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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

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.

The Salvador/Warts/Hippo (Hippo) signaling pathway defines a novel signaling cascade regulating cell contact inhibition, organ size control, cell growth, proliferation, apoptosis and cancer development in mammals. The upstream regulation of this pathway has been less well defined than the core kinase cassette. KIBRA has been shown to function as an upstream member of the Hippo pathway by influencing the phosphorylation of LATS and YAP, but functional consequences of these biochemical changes have not been previously addressed. We show that in MCF10A cells, loss of KIBRA expression displays epithelial-to-mesenchymal transition (EMT) features, which are concomitant with decreased LATS and YAP phosphorylation, but not MST1/2. In addition, ectopic KIBRA expression antagonizes YAP via the serine 127 phosphorylation site and we show that KIBRA, Willin and Merlin differentially regulate genes controlled by YAP. Finally, reduced KIBRA expression in primary breast cancer specimens correlates with the recently described claudin-low subtype, an aggressive sub-group with EMT features and a poor prognosis.

// 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.

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.

Triple-negative breast cancer (TNBC) is a heterogeneous disease that includes Basal-like and Claudin-low tumors. The Claudin-low tumors are enriched for features associated with epithelial-to-mesenchymal transition (EMT) and possibly for tumor initiating cells. Primary TNBCs respond relatively well to conventional chemotherapy; however, metastatic disease is virtually incurable. Thus, there is a great interest in identifying specific therapeutic targets for TNBC. The tumor suppressor RB1 is frequently lost in Basal-like breast cancer. To test for a causative role of RB1 gene loss in BC and for its effect on specific subtypes, we deleted mouse Rb in mammary stem/bipotent progenitor cells. This led to diverse mammary tumors including TNBC, with a subset of the latter containing p53 mutations and exhibiting features of Basal-like BC or EMT. Combined mutation of Rb and p53 in mammary stem/bipotent progenitors induced EMT type tumors. Here, we review our findings and those of others, which connect Rb and p53 to EMT in TNBC. Furthermore, we discuss how by understanding this circuit and its vulnerabilities, we may identify novel therapy for TNBC.

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.

The epithelial-to-mesenchymal transition (EMT) produces cancer cells that are invasive, migratory, and exhibit stem cell characteristics, hallmarks of cells that have the potential to generate metastases. Inducers of the EMT include several transcription factors (TFs), such as Goosecoid, Snail, and Twist, as well as the secreted TGF-β1. Each of these factors is capable, on its own, of inducing an EMT in the human mammary epithelial (HMLE) cell line. However, the interactions between these regulators are poorly understood. Overexpression of each of the above EMT inducers up-regulates a subset of other EMT-inducing TFs, with Twist, Zeb1, Zeb2, TGF-β1, and FOXC2 being commonly induced. Up-regulation of Slug and FOXC2 by either Snail or Twist does not depend on TGF-β1 signaling. Gene expression signatures (GESs) derived by overexpressing EMT-inducing TFs reveal that the Twist GES and Snail GES are the most similar, although the Goosecoid GES is the least similar to the others. An EMT core signature was derived from the changes in gene expression shared by up-regulation of Gsc, Snail, Twist, and TGF-β1 and by down-regulation of E-cadherin, loss of which can also trigger an EMT in certain cell types. The EMT core signature associates closely with the claudin-low and metaplastic breast cancer subtypes and correlates negatively with pathological complete response. Additionally, the expression level of FOXC1, another EMT inducer, correlates strongly with poor survival of 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.

Some breast cancers have been shown to contain a small fraction of cells characterized by CD44 + /CD24 −/low cell-surface antigen profile that have high tumor-initiating potential. In addition, breast cancer cells propagated in vitro as mammospheres (MSs) have also been shown to be enriched for cells capable of self-renewal. In this study, we have defined a gene expression signature common to both CD44 + /CD24 −/low and MS-forming cells. To examine its clinical significance, we determined whether tumor cells surviving after conventional treatments were enriched for cells bearing this CD44 + /CD24 −/low -MS signature. The CD44 + /CD24 −/low -MS signature was found mainly in human breast tumors of the recently identified “claudin-low” molecular subtype, which is characterized by expression of many epithelial-mesenchymal-transition (EMT)-associated genes. Both CD44 + /CD24 −/low -MS and claudin-low signatures were more pronounced in tumor tissue remaining after either endocrine therapy (letrozole) or chemotherapy (docetaxel), consistent with the selective survival of tumor-initiating cells posttreatment. We confirmed an increased expression of mesenchymal markers, including vimentin ( VIM ) in cytokeratin-positive epithelial cells metalloproteinase 2 ( MMP 2), in two separate sets of postletrozole vs. pretreatment specimens. Taken together, these data provide supporting evidence that the residual breast tumor cell populations surviving after conventional treatment may be enriched for subpopulations of cells with both tumor-initiating and mesenchymal features. Targeting proteins involved in EMT may provide a therapeutic strategy for eliminating surviving cells to prevent recurrence and improve long-term survival in breast cancer patients.

