Absence Of Estrogen Receptors Research Articles

Predicting the ER Status Using the Gene Expression Profiles

Breast cancer is the most frequently diagnosed malignancy among women globally, making it the leading cause of cancer incidence worldwide. This type of cancer is particularly challenging due to its high degree of molecular heterogeneity, with many different subtypes exhibiting distinct genetic and biological characteristics. Estrogen receptors (ERs) play a crucial role in breast cancer by acting as receptors for the hormone estrogen, often referred to as the hormone receptor (HR). The presence or absence of ERs, known as ER status, significantly impacts treatment decisions, making accurate prediction of ER status essential for personalized treatment plans. Various methods have been proposed in the literature to predict ER status based on gene expression profiles using microarray genome-wide gene expression profiling. The issue of class imbalance, indicated by a notable variation in the number of samples per class and the curse of dimensionality, is a significant concern with microarray datasets. This paper introduces a novel technique, Fuzzy XGBoost, to predict ER status in breast cancer cases while addressing the class imbalance problem. The proposed algorithm enhances the traditional XGBoost method by incorporating fuzzy logic principles, improving its robustness, computational power, and accuracy in handling imbalanced datasets. Our research demonstrates the effectiveness of the proposed Fuzzy XGBoost algorithm in accurately predicting ER status across multiple gene expression profile datasets. This approach not only contributes to advancements in breast cancer treatment but also showcases the power of computational methods in bioinformatics. We evaluate our method using four datasets: GSE2990, GSE3494, GSE6532, and GSE7390, achieving predictive accuracies of 100%, 92%, 100%, and 96%, respectively. The successful application of Fuzzy XGBoost in this context highlights its potential for broader use in bioinformatics, where leveraging computational power to address challenges such as imbalanced data is critical for predictive accuracy and personalized treatment. DOI: https://doi.org/10.52783/pst.619

Selective Antineoplastic Potential of Fractionated Caribbean Native Ganoderma Species Extracts on Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor type 2 expression. It is known for its high malignancy, invasiveness, and propensity for metastasis, resulting in a poor prognosis due to the absence of beneficial therapeutic targets. Natural products derived from mushrooms have gained significant attention in neoplastic therapy due to their potential medicinal properties. The therapeutic potential of Ganoderma lucidum in breast cancer has been highlighted by our group, suggesting its use as an adjuvant treatment. The present study aims to assess the potential antineoplastic capacity of two Caribbean native Ganoderma species found in Puerto Rico, Ganoderma multiplicatum (G. multiplicatum) and Ganoderma martinicense (G. martinicense). Antiproliferative studies were conducted via cell viability assays after cultivation, harvesting, and fractionation of both species. The obtained results indicate that most of the fractions show some cytotoxicity against all cell lines, but 33% of the fractions (F1, F2, F7, F12) display selectivity towards cancer cell models. We demonstrate for the first time that native Ganoderma species can generate metabolites with anti-TNBC properties. Future avenues will focus on structure elucidation of the most active fractions of these Ganoderma extracts.

Chemotherapeutic Efficacy of Rutin in Triple Negative Breast Cancer

Cancer is a complex group of diseases characterized by the uncontrolled growth and spread of abnormal cells in the body. It causes millions of deaths each year and remaining a significant global health concern for both men and women. Effective treatment strategies are crucial for improving patient outcomes in breast cancer, particularly in the case of triple-negative breast cancer (TNBC), characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Chemotherapy, like paclitaxel and docetaxel, is the standard treatment for TNBC due to the lack of targeted therapies for this subtype. Paclitaxel (PTX) is a widely used chemotherapeutic medication that is particularly effective against lung, ovarian, and other cancers; nevertheless, its clinical use is limited due to its multi-organ toxicity. As a result, the current study aims to improve treatment efficacy and reduce PTX-induced toxicity through the concurrent use of the natural polyphenolic substance Rutin. Rutin hydrate (purity > 94%) and paclitaxel were utilized in in vitro studies with 4T1 and MDA MB-231 cell lines. In the proliferation assay, cells were treated with rutin and paclitaxel at varying concentrations. Cytochrome-c release and cell cycle analysis were conducted, and flow cytometry assessed apoptosis. According to the findings of this investigation, rutin in combination with PTX considerably (P<0.05) lowers the growth and proliferation of breast cancer cell lines in vitro. Furthermore, flow cytometry research revealed that combining rutin with PTX triggered GO/Gl cell cycle arrest and apoptosis in a breast cancer cell line. Furthermore, after co-administration of rutin and PTX, mitochondrial depolarization increased significantly (P<0.05). Thus, the current study convincingly established rutin’s sensitizing activity and suggests it could be a potential adjuvant in cancer chemotherapy.

