Basal-like Breast Tumors Research Articles

Alpha-6 integrin deletion delays the formation of Brca1/p53-deficient basal-like breast tumors by restricting luminal progenitor cell expansion

BackgroundThe aberrant amplification of mammary luminal progenitors is at the origin of basal-like breast cancers associated with BRCA1 mutations. Integrins mediate cell–matrix adhesion and transmit mechanical and chemical signals that drive epithelial stem cell functions and regulate tumor progression, metastatic reactivation, and resistance to targeted therapies. Consistently, we have recently shown that laminin-binding integrins are essential for the expansion and differentiation of mammary luminal progenitors in physiological conditions. As over-expression of the laminin-binding α6 integrin (Itgα6) is associated with poor prognosis and reduced survival in breast cancer, we here investigate the role of Itgα6 in mammary tumorigenesis.MethodsWe used Blg-Cre; Brca1F/F; Trp53F/F mice, a model that phenocopies human basal-like breast cancer with BRCA1 mutations. We generated mutant mice proficient or deficient in Itgα6 expression and followed tumor formation. Mammary tumors and pretumoral tissues were characterized by immunohistochemistry, flow cytometry, RT-qPCR, Western blotting and organoid cultures. Clonogenicity of luminal progenitors from preneoplastic glands was studied in 3D Matrigel cultures.ResultsWe show that Itga6 deletion favors activation of p16 cell cycle inhibitor in the preneoplastic tissue. Subsequently, the amplification of luminal progenitors, the cell of origin of Brca1-deficient tumors, is restrained in Itgα6-deficient gland. In addition, the partial EMT program operating in Brca1/p53-deficient epithelium is attenuated in the absence of Itgα6. As a consequence of these events, mammary tumor formation is delayed in Itgα6-deficient mice. After tumor formation, the lack of Itgα6 does not affect tumor growth but rather alters their differentiation, resulting in reduced expression of basal cell markers.ConclusionsOur data indicate that Itgα6 has a pro-tumorigenic role in Blg-Cre; Brca1F/F; Trp53F/F mice developing basal-like mammary tumors. In particular, we reveal that Itgα6 is required for the luminal progenitor expansion and the aberrant partial EMT program that precedes the formation of BRCA1 deficient tumors.

The VEGF-Hypoxia Signature Is Upregulated in Basal-like Breast Tumors from Women of African Ancestry and Associated with Poor Outcomes in Breast Cancer.

Black women experience the highest breast cancer mortality rate compared with women of other racial/ethnic groups. To gain a deeper understanding of breast cancer heterogeneity across diverse populations, we examined a VEGF-hypoxia gene expression signature in breast tumors from women of diverse ancestry. We developed a NanoString nCounter gene expression panel and applied it to breast tumors from Nigeria (n = 182) and the University of Chicago (Chicago, IL; n = 161). We also analyzed RNA sequencing data from Nigeria (n = 84) and The Cancer Genome Atlas (TCGA) datasets (n = 863). Patient prognosis was analyzed using multiple datasets. The VEGF-hypoxia signature was highest in the basal-like subtype compared with other subtypes, with greater expression in Black women compared with White women. In TCGA dataset, necrotic breast tumors had higher scores for the VEGF-hypoxia signature compared with non-necrosis tumors (P < 0.001), with the highest proportion in the basal-like subtype. Furthermore, necrotic breast tumors have higher scores for the proliferation signature, suggesting an interaction between the VEGF-hypoxia signature, proliferation, and necrosis. T-cell gene expression signatures also correlated with the VEGF-hypoxia signature when testing all tumors in TCGA dataset. Finally, we found a significant association of the VEGF-hypoxia profile with poor outcomes when using all patients in the METABRIC (P < 0.0001) and SCAN-B datasets (P = 0.002). These data provide further evidence for breast cancer heterogeneity across diverse populations and molecular subtypes. Interventions selectively targeting VEGF-hypoxia and the immune microenvironment have the potential to improve overall survival in aggressive breast cancers that disproportionately impact Black women in the African Diaspora.

