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Background: Molecular subtyping of breast cancer has provided a new perspective on the pathogenesis of the disease and a foundation for building a clinical classification for this heterogeneous disease. The initial classification categorizing breast cancers into five groups, luminal A, luminal B, ERBB2-overexpressing, basal-like and normal-like, was later supplemented by an additional group, claudin-low tumors. However, the claudin-low group has been more difficult to align with clinically used immunohistochemical categories. The identity of this group among clinical cases remains ill defined. Methods: The METABRIC cohort comprising more than 1700 breast cancers and providing information for classifying them in both clinical groups and the genomic PAM50/claudin-low groups was analyzed to derive relationships and clarify potential pathogenic ramifications. Comparisons of the claudin-low cases bearing different clinical group classifications and of the respective cases with the same clinical non-claudin-low classifications were performed. Results: ER-negative/HER2-negative breast cancers are predominantly (88.4%) basal-like and claudin low. Conversely, most basal-like cancers (83.6%) are ER negative/HER2 negative. However, claudin-low breast cancers are only in 68.4% of cases ER negative/HER2 negative and the other clinical phenotypes, mostly ER positive/HER2 negative/low proliferation, are also represented in more than 30% of claudin-low cancers. These claudin-low non-ER-negative/HER2-negative breast cancers differ from claudin-low ER-negative/HER2-negative cases in grade, prevalence of integrative clusters, and prevalence of common mutations and common amplifications. Differences also exist between the two groups classified clinically as ER negative/HER2 negative, that are genomically basal-like or claudin-low, including in menopause status, grade, histology, prevalence of high tumor mutation burden, distribution of integrative clusters, prevalence of TP53 mutations and of amplifications in the MYC and MCL1 loci. Furthermore, distinct characteristics are observed between the luminal A and claudin-low groups within the clinical ER-positive/HER2-negative/low proliferation group. Conclusion: Within genomically claudin-low breast cancers, the ER-negative/HER2-negative group is distinct from the group with either ER or HER2 positivity. Conversely, within clinical phenotypes, claudin-low and non-claudin-low breast cancers differ in clinical characteristics and molecular attributes.

BackgroundRecent studies have suggested that fatty acid oxidation (FAO) is a key metabolic pathway for the growth of triple negative breast cancers (TNBCs), particularly those that have high expression of MYC. However, the underlying mechanism by which MYC promotes FAO remains poorly understood.MethodsWe used a combination of metabolomics, transcriptomics, bioinformatics, and microscopy to elucidate a potential mechanism by which MYC regulates FAO in TNBC.ResultsWe propose that MYC induces a multigenic program that involves changes in intracellular calcium signalling and fatty acid metabolism. We determined key roles for fatty acid transporters (CD36), lipases (LPL), and kinases (PDGFRB, CAMKK2, and AMPK) that each contribute to promoting FAO in human mammary epithelial cells that express oncogenic levels of MYC. Bioinformatic analysis further showed that this multigenic program is highly expressed and predicts poor survival in the claudin-low molecular subtype of TNBC, but not other subtypes of TNBCs, suggesting that efforts to target FAO in the clinic may best serve claudin-low TNBC patients.ConclusionWe identified critical pieces of the FAO machinery that have the potential to be targeted for improved treatment of patients with TNBC, especially the claudin-low molecular subtype.

Purpose Claudin-low molecular subtypes have been identified in breast and bladder cancers and are characterized by low expression of claudins, enrichment for epithelial-to-mesenchymal transition (EMT), and tumor-initiating cell (TIC) features. We evaluated whether the claudin-low subtype also exists in gastric cancer. Materials and Methods Four hundred fifteen tumors from The Cancer Genome Atlas (TCGA) gastric cancer mRNA data set were clustered on the claudin, EMT, and TIC gene sets to identify claudin-low tumors. We derived a 24-gene predictor that classifies gastric cancer into claudin-low and non–claudin-low subtypes. This predictor was validated with the Asian Cancer Research Group (ACRG) data set. We characterized molecular and clinical features of claudin-low tumors. Results We identified 46 tumors that had consensus enrichment for claudin-low features in TCGA data set. Claudin-low tumors were most commonly diffuse histologic type (82%) and originally classified as TCGA genomically stable (GS) subtype (78%). Compared with GS subtype, claudin-low subtype had significant activation in Rho family of GTPases signaling, which appears to play a key role in its EMT and TIC properties. In the ACRG data set, 28 of 300 samples were classified as claudin-low tumors by the 24-gene predictor and were phenotypically similar to the initially derived claudin-low tumors. Clinically, claudin-low subtype had the worst overall survival. Of note, the hazard ratios that compared claudin-low versus GS subtype were 2.10 (95% CI, 1.07 to 4.11) in TCGA and 2.32 (95% CI, 1.18 to 4.55) in the ACRG cohorts, with adjustment for age and pathologic stage. Conclusion We identified a gastric claudin-low subtype that carries a poor prognosis likely related to therapeutic resistance as a result of its EMT and TIC phenotypes.

MALT1 is the effector protein of the CARMA/Bcl10/MALT1 (CBM) signalosome, a multiprotein complex that drives pro-inflammatory signaling pathways downstream of a diverse set of receptors. Although CBM activity is best known for its role in immune cells, emerging evidence suggests that it plays a key role in the pathogenesis of solid tumors, where it can be activated by selected G protein–coupled receptors (GPCR). Here, we demonstrated that overexpression of GPCRs implicated in breast cancer pathogenesis, specifically the receptors for Angiotensin II and thrombin (AT1R and PAR1), drove a strong epithelial-to-mesenchymal transition (EMT) program in breast cancer cells that is characteristic of claudin-low, triple-negative breast cancer (TNBC). In concert, MALT1 was activated in these cells and contributed to the dramatic EMT phenotypic changes through regulation of master EMT transcription factors including Snail and ZEB1. Importantly, blocking MALT1 signaling, through either siRNA-mediated depletion of MALT1 protein or pharmacologic inhibition of its activity, was effective at partially reversing the molecular and phenotypic indicators of EMT. Treatment of mice with mepazine, a pharmacologic MALT1 inhibitor, reduced growth of PAR1+, MDA-MB-231 xenografts and had an even more dramatic effect in reducing the burden of metastatic disease. These findings highlight MALT1 as an attractive therapeutic target for claudin-low TNBCs harboring overexpression of one or more selected GPCRs. Implications: This study nominates a GPCR/MALT1 signaling axis as a pathway that can be pharmaceutically targeted to abrogate EMT and metastatic progression in TNBC, an aggressive form of breast cancer that currently lacks targeted therapies.

Proteogenomics is the field of integrating data from mass spectrometry-based shotgun proteomics, and phosphoproteomics into next-generation RNA and DNA sequencing data analysis pipelines that promises new insights into cancer biology and therapeutic targeting. As well as analyses of clinical samples for disease phenotype association analysis, the application of proteogenomics to model systems also has considerable potential. A Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteogenomic analysis prioritized dihydropyrimidinase-like-3 (DPYSL3) as a multi-level (RNA/Protein/Phosphoprotein) expression outlier specific to the Claudin-Low (CLOW) subset of triple negative breast cancers. A Pubmed informatics tool indicated a paucity of data in the context of breast cancer which further prioritized DPYSL3 for study. DPYSL3 was identified as a protein that is regulated during neuronal differentiation in the cerebral cortex and in neuronal cell lines and plays a role in regulating neurite outgrowth somehow through an association with vesicles in the growth cone. In addition, DPYSL3 expression has been observed in several malignant tumors, including prostate cancer, pancreatic cancer, gastric cancer and neuroblastoma. DPYSL3 is reported to play a role in cell migration and metastasis suppression in prostate cancer. However, in pancreatic cancer, DPYSL3 is positively associated with liver metastasis and poor outcome. DPYSL3 knock-down in DPYSL3 (+) CLOW cell lines demonstrated reduced proliferation, yet enhanced motility and increased expression of Epithelial to Mesenchymal Transition (EMT) markers suggesting that DPYSL3 is a multi-functional signaling modulator. Slower proliferation in DPYSL3 (-) CLOW cells was associated with accumulation of multi-nucleated cells indicating a mitotic defect that was associated with a collapse of the vimentin (VIM) microfilament networkinduced by VIM hyperphosphorylation. On the other hand, DPYSL3 suppressed the expression of EMT regulators TWIST and SNAIL and opposed p21 activated kinase 2 (PAK2) dependent migration, but these EMT regulators in turn induced DPYSL3 expression, suggesting DPYSL3 participates in negative feedback in EMT. Cell migration in DPYSL3 (-) cells correlated with increased phosphorylation of PAK2 on Ser20 and was sensitive to PAK2 siRNA and pharmacological PAK inhibition.Immunoprecipitation and mass spectrometry-based proteomics or western blotting strongly suggests that PAKs interact such that DPYSL3 may function as a direct negative regulator of PAK family kinases. Thus, a PAK inhibitor could potentially mitigate increase migration as an adverse effect of DPYSL3 suppression. In conclusion, DPYSL3 is a remarkable multifunctional signaling scaffold that should be examined further to provide insights into the stem cell-like state of claudin-low breast cancers, particularly in terms of their cell cycle dependencies, migratory activity and capacity for EMT. Citation Format: Matsunuma R, Chan DW, Kim B-J, Singh P, Han A, Saltzman A, Cheng C, Lei JT, Sahin E, Leng M, Fan C, Perou CM, Malovannaya A, Ellis MJ. DPYSL3 modulates mitosis, migration and epithelial to mesenchymal transition in claudin-low breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-08-01.

Simple Summary Here, we identified and characterized a claudin-low subtype of high-grade serous ovarian cancer. This rare variant of undifferentiated neoplasm shares transcriptional features with the homonym subtype of breast cancer, including low epithelial differentiation, high mesenchymal signature, and enrichment for stem cell features. Since the claudin-low transcriptional signature is associated with poor prognosis, we believe that the identification of the claudin-low molecular profile may have important clinical implications, paving the way for personalized medicine in ovarian cancer patients. Abstract Claudin-low cancer (CL) represents a rare and biologically aggressive variant of epithelial tumor. Here, we identified a claudin-low molecular profile of ovarian high-grade serous carcinoma (HGSOC), which exhibits the main characteristics of the homonym breast cancer subtype, including low epithelial differentiation and high mesenchymal signature. Hierarchical clustering and a centroid based algorithm applied to cell line collection expression dataset labeled 6 HGSOC cell lines as CL. These have a high energy metabolism and are enriched in CD44+/CD24− mesenchymal stem-like cells expressing low levels of cell-cell adhesion molecules (claudins and E-Cadherin) and high levels of epithelial-to-mesenchymal transition (EMT) induction transcription factors (Zeb1, Snai2, Twist1 and Twist2). Accordingly, the centroid base algorithm applied to large retrospective collections of primary HGSOC samples reveals a tumor subgroup with transcriptional features consistent with the CL profile, and reaffirms EMT as the dominant biological pathway functioning in CL-HGSOC. HGSOC patients carrying CL profiles have a worse overall survival when compared to others, likely to be attributed to its undifferentiated/stem component. These observations highlight the lack of a molecular diagnostic in the management of HGSOC and suggest a potential prognostic utility of this molecular subtyping.