Clinical and histological profiling of triple-negative breast cancer at Chihuahua´s state cancer center: An observational study.

e22529 Background: Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by the absence of estrogen receptor, progesterone receptor, and HER2 expression. This subtype is associated with a poorer prognosis and higher recurrence rates compared to other breast cancer subtypes. Understanding the histopathological and clinical characteristics of TNBC is crucial for improving early detection, clinical management, and treatment outcomes. In this context, a descriptive analysis was conducted to explore the association between TNBC and histological grade, histopathological type, and clinical stage at the State Cancer Center. Methods: A retrospective, single-center study was conducted at Chihuahua's State Cancer Center, where medical records of patients aged ≥ 18 years old diagnosed with TNBC between January 2018 and December 2022 were retrospectively reviewed. Data related to weight, histological grade, histopathological type, and clinical stage at TNBC diagnosis were collected. Frequency analysis was performed to determine the distribution of these characteristics in the TNBC patient population. Additionally, the association between histological grade and clinical stage at diagnosis was examined using appropriate statistical tests. Results: During the study period, 753 patients were diagnosed with breast cancer. Of these, 84 (11%) were diagnosed with triple-negative breast cancer and were included in the study. The mean age was 49 ± 13 years, with a mean BMI of 28 ± 5. The most common histologic subtype in this group was invasive ductal carcinoma, accounting for 78 (93%) of the TNBC cases. At the time of diagnosis, 19 (23%) were diagnosed with stage IIA, 17 (20%) with stage IIIA, 9 (11%) with stage IV, and 4 (5%) were detected in situ. Overall, the majority were identified as grade 3 tumors (44; 52%). Finally, within the stage IIA group, the majority were grade 3 tumors (58%). Conclusions: As seen in other cohorts, we found a similar frequency of TNBC among patients with breast cancer in our facility in the northwest of Mexico. The distribution of clinical stages highlights the varying degrees of disease progression at the time of diagnosis, with a considerable portion presenting at advanced stages. Moreover, these insights underscore the urgent need for improved screening strategies and early detection methods.

Exploring the neutrophil-to-lymphocyte ratio as a prognostic biomarker in triple-negative breast cancer

Triple-negative breast cancer is a subtype characterized by the absence of estrogen receptor, progesterone Receptor, and Human Epidermal Growth Factor Receptor 2 receptors, limiting treatment options primarily to chemotherapy due to lack of targeted therapies typically used for other breast cancer types, hence necessitating research for improved therapeutic strategies and outcomes. The neutrophil and lymphocyte percentages, absolute counts, and the ratio of neutrophils to lymphocytes were evaluated in peripheral blood of 81 patients with triple-negative breast cancer (TNBC) and 25 healthy controls. This study observed trend of high neutrophil percentage was observed in patients with positive lymph node status as compared to negative lymph node status. Significant high absolute neutrophil count was observed in patients with positive LN status, High BR score and presence of metastasis. A trend of high neutrophil to lymphocyte ratio was observed in patients with presence of perineural invasion, presence of necrosis, and patients with presence of metastasis.

Abstract PO5-27-07: A novel ROR1 inhibitor CPD86 suppresses Triple-Negative Breast Cancer cells via regulation of AKT/GSK3β pathway