A novel bioinformatic approach reveals cooperation between Cancer/Testis genes in basal-like breast tumors

Breast cancer is the most prevalent type of cancer in women worldwide. Within breast tumors, the basal-like subtype has the worst prognosis, prompting the need for new tools to understand, detect, and treat these tumors. Certain germline-restricted genes show aberrant expression in tumors and are known as Cancer/Testis genes; their misexpression has diagnostic and therapeutic applications. Here we designed a new bioinformatic approach to examine Cancer/Testis gene misexpression in breast tumors. We identify several new markers in Luminal and HER-2 positive tumors, some of which predict response to chemotherapy. We then use machine learning to identify the two Cancer/Testis genes most associated with basal-like breast tumors: HORMAD1 and CT83. We show that these genes are expressed by tumor cells and not by the microenvironment, and that they are not expressed by normal breast progenitors; in other words, their activation occurs de novo. We find these genes are epigenetically repressed by DNA methylation, and that their activation upon DNA demethylation is irreversible, providing a memory of past epigenetic disturbances. Simultaneous expression of both genes in breast cells in vitro has a synergistic effect that increases stemness and activates a transcriptional profile also observed in double-positive tumors. Therefore, we reveal a functional cooperation between Cancer/Testis genes in basal breast tumors; these findings have consequences for the understanding, diagnosis, and therapy of the breast tumors with the worst outcomes.

Programming a Ferroptosis-to-Apoptosis Transition Landscape Revealed Ferroptosis Biomarkers and Repressors for Cancer Therapy.

Ferroptosis and apoptosis are key cell-death pathways implicated in several human diseases including cancer. Ferroptosis is driven by iron-dependent lipid peroxidation and currently has no characteristic biomarkers or gene signatures. Here a continuous phenotypic gradient between ferroptosis and apoptosis coupled to transcriptomic and metabolomic landscapes is established. The gradual ferroptosis-to-apoptosis transcriptomic landscape is used to generate a unique, unbiased transcriptomic predictor, the Gradient Gene Set (GGS), which classified ferroptosis and apoptosis with high accuracy. Further GGS optimization using multiple ferroptotic and apoptotic datasets revealed highly specific ferroptosis biomarkers, which are robustly validated in vitro and in vivo. A subset of the GGS is associated with poor prognosis in breast cancer patients and PDXs and contains different ferroptosis repressors. Depletion of one representative, PDGFA-assaociated protein 1(PDAP1), is found to suppress basal-like breast tumor growth in a mouse model. Omics and mechanistic studies revealed that ferroptosis is associated with enhanced lysosomal function, glutaminolysis, and the tricarboxylic acid (TCA) cycle, while its transition into apoptosis is attributed to enhanced endoplasmic reticulum(ER)-stress and phosphatidylethanolamine (PE)-to-phosphatidylcholine (PC)metabolic shift. Collectively, this study highlights molecular mechanisms underlying ferroptosis execution, identified a highly predictive ferroptosis gene signature with prognostic value, ferroptosis versus apoptosis biomarkers, and ferroptosis repressors for breast cancer therapy.

The expression of O-linked glycosyltransferase GALNT7 in breast cancer is dependent on estrogen-, progesterone-, and HER2-receptor status, with prognostic implications.

GALNT7 is a glycosyltransferase enzyme transferring N-acetylgalactosamine to initiate O-linked glycosylation in the Golgi apparatus. Breast cancer is the most common cancer in women globally. Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2; ERBB2) are important biomarkers in the prognosis and molecular subtyping of breast cancer. Here, we showed that ER-positive, PR-positive or HER2-positive breast tumors have higher expression of GALNT7 compared to ER-negative, PR-negative or HER2-negative breast tumors, respectively. We found that CpG-aggregated methylation of GALNT7 gene is decreased, and in parallel, its transcript levels are increased in breast cancer compared to healthy breast tissue. We observed that the difference in the expression of GALNT7 between negative and positive status of the receptors is the highest for HER2, followed by ER and PR, pointing that HER2 might be relatively more influential than ER and PR on the expression of GALNT7 in breast cancer. We reported that basal-like breast tumors have decreased expression of GALNT7 compared to non-basal-like tumors, and that high GALNT7 expression is associated with favorable relapse-free and distant metastasis-free survival in HER2 status-dependent manner in breast cancer patients. Moreover, we showed that GALNT7 expression in breast cancer is cell type- (epithelial vs stromal cells), tumor grade- and ethnicity-dependent. Combined, we propose that GALNT7 might contribute to different clinical outcomes depending on the receptor status in breast cancer, and that a better understanding of GALNT7 and its function in the context of breast cancer is needed.

MicroRNAs as Molecular Biomarkers for the Characterization of Basal-like Breast Tumor Subtype.

