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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.

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.

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.

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.

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.

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

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

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

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

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

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

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

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.

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.

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 MatrigelTM. 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 with low CDH1/Claudins expression and enriched in EMT markers 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. Note: This abstract was not presented at the meeting. Citation Format: Shreya Mitra, Lorenzo Federico, Tapashree Roysarkar, Sendurai Mani, Gordon B. Mills. Transcription factor Snail mediates EMT by altering vesicular trafficking protein Rab25. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4040. doi:10.1158/1538-7445.AM2015-4040

Increased MAPK pathway activity is a major hallmark of acquired resistance of breast cancers to therapies and disease recurrence, and this oncogenic pathway is targeted in the clinic using MEK inhibitors. However, designing combination therapies is a challenge due to variability in the expression of potential partners of these kinases in different tumor types. For example, we have shown previously in ERα(+) breast cancer cell lines that ERK2 localization to chromatin was dictated by ERα, and that it marked those genes associated with ERα signaling (Madak-Erdogan et al., Mol Cell Biol 31:226-236, 2011). In this study, we hypothesized that in different subtypes of ERα(-) breast tumors, ERK2 might be partnering with different transcription factors, thereby regulating the signaling pathways that control the phenotype of that particular tumor subtype. Furthermore, by identifying new cellular and molecular mechanisms leading to the development of resistance to therapies in specific tumor subtypes, it might be possible to develop combination therapies to improve the clinical effectiveness of treatments of these subtypes. Toward this goal, we obtained ERK2 cistrome information (by ChIP-Seq) from our previously established dissociated primary breast tumor cultures. These cultures represented several different breast cancer subtypes, including Her2 enriched, triple negative (basal), triple negative (Luminal B like), triple negative (claudin low) and triple negative (basal epithelial). We found that tumor-type specific “ERK2 Functional Chromatin Footprints” marked those genes that delineated the subtype of the tumor as well as patient clinical outcome, based on Oncomine and TCGA datasets. In addition, transcription factor binding motif enrichment analysis identified new partners and potential druggable targets for chromatin-associated ERK2. Comparison of the ERK2 cistrome revealed Hedgehog and NRF1 signaling pathways as major pathways associated with MAPK activity in the different tumor subtypes. We then tested several inhibitors targeting these pathways and showed that ERK2 occupancy indeed predicted response to these therapies in different tumors. Our studies reveal that monitoring MAPK activity on chromatin is predictive of patient outcome and tumor response to different therapies. This systems approach can be used to precisely identify the subtype of the tumor, predict outcome, and design personalized therapies, based on the functional partners of ERK2 in a particular breast tumor subtype. (Supported by grants from The Breast Cancer Research Foundation (BSK), NIH P50 AT006268 (BSK), NIH R01 CA113674 (DEA), and Bankhead Coley Cancer Research Challenge Grant 09BW-04 (DEA)) Citation Format: Zeynep Madak-Erdogan, Dorraya El-Ashry, Benita S. Katzenellenbogen. ERK2 functional chromatin footprints in patient-derived breast tumor cells predict molecular subtype, clinical outcome, and drug response in breast cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4169. doi:10.1158/1538-7445.AM2014-4169

