Breast Cancer Prevention Research Articles

A single-cell atlas enables mapping of homeostatic cellular shifts in the adult human breast

Here we use single-cell RNA sequencing to compile a human breast cell atlas assembled from 55 donors that had undergone reduction mammoplasties or risk reduction mastectomies. From more than 800,000 cells we identified 41 cell subclusters across the epithelial, immune and stromal compartments. The contribution of these different clusters varied according to the natural history of the tissue. Age, parity and germline mutations, known to modulate the risk of developing breast cancer, affected the homeostatic cellular state of the breast in different ways. We found that immune cells from BRCA1 or BRCA2 carriers had a distinct gene expression signature indicative of potential immune exhaustion, which was validated by immunohistochemistry. This suggests that immune-escape mechanisms could manifest in non-cancerous tissues very early during tumor initiation. This atlas is a rich resource that can be used to inform novel approaches for early detection and prevention of breast cancer.

Antiprogestins for breast cancer treatment: We are almost ready

The development of antiprogestins was initially a gynecological purpose. However, since mifepristone was developed, its application for breast cancer treatment was immediately proposed. Later, new compounds with lower antiglucocorticoid and antiandrogenic effects were developed to be applied to different pathologies, including breast cancer. We describe herein the studies performed in the breast cancer field with special focus on those reported in recent years, ranging from preclinical biological models to those carried out in patients. We highlight the potential use of antiprogestins in breast cancer prevention in women with BRCA1 mutations, and their use for breast cancer treatment, emphasizing the need to elucidate which patients will respond. In this sense, the PR isoform ratio has emerged as a possible tool to predict antiprogestin responsiveness. The effects of combined treatments of antiprogestins together with other drugs currently used in the clinic, such as tamoxifen, CDK4/CDK6 inhibitors or pembrolizumab in preclinical models is discussed since it is in this scenario that antiprogestins will be probably introduced. Finally, we explain how transcriptomic or proteomic studies, that were carried out in different luminal breast cancer models and in breast cancer samples that responded or were predicted to respond to the antiprogestin therapy, show a decrease in proliferative pathways. Deregulated pathways intrinsic of each model are discussed, as well as how these analyses may contribute to a better understanding of the mechanisms involved.

Bibliometric Analysis of the Relationship Between Breast Cancer and Exercise Based on Citespace

This study aims to conduct a bibliometric analysis of the literature on the relationship between breast cancer and exercise using Citespace software, in order to explore the research hotspots, trends, and deficiencies in this field. Relevant literature including keywords such as “breast cancer” and “exercise” was retrieved from the Web of Science database and analyzed using Citespace software for data processing and visualization. The main conclusions of this study are as follows: In co-occurrence analysis of keywords, researchers mainly focus on the prevention, rehabilitation, and treatment of breast cancer, as well as the effects of different types of exercise on breast cancer. Meanwhile, the cooccurrence patterns of keywords in studies related to exercise and breast cancer indicate an increasing attention to the relationship between breast cancer and exercise, making it gradually one of the hotspots in breast cancer research. The timeline of keywords and burst detection analysis show that researchers have increasingly focused on the relationship between breast cancer and exercise in recent years, with research intensity showing an increasing trend year by year. Furthermore, in recent years, researchers have begun to emphasize the role of exercise in breast cancer rehabilitation, rather than just prevention and treatment. Discipline analysis indicates that research in this field involves multiple disciplinary areas, including medicine, oncology, sports science, nutrition, etc. Additionally, an analysis of papers published by country reveals that the United States leads in research on the relationship between breast cancer and exercise, followed closely by the United Kingdom, China, Canada, Australia, and other countries. Conclusion: Literature on the relationship between breast cancer and exercise shows a trend of increasing quantity year by year. However, there are still some deficiencies in this field, such as lack of uniform research methods, overly limited research samples, and insufficient rigor in research results. Therefore, further strengthening research on the relationship between breast cancer and exercise is highly necessary.

Fenretinide in Young Women at Genetic or Familial Risk of Breast Cancer: A Placebo-Controlled Biomarker Trial.

Fenretinide exhibits beneficial effects on the metabolic profile, supporting its clinical use in breast cancer prevention especially in premenopausal women with a positive family history and pathogenic variants in BRCA1/2 genes. This finding requires further investigations in larger trials to confirm its role in breast cancer prevention.

The comparison of effectivity in breast cancer prevention between skin sparing and subcutaneous mastectomy - 20 years of experience.

Breast cancer is the leading cause of neoplasm mortality among women. Several prevention strategies have been implemented to early detect and prevent the cancer occurrence. The most effective protocol includes prevention mastectomy for the high-risk patients. In our study, we have compared the efficacy of subcutaneous mastectomy (SCM) and skin sparing mastectomy (SSM) in long-term follow up. We have included 201 female patients who have been treated at our department over the course of 20 years between 2000 and 2019. All the patients were at high risk of developing breast cancer and therefore were indicated for the prophylactic mastectomy. The main indication was the presence of the mutation in the BRCA1 or BRCA2 cluster, however, even in the lack of such mutation, the family history was sufficient for the mastectomy indication. Patients underwent either SCM, SSM or areola sparing mastectomy (ASM), and were allocated to aforementioned groups, respectively. We have collected the data regarding the reconstruction method along with age, weight, height, body mass index (BMI) and presence of predisposing genetic mutations such as BRCA positivity. The patients who underwent SSM compared to those who underwent SCM were of higher age, with higher BMI and body mass. The patients in SSM group had statistically significantly higher BMI than in ASM. There was no difference in efficacy between patients who underwent SSM and SCM. The majority of patients (91.5%) were positive for BRCA1 or BRCA2 mutation. In our study, only four patients were tested negative for known breast cancer inducing mutation (three in SCM and one in SSM). The most common reconstruction method was an abdominal flap and breast implant. Prophylactic mastectomy is a reliable strategy for significantly reducing the number of breast cancer incidence in high-risk patients regardless of the selected method of mastectomy. These operations allow for the subsequent reconstruction with the whole spectrum of reconstructive options.