BACKGROUND: Metaplastic carcinoma is an aggressive, triple-negative breast cancer (TNBC) with differentiation towards squamous, spindle, or mesenchymal cell types. The molecular underpinnings of the histological subtypes are unclear. Our lab discovered a cytoplasmic function of EZH2, a transcriptional repressor, whereby pEZH2 T367 binds to cytoplasmic proteins in TNBC cells and enhances invasion and metastasis. Here, we investigated the expression and subcellular localization of pEZH2 T367 protein in metaplastic carcinomas. METHODS: Thirty-five metaplastic carcinomas (17 squamous, 10 mesenchymal, and 8 spindle) were evaluated and immunostained with anti-pEZH2 T367. We analyzed staining intensity (score 1–4), subcellular localization (nuclear/cytoplasmic), and localization within the tumor (center/invasive edge). Protein expression of pEZH2 T367-binding partners was measured from a quantitative multiplex proteomics analysis performed in our lab. RESULTS: Cytoplasmic pEZH2 T367 was significantly upregulated in squamous (14 of 17, 82%) compared to mesenchymal (4 of 10, 40%) and spindle (2 of 6, 33%) subtypes (p=0.011). Twenty-five of 34 (73%) tumors with available tumor–normal interface showed accentuated cytoplasmic pEZH2 T367 at the infiltrative edge. Cytoplasmic pEZH2 T367 was upregulated in 9 of 10 (90%) tumors with lymph node metastasis (p=0.05). Bioinformatics analyses identified an EZH2 protein network in metaplastic carcinomas (p value

Claudin-low triple negative breast cancer (CL-TNBC) is a clinically aggressive molecular TNBC subtype characterized by a propensity to metastasize, recur and acquire chemoresistance. CL-TNBC has a diverse intra- and extracellular composition and microenvironment, and currently there are no clinically approved targeted therapies. Histone deacetylase inhibitors (HDACi) have been investigated as therapeutic agents targeting invasive TNBC phenotypes. However, further studies are required to evaluate HDAC inhibition in CL-TNBC. Here, we utilize a novel CL- TNBC patient-derived xenograft model to study the various and diverse therapeutic potential targets within CL-TNBC tumors. To evaluate effects of the pan-HDACi panobinostat on metastasis and the mesenchymal phenotype of CL-TNBC, we utilize immunohistochemistry staining and qRT-PCR in in vitro, ex vivo and in vivo studies. Further, we evaluate pan-HDAC inhibition on stem-like subpopulations using 3D mammosphere culture techniques and quantification. Finally, we show that pan- HDACi suppresses collagen expression in CL-TNBC. In this study, we provide evidence that pan-HDAC inhibition has effects on various components of the CL-TNBC subtype, and we demonstrate the potential of our novel CL-TNBC PDX model in therapeutic discovery research.

Breast tumors display highly heterogeneous characteristics both at transcriptional level and in term of genomic landscape. We recently reported that the differentiation status of the cell-of-origin influences the genetic route toward tumorigenesis1. Indeed, mammary stem cells exhibit the intrinsic property to tolerate the oxidative stress inherent to the oncogenic transformation through the expression of a preemptive program driven by ZEB1, an epithelial-to-mesenchymal transition (EMT)-related transcription factor, and MSRB3, a methionine sulfoxide reductase. Thereby, ZEB1-expressing tumors exhibit low level of DNA damage associated with few genomic alterations and low level of TP53 mutations. Importantly, these tumors share several transcriptional characteristics and features with normal stem cells, and with Claudin-Low (CL) molecular subtype of breast cancers. In the present work, we attempt to decipher molecular traits of CL breast tumors through multi-omics analyses of publicly available databases. This global approach allows us to highlight various CL breast tumors features as clinical attributes, gene expression, copy number alterations (CNA), somatic mutations or drug responses by differential analyses of breast tumors and cancer cell lines. Preliminary results indicate that, independently of tumor purity, CL breast tumors are mostly diploids, present a paucity of genomic rearrangements and a lower mutation rate compared to other breast tumors. They mainly, but not exclusively, show basal features and belong to the integrative cluster 4 (CNA-devoid) described by Christina Curtis and colleagues2. Concerning gene expression, CL breast tumors exhibit a frequent activation of RAS signaling pathway, an observation consistent with their sensitivity to MAPK inhibitors. Other analyses are currently ongoing and aim to better understand the biology of CL breast tumors in order to improve both the diagnosis and the therapeutic strategy used. 1. Morel A-P et al. A stemness-related ZEB1-MSRB3 axis governs cellular pliancy and breast cancer genome stability. Nature Medicine. 2017 Apr 10. 2. Curtis C et al. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature. 2012 Apr 18. Citation Format: Pommier RM, Morel A-P, Tissier A, Thomas E, Kielbassa J, Tonon L, Sohier E, Viari A, Saintigny P, Puisieux A. Multi-omics characterization of claudin-low breast tumors [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-07-06.

Molecular profiling of tumors shows that triple-negative breast cancer (TNBC) can be stratified into mesenchymal (claudin-low breast cancer, CLBC) and epithelial subtypes (basal-like breast cancer, BLBC). Subtypes differ in underlying genetics and in response to therapeutics. Several reports indicate that therapeutic strategies that induce lipid peroxidation or proteotoxicity may be particularly effective for various cancers with a mesenchymal phenotype such as CLBC, for which no specific treatment regimens exist and outcomes are poor. We hypothesized that silver nanoparticles (AgNPs) can induce proteotoxic stress and cause lipid peroxidation to a greater extent in CLBC than in BLBC. We found that AgNPs were lethal to CLBCs at doses that had little effect on BLBCs and were non-toxic to normal breast epithelial cells. Analysis of mRNA profiles indicated that sensitivity to AgNPs correlated with expression of multiple CLBC-associated genes. There was no correlation between sensitivity to AgNPs and sensitivity to silver cations, uptake of AgNPs, or proliferation rate, indicating that there are other molecular factors driving sensitivity to AgNPs. Mechanistically, we found that the differences in sensitivity of CLBC and BLBC cells to AgNPs were driven by peroxidation of lipids, protein oxidation and aggregation, and subsequent proteotoxic stress and apoptotic signaling, which were induced in AgNP-treated CLBC cells, but not in BLBC cells. This study shows AgNPs are a specific treatment for CLBC and indicates that stratification of TNBC subtypes may lead to improved outcomes for other therapeutics with similar mechanisms of action.

Triple negative breast cancer (TNBC) is a lethal molecular subtype of invasive breast cancer (BC) that is ER-, PR- and HER2-. Claudin low (CL) TNBC, a distinct molecular subtype of TNBC, has the worst prognosis compared to other BC subtypes. Aberrant expression of claudin proteins disrupts the function of tight junctions. Consequently, inducing epithelial-to-mesenchymal transition (EMT) in cancers, an important factor for enhanced motility and metastasis. Therefore, understanding the molecular mechanisms that modulate claudin expression in TNBC is crucial. The 8q24 genomic locus is an important cancer susceptibility locus that is associated with poor clinical outcomes in cancer due to common amplification events that occur in many malignant diseases. This locus contains the PVT1 gene, which encodes a long noncoding RNA (lncRNA) that has been implicated in multiple cancers including BC. PVT1 consists of 12 exons that are alternatively spliced to generate lncRNAs. Although previous research has implicated PVT1 as an important player in BC, the underlying molecular mechanisms of PVT1 in CL TNBC is unknown. To examine the role of PVT1 in CL TNBC, we assessed PVT1 expression in T47D (ER+), MDA MB 231 (CL) and MDA MB 468 (claudin high (CH)) BC cells. We observed that PVT1 exons 4A, 4B, and 9 are significantly overexpressed in CL MDA MB 231 and significantly underexpressed in CH MDA MB 468 in comparison to T47D cells, suggesting a protumorigenic role of PVT1 in CL TNBC. We analyzed the functional consequences of siRNA targeting of PVT1 exon 9 expression in CL TNBC cells. siRNA targeting of PVT1 exon 9 expression in the MDA MB 231 CL TNBC cells led to a significant reduction in migration and the re-expression of claudin 4. To determine the role of PVT1 on EMT, we assessed the expression of EMT markers (vimentin, fibronectin, and E-cadherin) in MDA MB 231 cells. We observed no changes in the expression of EMT markers when PVT1 exon 9 is knocked down. However, our data show that mesenchymal markers are more highly expressed in MDA MB 231 cells in comparison to MDA MB 468 cells. Further, knockdown of PVT1 exon 9 did not induce apoptosis in MDA MB 231 cells as assessed by caspase 3 and caspase 9 expression. Taken together, our data indicate that PVT1 exon 9 regulates claudin expression and migration in CL TNBC, and may have implications for clinical outcomes in TNBC. Citation Format: Fayola A. Levine, Olorunseun O. Ogunwobi. Targeting PVT1 exon 9 transcript is not pro-apoptotic but induces claudin 4 expression and inhibits migration in triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2387.

Elevated overexpression of the lncRNA HOTAIR mediates invasion and metastasis in breast cancer. In an apparent paradox, we observed low expression of HOTAIR in the invasive Claudin‐low MDA‐MB‐231 and Hs578T cells in two‐dimensional culture (2D). However, HOTAIR expression exhibited robust induction in laminin‐rich extracellular matrix‐based three‐dimensional organotypic culture (lrECM 3D) over that in 2D culture. Induction of HOTAIR required intact ECM signaling, namely integrin α2 and SRC kinase activity. Moreover, invasive growth was suppressed by HOTAIR‐specific siRNA. Induction of HOTAIR in lrECM 3D culture resulted from the activation of a novel isoform of HOTAIR (HOTAIR‐N) whose transcription is started from the first intron of the HOXC11 gene. The HOTAIR‐N promoter exhibited increased trimethylation of histone H3 lysine 4, a histone marker of active transcription, and binding of BRD4, a reader of transcriptionally active histone markers. Inhibition of BRD4 substantially reduced the expression of HOTAIR in lrECM 3D culture. In summary, our results indicate that HOTAIR expression is activated by BRD4 binding to a novel HOTAIR‐N promoter in Claudin‐low breast cancer cells that are attached to ECM. Induction of HOTAIR is required for invasive growth of Claudin‐low breast cancer cells in lrECM 3D culture.

Exposure to exercise following a breast cancer diagnosis is associated with reductions in the risk of recurrence. However, it is not known whether breast cancers within the same molecular-intrinsic subtype respond differently to exercise. Syngeneic mouse models of claudin-low breast cancer (i.e., EO771, 4TO7, and C3(1)SV40Tag-p16-luc) were allocated to a uniform endurance exercise treatment dose (forced treadmill exercise) or sham-exercise (stationary treadmill). Compared to sham-controls, endurance exercise treatment differentially affected tumor growth rate: 1- slowed (EO771), 2- accelerated (C3(1)SV40Tag-p16-luc), or 3- was not affected (4TO7). Differential sensitivity of the three tumor lines to exercise was paralleled by effects on intratumoral Ki-67, Hif1-α, and metabolic programming. Inhibition of Hif1-α synthesis by the cardiac glycoside, digoxin, completely abrogated exercise-accelerated tumor growth in C3(1)SV40Tag-p16-luc. These results suggest that intratumoral Hif1-α expression is an important determinant of claudin-low breast cancer adaptation to exercise treatment.