Basal-like breast cancers arising in women carrying mutations in the BRCA1 gene, encoding the tumor suppressor protein BRCA1, are thought to develop from the mammary stem cell. To explore early cellular changes that occur in BRCA1 mutation carriers, we have prospectively isolated distinct epithelial subpopulations from normal mammary tissue and preneoplastic specimens from individuals heterozygous for a BRCA1 mutation. We describe three epithelial subsets including basal stem/progenitor, luminal progenitor and mature luminal cells. Unexpectedly, we found that breast tissue from BRCA1 mutation carriers harbors an expanded luminal progenitor population that shows factor-independent growth in vitro. Moreover, gene expression profiling revealed that breast tissue heterozygous for a BRCA1 mutation and basal breast tumors were more similar to normal luminal progenitor cells than any other subset, including the stem cell-enriched population. The c-KIT tyrosine kinase receptor (encoded by KIT) emerged as a key marker of luminal progenitor cells and was more highly expressed in BRCA1-associated preneoplastic tissue and tumors. Our findings suggest that an aberrant luminal progenitor population is a target for transformation in BRCA1-associated basal tumors .

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]

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

Triple negative breast cancer (TNBC) is highly aggressive and has a poor prognosis when compared to other molecular subtypes. In particular, the claudin-low subtype of TNBC exhibits tumor-initiating/cancer stem cell like properties. Here, we seek to find new biomarkers to discriminate different forms of TNBC by characterizing their bioimpedance. A customized bioimpedance sensor with four identical branched microelectrodes with branch widths adjusted to accommodate spreading of individual cells was fabricated on silicon and pyrex/glass substrates. Cell analyses were performed on the silicon devices which showed somewhat improved inter-electrode and intra-device reliability. We performed detailed analysis of the bioimpedance spectra of four TNBC cell lines, comparing the peak magnitude, peak frequency and peak phase angle between claudin-low TNBC subtype represented by MDA-MB-231 and Hs578T with that of two basal cells types, the TNBC MDA-MB-468, and an immortalized non-malignant basal breast cell line, MCF-10A. The claudin-low TNBC cell lines showed significantly higher peak frequencies and peak phase angles than the properties might be useful in distinguishing the clinically significant claudin-low subtype of TNBC.

Recently completed cancer genomics projects identified intrinsic subtypes in muscle-invasive bladder cancers. Here we will describe the studies that led to their discovery and review their biological and clinical properties.Whole genome mRNA expression profiling and unsupervised hierarchical cluster analyses identified intrinsic basal and luminal subtypes in muscle-invasive bladder cancers that are similar to the ones found in breast cancer. Tumors within each subtype have distinct responses to conventional cisplatin-based combination chemotherapy, and they contain gene expression signatures and DNA alterations that may render them vulnerable to clinically available targeted therapies.Like their breast cancer counterparts, basal bladder cancers are characterized by poor clinical outcomes in the absence of effective systemic therapy, but a large fraction of them do respond to neoadjuvant chemotherapy, suggesting that the tumors should be managed aggressively. On the contrary, tumors that belong to the 'p53-like' subtype tend to be chemoresistant, so patients with these tumors should probably be managed differently. It seems likely that prospective identification of tumor intrinsic subtype membership could complement the use of DNA-based biomarkers to identify the groups of patients who will benefit the most from chemotherapy and targeted agents.

Breast cancer is the principal cancer in women worldwide. Although there are serum tumor markers such as CEA and HER2, they are detected in advanced stages of the disease and used as progression and recurrence markers. Therefore, there is a necessity for the identification of new markers that might lead to an early detection and also provide evidence of an effective treatment. The aim of this work was to determine the differential protein expression profiles of four breast cancer cell lines in comparison to a normal control cell line by iTRAQ labelling and tandem mass spectrometry, in order to identify putative biomarkers of the disease. We identified 1,020 iTRAQ-labelled polypeptides with at least one peptide identified with more than 95% in confidence. Overexpressed polypeptides in all cancer cell lines were 78, whilst the subexpressed were 128. We categorised them with PANTHER program into biological processes, being the metabolic pathways the most affected. We detected six groups of proteins with the STRING program involved in DNA topology, glycolysis, translation initiation, splicing, pentose pathway, and proteasome degradation. The main subexpressed protein network included mitochondrial proteins involved in oxidative phosphorylation. We propose BAG6, DDX39, ANXA8 and COX4 as putative biomarkers in breast cancer. Biological significance We report a set of differentially expressed proteins in the MCF7 and T47D (Luminal A), MDA-MB-231 (Claudin low) and SK-BR-3 (HER2(+)) breast cancer cell lines that have not been previously reported in breast cancer disease. From these proteins, we propose BAG6, DDX39, ANXA8 and COX4 as putative biomarkers in breast cancer. On the other hand, we propose sets of unique polypeptides in each breast cancer cell line that can be useful in the classification of different subtypes of breast cancer. (C) 2015 Elsevier B.V. All rights reserved.

