Breast Tumor Tissues Research Articles

Dissection of triple-negative breast cancer microenvironment and identification of potential therapeutic drugs using single-cell RNA sequencing analysis

Breast cancer remains a leading cause of mortality in women worldwide. Triple-negative breast cancer (TNBC) is a particularly aggressive subtype characterized by rapid progression, poor prognosis, and lack of clear therapeutic targets. In the clinic, delineation of tumor heterogeneity and development of effective drugs continue to pose considerable challenges. Within the scope of our study, high heterogeneity inherent to breast cancer was uncovered based on the landscape constructed from both tumor and healthy breast tissue samples. Notably, TNBC exhibited significant specificity regarding cell proliferation, differentiation, and disease progression. Significant associations between tumor grade, prognosis, and TNBC oncogenes were established via pseudotime trajectory analysis. Consequently, we further performed comprehensive characterization of the TNBC microenvironment. A crucial epithelial subcluster, E8, was identified as highly malignant and strongly associated with tumor cell proliferation in TNBC. Additionally, epithelial-mesenchymal transition (EMT)-associated fibroblast and M2 macrophage subclusters exerted an influence on E8 through cellular interactions, contributing to tumor growth. Characteristic genes in these three cluster cells could therefore serve as potential therapeutic targets for TNBC. The collective findings provided valuable insights that assisted in the screening of a series of therapeutic drugs, such as pelitinib. We further confirmed the anti-cancer effect of pelitinib in an orthotopic 4T1 tumor-bearing mouse model. Overall, our study sheds light on the unique characteristics of TNBC at single-cell resolution and the crucial cell types associated with tumor cell proliferation that may serve as potent tools in the development of effective anti-cancer drugs.

Immunohistochemical findings and clinicopathological features of breast cancers with pathogenic germline mutations in Non-BRCA genes

Deleterious germline mutations in multiple genes confer an increased breast cancer (BC) risk. Immunohistochemical (IHC) expression of protein products of mutated high-risk genes has not been investigated in BC. We hypothesized that pathogenic mutations may lead to an abnormal IHC expression pattern in the tumor cells. BCs with deleterious germline mutations in CHEK2, ATM, PALB2 &PTEN were identified. Immunohistochemistry was performed using Dako staining platform on formalin fixed paraffin embedded tumor tissue. Primary antibodies for PALB2 (ab202970), ATM [2C1(1A10)}, CHK2 (EPR4325), and PTEN (138G6) proteins were used for BCs with respective deleterious mutations. IHC expression was assessed in tumor and adjacent benign breast tissue. Total 27 BCs with 10 CHEK2, 9 ATM, 6 PALB2 & 2 PTEN deleterious germline mutations were identified. IHC staining was performed on 8 CHEK2, 7 ATM, 6 PALB2 & 2 PTEN cases. Abnormal CHEK2 IHC staining was identified in 7/8(88%) BCs. Three distinct CHK2 IHC patterns were noted: 1) Strong diffuse nuclear positivity (5 BC), 2) Null-pattern (2 BC), & 3) Normal breast–like staining in 1 BC Four of 5 (80%) strong CHK2 staining BC had missense CHEK2 mutations. Null-pattern was present with a missense & a frameshift mutation. Normal breast-like CHEK2 IHC staining pattern was present in 1 BC with CHEK2 frameshift mutation. Loss of nuclear/cytoplasmic PTEN IHC expression was noted in 2 in-situ carcinomas. Abnormal PTEN and CHK2 IHC were present in atypical ductal hyperplasia and flat epithelial atypia. ATM and PALB2 IHC expression patterns were similar in tumor cells and benign breast epithelium: mild to moderate intensity nuclear and cytoplasmic staining. We report abnormal CHEK2 IHC expression in 88% of BCs with pathogenic CHEK2 mutations. With PTEN and CHEK2 pathogenic mutations, abnormal IHC patterns are seen in early atypical proliferative lesions. IHC may be applied to identify CHEK2 &PTEN mutated BCs and precursor lesions.

Breast Tumor Tissue Image Classification Using Single-Task Meta Learning with Auxiliary Network.