Breast cancer is a heterogeneous disease highlighted by the presence of multiple tumor variants and the basal-like breast cancer (BLBC) is considered to be the most aggressive variant with limited therapeutics and a poor prognosis. Though the absence of detectable protein and hormonal receptors as biomarkers hinders early detection, the integration of genomic and transcriptomic profiling led to the identification of additional variants in BLBC. The high-throughput analysis of tissue-specific micro-ribonucleic acids (microRNAs/miRNAs) that are deemed to have a significant role in the development of breast cancer also displayed distinct expression profiles in each subtype of breast cancer and thus emerged to be a robust approach for the precise characterization of the BLBC subtypes. The classification schematic of breast cancer is still a fluid entity that continues to evolve alongside technological advancement, and the transcriptomic profiling of tissue-specific microRNAs is projected to aid in the substratification and diagnosis of the BLBC tumor subtype. In this review, we summarize the current knowledge on breast tumor classification, aim to collect comprehensive evidence based on the microRNA expression profiles, and explore their potential as prospective biomarkers of BLBC.

Human Breast Tissue Microbiota Reveals Unique Microbial Signatures that Correlate with Prognostic Features in Adult Ethiopian Women with Breast Cancer.

Breast cancer (BC) is the leading cause of cancer mortality among women in Ethiopia. Overall, women of African ancestry have the highest death toll due to BC compared to other racial/ethnic groups. The cause of the disparity in mortality is unclear. Recently, studies conducted in the United States and other high-income countries highlighted the role of microbial dysbiosis in BC initiation, tumor growth, and treatment outcome. However, the extent to which inter-individual differences in the makeup of microbiota are associated with clinical and histopathological outcomes in Ethiopian women has not been studied. The goal of our study was to profile the microbiome in breast tumor and normal adjacent to tumor (NAT) tissues of the same donor and to identify associations between microbial composition and abundance and clinicopathological factors in Ethiopian women with BC. We identified 14 microbiota genera in breast tumor tissues that were distinct from NAT tissues, of which Sphingobium, Anaerococcus, Corynebacterium, Delftia, and Enhydrobacter were most significantly decreased in breast tumors compared to NAT tissues. Several microbial genera significantly differed by clinicopathological factors in Ethiopian women with BC. Specifically, the genus Burkholderia more strongly correlated with aggressive triple negative (TNBC) and basal-like breast tumors. The genera Alkanindiges, Anoxybacillus, Leifsonia, and Exiguobacterium most strongly correlated with HER2-E tumors. Luminal A and luminal B tumors also correlated with Anoxybacillus but not as strongly as HER2-E tumors. A relatively higher abundance of the genus Citrobacter most significantly correlated with advanced-stage breast tumors compared to early-stage tumors. This is the first study to report an association between breast microbial dysbiosis and clinicopathological factors in Ethiopian women.

Abstract P4-03-14: Proinflammatory Dietary Patterns and Risk of Total and Subtypes of Breast Cancer Among US Women

Abstract Background: Dietary patterns promoting chronic inflammation, including the empirical dietary inflammatory pattern (EDIP), have been shown to strongly influence risk of weight gain, type 2 diabetes, cardiovascular disease, and colorectal cancer. However, it is unclear if this dietary pattern is associated with other tumors in which the mechanisms are not totally understood such as breast cancer. Methods: We prospectively followed 76,295 women from the Nurses’ Health Study (NHS, 1984-2016) and 91,078 women from the Nurses’ Health Study II (NHSII, 1991-2017). Diet was assessed by food frequency questionnaires (FFQs) every 4 years. The inflammatory potential of diet was evaluated using the previously established EDIP based on plasma C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor alpha receptor 2 (TNF-αR2). Higher scores indicate higher inflammatory potential of the diet. Results: During 4,153,676 person-years of follow-up, we documented 10,632 invasive breast cancer cases (6,807 NHS; 3,825 NHSII). In the pooled multivariable-adjusted analyses, women in the highest, compared with the lowest, EDIP quintile were at higher breast cancer risk (HRQ5vsQ1=1.12; 95% CI 1.05, 1.20; P-trend&amp;lt; 0.001). This association was attenuated after adjusting for weight change since age 18 y, although it remained significant (HRQ5vsQ1=1.07; 95% CI 1.00, 1.14; P-trend=0.01). In subtype analyses, we found evidence that the inflammatory potential of diet influenced breast cancer risk differentially by ER status (P-heterogeneity=0.038) and by molecular phenotype (P-heterogeneity=0.007), with the association between EDIP and breast cancer limited to ER-negative tumors (HRQ5vsQ1=1.31; 95% CI: 1.11, 1.55; P-trend=0.002; for ER-positive tumors, HR Q5vsQ1=1.03; 95% CI, 0.96, 1.12;P-trend=0.10) and basal-like tumors (HRQ5vsQ1=1.78; 95% CI: 1.19, 2.65; P-trend=0.004). Further adjustment for weight change since age 18 y did not materially alter the association for these subtypes. Conclusions: Dietary patterns with high potential to contribute to chronic systemic inflammation, based on higher EDIP scores, were associated with a modestly increased risk of breast cancer, which was more pronounced for ER-negative and basal-like breast tumors. Citation Format: Andrea Romanos-Nanclares, Walter C Willett, Bernard A Rosner, Daniel G Stover, Sarah Asad, Sagar Sardesai, Michelle D Holmes, Wendy Y Chen, Rulla M. Tamimi, Fred K Tabung, A Heather Eliassen. Proinflammatory Dietary Patterns and Risk of Total and Subtypes of Breast Cancer Among US Women [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 P4-03-14.