Abstract 7311: Lipid soluble vitamin B1 derivatives, benfotiamine and fursultiamine, prevent breast cancer cells growth

Abstract Breast cancer is the leading cause of cancer death in women in the US and the second leading cancer after skin cancer in women worldwide. According to the CDC, women from 45-84 years of age are most at risk, with approximately 30% of deaths in this group being from cancer. Increased free radical formation, and oxidative and inflammatory responses have been shown to be major contributing factors for the progression of breast cancer. Several antioxidants and vitamins have been tested for their efficacy in preventing breast cancer. However, the role of vitamin B1 derivatives in the prevention of breast cancer is not known. Therefore, we hypothesize that with potent anti-oxidative and anti-inflammatory actions, lipid-soluble derivatives of vitamin B1 (thiamine), such as benfotiamine and fursultiamine, could prevent breast cancer growth and spread. We have treated human breast cancer cells such as MCF-7 and T-4D1 and mouse breast cancer cells 4T1 with vitamin B1 derivatives and examined the cancer cells growth. Our results indicate that treatment of various breast cancer cells with benfotiamine and fursultiamine prevents the proliferation of cancer cells in a dose-dependent manner. Further, both benfotiamine and fursultiamine by regulating the activation of caspase-3 and cleavage of PARP protein could prevent breast cancer cells growth. Benfotiamine and fursultiamine also regulates generation of reactive oxygen species in breast cancer cells. Further, vitamin B1 derivatives regulate the expression of various anti-apoptotic, pro-apoptotic, survival and inflammatory factors in both human and mouse breast cancer cells. We next planned to examine the effect of vitamin B1 derivatives on breast cancer growth in vivo using athymic nude mouse models. In conclusion, our in vitro results suggest that vitamin B1 derivatives, benfotiamine and fursultiamine, by promoting the apoptotic pathways prevent breast cancer cells growth and could be further developed as chemopreventive drugs. Citation Format: Caroline Ciaciuch O'Neill, Aveline P. Langmead, Kota V. Ramana. Lipid soluble vitamin B1 derivatives, benfotiamine and fursultiamine, prevent breast cancer cells growth [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 7311.

Abstract 4773: Interferon regulatory factor 8 (IRF8) as a biomarker for early detection and prevention of axillary lymph node-positive breast cancer

Abstract Breast cancer (BC) remains a leading health concern worldwide and is often undetected until the cancer metastasizes. Early detection can limit the need for aggressive treatments and improve prognosis. We analyzed early immune and molecular markers in benign breast disease (BBD) biopsies prior to BC development to identify women at elevated risk of developing node-positive invasive BC, which might direct surveillance and prevention efforts aimed at reducing incidence of this disease. Utilizing the Mayo Clinic BBD cohort, we compared women who developed node-positive BC following their BBD biopsy (cases; n=42) with those who remained cancer-free (controls; n=37), matched on patient age and biopsy date. We used NanoString nCounter system to identify differentially expressed genes (DEGs) between cases and controls. We also developed a multiplex immunofluorescence (mIF) approach using Opal system by Akoya Biosciences for comprehensive marker detection in FFPE tissue, enabling correlation of cells expressing DEGs with innate and adaptive immune cells. Digitized images were segmented, and cell phenotyping, and spatial relationships were compared between cases and controls. Our findings revealed significantly increased expression levels of IRF8 (interferon regulatory factor 8, a factor involved in immune cell differentiation) in controls as compared to cases and found that increased IRF8 expression in cases is correlated with delayed cancer onset. Additionally, controls exhibited higher frequencies of CD4+, CD8+, CD68+, CD20+ and CD11c+ immune cells, and lower frequency of Ki67+ staining, with a notable increase of CD11c+/CD68+ cells, a marker for M1 type macrophages, indicating a pro-inflammatory, antitumorigenic immune microenvironment polarization. Furthermore, our mIF analyses offer new insights into spatial biomarker localization, enhancing prognostic accuracy. Our findings suggest that decreased numbers of immune cells in BBD biopsies may be associated with increased risk of developing axillary node-positive BC. This research underscores the importance of dissecting molecular and immune interactions in BBD for assessing the risk of this disease. Our results have the potential to improve individualized risk assessment, leading to more targeted surveillance and informed screening, potentially reducing BC incidence and mortality through early intervention. This study not only contributes to our understanding of BC pathogenesis but also opens avenues for developing novel preventive strategies. Citation Format: Matilde Rossi, Nicole Cruz-Reyes, Melody L. Stallings Mann, Bryan M. McCauley, Tanya L. Hoskin, Robert A. Vierkant, Stacey J. Winham, Amy C. Degnim, Mark E. Sherman, Derek C. Radisky. Interferon regulatory factor 8 (IRF8) as a biomarker for early detection and prevention of axillary lymph node-positive breast cancer [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 4773.

Abstract 2179: Microbiome-derived metabolites mediate carcinogenic alterations of breast tissue in the context of obesity