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.

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.

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.

Breast cancer is the most common malignancy in United States women, accounting for >40,000 deaths each year. These breast tumors are comprised of phenotypically diverse populations of breast cancer cells. Using a model in which human breast cancer cells were grown in immunocompromised mice, we found that only a minority of breast cancer cells had the ability to form new tumors. We were able to distinguish the tumorigenic (tumor initiating) from the nontumorigenic cancer cells based on cell surface marker expression. We prospectively identified and isolated the tumorigenic cells as CD44 + CD24 −/low Lineage − in eight of nine patients. As few as 100 cells with this phenotype were able to form tumors in mice, whereas tens of thousands of cells with alternate phenotypes failed to form tumors. The tumorigenic subpopulation could be serially passaged: each time cells within this population generated new tumors containing additional CD44 + CD24 −/low Lineage − tumorigenic cells as well as the phenotypically diverse mixed populations of nontumorigenic cells present in the initial tumor. The ability to prospectively identify tumorigenic cancer cells will facilitate the elucidation of pathways that regulate their growth and survival. Furthermore, because these cells drive tumor development, strategies designed to target this population may lead to more effective therapies.

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.

Introduction The recently identified claudin-low subtype of breast cancer is enriched for cells with stem-like and mesenchymal-like characteristics. This subtype is most often triple-negative (lacking the estrogen and progesterone receptors (ER, PR) as well as lacking epidermal growth factor 2 (HER2) amplification) and has a poor prognosis. There are few targeted treatment options available for patients with this highly aggressive type of cancer. Methods Using a high throughput inhibitor screen, we identified high expression of glioma-associated oncogene homolog 1 (GLI1), the effector molecule of the hedgehog (Hh) pathway, as a critical determinant of cell lines that have undergone an epithelial to mesenchymal transition (EMT). Results High GLI1 expression is a property of claudin-low cells and tumors and correlates with markers of EMT and breast cancer stem cells. Knockdown of GLI1 expression in claudin-low cell lines resulted in reduced cell viability, motility, clonogenicity, self-renewal, and reduced tumor growth of orthotopic xenografts. We observed non-canonical activation of GLI1 in claudin-low and EMT cell lines, and identified crosstalk with the NFκB pathway. Conclusions This work highlights the importance of GLI1 in the maintenance of characteristics of metastatic breast cancer stem cells. Remarkably, treatment with an inhibitor of the NFκB pathway reproducibly reduces GLI1 expression and protein levels. We further provide direct evidence for the binding of the NFκB subunit p65 to the GLI1 promoter in both EMT and claudin-low cell lines. Our results uncover crosstalk between NFκB and GLI1 signals and suggest that targeting these pathways may be effective against the claudin-low breast cancer subtype. Electronic supplementary material The online version of this article (doi:10.1186/s13058-014-0444-4) contains supplementary material, which is available to authorized users.

Fibromatosis-like metaplastic carcinoma (FLMCa) of the breast is a rare low-grade spindle cell carcinoma, of which the biological characteristics have not been well studied. This study aims to assess, in FLMCa, immunohistochemical expression of claudins (CLDN) and features connected with the claudin-low subtype, such as the presence of tumor initiating cells (TIC), epithelial-mesenchymal transition (EMT) phenotype, as well as EGFR activating mutations. Three cases of FLMCa were retrieved from our hospital archives. Histological and immunohistochemical characteristics were reviewed. Expression of CLDN-1, CLDN-3, CLDN-4 and CLDN-7, CD44 and CD24 (TIC phenotype), and vimentin and E-cadherin (EMT features) were studied. EGFR mutations on exons 18, 19, 20, and 21 were investigated by real-time PCR. In all cases, the low-grade spindle cell component was predominant, with two cases presenting

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.