KLF5 inhibition potentiates anti-PD1 efficacy by enhancing CD8+ T-cell-dependent antitumor immunity.

Background: Immune checkpoint blockers (ICBs) are revolutionized therapeutic strategies for cancer, but most patients with solid neoplasms remain resistant to ICBs, partly because of the difficulty in reversing the highly immunosuppressive tumor microenvironment (TME). Exploring the strategies for tumor immunotherapy is highly dependent on the discovery of molecular mechanisms of tumor immune escape and potential therapeutic target. Krüppel-like Factor 5 (KLF5) is a cell-intrinsic oncogene to promote tumorigenesis. However, the cell-extrinsic effects of KLF5 on suppressing the immune response to cancer remain unclear. Methods: We analyzed the immunosuppressive role of KLF5 in mice models transplanted with KLF5-deleted/overexpressing tumor cells. We performed RNA sequencing, immunohistochemistry, western blotting, real time-PCR, ELISA, luciferase assay, chromatin immunoprecipitation (ChIP), and flow cytometry to demonstrate the effects of KLF5 on CD8+ T cell infiltration and related molecular mechanism. Single-cell RNA sequencing and spatial transcriptomics analysis were applied to further decipher the association between KLF5 expression and infiltrating immune cells. The efficacy of KLF5/COX2 inhibitors combined with anti-programmed cell death protein 1 (anti-PD1) therapy were explored in pre-clinical models. Finally, a gene-expression signature depending on KLF5/COX2 axis and associated immune markers was created to predict patient survival. Results: KLF5 inactivation decelerated basal-like breast tumor growth in a CD8+ T-cell-dependent manner. Transcriptomic profiling revealed that KLF5 loss in tumors increases the number and activated function of T lymphocytes. Mechanistically, KLF5 binds to the promoter of the COX2 gene and promotes COX2 transcription; subsequently, KLF5 deficiency decreases prostaglandin E2 (PGE2) release from tumor cells by reducing COX2 expression. Inhibition of the KLF5/COX2 axis increases the number and functionality of intratumoral antitumor T cells to synergize the antitumorigenic effects of anti-PD1 therapy. Analysis of patient datasets at single-cell and spatial resolution shows that low expression of KLF5 is associated with an immune-supportive TME. Finally, we generate a KLF5/COX2-associated immune score (KC-IS) to predict patient survival. Conclusions: Our results identified a novel mechanism responsible for KLF5-mediated immunosuppression in TME, and targeting the KLF5/COX2/PGE2 axis is a critical immunotherapy sensitizer.

PTEN alterations in sporadic and BRCA1-associated triple negative breast carcinomas

The similarities between sporadic basal-like breast cancer (BLBC) and BRCA1-mutated breast tumours raise the possibility that deregulation of the same pathway may underlie these tumour types. The aim of this study was to determine if PTEN aberrations are characteristic of both BRCA1 tumours and sporadic TN breast carcinomas with low BRCA1 expression, and can thus be used to identify sporadic tumours potentially sensitive to PARP inhibitors. Twelve BRCA1 tumours, 19 non-BRCA familial breast tumours and 71 unselected TN breast carcinomas were screened for PTEN mutations and assessed for PTEN expression and BRCA1 mRNA expression. Loss of PTEN expression was observed in 67% of BRCA1 tumours and more specifically in 89% of TN BRCA1 tumours highlighting the link between PTEN loss and BLBC in the context of germline BRCA1 mutations. Regarding unselected TN tumours, 56% showed PTEN expression loss and 35% displayed low BRCA1 mRNA expression. Unlike familial breast cancers with low BRCA1 mRNA expression, no significant correlation was observed between the loss of PTEN expression and low BRCA1 mRNA expression in this unselected TN tumours panel. Our data suggest that, unlike the germinal context, PTEN and BRCA1 alterations in sporadic TN breast tumours are independent events.