Abstract Breast cancer is the second most commonly diagnosed cancer in women in the United States. The most aggressive subtype is triple negative breast cancer (TNBC), which accounts for 15-20% of new cases on average each year. Histologically, it is characterized by the absence of estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor 2 (HER2), which are typically targeted by hormone therapies such as tamoxifen, fulvestrant, or letrozole. Standard of care for TNBC includes traditional chemotherapy and radiation, which are detrimental to both cancerous and normal cells. Therefore, the lack of all hormone receptors in TNBC calls for the investigation of novel anti-cancer compounds that specifically target TNBC with minimal damage to non-cancerous tissues. Receptor orphan tyrosine kinase-like receptor 1 (ROR1) is an oncoprotein that is overexpressed in several human malignancies such as lung, breast, prostate and pancreatic cancers, but not in normal tissues. Inhibition of ROR1 signaling has shown to repress proliferation and induce apoptosis of cancer cells. Through in-silico docking and isothermal titration calorimetry, we identified a novel compound CPD86 that interacts with and inhibits ROR1. We hypothesize that CPD86 selectively targets TNBC cells by reducing the phosphorylation of AKT/GSK3β via the inhibition of ROR1. Our in vitro results suggest that CPD86 inhibits cell viability and induces intrinsic apoptosis of TNBC cells at a half-maximal inhibitory concentration of 2-5 µM, but not in normal breast epithelial cells with minimal ROR1 expression. CPD86 also represses TNBC cell migration and invasion while leaving non-malignant cells unharmed. This study highlights that ROR1 inhibitors could be developed as a potential therapeutic for TNBC and the data would serve as proof-of-concept justification for evaluation in other ROR1-upregulated cancers. Citation Format: Tram Ta, Victoria Reed, Nikhil Chandra, Nick Dwyer, Norman Fultang, Shradheya Gupta, Indrakant Singh, Bela Peethambaran. A novel ROR1 inhibitor CPD86 suppresses Triple-Negative Breast Cancer cells via regulation of AKT/GSK3β pathway [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-27-07.

Abstract PO4-26-06: Identifying mutation-independent signaling pathway activation in molecular subtypes of triple negative breast cancers

Abstract Introduction Triple negative breast cancer (TNBC) is an aggressive cancer defined by the absence of estrogen receptors (ER), progesterone receptors (PR), and lack of human epidermal growth factor receptor 2 (HER-2). We previously identified molecular subgroups with distinct mutational, epigenetic, and clinicopathologic signatures. Here, we sought to identify signaling pathways using targeted RNA expression-based pathway analysis. Methods We analyzed 44 TNBC cases diagnosed at NYU Langone Health between March 2011 and April 2018. Samples were compared based on histology, immunohistochemistry, and epigenetics. We assessed mRNA expression levels of pathway-specific target genes with RT-qPCR using the OncoSIGNal pathway profiling assay. Signal transduction pathway analyses were used to measure activity of the estrogen receptor (ER), androgen receptor (AR), phosphoinositide-3-kinase (PI3K), mitogen-activated protein kinase (MAPK), Hedgehog (HH), Notch, and transforming growth factor beta (TGF- β) pathways. Results DNA methylation Cluster 1, which is enriched for apocrine histology and PI3K/Akt mutations, showed no increased activity in PI3K pathway by RNA expression, but showed increase of AR and MAPK signaling, with a single case of increased ER signaling in the absence of ESR1 activating mutations. In contrast, both DNA methylation Cluster 2 (no recurrent mutations) and Cluster 3 (DNA instability cluster) showed upregulation in PI3K pathway in 4/7 (57%) and 11/25 (44%) of cases, respectively. PI3K pathway activation was invariably associated with high Ki-67 >15%, however not all high Ki-67 cases showed high PI3K activity. Invasive ductal carcinoma (IDC) morphology was characterized by a nearly even split between MAPK (13/28, 46%) and PI3K (12/28, 44%), with single HH and TGF- β driven cases. In 5/28 (18%) of IDC we did not identify an upregulated pathway. Discussion We show that RNA expression-based pathway activity and mutational and epigenetic analyses provide complimentary information about the molecular landscape of TNBC. PI3K mutations are early drivers and lead to distinct epigenetic signatures, however they are not associated with increased PI3K pathway activity by RNA expression. In contrast, TNBC without PI3K mutations may show activation of the PI3K pathway in the absence of PI3K/Akt mutations. Our observation suggests that distinct biomarkers may be differently suited to predict response to PI3K inhibitors in TNBC. This may include expanding the number of patients for which PI3K/Akt pathway inhibition might be a therapeutic option, as well as explaining the lack of response in PI3K mutated tumors. Citation Format: Chanel Schroff, Lawrence Lin, Dianne van Strijp, Daniel Roses, Sigi Neerken, Freya Schnabel, Farbod Darvishian, Matija Snuderl. Identifying mutation-independent signaling pathway activation in molecular subtypes of triple negative breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-26-06.