Abstract Obesity increases the relative risk for breast cancer incidence. Multiple molecular mechanisms linked to obesity drive breast cancer progression. Obesity shifts the gut microbiome in ways that may increase breast cancer risk. Microbial-associated molecular pattern (MAMP)-proteins and metabolites could directly affect breast epithelial cell signaling. In this study, we focused on lipopolysaccharide (LPS), a structural component of the outer membranes of gram-negative bacteria and a toll-like receptor 4 (TLR4)-agonist. Systemic LPS levels are known to increase in obesity. We hypothesize that chronic low-grade inflammation caused by LPS contributes to breast cancer initiation. To test this hypothesis, we first quantified levels of LPS, along with a panel of adipokines/cytokines, in serum samples from women (n= 33; BMI range= 18.3-45.3 Kg/m2; Age range= 18-51 years). Women with elevated serum LPS had significantly higher BMI, leptin, and leptin/adiponectin ratio, confirming a link between systemic LPS and metabolic markers of obesity. Other biomarkers of breast cancer risk include DNA damage. Analyses of non-cancerous breast tissue sections from matched donors revealed higher densities of DNA double-strand breaks (estimated based on 53BP1 foci counts) in women with high serum LPS. Experiments with HMT-3522 S1 breast acini showed that LPS increases DNA break frequencies and oxidative stress levels in 3D structures of mammary epithelium. Moreover, LPS increases oxidative lesions in the DNA (estimated based on 8-oxoguanine fluorescence in MCF-10A cells) that may lead to increased LPS-mediated DNA damage. Patient obesity status was associated with alterations in oxidative modifications in the breast proteome. We also found that LPS disrupts epithelial polarity, a hallmark of breast tissue homeostasis. In mammary epithelium, LPS activates the nuclear factor-kappa B (NFκB) pathway by binding to the TLR4 receptor, leading to an increased expression of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α). Interestingly, TLR4 blockade prevented the loss of apical polarity and DNA damage induced by LPS. Last, but not least, we show an association between LPS immunogenicity (anti-LPS IgA concentrations) and participant BMI in non-cancerous breast tissue samples. These novel findings show that LPS is a key systemic and local mediator of the obesity-modulated microbiome that increases breast cancer risk. The outcomes of our study underscore the importance of considering the microbiome in the prevention of breast cancer. Citation Format: Mohamed Gaber, Kayla Neary, Julia Holmes, Adam S. Wilson, Adam J. Katz, Pierre-Alexandre Vidi, Katherine L. Cook. Microbiome-derived metabolites mediate carcinogenic alterations of breast tissue in the context of obesity [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 2179.

Guidelines for clinical diagnosis and treatment of advanced triple negative breast cancer in China（2024 edition）

Breast cancer ranks as the most common female malignancy worldwide. Data from GLOBOCAN 2020 showed that breast cancer surpassed lung cancer and become the leading malignancy globally which was a serious threat to women's health. Different from Caucasian, there is a lower prevalence of breast cancer in Chinese women. But the age-standardized incidence rate of breast cancer in China has reached to 39.1 per 100,000 in 2020, with 416,000 new cases, accounting for 18.4% of global breast cancer burden, due to multiple factors such as lifestyle and dietary habits changes in recent years[1]. The treatment and prevention of breast cancer are becoming increasingly a tuff task. In addition, triple-negative breast cancer (TNBC) is a subtype with higher malignancy and worse prognosis. Due to lack of specific therapeutic targets, less treatment progress has been made in recent years. There is even less progress in the therapy of advanced TNBC, but huge unmet needs exist for further therapeutic optimization. Echoing advocacy from The Society of Breast Cancer China Anti-Cancer Association; International Medical Exchange Society, Chinese Anti-Cancer Association; Breast Cancer Group, Branch of Oncologist, Chinese Medical Doctor Association, top experts from multidisciplinary departments of breast cancer in China have consensus on this "Guidelines for Clinical Diagnosis and Treatment of Advanced Triple-Negative Breast Cancer in China (2024 Edition)" according to the latest evidence based updates. We hope this pater can guide or optimize the precise therapeutic decision-making for advanced triple-negative breast cancer in China.

Abstract 3429: Using metabolomics to identify biomarkers mediating the associations of adiposity in childhood and early adulthood with mammographic breast density in premenopausal women

Abstract Introduction: Mammographic breast density (MBD), a strong predictor of breast cancer risk, is highly influenced by body mass index (BMI) in childhood and early adulthood, but the mechanisms elucidating this relationship are undetermined. To our knowledge, no study has explored the mediating role of biomarkers on the relationship between early-life adiposity and MBD. The goal of this study is to discover metabolites that mediate the relationship between BMI at ages 10 and 18 with MBD in premenopausal women. Methods: This study includes premenopausal women who had their screening mammogram at Washington University in St. Louis, MO, and provided a fasting blood sample. Metabolon performed untargeted metabolomic profiling, detecting 1,074 metabolites. Metabolites missing >300 observations were excluded, leaving 828 metabolites; the other missing values were imputed using the nearest neighbor method. To mitigate batch effect, we normalized the metabolite peak area data using ComBat. Volumetric percent density (VPD) was calculated in 700 women using Volpara software. BMI at age 10 was estimated using the Stunkard pictogram, and BMI at age 18 was calculated from self-reported weight at 18 and height at study initiation. To assess the mediating role of the 828 metabolites, we performed high dimensional mediation analysis using the hima R package adjusting for potential confounders. Missing values in the covariates and BMI measures included in the mediation analysis were imputed using multivariate imputation by chain equations. Associations were considered significant if FDR p-value <0.1. Results: Four metabolites (glutamate, beta-cryptoxanthin, phytanate, and cortolone glucuronide (1)) mediated the relationship between BMI at age 10 and VPD; and 2 metabolites (glutamate, beta-cryptoxanthin) mediated the relationship between BMI at age 18 and VPD. Glutamate and beta-cryptoxanthin significantly mediated the relationship between both BMI measures and VPD, but glutamate was the strongest mediator across both time points. Glutamate mediated 6.7% (FDR p-value=0.06) and 9.3% (FDR p-value=0.008) of the total effect between BMI at age 10 and 18, respectively, on VPD. Beta-cryptoxanthin mediated 4.1% (FDR p-value=0.06) and 6.3% (FDR p-value=0.04), of the total effect between BMI at age 10 and 18, respectively, on VPD. Additionally, phytanate mediated 2.6% (FDR p-value=0.06) and cortolone glucuronide (1) mediated 2.0% (FDR p-value=0.07) of the total effect between BMI at age 10 and VPD. Conclusion: Amino acid, lipid, cofactor/vitamin, and xenobiotic metabolites mediated the associations of BMI in early-life and VPD in premenopausal women. This innovative study offers insight into the biological mechanisms underlying the link between early-life adiposity and MBD, which can support future research into breast cancer prevention. Citation Format: Kayla R. Getz, Myung Sik Jeon, Lili Liu, Lei Liu, Chongliang Luo, Jingqin Luo, Adetunji T. Toriola. Using metabolomics to identify biomarkers mediating the associations of adiposity in childhood and early adulthood with mammographic breast density in premenopausal women [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 3429.