Breast cancer has a high mortality rate among cancers. If the type of breast tumor can be correctly diagnosed at an early stage, the survival rate of the patients will be greatly improved. Considering the actual clinical needs, the classification model of breast pathology images needs to have the ability to make a correct classification, even in facing image data with different characteristics. The existing convolutional neural network (CNN)-based models for the classification of breast tumor pathology images lack the requisite generalization capability to maintain high accuracy when confronted with pathology images of varied characteristics. Consequently, this study introduces a new classification model, STMLAN (Single-Task Meta Learning with Auxiliary Network), which integrates Meta Learning and an auxiliary network. Single-Task Meta Learning was proposed to endow the model with generalization ability, and the auxiliary network was used to enhance the feature characteristics of breast pathology images. The experimental results demonstrate that the STMLAN model proposed in this study improves accuracy by at least 1.85% in challenging multi-classification tasks compared to the existing methods. Furthermore, the Silhouette Score corresponding to the features learned by the model has increased by 31.85%, reflecting that the proposed model can learn more discriminative features, and the generalization ability of the overall model is also improved.

Is fibroblast growth factor 11 (FGF11) a predictive marker for breast cancer?

The prognostic role of fibroblast growth factor 11 (FGF11) has only been reported in cancers such as nasopharyngeal carcinoma and prostate cancer. The role of FGF11 in breast cancer is not fully known. It was aimed to compare FGF11 expression levels in de novo metastatic hormone receptor-positive, human epidermal reseptor-2-negative breast tumor tissue and healthy breast tissue and investigate the effect of the FGF11 expression on survival in breast cancer patients. To determine the FGF11 expression rate, breast tumor tissue of breast cancer patients diagnosed by breast biopsy and healthy breast tissue of healthy individuals who underwent breast biopsy due to benign lesions were used. The study population included 38 breast cancer patients and 24 healthy controls. The number of patients with a FGF11 expression level score of 1 (15.8% vs 12.5%), score of 2 (18.4% vs 12.5%), and score of 3 (31.6% vs 0%) was significantly higher in the patient group compared to the healthy control group. The median overall survival and progression-free survival were numerically better in the group with a FGF11 expression score of 0 to 1 than the group with a FGF11 expression score of 2 and 3, but this difference was not statistically significant. FGF11 may be a predictive marker for breast cancer formation. Additionally, with new FGF11-targeted treatment agents to be developed, endocrine resistance may be reduced, and better survival results may be achieved in hormone receptor-positive, human epidermal reseptor-2-negative breast cancer.

Anti-IL-8 monoclonal antibodies inhibits the autophagic activity and cancer stem cells maintenance within breast cancer tumor microenvironment.

Breast cancer tumor microenvironment (TME) is a promising target for immunotherapy. Autophagy, and cancer stem cells (CSCs) maintenance are essential processes involved in tumorigenesis, tumor survival, invasion, and treatment resistance. Overexpression of angiogenic chemokine interleukin-8 (IL-8) in breast cancer TME is associated with oncogenic signaling pathways, increased tumor growth, metastasis, and poor prognosis. Thus, we aimed to investigate the possible anti-tumor effect of neutralizing antibodies against IL-8 by evaluating its efficacy on autophagic activity and breast CSC maintenance. IL-8 monoclonal antibody supplemented tumor tissue culture systems from 15 females undergoing mastectomy were used to evaluate the expression of LC3B as a specific biomarker of autophagy and CD44, CD24 as cell surface markers of breast CSCs using immunofluorescence technique. Our results revealed that anti-IL-8 mAb significantly decreased the level of LC3B in the cultured tumor tissues compared to its non-significant decrease in the normal breast tissues.Anti-IL-8 mAb also significantly decreased the CD44 expression in either breast tumors or normal cultured tissues. While it caused a non-significant decrease in CD24 expression in cultured breast tumor tissue and a significant decrease in its expression in the corresponding normal ones. Anti-IL-8 monoclonal antibody exhibits promising immunotherapeutic properties through targeting both autophagy and CSCs maintenance within breast cancer TME.

Abstract 3437: Stromal tumor-infiltrating lymphocytes in association with body size and composition among Black and White women with breast cancer