Abstract 6283: Tumor repressive effects of estrogen receptor β mRNA-based therapy in aggressive breast cancers

Abstract Inflammatory breast cancer (IBC) and triple-negative breast cancer (TNBC) are the most aggressive forms of breast cancer due to their high propensity to develop distant metastases and the lack of specific effective targeted therapies. The failure of previous research to produce viable therapeutic targets combined with the absence of estrogen receptor α (ERα) in TNBC and the majority of IBC led us to explore the second ER (ERβ) as a molecule with targeting potential. We previously reported the anti-metastatic effects of ERβ in both TNBC and IBC. Considering the anti-tumor activity together with the decreased expression of ERβ in IBC and TNBC tumors, we employed an mRNA-based approach to induce tumor-specific upregulation of the receptor and restore its anti-metastatic activity. Our findings show that complexes of ERβ mRNA with lipid nanoparticles (LNPs) enable in vitro translation of a functional protein with strong anti-invasive activity. More importantly, direct administration of ERβ mRNA-LNPs in orthotopically grown IBC and TNBC tumors results in high tumor levels of the receptor and an evident inhibition of metastasis demonstrating for the first time the ability of ERβ mRNA to prevent progression of the disease. Our study may have important clinical implications by establishing ERβ mRNA as a novel and specific therapy intervention that either alone or in combination with existing treatments can substantially repress metastasis, eliminate associated mortality and benefit patients with aggressive breast cancer. Citation Format: Kim Cuong Cap, Aireana Phillips, Karem A. Court Pinto, Kristina D. Zambo, Wei Qian, Jianying Zhou, Jenny Chang, Biana Godin, Christoforos Thomas. Tumor repressive effects of estrogen receptor β mRNA-based therapy in aggressive breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6283.

Exploring the multifaceted impact of lanthanides on physiological pathways in human breast cancer cells

Lanthanum (La) and cerium (Ce), rare earth elements with physical properties similar to calcium (Ca), are generally considered non-toxic when used appropriately. However, their ions possess anti-tumor capabilities. This investigation explores the potential applications and mechanisms of LaCl3 or CeCl3 treatment in triple-negative breast cancer (TNBC) cell lines. TNBC, characterized by the absence of estrogen receptor (ERα), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2) expression, is prone to early metastasis and resistant to hormone therapy. Our results demonstrate that La/Ce treatment reduces cell growth, and when combined with cisplatin, it synergistically inhibits cell growth and the PI3K/AKT pathway. La and Ce induce oxidative stress by disrupting mitochondrial function, leading to protein oxidation. Additionally, they interfere with protein homeostasis and induce nucleolar stress. Furthermore, disturbance in F-actin web formation impairs cell migration. This study delves into the mechanism by which calcium-like elements La and Ce inhibit breast cancer cell growth, shedding light on their interference in mitochondrial function, protein homeostasis, and cytoskeleton assembly.

Extracellular Vesicles in Triple-Negative Breast Cancer: Immune Regulation, Biomarkers, and Immunotherapeutic Potential.

Triple-negative breast cancer (TNBC) is an aggressive subtype accounting for ~10-20% of all human BC and is characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) amplification. Owing to its unique molecular profile and limited targeted therapies, TNBC treatment poses significant challenges. Unlike other BC subtypes, TNBC lacks specific molecular targets, rendering endocrine therapies and HER2-targeted treatments ineffective. The chemotherapeutic regimen is the predominant systemic treatment modality for TNBC in current clinical practice. However, the efficacy of chemotherapy in TNBC is variable, with response rates varying between a wide range of patients, and the emerging resistance further adds to the difficulties. Furthermore, TNBC exhibits a higher mutational burden and is acknowledged as the most immunogenic of all BC subtypes. Consequently, the application of immune checkpoint inhibition has been investigated in TNBC, yielding promising outcomes. Recent evidence identified extracellular vesicles (EVs) as an important contributor in the context of TNBC immunotherapy. In view of the extraordinary ability of EVs to transfer bioactive molecules, such as proteins, lipids, DNA, mRNAs, and small miRNAs, between the cells, EVs are considered a promising diagnostic biomarker and novel drug delivery system among the prospects for immunotherapy. The present review provides an in-depth understanding of how EVs influence TNBC progression, its immune regulation, and their contribution as a predictive biomarker for TNBC. The final part of the review focuses on the recent key advances in immunotherapeutic strategies for better understanding the complex interplay between EVs and the immune system in TNBC and further developing EV-based targeted immunotherapies.

