ER-negative Breast Cancer Research Articles

The Clinical and Biological Significance of Estrogen Receptor-Low Positive Breast Cancer

Estrogen receptor (ER) status in breast cancer (BC) is determined using immunohistochemistry (IHC) with nuclear expression in ≥1% of cells defined as ER-positive. BC with 1%-9% expression (ER-low-positive), is a clinically and biologically unique subgroup. In this study, we hypothesized that ER-low-positive BC represents a heterogeneous group with a mixture of ER-positive and ER-negative tumor, which may explain their divergent clinical behavior. A large BC cohort (n = 8171) was investigated and categorized into 3 groups: ER-low-positive (1%-9%), ER-positive (≥10%), and ER-negative (<1%) where clinicopathological and outcome characteristics were compared. A subset of ER-low-positive cases was further evaluated using IHC, RNAscope, and RT-qPCR. PAM50 subtyping and ESR1 mRNA expression levels were assessed in ER-low-positive cases within The Cancer Genome Atlas data set. The reliability of image analysis software in assessment of ER expression in the ER-low-positive category was also assessed. ER-low-positive tumors constituted <2% of BC cases examined and showed significant clinicopathological similarity to ER-negative tumors. Most of these tumors were nonluminal types showing low ESR1 mRNA expression. Further validation of ER status revealed that 45% of these tumors were ER-negative with repeated IHC staining and confirmed by RNAscope and RT-qPCR. ER-low-positive tumors diagnosed on needle core biopsy were enriched with false-positive ER staining. BCs with 10% ER behaved similar to ER-positive, rather than ER-negative or low-positive BCs. Moderate concordance was found in assessment of ER-low-positive tumors, and this was not improved by image analysis. Routinely diagnosed ER-low-positive BC includes a proportion of ER-negative cases. We recommend repeat testing of BC showing 1%-9% ER expression and using a cutoff ≥10% expression to define ER positivity to help better inform treatment decisions.

Identification of PIMREG as a novel prognostic signature in breast cancer via integrated bioinformatics analysis and experimental validation.

Phosphatidylinositol binding clathrin assembly protein interacting mitotic regulator (PIMREG) expression is upregulated in a variety of cancers. However, its potential role in breast cancer (BC) remains uncertain. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were used to gather relevant information. The expression of PIMREG and its clinical implication in BC were assessed by using Wilcoxon rank-sum test. The prognostic value of PIMREG in BC was evaluated through the Cox regression model and nomogram, and visualized by Kaplan-Meier survival curves. Genes/proteins that interact with PIMREG in BC were also identified through GeneMANIA and MaxLink. Gene set enrichment analysis (GSEA) was then performed. The correlations of the immune cell infiltration and immune checkpoints with the expression of PIMREG in BC were explored via TIMER, TISIDB, and GEPIA. Potential drugs that interact with PIMREG in BC were explored via Q-omic. The siRNA transfection, CCK-8, and transwell migration assay were conducted to explore the function of PIMREG in cell proliferation and migration. PIMREG expression was significantly higher in infiltrating ductal carcinoma, estrogen receptor negative BC, and progestin receptor negative BC. High expression of PIMREG was associated with poor overall survival, disease-specific survival, and progression-free interval. A nomogram based on PIMREG was developed with a satisfactory prognostic value. PIMREG also had a high diagnostic ability, with an area under the curve of 0.940. Its correlations with several immunomodulators were also observed. Immune checkpoint CTLA-4 was significantly positively associated with PIMREG. HDAC2 was found as a potentially critical link between PIMREG and BRCA1/2. In addition, PIMREG knockdown could inhibit cell proliferation and migration in BC. The high expression of PIMREG is associated with poor prognosis and immune checkpoints in BC. HDAC2 may be a critical link between PIMREG and BRCA1/2, potentially a therapeutic target.

Small change – big consequence: The impact of C15-C16 double bond in a D‑ring of estrone on estrogen receptor activity

Estrogen receptor alpha (ER) is a key biomarker for breast cancer, and the presence or absence of ER in breast and other hormone-dependent cancers decides treatment regimens and patient prognosis. ER is activated after ligand binding - typically by steroid. 2682 steroid compounds were used in a molecular docking study to identify novel ligands for ER and to predict compounds that may show anticancer activity. The effect of the most promising compounds was determined by a novel luciferase reporter assay. Two compounds, 7 and 12, showing ER inhibitory activity comparable to clinical inhibitors such as tamoxifen or fulvestrant were selected. We propose that the inhibitory effect of compounds 7 and 12 on ER is related to the presence of a double bond in their D-ring, which may protect against ER activation by reducing the electron density of the keto group, or may undergo metabolism leading to an active compound. Western blotting revealed that compound 12 decreased the level of ER in the breast cancer cell line MCF7, which was associated with reduced expression of both isoforms of the progesterone receptor, a well-known downstream target of ER. However, compound 12 has a different mechanism of action from fulvestrant. Furthermore, we found that compound 12 interferes with mitochondrial functions, probably by disrupting the electron transport chain, leading to induction of the intrinsic apoptotic pathway even in ER-negative breast cancer cells. In conclusion, the combination of computational and experimental methods shown here represents a rapid approach to determine the activity of compounds towards ER. Our data will not only contribute to research focused on the regulation of ER activity but may also be useful for the further development of novel steroid receptor-targeted drugs applicable in clinical practice.

