Differential response of luminal and basal breast cancer cells to acute and chronic hypoxia.

Hypoxia is linked to disease progression and poor prognosis in several cancers, including breast cancer. Cancer cells can encounter acute, chronic, and/or intermittent periods of oxygen deprivation and it is poorly understood how the different breast cancer subtypes respond to such hypoxia regimes. Here, we assessed the response of representative cell lines for the luminal and basal A subtype to acute (24 h) and chronic hypoxia (5 days). High throughput targeted transcriptomics analysis showed that HIF-related pathways are significantly activated in both subtypes. Indeed, HIF1⍺ nuclear accumulation and activation of the HIF1⍺ target gene CA9 were comparable. Based on the number of differentially expressed genes: (i) 5 days of exposure to hypoxia induced a more profound transcriptional reprogramming than 24 h, and (ii) basal A cells were less affected by acute and chronic hypoxia as compared to luminal cells. Hypoxia-regulated gene networks were identified of which hub genes were associated with worse survival in breast cancer patients. Notably, while chronic hypoxia altered the regulation of the cell cycle in both cell lines, it induced two distinct adaptation programs in these subtypes. Mainly genes controlling central carbon metabolism were affected in the luminal cells whereas genes controlling the cytoskeleton were affected in the basal A cells. In agreement, in response to chronic hypoxia, lactate secretion was more prominently increased in the luminal cell lines which were associated with the upregulation of the GAPDH glycolytic enzyme. This was not observed in the basal A cell lines. In contrast, basal A cells displayed enhanced cell migration associated with more F-actin stress fibers whereas luminal cells did not. Altogether, these data show distinct responses to acute and chronic hypoxia that differ considerably between luminal and basal A cells. This differential adaptation is expected to play a role in the progression of these different breast cancer subtypes.

Acquisition of a hybrid E/M state is essential for tumorigenicity of basal breast cancer cells.

Carcinoma cells residing in an intermediate phenotypic state along the epithelial-mesenchymal (E-M) spectrum are associated with malignant phenotypes, such as invasiveness, tumor-initiating ability, and metastatic dissemination. Using the recently described CD104+/CD44hi antigen marker combination, we isolated highly tumorigenic breast cancer cells residing stably-both in vitro and in vivo-in an intermediate phenotypic state and coexpressing both epithelial (E) and mesenchymal (M) markers. We demonstrate that tumorigenicity depends on individual cells residing in this E/M hybrid state and cannot be phenocopied by mixing two cell populations that reside stably at the two ends of the spectrum, i.e., in the E and in the M state. Hence, residence in a specific intermediate state along the E-M spectrum rather than phenotypic plasticity appears critical to the expression of tumor-initiating capacity. Acquisition of this E/M hybrid state is facilitated by the differential expression of EMT-inducing transcription factors (EMT-TFs) and is accompanied by the expression of adult stem cell programs, notably, active canonical Wnt signaling. Furthermore, transition from the highly tumorigenic E/M state to a fully mesenchymal phenotype, achieved by constitutive ectopic expression of Zeb1, is sufficient to drive cells out of the E/M hybrid state into a highly mesenchymal state, which is accompanied by a substantial loss of tumorigenicity and a switch from canonical to noncanonical Wnt signaling. Identifying the gatekeepers of the various phenotypic states arrayed along the E-M spectrum is likely to prove useful in developing therapeutic approaches that operate by shifting cancer cells between distinct states along this spectrum.

Schlafen-11 expression is associated with immune signatures and basal-like phenotype in breast cancer.