Adiponectin-expressing Treg-containing T cell fraction inhibits tumor growth in orthotopically implanted triple-negative breast cancer.

In our previous study, we identified a population of adiponectin expressing regulatory T cells (Tregs) residing within thymic nurse cell complexes, which were capable of inhibiting the development of breast cancer in vitro. Triple-negative breast cancer (TNBC) with no proper treatment at present is characterized by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2. In this study, we aimed to investigate the potential of a cultured T cell fraction comprising adiponectin-expressing Tregs, referred to as A-TregTF (adiponectin-expressing Treg-containing T cell fraction), in inhibiting the progression of TNBC in vivo. The efficacy of a spontaneously expanding T cell fraction comprising adiponectin-expressing Treg in inhibiting tumor growth was analyzed in a murine orthotopic 4 T1-Luc TNBC model. The treatment with T cell fraction containing adiponectin-expressing Tregs significantly inhibited the growth and metastasis of orthotopically transplanted 4 T1-Luc tumor cells. Histopathological examination further revealed that the adiponectin-expressing Tregs infiltrated the tumor tissue via a cell-in-cell mechanism and were found to be specifically localized around the necrotic areas. Based on our findings, the T cell fraction comprising adiponectin-expressing Tregs, represents a potential candidate for adoptive cell therapy against TNBC.

Molecular Biology Mechanisms and Emerging Therapeutics of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is conventionally characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2),accounting for approximately 15-20% of all breast cancers. Compared to other molecular phenotypes, TNBC is typically associated with high malignancy and poor prognosis. Cytotoxic agents have been the mainstay of treatment for the past few decades due to the lack of definitive targets and limited therapeutic interventions. However, recent developments have demonstrated that TNBC has peculiar molecular classifications and biomarkers, which provide the possibility of evolving treatment from basic cytotoxic chemotherapy to an expanding domain of targeted therapies. This review presents a framework for understanding the current clinical experience surrounding molecular biology mechanisms in TNBC (Figure 1). Including immunotherapy, polymerase (PARP) and PI3K/AKT pathway inhibitors, antibody-drug conjugates, and androgen receptor (AR) blockade. Additionally, the role of miRNA therapeutics targeting TNBC and potential strategies targeting cancer stem cells (CSCs) are discussed and highlighted. As more and more treatments arise on the horizon, we believe that patients with TNBC will have a new sense of hope.

Prognostic Significance of Microvessel Density in Invasive Ductal Carcinoma of Breast.

Background: Breast cancer is the most common malignant tumor and cause of death in women. Factors that play role in tumor metastasis are lymph node involvement, lack of tumor differentiation and hormone receptor expression, high proliferation rate, and angiogenesis. In the present study, we tried to evaluate the microvessel density (MVD) using Immunohistochemistry for the CD34 marker to investigate the amount of angiogenesis in breast cancer and its relationship with other histopathological parameters and compare it with normal tissue. Materials and Methods: 58 paraffin-embedded samples of breast cancer were enrolled. All blocks were sectioned and stained for estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor 2(HER 2/neu), ki67, and CD34 by immunohistochemistry (IHC) method. Results: The mean age of patients in this study was 49.6 ± 10.6 years. Statistically, there was a significant relationship between the grade of the tumor (P = 0.01), absence of expression of estrogen receptor (P = 0.008), and progesterone receptor (P = 0.003) with MVD. Conclusion: Due to the association between MVD, tumor grade, and absence of ER and PR expression, this valuable marker can play an important role in the prediction of prognosis in breast cancer patients and can lead to new-targeted therapy in the future.