Molecular features of androgen-receptor low, estrogen receptor-negative breast cancers in the Carolina breast cancer study

PurposeAndrogen receptor (AR) expression is absent in 40–90% of estrogen receptor (ER)-negative breast cancers. The prognostic value of AR in ER-negative patients and therapeutic targets for patients absent in AR remains poorly explored.MethodsWe used an RNA-based multigene classifier to identify AR-low and AR-high ER-negative participants in the Carolina Breast Cancer Study (CBCS; N = 669) and The Cancer Genome Atlas (TCGA; N = 237). We compared AR-defined subgroups by demographics, tumor characteristics, and established molecular signatures [PAM50 risk of recurrence (ROR), homologous recombination deficiency (HRD), and immune response].ResultsAR-low tumors were more prevalent among younger (RFD = + 10%, 95% CI = 4% to 16%) participants in CBCS and were associated with HER2 negativity (RFD = − 35%, 95% CI = − 44% to − 26%), higher grade (RFD = + 17%, 95% CI = 8% to 26%), and higher risk of recurrence scores (RFD = + 22%, 95% CI = 16.1% to 28%), with similar results in TCGA. The AR-low subgroup was strongly associated with HRD in CBCS (RFD = + 33.3%, 95% CI = 23.8% to 43.2%) and TCGA (RFD = + 41.5%, 95% CI = 34.0% to 48.6%). In CBCS, AR-low tumors had high adaptive immune marker expression.ConclusionMultigene, RNA-based low AR expression is associated with aggressive disease characteristics as well as DNA repair defects and immune phenotypes, suggesting plausible precision therapies for AR-low, ER-negative patients.

Montmorillonite an efficient oral nanocarrier for punicalagin-rich pomegranate peel extract: An in vitro study

Pomegranate (Punica granatum L.) bioactive phenolics have multiple beneficial effects on human health and play a protective role against many degenerative diseases. Pomegranate peel is rich in bioactive phenolics, mainly punicalagin and ellagic acid, which cannot be uptaken by human intestinal epithelial cells (enterocytes) efficiently. This study is aimed to improve the cellular uptake of the bioactive phenolics using Food and Drug Administration-approved mucoadhesive montmorillonite (Mt) as a nanocarrier. Pomegranate peel extract (PPE) soluble in water (W-PPE, rich in punicalagin), methanol (M-PPE, rich in ellagic acid), and 75% aqueous methanol (WM-PPE) was prepared by maceration method, and corresponding PPE/Mt composites were made by intercalation technique. The thermogravimetry, X-Ray diffraction and Fourier transform infrared spectroscopy data demonstrated that the compounds of all the extracts were selectively adsorbed on the interlayer surfaces of Mt, leading to a 1.8- to 2.7-fold increase in their thermal stability and an increase and decrease in the basal space and interlayer water of Mt, respectively. Cell assays using MCF-7 (estrogen receptor-positive breast cancer), MDA-MB-231 (estrogen receptor-negative breast cancer), HepG2 (liver cancer) and HT-29 (colon cancer) cells showed that after intercalation into Mt, the cellular uptake of punicalagin-rich W-PPE and WM/PPE, but not ellagic acid-rich M-PPE, was considerably improved especially in enterocyte-like HT-29 cells. High performance liquid chromatography data also confirmed that Mt effectively adsorbed and carried hydrophilic phenolics, mainly punicalagin, but not ellagic acid. The adsorbed phenolics were released from Mt in a controlled manner which was fitted to the Bhaskar equation and parabolic diffusion kinetic models with R2 values of 0.995, implying that the release mechanism was diffusion and the diffusion through the particle was the rate limiting step. These results suggest that Mt can be an efficient oral nanocarrier for poorly absorbed punicalagin-rich W-PPE as a multifunctional traditional medicine.

Spectrum and Frequency of Germline FANCM Protein-Truncating Variants in 44,803 European Female Breast Cancer Cases.

FANCM germline protein truncating variants (PTVs) are moderate-risk factors for ER-negative breast cancer. We previously described the spectrum of FANCM PTVs in 114 European breast cancer cases. In the present, larger cohort, we report the spectrum and frequency of four common and 62 rare FANCM PTVs found in 274 carriers detected among 44,803 breast cancer cases. We confirmed that p.Gln1701* was the most common PTV in Northern Europe with lower frequencies in Southern Europe. In contrast, p.Gly1906Alafs*12 was the most common PTV in Southern Europe with decreasing frequencies in Central and Northern Europe. We verified that p.Arg658* was prevalent in Central Europe and had highest frequencies in Eastern Europe. We also confirmed that the fourth most common PTV, p.Gln498Thrfs*7, might be a founder variant from Lithuania. Based on the frequency distribution of the carriers of rare PTVs, we showed that the FANCM PTVs spectra in Southwestern and Central Europe were much more heterogeneous than those from Northeastern Europe. These findings will inform the development of more efficient FANCM genetic testing strategies for breast cancer cases from specific European populations.

