is unknown. Our study aims to address this gap by systematically comparing the DNA methylation profiles across these different tissue types. Methods: A total of 72 cancer patients were selected from Northwestern University, while data from 182 cancer-free women was obtained from tissue donated to the Komen Tissue Bank. From each individual cancer patient, four regional samples were collected: tumor tissue (TU), tumor-adjacent normal tissue (AN), benign tissue from the opposite quadrant of the involved breast (OQ), and benign tissue from the contralateral uninvolved breast (CUB). From cancer-free patients, normal breast tissue was collected (CFN). From these, we created a “tumor proximity axis”: CFN→CUB →OQ→AN→TU. Methylation profiles were assayed using Illumina’s Infinium Methylation EPIC v1.0 BeadChip. Differential methylation analysis was conducted in two sets of four pairwise comparisons: (1) comparing tumor samples to the four benign tissue categories, and (2) comparing tissue categories that are adjacent along the tumor proximity axis. The differentially methylated CpGs were analyzed for enrichment of transcription factor binding sites (TFBS) and genes. Results: Following data processing and quality control, there were 72 TU, 63 AN, 70 OQ, 72 CUB, and 182 CFN samples for analysis. Differential methylation analysis showed that TU samples had distinct methylation profiles, with more hypomethylation events compared to hypermethylation events relative to benign tissues. Case-benign tissues (AN, OQ, CUB) exhibited similar methylation profiles, distinct from CFN. Transcription factor binding site (TFBS) enrichment analysis revealed that case-benign samples, even those distant from tumor, i.e. CUB, showed breast cancer-related methylation changes. Hypomethylated sites in CUB compared to CFN were enriched for TF binding sites TP63, GATA3, ESR1, PR, AR, NR3C1, and GREB1. TU hypermethylation events were enriched for Polycomb-repressive complex 2 (PRC2) binding, including EZH2, SUZ12, and JARID2. ER+ and ER- tumors had distinct methylation profiles. Both ER+ and ER- tumors showed hypermethylation enrichment for PRC2-related binding motifs. However, hypomethylation events differed, with ER+ tumors enriched for hormone receptor-related pathways and ER- tumors enriched for hematopoiesis/immune-related pathways. We did not find any differential methylation between benign tissues from patients with ER+ vs. ER- tumors. Conclusions: DNA methylation changes profoundly at two points on the tumor proximity axis: CFN to CUB and AN to TU. Specifically, all case-benign tissues, including CUB, exhibited changes associated with breast cancer when compared to CFN. Although the methylation profiles of ER+ and ER- tumors differed, benign tissues showed no differences as a function of ER status of the tumor. These findings demonstrate that benign case tissue is not epigenetically “normal” and provide insights for further investigation into the process of tumorigenesis. Citation Format: Saya Dennis, Takahiro Tsukioki, Gannon Cottone, Wanding Zhou, Yuan Luo, Patricia A. Ganz, Mary E. Sehl, Seema Khan, Susan Clare. DNA methylation patterns are similar in benign tissue from ipsilateral and contralateral breast while different from matched breast cancer, and healthy controls [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 RF1-05.

