Basal-like Breast Cancer Patients Research Articles

Abstract PO2-03-08: Uncovering Novel Potential Prognostic Biomarkers in Basal-Like Breast Cancer using Transcriptomic Data of 1,899 Patients

Abstract Introduction: Patients with basal-like breast cancer (BLBC) predominantly represented by triple-negative breast cancer have shown a high recurrence rate and are characterized by poor prognosis. There is an urgent need to undercover reliable prognostic biomarkers that can help in the clinical management of such patients and identify additional therapeutic targets. The objective of this study was to create a comprehensive transcriptomic database on a large scale and leverage it to identify and prioritize cancer-related genes associated with BLBC patients’ outcomes. Methods: We identified breast cancer cohorts from public repositories that contained gene expression data at the transcriptome level, along with clinical follow-up information. BLBC were identified using the PAM50 signature. All samples were standardized using a standard array normalization coupled with scaling to have a mean expression across all genes of 1000 in each sample and incorporated into a unified database. Redundant samples were removed. For each gene, Cox univariate survival analysis was conducted, to account for multiple hypothesis testing, the false discovery rate was computed, and a significant cutoff of 1% was employed to determine the highest statistical significance. Association with RFS and OS was performed. Multivariate analysis was performed for selected genes involving clinical and pathological variables. To uncover higher-level functions related to altered RFS, gene ontology analysis was performed using the enrichGO function in the TNM plotter (http://www.tnmplot.com). Results: The complete integrated database comprises 1,899 samples from 52 breast cancer datasets. Altogether, 2,342 genes were correlated with relapse-free survival (RFS), and 1,149 genes were correlated with overall survival (OS). 619 genes were statistically significant for both RFS and OS. The most significant genes were ANGPTL4 (p=4.25E-08, HR=2.02), NHP2 (p=5.98E-10, HR=1.93), STK3 (p=4.86E-10, HR=1.93), GBE1 (p=2.77E-09, HR=1.86), and PMVK (p=3.65E-09, HR=1.85) for RFS and PINK1 (p=1.64E-05 , HR=3.31), CAMK2N1 (p=1.06E-07 , HR=2.93), CACFD1 (p=4.79E-04 , HR=2.61), SCAP (p=3.29E-04 , HR=2.6), SDC1 (p=2.81E-04 , HR=2.57), for OS. The most significant gene ontology biological processes upregulated in tumors with a worse prognosis include GO:0000184, nuclear-transcribed mRNA catabolic process, nonsense-mediated decay (p=6.64E-18); GO:0045047 , protein targeting to ER (p=6.64E-18); GO:0006614, SRP-dependent cotranslational protein targeting to membrane (p=9.08E-18); GO:0072599, establishment of protein localization to endoplasmic reticulum (p=1.30E-17); and GO:0006613, cotranslational protein targeting to membrane (p=1.90E-17). Conclusions: Our results help to prioritize genes and to neglect those which are most likely to fail in studies aiming to establish new clinically useful biomarkers and therapeutic targets in BLBC. Citation Format: Balazs Gyorffy, Libero Santarpia. Uncovering Novel Potential Prognostic Biomarkers in Basal-Like Breast Cancer using Transcriptomic Data of 1,899 Patients [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 PO2-03-08.

The A986S calcium sensing receptor variant supports the adaptation of breast cancer cells to high calcium — a novel paradigm in breast cancer progression