Effects of 7-ketocholesterol on tamoxifen efficacy in breast carcinoma cell line models in vitro

Oxysterols play significant roles in many physiological and pathological processes including cancer. They modulate some of the cancer hallmarks pathways, influence the efficacy of anti-cancer drugs, and associate with patient survival. In this study, we aimed to analyze the role of 7-ketocholesterol (7-KC) in breast carcinoma cells and its potential modulation of the tamoxifen effect. 7-KC effects were studied in two estrogen receptor (ER)-positive (MCF-7 and T47D) and one ER-negative (BT-20) breast cancer cell lines. First, we tested the viability of cells in the presence of 7-KC. Next, we co-incubated cells with tamoxifen and sublethal concentrations of 7-KC. We also tested changes in caspase 3/7 activity, deregulation of the cell cycle, and changes in expression of selected genes/proteins in the presence of tamoxifen, 7-KC, or their combination. Finally, we analyzed the effect of 7-KC on cellular migration and invasion. We found that the presence of 7-KC slightly decreases the efficacy of tamoxifen in MCF-7 cells, while an increased effect of tamoxifen and higher caspase 3/7 activity was observed in the BT-20 cell line. In the T47D cell line, we did not find any modulation of tamoxifen efficacy by the presence of 7-KC. Expression analysis showed the deregulation in CYP1A1 and CYP1B1 with the opposite trend in MCF-7 and BT-20 cells. Moreover, 7-KC increased cellular migration and invasion potential regardless of the ER status. This study shows that 7-KC can modulate tamoxifen efficacy as well as cellular migration and invasion, making 7-KC a promising candidate for future studies.

Association of Medicaid expansion with treatment receipt, delays in treatment initiation, and survival among young adult women with breast cancer.

1509 Background: Medicaid expansion under the Affordable Care Act has been shown to increase insurance coverage and early-stage cancer diagnoses for young adults (YAs), the age group with the highest uninsured rate. We examined whether Medicaid expansion was associated with increased receipt of timely, guideline-concordant treatment and survival among YA women newly diagnosed with breast cancer, the most common YA cancer diagnosis. Methods: Using the National Cancer Database, we identified 51,675 women aged 18-39 years who were diagnosed with breast cancer in 2011-2018. We applied the difference-in-differences (DD) method to estimate outcome changes pre vs. post Medicaid expansion, in expansion- vs. non-expansion states. Linear probability models estimated associations of Medicaid expansion with receipt of guideline-concordant treatment – including any endocrine therapy among women with ER-positive or PR-positive breast cancer, and any chemotherapy or targeted therapy among women with ER-negative and PR-negative breast cancer – and initiation of guideline-concordant treatment (surgery, chemotherapy, or targeted therapy for stage I-III diagnoses, and systemic therapy for stage IV diagnoses) &lt;60 days after diagnosis. Flexible parametric survival models were used to estimate 2-year overall survival. DD models also adjusted for age, race/ethnicity, rurality, zip code-level income, comorbidity, and diagnosis year, with standard errors clustered at the state level. Analyses were stratified by stage at diagnosis (stage I-III vs. IV). Results: Of the subset of women with stage I-III ER-positive or PR-positive breast cancer, the percentage receiving endocrine therapy increased in expansion states (85.20% pre-expansion to 86.46% post-expansion), but decreased in non-expansion states (84.32% to 82.79%), resulting in a net increase of 2.42 percentage points (ppt; 95% CI = 0.56 to 4.28) associated with Medicaid expansion in the adjusted DD model. Among all women with stage I-III breast cancer, the percentage with treatment initiation &lt;60 days after diagnosis decreased less in expansion states (84.10% pre-expansion to 82.36% post-expansion) than in non-expansion states (86.95% to 83.98%), resulting in a net reduction of 1.61 ppt (95% CI = 0.05 to 3.18) in treatment delays associated with Medicaid expansion. Notably, the 2-year overall survival increased in expansion states (96.84% pre-expansion to 96.99% post-expansion), but decreased in non-expansion states (97.24% to 96.30%), resulting in a net survival increase of 1.00 ppt (95% CI = 0.21 to 1.79) associated with Medicaid expansion. Conclusions: Among YA women with stage I-III breast cancer, Medicaid expansion was associated with increased receipt of guideline-concordant treatment, reduced delays in treatment initiation, and improved 2-year survival.

Prostate-specific membrane antigen (PSMA) expression in patients with metastatic triple negative breast cancer: Initial results of the PRISMA study.

