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Cytochrome P4501B1 (CYP1B1) is elevated in breast cancer. Studies indicate a relationship between CYP1B1 and aggressive cancer phenotypes. Here, we report on in vitro studies in triple-negative breast cancer cell lines, where knockdown (KD) of CYP1B1 was used to determine the influence of its expression on invasive cell phenotypes. CYP1B1 KD in MDA-MB-231 cells resulted in the loss of mesenchymal morphology, altered expression of epithelial–mesenchymal genes, and increased claudin (CLDN) RNA and protein. CYP1B1 KD cells had increased cell-to-cell contact and paracellular barrier function, a reduced rate of cell proliferation, abrogation of migratory and invasive activity, and diminished spheroid formation. Analysis of clinical breast cancer tumor samples revealed an association between tumors exhibiting higher CYP1B1 RNA levels and diminished overall and disease-free survival. Tumor expression of CYP1B1 was inversely associated with CLDN7 expression, and CYP1B1HI/CLDN7LOW identified patients with lower median survival. Cells with CYP1B1 KD had an enhanced chemosensitivity to paclitaxel, 5-fluorouracil, and cisplatin. Our findings that CYP1B1 KD can increase chemosensitivity points to therapeutic targeting of this enzyme. CYP1B1 inhibitors in combination with chemotherapeutic drugs may provide a novel targeted and effective approach to adjuvant or neoadjuvant therapy against certain forms of highly metastatic breast cancer.

CD44 is a transmembrane glycoprotein that can regulate the oncogenic process. This is known to be a marker of the claudin-low subtype of breast cancer, as well as a cancer stem cell marker. However, its functional regulatory roles are poorly understood in claudin-low breast cancer. To gain comprehensive insight into the function of CD44, we performed an in-depth tandem mass tag-based proteomic analysis of two claudin-low breast cancer cell lines (MDA-MB-231 and Hs 578T) transfected with CD44 siRNA. As a result, we observed that 2736 proteins were upregulated and 2172 proteins were downregulated in CD44-knockdown MDA-MB-231 cells. For Hs 578T CD44-knockdown cells, 412 proteins were upregulated and 443 were downregulated. Gene ontology and network analyses demonstrated that the suppression of this marker mediates significant functional alterations related to oncogenic cellular processes, including proliferation, metabolism, adhesion, and gene expression regulation. A functional study confirmed that CD44 knockdown inhibited proliferation by regulating the expression of genes related to cell cycle, translation, and transcription. Moreover, this promoted the expression of multiple cell adhesion-associated proteins and attenuated cancer cell migration. Finally, our proteomic study defines the landscape of the CD44-regulated proteome of claudin-low breast cancer cells, revealing changes that mediate cell proliferation and migration. Our proteomics data set has been deposited to the ProteomeXchange Consortium via the PRIDE repository with the data set identifier PXD015171.

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.

Several molecular subtypes of bladder cancer were identified with differing clinical behavior and responses to platinum-based chemotherapy. But so far, their urothelial histomorphologic features, besides association with some variant histologies, have remained fully undefined. We sought to characterize the histological features of genomically classified bladder cancers more extensively to tumor in radical cystectomy (RC) specimens. Forty-eight bladder cancers submitted to The Cancer Genome Atlas (TCGA) were classified using the BASE47 genomic classifier into luminal subtype (LS) (14 cases), basal subtype (BS) (18 cases), and claudin-low subtype (CLS) (16 cases), and TCGA samples and the corresponding RC specimens were histologically assessed. Marked pleomorphism was more extensive in CLS tumors (87.5% had15% extent) than in LS tumors (21.4%) (p = 0.0006), whereas the extent in BS tumors was in between LS and CLS tumors. Pleomorphism in distant carcinoma in situ appeared to correlate with that in the main tumor. Ki-67 proliferation was higher in CLS tumors (mean = 61%) than in LS tumors (mean = 29%) or BS (mean = 30%) (p 0.001). Squamous differentiation was more extensive in BS and CLS tumors (38.2% of BS and CLS tumors versus 7.1% of LS tumors had30% squamous, p = 0.040). Sarcomatoid change was present in BS and CLS tumors only. The micropapillary variant was identified in LS (3/14) and BS (4/18) tumors only. Histologic features associated with aggressiveness (eg, marked pleomorphism, high proliferation, and sarcomatoid change) are enriched in CLS tumors, correlating with its known poorer outcome that may provide hints in their microscopic distinction. Features more associated with BS than with LS tumors (eg, squamous, marked pleomorphism, and sarcomatoid change) are also identified or enhanced in CLS tumors, supporting the genomic findings suggesting CLS tumor as a hyperbasal form of BS tumor.

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.