Abstract 828: Associations of perfluoroalkyl substances with breast cancer incidence risk and mediation role of mitochondrial DNA copy number: A prospective case-cohort study

Abstract Background: Perfluorinated carboxylic acids (PFCAs) and perfluorinated sulfonic acids (PFSAs), notable representatives of perfluoroalkyl substances (PFASs), are synthetic persistent chemicals widely accumulated and detected in humans. Short-chain PFASs [PFCAs with carbon number <8 and PFSAs <6, e.g. perfluoroheptanoic acid (PFHpA)] are considered less bioaccumulative and increasingly used in commercial products, yet their health impacts are rarely investigated. Perfluorooctanoic acid (PFOA) has been categorized as a possible human carcinogen (group 2B) and a potential mammary toxicant. However, few population studies explored the prospective associations of PFOA and other common PFASs with breast cancer risk nor the underlying mechanisms. Methods: Hence, we conducted a case-cohort study within the DongFeng-Tongji Cohort, compromising 226 incident breast cancer cases and a random sub-cohort of 990 individuals. Plasma concentrations of four PFCAs [PFOA, perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), and PFHpA] and two PFSAs [perfluorooctane sulfonic acid (PFOS) and perfluorohexane sulfonic acid (PFHxS)], as well as peripheral blood mitochondrial DNA copy number (mtDNAcn) levels, were measured at baseline. Results: Using Barlow-weighted Cox regression, we found 35% and 20% increased incident risk of breast cancer for each 1-unit ln-transformed increase in PFOA and PFHpA, respectively [HR (95% CI) = 1.35 (1.03, 1.78) and 1.20 (1.02, 1.40)]. Quantile g-computation analysis revealed a 19% increased incident risk of breast cancer along with each simultaneous quartile increase in all ln-transformed PFCA concentrations [HR (95% CI) = 1.19 (1.01, 1.41)], driven mainly by contributions from PFOA (56%), PFNA (24%), and PFHpA (20%). Besides, mtDNAcn was positively associated with PFHpA levels [β (95% CI) =0.027 (0.005, 0.048)], but not with PFOA or PFNA (P>0.05). We also found significantly higher breast cancer risk in participants with the highest mtDNAcn (Q4, ≥1.52) than those with the lowest (Q1, <0.87) [HR (95% CI) =2.92 (1.69, 5.04)], and that mtDNAcn mediated 12.77% of PFHpA-breast cancer association [Indirect effect, HR (95% CI) =1.02 (1.00, 1.04)]. No significant mediation effect of mtDNAcn was observed in associations of PFOA and PFNA with breast cancer (P>0.05). Conclusions: Our work unveiled the relationship of short-chain PFHpA with increased incident risk of breast cancer for the first time and highlighted the intermediate role of mtDNAcn in this association, providing a research clue for exploring the potential mechanisms of PFAS-induced breast cancer. Moreover, co-exposure to PFASs was observed as a risk factor for breast cancer, shedding light on breast cancer prevention by regulating PFASs as a chemical class. Citation Format: Yue Feng, Huan Guo. Associations of perfluoroalkyl substances with breast cancer incidence risk and mediation role of mitochondrial DNA copy number: A prospective case-cohort study [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 828.

Abstract 3332: Kalkitoxin thiol amide suppresses the breast cancer metastasis to bone and brain

Abstract Introduction: Breast cancer is the primary frequently diagnosed cancer worldwide, with a high fatality rate. Metastasis resulting from advanced breast cancer increases mortality in patients. MDA-MB-231 is triple-negative breast cancer that does not have any of the receptors that are commonly found in breast cancer. The lipopeptide toxin kalkitoxin thiol amide (KTA), generated from the marine cyanobacterium Moorena producens (formerly Lyngbya majuscule), has a wide range of biological and pharmacological effects. Its promise as a therapeutic agent for treating bone metastases from breast cancer is still uncertain. This study aimed to determine whether KTA could prevent breast cancer from metastasis to mice's bones and to elucidate any potential mechanisms causing this inhibition. Experimental Procedures: MDA-MB-231 cells were transfected with pGL4.50 (Luc2/CMV/Hygro) vector using LipofectamineTM 2000. Positive clones of MDA-MB-231 cells were selected after treatment with 300 µg/ml hygromycin for one week. After cancer implantation to the heart, bioluminescent imaging of femurs was measured upto 5 weeks and signal intensity of KTA-treated groups was compared with the vehicle group. Summary: KTA decreased breast cancer cell proliferation, migration, and invasion. KTA prevented MDA-MB-231 cells from breast cancer metastasizing to the bone and brain via inhibiting MAPK and STAT signaling. The epithelial-mesenchymal transition markers, which are responsible for distant metastasis, are highly opposed by KTA. Compared to mice in the control group, mice receiving KTA displayed considerably less bone and brain metastasis, a lower tumor burden, and less bone destruction in the murine breast cancer bone metastasis model. Conclusion: KTA has new inhibitory effects on cancer cell invasion and migration both in vitro and in vivo. From these findings, KTA may have therapeutic promise for treating breast cancer patients with distant metastases. Citation Format: Yunjo Soh, Saroj Kumar Shrestha, Se woong Kim. Kalkitoxin thiol amide suppresses the breast cancer metastasis to bone and brain [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 3332.