1025 Background: Radioligand therapy (RLT) targeting prostate-specific membrane antigen (PSMA) prolongs survival of patients (pts) with prostate cancer. Preliminary immunohistochemistry (IHC)-based studies suggest that PSMA is highly expressed in triple negative breast cancer (TNBC). The PRISMA study aims to assess PSMA expression in pts with metastatic TNBC (mTNBC) via positron-emission tomography/computed tomography (PET/CT) to evaluate the feasibility of PSMA-targeted RLT for TNBC. Methods: PRISMA is a prospective, single-center study that enrolled pts with progressive mTNBC and measurable disease on 18F-FDG PET/CT to undergo 68Ga-PSMA-11 PET/CT. TNBC definition (ASCO/CAP) was based on the IHC of the primary breast cancer (BC) or metastasis. Target lesions (TL) were defined as metastatic lesions ≥1.5 cm with significant uptake in 18FDG-PET/CT. The same TL regions were propagated to the 68Ga-PSMA-PET/CT. 68Ga-PSMA-11 maximum standard uptake value (SUVmax) was measured for each TL. PSMA patterns A and B were defined according to presence of a majority (pattern A) or a minority (pattern B) of positive TL on 68Ga-PSMA (ie. uptake greater than that of normal liver parenchyma). Mann-Whitney U test was used to assess differences in median SUVmax (mSUVmax) between subgroups. Results: Ten pts with progressive mTNBC were enrolled. One pt was excluded due to lack of TL on 18FDG-PET/CT. Median age was 48 years (range 36-68), median number of TL per patient was 8 (5-18). Five pts had a TNBC IHC since the initial BC diagnosis, while 4 had a switch to TNBC (4 with initial estrogen receptor [ER]-positive and 1 with HER2-positive BC). All evaluable pts had inter- and intra-organ heterogeneity in PSMA uptake in TL and 2 pts had some mismatch with FDG-positive and PSMA-negative TL. mSUVmax was 2.99 (range 1.7-6.3) in the overall population and was higher in pts with pattern A (5.1 vs 2.6 in patterns A and B, respectively, p = 0.03). mSUVmax was numerically higher in younger pts (5.0 vs 2.6 in pts &lt;50y and ≥50y, p = 0.28), PD-L1-positive (5.0 vs 2.7 in PD-L1-positive and -negative, p = 0.14), de novo metastatic disease (5.0 vs 2.9 in de novo and recurrent BC, p = 0.38) and those with ER-negative primary breast tumor (3.4 vs 3.0 in ER-negative and ER-positive primary BC, p = 0.9). Conclusions: A significant PSMA uptake was observed in a proportion of pts with mTNBC. Inclusion of additional pts and translational analyses will allow the identification of PSMA expression determinants. PSMA-targeted RLT may be considered as an innovative therapeutic strategy to be explored in selected pts with mTNBC. [Table: see text]

The Causal Effect of Dietary Composition on the Risk of Breast Cancer: A Mendelian Randomization Study.

Breast cancer has become the most common malignancy among women, posing a severe health risk to women worldwide and creating a heavy social burden. Based on current observational studies, the dietary factor may have a causal relationship with breast cancer. Therefore, exploring how dietary composition affects breast cancer incidence will provide nutrition strategies for clinicians and women. We performed a two-sample Mendelian randomization (MR) analysis to find the causal effect of four kinds of relative macronutrient intake (protein, carbohydrate, sugar, and fat) on the risk of breast cancer and its subtypes [Luminal A, Luminal B, Luminal B HER2-negative, HER2-positive, Triple-negative, Estrogen receptor (ER) positive, and ER-negative breast cancer]. The Mendelian randomization pleiotropy residual sum and outlier (MR-PRESSO) test, MR-Egger intercept test, Cochran's Q statistic, funnel plot, and leave-one-out (Loo) analysis were all used in a sensitivity analysis to test the robustness of MR. Genetically, a higher relative protein intake was found as a protective factor for Luminal A and overall breast cancer, which was inconsistent with recent findings. A higher relative sugar intake could genetically promote the risk of Luminal B and HER2-positive breast cancer. Conclusions: A higher protein proportion in diet genetically reduces the risk of breast cancer, while higher relative sugar intake does the opposite.

Risk of estrogen receptor-specific breast cancer by family history of estrogen receptor subtypes and other cancers.

The extent to which the risk of estrogen receptor (ER)-specific breast cancer is associated with ER status of breast cancer and other cancers among first-degree relatives is unclear. This population-based cohort included 464 707 cancer-free women in Stockholm, Sweden, during 1978-2019. For ER-negative and ER-positive breast cancers, we estimated hazard ratios (HRs) associated with ER status of female first-degree relatives with breast cancer and of other cancers in all first-degree relatives. Associations between ER-negative and ER-positive status by family cancer history were estimated using logistic regression in a case-only design. Women with familial ER-positive breast cancer had 1.87 times (95% confidence interval [CI] = 1.77 to 1.97) higher risk of ER-positive subtype, whereas the corresponding hazard ratio for ER-negative was 2.54 (95% CI = 2.08 to 3.10) when having familial ER-negative breast cancer. The risk increased with an increasing number of female first-degree relatives having concordant subtypes and younger age at diagnosis (Ptrend <.001 for both). Nonbreast cancers among first-degree relatives were associated with both ER-positive (HR = 1.14, 95% CI = 1.10 to 1.17) and ER-negative (HR = 1.08, 95% CI = 1.01 to 1.16) breast cancers. Compared with women with ER-positive breast cancer, women with ER-negative breast cancer were more likely to have family history of liver (odds ratio [OR] = 1.33, 95% CI = 1.05 to 1.67), ovary (OR = 1.28, 95% CI = 1.01 to 1.61), and testicle cancer (OR = 1.79, 95% CI = 1.01 to 3.16) but less likely to have family history of endometrial cancer (OR = 0.77, 95% CI = 0.60 to 1.00) and leukemia (OR = 0.72, 95% CI = 0.56 to 0.91). Risk of ER-specific breast cancer differs according to ER status of female first-degree relatives with breast cancer and some other cancers of first-degree relatives. This family history information should be considered in the individual risk prediction for ER subtypes.