Breast cancer is an extremely heterogeneous disease comprising at least ten different subtypes, each exhibiting different characteristics in progression, prognosis and response to treatment. Transforming growth factor �� (TGF��) a member of the TGF superfamily of cytokines differentially regulates breast tumour-initiating cells (BTICs). In the claudin-low breast cancer subtype, TGF�� increases the tumour initiating capacity through the ability of the scaffolding protein NEDD9 to unite the TGF��/SMAD and Rho-Actin-SRF pathways. Previously, oncogenicity has been ascribed to the level of NEDD9 protein expression. However, my analysis indicates this mediation by NEDD9 is irrespective of NEDD9 protein expression, which is ubiquitously overexpressed in the majority of cancer types including breast cancer. In this thesis, I demonstrate how in the Claudin-low breast cancer subtype NEDD9 protein expression and post-translational modification are influenced by TGF�� pathway activation. I identify significant NEDD9 interacting proteins and their downstream effectors which contribute towards oncogenic TGF�� signalling pathways. A key TGF�� specific NEDD9 interactor identified in this subtype is the metabolic isoenzyme PKM2. Through a variety of techniques, I explore the known roles of PKM2 in the regulation of oncogenic metabolic reprogramming and demonstrate how these processes are influenced by its association with NEDD9. Finally, I investigate potential translational applications and biomarkers of TGF�� mediated, NEDD9/PKM2 dependent downstream signalling pathways, using large clinical breast cancer datasets and patient-derived tumour xenograft (PDTX) models. These data suggest a novel mechanism by which oncogenic TGF�� signalling regulates cellular proliferation and self-renewal via ��-Catenin/c-Myc regulation of altered metabolism in the Claudin-low breast cancer subtype, a process which is dependent upon the scaffolding protein NEDD9 and the metabolic enzyme PKM2. Together, these data suggest that a combined biomarker of TGF�� signalling and c-Myc expression may be useful in identifying a subset of Claudin-low breast cancer patients who would be sensitive to inhibition of Wnt/��-Catenin signalling. Additionally, due to the dependence of these tumours on c-Myc driven glutamine dependent metabolic processes, metabolic magnetic resonance imaging may be useful for monitoring response in these patients.

HER2-positive breast cancers may lose HER2 expression in recurrences and metastases. In this work, we studied cell lines derived from two transgenic mammary tumors driven by human HER2 that showed different dynamics of HER2 status. MamBo89HER2stable cell line displayed high and stable HER2 expression, which was maintained upon in vivo passages, whereas MamBo43HER2labile cell line gave rise to HER2-negative tumors from which MamBo38HER2loss cell line was derived. Both low-density seeding and in vitro trastuzumab treatment of MamBo43HER2labile cells induced the loss of HER2 expression. MamBo38HER2loss cells showed a spindle-like morphology, high stemness and acquired in vivo malignancy. A comprehensive molecular profile confirmed the loss of addiction to HER2 signaling and acquisition of an EMT signature, together with increased angiogenesis and migration ability. We identified PDGFR-B among the newly expressed determinants of MamBo38HER2loss cell tumorigenic ability. Sunitinib inhibited MamBo38HER2loss tumor growth in vivo and reduced stemness and IL6 production in vitro. In conclusion, HER2-positive mammary tumors can evolve into tumors that display distinctive traits of claudin-low tumors. Our dynamic model of HER2 status can lead to the identification of new druggable targets, such as PDGFR-B, in order to counteract the resistance to HER2-targeted therapy that is caused by HER2 loss.

Description, EMT induces cilium assembly and signaling to promote mammogenesis and tumorigenesis., The epithelial-mesenchymal transition (EMT) and primary ciliogenesis induce stem cell properties in basal mammary stem cells (MaSCs) to promote mammogenesis, but the underlying mechanisms remain incompletely understood. Here, we show that EMT transcription factors promote ciliogenesis upon entry into intermediate EMT states by activating ciliogenesis inducers, including FGFR1. The resulting primary cilia promote ubiquitination and inactivation of a transcriptional repressor, GLIS2, which localizes to the ciliary base. We show that GLIS2 inactivation promotes MaSC stemness, and GLIS2 is required for normal mammary gland development. Moreover, GLIS2 inactivation is required to induce the proliferative and tumorigenic capacities of the mammary tumor–initiating cells (MaTICs) of claudin-low breast cancers. Claudin-low breast tumors can be segregated from other breast tumor subtypes based on a GLIS2-dependent gene expression signature. Collectively, our findings establish molecular mechanisms by which EMT programs induce ciliogenesis to control MaSC and MaTIC stemness, mammary gland development, and claudin-low breast cancer formation.

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.