Claudins (CLDNs) are key cell adhesion molecules, which compose tight junctions (TJs), and the disruption of TJs is associated with cancer development. Here we immunohistochemically studied expression patterns of CLDNs in 222 primary invasive breast cancers including 68 triple-negative breast cancers (TNBCs), and examined their correlation with epithelial-to-mesenchymal transition (EMT)-related markers, breast cancer stem cell (BCSC) markers, and clinicopathological features including patients' clinical outcome. Tumor margins were classified as three infiltrating growth patterns (expanding, intermediate and infiltrating). For CLDN1, 3, 4, and 7, their expression rates were more frequent in TNBCs than in other subtypes (11.8% vs 0.7%, 26.5% vs 2.0%, 48.5% vs 11.1%, and 32.4% vs 8.7%, respectively; P ≤ 0.001). In 68 TNBCs, we identified high Ki67 labeling index (LI) and the combination of CLDN4 high/CLDN7 low expression as independent predictors of axillary nodal metastasis (P = 0.019; OR, 4.36; 95%CI, 1.28-14.90 and P = 0.007; OR, 5.33; 95%CI, 1.58-17.90). Moreover, the combination of CLDN1 low/CLDN7 low/E-cadherin negative as well as tumor infiltrating patterns were predictors for worse recurrence-free survival by univariate analyses in TNBCs (P = 0.005 and P = 0.011). Our analyses provide further evidence that CLDNs would be valuable prognostic markers in TNBCs.

// Robert Jones 1 , Katrina Watson 1 , Anthony Bruce 1 , Sarah Nersesian 1 , Jenna Kitz 1 , Roger Moorehead 1 1 Department of Biomedical Science, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada Correspondence to: Roger Moorehead, email: rmoorehe@uoguelph.ca Keywords: microRNA, miR-200f, miR-200c, mammary tumors, claudin-low Received: August 15, 2016 Accepted: February 06, 2017 Published: March 02, 2017 ABSTRACT Claudin-low breast cancer is a relatively rare breast cancer subtype. These cancers are typically ER - /PR - /HER2 - and express high levels of mesenchymal genes as well as genes associated with inflammation, angiogenesis and stem cell function. In addition to alterations in gene expression, it was recently demonstrated that claudin-low breast cancers express very low levels of the miR-200 family of miRNAs. Given that each miRNA can regulate tens, hundreds or even thousands of genes, miRNAs are being evaluated as therapeutic targets. In this study we show that mammary tumors from MTB-IGFIR transgenic mice and cell lines derived from these tumors represent a model of human claudin-low breast cancer and murine claudin-low mammary tumors and cell lines express only very low levels of all five members of the miR-200 family. Reduced miR-200 family expression appears to be regulated via methylation as cells and tumors expressing low levels of miR-200 family members had higher levels of CpG methylation in a putative promoter region than tumors and cells expressing high levels of miR-200 family members. Re-expression of miR-200c in murine claudin-low mammary tumor cells inhibited tumor cell proliferation and colony formation in vitro and tumor growth in vivo . With respect to tumor growth in vivo , re-expression of miR-200c was associated with a reduction in tumor vasculature and expression of Flt1 and Vegfc . Therefore, miR-200c is an important regulator of mesenchymal tumor cell growth.

Dedicator of cytokinesis 1 (DOCK1) is a critical regulator of cancer metastasis. Claudins are transmembrane proteins that play a role in epithelial barrier integrity. Due to a loss or low expression of claudins (CLDN), the claudin-low type of triple-negative breast cancer (TNBC) is characterized by a mesenchymal-like phenotype with strong metastatic potential. In order to elucidate the mechanism of DOCK1 in cancer metastasis, we first analyzed the transcriptomic changes using a clinical database of human TNBC and found that the increase in DOCK1 expression was highly correlated with the poor survival rate of TNBC patients. Interference with DOCK1 expression by shRNA resulted in re-expression of claudin-1 in conjunction with significant inhibition of cell viability and motility of claudin-low breast cancer cells. Accordingly, overexpression of claudin-1 suppressed cell viability and migration. Genetic knockdown and pharmacological blockade of Rac1/Rac2 up-regulated claudin-1. DOCK1 knockdown also caused a decrease in DNA methyltransferase (DNMT) expression and an increase in claudin-1 transcript and promoter activity. Furthermore, RRP1B mediated DOCK1 depletion, which up-regulated claudin-1 expression, cell viability, and motility in claudin-low breast cancer cells. This study demonstrated that DOCK1 mediates growth and motility through down-regulated claudin-1 expression via the RRP1BDNMTclaudin-1 pathway and that claudin-1 serves as an important effector in DOCK1-mediated cancer progression and metastasis in claudin-low breast cancer cells.

Increased expression of GLI1, the main Hedgehog signalling pathway effector, is related to unfavourable prognosis and progressive disease of certain breast cancer subtypes. We used conditional transgenic mice induced to overexpress GLI1 in the mammary epithelium either alone or in combination with deletion of one Trp53 allele to address the role of elevated GLI1 expression in breast tumour initiation and progression. Induced GLI1 expression facilitates mammary gland tumour formation and this was further increased upon heterozygous deletion of Trp53. The GLI1-induced primary tumours were of different murine molecular subtypes, including Normal-likeEx , Class8Ex , Claudin-LowEx and Erbb2-likeEx . The gene expression profiles of some of the tumours correlated well with the PAM50 subtypes for human breast cancer. Whole-exome sequencing revealed somatic mutation profiles with only little overlap between the primary tumours. Orthotopically serially transplanted GLI1-induced tumours maintained the main morphological characteristics of the primary tumours for ≥10 generations. Independent of Trp53 status and molecular subtype, the serially transplanted GLI1-induced tumours were able to grow both in the absence of transgenic GLI1 expression and in the presence of the GLI1 inhibitor GANT61. These data suggest that elevated GLI1 expression has a determinant role in tumour initiation; however, additional genetic events are required for tumour progression.

Simple Summary Triple-negative breast cancer (BRCA) cells overexpress the cytokines GM-CSF, G-CSF, MCP-1, and RANTES. We have previously reported that monocytes in 3-D co-culture with BRCA cells generate M1-like macrophages with the ability to induce aggressive features in luminal BRCA cells. Here, we stimulated peripheral blood monocytes with the four cytokines, confirming their capacity to generate protumoral M1-like macrophages. We used the BRCA database to generate an M1-like macrophage gene expression signature related to these cytokines. We observed that the M1-like macrophage, Th1, and immunosuppressive signatures all coincide in claudin-low BRCA but also in mesenchymal carcinomas of colon (COAD) and bladder (BLCA), where they are associated with decreased overall survival in patients. Claudin-low is a tumor subtype with an adverse clinical outcome that remains poorly understood. This study indicates that M1 macrophages may be potential protumoral drivers in already established cancers, and may contribute to the aggressiveness and poor prognosis of claudin-low tumors. These results add to the knowledge of the claudin-low tumor microenvironment and could open a window to immunotherapy strategies to improve patient prognosis. Abstract We previously reported that triple-negative breast cancer (BRCA) cells overexpress the cytokines GM-CSF, G-CSF, MCP-1, and RANTES, and when monocytes were 3-D co-cultured with them, M1-like macrophages were generated with the ability to induce aggressive features in luminal BRCA cell lines. These include upregulation of mesenchymal and stemness markers and invasion. In this study, we stimulated peripheral blood monocytes with the four cytokines and confirmed their capacity to generate protumoral M1-like macrophages. Using the METABRIC BRCA database, we observed that GM-CSF, MCP-1, and RANTES are associated with triple-negative BRCA and reduced overall survival, particularly in patients under 55 years of age. We propose an extended M1-like macrophage proinflammatory signature connected with these three cytokines. We found that the extended M1-like macrophage signature coexists with monocyte/macrophage, Th1 immune response, and immunosuppressive signatures, and all are enriched in claudin-low BRCA samples, and correlate with reduced patient overall survival. Furthermore, we observed that all these signatures are also present in mesenchymal carcinomas of the colon (COAD) and bladder (BLCA). The claudin-low tumor subtype has an adverse clinical outcome and remains poorly understood. This study places M1 macrophages as potential protumoral drivers in already established cancers, and as potential contributors to claudin-low aggressiveness and poor prognosis.

Simple Summary Triple negative breast cancer accounts for 10–15% of all breast cancers. Specific molecular characteristics have led to the identification of six subtypes of triple negative breast cancer, with one in particular being claudin-low. PVT1, a non-protein coding gene, has been demonstrated to play an oncogenic role in various cancers. Specifically, PVT1 exon 9 has been shown to have oncogenic capability. In this study, we aimed to assess the role of PVT1 exon 9 in triple negative breast cancer cells. We have observed that siRNA targeting of PVT1 exon 9 in claudin-low triple negative breast cancer cells resulted in re-expression of claudin 4 protein, and inhibition of migration. These findings indicate that PVT1 exon 9 regulates claudin 4 expression and migration in triple negative breast cancer cells. Abstract PVT1 is a long non-coding RNA transcribed from a gene located at the 8q24 chromosomal region that has been implicated in multiple cancers including breast cancer (BC). Amplification of the 8q24 chromosomal region is a common event in BC and is associated with poor clinical outcomes. Claudin–low (CL) triple negative breast cancer (TNBC) is a subtype of BC with a particularly dismal outcome. We assessed PVT1 exon 9 expression in the T47D estrogen receptor positive BC cell line, and in the MDA MB 468 and MDA MB 231 TNBC cell lines, followed by the assessment of the expression of claudins 1, 3, 4 and 7, in MDA MB 468 and MDA MB 231 (TNBC) cells. We found that MDA MB 231 TNBC cells significantly express less claudin 1, 3, 4, and 7 than MDA MB 468 TNBC cells. PVT1 exon 9 is significantly upregulated in MDA MB 231 CL TNBC cells, and significantly downregulated in MDA MB 468 claudin high (CH) TNBC cells, in comparison to T47D estrogen receptor positive BC cells. We then analyzed the functional consequences of siRNA targeting of PVT1 exon 9 expression in the MDA MB 231 CL TNBC cells. Notably, siRNA targeting of PVT1 exon 9 expression in the MDA MB 231 CL TNBC cells led to a significant reduction in migration and the re-expression of claudin 4. Taken together, our data indicate that PVT1 exon 9 regulates claudin 4 expression and migration in CL TNBC cells, and may have clinical implications in CL TNBC.