Causal relationships between serum matrix metalloproteinases and estrogen receptor-negative breast cancer: a bidirectional mendelian randomization study

To better clarify the causal effects between matrix metalloproteinases (MMPs) and estrogen-receptor (ER)-negative breast cancer (BC), we investigated the bidirectional causal relationship between MMPs and ER-negative BC by mendelian randomization (MR) analysis. Summary statistic data of five MMPs were extracted from European participants in 13 cohorts. Data of ER-negative BC collected from one of genome-wide association studies of European ancestry was used as experimental datasets and another four ER-negative BC datasets were used as validation sets. Inverse variance weighted method was used for main MR analysis and sensitivity analysis was also conducted. Serum level of MMP-1 has negative effect on ER-negative BC (odds ratio = 0.92, P = 0.0008) but the latter one was not the cause of the former one, which was supported by validation sets. No bidirectional causal effect was detected between the other four types of MMPs and ER-negative BC (P > 0.05). Sensitivity analysis indicated robustness of the above results without remarkable bias. To conclude, serum MMP-1 may be a protective factor against ER-negative BC. No reciprocal causality was found between the other kinds of MMPs and ER-negative BC. MMP-1 was indicated as a biomarker for risk of ER-negative BC.

Relationship between food-derived antioxidant vitamin intake and breast cancer risk: a mendelian randomized study.

In previous observational studies, food-derived antioxidant vitamins have been suggested to be associated with breast cancer. However, the findings were inconsistent and the causal relationship could not be clearly elucidated. To confirm the potential causal relationship between food-derived antioxidants (retinol, carotene, vitamin C and vitamin E) and the risk of breast cancer, we conducted a two-sample Mendelian randomization (MR) study. The instrumental variables (IVs) as proxies of genetic liability to food-derived antioxidant vitamins were obtained from the UK Biobank Database. We extracted breast cancer data (122,977 cases and 105,974 controls) from the Breast Cancer Consortium (BCAC). In addition, we studied estrogen expression status categorically, including estrogen receptor positive (ER+) breast cancer (69,501 cases and 105,974 controls) and versus estrogen receptor (ER-) negative breast cancer (21,468 cases and 105,974 controls). We performed two-sample Mendelian randomization study, and inverse variance-weighted (IVW) test was regarded as main analysis. Sensitivity analyses were further conducted to assess heterogeneity and horizontal pleiotropy. The results of IVW showed that among the four food-derived antioxidants, only vitamin E had protective effect on the risk of overall breast cancer (OR = 0.837, 95% CI 0.757-0.926, P = 0.001) and ER+ breast cancer (OR = 0.823, 95% CI 0.693-0.977, P = 0.026). However, we found no association between food-derived vitamin E and ER- breast cancer. Our study suggested food-derived vitamin E can decrease the risk of breast cancer overall and ER+ breast cancer, and the robustness of our results was confirmed by sensitivity analyses.

SNORA71C promotes development and metastasis of breast cancer by regulating RUNX1 and ferroptosis.

SNORA71C promotes development and metastasis of breast cancer by regulating RUNX1 and ferroptosis.

Abstract 4176: Timing of pregnancy-related factors and breast cancer risk for women across a range of absolute risk

Abstract Background: Studies in women with pathogenic variants in BRCA1 or BRCA2 suggest that underlying breast cancer (BC) risk modifies the associations between pregnancy-related factors and BC risk. Less is known about whether the associations are modified across a range of predicted absolute BC risk based on age and the extent of BC family history. Objective: Examine the association between pregnancy-related factors and BC risk and modification by predicted absolute BC risk in the Prospective Family Study Cohort (ProF-SC) with women enrolled between 1992-2011 and a median 10.0 years of follow-up time. Methods: ProF-SC includes the six international sites of the Breast Cancer Family Registry [USA, Canada, and Australia] and the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab). 17,274 women with 943 prospectively ascertained BC cases were eligible for the prospective analysis with 87% of cases self-identifying as non-Hispanic White. Questionnaires at study entry assessed self-reported pregnancy-related factors (parity, number of full-term pregnancies (FTP), age at first FTP, years since last FTP, breastfeeding, and age at menarche). We used Cox proportional hazards regression models with age as a time scale to estimate Hazard Ratios (HR) and the 95% Confidence Intervals (CI) for each reproductive variable (main effect) and examined modification by predicted absolute risk of BC, measured by a 1-year absolute risk score (continuous) estimated by the BOADICEA (interaction effect). We also examined associations stratified by estrogen receptor (ER) subtype. Results: Women with a first FTP at age ≥30 vs. &amp;lt;20 years had an increased risk of BC (HR=1.49, 95% CI 1.10, 2.02); no other pregnancy-related factors were associated with BC risk. Compared to nulliparous women, the main effect for higher parity was associated with increased ER-negative BC (PARS*≥4 FTP HR 2.34, 95% CI 1.09, 5.01). In contrast, increasing absolute BC risk for women with higher parity was associated with reduced risk of ER-negative BC (PARS*≥4 FTP HRinteraction 0.66, 95% CI 0.48, 0.90; pinteraction=0.01). Increasing absolute BC risk for nulliparous or first FTP at age ≥25 years had a &amp;gt;20% increased risk of ER-negative BC compared to first FTP at age &amp;lt;20 years (pinteraction=0.002); the main effects for nulliparity and first FTP at age ≥25 years and ER-negative BC were null (p-values=0.2-1.0). Compared to nulliparous women, increasing absolute risk for women who were within 5 years of their last FTP was positively associated with ER-negative BC (HRinteraction=1.54, 95% CI 1.03, 2.31); main effects were null (HR=1.00, 95% CI 0.39, 2.54). Conclusion: The association between pregnancy-related factors and BC risk are modified by predicted absolute BC risk, with stronger associations observed for ER-negative BC. Women at higher absolute BC risk may benefit from more frequent BC monitoring following childbirth. Citation Format: Jasmine A. McDonald, Yuyan Liao, Esther M. John, Allison W. Kurian, Jeanine M. Genkinger, Saundra S. Buys, John L. Hopper, kConFab Investigators, Mary Beth Terry. Timing of pregnancy-related factors and breast cancer risk for women across a range of absolute risk. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4176.