PURPOSE: Breast cancer (BC) is a heterogeneous disorder, with variable response to systemic chemotherapy. Likewise, BC shows highly complex immune activation patterns, only in part reflecting classical histopathological subtyping. Schlafen-11 (SLFN11) is a nuclear protein we independently described as causal factor of sensitivity to DNA damaging agents (DDA) in cancer cell line models. SLFN11 has been reported as a predictive biomarker for DDA and PARP inhibitors in human neoplasms. SLFN11 has been implicated in several immune processes such as thymocyte maturation and antiviral response through the activation of interferon signaling pathway, suggesting its potential relevance as a link between immunity and cancer. In the present work, we investigated the transcriptional landscape of SLFN11, its potential prognostic value, and the clinico-pathological associations with its variability in BC. METHODS: We assessed SLFN11 determinants in a gene expression meta-set of 5061 breast cancer patients annotated with clinical data and multigene signatures. RESULTS: We found that 537 transcripts are highly correlated with SLFN11, identifying "immune response", "lymphocyte activation", and "T cell activation" as top Gene Ontology processes. We established a strong association of SLFN11 with stromal signatures of basal-like phenotype and response to chemotherapy in estrogen receptor negative (ER-) BC. We identified a distinct subgroup of patients, characterized by high SLFN11 levels, ER- status, basal-like phenotype, immune activation, and younger age. Finally, we observed an independent positive predictive role for SLFN11 in BC. CONCLUSIONS: Our findings are suggestive of a relevant role for SLFN11 in BC and its immune and molecular variability.

miRNA-135b Contributes to Triple Negative Breast Cancer Molecular Heterogeneity: Different Expression Profile in Basal-like Versus non-Basal-like Phenotypes.

The clinical and genetic heterogeneity of Triple Negative Breast Cancer (TNBC) and the lack of unambiguous molecular targets contribute to the inadequacy of current therapeutic options for these variants. MicroRNAs (miRNA) are a class of small highly conserved regulatory endogenous non-coding RNA, which can alter the expression of genes encoding proteins and may play a role in the dysregulation of cellular pathways. Our goal was to improve the knowledge of the molecular pathogenesis of TNBC subgroups analyzing the miRNA expression profile, and to identify new prognostic and predictive biomarkers. We conducted a human miRNome analysis by TaqMan Low Density Array comparing different TNBC subtypes, defined by immunohistochemical basal markers EGFR and CK5/6. RT-qPCR confirmed differential expression of microRNAs. To inspect the function of the selected targets we perform Gene Ontology and KEGG enrichment analysis. We identified a single miRNA signature given by miR-135b expression level, which was strictly related to TNBC with basal-like phenotype. miR-135b target analysis revealed a role in the TGF-beta, WNT and ERBB pathways. A significant positive correlation was identified between neoplastic proliferative index and miR-135b expression. These findings confirm the oncogenic roles of miR-135b in the pathogenesis of TNBC expressing basal markers. A potential negative prognostic role of miR-135b overexpression might be related to the positive correlation with high proliferative index. Our study implies potential clinical applications: miR-135b could be a potential therapeutic target in basal-like TNBCs.

Molecular pathogenesis of human prostate basal cell hyperplasia.

BACKGROUND: Understanding the molecular pathogenesis of distinct phenotypes in human benign prostatic hyperplasia (BPH) is essential to improving therapeutic intervention. Current therapies target smooth muscle and luminal epithelia for relief of lower urinary tract symptoms (LUTS) due to BPH, but basal cell hyperplasia (BCH) remains untargeted. The incidence of has been reported at 8-10%, but a molecular and cellular characterization has not been performed on this phenotype. METHODS: Using freshly digested tissue from surgical specimens, we performed RNA-seq analysis of flow cytometry-purified basal epithelia from 3 patients with and 4 patients without a majority BCH phenotype. qPCR was performed on 28 genes identified as significant from 13 non-BCH and 7 BCH specimens to confirm transcriptomic analysis. IHC was performed on several non-BCH and BCH specimens for 3 proteins identified as significant by transcriptomic analysis. RESULTS: A total of 141 human BPH specimens were analyzed for the presence of BCH. Clinical characteristics of non-BCH and BCH cohorts revealed no significant differences in age, PSA, prostate volume, medical treatment, or comorbidities. Quantitation of cellular subsets by flow cytometry in 11 BCH patients vs. 11 non-BCH patients demonstrated a significant increase in the ratio of basal to luminal epithelia in patients with BCH (P <0.05), but no significant differences in the total number of leukocytes. RNA-seq data from flow cytometry isolated basal epithelia from patients with and without BCH were subjected to gene set enrichment analysis of differentially expressed genes, which revealed increased expression of members of the epidermal differentiation complex. Transcriptomic data were complemented by immunohistochemistry for members of the epidermal differentiation complex, revealing a morphological similarity to other stratified squamous epithelial layers. CONCLUSIONS: Increased expression of epidermal differentiation complex members and altered epithelial stratification resembles the progression of other metaplastic diseases. These data provide insight into the plasticity of the human prostate epithelium and suggest a classification of basal cell hyperplasia as a metaplasia.