Claudin-low breast cancer represents an aggressive molecular subtype that is comprised of mostly triple-negative mammary tumor cells that possess stem cell-like and mesenchymal features. Little is known about the cellular origin and oncogenic drivers that promote claudin-low breast cancer. In this study, we show that persistent oncogenic RAS signaling causes highly metastatic triple-negative mammary tumors in mice. More importantly, the activation of endogenous mutant KRAS and expression of exogenous KRAS specifically in luminal epithelial cells in a continuous and differentiation stage-independent manner induces preneoplastic lesions that evolve into basal-like and claudin-low mammary cancers. Further investigations demonstrate that the continuous signaling of oncogenic RAS, as well as regulators of EMT, play a crucial role in the cellular plasticity and maintenance of the mesenchymal and stem cell characteristics of claudin-low mammary cancer cells., The cellular origin and oncogenic drivers promoting claudin-low breast cancer are undefined. Here, the authors report that the consistent activation of oncogenic RAS signaling, as well as regulators of EMT, play a crucial role in the cellular plasticity and maintenance of the mesenchymal and stem cell characteristics of claudin-low mammary cancer cells.

The Epithelial–Mesenchymal Transition (EMT) and primary ciliogenesis induce stem cell properties in basal Mammary Stem Cells (MaSCs) to promote mammogenesis, but the underlying mechanisms remain incompletely understood. Here, we show that EMT transcription factors promote ciliogenesis at intermediate EMT transition states by activating ciliogenesis inducers, including FGFR1. The resulting primary cilia promote BBS11-dependent ubiquitination and inactivation of a central signaling node, GLIS2. We show that GLIS2 inactivation promotes MaSC stemness, and GLIS2 is required for normal mammary gland development. Moreover, GLIS2 inactivation is required to induce the proliferative and tumorigenic capacities of the Mammary-Tumor-initiating cells (MaTICs) of claudin-low breast cancers. Claudin-low breast tumors can be segregated from other breast tumor subtypes based on a GLIS2-dependent gene expression signature. Collectively, our findings establish molecular mechanisms by which EMT programs induce ciliogenesis to control MaSC and MaTIC biology, mammary gland development, and claudin-low breast cancer formation.

Background Metastasis and mortality remain high among breast cancer patients with the claudin-low subtype because these tumors are aggressive, chemoresistant, and lack targeted therapies. Our objective was to utilize discovery-based proteomics to identify proteins associated with claudin-low primary and metastatic tumors to gain insight into pathways and mechanisms of tumor progression. Methods We used nano-LC-MS/MS proteomics to analyze orthotopic and metastatic tumors from the syngeneic murine T11 tumor model, which displays gene expression profiles mirroring human claudin-low tumors. Galectin-1 identity, expression and spatial distribution were investigated by biochemical and immunochemical methods and MALDI/IMS. RNA seq data from mouse and human tumors in our study and publicly available microarray data were analyzed for differential galectin-1 expression across breast cancer subtypes. Results Galectin-1, an N-acetyllactosamine-binding protein, exhibited the highest sequence coverage and high abundance rank order among nano-LC-MS/MS-identified proteins shared by T11 claudin-low tumors but not normal tissue. Label-free quantitation, Western immunoblot and ELISA confirmed galectin-1 identity and significant differential expression. MALDI/IMS spatial mapping and immunohistochemistry detected galectin-1 in T11 metastatic lung foci. Immunohistochemistry of human claudin-low tumors demonstrated intermediate-to-high intensity galectin-1 staining of tumor and stroma. Gene expression analysis of mouse and human tumors found the highest galectin-1 levels in the claudin-low breast cancer subtype. Conclusions Proteomics and genomics reveal high expression of galectin-1 protein and RNA in primary and metastatic claudin-low breast cancer. General significance This work endorses proteomic approaches in cancer research and supports further investigations of the function and significance of galectin-1 overexpression in claudin-low tumor progression.

BackgroundBreast cancer is a heterogeneous disease. Genomic studies have revealed five different intrinsic subtypes - Luminal A, Luminal B, HER2-enriched, Claudin-low, and Basal-like. Patients diagnosed with Basal-like or Claudin-low breast cancer (BL-CL) suffer from poor prognosis and limited treatment options. Hence, there is an urgent need to identify a new therapeutic lead that can benefit patients with BL-CL breast cancer.MethodsWe used a step-wise screening approach to screen 2778 HP20 fractions from our Marine Invertebrate Compound Library (MICL) to identify compounds that specifically target BL-CL breast cancer. We also performed biochemical investigations to study the effect of the compound in the signaling pathway. Finally, we generated a drug response gene-expression signature and projected it against a human tumor panel of 12 different cancer types to identify other cancer types sensitive to the compound.ResultsWe identified a previously unreported trisulfated sterol, topsentinol L trisulfate (TLT) that exhibits increased efficacy against BL-CL relative to Luminal/HER2+ breast cancer. Biochemical investigation of the effects of TLT on BL-CL revealed its ability to inhibit activation of AMPK and CHK1 and promote activation of p38. The importance of targeting AMPK and CHK1 in BL-CL was validated by treating a panel of breast cancer cell lines with known small molecule inhibitors of AMPK (Dorsomorphin) and CHK1 (Ly2603618) and recording the increased effectiveness against BL-CL compared to Luminal/HER2+ breast cancer. The TLT sensitivity gene-expression signature identified breast and bladder cancer as the most sensitive to TLT while glioblastoma multiforme as least sensitive.ConclusionsOur study identified TLT, a previously uncharacterized trisulfated sterol, as a potential therapeutic selective against BL-CL. Our results also showed that inhibition of AMPK and/or CHK1 might be an effective therapy in BL-CL.