Breast cancer and other solid tumors that metastasize to bone are frequently accompanied by a progressive increase in circulating calcium as the disease becomes more malignant. The increase in circulating calcium not only disrupts systemic calcium homeostasis but also alters the ability of cells to sense slight changes in extracellular Ca2+ via the ubiquitous calcium sensing receptor (CaSR). Of the CASR polymorphisms at rs1801725 (p.A986S), rs1801726 (p.Q1011E), and rs1042636 (p.R990G) in exon 7 of the receptor, previous studies have shown that only the A986S CaSR variant is associated with calcium in breast, prostate and skin cancers. In this study, we assessed if exon 7 inactivating A986S (AS-CaSR) and Q1011E (QE-CaSR) variants of the receptor influence the survival of breast tumor cells at higher Ca2+. We generated isogenic HEK293T and BT-549 cells expressing these CaSR variants and carried out bulk RNA sequencing of high Ca2+ adapted control and breast cancer cells. CaSR expression was detected by western blotting using anti-flag M2 (Sigma) antibody. By using phospho-ERK1/2 as the readout, the activation of AS-CaSR required higher concentrations of Ca2+ (EC50 ~5.28 mM) compared to the QE-CaSR (EC50 of ~3.81 mM) and the wild type (WT-CaSR; EC50 ~3.69 mM) receptor. When the CaSR variants transfected cells initially cultured at 7.5 mM for 6 days, and then sub-cultured in medium containing 7.5 to 20 mM Ca2+, the viability of empty vector control and WT-CaSR transfected cells strongly decreased at Ca2+ levels beyond 7.5 mM and these cells were mostly necrotic at ≥15 mM Ca2+. The QE-CaSR variant transfected cells on the other hand exhibited limited cell death beyond 10 mM Ca2+, while the surviving colonies of the AS-CaSR variant were viable at up to 15 mM Ca2+ with invasive tendencies. Analysis of differentially expressed genes in high Ca2+ adapted mesenchymal-like BT-549 and epithelial MDA-MB-468 breast cancer cells compared to MCF10A control cells revealed a small set of differentially expressed genes that were significantly associated with relapse free survival of basal-like breast cancer patients. Among these genes are ligands and negative regulators of the WNT/FZD pathway. Our data suggest that the A986S-CaSR variant with reduced sensitivity to Ca2+ supported adaptation of TNBC cells at high Ca2+, and that high Ca2+ adaptation of breast cancer cells may be mediated via a panel of high Ca2+ inducible genes with implications in bone metastasis. This study is supported by NIH SC1GM139814, T32AI007281, T32GM144927, T32HL007737; and the American Cancer Society. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 1740: Histone proteomic profiling of EMT-transformed MCF10A breast cells demonstrates a loss in novel histone arginine methylation sites via dual p53 and PTEN deletion

Abstract Metastatic potential of basal-like breast cancers typically is initiated by genetic alterations that lead to a process known as epithelial-mesenchymal transition (EMT). However, much is currently not understood regarding the role of epigenetic modifications that lead to invasive characteristics and EMT phenotype of metastatic breast cancers. Based on the previous notion connecting epigenetic changes to breast cancer metastasis, we performed DIA-based mass spectrometry of isolated histones from an isogenic panel of MCF10A breast cell lines where the tumor suppressor genes TP53 and PTEN were silenced and induced EMT. With this approach, we determined which histone modifications were differentially enriched in the non-EMT and EMT-induced MCF10A cell lines. From approximately 72 histone modifications identified and annotated from our mass spectrometry results, we were able to identify 5 histone events that were differentially enriched in our MCF10A cell line panel. Two events of note were histone H3 lysine-14 acetylation (H3K14ac) significantly increasing and histone H4 arginine 55 dimethylation (H4R55me2) significantly decreasing in our EMT-transformed MCF10A p53-/PTEN- cell lines when compared to the parental, non-tumorigenic MCF10A cell line, showing these events are differentially affected during the EMT process in breast cancer cells. Furthermore, significant arginine demethylation of H4R55me2 & H3.1R83me in the EMT-transformed MCF10A p53-/PTEN- cell lines corresponded with JMJD6, an established histone arginine demethylase, being overexpressed in basal-like breast cancer cell lines as well as basal-like breast cancer patients from The Cancer Genome Atlas (TCGA) and METABRIC datasets. Based on histone proteomic profiling of our isogenic cell line model, the loss of specific histone arginine methylation events corresponding with JMJD6 overexpression could highlight the potential for a targetable epigenetic mechanism in breast cancer metastasis. Citation Format: Charlotte B. McGuinness, Megan A. Wilson, Margaret V. Leonard, Maria Ouzounova, Hasan Korkaya, Austin Y. Shull. Histone proteomic profiling of EMT-transformed MCF10A breast cells demonstrates a loss in novel histone arginine methylation sites via dual p53 and PTEN deletion [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 1740.

Abstract 3087: The m6A methyltransferase RBM15 drives the growth of triple-negative breast cancer cells through the stimulation of serine and glycine metabolism

Abstract N6-adenosine methylation (m6A) is critical for controlling cancer cell growth and tumorigenesis. However, the function and detailed mechanism of how m6A methyltransferases modulate m6A levels on specific targets remains unknown. In the current study, we identified significantly elevated levels of RBM15, an m6A writer, in basal-like breast cancer (BC) patients compared to non-basal like BC and linked it to worse clinical outcome. Gene expression profiling uncovered correlations between RBM15 and serine and glycine metabolism genes including PHGDH, PSAT1, PSPH, and SHMT2. RBM15 influences overall m6A levels and, specifically, m6A of serine and glycine metabolism genes via direct binding to target RNA. Further RBM15 effects on cell growth were largely dependent on serine and glycine metabolism. Thus, RBM15 coordinates cancer cell growth through altered serine and glycine metabolism suggesting RBM15 as a new therapeutic target in BC. Citation Format: Su Hwan Park, Yun-Yong Park, Jong-Ho Lee. The m6A methyltransferase RBM15 drives the growth of triple-negative breast cancer cells through the stimulation of serine and glycine metabolism [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 3087.