Abstract 849: Deciphering the link between aging, lobular involution, and breast cancer risk: The role of LIFR/pSTAT3 signaling

Abstract Background: Age-related lobular involution (ARLI), characterized by regression of breast epithelial tissue during the peri-and post-menopausal period, plays a crucial role in breast cancer risk. Delayed or incomplete ARLI is linked with increased postmenopausal breast cancer susceptibility.This study focuses on unraveling the molecular mechanisms of ARLI, drawing parallels with the well-characterized postpartum involution (PPI). Our aim was to discover biomarkers differentiating women who progressed through ARLI from those with stagnant ARLI. Methods: We utilized the Mayo Clinic Benign Breast Disease (BBD) cohort, analyzing 108 perimenopausal women aged 45-55 who had a subsequent benign biopsy between 2-15 years later. We assessed Lobular Involution (Ll) status by comparing the number and size of lobules in initial and subsequent biopsies. RNA profiling of initial biopsies was performed using NanoString 10360 and BC360, identifying differentially expressed genes between ARLI progression and stagnation. Immunohistochemistry (IHC) mapped the expression of these biomarkers in epithelial lobules. Results: We identified 37 genes with significant differential expression, highlighting Leukemia Inhibitory Factor Receptor (LIFR) as upregulated in ARLI stagnant samples. LIFR, part of the IL-6 cytokine signaling pathway, is known for its role in the induction of apoptosis during PPI via pSTAT3 activation. Our IHC analyses of initial biopsies of women who differentially progress through ARLI confirmed differential expression of LIFR that is coordinately regulated with pSTAT3. Conclusion: Our findings reveal significant parallels between ARLI and PPI. Future research will assess how LIFR/pSTAT3 signaling is linked to induction of apoptotic markers in perimenopausal women and will investigate how anomalies in the LIFR/pSTAT3 pathway might contribute to heightened breast cancer risk in women with delayed or incomplete ARLI. This study not only sheds light on the complex biological processes of aging in breast tissue but also opens new avenues for breast cancer risk assessment and prevention strategies. Citation Format: Jaida C. Lue, Derek Radisky, Melody Stallings-Mann, Mark Sherman, Amy Degnim, Bryan McCauley, Tanya Hoskin. Deciphering the link between aging, lobular involution, and breast cancer risk: The role of LIFR/pSTAT3 signaling [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 849.

Abstract 1072: The EsomiR project: A case control study to assess the role of exosomal miRNAs in breast cancer cancerogenesis

Abstract Introduction: One of the unmet needs in Breast Cancer (BC) prevention is how to identify women at higher risk of developing this disease, who might benefit from periodic surveillance and/or from chemoprevention. The goal of this retrospective study is to assess the role miRNAs isolated from extracellular vesicles (EV-miRNAs) as possible early biomarkers for identifying patients with higher risk of developing BC, in order to personalize their screening programme. To this goal, EV-miRNAs from cyst fluid derived from women with Gross Cyst Disease of the Breast (GCDB - a benign disease of the mammary gland associated with a 2-4-fold increase in the risk of developing BC) have been analysed. Methodology: Cyst fluids have been selected among samples from a cohort of 600 patients diagnosed with GCDB between 1985 and 1993, some of whom developed BC during follow-up. EV-miRNAs have been extracted from cyst fluid using the exoRNeasy midi kit (Qiagen), qualitatively evaluated by Bioanalyzer (Agilent) and quantified by Qubit™ using the microRNA Assay Kit (Thermo Fisher Scientific). The MiRNome profiles have been assessed by microarray using an optimised protocol on Agilent platform (labelling and hybridization on SurePrint Human miR Microarrays 8 × 60 K and images acquisition by G2565CA scanner). Results: A total of 117 samples (58 cases and 59 controls, paired on the bases of clinical and pathological variables) have been analysed. Single variable logistic regression analysis on the most expressed EV-miRNAs (logFC >= 6 (n=329)), selected six EV-miRNAs (miR-6076, miR-202-3p, miR-6872-3p, miR-769-3p, miR-5195-3p, miR-4443) and one (miR-4713-3p) negatively and positively associated with BC development, respectively (raw p-value <0.05). A risk score combining the seven EV-miRNAs was derived by multivariable logistic regression modelling and reported an AUC=0.73 (95% [CI] 0.6427-0.8231). The model was refined including clinical variables and applying step-wise logistic regression analysis. The combination of 3 EV-miRNAs (miR-6076, miR-6872-3p, miR-5195-3p) with menopausal status, familiarity and type of cyst yielded an AUC of 0.8 (95% [CI] 0.7015-0.8847). Logistic regression was also performed on Type I cysts (n=78), that are correlated with a higher risk of developing BC, pointing in evidence 3 EV-miRNAs with a negative (miR-6076, miR-6872-3p) or positive (miR-4515) correlation with BC development risk (AUC = 0.74, 95% [CI] 0.6278-0.8475). In the model comprehensive of clinical variables, two of those EV-miRNAs (miR-6872-3p, miR-4515) together with familiarity and menopausal status were selected, giving an AUC of 0.8 (95% [CI] 0.6961-0.8991). Conclusion: This study allowed the identification of a EV-miRNA-based BC risk signature obtained by liquid biopsy on cyst fluid. The final purpose of this project is to transfer this signature in the clinic by testing it prospectively on plasma samples. Citation Format: Barbara Cardinali, Patrizia Piccioli, Francesco Boccardo, Alessandra Rubagotti, Linda Zinoli, Andrea Sciutto, Simona Coco, Giovanna Chiorino, Paola Ostano, Emir Sehovic, Zita Cavalieri, Cristina Bruzzo, Silvia Marconi, Roberta Tasso, Rodolfo Quarto, Davide Ceresa, Paolo Malatesta, Lucia Del Mastro. The EsomiR project: A case control study to assess the role of exosomal miRNAs in breast cancer cancerogenesis [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 1072.