Abstract 5989: Targeting RRM2 &amp; cell cycle for breast cancer treatment

Abstract Endocrine treatment is an effective first-line therapy for targeting ER positive, HER2 negative breast cancer. But success is limited by development of acquired resistance due to long-term therapy. Cyclin D1 and cyclin dependent kinase 4/6 (CDK4/6) complex causes phosphorylation and subsequent inactivation of retinoblastoma (Rb) tumor suppressor protein which promotes progression of the cell cycle from G1 to S phase. This observation led the development of the first CDK4/6 inhibitor palbociclib (Ibrance; Pfizer) which induces cell cycle arrest at G1 phase in cancer cells. Owing to intrinsic and acquired drug resistance development, success is limited despite promising clinical outcomes. This situation necessitates the development of potential combination strategies to overcome drug resistance. Ribonucleotide reductase (RR) is a rate limiting enzyme in DNA synthesis consisting of two subunits RRM1 and RRM2. Didox inhibits ribonucleotide reductase subunit 2 (RRM2) which ultimately blocks DNA synthesis. Combination of didox with palbociclib is a potential strategy to target ER positive and ER negative/triple negative breast cancer. Previously, we have shown that didox (DDX) can significantly halt malignant breast cancer cell division in combination with the anthracycline drug doxorubicin by targeting RRM2, mutant p53 and NFkB regulatory proteins. Recently, we confirmed that didox in combination with palbociclib significantly lowers ER positive and ER negative breast cancer and their palbociclib resistant counterparts’ growth compared to no treatment or palbociclib treatment. We also confirmed that ER positive MCF7 and ER negative MDA-MB-468 parental breast cancer cells IC50 of palbociclib drug is lower than their palbociclib resistant counterparts. Here, we are reporting that didox decreases cell cycle proteins alone or in combination with palbociclib in ER positive MCF7 and ER negative MDA-MB-468 parental and palbociclib resistant breast cancer cells. This finding opens a novel approach for targeting both ER positive as well as ER negative breast cancer treatment. We are also reporting that didox treatment modulates cyclin D1 (CCND1) and RRM2 expression in MCF7 and MDA-MB-468 breast cancer along with their palbociclib resistant counterparts. Additionally, we observed that didox alone or in combination with palbociclib alters the cell cycle of MCF7 and MDA-MB-468 parental and palbociclib resistant breast cancer cells. Our data presents a novel and promising approach for the treatment of ER positive and ER negative breast cancer that involves inhibition of RRM2 and cell cycle that merits further clinical investigation in animal and human models. Citation Format: Nahid Sultana, Howard L. Elford, Jesika S. Faridi. Targeting RRM2 &amp; cell cycle for breast cancer treatment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5989.

Abstract 6470: Coffee and tea consumption, coffee-related metabolites, and breast cancer risk in US Women