// Yirong Zhu 1 , Mark D. Aupperlee 2 , Yong Zhao 2 , Ying Siow Tan 2 , Erin L. Kirk 4 , Xuezheng Sun 4 , Melissa A. Troester 4, 5, 6 , Richard C. Schwartz 3 , Sandra Z. Haslam 2 1 Cell and Molecular Biology Program and Breast Cancer and the Environment Research Program, Michigan State University, East Lansing, MI, USA 2 Department of Physiology and Breast Cancer and the Environment Research Program, Michigan State University, East Lansing, MI, USA 3 Department of Microbiology and Molecular Genetics and Breast Cancer and the Environment Research Program, Michigan State University, East Lansing, MI, USA 4 Department of Epidemiology, University of North Carolina at Chapel Hill, NC, USA 5 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, NC, USA 6 Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, NC, USA Correspondence to: Sandra Z. Haslam, email: shaslam@msu.edu Richard C. Schwartz, email: schwart9@msu.edu Keywords: dietary animal fat, breast cancer, puberty, adulthood, Trp53-null Received: July 22, 2016 Accepted: October 19, 2016 Published: November 04, 2016 ABSTRACT Premenopausal breast cancer is associated with increased animal fat consumption among normal weight, but not overweight women (Farvid et al., 2014). Our previous findings in obesity-resistant BALB/c mice similarly showed promotion of carcinogen-induced mammary tumorigenesis by a diet high in saturated animal fat (HFD). This effect was specific to pubertal versus adult HFD. This study identifies the effects of HFD during puberty versus adulthood in Trp53-null transplant BALB/c mice and investigates its mechanism of enhancing tumorigenesis. Either pubertal or adult HFD is sufficient to increase incidence of Trp53-null mammary tumors. Puberty-restricted HFD exposure promoted tumor cell proliferation, increased angiogenesis, and increased recruitment of total and M2 macrophages in epithelial tumors. Adult-restricted exposure to HFD similarly increased proliferation, angiogenesis, recruitment of total and M2 macrophages, and additionally reduced apoptosis. Adult HFD also increased incidence of spindle cell carcinomas resembling claudin-low breast cancer, and thus adult HFD in the Trp53-null transplantation system may be a useful model for human claudin low breast cancer. Importantly, these results on Trp53-null and our prior studies on DMBA-induced mammary tumorigenesis demonstrate a pubertal window of susceptibility to the promotional effects of HFD, indicating the potential of early life dietary intervention to reduce breast cancer risk.

// Miao Li 1 , Xi Li 2 , Yan Zhuang 3 , Yan Wang 4 , Matthew E. Burow 3 , Bridgette Collins-Burow 3 , Min Xue 5 , Chengjie Song 5 , Bin Shan 6 1 Department of Microbiology and Parasitology, College of Basic Medical Sciences, China Medical University, Shenyang, China 2 Department of Sports Medicine and Joint Surgery, The People’s Hospital of Liaoning Province, Shenyang, China 3 Department of Medicine, Tulane University School of Medicine, New Orleans, LA, USA 4 Department of Biological Engineering, Zunyi Medical College Zhuhai Campus, Zhuhai, China 5 Department of Physiology, Xuzhou Medical College, Xuzhou, China 6 Department of Biomedical Sciences, Elson S. Floyd College of Medicine, Washington State University Spokane, Spokane, WA, USA Correspondence to: Miao Li, email: sendtolm@126.com Bin Shan, email: bin.shan@wsu.edu Keywords: breast cancer, extracellular matrix, three-dimensional organotypic culture, gene expression, homeobox gene Received: February 05, 2016 Accepted: June 29, 2016 Published: July 08, 2016 ABSTRACT The gene expression signatures of the molecular intrinsic subtypes of breast cancer are regulated by epigenetic mechanisms such as methylation of CpG islands in gene promoters. Epigenetic codes can be regulated by the tumor microenvironment. The Claudin-low subtype is associated with triple-negative invasive ductal carcinomas in patients. Herein we explored epigenetic regulation of gene expression in the Claudin-low breast cancer cells by extracellular matrix (ECM), a key component of the tumor microenvironment. We modeled attachment to ECM using laminin rich ECM three-dimensional organotypic culture (lrECM 3D). In 2D and lrECM 3D cultures we examined expression of the homeobox (HOX) genes that epigenetically regulated in development and cancer. We demonstrated induction of the selected HOX genes in lrECM 3D culture of the Claudin-low breast cancer cells MDA-MB-231 and Hs578T. In particular activation of HOXA9 expression in lrECM 3D culture required binding of bromodomain containing 4 to the HOXA9 promoter and involved CpG hypomethylation. Our findings warrant further investigation of the ECM-regulated epigenetic coding of gene expression in the Claudin-low breast cancer.

// Shreya Mitra 1 , Lorenzo Federico 1 , Wei Zhao 1 , Jennifer Dennison 1 , Tapasree Roy Sarkar 2 , Fan Zhang 1 , Vinita Takiar 3 , Kwai W. Cheng 4 , Sendurai Mani 5 , Ju Seog Lee 1 , Gordon B. Mills 1 1 Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Center for Statistical Bioinformatics, Texas A&M University, College Station, TX, USA 3 Department of Radiation Oncology, University of Cincinnati, Cincinnati, OH, USA 4 Cardiac Catheterization Laboratory, Michael and DeBakey Medical Center, Houston, TX, USA 5 Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Correspondence to: Shreya Mitra, email: smitra@mdanderson.org Keywords: Rab25, Rab coupling protein, breast cancer, luminal B, claudin low Received: March 30, 2016 Accepted: May 10, 2016 Published: May 31, 2016 ABSTRACT The Rab GTPases regulate vesicular trafficking machinery that transports and delivers a diverse pool of cargo, including growth factor receptors, integrins, nutrient receptors and junction proteins to specific intracellular sites. The trafficking machinery is indeed a major posttranslational modifier and is critical for cellular homeostasis. Deregulation of this stringently controlled system leads to a wide spectrum of disorders including cancer. Herein we demonstrate that Rab25, a key GTPase, mostly decorating the apical recycling endosome, is a dichotomous variable in breast cancer cell lines with higher mRNA and protein expression in Estrogen Receptor positive (ER+ve) lines. Rab25 and its effector, Rab Coupling Protein (RCP) are frequently coamplified and coordinately elevated in ER+ve breast cancers. In contrast, Rab25 levels are decreased in basal-like and almost completely lost in claudin-low tumors. This dichotomy exists despite the presence of the 1q amplicon that hosts Rab25 across breast cancer subtypes and is likely due to differential methylation of the Rab25 promoter. Functionally, elevated levels of Rab25 drive major hallmarks of cancer including indefinite growth and metastasis but in case of luminal B breast cancer only. Importantly, in such ER+ve tumors, coexpression of Rab25 and its effector, RCP is significantly associated with a markedly worsened clinical outcome. Importantly, in claudin-low cell lines, exogenous Rab25 markedly inhibits cell migration. Similarly, during Snail-induced epithelial to mesenchymal transition (EMT) exogenous Rab25 potently reverses Snail-driven invasion. Overall, this study substantiates a striking context dependent role of Rab25 in breast cancer where Rab25 is amplified and enhances aggressiveness in luminal B cancers while in claudin-low tumors, Rab25 is lost indicating possible anti-tumor functions.

Breast cancer (BC) is a heterogeneous disease that is classically driven by the estrogen receptor (ER), progesterone receptor (PR) and human epithelial growth factor receptor 2 (EGFR2/HER2) signaling pathways. Triple negative breast cancer (TNBC), is a lethal subtype of invasive BC tumors that are ER-, PR- and HER2-. A subtype of TNBC is claudin low (CL). Dysregulation of claudin proteins disrupts tight junctions, consequently inducing the epithelial-to-mesenchymal transition (EMT) in cancers. This leads to enhanced motility and metastasis. Patients with CL TNBC have worse prognosis than patients with other BC subtypes. PVT1 is a long noncoding RNA (lncRNA) transcribed from the 8q24 genomic locus that has been implicated in multiple cancers including BC. Amplification of the 8q24 gene locus is a common event in many malignant diseases and is associated with poor clinical outcomes. Although previous research has implicated PVT1 as an important player in BC, the underlying molecular mechanisms of PVT1 in CL TNBC was previously unknown. We assessed PVT1 expression in BC, and we observed that PVT1 exons 4A, 4B, and 9 are significantly upregulated in MDA MB 231 cells (claudin low) and significantly downregulated in MDA MB 468 cells (claudin high), in comparison to T47D (ER+). We have confirmed that claudin expression, specifically claudins 1, 3, 4 and 7, are significantly higher in MDA MB 468 cells and significantly lower in MDA MB 231 cells. Knockdown of PVT1 exon 9 expression in the MDA MB 231 cell line, led to a significant reduction in migration when compared to cells transfected with a control scramble siRNA, indicating that PVT1 exon 9 regulates migration in CL TNBC. Interestingly, we observed that claudin 4 expression, and not claudins 1, 3 and 7, was increased in cells where PVT1 exon 9 was knocked down when compared to the control cells. This indicates that PVT1 exon 9 regulates claudin 4 protein stability in CL TNBC. We also assessed the expression of EMT markers (vimentin, fibronectin, and E-cadherin) in MDA MB 231 cells. We observed no changes in EMT markers when PVT1 exon 9 is knocked down; however, our data suggests that EMT markers are more highly expressed in MDA MB 231 cells in comparison to MDA MB 468 cells. Taken together, our data indicate that PVT1 exon 9 regulates claudin expression and migration in CL TNBC, and may have implications for clinical outcomes in TNBC. Citation Format: Fayola Levine, Olorunseun Ogunwobi. PVT1 exon 9 transcript regulates claudin 4 expression and migration in triple negative breast cancer [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-126.

Recent whole genome characterizations of primary human bladder cancers revealed that they can be grouped into “intrinsic” basal and luminal molecular subtypes. Here, we provide an overview of the subtypes and discuss their biological and clinical properties. Basal cancers are characterized by advanced stage and metastatic disease at presentation. They tend to be enriched with squamous and small cell/neuroendocrine features and inactivating mutations and deletions of TP53 and RB1. Basal cancers can be divided into “epithelial” and “mesenchymal” (also known as “claudin low”) subsets, and a portion of the latter form a “neuroendocrine/neuronal” subset that is associated with particularly poor survival. Luminal cancers are often enriched with papillary histopathological features and activating mutations in FGFR3, and they can also be divided into additional subsets based on differential stromal cell infiltration, relative genomic instability, and high- versus low-level expression of carcinoma in situ (CIS) gene expression signatures. Importantly, the bladder cancer molecular subtypes display differential sensitivities to neoadjuvant chemotherapy and immune checkpoint blockade, and preliminary data also suggest that they respond differently to radiation with or without hypoxia modulation. Ongoing studies are investigating the relevance of the molecular subtypes to the bladder cancer histopathological variants and to upper tract urothelial cancer. The bladder cancer molecular subtypes were associated with different prognoses and responses to conventional and targeted therapies in retrospective studies. If validated in prospective studies, molecular subtyping will be integrated into bladder cancer clinical management.