Abstract 1054: Reciprocal circRNA-miRNA expression axes indicative of early tumor initiation in breast

Abstract Cancer prevention and early detection are the most effective tools for cancer control. In this study, we adopt a novel approach of identifying a network of proteins/mRNA-circRNA-miRNA axes. We hypothesize that mutation of oncogenes or tumor suppressor genes during pre-oncogenic stage result in aligned change in levels of circular RNAs-derived genes, as well as, reciprocally, in corresponding sponged miRNAs. These epigenetic risk predictive proteins/mRNA-circRNA-miRNA axes serve as potential tool for early detection of changes leading to breast cancer (BC) and can be further used as non-invasive screening biomarkers of patients’ blood in populations susceptible to cancer. BC patients in GDC Data Portal, TCGA-BRCA project are selected based on several criteria such as their age at diagnosis (20-50 years old), early clinical stage of cancer (stage I) and disease type (ductal and lobular neoplasms). Differentially expressed genes are acquired from mRNA sequencing data by comparing Stage I Breast cancer versus normal primary tissues using R Studio. Then, mRNAs whose expression is aligned with the differentially expressed circRNAs and their host genes acquired from Gene Expression Omnibus (GEO) (GSE182471, Triple Negative Breast cancer tissues versus normal tissues) are selected. Differentially expressed miRNAs, in reciprocal expression with circRNAs, are defined using miRNA sequencing data of the same previously chosen TCGA BRCA patients. Selected miRNAs are hypothesized to be sponged with high confidence by selected circRNAs based on a context percentile binding score in CircInteractome. Using KM Plotter, the survival analysis of corresponding miRNAs in grade I BC patients is examined. The predicted miRNA expression was correlated with lower survival rates among stage I BC patients in KM Plotter. MiRNA-enrichment analysis is used to further validate the involvement of selected miRNAs in early tumorigenic events through exploring in which molecular pathways their downstream genes are enriched. We have constructed more than 20 potential proteins/mRNA-circRNA-miRNA axes for non-invasive screening of patients’ body fluid (i.e. Blood) in populations susceptible to BC using RNA and miRNA sequencing data available in online databases and further validate the proposed axes using in-silico tools. The described axes, once validated in clinical samples could provide a potential tool for early-stage BC prediction and prevention. Citation Format: Nour Abbas Maatouk, Sarah Hussein Marei, Abdallah Bilal Kurdi, Nadim Shady Hamade, Rihab Raif Nasr, Rabih Shakib Talhouk. Reciprocal circRNA-miRNA expression axes indicative of early tumor initiation in breast [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 1054.

Abstract 778: Atorvastatin modulates serum biomarkers in women at increased risk for breast cancer

Abstract Background Tamoxifen and Aromatase inhibitors have been shown to be highly effective at reducing the incidence of ER-positive breast cancers in women at increased risk, but there are no approved agents for the risk reduction of ER-negative cancers. Evidence from observational and preclinical studies supports the study of statins for breast cancer risk reduction. Objective This study aimed to investigate the effects of Atorvastatin treatment on serum and tissue biomarkers in women at increased risk for breast cancer. Methods Women at increased risk for breast cancer were accrued prospectively at UTMDACC, Breast Medical Oncology High-Risk Clinic. The study was approved by the IRB and all participants signed informed consent. High risk was defined as having a previous history of DCIS, LCIS, or atypical hyperplasia, 5-year projected Gail risk >1.67 %, or lifetime risk >20 % calculated by clinical models. Patients were randomized to no treatment (n=15) or daily 10mg (n=15), 20mg (n=14), or 40mg (n=16) Atorvastatin for 3 months. Fasting blood and breast tissue samples via fine needle aspiration (FNA) were collected at baseline and at study completion. Serum biomarkers were analyzed with ELISA (R&D Systems). The IGF1/IGFBP3 molar ratio was calculated using [IGF1 (ng/mL) x 0.13]/[IGFBP-3 (ng/mL) x 0.035]. The Wilcoxon matched-pairs signed rank test was used to determine significance. Results Tissue biomarker evaluation was previously reported. Serum biomarker levels of IGF1 and its main binding proteins IGFBP1 and IGFBP3 were compared in baseline and post-intervention samples by ELISA analysis. The average change in serum IGF1 (ng/mL) levels between pre- and post-treatment samples (represented as average difference with interquartile range) was +3.5 (-16.8 to 8.7) p=0.98, -0.6 (-23.6 to 15.4) p=0.64, -1.76 (-27.7 to 28.6) p=0.81, and +2.6 (-14.5 to 32.2) p=0.67 in the 0, 10, 20, and 40mg groups respectively. The average change in serum IGFBP1 (pg/mL) was -23.2 (-2372 to 4487) p=0.33, -480 (-4567 to 3008) p=0.72, 429 (-1259 to 3039) p=0.54, and -250 (-3581 to 2492) p=0.94 in the 0, 10, 20, and 40mg groups. The average change in serum IGFBP3 (ng/mL) was -24 (-193 to 98) p=0.72, -119 (-353 to 111) p=0.17, -190 (-433 to 14) p=0.07, and -234 (-369 to -79) p=0.002 in the 0, 10, 20, and 40mg groups. Serum IGF1/IGFBP3 molar ratio was +0.012 (-0.018 to 0.026) p=,0.76, +0.0065 (-0.013, to 0.047) p=0.53, +.0073 (-0.036 to 0.045) p=0.86, and +.027 (-.011 to .062) p=0.058 in the 0, 10, 20, and 40mg groups. Conclusions Serum IGFBP3, which has been associated with breast cancer risk, was decreased in a dose-dependent manner in patients treated with Atorvastatin. The IGF1/IGFBP3 molar ratio was increased in the 40mg group, although not statistically significant (p=.0577). This shows that treatment with Atorvastatin induced changes in circulating biomarkers and that Atorvastatin could be further studied in larger prospective breast cancer prevention trials. Citation Format: Amanda L. Lanier, Johannes Fahrmann, Fatma N. Mustafayev, Angelica Gutierrez Barrera, Powel H. Brown, Banu Arun. Atorvastatin modulates serum biomarkers in women at increased risk for breast cancer [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 778.