Abstract Background: Obesity is a major risk factor for breast cancer and negatively affects disease prognosis. Tumor-infiltrating lymphocytes (TILs) in the stroma of breast tissue play a major role in eliminating cancer cells and modulating immunotherapy efficacy. We aim to study the association between body size and composition with stromal TILs in breast tumor tissue by race. Methods: The study included 920 Black and 395 White women with newly diagnosed invasive breast cancer who have stromal TIL scores from the Women’s Circle of Health Study (WCHS). Body mass index (BMI), waist circumference (WC), and waist-to-hip ratio (WHR) were measured during the home interview. Pre-treatment H&E-stained tumor tissue sections were reviewed by our study pathologist and stromal TILs were scored on a scale of 0-100% at 10% increment according to the recommendations of the International TILs Working Group. Linear regressions adjusting for age at diagnosis, tumor grade, stage, and molecular subtype were used to test the differences in TILs across the categories of body size and composition indicators separately for Black and White women. Results: A higher stromal TILs score was associated with more aggressive breast cancer features, including higher grade, larger tumor size, more advanced stage, hormone receptor (HR) negativity, and human epidermal growth factor receptor 2 (HER2) positivity in both Black and White women (all P<0.05). Among Black women, WHR >0.90 vs ≤0.85 was associated with lower TILs overall (β = -3.5%, P = 0.045) and in triple-negative tumors (β = -12.3%, P = 0.003). Among White women, BMI 25.0 - 29.9 vs <25 was associated with higher TILs in HR-/HER2+ tumors (β = 28.1%, P = 0.031). A pattern suggested that BMI ≥30 vs <25 was associated with lower TILs in both HR-/HER2+ (β = -10.2, P=0.52) and triple-negative (β = -12.8, P=0.14) tumors, although the associations were not significant. No association was observed between TILs and any of the body composition parameters in women with HR+ tumors. Conclusions and Relevance: Body size and composition correlate with stromal TILs mainly in patients with HR- tumors, and this relation may be race-dependent. These findings suggest that body size and composition potentially modulate breast cancer treatment outcomes at least in part through TILs-dependent mechanisms. Funding: This work was in part supported by grants from the US National Institutes of Health (P01CA151135, R01CA100598, R01CA185623, P30CA016056, P30CA072720, K07CA201334, and R37CA248371), US Army Medical Research and Material Command (DAMD-17-01-1-0334), the Breast Cancer Research Foundation (CBA), and the Philip L. Hubbell family. Citation Format: Rand T. Akasheh, Thaer Khoury, Song Yao, Angela Omilian, Chi-Chen Hong, Elisa V. Bandera, Bonnie Qin, Nur Zeinomar, Christine Ambrosone, Ting-Yuan David Cheng. Stromal tumor-infiltrating lymphocytes in association with body size and composition among Black and White women with 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 3437.

Abstract 6698: Tumor microbiota associated with immune enrichment in breast tumor

Abstract Asian cancer patients have enriched immune profiles when compared to Caucasians. This is associated with improved overall survival and potentially better response to immunotherapy. Yet the reason behind enriched Asian immune profiles is unclear. This study investigated microbes found in Malaysian breast tumors in association with immune profiles. We used an existing dataset of RNA-seq data from fresh frozen breast tumor tissue (n=978). Sequencing reads were trimmed and filtered to remove human reads. Non-human reads were mapped to Kraken2 and relative abundance was estimated using Bracken. Microbiome analysis was conducted on R following Microbiome OMA guidebook. The top five genera found in the Malaysian Breast Cancer (MyBrCa) cohort were Pseudomonas, Siphoviridae, Bacillus, Escherichia and Klebsiella. Alpha diversity of basal subtype microbiota was significantly higher than Her2 subtype (Shannon index, p=0.027) whilst the other subtypes were more homogenous. MyBrCa patients with low immune scores were significantly different from high immune scores while controlling for age, stage, grade, ethnicity and PAM50 subtype (PERMANOVA, F-statistic = 1.656, p=0.05). Differential abundance analysis revealed Sulfidibacter and Priestia to be significantly different in abundance between high vs low IFNg groups (FDR-adjusted p-value < 0.05) using ALDEx2, ANCOM-BC2 and ZicoSeq and LinDA algorithms. Sulfidibacter was significantly different between high vs low immune group for Bindea combined immune score and ESTIMATE score groups (FDR-adjusted p-value <0.05). An independent breast cancer cohort (n=419) was used as validation where Sulfidibacter and Priestia were also significantly different in abundance for immune high vs low groups. Machine learning using Random Forest model predicted immune group with an AUC of 0.79 (CI = 0.74, 0.83; p-value < 0.01). The top five most important features contributing to the random forest model were Sulfidibacter, Prestia, Cutibacterium, Acinetobacter and Rhizobium. This study is the largest to date that characterize the tumor microbiota of an Asian cohort of breast cancers, where Sulfidibacter and Priestia, both relatively new and uncharacterized bacteria, were differentially abundant among patients with high vs low immune scores. Citation Format: Li-Fang Yeo, Bernard KB Lim, Tania Islam, Li-Ying Teoh, Suniza Jamaris, Mee-Hoong See, Lai-Meng Looi, Nur Aishah Mohd Taib, Cheng-Har Yip, Pathmanathan Rajadurai, Soo-Hwang Teo, Jia Wern Pan. Tumor microbiota associated with immune enrichment in breast tumor [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 6698.