Retraction: Kinesin family member 2A serves as a potential biomarker reflecting more frequent lymph node metastasis and tumor recurrence risk in basal-like breast cancer patients.

[This retracts the article DOI: 10.3389/fsurg.2022.889294.].

Multidimensional quantitative phenotypic and molecular analysis reveals neomorphic behaviors of p53 missense mutants

Mutations in the TP53 tumor suppressor gene occur in >80% of the triple-negative or basal-like breast cancer. To test whether neomorphic functions of specific TP53 missense mutations contribute to phenotypic heterogeneity, we characterized phenotypes of non-transformed MCF10A-derived cell lines expressing the ten most common missense mutant p53 proteins and observed a wide spectrum of phenotypic changes in cell survival, resistance to apoptosis and anoikis, cell migration, invasion and 3D mammosphere architecture. The p53 mutants R248W, R273C, R248Q, and Y220C are the most aggressive while G245S and Y234C are the least, which correlates with survival rates of basal-like breast cancer patients. Interestingly, a crucial amino acid difference at one position—R273C vs. R273H—has drastic changes on cellular phenotype. RNA-Seq and ChIP-Seq analyses show distinct DNA binding properties of different p53 mutants, yielding heterogeneous transcriptomics profiles, and MD simulation provided structural basis of differential DNA binding of different p53 mutants. Integrative statistical and machine-learning-based pathway analysis on gene expression profiles with phenotype vectors across the mutant cell lines identifies quantitative association of multiple pathways including the Hippo/YAP/TAZ pathway with phenotypic aggressiveness. Further, comparative analyses of large transcriptomics datasets on breast cancer cell lines and tumors suggest that dysregulation of the Hippo/YAP/TAZ pathway plays a key role in driving the cellular phenotypes towards basal-like in the presence of more aggressive p53 mutants. Overall, our study describes distinct gain-of-function impacts on protein functions, transcriptional profiles, and cellular behaviors of different p53 missense mutants, which contribute to clinical phenotypic heterogeneity of triple-negative breast tumors.

Genome-Wide Characterization of TAZ Binding Sites in Mammary Epithelial Cells.

The transcriptional co-activator with PDZ binding motif (TAZ) is a key effector of the Hippo signaling pathway. We and others previously reported that high expression levels of TAZ are positively associated with decreased survival rates and shorter times to relapse in basal-like breast cancer (BLBC) patients. The oncogenic activity of TAZ involves the regulation of diverse signal transduction pathways that direct processes such as cell proliferation, migration, and resistance to apoptosis, albeit through poorly characterized gene expression programs. Here, using a tet-inducible system in mammary epithelial MCF10A cells, we have characterized the TAZ-regulated transcription program using RNA sequencing in a temporal and spatial manner. We further identified global TAZ binding sites at different TAZ activation time points by chromatin immunoprecipitation (ChIP) sequencing analysis. We found that the vast majority of TAZ was rapidly localized in enhancer regions at the early TAZ activation time point and then gradually spread to promoter regions. TAZ bound to enhancer regions following a switch in potential TEAD and FOSL2 transcription factor motifs. Furthermore, the ATAC sequencing analysis indicated that TAZ activation led to chromatin structural alterations. Together, our results have revealed the landscape of genome-wide TAZ binding sites and may lead to improvements in the current understanding of how TAZ regulates the gene expression program that contributes to the development of breast cancer.

A positive TGF-β/miR-9 regulatory loop promotes the expansion and activity of tumour-initiating cells in breast cancer.

MicroRNA-9 (miR-9) has previously been described as a dual-functional RNA during breast cancer progression and its roles need to be clarified thoroughly. A miR-9 knockout mode of mouse breast cancer, the MMTV-PyMT model (PyMT-miR-9-/- ), combined with different human breast cancer cell lines were used to evaluate the effects of miR-9 on breast cancer initiation, progression and metastasis. Lin-NECs (Neoplastic mammary epithelial cells) and pNECs (Pre-neoplastic mammary epithelial cells) were isolated and subjected to tumour-initiation assay. Whole-mount staining of mammary gland and histology was performed to determine mammary gland growth. Tumour-initiating analysis combining a series of in vitro experiments were carried out to evaluate miR-9 roles in tumour-initiating ability. RNA-sequencing of human breast cancer cells, and mammary glands at hyperplastic stages and established tumours in PyMT and PyMT-miR-9-/- mice, ChIP and luciferase report assays were conducted to reveal the underlying mechanisms. MiR-9 is ectopically expressed in breast cancer and its level is negatively correlated with the prognosis, especially in basal-like breast cancer patients. Additionally, miR-9 is essential for breast cancer progression by promoting the expansion and activity of tumour-initiating cells (TICs) in preneoplastic glands, established tumours and xenograft modes. Mechanistically, the activity of TICs hinges on a positive TGF-β/miR-9 regulatory loop mediated by the STARD13/YAP axis. These findings demonstrate that miR-9 is an oncogenic miRNA rather than a tumour-suppressor in breast cancer, calling for rectification of the model for this conserved and highly abundant miRNA.