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.

Triple negative breast cancer (TNBC) is a heterogeneous disease entity constituting about 15% of breast cancer cases worldwide. TNBC is associated with poor prognosis and lack of sustained response to conventional chemotherapeutic agents. Tumoral heterogeneity and the presence of several subtypes of TNBC such as Basal like (BL)-1, BL-2, immune-modulatory, luminal androgen receptor, mesenchymal, and mesenchymal/stem like subtype and claudin low subtype, may explain some of the difficulties faced in managing this challenging disease subgroups. Although no approved targeted therapy is available for TNBCs, molecular-profiling efforts have revealed promising molecular targets such as the vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), polyadenosine ribose polymerase inhibitors (PARPi) and DNA repair pathway, androgen pathway, and NOTCH pathway. TNBC is subject to intense research activities aiming at dissecting potential pathways, identifying potential molecular signatures and biomarkers in order to properly develop new targeted biologic modifiers. Despite this, there is a lack of approved predictive and prognostic biomarkers, and keeping in view the complexity of TNBC biology, research should be targeted towards identifying multi-factorial signatures rather than single markers. This review aims to summarize the current evidence, ongoing research and discuss future strategies for the treatment of patients with TNBC. In addition we have reviewed the recent advances in detecting predictive and prognostic biomarkers and identifying surrogate markers for early identification of potential responders to the new therapies.

// 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.

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.

Mesenchymal stem-like/claudin-low (MSL/CL) breast cancers are highly aggressive, express low cell-cell adhesion cluster containing claudins (CLDN3/CLDN4/CLDN7) with enrichment of epithelial-to-mesenchymal transition (EMT), immunomodulatory, and transforming growth factor-beta (TGF-β) genes. We examined the biological, molecular and prognostic impact of TGF-β upregulation and/or inhibition using in vivo and in vitro methods. Using publically available breast cancer gene expression databases, we show that upregulation and enrichment of a TGF-β gene signature is most frequent in MSL/CL breast cancers and is associated with a worse outcome. Using several MSL/CL breast cancer cell lines, we show that TGF-β elicits significant increases in cellular proliferation, migration, invasion, and motility, whereas these effects can be abrogated by a specific inhibitor against TGF-β receptor I and the anti-diabetic agent metformin, alone or in combination. Prior reports from our lab show that TNBC is exquisitely sensitive to metformin treatment. Mechanistically, metformin blocks endogenous activation of Smad2 and Smad3 and dampens TGF-β-mediated activation of Smad2, Smad3, and ID1 both at the transcriptional and translational level. We report the use of ID1 and ID3 as clinical surrogate markers, where high expression of these TGF-β target genes was correlated to poor prognosis in claudin-low patients. Given TGF-β’s role in tumorigenesis and immunomodulation, blockade of this pathway using direct kinase inhibitors or more broadly acting inhibitors may dampen or abolish pro-carcinogenic and metastatic signaling in patients with MCL/CL TNBC. Metformin therapy (with or without other agents) may be a heretofore unrecognized approach to reduce the oncogenic activities associated with TGF-β mediated oncogenesis.

Aim: To unravel the multimodal nanotheranostic ability of Fe3O4-saturated bovine lactoferrin nanocapsules (FebLf NCs) in claudin-low, triple-negative breast cancer model. Materials & methods: Xenograft study was performed to examine biocompatibility, antitumor efficacy and multimodal nanotheranostic action in combination with near-infrared live mice imaging. Results: FebLf NCs exhibited a size range of 80 nm ± 5 nm with observed superparamagnetism. FebLf NCs successfully internalized into breast cancer cells through receptor-mediated endocytosis and induced apoptosis through the downregulation of inhibitor of apoptosis survivin and livin proteins. Investigations revealed a remarkable biocompatibility, anticancer efficacy of the FebLf NCs. Near-infrared imaging observations confirmed selective localization of multimodal FebLf NCs at the tumor site and lead to time-dependent reduction of tumor growth. Conclusion: FebLf NCs can be safe, biocompatible nanotheranostic approach for real-time imaging and monitoring the effect of drugs in real time and have potentials in future clinical trials.