A subset of human breast cancer cell lines exhibits aberrant DNA hypermethylation that is characterized by hyperactivity of the DNA methyltransferase enzymes, overexpression of DNMT3b, and concurrent methylation-dependent silencing of numerous epigenetic biomarker genes. The objective of this study was to determine if this aberrant DNA hypermethylation (i) is found in primary breast cancers, (ii) is associated with specific breast cancer molecular subtypes, and (iii) influences patient outcomes. Analysis of epigenetic biomarker genes (CDH1, CEACAM6, CST6, ESR1, GNA11, MUC1, MYB, SCNN1A, and TFF3) identified a gene expression signature characterized by reduced expression levels or loss of expression among a cohort of primary breast cancers. The breast cancers that express this gene expression signature are enriched for triple-negative subtypes - basal-like and claudin-low breast cancers. Methylation analysis of primary breast cancers showed extensive promoter hypermethylation of epigenetic biomarker genes among triple-negative breast cancers, compared to other breast cancer subclasses where promoter hypermethylation events were less frequent. Furthermore, triple-negative breast cancers either did not express or expressed significantly reduced levels of protein corresponding to methylation-sensitive biomarker gene products. Together, these findings suggest strongly that loss of epigenetic biomarker gene expression is frequently associated with gene promoter hypermethylation events. We propose that aberrant DNA hypermethylation is a common characteristic of triple-negative breast cancers and may represent a fundamental biological property of basal-like and claudin-low breast cancers. Kaplan-Meier analysis of relapse-free survival revealed a survival disadvantage for patients with breast cancers that exhibit aberrant DNA hypermethylation. Identification of this distinguishing trait among triple-negative breast cancers forms the basis for development of new rational therapies that target the epigenome in patients with basal-like and claudin-low breast cancers.

TP53 is one of the most commonly mutated genes in cancer. In breast cancer, it is mutated in about 40% of primary clinical tumors and is associated with poor survival. The mammotrophic hormone, prolactin (PRL), and/or its receptor are also expressed in many breast cancers, and accumulating epidemiologic data link PRL to breast cancer development and progression. Like TP53 mutations, evidence for PRL activity is evident across several molecular cancer subtypes, and elevated PRL expression and loss of p53 have been observed in some of the same clinical tumors. In order to examine the interaction of these factors, we used genetically modified mouse models of mammary-specific p53 loss and local overexpression of PRL. We demonstrated that mammary PRL decreased the latency of tumors in the absence of p53, and increased the proportion of triple-negative claudin-low carcinomas, which display similarities to human clinical metaplastic carcinomas. Moreover, PRL/p53(-/-) carcinomas displayed higher rates of proliferation and more aggressive behavior. Transcripts associated with cell cycle progression, invasion and stromal reactivity were differentially expressed in carcinomas that developed in the presence of elevated PRL. PRL/p53(-/-) carcinomas also exhibited selectively altered expression of activating protein-1 components, including higher levels of c-Jun and FosL1, which can drive transcription of many of these genes and the epithelial-mesenchymal transition. The ability of PRL to promote claudin-low carcinomas demonstrates that PRL can influence this subset of triple-negative breast cancers, which may have been obscured by the relative infrequency of this cancer subtype. Our findings suggest novel therapeutic approaches, and provide a preclinical model to develop possible agents.

MEK1/2 inhibitors such as AZD6244 are in clinical trials for the treatment of multiple cancers, including breast cancer. Targeted kinase inhibition can induce compensatory kinome changes, rendering single therapeutic agents ineffective. To identify target proteins to be used in a combinatorial approach to inhibit tumor cell growth, we used a novel strategy that identified microRNAs (miRNAs) that synergized with AZD6244 to inhibit the viability of the claudin-low breast cancer cell line MDA-MB-231. Screening of a miRNA mimic library revealed the ability of miR-9-3p to significantly enhance AZD6244-induced extracellular signal-regulated kinase inhibition and growth arrest, while miR-9-3p had little effect on growth alone. Promoter methylation of mir-9 genes correlated with low expression of miR-9-3p in different breast cancer cell lines. Consistent with miR-9-3p having synthetic enhancer tumor suppressor characteristics, miR-9-3p expression in combination with MEK inhibitor caused a sustained loss of c-MYC expression and growth inhibition. The β1 integrin gene (ITGB1) was identified as a new miR-9-3p target, and the growth inhibition seen with small interfering RNA knockdown or antibody blocking of ITGB1 in combination with MEK inhibitor phenocopied the growth inhibition seen with miR-9-3p plus AZD6244. The miRNA screen led to identification of a druggable protein, ITGB1, whose functional inhibition synergizes with MEK inhibitor.