The interplay between IGF-1R signaling and Hippo-YAP in breast cancer stem cells

BackgroundBoth IGF-1R/PI3K/AKT/mTOR and Hippo pathways are crucial for breast cancer stem cells (BCSCs). However, their interplay remains unclear.MethodsFour triple negative breast cancer cell lines derived from CSC of two patient-derived xenografts (PDXs), AS-B145, AS-B145-1R, AS-B244, and AS-B244-1R, were used to elucidate the role of YAP in BCSCs. YAP silenced BCSCs were analyzed by cell proliferation, aldehyde dehydrogenase (ALDH) activity, mammosphere formation, and tumorigenesis. The effects of modulating IGF-1R and IGF-1 on YAP expression and localization were evaluated. The clinical correlation of YAP and IGF-1R signaling with the overall survival (OS) of 7830 breast cancer patients was analyzed by KM plotter.ResultsKnockdown of YAP abates the viability and stemness of BCSCs in vitro and tumorigenicity in vivo. Depletion of IGF-1R by shRNA or specific inhibitor decreases YAP expression. In contrast, IGF-1 addition upregulates YAP and enhances its nuclear localization. YAP overexpression increased the mRNA level of IGF-1, but not IGF-1R. Data mining of clinical breast cancer specimens revealed that basal-like breast cancer patients with higher level of IGF-1 and YAP exhibit significantly shorter OS.ConclusionsYAP contributes to stemness features of breast cancer in vitro and in vivo. The expression and localization of YAP was regulated by IGF-1R and YAP expression in turns upregulates IGF-1, but not IGF-1R. Clinically, higher level of YAP and IGF-1 significantly correlated with shorter OS in basal-like breast cancer. Taken together, these findings suggest the clinical relevance of interplay between YAP and IGF-1/IGF-1R pathway in sustaining the properties of BCSCs.DsuDGZeKddKHpMfgDukzC_Video

Basal-like mammary carcinomas stimulate cancer stem cell properties through AXL-signaling to induce chemotherapy resistance.

Basal-like breast cancer (BLBC) is the most aggressive and heterogeneous breast cancer (BC) subtype. Conventional chemotherapies represent next to surgery the most frequently employed treatment options. Unfortunately, resistant tumor phenotypes often develop, resulting in therapeutic failure. To identify the early events occurring upon the first drug application and initiating chemotherapy resistance in BLBC, we leveraged the WAP-T syngeneic mammary carcinoma mouse model and we developed a strategy combining magnetic-activated cell sorting (MACS)-based tumor cell enrichment with high-throughput transcriptome analyses. We discovered that chemotherapy induced a massive gene expression reprogramming toward stemness acquisition to tolerate and survive the cytotoxic treatment in vitro and in vivo. Retransplantation experiments revealed that one single cycle of cytotoxic drug combination therapy (Cyclophosphamide, Adriamycin and 5-Fluorouracil) suffices to induce resistant tumor cell phenotypes in vivo. We identified Axl and its ligand Pros1 as highly induced genes driving cancer stem cell (CSC) properties upon chemotherapy in vivo and in vitro. Furthermore, from our analysis of BLBC patient datasets, we found that AXL expression is also strongly correlated with CSC-gene signatures, a poor response to conventional therapies and worse survival outcomes in those patients. Finally, we demonstrate that AXL inhibition sensitized BLBC-cells to cytotoxic treatment in vitro. Together, our data support AXL as a promising therapeutic target to optimize the efficiency of conventional cytotoxic therapies in BLBC.

GRHL2 Regulation of Growth/Motility Balance in Luminal versus Basal Breast Cancer.