Amplification of SOX4 promotes PI3K/Akt signaling in human breast cancer.

PURPOSE: The PI3K/Akt signaling axis contributes to the dysregulation of many dominant features in breast cancer including cell proliferation, survival, metabolism, motility, and genomic instability. While multiple studies have demonstrated that basal-like or triple-negative breast tumors have uniformly high PI3K/Akt activity, genomic alterations that mediate dysregulation of this pathway in this subset of highly aggressive breast tumors remain to be determined. METHODS: In this study, we present an integrated genomic analysis based on the use of a PI3K gene expression signature as a framework to analyze orthogonal genomic data from human breast tumors, including RNA expression, DNA copy number alterations, and protein expression. In combination with data from a genome-wide RNA-mediated interference screen in human breast cancer cell lines, we identified essential genetic drivers of PI3K/Akt signaling. RESULTS: Our in silico analyses identified SOX4 amplification as a novel modulator of PI3K/Akt signaling in breast cancers and in vitro studies confirmed its role in regulating Akt phosphorylation. CONCLUSIONS: Taken together, these data establish a role for SOX4-mediated PI3K/Akt signaling in breast cancer and suggest that SOX4 may represent a novel therapeutic target and/or biomarker for current PI3K family therapies.

LMO2 Enhances Lamellipodia/Filopodia Formation in Basal-Type Breast Cancer Cells by Mediating ARP3-Profilin1 Interaction.

BACKGROUND The human LMO2 gene was first cloned from an acute T lymphocytic leukemia patient; it is primarily expressed in hematopoietic and vascular endothelial systems, and functions as a pivotal transcriptional regulator during embryonic hematopoiesis and angiogenesis. However, some recent reports indicated that LMO2 is widely expressed in many tissues and tumors, predominantly in cytoplasm, and revealed complicated functions on tumor behaviors in a variety of cancer types. As an adaptor molecule, binding partners and function details of LMO2 in these solid tumors need to be further investigated. MATERIAL AND METHODS In this study, we used yeast two-hybrid method to screen potential LMO2 interacting partners, MBP-pulldown, and co-immunoprecipitation assay to confirm protein-protein interactions, and confocal microscopy to reveal the subcellular localization of relevant proteins and actin cytoskeleton changes in relevant cells. RESULTS We found that ARP3 and profilin1 were 2 binding partners of LMO2, primarily in cytoplasm. LMO2. Functionally, LMO2 mediated the assembly of a complex including ARP3, profilin1, and actin monomer, increased actin monomer binding to profilin1, and promoted lamellipodia/filopodia formation in basal-type breast cancer cells. CONCLUSIONS Our data indicate a novel functional mechanism of LMO2 in facilitating the delivery of actin monomers to the branched microfilament and increasing lamellipodia/filopodia formation in basal-type breast cancer cells, suggesting a cancer-promoting role of LMO2 in a subtype-dependent manner and its potential as a subtype-specific biomarker for clinical treatment of breast cancers.

The co-factor of LIM domains (CLIM/LDB/NLI) maintains basal mammary epithelial stem cells and promotes breast tumorigenesis.

Mammary gland branching morphogenesis and ductal homeostasis relies on mammary stem cell function for the maintenance of basal and luminal cell compartments. The mechanisms of transcriptional regulation of the basal cell compartment are currently unknown. We explored these mechanisms in the basal cell compartment and identified the Co-factor of LIM domains (CLIM/LDB/NLI) as a transcriptional regulator that maintains these cells. Clims act within the basal cell compartment to promote branching morphogenesis by maintaining the number and proliferative potential of basal mammary epithelial stem cells. Clim2, in a complex with LMO4, supports mammary stem cells by directly targeting the Fgfr2 promoter in basal cells to increase its expression. Strikingly, Clims also coordinate basal-specific transcriptional programs to preserve luminal cell identity. These basal-derived cues inhibit epidermis-like differentiation of the luminal cell compartment and enhance the expression of luminal cell-specific oncogenes ErbB2 and ErbB3. Consistently, basal-expressed Clims promote the initiation and progression of breast cancer in the MMTV-PyMT tumor model, and the Clim-regulated branching morphogenesis gene network is a prognostic indicator of poor breast cancer outcome in humans.