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.

Background: Breast cancer is a heterogeneous disease. Based on gene expression profiling, it can be classified into at least 5 subtypes. They are luminal A and B, normal breast-like, Her2-positive, basal-like, and newly described claudin-low. Among all these subtypes, basal-like and claudin-low tumors have relatively poor prognosis. Claudin-low tumors are characterized by the low expression of cell-cell junction proteins like claudin 3 and E-cadherin along with an enrichment of EMT markers such as SNAI1 and SNAI2 and high levels of mesenchymal markers such as vimentin. Claudin-low tumors are generally more aggressive and the rate of response to primary chemotherapy is around 32%. Initial data have shown that p-53 null claudin-low tumors are sensitive to cyclophosphamide treatment. Cyclophosphamide (CTX) is a chemotherapy drug that has been used in lymphoma, leukemia and some solid tumors. At low and continuous doses, it shows immunostimulatory properties. The overall goal of this proposal is to test the therapeutic response of claudin-low tumors to cyclophosphamide treatment and define the functional role of the immune system in a mouse model of claudin-low breast cancer. Preliminary data have shown that tumor cells in a p53 null claudin-low murine model (T12) possess tumor-initiating abilities and have STAT3 activation. Initial data in claudin-low T12 tumors also suggest that CTX induces CD4+ and CD8+ T-cell homing to the primary tumor. Additionally, wild-type Balb/c mice implanted with T12 tumors respond favorably to CTX treatment as compared to T cell-deficient NSG mice. Our studies have shown that treatment-resistant claudin-low tumors have a high tumor-associated macrophage (TAM) infiltrate. Tumors with poor response to CTX show higher levels of vimentin staining as compared to responsive tumors. Depletion of specific T-cell subsets to determine their functional importance in the response to CTX shows that while CD8+ T-cell depletion does not have a significant effect on treatment response, depletion of CD4+ T cells results in impaired tumor response to CTX treatment. Additionally, TAMs increase upon CD4+ T-cell depletion. We hypothesize that CTX treatment can sensitize T12 primary tumors to CD4+ T-cell mediated apoptosis. We further hypothesize that TAMS can suppress T-cell function and this, along with changes in the tumor EMT status, can promote chemoresistance. TAMs can also interact with tumor-initiating cells (TIC) within claudin-low tumors via the STAT3 pathway to promote survival and proliferation of chemotherapy resistant TICs. Furthermore, depleting immunosuppressive cells such as TAMs may provide us with an additional therapeutic modality for chemotherapy-resistant breast cancer in combination with checkpoint immunotherapy. Reference: 1. Prat A et al. Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res 2010;12:R68. Citation Format: Swarnima Singh, Se-Jin Kim, Xiang Zhang, Jeffrey Rosen. Immune cells, tumor-initiating cells, and drug sensitivity in claudin-low TNBC: A delicate balance [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B13.

Breast cancer is a heterogeneous disease. It can be classified into at least five subtypes: luminal A and B, normal breast like, Her2 positive, basal like, and claudin low. Among these, claudin-low tumors have relatively poor prognosis. These tumors are characterized by the low expression of cell-cell junction proteins along with an enrichment of EMT markers. They are more aggressive and the rate of response to primary chemotherapy is around 32%. Initial data have shown that murine p53 null claudin-low tumors are only sensitive to cyclophosphamide treatment. Cyclophosphamide (CTX) is a chemotherapy drug that has been used in lymphoma, leukemia, and some solid tumors. At low and continuous doses, it shows immunostimulatory properties. In this study we describe the implementation of a novel combination treatment for claudin-low TNBC as informed by combined scRNA-seq and V(D)J seq. Immune profiles of a murine claudin-low tumor model (T12) shows heavy infiltration by tumor-associated macrophages (TAMs) pretreatment. Initial data in T12 tumors also suggest that CTX induces CD4+ and CD8+ T-cell homing to the primary tumor. Wild-type Balb/c mice implanted with T12 tumors respond favorably to CTX treatment as compared to T cell-deficient NSG mice. We combined 5´ scRNA-seq with V(D)J seq to gain an insight into how immune cell subpopulations change during the course of treatment. Combining tSNE plots for untreated and treated tumors revealed the emergence of new transcriptionally distinct populations in the CTX treated group, which included six T cell subpopulations. Projection of T cells along pseudotime using Monocle3 revealed a shift from naive CD8+ and CD4+ T cells to a terminal effector CD8+ T-cell phenotype and CD4+ effector memory cells in treated tumors while untreated tumors have higher numbers of exhausted Tigit+ CD8+ T cells and Tregs. V(D)J analysis revealed higher numbers of unique TCRs in CTX-treated tumors and greater clonal expansion of T cells. However, we noted a persistence of TAMs in our model even after treatment as well as high recurrence rates upon cessation of treatment. We then examined markers specific to the TAM subsets in the treated and untreated tumors and observed that TAMs expressed high levels of Csf1r. A chow containing PLX3397(iCSF1R) was employed to inhibit CSF1R, a macrophage differentiation/recruitment factor. Combination treatment with CTX and the iCSF1R chow lead to long-term regression of T12 tumors. Rechallenging these mice resulted in much slower tumor growth, indicating the formation of T cell memory. scRNA-seq and V(D)J seq is being performed on these samples to identify the clonal dynamics of effector T cells that assist in this striking regression. We hypothesize that CTX treatment can sensitize T12 primary tumors to T cell-mediated apoptosis, and that TAMS can suppress T-cell function and promote chemoresistance. Furthermore, depleting these TAMs in combination with CTX treatment may lead to long-term durable regression of both the primary tumor and metastases. Citation Format: Swarnima Singh, Ying-Wooi Wan, Nigel Lee, Se-Jin Kim, Zhandong Liu, Charles M. Perou, Xiang Zhang, Jeffrey Rosen. Single-cell analytics in claudin-low TNBC reveals immunostimulatory effects of low-dose metronomic cyclophosphamide treatment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B54.