The transcription factor Grainyhead-like 2 (GRHL2) is a critical transcription factor for epithelial tissues that has been reported to promote cancer growth in some and suppress aspects of cancer progression in other studies. We investigated its role in different breast cancer subtypes. In breast cancer patients, GRHL2 expression was increased in all subtypes and inversely correlated with overall survival in basal-like breast cancer patients. In a large cell line panel, GRHL2 was expressed in luminal and basal A cells, but low or absent in basal B cells. The intersection of ChIP-Seq analysis in 3 luminal and 3 basal A cell lines identified conserved GRHL2 binding sites for both subtypes. A pathway analysis of ChIP-seq data revealed cell-cell junction regulation and epithelial migration as well as epithelial proliferation, as candidate GRHL2-regulated processes and further analysis of hub genes in these pathways showed similar regulatory networks in both subtypes. However, GRHL2 deletion in a luminal cell line caused cell cycle arrest while this was less prominent in a basal A cell line. Conversely, GRHL2 loss triggered enhanced migration in the basal A cells but failed to do so in the luminal cell line. ChIP-Seq and ChIP-qPCR demonstrated GRHL2 binding to CLDN4 and OVOL2 in both subtypes but not to other GRHL2 targets controlling cell-cell adhesion that were previously identified in other cell types, including CDH1 and ZEB1. Nevertheless, E-cadherin protein expression was decreased upon GRHL2 deletion especially in the luminal line and, in agreement with its selectively enhanced migration, only the basal A cell line showed concomitant induction of vimentin and N-cadherin. To address how the balance between growth reduction and aspects of EMT upon loss of GRHL2 affected in vivo behavior, we used a mouse basal A orthotopic transplantation model in which the GRHL2 gene was silenced. This resulted in reduced primary tumor growth and a reduction in number and size of lung colonies, indicating that growth suppression was the predominant consequence of GRHL2 loss. Altogether, these findings point to largely common but also distinct roles for GRHL2 in luminal and basal breast cancers with respect to growth and motility and indicate that, in agreement with its negative association with patient survival, growth suppression is the dominant response to GRHL2 loss.

Obesity Influences the Expression of Key Immunomodulators in Normal Human Breast Tissue, Basal-like Breast Cancer Patients, and Cell Lines.

Among the different components of the breast cancer microenvironment are adipocytes, which are mainly composed of differentiated adipocytes and adipose progenitors. The role of obesity in tumor progression has become a key topic in clinical studies, but the mechanics of this are still misunderstood. There is significant evidence of serum amyloid (SAA1), an acute-phase protein, being heavily expressed in inflamed, septic conditions. VTCN1 and VSIR, members of the immunoglobulin family, are key players in T-cell regulation. The present study investigates the differentially expressed genes caused by adipose-conditioned media on the novel triple-negative breast cancer cell lines MDA MB 231 and MDA MB 468. RNA sequencing of adipocyte-conditioned media (ACM)-treated MDA MB 231 and MDA MB 468 cells were analyzed and compared using the gene sequencing enrichment analysis database (GSEA). GSEA was also done on microarray data from obese, non-tumorous breast tissue patients (GSE:33526) to show significantly upregulated immunomodulators. Obesity was also shown to influence gene expression related to immune sensing and evasion in a dataset analysis of basal-like obese patients (GSE:79858). We showed obesity significantly upregulated immunomodulators related to immune suppression in non-tumorous, basal-like patients, as well as in novel basal-like TNBC cell lines.

DNA barcoding reveals ongoing immunoediting of clonal cancer populations during metastatic progression and immunotherapy response

Cancers evade the immune system through the process of cancer immunoediting. While immune checkpoint inhibitors are effective for reactivating tumour immunity in some cancer types, many other solid cancers, including breast cancer, remain largely non-responsive. Understanding how non-responsive cancers evade immunity and whether this occurs at the clonal level will improve immunotherapeutic design. Here we use DNA barcoding to track murine mammary cancer cell clones during immunoediting and determine clonal transcriptional profiles that allow immune evasion following anti-PD1 plus anti-CTLA4 immunotherapy. Clonal diversity is significantly restricted by immunotherapy treatment in both primary tumours and metastases, demonstrating selection for pre-existing breast cancer cell populations and ongoing immunoediting during metastasis and treatment. Immunotherapy resistant clones express a common gene signature associated with poor survival of basal-like breast cancer patient cohorts. At least one of these genes has an existing small molecule that can potentially be used to improve immunotherapy response.

Investigation of Transcript Variant 6 of TPD52L2 as a Prognostic and Predictive Biomarker in Basal-Like MDA-MB-231 and MDA-MB-453 Cell Lines for Breast Cancer.

Background Basal-like breast cancer (BLBC) exhibits worse pathological features than other breast cancer subtypes, and patients diagnosed with BLBC have short disease-free and overall survival times. Thus, the identification of novel biomarkers and therapeutic targets for BLBC is of upmost importance. Although TPD52L2 is upregulated in multiple cancers, little is known about its roles in BLBC. Methods RNA levels were analyzed between breast cancer tissues and paired adjacent normal tissues using RNA-seq data from The Cancer Genome Atlas (TCGA). TPD52L2 stable knockdown and inducible knockout cell lines were established using basal-like MDA-MB-231 and MDA-MB-453 cell lines. Cell proliferation assays in vitro and tumor growth analysis in vivo were performed to determine the function of TPD52L2 during BLBC progression. Transwell assays were used to estimate the regulatory effect of TPD52L2 on BLBC cell migration. The expression profile of all tpd52l2 transcripts was analyzed to assess the functional protein isoform. Association of transcript variant 6 (V6) expression with pathological parameters was carried out using the clinical data of the BRCA cohort. Results We identified V6 of TPD52L2 as a novel biomarker and regulator of BLBC progression. TPD52L2 is upregulated in BLBCs and associated with patient outcomes. TPD52L2 knockdown suppresses tumor growth, and V6 correlates with cancer-related phenotypes in BLBC. Clinical data further proved that V6 is associated with different pathological features, such as pathological stage and pathological tumor status, and independently predicts patient outcomes and responses to therapies. Conclusions Our findings demonstrate that V6 of TPD52L2 is a novel biomarker for BLBC patients. V6 promotes cell proliferation and migration and has marked oncogenic roles in determining the malignant phenotypes of BLBC.