Bioinformatic analysis reveals a pattern of STAT3-associated gene expression specific to basal-like breast cancers in human tumors.

Signal transducer and activator of transcription 3 (STAT3), a latent transcription factor associated with inflammatory signaling and innate and adaptive immune responses, is known to be aberrantly activated in a wide variety of cancers. In vitro analysis of STAT3 in human cancer cell lines has elucidated a number of specific targets associated with poor prognosis in breast cancer. However, to date, no comparison of cancer subtype and gene expression associated with STAT3 signaling in human patients has been reported. In silico analysis of human breast cancer microarray and reverse-phase protein array data was performed to identify expression patterns associated with STAT3 in basal-like and luminal breast cancers. Results indicate clearly identifiable STAT3-regulated signatures common to basal-like breast cancers but not to luminal A or luminal B cancers. Furthermore, these differentially expressed genes are associated with immune signaling and inflammation, a known phenotype of basal-like cancers. These findings demonstrate a distinct role for STAT3 signaling in basal breast cancers, and underscore the importance of considering subtype-specific molecular pathways that contribute to tissue-specific cancers.

DNA Methylation Affects the SP1-regulated Transcription of FOXF2 in Breast Cancer Cells.

FOXF2 (forkhead box F2) is a mesenchyme-specific transcription factor that plays a critical role in tissue homeostasis through the maintenance of epithelial polarity. In a previous study, we demonstrated that FOXF2 is specifically expressed in basal-like breast cancer (BLBC) cells and functions as an epithelial-mesenchymal transition suppressor. FOXF2 deficiency enhances the metastatic ability of BLBC cells through activation of the epithelial-mesenchymal transition program, but reduces cell proliferation. In this study, we demonstrate that CpG island methylation of the FOXF2 proximal promoter region is involved in the regulatory mechanism of the subtype-specific expression of FOXF2 in breast cancer cells. DNMT1, DNMT3A, and DNMT3B commonly or individually contributed to this DNA methylation in different breast cancer cells. SP1 regulated the transcriptional activity of FOXF2 through direct binding to the proximal promoter region, whereas this binding was abrogated through DNA methylation. FOXF2 mediated the SP1-regulated suppression of progression and promotion of proliferation of non-methylated BLBC cells. Thus, we conclude that the subtype-specific expression and function of FOXF2 in breast cancer cells are regulated through the combined effects of DNA methylation and SP1 transcriptional regulation.

Targeted Pten deletion plus p53-R270H mutation in mouse mammary epithelium induces aggressive claudin-low and basal-like breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC), an aggressive disease comprising several subtypes including basal-like and claudin-low, involves frequent deletions or point mutations in TP53, as well as loss of PTEN. We previously showed that combined deletion of both tumor suppressors in the mouse mammary epithelium invariably induced claudin-low-like TNBC. The effect of p53 mutation plus Pten deletion on mammary tumorigenesis and whether this combination can induce basal-like TNBC in the mouse are unknown. METHODS: WAP-Cre:Pten(f/f):p53(lox.stop.lox_R270H) composite mice were generated in which Pten is deleted and a p53-R270H mutation in the DNA-binding domain is induced upon expression of Cre-recombinase in pregnancy-identified alveolar progenitors. Tumors were characterized by histology, marker analysis, transcriptional profiling [GEO-GSE75989], bioinformatics, high-throughput (HTP) FDA drug screen as well as orthotopic injection to quantify tumor-initiating cells (TICs) and tail vein injection to identify lung metastasis. RESULTS: Combined Pten deletion plus induction of p53-R270H mutation accelerated formation of four distinct mammary tumors including poorly differentiated adenocarcinoma (PDA) and spindle/mesenchymal-like lesions. Transplantation assays revealed highest frequency of TICs in PDA and spindle tumors compared with other subtypes. Hierarchical clustering demonstrated that the PDA and spindle tumors grouped closely with human as well as mouse models of basal and claudin-low subtypes, respectively. HTP screens of primary Pten(∆):p53(∆) vs. Pten(∆):p53(R270H) spindle tumor cells with 1120 FDA-approved drugs identified 8-azaguanine as most potent for both tumor types, but found no allele-specific inhibitor. A gene set enrichment analysis revealed increased expression of a metastasis pathway in Pten(∆):p53(R270H) vs. Pten(∆):p53(∆) spindle tumors. Accordingly, following tail vein injection, both Pten(∆):p53(R270H) spindle and PDA tumor cells induced lung metastases and morbidity significantly faster than Pten(∆):p53(∆) double-deletion cells, and this was associated with the ability of Pten(∆):p53(R270H) tumor cells to upregulate E-cadherin expression in lung metastases. CONCLUSIONS: Our results demonstrate that WAP-Cre:Pten(f/f):p53(lox.stop.lox_R270H) mice represent a tractable model to study basal-like breast cancer because unlike p53 deletion, p53(R270H) mutation in the mouse does not skew tumors toward the claudin-low subtype. The WAP-Cre:Pten(f/f):p53(lox.stop.lox_R270H) mice develop basal-like breast cancer that is enriched in TICs, can readily form lung metastasis, and provides a preclinical model to study both basal-like and claudin-low TNBC in immune-competent mice.