Breast cancer (BC) risk reducing drugs have minimal impact on cancer incidence due to their adverse effects and low acceptance by risk-eligible women. Further, they are ineffective against hormone receptor negative (HR-) BC. Novel non-endocrine agents with reduced toxicity and efficacy not limited to HR+ BC are needed. Sterol regulatory element binding protein 1 (SREBP1) has been shown to have a key role in tumorigenesis in the breast through reprogramming immune and microenvironmental factors, epithelial to mesenchymal transition, cell cycle, and programmed cell death. It has a prognostic value in breast cancer patients and is an independent factor of 5-year overall and disease specific survival. Targeting this metabolic vulnerability presents opportunities to reverse tumorigenesis in several tissues such as breast and endometrium. We recently presented data which revealed that in the microstructures from the contralateral unaffected breast (CUB) of postmenopausal women with unilateral BC, LicA significantly upregulated antioxidant pathways and downregulated NF-kB dependent inflammatory pathways as well as SREBP1-dependent lipogenesis. We also showed that metabolic flux in the CUBs treated with LicA supports these effects. In addition, LicA exhibited suppression of proliferation in pre-malignant and malignant HR+ and HR- breast cell lines and in their xenografts in mice. Now we present confirmatory evidence that the mechanism through which LicA prevents HR+ and HR- BC is by reducing SREBP1-dependent metabolism and inflammation. We also show the promising PK profiles of novel oral formulations we have developed. We performed NanoString metabolism panel analysis in MDA-MB-231 (HR-) and MCF-7 (HR+) breast cancer cells treated with LicA (5 µM) for 24 h. We also performed western blots to confirm the significantly modulated metabolic pathways. In addition, we evaluated spatiotemporal changes in the concentrations of cholesterol in the inner leaflet of plasma membrane of these cells. Four proprietary novel formulations of LicA were developed and administered orally to female BALB/c mice and Sprague-Dawley rats followed by LC-MS/MS analysis of the plasma and mammary tissue. Using SAAM II we modeled their pharmacokinetic (PK) profiles. We observed significant (adj P < 0.05) upregulation of antioxidant (up to 9-fold), and downregulation (up to 6-fold) of NF-kB-dependent inflammatory pathways such as prostaglandin E2 synthesis. These results were consistent with our previous observations in animal models and in the high-risk women’s CUBs. We also observed significant downregulation of SREBP1 dependent lipogenesis genes such as ACAT2, FASN, and SCD in these cells, which is in line with our data in the CUBs. Our western blot analysis showed a significant suppression of SREBP1 (4-fold) particularly in HR- cells, along with the reduced phosphorylation of PI3K (5-fold) and AKT (6-fold, on Ser 473). These results were confirmed by the depletion of cholesterol in the inner leaflet of plasma membrane in HR- (8-fold) and HR+ (4-fold) cells. Consistent with our data from the CUBs and in vivo models, we observed 2- to 3-fold reduction in proliferative markers such as MKI67, BCL2, and RRM2 in HR+ and HR- cells after 24 h exposure to LicA. These results provided additional evidence that LicA exerts its antiproliferative effects by suppressing SREBP1-dependent lipogenesis and inflammation. Two of the novel LicA oral formulations showed promising PK, sufficient for efficacy with less frequent dosing which make them suitable for BC prevention. Our data suggests that suppression of SREBP1 dependent metabolism and inflammation can prevent BC and presents evidence that LicA is an excellent candidate for HR+ and HR- BC prevention through this mechanism. We will test LicA’s oral formulation in intraductal models of precancer lesions in immunocompetent animals to further establish its preventive efficacy. Citation Format: Atieh Hajirahimkhan, Elizabeth T Bartom, Carolina H Chung, Xingyu Guo, Kyli Berkley, Ruohui Chen, Wonhwa Cho, Sriram Chandrasekaran, Susan E Clare, Seema A Khan. Reprogramming SREBP1-dependent metabolism and inflammation in high-risk breast to prevent cancer: the example of licochalcone A [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 P1-03-12.