We report the discovery of a claudin-low molecular subtype of high-grade bladder cancer that shares characteristics with the homonymous subtype of breast cancer. Claudin-low bladder tumors were enriched for multiple genetic features including increased rates of RB1, EP300, and NCOR1 mutations; increased frequency of EGFR amplification; decreased rates of FGFR3, ELF3, and KDM6A mutations; and decreased frequency of PPARG amplification. While claudin-low tumors showed the highest expression of immune gene signatures, they also demonstrated gene expression patterns consistent with those observed in active immunosuppression. This did not appear to be due to differences in predicted neoantigen burden, but rather was associated with broad upregulation of cytokine and chemokine levels from low PPARG activity, allowing unopposed NFKB activity. Taken together, these results define a molecular subtype of bladder cancer with distinct molecular features and an immunologic profile that would, in theory, be primed for immunotherapeutic response.

Background: BRCA mutation-associated (BRCAmut) breast cancer represents a heterogeneous group displaying certain molecular features. Claudin-low breast cancers (CLBC) overlap with characteristics of BRCAmut tumors; therefore, we have investigated whether these are identical subtypes. Methods: Using public gene expression data, CLDN, CDH1, 9-cell line claudin-low predictor (9CLCLP) and PAM50 expression was evaluated in BRCAmut and BRCA wild-type (BRCAwt) breast cancer cases focusing on their possible overlap with the CLBC subtype. A separate formalin-fixed, paraffin-embedded (FFPE) cohort of 22 BRCAmut and 19 BRCAwt tumor tissues was used for immunohistochemical examination of AR, CD24, CD44, CK5/6, claudin-1, -3, -4 and -7, E-cadherin, EGFR, estrogen receptor (ER), EZH2, HER2, Ki67, p53, progesterone receptor (PgR) and vimentin expression. Results: In the data sets, CLDN1 (ROC = 0.785, p < 0.001), CDH1 (ROC = 0.785, p < 0.001), CLDN7 (ROC = 0.723, p < 0.001), CLDN3 (ROC = 0.696, p = 0.020) and CLDN4 (ROC = 0.685, p = 0.027) were expressed at higher level in BRCAmut than BRCAwt tumor tissue. The PAM50 subtype differed from the assigned immunohistochemistry (IHC)-based subtype in 30%. Based on accessible 9CLCLP predictor genes, BRCAmut breast cancer does not display the claudin-low phenotype. Utilizing FFPE samples, claudins were evidently expressed in both BRCAmut and BRCAwt cases. However, at the protein level, only claudin-3 expression was higher in BRCAmut tumors, while claudin-1, -4 and -7 and E-cadherin expression was lower compared to BRCAwt cases. A CD24low/CD44high phenotype was found in BRCAmut tumors upon comparison with BRCAwt cases (p < 0.001 and p = 0.001, respectively). Conclusions: There is a prominent correlation between the genes under focus herein and BRCA mutation status. BRCAmut tumors bear stem cell characteristics displaying a distinct cell adhesion molecule profile characterized by high expression of CDH1 and CLDN4 according to public gene expression data set analysis, and higher claudin-3 expression as detected by IHC; thus, BRCAmut breast carcinomas are not identical with the previously identified claudin-low subtype of breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) subtypes are clinically aggressive and are treated with targeted therapeutics commonly used in other BC subtypes. The claudin-low (CL) molecular subtype of TNBC has high rates of metastases, chemoresistance and recurrence. There exists an urgent need to identify novel therapeutic targets in TNBC; however, existing models utilized in target discovery research have limitations. Patient-derived xenograft (PDX) models have emerged as superior models for target discovery experiments because they recapitulate features of patient tumors that are limited by xenograft methods. METHODS: We utilize immunohistochemistry, qRT-PCR and Western Blot to visualize tumor architecture, cellular composition, genomic and protein expressions of a new TNBC PDX model (TU-BcX-2O0). We utilize tissue decellularization techniques to examine extracellular matrix composition of TU-BcX-2O0. RESULTS: Our laboratory successfully established a TNBC PDX tumor, TU-BCX-2O0, which represents a CL-TNBC subtype and maintains this phenotype throughout subsequent passaging. We dissected TU-BCx-2O0 to examine aspects of this complex tumor that can be targeted by developing therapeutics, including specific cell populations within the tumor, the extracellular matrix, and cancer stem-like cell populations. CONCLUSIONS: Here we characterize a claudin-low TNBC patient-derived xenograft model that can be utilized for therapeutic research studies.

Background ADAM12 is upregulated in human breast cancers and is a predictor of chemoresistance in estrogen receptor-negative tumors. ADAM12 is induced during epithelial-to-mesenchymal transition, a feature associated with claudin-low breast tumors, which are enriched in cancer stem cell (CSC) markers. It is currently unknown whether ADAM12 plays an active role in promoting the CSC phenotype in breast cancer cells. Methods ADAM12 expression was downregulated in representative claudin-low breast cancer cell lines, SUM159PT and Hs578T, using siRNA transfection or inducible shRNA expression. Cell characteristics commonly associated with the CSC phenotype in vitro (cell migration, invasion, anoikis resistance, mammosphere formation, ALDH activity, and expression of the CD44 and CD24 cell surface markers) and in vivo (tumor formation in mice using limiting dilution transplantation assays) were evaluated. RNA sequencing was performed to identify global gene expression changes after ADAM12 knockdown. Results We found that sorted SUM159PT cell populations with high ADAM12 levels had elevated expression of CSC markers and an increased ability to form mammospheres. ADAM12 knockdown reduced cell migration and invasion, decreased anoikis resistance, and compromised mammosphere formation. ADAM12 knockdown also diminished ALDEFLUOR+ and CD44hi/CD24-/lo CSC-enriched populations in vitro and reduced tumorigenesis in mice in vivo. RNA sequencing identified a significant overlap between ADAM12- and Epidermal Growth Factor Receptor (EGFR)-regulated genes. Consequently, ADAM12 knockdown lowered the basal activation level of EGFR, and this effect was abolished by batimastat, a metalloproteinase inhibitor. Furthermore, incubation of cells with exogenously added EGF prevented the downregulation of CD44hi/CD24-/lo cell population by ADAM12 knockdown. Conclusions These results indicate that ADAM12 actively supports the CSC phenotype in claudin-low breast cancer cells via modulation of the EGFR pathway. Electronic supplementary material The online version of this article (doi:10.1186/s12943-017-0599-6) contains supplementary material, which is available to authorized users.

Metastasis and mortality among patients with triple negative breast cancer (TNBC) of the claudin-low subtype remain high because these tumors are clinically aggressive, chemoresistant, and lack targeted therapies. We have previously used proteomic approaches to analyze orthotopic and metastatic tumors from the syngeneic murine T11 tumor model, which displays gene expression profiles that mirror human claudin-low TNBC. These studies identified Galectin-1 (Gal-1), an N-acetyllactosamine-binding protein, as an overexpressed protein in cultured T11 cells, primary tumors, and lung metastases compared to normal lung and mammary fat pad tissue from healthy control mice. These results were confirmed by Western immunoblot and immunohistochemistry and were further supported by analysis of murine tumor microarray data sets, which revealed Gal-1 overexpression in the claudin-low subtype compared to other breast cancer subtypes. Here we extend our murine studies to investigate Gal-1 expression in breast cancer patients, with specific inclusion of claudin-low tumors. To compare differential Gal-1 expression across breast cancer subtypes, we initially analyzed published human microarray data sets from normal breast and primary breast tumors (total n=423, of which 51 are the claudin-low subtype): the highest Gal-1 expression was observed in claudin-low breast cancer. In order to validate these findings, we are examining breast cancer specimens from a unique cohort of ethnically diverse patients from rural Eastern North Carolina with a median follow-up of over 8 years. RNASeq data from a subset of this cohort (n=121) was used to assign subtypes (using PAM50 and the claudin-low classifier). Further studies are in progress to compare Gal-1 expression between subtypes and correlate with clinicopathologic factors and outcome. Immunohistochemical Gal-1 staining of primary tumor tissue blocks is underway to confirm expression in each subtype at the protein level. Immunohistochemistry will also define Gal-1 expression in the cell types comprising the heterogeneous tumor microenvironment (tumor, stromal, immune cells) and its subcellular localization (nuclear, cytoplasmic, membrane-associated). Through this ongoing work evaluating Gal-1 across breast cancer subtypes, we will help corroborate initial findings from the T11 claudin-low mouse model and yield further insights into the role of Gal-1 in breast cancer progression. Citation Format: Kassondra Balestrieri, Moses McDaniel, Christiana Shoopman, Nasreen Vohra, Kathryn Verbanac. Galectin-1: A marker for claudin-low breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2147.

Claudin-low breast cancer is a molecular type of breast cancer originally identified by gene expression profiling and reportedly associated with poor survival. Claudin-low tumors have been recognised to preferentially display a triple-negative phenotype, however only a minority of triple-negative breast cancers are claudin-low. We sought to identify an immunohistochemical profile for claudin-low tumors that could facilitate their identification in formalin fixed paraffin embedded tumor material. First, an in silico collection of ~1600 human breast cancer expression profiles was assembled and all claudin-low tumors identified. Second, genes differentially expressed between claudin-low tumors and all other molecular subtypes of breast cancer were identified. Third, a number of these top differentially expressed genes were tested using immunohistochemistry for expression in a diverse panel of breast cancer cell lines to determine their specificity for claudin-low tumors. Finally, the immunohistochemical panel found to be most characteristic of claudin-low tumors was examined in a cohort of 942 formalin fixed paraffin embedded human breast cancers with >10 years clinical follow-up to evaluate the clinico-pathologic and survival characteristics of this tumor subtype. Using this approach we determined that claudin-low breast cancer is typically negative for ER, PR, HER2, claudin 3, claudin 4, claudin 7 and E-cadherin. Claudin-low tumors identified with this immunohistochemical panel, were associated with young age of onset, higher tumor grade, larger tumor size, extensive lymphocytic infiltrate and a circumscribed tumor margin. Patients with claudin-low tumors had a worse overall survival when compared to patients with luminal A type breast cancer. Interestingly, claudin-low tumors were associated with a low local recurrence rate following breast conserving therapy. In conclusion, a limited panel of antibodies can facilitate the identification of claudin-low tumors. Furthermore, claudin-low tumors identified in this manner display similar clinical, pathologic and survival characteristics to claudin-low tumors identified from fresh frozen tumor material using gene expression profiling.

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:Ptenf/f:p53lox.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∆:p53R270H 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∆:p53R270H vs. Pten∆:p53∆ spindle tumors. Accordingly, following tail vein injection, both Pten∆:p53R270H 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∆:p53R270H tumor cells to upregulate E-cadherin expression in lung metastases. Conclusions Our results demonstrate that WAP-Cre:Ptenf/f:p53lox.stop.lox_R270H mice represent a tractable model to study basal-like breast cancer because unlike p53 deletion, p53R270H mutation in the mouse does not skew tumors toward the claudin-low subtype. The WAP-Cre:Ptenf/f:p53lox.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. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0668-y) contains supplementary material, which is available to authorized users.