Tumor Evolution in Two Patients with Basal-like Breast Cancer: A Retrospective Genomics Study of Multiple Metastases.

BACKGROUND: Metastasis is the main cause of cancer patient deaths and remains a poorly characterized process. It is still unclear when in tumor progression the ability to metastasize arises and whether this ability is inherent to the primary tumor or is acquired well after primary tumor formation. Next-generation sequencing and analytical methods to define clonal heterogeneity provide a means for identifying genetic events and the temporal relationships between these events in the primary and metastatic tumors within an individual. METHODS AND FINDINGS: We performed DNA whole genome and mRNA sequencing on two primary tumors, each with either four or five distinct tissue site-specific metastases, from two individuals with triple-negative/basal-like breast cancers. As evidenced by their case histories, each patient had an aggressive disease course with abbreviated survival. In each patient, the overall gene expression signatures, DNA copy number patterns, and somatic mutation patterns were highly similar across each primary tumor and its associated metastases. Almost every mutation found in the primary was found in a metastasis (for the two patients, 52/54 and 75/75). Many of these mutations were found in every tumor (11/54 and 65/75, respectively). In addition, each metastasis had fewer metastatic-specific events and shared at least 50% of its somatic mutation repertoire with the primary tumor, and all samples from each patient grouped together by gene expression clustering analysis. TP53 was the only mutated gene in common between both patients and was present in every tumor in this study. Strikingly, each metastasis resulted from multiclonal seeding instead of from a single cell of origin, and few of the new mutations, present only in the metastases, were expressed in mRNAs. Because of the clinical differences between these two patients and the small sample size of our study, the generalizability of these findings will need to be further examined in larger cohorts of patients. CONCLUSIONS: Our findings suggest that multiclonal seeding may be common amongst basal-like breast cancers. In these two patients, mutations and DNA copy number changes in the primary tumors appear to have had a biologic impact on metastatic potential, whereas mutations arising in the metastases were much more likely to be passengers.

Transcriptomic analyses identify association between mitotic kinases, PDZ-binding kinase and BUB1, and clinical outcome in breast cancer.

Protein kinases are important components in oncogenic transformation of breast cancer. Evaluation of upregulated genes that codify for protein kinases could be used as biomarkers to predict clinical outcome. Gene expression and functional analyses using public datasets were performed to identify differential gene expression and functions in basal-like tumors compared with normal breast tissue. Overall survival (OS) associated with upregulated genes was explored using the KM Plotter online tool. The prognostic influence of these genes in luminal tumors and systemically untreated patients was also assessed. Of the 426 transcripts identified in basal-like tumors, 11 genes that coded for components of protein kinases were upregulated with more than a fourfold change. Regulation of cell cycle was an enriched function containing 10 of these 11 identified genes. Among them, expression of four genes, BUB1ß, CDC28, NIMA, and PDZ binding kinase, were all associated with improved OS when using at least one probe in the basal-like subtype. Two genes, BUB1ß and PDZ binding kinase, showed consistent association with improved OS irrespective of the gene probe used for the analysis. No association was observed for these genes with relapse-free survival. In contrast, both BUB1ß and PDZ binding kinase showed worse OS in luminal tumors and in a cohort of systemically untreated patients. BUB1ß and PDZ binding kinase are associated with improved OS in basal-like tumors and worse OS in luminal and untreated patients. The association with a better outcome in basal-like tumors could be due to a more favorable response to chemotherapy.