Abstract 6108: Investigation of breast tumor biology and microenvironment in women of African descent using a single cell multiomic approach

Abstract Women of African descent are at an increased risk of developing and dying from aggressive subtypes of breast cancer. A connection between aggressive disease and Western Sub-Saharan African ancestry has been postulated, but it remains largely unknown to what extent breast cancer in Africa is reminiscent of breast cancer in U.S. African American (AA) women who experience disproportionately high mortality rates. We performed ATAC- and RNA-sequencing on 9 human triple-negative breast cancer cell lines of U.S. origin and discovered that African ancestry influences the chromatin landscape, leading to disparate transcription factor (TF) activity and downstream gene expression patterns indicative of an aggressive tumor biology. Here, we describe an ambitious study that employs single-nucleus (sn) ATAC- and RNA-sequencing (snMultiome) of frozen breast tumors to characterize chromatin accessibility and gene expression patterns with single-cell resolution in AA (n=33), Kenyan (n=25), and European American (EA, n=24) women in relation to genetic ancestry, risk factor exposures, clinical characteristics, and 5-year survival. To achieve this, we successfully isolated intact, high-quality single nuclei from archival frozen breast tumor tissue through an optimized combination of enzymatic digestion and automated tissue homogenization. We performed snMultiome sequencing of 82 tumors using the 10x Genomics platform. Following filtering, normalization (SCT for snRNA; LSI for snATAC), peak calling (MACS2), and integration (Harmony), our dataset includes a total of 296,557 nuclei. Cancerous (163,419 nuclei) and non-cancerous (133,138 nuclei) cells were distinguished based on DNA copy number (CopyKat). Within the microenvironment, 11 major immune, epithelial, and stromal cell types were successfully annotated, exhibiting distinct patterns by population group (e.g. AA tumors showed markedly increased abundance of myeloid and T-cells, while Kenyan tumors showed increased abundance of pericytes and fibroblasts). A large number of enriched TFs within each cell type varied significantly by population group, suggesting distinct chromatin accessibility patterns related to genetic ancestry. Within cancerous cells, striking intra- and inter-tumoral heterogeneity was observed by genetic ancestry even within molecular subtype groups. Current efforts focus on in-depth molecular characterization of ancestry- and risk factor-related differences in the tumor epithelium and microenvironment and distinct signatures present in lethal disease. This project holds the potential to yield crucial insights into how ancestry or other factors may influence the etiology of different breast cancer subtypes, as well as produce clinically actionable biomarkers and therapeutic targets to enhance precision medicine within patient populations at high risk for aggressive disease. Citation Format: Alexandra R. Harris, Huaitian Liu, Brittany Jenkins-Lord, Tiffany H. Dorsey, Francis Makokha, Shahin Sayed, Gretchen Gierach, Stefan Ambs. Investigation of breast tumor biology and microenvironment in women of African descent using a single cell multiomic approach [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 6108.

Study on breast cancer image detection and classification based on residual connected convolutional neural network (CNN)

This paper presents a method for breast cancer image detection and classification based on convolutional neural networks. While traditional breast cancer detection methods carry the risk of subjectivity and misclassification, machine learning can improve the accuracy and efficiency of detection by training algorithms to automatically identify breast cancer lesions in patients. The dataset of this paper includes 7909 microscopic images of breast tumor tissues with different magnifications collected from 82 patients, which are divided into two categories: benign and malignant tumors. In this paper, residual connection is designed for convolutional neural networks, which is a cross-layer connection method that can effectively solve the gradient vanishing and gradient explosion problems in deep neural networks, and at the same time, improve the generalization ability and training speed of the model. In this paper, the dataset is divided into training set, validation set and test set according to the ratio of 6:2:2. Through training and validation, the prediction accuracy of the obtained model on the test set reaches 85.5%, which achieves good prediction results in breast cancer image detection and classification. Eventually, the model's loss converged at 0.53 and the AUC was 0.832. The research results in this paper are of great significance for the early diagnosis and treatment of breast cancer. Automatic identification and classification of breast cancer images by machine learning algorithms can reduce the subjective judgment of doctors, improve the accuracy and efficiency of detection, and provide important support for the early diagnosis and treatment of breast cancer. Meanwhile, the research method and results of this paper also provide reference and reference for image detection and classification of other malignant tumors.

Immunohistochemical and molecular evaluation of TUSC2 expression in breast cancer.