A lipid metabolism gene signature is enriched in breast tissue at risk for estrogen receptor negative (ERneg) breast cancer (BC). Fatty acid (FA) exposure alters histone methylation, gene expression and increases metabolic flux through serine, one-carbon, glycine (SOG) and methionine pathways. We hypothesize that FA exposure induces a metabolic shift towards the SOG, increasing S-adenosylmethionine (SAM), altering histone methylation, gene expression, and promoting ERneg BC. Proteomics, metabolomics, Reactive Oxygen Species (ROS) measurement, comet assay and H3K4me3 CUT&RUN were performed in MCF-10A cells exposed to octanoic acid (OA). Single-cell RNA-seq (scRNAseq) was performed in breast tissue derived microstructures exposed to OA. Intracellular communication was analyzed using CellChat, and metabolic flux with Compass. OA increased SAM, glutathione (GSH) and 2-hydroxyglutarate (2-HG); blocking the serine pathway (SSP) prevented these increases. ScRNAseq revealed that OA increased expression of the SSP transcription factor ATF3 and genes PHGDH and PSAT1 in epithelial and stromal compartments. Metabolic flux analysis revealed a significant increase in flux through SSP in Basal BSL1, Luminal Progenitor LP3, and Hormone Sensing HS1 cells after OA exposure. Differential proteomics reveals PHGDH overexpression and downregulation of proteins involved in extracellular matrix (ECM)-receptor interaction and focal adhesion post-OA exposure, along with significant increase in mitochondrial and nuclear ROS (p < 0.01). OA exposure also induced DNA damage, likely due to elevated nuclear ROS. OA increased GSH metabolism and ROS detoxification in BSL1. CUT&RUN identified 661 peaks significantly enriched upon OA (FDR < 0.01) in regulatory regions of OA-induced genes involved in neural pathways and BC, including MDK, NGF, and NGFR. CellChat predicted a decrease in ECM-cell interactions, a reduction in cell-cell adhesions, and an increase of secreted signaling upon OA exposure. The strongest secreted signals in OA were AREG (linked to proliferation, growth, and invasiveness), GDF15 (involved in EMT, invasion, and aging), and MDK (linked to neurogenesis, and aging). We demonstrate an FA-induced shift towards the SOG and methionine pathways that promotes epigenetic plasticity, regulates ROS, and supports the survival of cells with 'inappropriate' phenotypes. These accumulate DNA damage, leading to age-related changes in the mammary gland (elevated ROS, disrupted junctions, altered ECM interactions, and increased MDK/GDF15 expression), all supporting carcinogenesis. Our findings also provide a metabolic explanation for the elevation of PHGDH in 70% of ERneg BCs, despite gene amplification in only 6%, and point to preventive strategies targeting the SSP. Mariana Bustamante Eduardo, Gannon Cottone, Curtis McCloskey, Flavio Palma, Shiyu Liu, Maria Paula Zappia, Abul B.M.M.K. Islam, Elizaveta Benevolenskaya, Maxim Frolov, Jason Locasale, Marcelo Bonini, Rama Khokha, Navdeep Chandel, Seema A. Khan, Susan E. Clare. Metabolic shift towards the de novo serine pathway in non-transformed breast cells drives epigenetic plasticity, oxidative DNA damage, and pro-tumorigenic cChanges associated with aging [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 5402.