Triple-negative breast cancer (TNBC) accounts for ∼20% of cases and contributes to basal and claudin-low molecular subclasses of the disease. TNBCs have poor prognosis, display frequent mutations in tumor suppressor gene p53 (TP53), and lack targeted therapies. The MET receptor tyrosine kinase is elevated in TNBC and transgenic Met models (Met(mt)) develop basal-like tumors. To investigate collaborating events in the genesis of TNBC, we generated Met(mt) mice with conditional loss of murine p53 (Trp53) in mammary epithelia. Somatic Trp53 loss, in combination with Met(mt), significantly increased tumor penetrance over Met(mt) or Trp53 loss alone. Unlike Met(mt) tumors, which are histologically diverse and enriched in a basal-like molecular signature, the majority of Met(mt) tumors with Trp53 loss displayed a spindloid pathology with a distinct molecular signature that resembles the human claudin-low subtype of TNBC, including diminished claudins, an epithelial-to-mesenchymal transition signature, and decreased expression of the microRNA-200 family. Moreover, although mammary specific loss of Trp53 promotes tumors with diverse pathologies, those with spindloid pathology and claudin-low signature display genomic Met amplification. In both models, MET activity is required for maintenance of the claudin-low morphological phenotype, in which MET inhibitors restore cell-cell junctions, rescue claudin 1 expression, and abrogate growth and dissemination of cells in vivo. Among human breast cancers, elevated levels of MET and stabilized TP53, indicative of mutation, correlate with highly proliferative TNBCs of poor outcome. This work shows synergy between MET and TP53 loss for claudin-low breast cancer, identifies a restricted claudin-low gene signature, and provides a rationale for anti-MET therapies in TNBC.

Rationale: High-grade urothelial carcinoma of the bladder is a heterogeneous disease, with molecular subtypes characterized by distinct tumor biologies and prognoses. Our group and others have described the basal and luminal subtypes, and we now report on the discovery of the claudin-low subtype, all of which resemble analogous subtypes in breast cancer. Methods: We analyzed mRNA sequencing and whole exome sequencing data for The Cancer Genome Atlas (TCGA) bladder tumors and tumors collected at UNC. We performed unsupervised hierarchical clustering on relative gene expression values with significance testing using SigClust to identify the claudin-low subtype. Basal, luminal, and claudin-low bladder tumors were compared for enrichment of genetic features (single nucleotide and copy number variation), gene set expression, immune gene signature expression, T cell receptor (TCR) and B cell receptor (BCR) gene segment expression, and number of predicted MHC Class I and Class II neoantigens. We further report on the first use of our VDJician software to reconstruct full-length rearranged BCR sequences from short-read RNA sequencing data in bladder cancer. Results: Claudin-low bladder tumors were defined by low expression of tight junction claudin-proteins, high expression of epithelial-to-mesenchymal transition genes and immune gene signatures, and were associated with reduced overall survival compared to luminal tumors. A minimal gene set classifier to identify claudin-low tumors showed some but not complete overlap with an optimal classifier derived in breast cancer. Claudin-low bladder tumors were enriched for multiple genetic features: 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. Claudin-low tumors showed the highest expression of immune gene signatures (including an immunosuppression signature), however in contrast to basal tumors, increased immune gene signature expression was not associated with prolonged survival. Claudin-low tumors also showed the highest overall expression of rearranged BCR sequences but lowest BCR repertoire diversity. Predicted neoantigen burden did not vary significantly by subtype, however broad cytokine and chemokine expression levels were elevated in claudin-low tumors, potentially related to low PPARG activity driving increased NFKB activity. Conclusions: Claudin-low bladder cancer is a novel molecular subtype with distinct molecular and immunologic features and prognostic significance. Given the presence of dense immune infiltrates, BCR repertoire characteristics consistent with an antigen-driven response, and high expression of immunosuppression genes, claudin-low bladder tumors may be primed to respond to immune checkpoint inhibitor therapy. Citation Format: Benjamin G. Vincent, William Kim, Jordan Kardos, Shengjie Chai, Joel Parker, Lisle Mose, Sara Selitsky, Michael Iglesia, Matthew Milowsky. The novel claudin-low molecular subtype of high-grade urothelial bladder cancer is highly immunogenic yet immunosuppressed. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5120.