Estimating DNA methylation levels by joint modeling of multiple methylation profiles from microarray data.

DNA methylation studies have been revolutionized by the recent development of high throughput array-based platforms. Most of the existing methods analyze microarray methylation data on a probe-by-probe basis, ignoring probe-specific effects and correlations among methylation levels at neighboring genomic locations. These methods can potentially miss functionally relevant findings associated with genomic regions. In this article, we propose a statistical model that allows us to pool information on the same probe across multiple samples to estimate the probe affinity effect, and to borrow strength from the neighboring probe sites to better estimate the methylation values. Using a simulation study, we demonstrate that our method can provide accurate model-based estimates. We further use the proposed method to develop a new procedure for detecting differentially methylated regions, and compare it with a state-of-the-art approach via a data application.

Cross-species DNA copy number analyses identifies multiple 1q21-q23 subtype-specific driver genes for breast cancer.

A large number of DNA copy number alterations (CNAs) exist in human breast cancers, and thus characterizing the most frequent CNAs is key to advancing therapeutics because it is likely that these regions contain breast tumor 'drivers' (i.e., cancer causal genes). This study aims to characterize the genomic landscape of breast cancer CNAs and identify potential subtype-specific drivers using a large set of human breast tumors and genetically engineered mouse (GEM) mammary tumors. Using a novel method called SWITCHplus, we identified subtype-specific DNA CNAs occurring at a 15% or greater frequency, which excluded many well-known breast cancer-related drivers such as amplification of ERBB2, and deletions of TP53 and RB1. A comparison of CNAs between mouse and human breast tumors identified regions with shared subtype-specific CNAs. Additional criteria that included gene expression-to-copy number correlation, a DawnRank network analysis, and RNA interference functional studies highlighted candidate driver genes that fulfilled these multiple criteria. Numerous regions of shared CNAs were observed between human breast tumors and GEM mammary tumor models that shared similar gene expression features. Specifically, we identified chromosome 1q21-23 as a Basal-like subtype-enriched region with multiple potential driver genes including PI4KB, SHC1, and NCSTN. This step-wise computational approach based on a cross-species comparison is applicable to any tumor type for which sufficient human and model system DNA copy number data exist, and in this instance, highlights that a single region of amplification may in fact harbor multiple driver genes.

Comparative microRNA profiling of sporadic and BRCA1 associated basal-like breast cancers.

BACKGROUND: While a number of studies have examined miRNA profiles across the molecular subtypes of breast cancer, it is unclear whether BRCA1 basal-like cancers have a specific miRNA profile. This study aims to compare grade independent miRNA expression in luminal cancers, sporadic and BRCA1 basal-type breast cancers. It also aims to ascertain an immunohistochemical profile regulated by BRCA1 specific miRNAs for potential diagnostic use. METHODS: miRNA expression was assessed in 11 BRCA1 basal, 16 sporadic basal, 17 luminal grade 3 cancers via microarrays. The expression of Cyclin D1, FOXP1, FIH-1, pan-ERß, NRP1 and CD99, predicted to be regulated by BRCA1 specific miRNAs by computer prediction algorithms, was assessed via immunohistochemistry in a cohort of 35 BRCA1 and 52 sporadic basal-like cancers. Assessment of cyclin D1, FOXP1, NRP1 and CD99 expression was repeated on a validation cohort of 82 BRCA1 and 65 sporadic basal-like breast cancers. RESULTS: Unsupervised clustering of basal cancers resulted in a "sporadic" cluster of 11 cancers, and a "BRCA1" cluster of 16 cancers, including a subgroup composed entirely of 10 BRCA1 cancers. Compared with sporadic basal cancers, BRCA1 cancers showed reduced positivity for proteins predicted to be regulated by miRNAs: FOXP1 (6/20[30 %] vs. 37/49[76 %], p < 0.001), cyclin D1 (8/22[36 %] vs. 30/46[65 %], p = 0.025), NRP1 (2/20[10 %] vs. 23/46[50 %], p = 0.002). This was confirmed in the validation cohort (all p < 0.001). Negative staining for 2 or more out of FOXP1, cyclin D1 and NRP1 predicts germline BRCA1 mutation with a sensitivity of 92 %, specificity of 44 %, positive predictive value of 38 % and a negative predictive value of 94 %. CONCLUSION: Sporadic and BRCA1 basal-like cancers have grade independent miRNA expression profiles. Furthermore miRNA driven differences in the expression of proteins in BRCA1 basal cancers may be detected via immunohistochemistry. These findings may have important diagnostic implications, as immunohistochemical assessment of basal cancers, in addition to the patient's family and clinical history, may potentially identify patients who may benefit from BRCA1 gene testing.