Abstract Background: Coffee intake may protect against breast cancer; however, previous studies have been inconsistent and potential underlying mechanisms remain unclear. We aimed to 1) examine the association between total coffee, tea, and caffeine intake and breast cancer risk and 2) identify plasma metabolites associated with coffee intake and examine these metabolites in relation to breast cancer risk. Methods: Data on breast cancer risk factors were collected from questionnaires administered biennially and at the time of blood collections (Nurses’ Health study, NHS, distant collection: 1989-1990, recent collection: 2000-2002; NHSII, 1996-1999). We evaluated the association between total coffee (caffeinated and decaffeinated), tea consumption and caffeine intake and breast cancer risk in the full cohort using Cox regression models. Leveraging dietary and metabolomic data, we identified and validated plasma metabolites associated with coffee intake in 2,033 women from the NHSII breast cancer sub-study. We then evaluated the prospective association of all prior (PMID: 32788283) and new coffee-related metabolites with breast cancer risk in a nested case-control study using conditional logistic regression models. Results: We documented 10,060 cases of invasive breast cancer during 3,901,211 person-years of follow-up. In pooled, multivariable-adjusted models, compared to never consumers (0 cups/d), women consuming ≥6 cups/d of total coffee were at lower risk of invasive breast cancer (HR, 0.91; 95% CI, 0.82, 1.00; P-trend&amp;lt;0.01). Stratified analysis showed a significant effect modification by BMI (P-interaction=0.03). Compared to never consumers, drinking ≥6 cups of coffee per day was inversely associated with risk of invasive breast cancer among lean women (HR, 0.83; 95% CI, 0.72, 0.97) but not among overweight or obese women. Moreover, a lower risk of ER-negative breast cancer was observed with total coffee (HR ≥6 cups/d vs. 0 cups/day 0.75; 95% CI, 0.59, 0.95; P-trend=0.01) and decaffeinated coffee consumption (HR ≥4 cups/d vs. o cups/day 0.66; 95% CI, 0.45, 0.95; P-trend=0.01). Among the 31 studied metabolites, no single metabolite was associated with breast cancer risk. Associations were generally consistent between ER-positive and ER-negative breast cancer, although trigonelline seemed to be suggestively stronger for ER-negative (OR, 95% CI, 0.87, 0.75-1.01; P-value=0.061). Furthermore, 1,7-dimethyluric acid, some cholesterol esters (C18:1 CE, C18:2 CE and C20:4 CE) and trigonelline, all positively associated with coffee, were indicative of an inverse association with breast cancer risk among lean women. Conclusions: Coffee consumption was associated with lower breast cancer risk, particularly among lean women and ER-negative breast cancer. Some metabolites that might explain some of the observed inverse associations. Nonetheless, these results merit further exploration. Citation Format: Andrea Romanos-Nanclares, Oana A. Zeleznik, Mingyang Song, Walter C. Willett, Rulla M. Tamimi, Wendy Y. Chen, Michelle D. Holmes, Jae Hee Kang, A. Heather Eliassen. Coffee and tea consumption, coffee-related metabolites, and breast cancer risk in US Women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6470.

Abstract 4744: Epigenetic reprogramming induced by lipids fosters mammary cell plasticity in non-transformed breast epithelial cells

Abstract Introduction. Lipid exposure of non-transformed breast epithelial cells results in increased flux modifications, and profound changes in gene expression. The upregulated genes are involved in stemness, neural development, and neural crest pathways. Neural genes are highly expressed in an estrogen receptor negative breast cancer (ERnegBC): the triple negative subtype. We aim to identify mechanisms that link lipids and epigenetic reprogramming to the genesis of ERnegBC. Methods. Non-transformed MCF-10A cells exposed to octanoic acid (OA) were utilized for U13C-glucose tracing. S-adenosylmethionine (SAM), 2-HG and other metabolites were analyzed following treatment with OA ± PHGDH inhibitor. CUT&amp;RUN for H3K4me3 and H3K27me3 was performed, and genes affected by OA (PMID: 28263391) were compared with OA-responsive peaks. OA-induced genes with CUT&amp;RUN peaks were measured in breast organoids derived from reduction mammoplasty samples exposed to OA. The Aldefluour assay was used to identify stem-like (ALDH+) cells. Results. U13C-glucose tracing in presence of OA showed decreased glucose uptake, glycolysis and pentose phosphate pathway flux, with little effect on the TCA cycle. One-carbon (1C)-THF was redirected to the methionine cycle increasing flux to methylation. The methyl donor SAM and the demethylase inhibitor 2-HG increased after 15- and 30-min OA exposure, respectively; PHGDH inhibitor blocked these increases likely via effects on 1C metabolism. H3K4me3 CUT&amp;RUN revealed 661 differential peaks (FDR &amp;lt; 0.05) comparing OA to control. The genes associated with these peaks are involved in neuron differentiation, neural crest, EMT and neural genes expressed in ERnegBC. 73% of H3K4me3 OA-associated peaks were in regulatory regions of OA-induced genes (FDR &amp;lt; 0.01), including NGFR (log2FC = 11.7), NGF (log2FC = 8), NTRK1 (log2FC = 2.1). OA-induced genes with enriched H3K4me3 peaks were also upregulated in breast organoids exposed to OA, such as NGFR (FC = 2.2), NGF (FC = 4.7), NTRK1 (FC = 6.6). Motif analysis revealed an overrepresentation of transcription factors (p &amp;lt; 0.05) associated with EMT (Zeb1, Slug), neural functions (E2A, AP1, JunB), neuronal-injury (Atf3) and stress response (Chop, Atf4). Twelve H3K27me3 peaks were enriched in control (FDR &amp;lt; 0.05) and associated with increased gene expression in OA, among them were LGR6 (log2FC = 1.9), a gene associated with ERnegBC and PLAG1 (log2FC = 2.8) a stem cell marker. ALDH+ cells’ percentage increased by at least 10% after OA exposure. Conclusions. Lipid exposure affects the production of SAM and 2-HG, which results in epigenetic fostered plasticity, leading to reprogramming/selecting cells with a multi-potential embryonic or stem-like state and/or differentiation to a neural/neural crest-like state which may facilitate ERnegBC genesis. Citation Format: Mariana Bustamante Eduardo, Gannon Cottone, Shiyu Liu, Seema Khan, Susan Clare. Epigenetic reprogramming induced by lipids fosters mammary cell plasticity in non-transformed breast epithelial cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4744.