The expression of the transcription factor SOX4 is increased in many human cancers, however, the pro-oncogenic capacity of SOX4 can vary greatly depending on the type of tumor. Both the contextual nature and the mechanisms underlying the pro-oncogenic SOX4 response remain unexplored. Here, we demonstrate that in mammary tumorigenesis, the SOX4 transcriptional network is dictated by the epigenome and is enriched for pro-angiogenic processes. We show that SOX4 directly regulates endothelin-1 (ET-1) expression and can thereby promote tumor-induced angiogenesis both in vitro and in vivo. Furthermore, in breast tumors, SOX4 expression correlates with blood vessel density and size, and predicts poor-prognosis in patients with breast cancer. Our data provide novel mechanistic insights into context-dependent SOX4 target gene selection, and uncover a novel pro-oncogenic role for this transcription factor in promoting tumor-induced angiogenesis. These findings establish a key role for SOX4 in promoting metastasis through exploiting diverse pro-tumorigenic pathways.

Background Epithelial mesenchymal plasticity (EMP) is deemed vital in breast cancer progression, metastasis, stemness and resistance to therapy. Therefore, characterizing molecular mechanisms contributing to EMP are in need enabling the development of more advanced therapeutics against breast cancer. While kinesin superfamily proteins (KIFs) are well known for their role in intracellular cargo movement, our knowledge of their function in breast tumorigenesis is still limited. Methods Various breast cancer cell lines representing different molecular subtypes were used to determine the role of kinesine-1 subunits KIF5B/KLC1 in regulation of EMP. Findings In breast cancer, we show that kinesin family member 5B (KIF5B) and its partner protein kinesin light chain 1 (KLC1), subunits of kinesin-1, to play differential roles in regulating EMP and tumorigenesis. Indeed, we found KIF5B to be expressed in triple negative (TN)-basal-like/claudin low breast cancer subtype and to be an inducer of epithelial-mesenchymal transition (EMT), stemness, invasiveness, tumor formation and metastatic colonization. Whereas, we found KLC1 to be expressed in epithelial/luminal breast cancer subtypes and to be a suppressor of EMT, invasion, metastasis and stem cell markers expression as well as to be an inducer of epithelial/luminal phenotype. Interestingly, in TN-basal-like/claudin low cells we found a novel nuclear accumulation of KIF5B and its interaction with the EMT transcriptional regulator Snail1 independent of KLC1. In addition, TGF-β mediated pro-invasive activity was found to be dependent on KIF5B expression. In contrast, the epithelial differentiation factor and EMT suppressor prolactin (PRL) was found to repress KIF5B gene expression and KIF5B-Snail1 nuclear accumulation, but enhanced KLC1 gene expression and KIF5B-KLC1 interaction. Interpretation Together, these results highlight a new paradigm for kinesin-1 function in breast tumorigenesis by regulating EMP programing and aggressiveness. Fund This work was supported by the Canadian Institutes of Health Research (operating grants #233437 and 233438) granted to Suhad Ali.

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.