Epithelial-to-mesenchymal transition (EMT), the process through which epithelial cells gain mesenchymal characteristics including reduced adhesion and enhanced invasion, is a critical step in progression of cancer cells toward metastasis. However, the molecular mechanisms governing this process remain poorly understood. Furthermore, though metastasis is the cause of 90% of cancer deaths, no targeted therapy is currently available for metastatic disease. Metabolic reprogramming is a key feature of most cancer cells, with oxidative phosphorylation often being switched off in favor of glycolytic and synthetic pathways. Autophagy is a catabolic process in which protein complexes, damaged organelles and other macromolecules are lysosomally degraded. Autophagy can promote cancer cell survival, providing protein, lipid, nucleic acid and membrane precursors as well as auxiliary energy during periods of energy stress. The role of metabolism in EMT and metastasis has not been well studied. We hypothesized that the transition to metastasis involves metabolic alterations, including activation of autophagy. Previously, we derived (from a spontaneous mammary tumor in an MMTV-Wnt transgenic mouse) epithelial-like (E-Wnt) and mesenchymal-like (M-Wnt) cell lines, which recapitulate basal-like and claudin-low and breast tumors, respectively. Here, a metastatic derivative of M-Wnt cells was generated from lung metastases following serial passages in a severe combined immunodeficient (SCID) mouse. metM-Wnt cells were more proliferative, invasive and formed more colonies in soft agar than their parental M-Wnt cell line. Tail vein injection or mammary fat pad orthotopic implantation of metM-Wnt cells resulted in metastatic tumor formation in either the lungs or liver as detected by IVIS imaging and histological analysis. metM-Wnt cells displayed higher rates of glycolysis and oxidative phosphorylation, indicating that these cells are highly energetic. Furthermore, M-Wnt and metM-Wnt cells and tumors were found to have increased autophagic flux, as measured by LC3B expression and cleavage compared to E-Wnt cells and tumors. metM-Wnt cells were more sensitive to autophagy inhibition (either via chloroquine treatment or knockout of Atg5, a key component of the autophagic machinery) than M-Wnt or E-Wnt cells. Conversely, metM-Wnt cells were resistant to treatment with rapamycin, concomitant with sustained activation of mTOR, which controls many metabolic pathways including autophagy and protein synthesis. These results indicate that metabolic alteration is a feature of EMT in claudin-low breast cancer, including increased glycolysis, oxidative phosphorylation and autophagy. Furthermore, metastatic breast cancer cells may be more reliant on autophagy than non-metastatic cells, and autophagy may therefore be a therapeutic target in which to treat metastatic breast cancers. Citation Format: Ciara H. O’Flanagan, Emily L. Rossi, Stephen D. Hursting. Autophagy forms part of a metabolic switch during epithelial-to-mesenchymal transition and metastasis in a murine claudin-low breast cancer model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1020.

The retinoic acid (RA) signalling pathway plays an important role in breast cancer progression and has either a pro-tumorigenic or tumor-suppressive role depending upon the effector function of RA-inducible genes that are expressed or epigenetically silenced. To study this paradigm in breast cancer, we focused on a controversial RA-inducible gene, the retinoic acid receptor responder 1 (RARRES1) protein that is often hypermethylated in cancer and has been reported to have tumor-suppressive function in prostate and nasopharyngeal carcinomas. However, in a study focused on a rare subtype of breast cancer, inflammatory breast cancer, RARRES1 is pro-tumorigenic. This functional discrepancy requires further investigation to determine its role in breast cancer in general. First, analysis of patient data sets revealed that RARRES1 is predominantly expressed in triple-negative breast cancers (TNBCs). Knockdown of RARRES1 in claudin-low MDA-MB-231 and basal-like MDA-MB-468 and HCC1937 significantly increased tumor growth and cell proliferation, suggesting RARRES1 has a tumor suppressive function in (TNBC), regardless of position on the differentiation hierarchy. Expression analyses of 24 breast cancer cell lines (including 18 TNBC and 2 normal-like cell lines) revealed that RARRES1 is predominantly expressed in basal-like TNBC cells We found that RARRES1 expression is dependent on, and strongly correlates with, the cancer stem cell marker, and RA-producing, ALDH1A3 in fixed breast cancer patient samples. Immunohistochemistry of the same patient tumor samples revealed RARRES1 expression is localized to the endoplasmic reticulum. Importantly, however, the presence of ALDH1A3 or RA is not sufficient to induce RARRES1 expression. RARRES1 is hypermethylated in claudin-low breast cancer cell lines, and release of this silencing is required for full induction of RARRES1 expression. We have identified sites of regulation by methylation in RARRES1 using Illumina 450K methylation arrays and 5-methylcytosine ChIP. We conclude that RARRES1 is an ALDH1A3/RA-inducible tumor suppressor in TNBC with methylation and expression profiles distinct to the differentiation hierarchy observed in breast cancer. Citation Format: Krysta M. Coyle, Patrick Murphy, Dejan Vidovic, Cheryl A. Dean, Margaret L. Thomas, Derek Clements, Mohammad Sultan, Ahmad Vaghar-Kashani, Carman Giacomantonio, Lucy Helyer, Ian Weaver, Shashi Gujar, Patrick WK Lee, Paola Marcato. ALDH1A3-inducible RARRES1 is a tumor suppressor in triple-negative breast cancer and is methylated in claudin-low breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3661.