Inhibition of SHP2 in basal-like and triple-negative breast cells induces basal-to-luminal transition, hormone dependency, and sensitivity to anti-hormone treatment.

BACKGROUND: The Src homology phosphotyrosyl phosphatase 2 (SHP2) is a positive effector of cell growth and survival signaling as well transformation induced by multiple tyrosine kinase oncogenes. Since the basal-like and triple-negative breast cancer (BTBC) is characterized by dysregulation of multiple tyrosine kinase oncogenes, we wanted to determine the importance of SHP2 in BTBC cell lines. METHODS: Short hairpin RNA-based and dominant-negative expression-based SHP2 inhibition techniques were used to interrogate the functional importance of SHP2 in BTBC cell biology. In addition, cell viability and proliferation assays were used to determine hormone dependency for growth and sensitivity to anti-estrogen treatment. RESULTS: We show that inhibition of SHP2 in BTBC cells induces luminal-like epithelial morphology while suppressing the mesenchymal and invasive property. We have termed this process as basal-to-luminal transition (BLT). The occurrence of BLT was confirmed by the loss of the basal marker alpha smooth muscle actin and the acquisition of the luminal marker cytokeratin 18 (CK18) expression. Furthermore, the occurrence of BLT led to estrogen receptor alpha (ERα) expression, hormone dependency, and sensitivity to tamoxifen treatment. CONCLUSIONS: Our data show that inhibition of SHP2 induces BLT, ERα expression, dependency on estrogen for growth, and sensitivity to anti-hormone therapy. Therefore, inhibition of SHP2 may provide a therapeutic benefit in basal-like and triple-negative breast cancer.

MicroRNA-18a inhibits hypoxia-inducible factor 1α activity and lung metastasis in basal breast cancers.

INTRODUCTION: In breast cancer, distinct expression profiles of microRNAs (miRNAs) have been associated with molecular subgroups and clinicopathological characteristics, implicating a diagnostic and prognostic role of miRNAs. However, the biological functions of deregulated miRNAs in tumor progression are not yet completely defined. In this study, we investigated the function of miR-18a in regulating breast cancer metastasis through the hypoxia-inducible factor 1α (HIF1A)-dependent hypoxic response. METHODS: An orthotopic metastatic breast cancer xenograft model (MDA-MB-231 cells) was used to identify miRNAs associated with spontaneous lung metastasis. The function of miR-18a in regulating HIF1A expression, as well as cellular responses to hypoxia and metastasis, were then studied in vitro and in vivo by assessing ectopic miR-18a expression or miR-18a inhibition. miRNA-mRNA interactions (AGO2 immunoprecipitation and 3' untranslated region Luciferase reporter assays), gene expression (quantitative PCR and microarray), cell migration and invasion, and cell growth were assessed under normoxic or hypoxic conditions, complemented by orthotopic xenograft of tumor cells to the mammary fat pad to investigate the effect of modulating miR-18a expression on primary tumor growth and lung metastasis. Last, clinically relevant correlations between miR-18a, HIF1A, hypoxia-responsive gene expression and distant metastasis-free survival (DMFS) were assessed using published expression array breast tumors data sets. RESULTS: miRNAs encoded by the MIR17HG gene were downregulated in lung metastases compared to primary tumors. Ectopic expression of miR-18a, a MIR17HG family member, in a metastatic variant of MDA-MB-231 cells reduced primary tumor growth and lung metastasis, whereas miR-18a inhibition in the parental cells promoted tumor growth and lung metastasis. We identified HIF1A as a direct target of miR-18a. Modulating miR-18a expression significantly affected hypoxic gene expression, cell invasiveness and sensitivity to anoikis and hypoxia in vitro in a HIF1A-dependent manner. Analysis of previously published data revealed that higher expression of HIF1A and a panel of hypoxic genes is associated with shorter DMFS interval in patients with basal-like breast tumors, and that, within this subtype, miR-18a expression is inversely correlated with hypoxic gene expression. Together, these data support a role of miR-18a in repressing distant metastasis through a HIF1A-dependent pathway. CONCLUSIONS: The results of this study reveal a novel role for miR-18a in targeting HIF1A and repressing metastasis of basal-like breast tumors.