A Hybrid Method Based on Feature Fusion for Breast Cancer Classification using Histopathological Images

Breast cancer is the most common type of cancer in women today, and it ranks second after lung cancer with a very high mortality rate. If it is detected late, the treatment of breast cancer becomes very difficult. Although there are various methods for the detection of breast cancer, there is still a need for auxiliary diagnosis and treatment methods. In this study, a hybrid method is proposed to investigate the development of basal-like breast tumors and classify basal-like breast cancer types using histopathological images. In the study, firstly, appropriate features that support the accurate classification between tumor and non-tumor regions are extracted from histopathological images. Then the dataset is created by combining the obtained features. In the last stage of the study, the classification of images is carried out by using bag of words (BoW) and deep neural networks (DNN) techniques in a hybrid manner. Generally, immunohistochemical markers are used for this classification, but the performance of these markers remains at 60%. The performance of the classification accuracy of the proposed system is increased with the proposed hybrid classifier based on feature fusion. As a result of the study, 94.5% classification accuracy is achieved on the training set, while 80.8% classification accuracy is succeed on the test set. As a result, it is verified that successful results are achieved in the classification of basal-like breast cancer on histopathological images using the proposed hybrid method based on feature fusion.

Increased gene expression variability in BRCA1-associated and basal-like breast tumours

PurposeInherited variants in the cancer susceptibility genes, BRCA1 and BRCA2 account for up to 5% of breast cancers. Multiple gene expression studies have analysed gene expression patterns that maybe associated with BRCA12 pathogenic variant status; however, results from these studies lack consensus. These studies have focused on the differences in population means to identified genes associated with BRCA1/2-carriers with little consideration for gene expression variability, which is also under genetic control and is a feature of cellular function.MethodsWe measured differential gene expression variability in three of the largest familial breast cancer datasets and a 2116 breast cancer meta-cohort. Additionally, we used RNA in situ hybridisation to confirm expression variability of EN1 in an independent cohort of more than 500 breast tumours.ResultsBRCA1-associated breast tumours exhibited a 22.8% (95% CI 22.3–23.2) increase in transcriptome-wide gene expression variability compared to BRCAx tumours. Additionally, 40 genes were associated with BRCA1-related breast cancers that had ChIP-seq data suggestive of enriched EZH2 binding. Of these, two genes (EN1 and IGF2BP3) were significantly variable in both BRCA1-associated and basal-like breast tumours. RNA in situ analysis of EN1 supported a significant (p = 6.3 × 10−04) increase in expression variability in BRCA1-associated breast tumours.ConclusionOur novel results describe a state of increased gene expression variability in BRCA1-related and basal-like breast tumours. Furthermore, genes with increased variability may be driven by changes in DNA occupancy of epigenetic effectors. The variation in gene expression is replicable and led to the identification of novel associations between genes and disease phenotypes.

Cholesterol-Induced Metabolic Reprogramming in Breast Cancer Cells Is Mediated via the ERRα Pathway.

Simple SummaryThere is increasing evidence that obesity and high circulating cholesterol levels are associated with an increased risk of recurrence and a higher mortality rate in breast cancer patients via altering the metabolic programming in breast cancer cells. However, the underlying molecular mechanism by which high cholesterol levels reprogram the metabolic pathways in breast cancer cells is not well-understood. We have previously demonstrated that cholesterol acts as an endogenous agonist of estrogen-related receptor α (ERRα), a strong regulator of cellular metabolism. The aim of the current study is to demonstrate whether cholesterol/obesity mediates its pathogenic effect in breast cancer cells via altering metabolic pathways in an ERRα-dependent manner. The findings of this study provide mechanistic insights into the link between cholesterol/obesity and metabolic reprogramming in breast cancer patients and reveal the metabolic vulnerabilities in such breast cancer patients that could be therapeutically targeted.The molecular mechanism underlying the metabolic reprogramming associated with obesity and high blood cholesterol levels is poorly understood. We previously reported that cholesterol is an endogenous ligand of the estrogen-related receptor alpha (ERRα). Using functional assays, metabolomics, and genomics, here we show that exogenous cholesterol alters the metabolic pathways in estrogen receptor-positive (ER+) and triple-negative breast cancer (TNBC) cells, and that this involves increased oxidative phosphorylation (OXPHOS) and TCA cycle intermediate levels. In addition, cholesterol augments aerobic glycolysis in TNBC cells although it remains unaltered in ER+ cells. Interestingly, cholesterol does not alter the metabolite levels of glutaminolysis, one-carbon metabolism, or the pentose phosphate pathway, but increases the NADPH levels and cellular proliferation, in both cell types. Importantly, we show that the above cholesterol-induced modulations of the metabolic pathways in breast cancer cells are mediated via ERRα. Furthermore, analysis of the ERRα metabolic gene signature of basal-like breast tumours of overweight/obese versus lean patients, using the GEO database, shows that obesity may modulate ERRα gene signature in a manner consistent with our in vitro findings with exogenous cholesterol. Given the close link between high cholesterol levels and obesity, our findings provide a mechanistic explanation for the association between cholesterol/obesity and metabolic reprogramming in breast cancer patients.