Abstract Background: Breast cancer risk reducing drugs with proven efficacy have adverse side effects, significantly minimizing their uptake and impact. Further, they do not prevent ER- breast cancer. Effective alternative strategies with lower toxicity are needed. Previously, we have shown that licochalcone A (LicA) suppresses aromatase expression and activity, enhances the activity of detoxifying enzymes, and reduces estrogen genotoxic metabolism in cell lines and animal models. These data led us to hypothesize that LicA creates a tumor preventive environment in the breast by reprogramming metabolism and antioxidant/anti-inflammatory responses in the breast leading to decreased proliferation and tumor suppression. We now report on the breast tumor preventive effects of LicA in xenograft models, its oral bioavailability, and its biologic effects on human breast microstructures from women at increased risk of breast cancer. Methods: We prepared microstructures from the fresh tissue of contralateral unaffected mastectomy specimens of 6 postmenopausal women with incident unilateral breast cancer. After exposing them to DMSO (control) or LicA (5 µM), we performed total RNA sequencing. Differentially expressed genes were identified and analyzed by gene ontology and pathway membership. The RNA-seq data was also utilized to conduct metabolism flux analysis. Combined enrichment scores > 4 and FDR < 0.05 was considered significant. The NanoString metabolism panel was employed in 6 additional subjects. We performed live cell imaging to monitor proliferation of pre-malignant DCIS.COM, DCIS.COM/ER+ PR+; and malignant MDA-MB-231 (ER- PR-), MCF-7 (ER+ PR+), MCF-7aro, and BRCA1 defective HCC-1937, and HCC3153 cells. Xenograft models of MCF-7aro and MDA-MB-231 tumors were established in female nude mice and the animals treated for 28 days with vehicle or LicA (80 mg/kg.day, s.c.). We measured the rate of tumor growth. We also conducted a PK/PD study with oral LicA (100 mg/kg) in intact BALB/c female mice. Results: We observed significant (FDR < 0.05) upregulation of antioxidant genes (up to 8-fold), consistent with upregulation of NRF2 and the thioredoxin system, the major regulators of antioxidant pathways. This was accompanied by significant downregulation of RELA- and NF-kB1-dependent inflammatory pathways. In addition, we observed decreased expression of PI3K-AKT genes and the pro-adipogenic transcription factors SREBF1 and SREBF2, which may explain the downregulation (4 to 32-fold) of cholesterol biosynthesis and transport, and lipid metabolism genes. Metabolism studies confirmed these data and demonstrated a robust increase in the pentose phosphate shunt and NAD(P)H generation without enhancing ribose 5 phosphate formation, suggesting an antioxidant and anti-proliferative environment. LicA also suppressed proliferation of pre-malignant and malignant cells, with sustained effects on aggressive cells at doses < 10 µM. LicA significantly reduced tumor growth in luminal (P = 0.008) and triple negative (P = 0.001) in vivo models (unpaired t-test with Welch’s correction for unequal variances). Promising serum and breast bioavailability, equivalent to low micromolar concentrations sufficient to show efficacy was demonstrated as well. Conclusion: Our data suggest that LicA is a good candidate for breast cancer prevention in both ER+ and ER- breast cancers through reprogramming metabolism and antioxidant pathways leading to decreased proliferation. We will study LicA in intraductal models of ER+ and ER- precancer lesions in immunocompetent mice and will monitor their progression to invasive breast cancer to further establish its preventive efficacy. Citation Format: Atieh Hajirahimkhan, Elizabeth Bartom, Sriram Chandrasekaran, Susan Clare, Seema Khan. Licochalcone A as a risk reducing agent against luminal and non-luminal breast cancers [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 PO3-08-07.

Abstract Breast cancer (BC) risk reducing drugs have minimal impact due to their adverse effects and low acceptance by risk-eligible women. Further, they are ineffective against hormone receptor negative (HR-) BC. Novel agents with reduced toxicity and sufficient efficacy that extends beyond HR+ BC are needed. We showed previously that licochalcone A (LicA) from licorice has antioxidant/anti-inflammatory effects, inhibits aromatase, and blocks estrogen genotoxic metabolism. We hypothesize that LicA prevents HR+ and HR- BC through reprogramming metabolism and antioxidant pathways. We prepared microstructures from the fresh tissue of contralateral unaffected breast of 6 postmenopausal women with unilateral BC. After exposing them to DMSO (control) or LicA (5 µM), we performed total RNA sequencing followed by differential gene expression, pathway identification, and metabolism flux analyses. The NanoString metabolism panel was employed in 6 additional subjects. We performed live cell imaging to monitor proliferation of HR- and HR+ pre-malignant and malignant, as well as BRCA mutated cells. Xenograft models of HR+ and HR- tumors were established in female nude mice followed by 28-day treatment with vehicle or LicA (80 mg/kg.day, s.c.) and monitoring the tumor growth. Using LC-MS/MS we measured LicA in the blood and various tissues of intact BALB/c female mice receiving oral LicA (100 mg/kg) and assessed PK using Phoenix Platform. We observed significant (combined enrichment scores > 4 and FDR < 0.05) upregulation of antioxidant genes (up to 8-fold), consistent with upregulation of NRF2 and the thioredoxin system. This was accompanied by significant downregulation of RELA- and NF-kB1-dependent inflammatory pathways. In addition, we observed decreased expression of PI3K-AKT genes and the pro-adipogenic transcription factors SREBF1 and SREBF2, which may explain the downregulation (4 to 32-fold) of cholesterol biosynthesis, transport, and lipid metabolism genes. Metabolism studies confirmed these data and demonstrated a robust increase in the pentose phosphate shunt and NAD(P)H generation without enhancing ribose 5 phosphate formation, suggesting an antioxidant and antiproliferative environment. LicA also suppressed proliferation of pre-malignant and malignant cells, with sustained effects on aggressive cell lines at doses <10 µM. It reduced tumor growth in luminal (P = 0.008) and triple negative (P = 0.001) xenograft models. Oral bioavailability in serum and breast was in the low micromolar range and was sufficient to show efficacy.Our data suggest that LicA is an excellent candidate for both HR+ and HR- BC prevention by reprogramming metabolism and antioxidant pathways leading to decreased proliferation. Our next study will test an optimized oral formulation of LicA in intraductal models of HR+ and HR- precancer lesions in immunocompetent mice to further establish its preventive efficacy. Citation Format: Atieh Hajirahimkhan, Elizabeth T. Bartom, Sriram Chandrasekaran, Ruohui Chen, Jeremy J. Johnson, Susan E. Clare, Seema A. Khan. Licochalcone A is an excellent candidate for preventing luminal and non-luminal breast cancers [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 7306.