Tumor suppressor candidate 2 has shown to be deleted in lung, colon, and bladder cancer types. In the present study, we aimed to investigate the expression of TUSC2 in breast cancer. A total of thirty patients with breast cancer were included in the study. Normal and tumor tissue samples from fresh mastectomy materials were stored at -80C until the number of cases was completed for gene expression analysis. Histopathological examination was carried out with routine hematoxylin & eosin method. TUSC2 staining was performed for immunohistochemical analysis. The tumors of thirteen patients were Luminal A, fourteen patients were Luminal B, one patient was cerbB2(+), and tumors of two patients were triple-negative. Ki67 proliferation index was less than 14% in fifteen cases and tumor size was less than 2cm in seven cases. Lymphovascular invasion and lymph node metastasis were present in thirteen cases. Statistically, TUSC2 expression significantly decreased or was lost in breast tumor tissues compared to normal tissues (p < 0.0001). TUSC2 expression decreased as the Ki67 proliferation index increased (p = 0.0003), and TUSC2 expression decreased as tumor size increased (p = 0.0483). The loss or decrease in the TUSC2 expression was significant as the tumor grade increased (p = 0.3740). Gene expression analysis correlated with immunohistochemistry results. The results of the present study demonstrated a decrease or loss of TUSC2 expression in breast cancer tissue compared to normal tissue. A correlation was found between TUSC2 expression and Ki67 proliferation index and tumor size.

Non-Contact Electric Field May Induced Higher CD4, CD8, Caspase-8, and Caspase-9 Protein Expression in Breast Tumor Tissue of Rats (Rattus norvegicus Berkenhout, 1769)

Non-contact electric field therapy has been studied as one of less invasive and safe alternative method in cancer therapy. Some studies reported that non-contact electric field therapy with low intensity (50 - 60 V/m) and medium frequency (150 kHz) could slower the rat’s breast tumor growth and did not cause significant damage on the rat's kidney and liver. However, further study needed to evaluate the potency of non-contact electric field to be developed into effective cancer therapy. Thus, we investigated the immune response of non-contact electric field therapy through the detection of CD4 and CD8 protein expression and the mechanism of apoptosis through the detection of caspase-8 and caspase-9 proteins on breast tumor tissue of rats. Twenty four rats were divided into 4 groups: 1. non-induction - non-therapy (NINT), 2. non-induction - therapy (NIT), 3. induction - non-therapy (INT), and 4. induction - therapy (IT). DMBA (7,12-dimethylbenz(α)anthracene) 20 mg/kg body weight was used to induced breast tumor formation. Rats with breast nodules that had reached 1 cm in size were exposed to an ECCT device with 150 kHz and 50 - 60 V/m electric fields. Further, the breast tissues were collected for routine histological and immunohistochemistry preparation for CD4, CD8, caspase 8, as well as caspase 9 detection. The data were statistically analyzed using Mann-Whitney U-Test method, with a significance level of p &lt; 0.05 using the SPSS 16.0 version. The scoring results were compared between the INT and IT groups. Our results showed that non-contact electric field therapy could suppress breast tumor growth and improved its histological structure. Interestingly, the breast tissue of IT group qualitatively had slightly more necrotic and apoptotic cells than that of INT group. Moreover, the IT group showed by higher CD4 and CD8 as well as higher caspase 8 and caspase 9 expression. Collecting all the data together, we concluded that non-contact electric field therapy potent to improve histological structure of breast tumor.

Induced Current Electro-Thermal Imaging for Breast Tumor Detection: A Numerical and Experimental Study.

This study proposes using magnetically induced currents in medical infrared imaging to increase the temperature contrast due to the electrical conductivity differences between tumors and healthy tissues. There are two objectives: (1) to investigate the feasibility of this active method for surface and deep tumors using numerical simulations, and (2) to demonstrate the use of this method through different experiments conducted with phantoms that mimic breast tissues. Tumorous breasts were numerically modeled and simulated in active and passive modes. At 750kHz, the applied current was limited for breast tissue-tumor conductivities (0.3S/m and0.75S/m) according to the local specific absorption rate limit of 10W/kg.Gelatin-based and mashed potato phantoms were produced to mimic tumorous breast tissues. In the simulation studies, the induced current changed the temperature contrast on the imaging surface, and the tumor detection sensitivity increased by 4mm.An 11-turn 70-mm-long solenoid coil was constructed, 20A current was applied for deep tumors, and a difference of up to 0.4 C was observed in the tumor location compared with the temperature in the absence of the tumor.Similarly, a 23-turn multi-layer coil was constructed, and a temperature difference of 0.4 C was observed. The temperature contrast on the body surface changed, and the tumor detection depth increased with the induced currents in breast IR imaging. The proposed active thermal imaging method was validated using numerical simulations and in vitro experiments.