Inflammation-Driven Regulation of PD-L1 and PD-L2, and Their Cross-Interactions with Protective Soluble TNFα Receptors in Human Triple-Negative Breast Cancer.

Simple SummaryImmune checkpoint blockades (ICBs) to PD-L1 have led to major breakthroughs in cancer therapy, but in triple-negative breast cancer (TNBC) success rates are rather limited. Following studies suggesting that chronic inflammation may limit ICB efficacy, we found that pro-inflammatory cytokines up-regulated the proportion of TNBC cells co-expressing the inhibitory immune checkpoint PD-L1 and its cognate PD-L2 molecule. Moreover, we demonstrated that in the context of inflammation-driven signals, PD-L1 down-regulated the cell-derived levels of sTNFR1 and sTNFR2, the soluble receptors of tumor necrosis factor α (TNFα); these soluble receptors were found to exert protective/anti-metastatic effects in TNBC cells, manifested by their ability to down-regulate TNFα-induced production of pro-metastatic chemokines by TNBC cells. Our findings possibly testify for a novel mechanism of PD-L1-mediated tumor progression in which PD-L1 prevents the anti-metastatic effects of sTNFR1 and sTNFR2 in TNBC cells. This mechanism may also act in vivo, in parallel to immune suppression under inflammatory conditions.Pro-inflammatory cytokines play key roles in elevating cancer progression in triple-negative breast cancer (TNBC). We demonstrate that specific combinations between TNFα, IL-1β and IFNγ up-regulated the proportion of human TNBC cells co-expressing the inhibitory immune checkpoints PD-L1 and PD-L2: TNFα + IL-1β in MDA-MB-231 cells and IFNγ + IL-1β in BT-549 cells; in the latter cells, the process depended entirely on STAT1 activation, with no involvement of p65 (CRISPR-Cas9 experiments). Highly significant associations between the pro-inflammatory cytokines and PD-L1/PD-L2 expression were revealed in the TCGA dataset of basal-like breast cancer patients. In parallel, we found that the pro-inflammatory cytokines regulated the expression of the soluble receptors of tumor necrosis factor α (TNFα), namely sTNFR1 and sTNFR2; moreover, we revealed that sTNFR1 and sTNFR2 serve as anti-metastatic and protective factors in TNBC, reducing the TNFα-induced production of inflammatory pro-metastatic chemokines (CXCL8, CXCL1, CCL5) by TNBC cells. Importantly, we found that in the context of inflammatory stimulation and also without exposure to pro-inflammatory cytokines, elevated levels of PD-L1 have down-regulated the production of anti-tumor sTNFR1 and sTNFR2. These findings suggest that in addition to its immune-suppressive activities, PD-L1 may promote disease course in TNBC by inhibiting the protective effects of sTNFR1 and sTNFR2.

Kinesin Family Member 2A Serves as a Potential Biomarker Reflecting More Frequent Lymph Node Metastasis and Tumor Recurrence Risk in Basal-Like Breast Cancer Patients.

BackgroundKinesin family member 2A (KIF2A) is reported as an oncogene and a potential biomarker for progression and prognosis in several cancers such as cervical, ovarian, and gastric. However, its clinical value in basal-like breast cancer (BLBC) is unclear. This study aims to evaluate KIF2A expression and its correlation with clinical features and survival rates in BLBC patients.MethodsKIF2A mRNA and protein expressions in tumor and adjacent tissues from 89 BLBC patients are assessed by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry assays, respectively.ResultsBoth KIF2A protein (p < 0.001) and mRNA expressions (p < 0.001) were higher in tumor than in adjacent tissue. Besides, tumor KIF2A protein expression was positively correlated with N (p = 0.028) and TNM (p = 0.014) stages; meanwhile, tumor KIF2A mRNA expression was positively correlated with N stage (p = 0.046), TNM stage (p = 0.006), and tumor size (p = 0.043). Additionally, both tumor KIF2A protein (p = 0.035) and mRNA (p = 0.039) high expressions were correlated with worse disease-free survival (DFS) but not with overall survival (both p > 0.05). Moreover, tumor KIF2A protein expression was higher in relapsed patients than in non-relapsed patients within 3 years (p = 0.015) and 5 years (p = 0.031), whereas no difference was found between the dead and survivors within 3 years (p = 0.057) or 5 years (p = 0.107). Lastly, after adjustment, tumor KIF2A mRNA high exhibited a trend that correlated with DFS but without statistical significance (p = 0.051).ConclusionKIF2A correlates with more frequent lymph node metastasis and worse DFS in BLBC patients, shedding light on its potency as a biomarker for BLBC.