Epithelial cells maintain intercellular communication directly by cell-cell, cell-ECM interaction and indirectly by paracrine secretion of extracellular messengers either as free molecules or via exosomes. The vesicular trafficking machinery is an integral part of such communication, paralleling digital cables that transmit the information bytes to the correct hub. During the process of EMT, derailing of the trafficking machinery leads to a systemic breakdown of intra and inter-cellular communication pathways. The purpose of the current study is to elucidate the functional role of Rab25, a key member of the vesicular trafficking machinery in mediating EMT-MET processes during breast cancer progression. Aberrant endocytosis is a new hallmark of cancer. Rab GTPases are the primary regulators of endocytosis. Many members of this large family are implicated in a wide array of cancers including breast cancer. Specifically, Rab25, a GTPase associated with the recycling endosomes, is deregulated in most epithelial cancers. Rab25 appears to function both as a tumor suppressor and an oncogene in a context dependent manner. We found that Rab25 is lost in Claudin Low tumors as well as in representative cell lines such as the MDA MB231. Rab25 is a robust and sufficient marker of breast cancers and cell lines with an increased EMT signature. Additionally, in immortalized human mammary epithelial cell lines (HMLE), induction of EMT by overexpression of Snail1 completely obliterates Rab25 protein. Exogenous re-expression of Rab25 dramatically rescues these cells from Snail driven invasion through Matrigel. However this Snail dependent regulation is unique only to TN and Claudin Low tumors and does not hold true for hormone receptor positive tumors. Further, analysis of RNA Seq data from a MMTV mouse model showed that tumors enriched in EMT markers and by corollary with low CDH and claudins, also lost Rab25 transcripts. Snail mediated transcriptional repression targets recycling endosomal proteins such as Rab25 to decrease delivery of junction proteins that facilitate cell-cell communication to the appropriate compartment. In parallel, it potentially enhances paracrine regulation by secreted exosomes. Overall, our study shows presence and absence of Rab25 markedly alters cell-cell communication, which is essential for maintenance of epithelial features of cells. Rab25 could be a prognostic marker and a therapeutic target for a subset of patients with increased risk of metastatic disease. Citation Format: Shreya Mitra, Lorenzo Federico, Gordon B. Mills. Transcription factor Snail mediates EMT by altering vesicular trafficking protein Rab25. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B40.

Triple-negative breast cancer (TNBC) is a highly diverse and typically aggressive group of cancers. Consequently, elucidating the biology of the various subtypes is necessary for more effective diagnosis and better targeted treatment of this disease. Recently, a novel claudin-low TNBC subtype has been identified. Patients with this subtype have a very poor prognosis, specifically young African-American females. The major focus of our study is to characterize a novel claudin-low cell line cultured from a “humanized” mouse model that is p53 and BRCA1 deficient (K14TRT cell line). Procedures: This cell line has been maintained for more than 40 months and for over 25 subcultures, and was characterized by microscopy, histopathology, genotyping, cytogenetics, and bioinformatics based techniques. Results: Histological analysis supports that the primary K14TRT tumors were highly penetrant, with some having spindloid morphology while others were undifferentiated adenocarcinoams, with large areas of necrosis. As compared to normal mammary cells, K14TRT cells possessed larger variable shaped nuclei with a higher nuclear-to-cytoplasmic ratio. The tumor cells also demonstrated a highly aneuploidy karyotype. When isolated via single cell suspension and subsequently passaged, the K14TRT cells were able to form colonies in soft agar. Tumorigenicity was additionally supported by induction of solid tumors following serial injections of the K14TRT cells into the flanks of nude mice. The primary K14TRT tumor overwhelmingly possessed a basal-like breast tumor profile, whereas the transplanted and serially passaged K14TRT cells diverged to a claudin-low profile, as demonstrated by microarray analysis. These serially passaged cells were highly proliferative, with low to absent expression of tight junction proteins (claudin 3, 4, & 7 and E-cadherin), and typically negative for estrogen receptors (ER-), progesterone receptors (PR-), and HER2/neu (HER2-). Since this cell line did demonstrate an ability to form new tumors in recipient mice we speculated they could possess tumor stem cell properties. There competence for self-renewal and capacity to reestablish tumor heterogeneity was supported by fluorescence-activating cell sorting (FACS) analysis of the primary BRCA1/p53 deficient mammary tumor cells in which two populations were identified. Cells that possess low levels of epithelial marker CD24 and high levels of the B1 integrin marker CD29 have been shown to regenerate entire mammary glands indicative of mammary STEM cells. Strikingly, FACS analysis of the K14TRT primary cell line identified two cell populations, one which expressed markers that were CD24high/CD29high (differentiated) and the other population CD24low/CD29high (undifferentiated stem cell-like profile). The sorted undifferentiated mammosphere population when analyzed by microarray analysis clustered with transplanted and serially passaged K14TRT cells identified as being claudin-low, whereas the differentiated sorted population clustered with the primary basal-like cells. Conclusions: Our current data has demonstrated that the K14TRT cell line is a good candidate for a “humanized” TNBC claudin-low cell line with stem cell properties, and could play a significant role in devising better diagnostic tools and chemotherapeutic strategies for this aggressive tumor subtype. Citation Format: Stephanie T. Dance-Barnes, Ebone Evans, Shanderys Steward, Xiaping He, Jerry Usary, Charles Perou. Characterization of a novel p53; BRCA1-deficient claudin-low mammary tumor cell line. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A24.