Quantitative Changes in Pro-Oncogenic and Tumor-Suppressing MicroRNA in the Regional Lymph Node during Photodynamic Therapy of Experimental Breast Cancer.

We studied quantitative changes in microRNAs in the axillary lymph node (regional lymph node of the mammary gland) and in the breast tumor tissue. The quantity of pro-oncogenic microRNAs (miR-21 and miR-221) in the regional lymph node decreased in the group after photodynamic therapy of the breast cancer. At the same time, the quantity of pro-oncogenic miR-27a remained decreased in this group, as well as in the group with breast cancer without treatment. The quantity of tumor-suppressing miR-429 in the regional lymph node after photodynamic therapy of breast cancer remained at the same level with the quantity in the group of rats with breast cancer without treatment.

Abstract A071: Identification and characterization of functionally distinct isoforms of estrogen receptor alpha in breast cancer

Abstract We used long- and short-read RNA sequencing of breast tumor tissue, breast cancer cell lines, and healthy tissues to create a comprehensive annotation of alternative mRNA isoforms of estrogen receptor alpha (ER, gene symbol ESR1) and combined this with functional studies of alternative protein isoforms. The ER is frequently overexpressed in breast cancer and is used in the clinic as a prognostic and treatment-predictive factor. It is also a drug target. The ER is a transcription factor and regulates gene expression in response to estrogen, a hormone that can stimulate breast cell proliferation and contribute to tumor growth. The gene has multiple promoters, and alternative mRNA isoforms have been linked to more aggressive breast tumors, suggesting that the splicing pattern of ER transcripts may influence breast cancer biology and clinical outcome. We cloned and characterized six alternative protein isoforms of ER. They were selected based on expression level in sequencing data, splice junction support, and only contained one alternative splicing event each compared to the full-length ER. Experimental characterization was done in cell lines and included transcription factor activity, alone and upon co-expression with full-length ER, response to the ER-targeting drugs tamoxifen and fulvestrant, as well as subcellular localization. Two alternative isoforms exhibited transcription factor activity, although at lower levels than the full-length ER, while isoforms with larger deletions in the DNA-binding domain or lacking parts of the ligand-binding domain were inactive. One isoform had a dominant negative effect on the activity of the full-length protein. Furthermore, full-length ER and three alternative isoforms were found in both cytoplasm and nucleus, while the remaining isoforms were only present in the cytoplasm. Isoforms capable of entering the nucleus showed increased nuclear localization upon estradiol treatment, with one isoform accumulating significantly higher and one lower than the full-length ER. Co-transfection of full-length ER and isoforms lacking transcription factor activity did not alter their distribution, suggesting a possible role of heterodimerization with full-length ER for the dominant negative isoform in the nucleus. In ongoing experiments, we are studying the regulation, expression, and function of alternative isoforms of ER in cell line models of drug resistance. These findings highlight the complexity and diversity of ER isoforms in breast cancer and suggest that specific isoforms may have distinct functional roles in tumor progression and response to therapy. Our work provides insights into the role of alternative mRNA splicing in breast cancer development and progression. Citation Format: Juliane Albrecht, Carlos Enrique Balcazar, Helena Persson, Völundur Hafstað, Cornelia Börjesson Freitag, Johan Vallon-Christersson, Jari Häkkinen. Identification and characterization of functionally distinct isoforms of estrogen receptor alpha in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A071.

Abstract B003: Investigating PHLDA2 as a Genetic Driver of Breast Cancer Metastasis