Single-Cell RNA Sequencing Reveals the Cellular Origin and Evolution of Breast Cancer in BRCA1 Mutation Carriers.

The cell of origin and the development of breast cancer are not fully elucidated in BRCA1 mutation carriers, especially for estrogen receptor (ER)-positive breast cancers. Here, we performed single-cell RNA sequencing (RNA-seq) on 82,122 cells isolated from the breast cancer tissues and adjacent or prophylactic normal breast tissues from four BRCA1 mutation carriers and three noncarriers. Whole-exome sequencing was performed on breast tumors from the four BRCA1 mutation carriers; for validation, bulk RNA-seq was performed on adjacent normal breast tissues from eight additional BRCA1 mutation carriers and 14 noncarriers. Correlation analyses suggested that breast cancers in BRCA1 mutation carriers might originate from luminal cells. The aberrant luminal progenitor cells with impaired differentiation were significantly increased in normal breast tissues in BRCA1 mutation carriers compared with noncarriers. These observations were further validated by the bulk RNA-seq data from additional BRCA1 mutation carriers. These data suggest that the cell of origin of basal-like breast tumors (ERneg) in BRCA1 mutation carriers might be luminal progenitor cells. The expression of TP53 and BRCA1 was decreased in luminal progenitor cells from normal breast tissue in BRCA1 mutation carriers, which might trigger the basal/mesenchymal transition of luminal progenitors and might result in basal-like tumor development. Furthermore, ERhigh luminal tumors might originate from mature luminal cells. Our study provides in-depth evidence regarding the cells of origin of different breast cancer subtypes in BRCA1 mutation carriers. SIGNIFICANCE: Single-cell RNA-seq data indicate that basal-like breast cancer (ERneg) might originate from luminal progenitors, and ERhigh luminal breast cancer might originate from mature luminal cells in BRCA1 mutation carriers.

Annexin A1 Is Required for Efficient Tumor Initiation and Cancer Stem Cell Maintenance in a Model of Human Breast Cancer.

Simple SummaryTriple-negative breast cancer (TNBC) has a poor outcome compared to the other major breast cancer subtypes and new therapies are needed. We sought to clarify the functions of a ubiquitous protein, Annexin A1, in the development and progression of TNBC. We found that Annexin A1 expression correlated with poor patient prognosis in basal-like breast tumors and also in the basal like-2 subset of TNBCs. Stable knockdown of Annexin A1 attenuated the growth of SUM149 xenografts, which model basal-like 2 tumors. In a polyoma middle T antigen-driven allograft model of breast cancer, Annexin A1 depletion markedly delayed tumor formation, induced epithelial to mesenchymal transition and upregulated basal markers. Finally, loss of Annexin A1 resulted in the loss of a discrete CD24+/Sca1− population containing putative tumor-initiating cells. Collectively, our data demonstrate a novel cell-autonomous role for Annexin A1 in the promotion of tumor-forming capacity in certain TNBC tumors.Triple-negative breast cancer (TNBC) has a poor outcome compared to other breast cancer subtypes, and new therapies that target the molecular alterations driving tumor progression are needed. Annexin A1 is an abundant multi-functional Ca2+ binding and membrane-associated protein. Reported roles of Annexin A1 in breast cancer progression and metastasis are contradictory. Here, we sought to clarify the functions of Annexin A1 in the development and progression of TNBC. The association of Annexin A1 expression with patient prognosis in subtypes of TNBC was examined. Annexin A1 was stably knocked down in a panel of human and murine TNBC cell lines with high endogenous Annexin A1 expression that were then evaluated for orthotopic growth and spontaneous metastasis in vivo and for alterations in cell morphology in vitro. The impact of Annexin A1 knockdown on the expression of genes involved in mammary epithelial cell differentia tion and epithelial to mesenchymal transition was also determined. Annexin A1 mRNA levels correlated with poor patient prognosis in basal-like breast tumors and also in the basal-like 2 subset of TNBCs. Unexpectedly, loss of Annexin A1 expression had no effect on either primary tumor growth or spontaneous metastasis of MDA-MB-231_HM xenografts, but abrogated the growth rate of SUM149 orthotopic tumors. In an MMTV-PyMT driven allograft model of breast cancer, Annexin A1 depletion markedly delayed tumor formation in both immuno-competent and immuno-deficient mice and induced epithelial to mesenchymal transition and upregulation of basal markers. Finally, loss of Annexin A1 resulted in the loss of a discrete CD24+/Sca1− population containing putative tumor initiating cells. Collectively, our data demonstrate a novel cell-autonomous role for Annexin A1 in the promotion of tumor-forming capacity in a model of human breast cancer and suggest that some basal-like TNBCs may require high endogenous tumor cell Annexin A1 expression for continued growth.