Breast cancer is a heterogeneous disease in terms of histology, therapeutic response, dissemination patterns to distant sites, and patient outcomes. To date, molecular characterization studies have started to elucidate the biology behind this heterogeneity. For example, 15 years of studies based on global gene expression analyses have helped identify 4 main intrinsic molecular subtypes of breast cancer (Luminal A, Luminal B, HER2-enriched, and basal-like). More recently, The Cancer Genome Atlas (TCGA) breast cancer project has further characterized this disease from a molecular perspective and provided important insights. In this chapter, we will describe the main molecular features of the intrinsic molecular subtypes and discuss how the combination of standard clinical–pathological markers with the information provided by these molecular entities might help further understand the biological complexity of this disease, increase the efficacy of current and novel therapies, and ultimately improve outcomes for breast cancer patients.

Background: Claudin-low breast carcinoma represents 19% of all breast cancer cases and is characterized by an aggressive progression with metastatic nature and high rates of relapse. Due to a lack of known specific molecular biomarkers for this breast cancer subtype, there are no targeted therapies available, which results in the worst prognosis of all breast cancer subtypes. Hence, the identification of novel biomarkers for this type of breast cancer is highly important for early diagnosis and targeted therapy. Methods: In this work, we propose the identification of peptides for the specific recognition of MDA-MB-231, a cell line representative of claudin-low breast cancers, using phage display (both conventional panning and BRASIL). Binding assays were performed to select the most interesting peptides and bioinformatics approaches were applied to putatively identify the biomarkers to which these peptides bind. Results: Two peptides were selected using this methodology specifically targeting MDA-MB-231 cells, as demonstrated by a 4 to 9 log higher affinity as compared to control cells. The use of bioinformatics approaches provided relevant insights into possible cell surface targets for each peptide identified. Conclusions: The peptides herein identified may contribute to an earlier detection of claudin-low breast carcinomas and possibly to develop more individualized therapies., This study was supported by the Portuguese Foundation for Science and Technology (FCT) and the European Community fund FEDER, through Program COMPETE, under the scope of the Projects FCOMP-01–0124-FEDER021053 (PTDC/SAU-BMA/121028/2010), RECI/BBB-EBI/0179/2012 (FCOMP-01– 0124-FEDER-027462), the strategic funding of UID/BIO/04469/2013 unit, and the Projects “BioHealth – Biotechnology and Bioengineering approaches to improve health quality”, REF. NORTE-07–0124-FEDER-000027, and “BioInd – Biotechnology and Bioengineering for improved Industrial and Agro-Food processes”, REF. NORTE-07–0124-FEDER-000028, co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER. Franklin L. Nóbrega acknowledges FCT for the grant SFRH/BD/86462/2012.

Breast cancer recurrence is believed to be caused by a subpopulation of cancer cells that possess the stem cell attribute of treatment resistance. Recently, we and others have reported the generation of breast cancer stem cells (BCSC) by epithelial–mesenchymal transition (EMT), although the physiologic process by which these cells may arise in vivo remains unclear. We show here that exposure of tumor cells to TGFβ and TNFα induces EMT and, more importantly, generates cells with a stable BCSC phenotype which is shown by increased self-renewing capacity, greatly increased tumorigenicity, and increased resistance to oxaliplatin, etoposide, and paclitaxel. Furthermore, gene expression analyses found that the TGFβ/TNFα-derived BCSCs showed downregulated expression of genes encoding claudin 3, 4, and 7 and the luminal marker, cytokeratin 18. These changes indicate a shift to the claudin-low molecular subtype, a recently identified breast cancer subtype characterized by the expression of mesenchymal and stem cell-associated markers and correlated with a poor prognosis. Taken together, the data show that cytokine exposure can be used to generate stable BCSCs ex vivo, and suggest that these cells may provide a valuable tool in the identification of stem cell-directed biomarkers and therapies in breast cancer. Cancer Res; 71(13); 4707–19. ©2011 AACR.

Breast cancer is a heterogeneous disease in terms of histology, therapeutic response, dissemination patterns to distant sites, and patient outcomes. Global gene expression analyses using high‐throughput technologies have helped to explain much of this heterogeneity and provided important new classifications of cancer patients. In the last decade, genomic studies have established five breast cancer intrinsic subtypes (Luminal A, Luminal B, HER2‐enriched, Claudin‐low, Basal‐like) and a Normal Breast‐like group. In this review, we dissect the most recent data on this genomic classification of breast cancer with a special focus on the Claudin‐low subtype, which appears enriched for mesenchymal and stem cell features. In addition, we discuss how the combination of standard clinical‐pathological markers with the information provided by these genomic entities might help further understand the biological complexity of this disease, increase the efficacy of current and novel therapies, and ultimately improve outcomes for breast cancer patients.

Our understanding of breast cancer as a clinical and biologic entity has been gaining granularity for several decades; in particular, the importance of hormone receptors and HER2 were realized long ago and have served as the impetus for therapeutic agents that have improved the cure rate of estrogen receptor-positive and HER2-positive breast cancer and the lives of thousands of women. The past decade brought even more understanding of the complexity of breast cancer biology through the development and clinical applications of array-based technologies for discovery and prognostication. We now realize that there are at least 5 intrinsic subtypes within breast cancer, at least one of which-the basal-like-currently lacks targeted therapies and is the most pressing therapeutic challenge for the next decade. We have several validated prognostic profiles that allow increased thoughtfulness in adjuvant decision making. With this understanding also comes the recognition that if breast cancer represents several biologically distinct entities, then breast cancer risk assessment and treatment must take this heterogeneity into account, which complicates trial design and interpretation. Despite therapeutic advances and the development of a number of targeted agents against hormone receptor signaling, HER2, and angiogenesis, we have significant challenges to overcome. These include the need for more tissue-based studies to allow us to understand the mechanisms of sensitivity and resistance within and across subtypes, and the need to revisit risk and prevention by subtype.

Cancer is often driven by deregulated protein kinases pathways including breast cancer. To profile the kinome at the protein level we are developing a kinase pulldown (KiP) proteomic platform that uses multiple kinase inhibitors conjugated to sepharose beads for protein kinase profiling. Mass spectrometry-base tryptic peptide sequencing is used to identify and quantify the kinome in an unbiased manner. To date, we have profiled and analyzed the kinomes of more than 50 breast cancer PDX model systems that include all five breast cancer subtypes. In these analyses we have begun to explore the kinome interactome because we have observed that KiP also pulls down breast cancer subtype specific kinase interacting proteins that may represent new biological insights. For example we have consistently identified dihydropyrimidinase-like 3 (DPYSL3) in p21-activated kinase (PAK) inhibitor KiP interactome in the claudin-low WHIM12 PDX model. DPYSL3 is an intracellular phosphoprotein known to play a role in cell migration and metastasis. However it remains to be seen if DPYSL3 functions as a suppressor or a promoter of metastasis. We therefore generated DPYSL3 knock-down cell lines to evaluate function in claudin-low breast cancer. Proliferation levels of DPYSL3 depleted Claudin-low WHIM12 cells (DPYSL3-) were lower than those of control WHIM 12 (DPYSL3+) cells. In contrast, migration levels of DPYSL3- WHIM12 were greater than those of control cells, suggesting a pivotal role for DPYSL3 in several cellular physiologies. Additionally the activity of a PAK inhibitor (FRAX597) on migration appeared to require DPYSL3, as the inhibitory effect of FRAX579 on migration in WHIM12 was lost when DPYSL3 expression was depleted. Additionally we find that only the long isoform of DPYSL3, not a short isoform, interacts with PAK to regulate migration, suggesting that alternative splicing of DPYSL3 may an important to the control of migration. Furthermore Snail is also negatively regulated DPYSL3 with elevated expression in DYPSL3- cells. In conclusion, the study of the kinome interactome in breast cancer PDX identified DPYSL3 as regulator of the PAK family of kinases in a claudin low breast cancer. DPYSL3 is necessary for PAK to promote migration and may play a pivotal role in complex cellular transitions from a proliferative state versus an EMT/cell migratory state. Citation Format: Matsunuma R, Chan DW, Kim B-J, Singh P, Han A, Malovannaya A, Perou CM, Ellis MJ. DPYSL3 modulates proliferation and migration in claudin-low breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-08.

Metaplastic breast cancers (MBC) are aggressive, chemoresistant tumors characterized by lineage plasticity. To advance understanding of their pathogenesis and relatedness to other breast cancer subtypes, 28 MBCs were compared with common breast cancers using comparative genomic hybridization, transcriptional profiling, and reverse-phase protein arrays and by sequencing for common breast cancer mutations. MBCs showed unique DNA copy number aberrations compared with common breast cancers. PIK3CA mutations were detected in 9 of 19 MBCs (47.4%) versus 80 of 232 hormone receptor–positive cancers (34.5%; P = 0.32), 17 of 75 HER-2–positive samples (22.7%; P = 0.04), 20 of 240 basal-like cancers (8.3%; P < 0.0001), and 0 of 14 claudin-low tumors (P = 0.004). Of 7 phosphatidylinositol 3-kinase/AKT pathway phosphorylation sites, 6 were more highly phosphorylated in MBCs than in other breast tumor subtypes. The majority of MBCs displayed mRNA profiles different from those of the most common, including basal-like cancers. By transcriptional profiling, MBCs and the recently identified claudin-low breast cancer subset constitute related receptor-negative subgroups characterized by low expression of GATA3-regulated genes and of genes responsible for cell-cell adhesion with enrichment for markers linked to stem cell function and epithelial-to-mesenchymal transition (EMT). In contrast to other breast cancers, claudin-low tumors and most MBCs showed a significant similarity to a “tumorigenic” signature defined using CD44+/CD24− breast tumor–initiating stem cell–like cells. MBCs and claudin-low tumors are thus enriched in EMT and stem cell–like features, and may arise from an earlier, more chemoresistant breast epithelial precursor than basal-like or luminal cancers. PIK3CA mutations, EMT, and stem cell-like characteristics likely contribute to the poor outcomes of MBC and suggest novel therapeutic targets. [Cancer Res 2009;69(10):4116–24]

// Alexander Ring 1, 2 , Neal Mineyev 3 , Weizhu Zhu 1, 2 , Emily Park 4 , Chip Lomas 5 , Vasu Punj 2, 6 , Min Yu 2, 7 , Dany Barrak 1, 2 , Victoria Forte 2 , Tania Porras 1, 2 , Debu Tripathy 8 , Julie E. Lang 1, 2 1 Department of Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA 2 Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, USA 3 Department of Surgery, Lenox Hospital New York, New York, NY 10065, USA 4 Advanced Cell Diagnostics, Research and Development, Hayward, CA 94545, USA 5 BD Biosciences, Research and Development, San Jose, CA 95131, USA 6 Division of Hematology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA 7 Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA 8 Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA Correspondence to: Julie E. Lang, e-mail: Julie.Lang@med.usc.edu Keywords: breast cancer, circulating tumor cells, IE/FACS, EpCAM Received: July 10, 2015 Accepted: October 09, 2015 Published: October 19, 2015 ABSTRACT Purpose: The potential utility of circulating tumor cells (CTCs) as liquid biopsies is of great interest. We hypothesized that CTC capture using EpCAM based gating is feasible for most breast cancer subtypes. Results: Cancer cells could be recovered from all intrinsic subtypes of breast cancer with IE/FACS, however, claudin-low cell lines showed very low capture rates compared to the four other groups ( p = 0.03). IE/FACS detection of CTC mimic cells was time sensitive, emphasizing controlling for pre-analytic variables in CTC studies. Median fluorescent intensity for flow cytometry and RNA flow cell type characterization were highly correlated, predicting for CTC isolation across molecular subtypes. RNA-Seq of IE/FACS sorted single cell equivalents showed high correlation compared to bulk cell lines, and distinct gene expression signatures compared to PB. Materials and Methods: Ten cell lines representing all major subtypes of breast cancer were spiked (as CTC mimics) into and recovered from peripheral blood (PB) using immunomagnetic enrichment followed by fluorescence-activated cell sorting (IE/FACS). Flow cytometry and RNA flow were used to quantify the expression of multiple breast cancer related markers of interest. Two different RNA-Seq technologies were used to analyze global gene expression of recovered sorted cells compared to bulk cell lines and PB. Conclusions: EpCAM based IE/FACS detected and captured a portion of spiked cells from each of the 10 cell lines representing all breast cancer subtypes, including basal-like but not claudin-low cancers. The assay allows for the isolation of high quality RNA suitable for accurate RNA-Seq of heterogeneous rare cell populations.