Abstract Metastasis is cancer’s lethal driver. Metastatic cancer cells leave the primary tumor and travel through the blood stream to distal sites. These cancer cells are aggressive, highly recurring and pose challenges for clinical treatment. There is a critical need to define drivers of metastasis in order to identify novel drug targets and improve patient prognosis. Our previous work identified a potential breast cancer metastasis promoting gene known as PHLDA2. By single cell RNA sequencing, PHLDA2 gene expression was elevated in metastatic lesions compared to primary breast tumor tissue. This gene has not previously been linked to cancer metastasis; therefore, the objective of the current study is to confirm PHLDA2 as a driver of breast cancer metastasis and define the mechanisms by which it exerts its pro-metastatic function. We manipulated multiple breast cancer models to have elevated or reduced PHLDA2 levels to investigate its role in metastasis. We have additionally explored the mechanism of PHLDA2 in metastasis at the RNA level by qPCR, the protein level by western blot analysis, and the organ level using immunofluorescence staining and bulk sequencing. We confirmed using our breast cancer models of elevated or reduced PHLDA2 expression that PHLDA2 has a pro-metastatic function. Bulk RNA sequencing data revealed that PHLDA2 promotes extracellular matrix (ECM) remodeling. This finding is significant as ECM remodeling plays a critical role in cancer progression and metastasis. Investigation of PHLDA2’s pro-metastatic function is necessary to gain deeper insights into the mechanisms of cancer cell spread and reveal novel drug targets to improve patient survival. Citation Format: Paige Halas, Hannah Savage, Dennis Ma, Jacob Insua-Rodriguez, Sharmila Mallya, Connie Chan, Radhika Domadia, Tatyana Lev, Devon Lawson. Investigating PHLDA2 as a Genetic Driver of Breast Cancer Metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B003.

Breast Cancer: Mitochondria-Centered Metabolic Alterations in Tumor and Associated Adipose Tissue.

The close cooperation between breast cancer and cancer-associated adipose tissue (CAAT) shapes the malignant phenotype, but the role of mitochondrial metabolic reprogramming and obesity in breast cancer remains undecided, especially in premenopausal women. Here, we examined mitochondrial metabolic dynamics in paired biopsies of malignant versus benign breast tumor tissue and CAAT in normal-weight and overweight/obese premenopausal women. Lower protein level of pyruvate dehydrogenase and citrate synthase in malignant tumor tissue indicated decreased carbon flux from glucose into the Krebs cycle, whereas the trend was just the opposite in malignant CAAT. Simultaneously, stimulated lipolysis in CAAT of obese women was followed by upregulated β-oxidation, as well as fatty acid synthesis enzymes in both tumor tissue and CAAT of women with malignant tumors, corroborating their physical association. Further, protein level of electron transport chain complexes was generally increased in tumor tissue and CAAT from women with malignant tumors, respective to obesity. Preserved mitochondrial structure in malignant tumor tissue was also observed. However, mitochondrial DNA copy number and protein levels of PGC-1α were dependent on both malignancy and obesity in tumor tissue and CAAT. In conclusion, metabolic cooperation between breast cancer and CAAT in premenopausal women involves obesity-related, synchronized changes in mitochondrial metabolism.

Unique macrophage phenotypes activated by BMP signaling in breast cancer bone metastases.

Metastatic breast cancer (mBC) tissue in bone was systematically profiled to define the composition of the tumor microenvironment. Gene expression identified a high myeloid signature of patients with improved survival outcomes. Bone metastases were profiled by spatial proteomics to examine myeloid populations within the stroma that correlated with macrophage functions. Single-cell spatial analysis uncovered macrophage activation in the stroma of mBC bone lesions. Matched BC patient samples of primary breast tumor and bone metastasis tissues were compared for gene expression in the bone, where bone morphogenetic protein 2 (BMP2) was most significantly upregulated. Immune cell changes from breast to bone demonstrated a loss of lymphoid cells but a consistent population of macrophages. BMP-activated macrophages were increased uniquely in bone. Bone marrow-derived macrophage activation coupled with BMP inhibition increased inflammatory responses. Using experimental mouse models of mBC bone metastasis and trained immunity, we found that BMP inhibition restricts progression of metastases early in the macrophage activation state but not after tumors were established in the bone. This study revealed unique myeloid BMP activation states that are distinctly integrated with bone metastases.

Constitutional BRCA1 Epimutations: A Key for Understanding Basal‐Like Breast and High‐Grade Serous Ovarian Cancer

Germline pathogenic genetic variants in the BRCA1 and BRCA2 genes are the most frequent causes of familial breast and ovarian cancer. Contrasting BRCA2, epimutations in the BRCA1 gene are frequently detected in tissue from triple‐negative breast (TNBC) and high‐grade serous ovarian cancers (HGSOC). While studies over the last decade have reported BRCA1 epimutations in white blood cells (WBC) from breast and ovarian cancer patients, the potential hazard ratio for incident TNBC and HGSOC was not formally assessed until recently.Conducting a prospective nested case‐control study on women participating in the American Women’s Health Initiative Study, we provided firm evidence that mosaic WBC BRCA1 epimutations, even at allele frequencies &lt; 0.1%, are associated with a significantly increased risk of both incident HGSOC and TNBC &gt; 5 years after WBC collection. In a second study assessing BRCA1 epimutations in WBC and matched tumor samples from TNBC, our results indicated such epimutations to be the underlying cause of around 20% of TNBC, far exceeding the percentage of cases carrying BRCA1 germline pathogenic genetic variants.We detected primary constitutional BRCA1 epimutations in tissues derived from all three germ layers. They occur independently of BRCA1 promoter haplotypes but are present on the same allele in all WBC within affected individuals. Moreover, epimutations are consistently found on the same allele in normal and tumor breast tissue as well as in WBC. This finding, together with BRCA1 epimutations detected in WBC from newborns, strongly indicates an early embryonic event with clonal expansion affecting all germ layers.Future work in the field must lead to an understanding of exactly when and how the BRCA1 epimutations occur and, most importantly, whether primary constitutional epimutations in genes other than BRCA1 may cause an elevated risk of other cancer types.