Genomic Mapping Identifies Mutations in RYR2 and AHNAK as Associated with Favorable Outcome in Basal-Like Breast Tumors Expressing PD1/PD-L1.

Treatment with anti-PD-L1 antibodies has shown efficacy in basal-like breast cancer. In this context, identification of pre-activated immune tumors is a main goal. Here we explore mutations in PD1 and PD-L1 high-expressing tumors to identify genomic correlates associated with outcome. To do so, RNA-seq and mutation data from 971 breast cancer patients from the TCGA dataset were used to identify most prevalent mutations in patients with high levels of PD1 and PD-L1. Transcriptomic signatures associated with the selected mutations were identified and analyzed in terms of outcome and immune cell infiltration. We identified co-occurrent mutations in RYR2 and AHNAK in 8% and 5% of basal-like tumors respectively, which conferred good prognosis in patients with high expression of PD1 and PD-L1 genes. The transcriptomic signature associated with these mutations, composed of CXCL9, GBP5, C1QA, IL2RG, CSF2RB, IDO1 and LAG3 genes, also conferred good prognosis and correlated with immune infiltrations within the tumors. The joint signature classified patients with favorable relapse-free survival (HR: 0.28; CI: 0.2–0.38; p = 1.7 × 10−16) and overall survival (HR: 0.18; CI: 0.09–0.34; p = 6.8 × 10−9), showing a stronger prediction capacity than previous reported signatures. In conclusion, we describe two novel mutations and their transcriptomic signature, both associated with a favorable outcome and immune infiltrates in PD1 and PD-L1 high-expressing basal-like tumors.

Triggering a switch from basal- to luminal-like breast cancer subtype by the small-molecule diptoindonesin G via induction of GABARAPL1

Breast cancer is a heterogeneous disease that includes different molecular subtypes. The basal-like subtype has a poor prognosis and a high recurrence rate, whereas the luminal-like subtype confers a more favorable patient prognosis partially due to anti-hormone therapy responsiveness. Here, we demonstrate that diptoindonesin G (Dip G), a natural product, exhibits robust differentiation-inducing activity in basal-like breast cancer cell lines and animal models. Specifically, Dip G treatment caused a partial transcriptome shift from basal to luminal gene expression signatures and prompted sensitization of basal-like breast tumors to tamoxifen therapy. Dip G upregulated the expression of both GABARAPL1 (GABAA receptor-associated protein-like 1) and ERβ. We revealed a previously unappreciated role of GABARAPL1 as a regulator in the specification of breast cancer subtypes that is dependent on ERβ levels. Our findings shed light on new therapeutic opportunities for basal-like breast cancer via a phenotype switch and indicate that Dip G may serve as a leading compound for the therapy of basal-like breast cancer.

Genomic mapping to identify mutations in RYR2 and AHNAK in basal-like breast tumors expressing PD-L1.