The miR-200 family of microRNAs consisting of miR-141, miR-200a, miR-200b, miR-200c and miR-429 are emerging as important regulators of breast cancer progression. This family of microRNAs maintain mammary epithelial identity and downregulation of miR-200 expression has been associated with epithelial-to-mesenchymal transition in mammary tumors. Therefore, re-expression of one or more miR-200 family members in mammary tumor cells with mesenchymal characteristics may restore an epithelial phenotype including growth and metastasis suppression. To test this hypothesis, the miR-200b/200a/429 cluster was re-expressed in a murine claudin-low cell line, RJ423. Re-expression of the miR-200b/200a/429 cluster in RJ423 cells significantly suppressed the expression of Vim, Snai1, Twist1, Twist2 and Zeb1, reverted RJ423 cells to a more epithelial morphology and significantly inhibited proliferation in vitro. Moreover, the miR-200b/200a/429 cluster prevented lung metastasis in an experimental metastasis model and although tumor initiation was not prevented, re-expression of the miR-200b/200a/429 cluster induced a dormancy-like state where mammary tumors failed to grow beyond ~150 mm3 or grew extremely slowly following intra-mammary injection. These dormant tumors contained elevated levels of collagen and were highly vascularized. Therefore, re-expression of the miR-200b/200a/429 cluster in the claudin-low mammary tumor cell line, RJ423, is sufficient to alter cell morphology, impair metastasis and induce tumor dormancy.

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.

Breast cancer is the most common and the leading cause of mortality in women worldwide. There is a dire necessity of the identification of novel molecules useful in diagnosis and prognosis. In this work we determined the differentially expression profiles of four breast cancer cell lines compared to a control cell line. We identified 1020 polypeptides labelled with iTRAQ with more than 95% in confidence. We analysed the common proteins in all breast cancer cell lines through IPA software (IPA core and Biomarkers). In addition, we selected the specific overexpressed and subexpressed proteins of the different molecular classes of breast cancer cell lines, and classified them according to protein class and biological process. Data in this article is related to the research article "Determination of the protein expression profiles of breast cancer cell lines by Quantitative Proteomics using iTRAQ Labelling and Tandem Mass Spectrometry" (Calderon-Gonzalez et al. [1] in press). (C) 2015 The Authors. Published by Elsevier Inc.

The complex signaling networks of the tumor microenvironment that facilitate tumor growth and progression toward metastatic disease are becoming a focus of potential therapeutic options. The chemokine IL-8 is overexpressed in multiple cancer types, including triple-negative breast cancer (TNBC), where it promotes the acquisition of mesenchymal features, stemness, resistance to therapies, and the recruitment of immune-suppressive cells to the tumor site. The present study explores the utility of a clinical-stage monoclonal antibody that neutralizes IL-8 (HuMax-IL8) as a potential therapeutic option for TNBC. HuMax-IL8 was shown to revert mesenchymalization in claudin-low TNBC models both in vitro and in vivo as well as to significantly decrease the recruitment of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) at the tumor site, an effect substantiated when used in combination with docetaxel. In addition, HuMax-IL8 enhanced the susceptibility of claudin-low breast cancer cells to immune-mediated lysis with NK and antigen-specific T cells in vitro. These results demonstrate the multifaceted way in which neutralizing this single chemokine reverts mesenchymalization, decreases recruitment of MDSCs at the tumor site, assists in immune-mediated killing, and forms the rationale for using HuMax-IL8 in combination with chemotherapy or immune-based therapies for the treatment of TNBC.

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 Breast cancer is a global public health problem. For some subtypes, such as Claudin-low, the prognosis is poorer and the treatment is still a challenge. Pyrazoles are an important class of heterocyclic compounds and are promising anticancer agents based on their chemical properties. The present study was aimed not only at testing pyrazoles previously prepared by our research group in two breast cancer cell lines characterized by intermediated response to conventional chemotherapy but also at analyzing the possible synergistic effect of these pyrazoles associated with doxorubicin. Methods Four 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-1H pyrazoles were tested for the first time in MCF-7 and MDA-MB-231 culture cells. The pyrazoles with best results in cytotoxicity were used in combination with doxorubicin and compared with this drug alone as standard. The synergic effect was analyzed using Combination Index method. In addition, cell death and apoptosis assays were carried out. Results Two pyrazoles with cytotoxic effect in MCF-7 and especially in MDA-MB-231 were identified. This activity was markedly higher in pyrazoles containing bromine and chlorine substituents. The combination of these pyrazoles with doxorubicin had a significant synergic effect in both cells tested and mainly in MDA-MB-231. These data were confirmed with apoptosis and cell death analysis. Conclusions The synergic effect observed with combination of these pyrazoles and doxorubicin deserves special attention in Claudin-low breast cancer subtype. This should be explored in order to improve treatment results and minimize side effects.