Role of Human Cytomegalo virus and High Risk Human Papilloma Virus in Breast Cancer

Background: In Iraq, breast tumor is the greatest prevalent form of female malignancy. Increasing research in the past ten years shows that many human malignancies, including breast tumors, are associated with Human Cytomegalovirus and high-risk Human Papilloma Virus. Objectives: The aim of this research was to determine the role of HPV and HCMV in benign and malignant breast tumors. Methods: This retrospective research used 140 chosen formalin-fixed, paraffin-embedded pieces of breast specimen taken from 100 patients of breast tumor (70 cases of ductal mammary, 15 patients of lobular cancer, five persons mixed from ductal and lobular cancer and ten persons of medullary cancer) and 20 patients infected with benign epithelial lesion As well as 20 pieces of normal breast tissue as control groups. The range of age was 16-72 years. The study was conducted in hospitals and private laboratories in Kirkuk and Tikrit city from 15th January 2022 to 15th December 2022 using the in situ hybridization (ISH) technique. Results: The archival samples collected were related to breast cancer patients ranging from 16 years to 70 years of age, with a maximum age of 73 years in breast cancer and 60 years in healthy women. ISH found that CMV was positive in (25 percent) breast cancer tissues (25 / 100) according to the control group were negative. While in HPV demonstrated (45%) 45/ 100 breast cancer tissues compared to the control group were negative. Of these, The findings revealed that the spreading of HPV-DNA genotypes in malignant and benign cancer groups were as follows: HPV16 10 (20.8%), HPV 18 10 (20.8%), and HPV 31 28 (58.33%). These results suggest that HPV genotype 31 appeared at a greater rate than other genotypes (16 and 18). In 25 (55.6 percent) cases, the largest ratio of co-infection with several high-risk genotypes of HPV 16 + 18 + 31 was observed. Co-infection in the malignant group with other high-danger HPV genotypes was as follows: HPV 16 +18 6(13.33%), HPV 16 + 31 6(13.33), and HPV 18+31 8(17.8%). Positive signs suggesting high-risk HPV infection were detected for 1/1 (100 percent) of benign breast tumor tissues; 30(66.7 percent) mixed HPV infection was found in 45 cases of malignant breast tumors. Conclusion: The results of this study showed a connection between HCMV and high-oncogenic genotypes of HPV in breast cancer patients.

Extracellular matrix viscoelasticity regulates TGFβ1-induced epithelial-mesenchymal transition and apoptosis via integrin linked kinase.

Transforming growth factor (TGF)-β1 is a multifunctional cytokine that plays important roles in health and disease. Previous studies have revealed that TGFβ1 activation, signaling, and downstream cell responses including epithelial-mesenchymal transition (EMT) and apoptosis are regulated by the elasticity or stiffness of the extracellular matrix. However, tissues within the body are not purely elastic, rather they are viscoelastic. How matrix viscoelasticity impacts cell fate decisions downstream of TGFβ1 remains unknown. Here, we synthesized polyacrylamide hydrogels that mimic the viscoelastic properties of breast tumor tissue. We found that increasing matrix viscous dissipation reduces TGFβ1-induced cell spreading, F-actin stress fiber formation, and EMT-associated gene expression changes, and promotes TGFβ1-induced apoptosis in mammary epithelial cells. Furthermore, TGFβ1-induced expression of integrin linked kinase (ILK) and colocalization of ILK with vinculin at cell adhesions is attenuated in mammary epithelial cells cultured on viscoelastic substrata in comparison to cells cultured on nearly elastic substrata. Overexpression of ILK promotes TGFβ1-induced EMT and reduces apoptosis in cells cultured on viscoelastic substrata, suggesting that ILK plays an important role in regulating cell fate downstream of TGFβ1 in response to matrix viscoelasticity.