1027 Background: Basal-like breast cancer is a specific subtype of breast tumors with limited therapeutic options. Although treatment with the anti-PD-L1 antibody atezolizumab has recently shown clinical activity in this setting, not all patients do respond even expressing high levels of PD-L1. In the present article we explored the presence of mutations in breast cancer tumors with high expression of PD1 and PD-L1 with the aim to identify molecular correlates associated with outcome. Methods: We used RNA-seq and mutational data from 971 breast cancer patients using the TCGA dataset, to identify mutations in patients with high levels of PD1 and PD-L1. Data analysis was performed using DESeq R and MAFTools Bioconductor packages. Transcriptomic signatures from the identified mutations were associated with outcome using the Kapplan-Meyer Plotter tool. We correlated the identified transcripts with immune populations using TIMER online tool and correlation between genes with Cancertool online platform. Results: We identified co-occurrent mutations in RYR2 and AHNAK in 8% and 5% of basal like tumors, respectively, in patients with high levels of PD1 and PD-1. The transcriptomic signature of these mutations conferred good prognosis for relapse free survival (RFS) and overall survival (OS). CXCL9 for RYR2 and GBP5, C1QA, IL2RG, CSF2RB and IDO1 for AHNAK were the most relevant genes identified in these signatures. Expression of CXCL9, GBP5, IL2RG and IDO1 correlated with the presence of immune cell populations mainly dendritic cells. This signature, including CXCL9, GBP5, C1QA, IL2RG, CSF2RB and IDO1 classified patients with favorable RFS (HR 0.27 CI 0.2-0.30; p = 1.1e-16) and OS (HR 0.18 CI 0.09-0.34; p = 6.8e-9). This signature showed a stronger prediction capacity compared with already described immunologic signatures. Finally we identify that LAG3 was the only gene commonly present in both signatures and correlated positively with the expression of PD1 and PD-L1. Conclusions: We describe two novel mutations which transcriptomic signatures associated with favorable outcome in basal-like tumors that express elevated levels of PD1 and PD-L1. Future studies should be performed to confirm the role of these mutations and signatures in relation with clinical activity of PD1/PD-L1 inhibitors.

Genomic Signatures of Immune Activation Predict Outcome in Advanced Stages of Ovarian Cancer and Basal-Like Breast Tumors.

There is an unmet need for new therapies in metastatic ovarian cancer and basal-like breast cancer since no curative therapies are currently available. Immunotherapy has shown to be active in several solid tumors, but particularly more in those where a pre-activated immune state does exist. In this work, we aim to identify biomarkers that could distinguish immune-activated tumors and predict response to therapies in ovarian and basal-like breast cancer, as well as their association with the level of tumor immune infiltration. We found that the combined expression of IFNG, CD30, CXCL13, and PRF1 correlated with better overall survival (OS) in advanced stage ovarian cancer. This was confirmed using an independent dataset from TCGA. Interestingly, we observed that this gene combination also predicted for better prognosis in ovarian tumors with low mutational load, which typically respond less to immunotherapy. Expression of IFNG, CD30, CXCL13, and PRF1 was associated with increased level of immune infiltrates (CD8+ T cells, dendritic cells, and neutrophils) within the tumor. Moreover, we found that these gene signature also correlated with an increased OS and with a higher level of tumor immune infiltrates (B cells, CD8+ T cells, CD4+ T cells, neutrophils, and dendritic cells) in basal-like breast cancer. In conclusion, our analysis identifies genes signatures with potential to recognize immune activated ovarian and basal-like breast cancers with favorable prognosis and with a remarkable predictive capacity in tumors with low mutational burden. The presented results led to a hypothesis being formulated, but prospective clinical studies are needed to support a potential clinical application.

A high expression ratio of RhoA/RhoB is associated with the migratory and invasive properties of basal-like Breast Tumors.

Basal-like breast cancer is among the most aggressive cancers and there is still no effective targeted treatment. In order to identify new therapeutic targets, we performed mRNA-Seq on eight breast cancer cell lines. Among the genes overexpressed in basal-like tumors, we focused on the RhoA and RhoB genes, which encode small GTPases known to play a role in the actin cytoskeleton, allowing cells to migrate.qRT-PCR and Western blotting were used for expression studies. Migratory and invasive properties were analysed by wound healing and Boyden chambers assays. Stress fibers formation was evaluated by fluorescent actin labeling. Rho siRNA, small inhibitor Rhosin treatment and BRCA1 transfection were performed to study the role of Rho and BRCA1 proteins.We showed that strong expression of RhoA and low expression of RhoB was associated with the basal-like subtype of breast cancer. Decreasing RhoA expression reduced the migratory and invasive capacities of basal-like cell lines, while decreasing RhoB expression increased these capacities. Rhosin, an inhibitor of RhoA, could also reduce the migration of basal-like cell lines. Rho proteins are involved in the formation of stress fibers, a conformation of the actin cytoskeleton found in migrating cells: inhibition of RhoA expression decreased the formation of these fibers. BRCA1, a gene frequently inactivated in basal-like tumors, appears to play a role in the differential expression of RhoA and RhoB in these tumors, as the restoration of BRCA1 expression in a BRCA1-mutated basal-like cell line decreased expression of RhoA and increased expression of RhoB, resulting in reduced migratory capacity.These results suggest Rho proteins as potential therapeutic targets for basal-like and BRCA1-mutated breast cancer, as migration and acquisition of mesenchymal properties are key functional pathways in these tumors with high metastatic potential.