MET is a potential target for use in combination therapy with EGFR inhibition in triple-negative/basal-like breast cancer.

MET, a cell surface receptor for hepatocyte growth factor, is involved in the development of triple-negative/basal-like breast cancer (TNBC/BLBC). However, its utility as a therapeutic target in this subtype of breast cancer is poorly understood. To evaluate MET fully as a potential therapeutic target for TNBC/BLBC, we investigated the relationship between MET expression and clinical outcomes of patients with breast cancer and the functional effect of MET inhibition. Using automated immunohistochemistry (Ventana), we analyzed MET expression in 924 breast cancer patients with relevant clinicopathologic parameters. BLBC showed the strongest relationship with MET expression (57.5%, p < 0.001). High expression of MET in breast cancer resulted in poor overall survival (p = 0.001) and disease-free survival (DFS, p = 0.010). MET expression was relatively high in TNBC cell lines, and the silencing of MET via small interfering RNA reduced cell proliferation and migration. We observed reduced TNBC cell viability after treatment with the MET inhibitor PHA-665752. In the most drug-resistant cell line, MDA-MB-468, which showed elevated epidermal growth factor receptor (EGFR) expression, silencing of EGFR resulted in increased sensitivity to PHA-665752 treatment. We confirmed that PHA-665752 synergizes with the EGFR inhibitor erlotinib to decrease the viability of MDA-MB-468 cells. TNBC patients coexpressing MET and EGFR showed significantly worse DFS than that in patients expressing EGFR alone (p = 0.021). Our findings strongly suggest that MET may be a therapeutic target in TNBC and that the combined therapy targeting MET and EGFR may be beneficial for the treatment of TNBC/BLBC patients.

Nuclear CSPP1 expression defined subtypes of basal-like breast cancer.

BACKGROUND: The multi-exon CSPP1 gene, encoding for centrosome and microtubule-associated proteins involved in ciliogenesis and cell division, is a candidate oncogene in luminal breast cancer but expression of CSPP1 proteins remained unexplored. METHODS: CSPP1 gene and protein expression was examined in normal mammary tissue, human breast cancer cell lines, and primary breast cancer biopsies from two patient cohorts. Cell type and epitope-dependent subcellular-specific CSPP1 staining pattern in normal mammary gland epithelium and cancer biopsies were correlated to molecular and clinical parameters. RESULTS: A novel, nuclear localised CSPP1 isoform was exclusively detected in luminal epithelial cells, whereas cytoplasmic CSPP-L was generally expressed in normal mammary epithelium. Luminal cell-related nuclear CSPP1 expression was preserved in type-matched cell lines and carcinomas, and correlated to gene copy number and mRNA expression. In contrast, basal-like carcinomas displayed generally lower CSPP1 mRNA expression. Yet, a subgroup of basal-like breast carcinomas depicted nuclear CSPP1 expression, displayed luminal traits, and differed from nuclear CSPP1 devoid counterparts in expression of eight genes. Eight-gene signature defined groups of basal-like tumours from an independent cohort showed significant differences in survival. CONCLUSIONS: Differential expression of a nuclear CSPP1 isoform identified biologically and clinically distinct subgroups of basal-like breast carcinoma.