Abstract 1784: Glucose metabolism and de novo palmitate synthesis under normoxia and hypoxia in breast cancer cells that preferentially metastasize to lung and liver

Abstract Cancer cells are exposed to variable oxygen and nutrient availability during the metastatic process which can influence metabolic adaptations of cancer cells to metastasize to distant sites. Thus, we investigated the metabolic adaptations of breast cancer cells that preferentially metastasize to lung (metM-WntLung cells; MLg) and liver (metM-WntLiver cells; MLr) in normoxia and hypoxia and at different glucose concentrations. In normoxia, 14C-glucose uptake is similar, but mRNA abundance of hexokinase, the initial rate-limiting step in glycolysis, was 22% higher in MLg cells. This is consistent with higher 13C6-glucose flux into glycolytic metabolites pyruvate (M+3) and lactate (M+3) in MLg compared to MLr. Also, de novo palmitate synthesis from 13C6-glucose was 7.6% higher in MLg and was accompanied by higher mRNA expression of ATP-citrate lyase (ACLY), which converts citrate to acetyl-CoA for fatty acid synthesis. These suggest greater glucose metabolism in MLg compared to MLr in normoxia, consistent with reduced viability of MLr by 39% in high glucose (25 mM) concentrations. Further, mRNA abundance of CPT1A, a rate-limiting step in fatty acid oxidation, is 23% higher in MLg. Moreover, the fatty acid oxidation inhibition (etomoxir) reduced viability of MLg by 26.7% compared to MLr, suggesting that fatty acid synthesis and oxidation are critical for MLg. Protein expression of HIF1α, a transcription factor that induces glycolysis during hypoxia, is 1.5-fold higher in MLr than MLg in normoxia, consistent with higher MLr mRNA abundance of pyruvate dehydrogenase kinase (PDK1), a target of HIF1α that inhibits pyruvate dehydrogenase (PDH) activity. Surprisingly, mRNA abundance of pyruvate carboxylase (PC), the enzyme that converts pyruvate to oxaloacetate, is also higher in MLr (39%) than MLg, thus supporting a potential alternative mechanism for glucose metabolites to enter the TCA cycle in normoxia in MLr. In hypoxia, viability of MLr, relative to MLg, increased by 11% at high glucose concentration. This was accompanied by higher hexokinase (33%) and PDK1 (83%) mRNA abundance suggesting MLr’s shift towards a glycolytic phenotype. However, de novo palmitate synthesis from 13C6-glucose is higher in MLg, suggesting that palmitate synthesis is maintained in MLg even in hypoxia. Altogether, results demonstrate that glucose utilization shifts in breast cancer cells that preferentially metastasize to lung vs liver, but de novo fatty acid synthesis is higher in MLg in different oxygen conditions. Thus, targeting breast cancer cells’ adaptation to glucose and de novo fatty acid synthesis may be a potential strategy to prevent breast cancer progression to distant organs. Citation Format: Marjorie Anne A. Layosa, Morgan Conrad, Stephen Hursting, Dorothy Teegarden. Glucose metabolism and de novo palmitate synthesis under normoxia and hypoxia in breast cancer cells that preferentially metastasize to lung and liver [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 1784.

Abstract 1091: Distinct mechanisms of orthotopic tumor control by alphavbeta3and HER2 CAR Ts support development of a bispecific CAR T cell therapy for pediatric rhabdomyosarcoma

Abstract Goal: Evaluate αvβ3 and HER2 as antigens for a bispecific chimeric antigen receptor T (CAR T) cell therapy in pediatric rhabdomyosarcoma. Background: Current treatments are ineffective against relapsed/refractory or metastatic rhabdomyosarcoma (RMS). Antigen loss or modulation on tumor cells commonly leads to tumor escape across diverse cancer types after CAR T therapy targeting different antigens. We hypothesize that simultaneously targeting two RMS antigens with a bispecific CAR T will reduce the likelihood of tumor escape. The integrin alphavbeta3 (αvβ3) plays a role in cancer progression, metastasis and neovascularization in several malignancies. HER2 also contributes to cancer progression and metastasis. Methods: Control of intramuscular RMS tumors by αvβ3 or HER2 CAR Ts was assessed in NSG mice, with non-specific CD19 CAR Ts as controls. Tumor burden was assessed weekly by bioluminescence imaging. Persistence and phenotype of CAR Ts were evaluated by flow cytometry. CAR T cell infiltration, tumor antigen expression and residual disease were determined by immunohistochemistry. Results: Six of six pediatric patient-derived RMS cell lines express αvβ3 and/or HER2. αvβ3 and HER2 CAR Ts kill RMS cells in vitro at low effector-to-target ratios. αvβ3 and HER2 CAR Ts independently control—and nearly eliminate—orthotopic RMS tumors. Fewer HER2 CAR Ts (5 × 106) are needed to control tumors than αvβ3 CAR Ts (10 × 106), but this is not due to lack of persistence or exhaustion of αvβ3 CAR Ts. Tumor control by αvβ3 CAR Ts is delayed (>21 days) compared to that by HER2 CAR Ts (7 days), and is robust, despite initial tumor progression following treatment. Importantly, despite strong tumor control by both CAR Ts, at 7 weeks post-treatment residual tumor nests are evident by histology. At this time, CAR Ts are still abundant, are not exhausted and express memory markers, suggesting that the remaining tumor cells are no longer susceptible to CAR T killing. We are currently evaluating the hypothesis that the remaining tumor cells have down-regulated antigen expression. Delayed tumor control by αvβ3 CAR Ts was ameliorated by engineering αvβ3 overexpression in RMS tumors. Conclusions: αvβ3 and HER2 CAR Ts control orthotopic RMS tumors by distinct mechanisms with changes in αvβ3 antigen expression, likely due to tumor progression, one prevalent factor. This is consistent with our prior reports of αvβ3 CAR Ts preventing breast cancer metastases, which naturally upregulate αvβ3 compared to the primary tumor. Despite near elimination of tumors, residual nests of tumor cells are likely to rebound as CAR T resistant tumors, similar to what is seen in patients treated with monospecific CAR Ts. These data support the incorporation of αvβ3 and HER2 CAR Ts into a bispecific CAR T approach to mitigate antigen escape. Our data also support investigation into the role of αvβ3 in RMS. Citation Format: Amanda M. Lulu, Nagyeong Park, Joseph J. Caruso, Dustin A. Cobb, Daniel W. Lee. Distinct mechanisms of orthotopic tumor control by alphavbeta3and HER2 CAR Ts support development of a bispecific CAR T cell therapy for pediatric rhabdomyosarcoma [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 1091.