IL32 overexpression is driven by DNA hypomethylation and contributes to an extracellular matrix (ECM) remodeling phenotype in EpCAM‐/CD49f‐enriched breast cancer cells

Basal‐like breast cancers typically correspond with increased enrichment of EpCAM‐/CD49f‐ cancer stem cells (CSC) and a propensity toward metastasis. However, the molecular mechanisms underlying these general characteristics are not well understood. To provide further insight concerning CSCs and their intrinsic metastatic mechanisms, we compared the 450K DNA methylation profile of EpCAM‐/CD49f‐ poor breast cancer cell lines to that of EpCAM‐/CD49f‐ enriched breast cancer cell lines. From our results, we were able to determine and highlight IL32 as a gene whose promoter is hypomethylated in EpCAM‐/CD49f‐ enriched cell lines. The hypomethylated IL32 promoter corresponded with increased IL32 expression in both cell lines and basal‐like breast cancer patients from The Cancer Genome Atlas (TCGA) database. Interestingly, increased IL32 expression preferentially occurred for the IL32‐beta transcript and corresponds with previous reports demonstrating that IL32‐beta is not secreted from the cell like other canonical interleukins and preferentially localizes to the mitochondria in breast cancer cells. Additionally, expression of the beta‐transcript could be suppressed when CSC‐enriched cells were treated with the BET‐bromodomain inhibitor JQ1. Because of this phenomenon, we sought to determine the effects of suppressing IL32 in the EpCAM‐/CD49f‐ enriched cell line SUM159PT via siRNA‐mediated knockdown and subsequent RNAseq differential expression analysis. From our results, we determined that transcripts involved in extracellular matrix (ECM) organization and collagen/integrin interaction were preferentially affected by IL32 silencing. Additionally, IL32 suppression decreased the invasiveness of SUM159PT based on an ECM‐matrix cell invasion assay. Collectively, our results reflect the notion that differential IL32 expression by promoter hypomethylation in breast CSCs plays a role in ECM remodeling for purposes of breast cancer cell invasion and metastasis.

NRG1/ERBB3/ERBB2 Axis Triggers Anchorage-Independent Growth of Basal-like/Triple-Negative Breast Cancer Cells.

Simple SummaryBreast cancer is a heterogeneous disease, categorized into distinct subgroups with different clinical prognoses and treatment strategies. This study aimed to evaluate the role of ERBB3 in different molecular subtypes of breast cancers. Despite ERBB3/HER3 and its partner ERBB2/HER2 showing low expression levels in basal-like/triple-negative breast cancers, stratification of basal-like patients according to ERBB3 mRNA expression levels highlighted a correlation between higher ERBB3 levels and shorter relapse-free patients’ survival. In vitro analyses unveiled that the activation of the NRG1/ERBB3/ERBB2 axis robustly induces anchorage-independent growth of basal-like/triple-negative breast cancer cellular models, without significant effects on cell proliferation, differentiation, and migration in adhesion. Overall, our data suggest that ERBB2/ERBB3 plays an oncogenic role in basal-like/triple-negative breast cancer patients, suggesting its neutralization as a therapeutic strategy for these breast cancer subtypes, which today have very limited treatment opportunities.ERBB3, also known as HER3, is a tyrosine kinase transmembrane receptor of the ERBB family. Upon binding to neuregulin 1 (NRG1), ERBB3 preferentially dimerizes with HER2 (ERBB2), in turn inducing aggressive features in several cancer types. The analysis of a dataset of breast cancer patients unveiled that higher ERBB3 mRNA expression correlates with shorter relapse-free survival in basal-like breast cancers, despite low ERBB3 expression in this breast cancer subtype. Administration of neuregulin 1 beta (NRG1β) significantly affected neither cellular proliferation nor the basal migratory ability of basal-like/triple-negative quasi-normal MCF10A breast cells, cultured in mono-layer conditions. Furthermore, no significant regulation in cell morphology or in the expression of basal/myoepithelial and luminal markers was observed upon stimulation with NRG1β. In non-adherent conditions, NRG1β administration to MCF10A cells did not significantly influence cell survival; however, it robustly induced cell growth as spheroids (3D growth). Intriguingly, a remarkable upregulation of ERBB3 and ERBB2 protein abundance was observed in 3D compared to 2D cell cultures, and NRG1β-induced 3D cell growth was efficiently prevented by the anti-HER2 monoclonal antibody pertuzumab. Similar results were obtained by the analysis of basal-like/triple-negative breast cancer cellular models, MDA-MB-468 and MDA-MB-231 cells, in which NRG1β induced anchorage-independent cell growth that in turn was prevented or reduced by the simultaneous administration of anti-HER2 neutralizing antibodies. Finally, the ability of pertuzumab in suppressing NRG1β-induced 3D growth was also evaluated and confirmed in MCF10A engineered with HER2-overexpression. We suggest that the NRG1/ERBB3/ERBB2 pathway promotes the anchorage-independent growth of basal-like breast cancer cells. Importantly, we provide evidence that ERBB2 neutralization, in particular by pertuzumab, robustly inhibits this process. Our results pave the way towards the development of novel anticancer strategies for basal-like breast cancer patients based on the interception of the NRG1/ERBB3/ERBB2 signaling axis.