Abstract P2-23-07: DNA Methylation Analysis of Triple Negative Breast Cancers

Abstract Background: Triple negative breast cancers (TNBC) are characterized by the absence of estrogen receptors, progesterone receptors, and HER2 receptors on immunohistochemical analysis (IHC). This subtype of breast cancer has been traditionally associated with unfavorable prognosis. TNBC includes morphologically diverse mammary carcinomas including apocrine, metaplastic, medullary and other less common variants with heterogeneous clinical behavior and outcome. Despite previous efforts to further characterize TNBC with genomic and transcriptomic techniques, there is no specific targeted therapy for TNBC. Cytotoxic chemotherapy remains the mainstay of systemic treatment for these patients. DNA methylation has proved to be a promising tool in classifying a variety of cancers. In this study, we aim to analyze TNBC using comprehensive DNA methylation profiling. Method: DNA methylation profiling was performed in TNBC cases from a single academic institution using Illumina Infinium MethylationEPIC Kit. The tumors were obtained from formalin-fixed paraffin-embedded tissue from surgical specimens. Unsupervised clustering analysis based on the methylation profile was performed. Clinical, pathological, and genetic features were analyzed between the clusters. Results: We analyzed 44 cases (all female; median age 61 years) with treatment naïve TNBC diagnosed from March 2011 to April 2018. Median follow-up time was 58 months. Thirty-four (77%) patients were identified as white with the remaining 10 (23%) as non-white. Thirty-four tumors (77%) were classified as invasive ductal carcinoma of no special type, six (14%) as apocrine carcinoma, and four (9%) as metaplastic carcinoma. Six tumors (14%) were grade 2 and 38 (86%) were grade 3. Lymphovascular invasion was noted in 7 patients (16%). Tumor size ranged from 4 to 45 mm (median: 20 mm). Lymph node involvement was identified in 8 (18%) patients. Eleven (25%) patients harbored germline BRCA1/2 mutations. During the follow up period, 7 patients developed recurrent disease: 3 had local recurrences, 4 patients were found to have metastatic disease, and 1 patient had both local and distant recurrence. At last follow-up, 34 patients (77%) showed no evidence of disease, while 4 (9%) were alive with disease and 5 (11%) had died of disease. In this cohort, we identified three distinct DNA methylation clusters. In Cluster 1 (n=9), the patients were significantly older (mean age: 72 years; p=0.008) and tumors were more likely to be of apocrine morphology (56%, p= 0.001), of lower grade (55% were grade 3; p=0.009), and showed lower proliferation index (mean ki-67: 32%; p= 0.002). Cluster 3 (n=28) included younger patients (mean: 55 years) and tumors with higher grade (92% were grade 3) and proliferation index (mean ki-67: 75%). Cluster 2 (n=7) represented cases with intermediate features between Clusters 1 and 3. All patients in Cluster 1 were white, while Clusters 2 and 3 included non-white women. Cluster 1 included a significantly higher percentage of HER2-low tumors (HER2 1+ or 2+ by IHC and negative fluorescence in situ hybridization) (p=0.03; cluster 1: 89%, cluster 2: 28%, cluster 3: 46%). The vast majority of patients with germline BRCA1/2 mutation were found in Cluster 3 (67% of patients with genetic testing in Cluster 3 had germline BRCA1/2 mutation). There was no difference between the clusters in relation to stage, recurrence, and outcome. Conclusion: DNA methylation profiling is a promising tool to classify TNBC patients into clinicopathologically relevant groups, and we are continuing to expand this cohort. Classification by DNA methylation profile may result in better risk stratification for TNBC patients, which can inform their system therapy. In addition, specific methylation profiles have the potential to lead to the development of specific targeted therapies to improve the prognosis and survival for these patients. Citation Format: Lawrence H. Lin, Ivy Tran, Yiying Yang, Paolo Cotzia, Daniel Roses, Freya Schnabel, Farbod Darvishian, Matija Snuderl. DNA Methylation Analysis of Triple Negative Breast Cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-07.

Histopathological View of Benign Essential Blepharospasm: Orbicularis Oculi Hormone Receptor Levels.

Benign essential blepharospasm (BEB) is a focal dystonia characterized by involuntary contractions of the orbicularis oculi and periocular muscles. We aimed to investigate the effects of muscle receptor levels on the etiopathogenesis of blepharospasm by evaluating the orbicularis oculi estrogen receptor (ER) and androgen receptor (AR) levels. Four blepharospasm patients (2 females and 2 males) who underwent upper lid blepharoplasty and/or orbicularis myomectomy and 4 healthy cases (2 females, 2 males) that had upper lid blepharoplasty were included. The pretarsal, preseptal, and orbital parts of the orbicularis muscles of the patients who underwent orbicularis myomectomy and the waste muscle tissue materials taken from the preseptal orbicularis muscles of the patients who had only upper blepharoplasty were analyzed. Immunohistochemical staining was performed with estrogen alpha and androgen. In healthy men, the orbicularis oculi muscle was stained with ER at a moderate intensity and with AR at a high intensity. In men with blepharospasm, the orbicularis oculi were not stained with ER at all, but at a high intensity with AR. In healthy women, the orbicularis oculi were stained with ER and AR at a high intensity (>50%). In women with blepharospasm, the staining intensities of both receptors were moderate. We determined a decrease in ER and AR in females and almost the absence of ER in males with BEB. This decrease in ER may be associated with a functional abnormality in mitochondria and the decrease in hormonal receptors may be associated with sarcopenia in orbicularis oculi muscle fibers.