Using novel murine models of claudin-low and basal-like breast cancer, we tested the hypothesis that diet-induced obesity (DIO) and calorie restriction (CR) differentially modulate progression of these aggressive breast cancer subtypes. For model development, we characterized two cell lines, “mesenchymal (M)-Wnt” and “epithelial (E)-Wnt,” derived from MMTV-Wnt-1 transgenic mouse mammary tumors. M-Wnt, relative to E-Wnt, cells were tumor-initiating cell (TIC)-enriched (62% vs. 2.4% CD44high/CD24low) and displayed enhanced ALDEFLUOR positivity, epithelial-to-mesenchymal transition (EMT) marker expression, mammosphere-forming ability, migration, invasion, and tumorigenicity (P < 0.001; each parameter). M-Wnt and E-Wnt cells clustered with claudin-low and basal-like breast tumors, respectively, in gene expression profiles and recapitulated these tumors when orthotopically transplanted into ovariectomized C57BL/6 mice. To assess the effects of energy balance interventions on tumor progression and EMT, mice were administered DIO, control, or CR diets for 8 weeks before orthotopic transplantation of M-Wnt or E-Wnt cells (for each cell line, n = 20 mice per diet) and continued on their diets for 6 weeks while tumor growth was monitored. Relative to control, DIO enhanced M-Wnt (P = 0.01), but not E-Wnt, tumor progression; upregulated EMT- and TIC-associated markers including N-cadherin,fibronectin, TGFβ, Snail, FOXC2, and Oct4 (P < 0.05, each); and increased intratumoral adipocytes. Conversely, CR suppressed M-Wnt and E-Wnt tumor progression (P < 0.02, each) and inhibited EMT and intratumoral adipocyte accumulation. Thus, dietary energy balance interventions differentially modulate EMT and progression of claudin-low and basal-like tumors. EMT pathway components may represent targets for breaking the obesity–breast cancer link, particularly for preventing and/or controlling TIC-enriched subtypes such as claudin-low breast cancer. Cancer Prev Res; 5(7); 930–42. ©2012 AACR.

Expression of c-Myc is highly prevalent in human breast cancer and stability of the oncoprotein is regulated through Ras-regulated phosphorylation at serine 62 and threonine 58. Previous studies have illustrated the importance of accumulation of KRas mutations in Myc-mediated tumor formation. To examine Myc dependence upon Ras mutations we have generated MMTV regulated Myc and Myc T58A transgenic mice. Expression of the more stable T58A Myc allele resulted in a reduction in KRas-activating mutations. However, in a low-level expression T58A Myc transgenic, the majority of the tumors were squamous or epithelial-to-mesenchymal in nature and accumulated KRas mutations at a higher frequency. Interestingly, we show that these mice develop similar gene expression patterns and signaling pathway utilization as a subtype of human claudin-low breast cancer. Indeed, our results demonstrate a clear division in human claudin-low tumors based on Myc pathway activation and target genes. Together, our results demonstrate that Myc expression and stability has critical effects on molecular heterogeneity in mouse mammary tumors that parallel subtypes of human breast cancer.

Breast tumor cells display great diversity in gene and protein expression, and genomic alterations, associated with striking differences in clinical parameters. Our approach to understanding the molecular pathways that lead to the different types of breast cancer in vivo has been to model this process in vitro. Thus far, almost all in vitro transformed HMEC lines represent a limited subset of in vivo cancer phenotypes. We hypothesized that this could result from restrictive and stressful culture conditions that don t support growth of the target cells. Our proposal has sought to generate lines more reflective of breast cancer phenotypes by using our less stressful HMEC culture methods that support long-term growth of cells of luminal, basal, and progenitor lineages, and to directly examine the effects of stress on vulnerability to oncogenic transformation. We have now shown that unstressed cells are more vulnerable to c-Myc and TERT induced transformation, that different early-stage pathways to transformation are associated with distinct molecular properties, and that normal cells from older women have altered lineage markers that may make their luminal cells more vulnerable to transformation. In the process, we have generated, and continue to characterize, new transformed HMEC lines that exhibit a greater diversity of phenotypes including luminal, progenitor and claudin low as well as basal.