In silico recognition of a prognostic signature in basal-like breast cancer patients.

BackgroundTriple-negative breast cancers (TNBCs) display poor prognosis, have a high risk of tumour recurrence, and exhibit high resistance to drug treatments. Based on their gene expression profiles, the majority of TNBCs are classified as basal-like breast cancers. Currently, there are not available widely-accepted prognostic markers to predict outcomes in basal-like subtype, so the selection of new prognostic indicators for this BC phenotype represents an unmet clinical challenge.ResultsHere, we attempted to address this challenging issue by exploiting a bioinformatics pipeline able to integrate transcriptomic, genomic, epigenomic, and clinical data freely accessible from public repositories. This pipeline starts from the application of the well-established network-based SWIM methodology on the transcriptomic data to unveil important (switch) genes in relation with a complex disease of interest. Then, survival and linear regression analyses are performed to associate the gene expression profiles of the switch genes with both the patients’ clinical outcome and the disease aggressiveness. This allows us to identify a prognostic gene signature that in turn is fed to the last step of the pipeline consisting of an analysis at DNA level, to investigate whether variations in the expression of identified prognostic switch genes could be related to genetic (copy number variations) or epigenetic (DNA methylation differences) alterations in their gene loci, or to the activities of transcription factors binding to their promoter regions. Finally, changes in the protein expression levels corresponding to the so far identified prognostic switch genes are evaluated by immunohistochemical staining results taking advantage of the Human Protein Atlas.ConclusionThe application of the proposed pipeline on the dataset of The Cancer Genome Atlas (TCGA)-Breast Invasive Carcinoma (BRCA) patients affected by basal-like subtype led to an in silico recognition of a basal-like specific gene signature composed of 11 potential prognostic biomarkers to be further investigated.

The expression of HOXC10 is correlated with tumor-infiltrating immune cells in basal-like breast cancer and serves as a prognostic biomarker.

BackgroundHomeobox gene C10 (HOXC10) plays a vital role in the occurrence and development of several cancers, but its effects and underlying mechanism in the prognosis of different subtypes of breast cancer remain unclear.MethodsFirst, we evaluated and compared the expression levels of HOXC10 cancer to normal tissues in the Oncomine and Tumor Immune Estimation Resource (TIMER) databases. Second, the correlation between HOXC10 and the survival of patients with different types of cancer, including breast cancer, was analyzed in the PrognoScan, Gene Expression Profiling Interactive Analysis (GEPIA), and Kaplan-Meier plotter databases. Finally, the relationship between HOXC10 and immune-infiltration levels or gene marker sets of immune cells in basal-like breast cancer (BLBC) was investigated in the TIMER and GEPIA databases.ResultsThe expression of HOXC10 was elevated in breast cancer tissues. High HOXC10 expression indicated a poor prognosis for breast cancer patients, and expression affected the survival time of lymph-node positive or grade III breast cancer patients. In BLBC, the median overall survival (OS) of patients with high HOXC10 expression was significantly shorter than that of patients with low HOXC10 expression. HOXC10 was positively correlated with the immune infiltration of macrophages in BLBC. Breast cancer patients with low HOXC10 expression in different enriched immune-cell subgroups had a favorable prognosis.ConclusionsThe level of HOXC10 expression increased significantly in breast cancer, and elevated HOXC10 was positively correlated with immune-cell infiltration and poor prognosis in BLBC. These findings shed light on the important role of HOXC10 in breast cancer. HOXC10 should be recognized as a prognostic biomarker for determining prognosis and immune infiltration in BLBC patients.