Understanding pathways leading to metastasis remains a primary goal of breast cancer research. Epithelial to mesenchymal transition (EMT) is thought to be critical in this process as tumor cells migrate through the stroma and undergo deformation while passing endothelial cells to enter the circulation. To examine the role of EMT in tumor metastasis, we have developed a number of stable cell lines in polyomavirus middle T (PyVmT) mammary tumors congenic in the C57Bl/6 background. In this study we characterized the metastatic properties of a luminal cell line Py230 and an EMT cell line Py8119, both of which have cancer stem cell properties of self-renewal, differentiation and tumor initiation. Py230 cells form well-differentiated epithelial adenocarcinomas typical of the spontaneous PyVmT model when injected orthotopically. Injection of 1-10 cells at a single site can give rise to 1-2 metastases over a period of approximately 20 weeks, however the number of metastases can be increased and the duration of the experiment reduced, by injecting 106 cells at 4 sites and removing the tumors at 5-6 weeks when they reach 7-8 mm diameter. After an additional 4-5 weeks multiple lung metastases are visible. Multiple lung metastases can also be achieved by tail vein injection of 5 x 105 cells and inflammation plays a role in the growth at this site as a significant reduction in metastasis is observed in mice lacking the inducible nitric oxide synthase. No Py230 tumor metastases are found in other tissues such as liver, kidney or spleen either with orthotopic or tail vein injection. In contrast, Py8119 cells form highly invasive, poorly differentiated claudin low EMT tumors upon injection into the mammary fat pad. The tumors grow rapidly, but excision does not result in lung metastasis despite their highly invasive phenotype, indicating that EMT alone is not sufficient for metastasis from the mammary fat pad. However, when the cells are injected into the tail vein, metastases develop at multiple tissue sites including lung, liver, kidney and spleen. Py8119 cells upregulate markers of EMT including twist, sna1, sna2 and MMP2 in comparison to Py230 cells, but whether Py230 cells undergo transient EMT and MET during metastasis is currently unknown. Additional luminal or EMT cell lines exhibit similar patterns of metastasis to the Py230 and Py8119 cell lines respectively. Since the majority of human breast cancers are luminal and the less frequent claudin low tumors have a poor prognosis, both the Py230 and Py8119 cell lines should be a useful in dissecting pathways of metastasis relevant to human breast cancer. Citation Format: Lesley G. Ellies, Christopher Kuo. PyVmT luminal and EMT cell lines exhibit characteristic patterns of orthotopic and tail vein metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C91.

The malignant transformation of human epithelial cells is generally described as a multistep process resulting from the accumulation of five to seven rate-limiting changes. Here, we challenge this dogma and demonstrate that this number is radically reduced when cells undergo an epithelial-mesenchymal transition (EMT). EMT is an embryonic transdifferentiation process that consists of the conversion of polarized epithelial cells into motile mesenchymal ones. EMT-inducing transcription factors, including Twist and Zeb proteins, are aberrantly expressed in multiple tumor types and are known to favor the metastatic dissemination process. We demonstrate that, beyond their prometastatic potential, EMT inducers also act as potent drivers of tumorigenesis. We indeed show that the Twist1 EMT-inducing transcription factor promotes breast and skin cancer development in vivo in cooperation with the K-RasG12D oncoprotein. Importantly, in the model of breast tumorigenesis, transgene expression in differentiated mammary epithelial cells leads to the development of undifferentiated tumors exhibiting all the characteristic features of the claudin-low subtype. These observations challenge the concept that this tumor subtype specifically arises from transformation of an early epithelial precursor with inherent stemness properties and metaplastic features. Consistently, oncogenic cooperation assays performed in human mammary epithelial cells with Twist or Zeb EMT-inducers in combination with H-RasG12V generate transformed cell lines displaying all characteristics of claudin-low tumors including mesenchymal features, undifferentiated traits, and stem-cell-like properties. EMT might thus drive the development of claudin-low tumors by exhibiting a dual role in cell transformation and dedifferentiation. In other terms, the claudin-low tumor subtype of breast cancers might thus constitute a first example of human adult malignancies driven by aberrant reactivation of an embryonic transdifferentiation program. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4813. doi:1538-7445.AM2012-4813

Background: Epidemiological evidence suggests a potential role of obesity in regulating clinical subtype, differentiation status, and prognosis of breast cancer. Specifically, an elevated waist-hip ratio is associated with increased risk and progression of basal-like breast cancer, an aggressive form characterized by heterogeneous tumors typically enriched in a putative tumor initiating cell (TIC) population. However, the exact mechanism of obesity-driven tumor progression remains unknown. Therefore, we hypothesized that obesity regulates a plastic population of multipotent malignant cells. Materials, Methods, and Results: To test this hypothesis, we generated and characterized two distinct murine mammary tumor cell lines derived from MMTV-Wnt-1 transgenic mice, designated M-Wnt and E-Wnt. M-Wnt cells displayed a mesenchymal morphology while E-Wnt cells had an epithelial morphology. M-Wnt cells harbored a large CD44+/CD24- putative TIC population (62% +/−7.8), had significant mammosphere forming capacity (>30% of cells form mammospheres, p Control, p=0.011; CR < Control, p=0.012), while E-Wnt tumors were only affected by CR (CR < Control p=0.001; no difference between DIO and Control tumors). DIO enhanced M-Wnt tumor progression, adipocyte infiltration, and central necrosis and drove EMT (decreased E-Cadherin and increased fibronectin and N-Cadherin expression) through modulation of key TIC associated genes, including increased TGF-β, SNAIL, FOXC2, and Oct4 (p Discussion: In conclusion, we found that the mesenchymal M-Wnt cell line is highly responsive to changes in dietary energy balance status relative to the E-Wnt differentiated epithelial cell line, which demonstrates that clinical subtypes of breast cancer are differentially regulated by energy balance modulation. Additionally, our data demonstrates, for the first time, that energy balance modulation (ie, CR and obesity) directly regulates EMT, in response to local upregulation of TGF-β signaling. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S6-5.