Introduction:The rapid development of artificial intelligence (AI) in healthcare has exposed the unmet need for growing a multidisciplinary workforce that can collaborate effectively in the learning health systems. Maximizing the synergy among multiple teams is critical for Collaborative AI in Healthcare. Methods:We have developed a series of data, tools, and educational resources for cultivating the next generation of multidisciplinary workforce for Collaborative AI in Healthcare. We built bulk-natural language processing pipelines to extract structured information from clinical notes and stored them in common data models. We developed multimodal AI/machine learning (ML) tools and tutorials to enrich the toolbox of the multidisciplinary workforce to analyze multimodal healthcare data. We have created a fertile ground to cross-pollinate clinicians and AI scientists and train the next generation of AI health workforce to collaborate effectively. Results:Our work has democratized access to unstructured health information, AI/ML tools and resources for healthcare, and collaborative education resources. From 2017 to 2022, this has enabled studies in multiple clinical specialties resulting in 68 peer-reviewed publications. In 2022, our cross-discipline efforts converged and institutionalized into the Center for Collaborative AI in Healthcare. Conclusions:Our Collaborative AI in Healthcare initiatives has created valuable educational and practical resources. They have enabled more clinicians, scientists, and hospital administrators to successfully apply AI methods in their daily research and practice, develop closer collaborations, and advanced the institution-level learning health system.

A lipid metabolism gene signature is associated with the risk of estrogen negative breast cancer (ER-BC). In vitro, lipid exposure alters histone methylation affecting gene expression and increasing flux through various metabolic reactions; but little is known about the mechanism(s) linking lipids and epigenetic reprogramming with the genesis of ER-BC. Here we show that the metabolism of the medium-chain fatty acid Octanoic Acid (OA) in preference to glucose and glutamine results in a metabolic shift toward the serine pathway increasing the production of SAM, glutathione, and 2-HG, with implications for oncogenesis: SAM production results in epigenetic fostered plasticity leading to reprogramming/selecting cells that express Neural, EMT and BC related genes. 2-HG exposure results in appearance of DNA breaks, potentially consequent to the inhibition of essential demethylases for HR repair. ROS increases shortly after OA exposure and is mitigated by antioxidant defenses, which favors/enables the survival of specific cell subtypes.### Competing Interest StatementThe authors have declared no competing interest.