RF03 | PMON290 Transcriptional regulation of immune-pathways by chromatin reader ZMYND8 in triple-negative breast cancer

Abstract Zinc Finger transcription factors are crucial in modulating various cellular processes, including inflammation and immune response. Zinc Finger MYND (Myeloid, Nervy, and DEAF-1) type containing 8 (ZMYND8), a putative chromatin reader. We have observed this chromatin reader's unique role in regulating immune genes expression in triple-negative breast cancer (TNBC). TNBC is a very aggressive subtype of breast cancer. It is characterized as a heterogeneous group of tumors defined by the absence of estrogen receptor, progesterone receptor, and overexpression of the human epidermal growth factor receptor 2 gene. Ectopic expression and genetic deletion of ZMYND8 in triple-negative breast cancer cells modulated the expression of several genes implicated in immune response and cancer. Mechanistically, ZMYND8 regulates transcription by epigenetically modulating key promoters of target genes. Collectively our results elucidate a novel role for ZMYND8 in triple-negative cancer biology. This work was supported by grants from the Cancer Prevention and Research Institute of Texas (RR170020) and the Lizanell and Colbert Coldwell Foundation to S.S.G. Presentation: Saturday, June 11, 2022 1:54 p.m. - 1:59 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Effects of Combined Pentadecanoic Acid and Tamoxifen Treatment on Tamoxifen Resistance in MCF-7/SC Breast Cancer Cells.

Estrogen receptors are indicators of breast cancer adaptability to endocrine therapies, such as tamoxifen. Deficiency or absence of estrogen receptor α (ER−α) in breast cancer cells results in reduced efficacy of endocrine therapy. Here, we investigated the effect of combined tamoxifen and pentadecanoic acid therapy on ER−α−under−expressing breast cancer cells. Drug resistance gene expression patterns were determined by RNA sequencing analysis and in vitro experiments. For the first time, we demonstrate that the combined treatment of pentadecanoic acid, an odd−chain fatty acid, and tamoxifen synergistically suppresses the growth of human breast carcinoma MCF−7 stem cells (MCF−7/SCs), which were found to be tamoxifen−resistant and showed reduced ER−α expression compared with the parental MCF−7 cells. In addition, the combined treatment synergistically induced apoptosis and accumulation of sub−G1 cells and suppressed epithelial−to−mesenchymal transition (EMT). Exposure to this combination induces re−expression of ER−α at the transcriptional and protein levels, along with suppression of critical survival signal pathways, such as ERK1/2, MAPK, EGFR, and mTOR. Collectively, decreased ER−α expression was restored by pentadecanoic acid treatment, resulting in reversal of tamoxifen resistance. Overall, pentadecanoic acid exhibits the potential to enhance the efficacy of endocrine therapy in the treatment of ER−α−under−expressing breast cancer cells.