Abstract 781: Impact of vitamin D and pyruvate carboxylase on hypoxia inducible lipid droplet associated (HILPDA) protein in metastatic breast cancer cells

Abstract Metastatic breast cancer, a critical health issue for women globally, is closely linked to aberrant lipid metabolism. One feature of this dysregulated metabolism is the increase in lipid storage in the form of cytoplasmic lipid droplets (CLDs), although what drives their accumulation or their role in metastasis is not clear. In prior studies, we demonstrated that the active form of vitamin D (1,25-dihydroxyvitamin D, 1,25(OH)2D) downregulates pyruvate carboxylase (PC), a key metabolic enzyme, thereby reducing lipid accumulation in metastatic breast cancer cells (MCF10CA1a). Additionally, we have demonstrated that both 1,25(OH)2D treatment and PC-inhibition reduce breast cancer cell migration. Therefore, the goal of the current research is to identify the proteins associated with CLDs in metastatic MCF10CA1a breast cancer cells following 1,25(OH)2D treatment or reduction in PC expression (shPC). Proteins from CLDs and whole cell lysates (WCL) were processed for untargeted proteomic analysis. In the CLD fractions, 1562 identified enriched proteins were common to both MCF10CA1a and 1,25(OH)2D treated cells, while 160 proteins were higher in the MCF10CA1a cells, and 550 proteins were higher in the 1,25(OH)2D-treated cells compared to MCF10CA1a CLD fraction. In the CLD fractions, MCF10CA1a and shPC cells had a similar level of 380 enriched proteins, although 65 were higher in the shPC compared with MCF10CA1a, whereas 1268 were more enriched in the MCF10CA1a CLD fraction. Hypoxia Inducible Lipid Droplet Associated (HILPDA, known to inhibit triacylglycerol lipolysis) was only enriched in the CLD fraction of the metastatic MCF10CA1a cells. HILPDA was not identified in the CLDs of the 1,25(OH)2D treated or the shPC cells, nor in any WCL fraction. Further investigation of HILPDA is important to determine if HILPDA plays a role in dysregulated cellular metabolism, including CLD accumulation, or migration, and may potentially be a target for future therapies to prevent breast cancer progression. Citation Format: Yazhen Song, Chaylen Andolino, Dorothy Teegarden. Impact of vitamin D and pyruvate carboxylase on hypoxia inducible lipid droplet associated (HILPDA) protein in metastatic breast cancer cells [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 781.

Abstract 2134: Disaggregating breast cancer mortality trends in Asian American women from 2005-2020

Abstract Background: Breast cancer (BC) is the second leading cause of death among women and has disproportionately higher rates of mortality in certain racial groups, including Asian Americans (AA). While differences in BC mortality between AA and other racial groups have been identified, few studies have disaggregated nationwide AA mortality data. Methods: Using National Vital Statistics System mortality data, BC-related deaths were analyzed among AA groups (Asian Indian, Chinese, Filipina, Japanese, Korean, Vietnamese), Native Hawiian and Pacific Islanders (NHPIs), and non-Hispanic Whites (NHWs) from 2005-2020. We calculated cancer proportional mortality ratios (PMRs), age-adjusted mortality rates (AAMRs), standard mortality ratios, and used JoinPoint regression for average annual percentage change (AAPC). Results: Among 13677 AA, 522 NHPI, 501282 NHW descendants, 14.3% cancer deaths were due to BC. PMRs increased for all racial groups except Filipina. AAMRs significantly decreased in NHWs (AAPC -1.61; CI -1.70 to -1.48) while significantly increasing for aggregate AAs (AAPC 0.45; CI 0.08 to 0.94). Among AAs, Filipinas had the highest AAMR. AAMRs significantly increased for Asian Indians (AAPC 2.05; CI 1.20 to 3.46), Filipinas (AAPC 0.61; CI 0.15 to 1.46), Koreans (AAPC 2.72; CI 1.57 to 4.65) and Vietnamese women (AAPC 3.63; CI 2.26 to 6.20), but significantly decreased for Japanese (AAPC -1.41; CI -2.22 to -0.53). Conclusions: We observed heterogenous and converging BC mortality trends across AA groups. These trends elucidate which populations within the broader AA group have disproportionately higher death rates and therefore should be targeted for BC prevention protocols (e.g., Asian Indian and Filipina). Identifying such trends can also help elucidate disparities in cancer care and the overall social determinants that affect women with BC. Understanding disaggregated trends can inform more inclusive screening practices and culturally tailored interventions and treatments to reduce BC deaths in vulnerable populations. Citation Format: Mia Marcotte, Yashi Chauhan, Vedha Penmetcha, Caroline Thompson, Candice Thompson, Malathi Srinivasan, Gloria Kim, Xinran Qi, Robert Huang, Adrian Bacong. Disaggregating breast cancer mortality trends in Asian American women from 2005-2020 [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 2134.