Breast cancer recurrence is believed to be caused by a potently tumorigenic sub-population of cancer cells possessing stem cell attributes of resistance to chemotherapy, radiation and other forms of treatment. Recently, we and others have reported the generation of breast cancer stem cell (BCSC) characteristics by inducers of epithelial to mesenchymal transition (EMT). Both EMT and acquisition of BCSC properties are therefore important steps in cancer progression, although the physiological process by which BCSCs arise in vivo remains unclear. We show here that exposure to TGFβ and TNFα induces a stable BCSC phenotype, characterized by self-renewal, greatly increased tumorigenicity, and increased resistance to oxaliplatin, etoposide and paclitaxel. We further found that the TGFβ/TNFα-derived cells showed downregulated expression of Claudins 3, 4 and 7 and the luminal marker KRT18, demonstrating 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 correlation with poor prognosis. Furthermore, pathway analyses of the generated BCSCs showed deregulation of a number of breast cancer-associated genes and pathways including inflammatory cytokine and receptors, chemokines, homeobox, hedgehog, notch and metastases genes. These results confirm the role of EMT and BCSCs in resistance of breast cancer to conventional chemotherapy and identify novel target genes and pathways for validation as potential therapeutic targets. Our demonstration that stable BCSCs can be produced by specific microenvironmental signals also provides a valuable tool for studying cancer stem cells, as well as a means for screening potential cancer drugs for efficacy of targeting cancer stem cells to prevent recurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3341. doi:10.1158/1538-7445.AM2011-3341

The claudin-low subtype is a recently identified new and rare molecular subtype of human breast cancer which we hypothesize may be enriched for cancer stem cells or tumor-initiating cells (TIC). These tumors express low levels of tight and adherens junction genes including claudin 3 and E-cadherin. Claudin-low tumors also often highly express markers associated with epithelial-mesenchymal transition (EMT) including EMT inducers Snail, Twist, Zeb1, and Zeb2. These tumors are also enriched in recently published signatures derived from human TICs and a sorted population enriched for human mammary stem cells. Using comparative genomics, we discovered that there are mouse mammary tumors that have similar gene expression characteristics as claudin-low human tumors and are also enriched in the human TIC signature. Tumors in this group were rare tumors which came from a number of distinct mouse models including the p53 null transplant model. We have now collected over forty more tumors from the p53 null model, a phenotypically heterogeneous tumor model, in order to identify tumors of different molecular subtypes including claudin-low/spindloid tumors that can be transplanted and expanded in vivo. Here we present a molecular characterization of the p53 null mammary tumor bank as compared to other mouse models and human breast tumor subtypes. Similar to human tumors, the p53 null tumors fall into multiple molecular groups, a basal-like subtype, a luminal subtype, and a claudin-low subtype. Like human breast tumors, the p53 null claudin-low tumors show high expression of EMT inducers. They also show low to absent expression of both claudin 3 and E-cadherin by microarray as well as immunostaining. These groups also contain distinct genomic copy number changes as assessed by array comparative genomic hybridization. Finally, we show by limiting dilution transplantation, that p53 null claudin-low tumors are significantly enriched for TICs as compared to the more common adenocarcinomas arising in the same model. In conclusion, p53 null claudin-low/spindloid tumors show features of human claudin-low tumors and they are enriched for TICs, which has yet to be demonstrated experimentally for human claudin-low tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4212.