Claudin proteins are components of epithelial tight junctions; a subtype of breast cancer has been defined by the reduced expression of mRNA for claudins and other genes. Here, we characterize the expression of glycoproteins in breast cell lines for the claudin-low subtype using liquid chromatography/tandem mass spectrometry. Unsupervised clustering techniques reveal a group of claudin-low cell lines that is distinct from nonmalignant, basal, and luminal lines. The claudin-low cell lines express F11R, EPCAM, and other proteins at very low levels, whereas CD44 is expressed at a high level. Comparison of mRNA expression to glycoprotein expression shows modest correlation; the best agreement occurs when the mRNA expression level is lowest and little or no protein is detected. These findings from cell lines are compared to those for tumor samples by the Clinical Proteomic Tumor Analysis Consortium (CPTAC). The CPTAC samples contain a group low in CLDN3. The samples low in CLDN3 proteins share many differentially expressed glycoproteins with the claudin-low cell lines. In contrast to the situation for cell lines or patient samples classified as claudin-low by RNA expression, however, most of the tumor samples low in CLDN3 protein express the estrogen receptor or HER2. These tumor samples express CD44 protein at low rather than high levels. There is no correlation between CLDN3 gene expression and protein expression in these CPTAC samples; hence, the claudin-low subtype defined by gene expression is not the same group of tumors as that defined by low expression of CLDN3 protein.

The tumor suppressors Pten and p53 are frequently lost in breast cancer, yet the consequences of their combined inactivation are poorly understood. Here, we show that mammary-specific deletion of Pten via WAP-Cre, which targets alveolar progenitors, induced tumors with shortened latency compared to those induced by MMTV-Cre, which targets basal/luminal progenitors. Combined Pten-p53 mutations accelerated formation of claudin-low, triple-negative-like breast cancer (TNBC) that exhibited hyper-activated AKT signaling and more mesenchymal features relative to Pten or p53 single-mutant tumors. Twenty-four genes that were significantly and differentially expressed between WAP-Cre:Pten/p53 and MMTV-Cre:Pten/p53 tumors predicted poor survival for claudin-low patients. Kinome screens identified eukaryotic elongation factor-2 kinase (eEF2K) inhibitors as more potent than PI3K/AKT/mTOR inhibitors on both mouse and human Pten/p53-deficient TNBC cells. Sensitivity to eEF2K inhibition correlated with AKT pathway activity. eEF2K monotherapy suppressed growth of Pten/p53-deficient TNBC xenografts in vivo and cooperated with doxorubicin to efficiently kill tumor cells in vitro. Our results identify a prognostic signature for claudin-low patients and provide a rationale for using eEF2K inhibitors for treatment of TNBC with elevated AKT signaling.

Patient data suggest that colony stimulating factor-1 (CSF1) and its receptor (CSF1R) play critical roles during breast cancer progression. We have previously shown that in human breast tumors expressing both CSF1 and CSF1R, invasion in vivo is dependent both on a paracrine interaction with tumor-associated macrophages and an autocrine regulation of CSF1R in the tumor cells themselves. Although the role of the paracrine interaction between tumor cells and macrophages has been extensively studied, very little is known about the mechanism by which the autocrine CSF1R signaling contributes to tumor progression. We show here that breast cancer patients of the claudin-low subtype have significantly increased expression of CSF1R. Using a panel of breast cancer cells lines, we confirm that CSF1R expression is elevated and regulated by TGFβ specifically in claudin-low cell lines. Abrogation of autocrine CSF1R signaling in MDA-MB-231 xenografts (a claudin-low cell line) leads to increased tumor size by enhanced proliferation, but significantly reduced invasion, dissemination and metastasis. Indeed, we show that proliferation and invasion are oppositely regulated by CSF1R downstream of TGFβ only in claudin-low cells lines. Intravital multiphoton imaging revealed that inhibition of CSF1R in the tumor cells leads to decreased in vivo motility and a more cohesive morphology. We show that, both in vitro and in vivo, CSF1R inhibition results in a reversal of claudin-low marker expression by significant upregulation of luminal keratins and tight junction proteins such as claudins. Finally, we show that artificial overexpression of claudins in MDA-MB-231 cells is sufficient to tip the cells from an invasive state to a proliferative state. Our results suggest that autocrine CSF1R signaling is essential in maintaining low claudin expression and that it mediates a switch between the proliferative and the invasive state in claudin-low tumor cells downstream of TGFβ.

In this study, we examined the association between the RKIP expression and the molecular subtypes of breast cancer. Microarray gene expression data of 2,333 human breast cancer from 26 different cohorts performed on Affymetrix U133A or U133Plus2 platforms were downloaded from Array Express and Gene Expression Omnibus and the molecular subtype of breast cancer for the samples was determined by single sample Gene Set Enrichment Analysis. Differences in recurrence-free survival (RFS) were tested using the Log-rank test in univariate analysis and displayed using Kaplan–Meier curves. Cox proportional-hazards model was used to calculate the hazard ratio using univariate and multivariate analysis. Loss or reduced RKIP expression was associated with reduced RFS in breast cancer using univariate and multivariate analyses, which was independent of lymph node (LN) metastasis status. Basal-like, Claudin-low, and Her-2-enriched tumors had significantly lower RKIP levels compared to other subclasses (P