Abstract Introduction. Oncogenic factors that are local/in-breast are of great interest as they may be more specifically targetable for breast cancer prevention than systemic factors. We have identified a lipid metabolism gene signature that is enriched in breast tissue at risk for estrogen negative breast cancer (ER- BC). Utilizing the medium chain fatty acid Octanoic acid (OA) to probe lipid metabolism in non-transformed breast epithelial cells, we observed increased flux through several metabolic reactions and altered histone methylation with consequent changes in gene expression (e.g. neural genes). Neuronal signaling and regulatory circuits are observed in cancer cells of multiple origins, not just ones with ontological relationships to neurons. We hypothesize that the first and rate limiting step in the de novo serine pathway, which is catalyzed by Phosphoglycerate Dehydrogenase (PHGDH) is key to these observations. In the forward direction, PHGDH participates in the serine, one-carbon, glycine (SOG) and methionine pathways that produce the methyl donor S-adenosylmethionine (SAM) and in the reverse direction produces the oncometabolite 2-Hydroxyglycerate (2-HG). Methods. Non-transformed MCF-10A cells exposed to OA were utilized for U13C-glucose tracing. SAM and 2-HG concentrations following treatment with OA ± PHGDH inhibitor were measured by liquid chromatography. CUT&RUN for H3K4me3 was performed and genes affected by OA (PMID: 28263391) were compared with OA-responsive peaks. Single cell RNA-sequencing was carried out using breast microstructures derived from reduction mammoplasty tissue exposed to vehicle or OA. Microstructures were dissociated into single cells and sequenced using the 10x Genomics platform. The digital expression matrix file containing UMIs were analyzed with Seurat. Alkaline comet assay was performed to detect DNA breaks. Results. U13C-glucose tracing in presence of OA revealed that one-carbon-THF was redirected to the methionine cycle increasing flux to methylation. Concentrations of SAM and 2-HG increased after 15- and 30-min OA exposure, respectively; PHGDH inhibitor blocked these increases. H3K4me3 CUT&RUN revealed 661 differential peaks (FDR < 0.05) comparing OA to control. 73% of H3K4me3 OA-associated peaks were in regulatory regions of OA-induced genes (FDR < 0.01), these genes are involved in neural pathways, EMT and ER- BC. Motif analysis revealed an overrepresentation of binding sites for transcription factors ATF3/4 (p < 0.05), which are regulators of the serine pathway. Single cell RNA-sequencing revealed OA not only affected the distribution of cell subpopulations but also modulated the expression of many genes within each subcluster. The percentage of luminal progenitor subcluster 3 increased upon OA from less than 1% to about 13%. Within basal subcluster 3, OA drives the expression of ATF3, along with two of the enzymes in the de novo serine pathway: PHGDH and PSAT1. Alkaline comet assay showed DNA breaks in OA- and control 2-HG- treated cells. Conclusions. Metabolism of OA in preference to glucose and glutamine results in a metabolic shift toward the serine pathway increasing the production of SAM and 2-HG, with implications for oncogenesis: 1. SAM production results in epigenetic fostered plasticity leading to reprogramming/selecting cells that express genes consistent with a neural/neural crest-like state. These co-opted neuronal regulatory mechanisms can make critical contributions to the acquired functional capabilities that drive cancer development. 2. 2-HG exposure results in appearance of DNA breaks, which are likely consequent to the inhibition of the alpha-ketoglutarate-dependent dioxygenases KDM 4A/B by 2-HG. Their catalytic activity is required for homologous recombination repair; inhibition results in metabolic “BRCAness”. Citation Format: Mariana Bustamante Eduardo, Gannon Cottone, Shiyu Liu, Maria Paula Zappia, Elizaveta V. Benevolenskaya, Abul Bashar Mir Md. Khademul Islam, Maxim V. Frolov, Seema Khan, Susan Clare. Metabolic shift to serine pathway induced by lipids confers oncogenic properties in non-transformed breast cells [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 PO5-08-06.

<p>Supplemental table 2. Differential gene expression and pathway enrichment analysis in placebo responders (excel file) -uploading separately.</p>

Supplemental Figure 3. Validation of RNA seq results of responders by Nanostring nCounter gene expression assay

Supplementary Data 2 from Progression-Specific Genes Identified by Expression Profiling of Matched Ductal Carcinomas In situ and Invasive Breast Tumors, Combining Laser Capture Microdissection and Oligonucleotide Microarray Analysis

<p>Supplemental Figure 1. Validation of RNA seq results of responders by Nanostring nCounter gene expression assay.</p>