Ceritinib is a novel triple negative breast cancer therapeutic agent

BackgroundTriple-negative breast cancers (TNBCs) are clinically aggressive subtypes of breast cancer. TNBC is difficult to treat with targeted agents due to the lack of commonly targeted therapies within this subtype. Androgen receptor (AR) has been detected in 12–55% of TNBCs. AR stimulates breast tumor growth in the absence of estrogen receptor (ER), and it has become an emerging molecular target in TNBC treatment.MethodsCeritinib is a small molecule inhibitor of tyrosine kinase and it is used in the therapy of non-small lung cancer patients. Enzalutamide is a small molecule compound targeting the androgen receptor and it is used to treat prostate cancer. Combination therapy of these drugs were investigated using AR positive breast cancer mouse xenograft models. Also, combination treatment of ceritinib and paclitaxel investigated using AR− and AR low mouse xenograft and patient derived xenograft models.ResultsWe screened 133 FDA approved drugs that have a therapeutic effect of AR+ TNBC cells. From the screen, we identified two drugs, ceritinib and crizotinib. Since ceritinib has a well- defined role in androgen independent AR signaling pathways, we further investigated the effect of ceritinib. Ceritinib treatment inhibited RTK/ACK/AR pathway and other downstream pathways in AR+ TNBC cells. The combination of ceritinib and enzalutamide showed a robust inhibitory effect on cell growth of AR+ TNBC cells in vitro and in vivo. Interestingly Ceritinib inhibits FAK-YB-1 signaling pathway that leads to paclitaxel resistance in all types of TNBC cells. The combination of paclitaxel and ceritinib showed drastic inhibition of tumor growth compared to a single drug alone.ConclusionsTo improve the response of AR antagonist in AR positive TNBC, we designed a novel combinational strategy comprised of enzalutamide and ceritinib to treat AR+ TNBC tumors through the dual blockade of androgen-dependent and androgen-independent AR signaling pathways. Furthermore, we introduced a novel therapeutic combination of ceritinib and paclitaxel for AR negative or AR-low TNBCs and this combination inhibited tumor growth to a great extent. All agents used in our study are FDA-approved, and thus the proposed combination therapy will likely be useful in the clinic.

Discovery of fused benzimidazole-imidazole autophagic flux inhibitors for treatment of triple-negative breast cancer

Triple-negative breast cancer (TNBC) with the absence of estrogen receptor (ER), progesterone receptor (PR) and HER2 ptotein, is the highly aggressive subtype of breast cancer that exhibits poor prognosis and high tumor recurrence. It is vital to develop effective agents regulating the core molecular pathway of TNBC. Through a medium throughput screening and iterative medicinal chemistry optimization, we identified compound 7h as an autophagic flux inhibitor, which showed potent activities against human TNBC (MDA-MB-231 and MDA-MB-468) cell lines with IC50 values of 8.3 μM, and 6.0 μM, respectively, which are comparable to the potency of 5-FU and Cisplatin, the first line therapies for TNBC. Extensive investigation of mechanisms of action indicated that 7h inhibits autophagic flux and sequential accumulation of p62, leading to DNA damage and disrepair in TNBC cells. Importantly, nuclear p62 accumulation induced by compound 7h results in the inhibition of RNF168-mediated chromatin ubiquitination and the degradation of HR-related proteins in regulating the DNA damage response (DDR) process. In in vivo studies, compound 7h completely suppressed tumor growth in the MDA-MB-231 xenograft model at a dose of 15 mg/kg/q.d. Our findings indicate that compound 7h is an autophagic flux inhibitor and induced the degradation of HR-related proteins. Compound 7h could be potentially developed as an anti-cancer therapeutics for TNBC.

EpCAM- and EGFR-Specific Antibody Drug Conjugates for Triple-Negative Breast Cancer Treatment.

Triple-negative breast cancer (TNBC) is a group of heterogeneous and refractory breast cancers with the absence of estrogen receptor (ER), progesterone receptor (PgR) and epidermal growth factor receptor 2 (HER2). Over the past decade, antibody drug conjugates (ADCs) have ushered in a new era of targeting therapy. Since the epidermal growth factor receptor (EGFR) and epithelial cell adhesion molecule (EpCAM) are over expressed on triple-negative breast cancer, we developed novel ADCs by conjugating benzylguanine (BG)-modified monomethyl auristatin E (MMAE) to EpCAM- and EGFR-specific SNAP-tagged single chain antibody fragments (scFvs). Rapid and efficient conjugation was achieved by SNAP-tag technology. The binding and internalization properties of scFv-SNAP fusion proteins were confirmed by flow cytometry and fluorescence microscopy. The dose-dependent cytotoxicity was evaluated in cell lines expressing different levels of EGFR and EpCAM. Both ADCs showed specific cytotoxicity to EGFR or EpCAM positive cell lines via inducing apoptosis at a nanomolar concentration. Our study demonstrated that EGFR specific scFv-425-SNAP-BG-MMAE and EpCAM-specific scFv-EpCAM-SNAP-BG-MMAE could be promising ADCs for the treatment of TNBC.