Epithelial Mesenchymal Transition (EMT) is the transdifferentiation of epithelial cells into a mesenchymal phenotype. This process occurs during embryogenesis but also in wound healing and in tumors. The neoplastic EMT is characterized by variably complete shedding of epithelial architectural features and acquisition of mesenchymal traits. In immunohistochemistry a variable coexpression of cytokeratins, vimentin or alpha-smooth muscle actin with loss of E-cadherin and other interepithelial adhesion molecules is characteristic. Such transition is associated with mutations both at the genetic (somatic) and epigenetic levels and is believed to confer a more advantageous phenotype for local and distant spread of cancer cells. Mammary carcinoma can exhibit EMT features in humans and mice and it tends to occur more frequently in women with tumors bearing a worse prognosis such as the claudin low subtype within the triple negative cancer. Missense mutation of TP53 is one of the most common mutations in cancer and it is frequently found in EMT tumor types, often with a more aggressive behavior. The current literature and survey of our mouse EMT cases in the Genomic Pathology Center image archives demonstrate a synergy between p53 and EMT that is independent of the initiating oncogene. However, p53 mutation is not sufficient or causal for EMT. Moreover, despite the local malignant behavior, processes such as spontaneous metastases and Mesenchymal Epithelial Transition (MET) appear not to be as frequent and obvious as previously hypothesized.

Significant morphological, clinical and biological prognostic factors vary according to molecular subtypes of breast tumors, yet comprehensive analysis of such factors linked to survival in each group is lacking. Clinicopathological and micro-environmental criteria, estrogen (ER), progesterone (PR) receptors, HER2, Ki67, basal markers, CD24, CD44, ALDH1, BCL2, E-Cadherin and Trio were assessed in 1070 primary operable breast cancers from a single center according to five main molecular subtypes and associations with distant metastasis-free survival (DMFS) were examined. There were 682 (64 %) luminal A (LA), 166 (16 %) Luminal B HER2 negative (LBH−), 47 (4 %) Luminal B HER2 positive (LBH+), 108 (10 %) triple negative (TN) and 67 (6 %) HER2-enriched tumors (H2+). Median follow-up was 13.7 years. At 5 years, DMFS in LA (90 %) was better than in LBH− (80.9 %), hazard ratio (HR) = 2.22 [1.44–3.43] P

Summary Recent studies suggest that thousands of genes may contribute to breast cancer pathophysiologies when deregulated by genomic or epigenomic events. Here, we describe a model ‘‘system’’ to appraise the functional contributions of these genes to breast cancer subsets. In general, the recurrent genomic and transcriptional characteristics of 51 breast cancer cell lines mirror those of 145 primary breast tumors, although some significant differences are documented. The cell lines that comprise the system also exhibit the substantial genomic, transcriptional, and biological heterogeneity found in primary tumors. We show, using Trastuzumab (Herceptin) monotherapy as an example, that the system can be used to identify molecular features that predict or indicate response to targeted therapies or other physiological perturbations.

Characteristic patterns of gene expression measured by DNA microarrays have been used to classify tumors into clinically relevant subgroups. In this study, we have refined the previously defined subtypes of breast tumors that could be distinguished by their distinct patterns of gene expression. A total of 115 malignant breast tumors were analyzed by hierarchical clustering based on patterns of expression of 534 “intrinsic” genes and shown to subdivide into one basal-like, one ERBB2 -overexpressing, two luminal-like, and one normal breast tissue-like subgroup. The genes used for classification were selected based on their similar expression levels between pairs of consecutive samples taken from the same tumor separated by 15 weeks of neoadjuvant treatment. Similar cluster analyses of two published, independent data sets representing different patient cohorts from different laboratories, uncovered some of the same breast cancer subtypes. In the one data set that included information on time to development of distant metastasis, subtypes were associated with significant differences in this clinical feature. By including a group of tumors from BRCA1 carriers in the analysis, we found that this genotype predisposes to the basal tumor subtype. Our results strongly support the idea that many of these breast tumor subtypes represent biologically distinct disease entities.

Introduction: The molecular architecture of tight junctions (TJ) has been a subject of extensive studies in a number of malignancies. Among the TJ components, the members of the protein family claudins (CLDN) have been found to be differentially expressed correlating with histological grade (HG) in a number of tissues. Furthermore, the expression of these junctional proteins (JP) have recently been implied in the identification of the claudin-low subtype by gene expression analysis. Material and methods: 261 breast cancer specimens diagnosed between 1999 and 2002 were collected and evaluated histopathologically and immunophenotypically by a single specialist. Tissue microarrays were constructed using a TMA-builder instrument (Histopathology Ltd., Pecs, Hungary). To identify the eventual prognostic role of the TJ proteins CLDN3, −4 and −7, and adherent junction protein E-cadherin (ECDH) their immunohistochemical (IHC) expression was analysed by 3 investigators separately, and a consensus score was reached. The IHC results were compared to the patients' follow-up data applying Kaplan-Meier analysis supported by log-rank test. Furthermore, we aimed at identifying the worse prognosis tumour group based on clustering of the expression of the above mentioned JPs. Results: The expression of CLDN3 correlated with vascular invasion (VI)/p = 0.029/, necrosis (NC)/p = 0.001/, HG/p = 0.001/ and mitotic index (MI)/p = 0.022/. CLDN4 and CLDN7 correlated with NC/p = 0.001, p = 0.012, respectively/ and HG/p = 0.034, p = 0.001, respectively/, while ECDH correlated with HG/p = 0.001/ and MI/p = 0.001/. In single protein analysis, groups displaying gradually increasing CLDN3, −7 and ECDH/p = 0.017/expression (split at mean expression ± SD) showed a trend of bearing poorer prognostic features. CLDN4, on the other hand, showed different tendency: gradually increasing expression also correlated with worse relapse-free survival (RFS), while CLDN4 negative/low tumors had the poorest prognosis/p = 0.034/. In multiple protein analysis, groups were created using k-means clustering and the following cohorts were created - appearing in prognostic sequence starting with the longest RFS: ECDH-low groups with 1) low/n=31/ and 2) high/n=42/ CLDN3 expression; 3) CLDN7-low (CLDN3, -4 and ECDH-high) group/n=35/, 4) “all-high” expression group/n=70/; 5) CLDN3-low (CLDN4, -7 and ECDH-high) group/n=68/ and 6) “all-low” group/n=15/. The latter was characterised by the CLDN4-low feature as compared to the other groups. Conclusion: Expression of JPs can have prognostic relevance in breast carcinomas. The groups with low expression of ECDH have good prognosis, while gradually increasing JP expression outlines poor prognostic tumours. Strikingly the drop in all JPs expression - most likely resembling the claudin-low subtype - specifies the subgroup with the poorest outcome, which still remains an infrequent disease entity. Grant support: TÁMOP-4.2.2/B-10/1-2010-0013. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-05-08.

Many cancers including breast cancer display hieratical organization and are driven by a cellular sub-population that displays stem cell properties. These cancer stem cells (CSC’s) mediate metastasis and contribute to treatment resistance highlighting the importance of elucidating CSC regulatory pathways. Nuclear proteins and lncRNAs involved in the maintenance of chromatin structure have been shown to play roles in CSC regulation. Among them the polycomb proteins bmi-1 and EZH2 are involved in epigenetic regulation of CSC’s in breast and other cancers. SHARP/SPEN, an RNA binding protein, has been previously been found to bind the lncRNA Xist which, by recruiting the PRC2 polycomb complex mediates transcriptional repression of the X chromosome. We utilized breast cancer cell lines and xenograft models to examine the role of the SHARP/Xist complex in the regulation of breast CSC’s. In a series of breast cancer cell lines including MCF7 (luminal )and SUM 159 (basal/claudin low) cells, SHARP mRNA and protein as well as the lncRNA Xist were more highly expressed in cells expressing aldehyde dehydrogenase as accessed by the Aldefluor assay than in ALDH -cells . Conditional knockdown of SHARP or Xist in SUM159 significantly decreased the ALDH+ CSC population without affecting cell growth in vitro. Furthermore, SHARP or Xist knockdown significantly reduced tumor initiating capacity and growth of SUM 159 cells in NOD/SCID mice. To determine the mechanism of breast CSC regulation by the SHARP complex we compared mRNA expression patterns of control and SHARP knockdown SUM 159 cells using RNAseq. There was significant overlap between the genes regulated by SHARP with genes regulated by the PCR1 and PRC2 polycomb complexes in addition to many histone genes. These studies suggest that the SHARP/Xist complex my regulate breast CSC’s through epigenetic regulatory histone and polycomb genes. Since SHARP expression is elevated in a number of cancers including breast cancer this pathway may represent a novel therapeutic target. Citation Format: Yongyou Zhu, Li Shang, Justin Colacino, Michael Brooks, Ramdane Harouaka, Chang-Ching Lin, Ming Luo, Max S. Wicha. A SHARP /Xist complex regulates breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4768. doi:10.1158/1538-7445.AM2017-4768