Abstract 5225: Expression- and splicing-based multi-tissue transcriptome-wide association studies identified multiple genes for estrogen receptor-negative breast cancer

Abstract Although several transcriptome-wide association studies (TWAS) have been performed to identify genes associated with overall breast cancer risk, few genes were found to be associated with estrogen receptor-negative (ER-) breast cancer. These studies were based on gene expression prediction models trained mainly in breast tissue, and they did not account for alternative splicing of genes. In this study, we utilized two approaches to perform multi-tissue TWASs of ER- breast cancer: 1) an expression-based TWAS that combined TWAS signals for each gene across multiple tissues and 2) a splicing-based TWAS that combined TWAS signals of all excised introns for each gene across tissues. To perform these two TWASs, we first obtained gene expression prediction models that were trained in 11 tissues from the Genotype-Tissue Expression including breast, ovary, uterus, vagina, EBV-transformed lymphocytes, whole blood, spleen, liver, subcutaneous adipose, visceral adipose, and cell-cultured fibroblasts. Furthermore, we obtained GWAS summary statistics for ER- breast cancer by performing a meta-analysis of 21,468 ER- cases and 105,974 controls from the Breast Cancer Association Consortium and 9414 BRCA1 mutation carriers who were breast cancer cases and 9494 BRCA1 mutation carriers who did not have breast cancer from the Consortium of Investigators of Modifiers of BRCA1 and BRCA2. For our expression-based TWAS, we utilized the Aggregated Cauchy Association Test (ACAT) to collate TWAS signals across all 11 tissues for each gene using equal weights for each tissue, while for our splicing-based TWAS, we utilized ACAT to collate TWAS signals for all excised introns in all tissues for each gene. Overall, we identified 63 genes in 29 loci that were significantly associated with ER- breast cancer, including 23 genes that were identified using both approaches, 27 that were uniquely identified in the expression-based TWAS, and 13 that were uniquely identified in the splicing-based TWAS. Of the 63 genes, 50 genes have not been previously reported in TWAS studies of breast cancer. Using a weighted ACAT method, in which breast tissue was given a 5-fold higher weight, we obtained similar results. In summary, our joint, multi-tissue TWAS corroborated previous GWAS loci for both ER- and overall breast cancer while highlighting how incorporating TWAS signals from multiple tissues and alternative splicing allowed us to discover new susceptibility genes for ER- breast cancer. Citation Format: James L. Li, Julian C. McClellan, Guimin Gao, Dezheng Huo. Expression- and splicing-based multi-tissue transcriptome-wide association studies identified multiple genes for estrogen receptor-negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5225.

Abstract 3011: Psychosocial stress, social support, and incident breast cancer by estrogen receptor status

Abstract Introduction: Chronic stress affects immune function and hormonal signaling and has been hypothesized to be associated with breast cancer, although results from the few prior studies are mixed and have not examined potential differences by estrogen receptor (ER) status. We investigated the relationship between stressful life events and incident breast cancer by ER status among postmenopausal women. We further evaluated whether this association is modified by social support. Methods: Our analysis included 76,951 postmenopausal women enrolled in the Women’s Health Initiative. Participants were followed for events for up to 26 years (median, 16.7). We generated Cox proportional hazards models adjusting for demographic, clinical, lifestyle/behavioral, and social factors to estimate hazard ratios (HR) and 95% confidence intervals (95%CI) for associations between baseline self-reported stressful life events and incident breast cancer by ER status. Results: The mean age was 63 (SD 7.3), and majority of participants were White race (83.5%) and married or in a marriage-like relationship (63.0%). In analyses stratified by ER status, there was no relationship between stressful life events and ER-positive breast cancer. In contrast, compared to women in the lowest quartile, those in higher quartiles had an increased risk of ER-negative breast cancer, where those in quartile 4 had the highest risk (Quartile 4 vs Quartile 1; HR=1.30; 95%CI 1.01-1.68; Ptrend=0.050). Additionally, associations were stronger for the highest versus lowest quartile of stressful life events among widowed women (HR=2.39; 95%CI 1.29-4.44; Pinteraction&amp;lt;0.001). Moreover, while the interaction was not statistically significant, we observed an elevated risk for ER-negative breast cancer among women in the highest quartile of stressful life events reporting lower social support. Conclusion: In this cohort of postmenopausal women, higher experiences of pre-diagnostic stressful life events were associated with increased risk of ER-negative breast cancer. Citation Format: Wayne R. Lawrence, Jasmine A. McDonald, Faustine Williams, Meredith S. Shiels, Neal D. Freedman, Ziqiang Lin, Jared W. Magnani. Psychosocial stress, social support, and incident breast cancer by estrogen receptor status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3011.