Introduction: Breast cancer can be separated into five intrinsic subtypes based on differences in the transcriptome of the tumor. We propose that the intrinsic differences of specific tumor subytpes lead to extrinsic differences in the tumor microenvironment. Methods: We utilized human clinical and genetically engineered mouse model (GEMM) samples of the intrinsic subtypes luminal A, basal-like, and claudin-low breast cancers to evaluate the immune landscape in the tumor microenvironment by histology and microarray analysis. Our claudin-low GEMM was derived from BRCA-/-/p53-/- mice. The HER-2 overexpressing, basal-like, and luminal A models have been previously descried. We utilized the FoxP3-DTR transgenic mouse model as a method of regulatory T cell (Treg) depletion to evaluate their function in these GEMMs. Results: The claudin-low human tumors were heavily infiltrated with immune cells, with CD4+ T cells being the most prominent, when compared to the luminal A subtype (P = 0.01). There were also increased focal areas of Tregs in human claudin-low tumors. To evaluate the mechanism for these findings, we utilized a GEMM of claudin-low tumors in addition HER-2 overexpressing, basal-like, and luminal A models. Mice with claudin-low tumors recruited elevated numbers of immune cells to the tumor microenvironment when compared to other breast cancer subtypes (P=0.01). Additionally, there was increased expression of multiple chemokine ligands in the tumor microenvironment among claudin-low tumors, with CXCL12 being the most highly overexpressed. Because the claudin-low tumors were heavily immune infiltrated, we hypothesized that blockade of the inhibitory checkpoint receptors programmed death-1 (PD-1) and cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) could delay tumor growth and improve anti-tumor immune response. Surprisingly, we saw no delay in tumor growth in the claudin-low model using checkpoint inhibition. To investigate if presence of Tregs limited the function of checkpoint inhibitors, mice with claudin-low tumors were treated with AMD3100, a CXCR4 inhibitor. This decreased Treg infiltration into the tumor but did not alter tumor growth. We then utilized the FoxP3-DTR transgenic mouse model, where depletion of Tregs alone resulted in a very modest decrease in tumor growth, while depletion of Tregs plus checkpoint inhibition significantly improved survival (P = 0.03) and increased cytokine production by CD8+ T cells. Conclusion: We found that an effective anti-tumor immune response in claudin-low tumors is inhibited by the recruitment of Tregs to the tumor microenvironment. These data highlight early Treg recruitment as a possible mechanism for the lack of response to immune checkpoint inhibition therapy for claudin-low breast cancer. Note: This abstract was not presented at the meeting. Citation Format: Sarah C. Vick, Nicholas A. Taylor, Michael D. Iglesia, W June Brickey, Lisa A. Carey, Bentley R. Midkiff, Karen P. McKinnon, Shannon Reisdorf, Joel S. Parker, Charles M. Perou, Benjamin G. Vincent, Jonathan S. Serody. Regulatory T cell recruitment limits the effectiveness of checkpoint inhibition for claudin-low breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 568. doi:10.1158/1538-7445.AM2017-568

Triple negative breast cancers (TNBC) are characterized by loss of expression of hormone receptors and decreased expression of the human epidermal growth factor receptor 2 (HER2). TNBC patients do not benefit from current targeted breast cancer (BC) treatments. Molecular profiling of breast cancer has found that TNBC is largely comprised of basal-like and claudin-low intrinsic molecular subtypes. Claudin-low breast cancer (CLBC) accounts for approximately one third of TNBCs, and early evidence suggests CLBC tumors may be more resistant to neoadjuvant anthracycline/taxane-based chemotherapy compared to basal-like tumors. For the development of novel breast cancer therapeutics, attention must be paid to therapeutic efficacy in specific sub-types of the disease. We discovered a type of silver nanoparticle (AgNP) that is selectively cytotoxic for treatment of CLBC. We find that the increased sensitivity of CLBC cells as compared to non-cancerous cells was independent of nanoparticle size, and CLBC cell lines (MDA-MB-231, BT-549, SUM-159) are more sensitive to AgNP exposure than luminal A BC (MCF-7), HER2 positive BC (SKBR3), and basal-like BC (MDA-MB-468, BT-20), or non-cancerous breast cells (MCF-10A, 184B5, HMT-3522 S1) via MTT assay. By treating CLBC cells and non-cancer breast cells with AgNPs or silver ions, in the form of silver nitrate, we demonstrated that intact AgNPs are necessary for selective cytotoxicity in CLBC. To determine the mechanism of cell death caused by AgNPs, annexin V (AnnV) and propidium iodide (PI) co-staining was performed on non-cancerous MCF-10A breast cells and MDA-MB-231 CLBC cells treated with AgNPs for 48 hours. AgNPs induced a dose-dependent increase in late-stage apoptosis and an increase in necrosis in MDA-MB-231 compared to vehicle control. Conversely, AgNPs had a minimal effect on late-stage apoptosis and necrosis in non-cancerous MCF-10A. Utilizing western blots, we showed that AgNPs induce oxidative damage to protein thiols and activate the unfolded protein response (UPR) in CLBC, but not in non-cancerous breast cells. To better recapitulate the tumor volume in an in vitro setting, multicellular tumor nodules from MDA-MB-231 TNBC cells or HMT-3522 S1 non-transformed mammary epithelial cells were formed by culturing the cells on basement membrane. When grown using 3D culture techniques, HMT-3522 S1 cells can develop growth arrested, polarized spherical structures containing lumens resembling a normal breast acinar structure characterized by a central lumen, cell-cell junctional complexes (including apical tight junctions, TJ), and basal expression of hemidesmosomal α6/β4 integrins ligating an endogenous basement membrane. 48h treatment with AgNPs did not prevent apical localization of the TJ marker ZO-1, alter the basal deposition of collagen-IV, induce proliferation of the growth arrested acini, or induce DNA damage. Conversely, significant amounts of DNA damage were observed in the MDA-MB-231 tumor nodules following 48h AgNP treatment. We conducted a 3 month in vivo safety/efficacy study in CLBC tumor-bearing mice where we showed that AgNPs can be delivered repeatedly at substantial doses intravenously and are effective for treatment of tumors without systemic toxicity. This is the first time any nanomaterial has been demonstrated subtype-specific therapeutic efficacy. This work demonstrates that AgNPs may provide a significant benefit for the CLBC patient population. Citation Format: Jessica L. Swanner, Iliana Tenvooren, Brian W. Bernish, Cale D. Fahrenholtz, Pierre A. Vidi, Katherine L. Cook, Ravi N. Singh. Silver nanoparticles exhibit subtype specific cytotoxic and therapeutic effects in claudin low breast cancer in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B47.

Claudin-low breast cancer (CLBC) is a poor prognosis disease biologically characterized by stemness and mesenchymal features. These tumors disproportionately affect younger patients and women with African ancestry, causing significant morbidity and mortality, and no effective targeted therapy exists at present. CLBC is thought to originate from mammary stem cells, but little is known on how or why these tumors express a stable epithelial-to-mesenchymal transition phenotype, or what are the driving forces of this disease. Here, we report that Manic Fringe (Mfng), which encodes an O-fucosylpeptide 3-β-N-acetylglucosaminyltransferase known to modify EGF repeats in the Notch extracellular domain, is highly expressed in CLBC and functions as an oncogene in this context. We show that Mfng modulates Notch activation in human and mouse CLBC cell lines, as well as in mouse mammary gland. Mfng silencing in CLBC cell lines reduced cell migration, tumorsphere formation, and in vivo tumorigenicity associated with a decrease in the stem-like cell population. Mfng deletion in the Lfngflox/flox;MMTV-Cre mouse model, in which one-third of mammary tumors resemble human CLBC, caused a tumor subtype shift away from CLBC. We identified the phosphoinositide kinase Pik3cg as a direct transcriptional target of Mfng-facilitated RBPJκ-dependent Notch signaling. Indeed, pharmacologic inhibition of PI3Kγ in CLBC cell lines blocked migration and tumorsphere formation. Taken together, our results define Mfng as an oncogene acting through Notch-mediated induction of Pik3cg. Furthermore, they suggest that targeting PI3Kγ may prove beneficial for the treatment of CLBC subtype. Cancer Res; 75(10); 1936–43. ©2015 AACR.

The vasculature serves as the main conduit for breast tumor metastases and is a target of therapeutics in many tumor types. In this study, we aimed to determine if tumor-associated vascular properties could help to explain the differences observed in metastagenicity across the intrinsic subtypes of human breast tumors. Analysis of gene expression signatures from more than 3,000 human breast tumors found that genomic programs that measured vascular quantity, vascular proliferation, and a VEGF/Hypoxia-signature were the most highly expressed in claudin-low and basal-like tumors. The majority of the vascular gene signatures added metastasis-predictive information to immunohistochemistry-defined microvessel density scores and genomically defined-intrinsic subtype classification. Interestingly, pure claudin-low cell lines, and subsets of claudin-low-like cells within established basal-like cancer cell lines, exhibited endothelial/tube-like morphology when cultured on Matrigel. In vivo xenografts found that claudin-low tumors, but not luminal tumors, extensively perfused injected contrast agent through paracellular spaces and non-vascular tumor-lined channels. Taken together, the endothelial-like characteristics of the cancer cells, combined with both the amount and the physiologic state of the vasculature contribute to breast cancer metastatic progression. We hypothesize that the genetic signatures we have identified highlight patients that should respond most favorably to anti-vascular agents. Electronic supplementary material The online version of this article (doi:10.1007/s10585-013-9607-4) contains supplementary material, which is available to authorized users.

Background The lastly identified claudin-low (CL) subtype of breast cancer (BC) remains poorly described as compared to the other molecular subtypes. We provide a comprehensive characterization of the largest series of CL samples reported so far. Methods From a data set of 5447 invasive BC profiled using DNA microarrays, we identified 673 CL samples (12,4%) that we describe comparatively to the other molecular subtypes at several levels: clinicopathological, genomic, transcriptional, survival, and response to chemotherapy. Results CL samples display profiles different from other subtypes. For example, they differ from basal tumors regarding the hormone receptor status, with a lower frequency of triple negative (TN) tumors (52% vs 76% for basal cases). Like basal tumors, they show high genomic instability with many gains and losses. At the transcriptional level, CL tumors are the most undifferentiated tumors along the mammary epithelial hierarchy. Compared to basal tumors, they show enrichment for epithelial-to-mesenchymal transition markers, immune response genes, and cancer stem cell–like features, and higher activity of estrogen receptor (ER), progesterone receptor (PR), EGFR, SRC and TGFβ pathways, but lower activity of MYC and PI3K pathways. The 5-year disease-free survival of CL cases (67%) and the rate of pathological complete response (pCR) to primary chemotherapy (32%) are close to those of poor-prognosis and good responder subtypes (basal and ERBB2-enriched). However, the prognostic features of CL tumors are closer to those observed in the whole BC series and in the luminal A subtype, including proliferation-related gene expression signatures (GES). Immunity-related GES valuable in basal breast cancers are not significant in CL tumors. By contrast, the GES predictive for pCR in CL tumors resemble more to those of basal and HER2-enriched tumors than to those of luminal A tumors. Conclusions Many differences exist between CL and the other subtypes, notably basal. An unexpected finding concerns the relatively high numbers of ER-positive and non-TN tumors within CL subtype, suggesting a larger heterogeneity than in basal and luminal A subtypes. Electronic supplementary material The online version of this article (doi:10.1186/1476-4598-13-228) contains supplementary material, which is available to authorized users.