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

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

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

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

The claudin-low breast cancer subtype is defined by gene expression characteristics and encompasses a remarkably diverse range of breast tumors. Here, we investigate genomic, transcriptomic, and clinical features of claudin-low breast tumors. We show that claudin-low is not simply a subtype analogous to the intrinsic subtypes (basal-like, HER2-enriched, luminal A, luminal B and normal-like) as previously portrayed, but is a complex additional phenotype which may permeate breast tumors of various intrinsic subtypes. Claudin-low tumors are distinguished by low genomic instability, mutational burden and proliferation levels, and high levels of immune and stromal cell infiltration. In other aspects, claudin-low tumors reflect characteristics of their intrinsic subtype. Finally, we explore an alternative method for identifying claudin-low tumors and thereby uncover potential weaknesses in the established claudin-low classifier. In sum, these findings elucidate the heterogeneity in claudin-low breast tumors, and substantiate a re-definition of claudin-low as a cancer phenotype., In breast cancer, the claudin-low breast cancer subtype is remarkably diverse. Here, the authors propose that claudin-low is not a classical intrinsic breast cancer subtype, but rather a complex additional phenotype that can occur across intrinsic subtypes.

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

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

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

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

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

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

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

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

Patients diagnosed with basal-like breast cancer suffer from poor prognosis and limited treatment options. There is an urgent need to identify new targets that can benefit patients with basal-like and claudin-low (BL-CL) breast cancers. We screened fractions from our Marine Invertebrate Compound Library (MICL) to identify compounds that specifically target BL-CL breast cancers. We identified a previously unreported trisulfated sterol, i.e., topsentinol L trisulfate (TLT), which exhibited increased efficacy against BL-CL breast cancers relative to luminal/HER2+ breast cancer. Biochemical investigation of the effects of TLT on BL-CL cell lines revealed its ability to inhibit activation of AMP-activated protein kinase (AMPK) and checkpoint kinase 1 (CHK1) and to promote activation of p38. The importance of targeting AMPK and CHK1 in BL-CL cell lines was validated by treating a panel of breast cancer cell lines with known small molecule inhibitors of AMPK (dorsomorphin) and CHK1 (Ly2603618) and recording the increased effectiveness against BL-CL breast cancers as compared with luminal/HER2+ breast cancer. Finally, we generated a drug response gene-expression signature and projected it against a human tumor panel of 12 different cancer types to identify other cancer types sensitive to the compound. The TLT sensitivity gene-expression signature identified breast and bladder cancer as the most sensitive to TLT, while glioblastoma multiforme was the least sensitive.

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

GalNAc-type O-glycosylation, initially catalyzed by polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), is one of the most abundant and complex post-translational modifications of proteins. Emerging evidence has proven that aberrant ppGalNAc-Ts are involved in malignant tumor transformation. However, the exact molecular functions of ppGalNAc-Ts are still unclear. Here, the role of one isoform, ppGalNAc-T4, in breast cancer cell lines was investigated. The expression of ppGalNAc-T4 was found to be negatively associated with migration of breast cancer cells. Loss-of function studies revealed that ppGalNAc-T4 attenuated the migration and invasion of breast cancer cells by inhibiting the epithelial-mesenchymal transition (EMT) process. Correspondingly, transforming growth factor beta (TGF-β) signaling, which is the upstream pathway of EMT, was impaired by ppGalNAc-T4 expression. ppGalNAc-T4 knock-out decreased O-GalNAc modification of TGF-β type Ⅰ and Ⅱ receptor (TβR Ⅰ and Ⅱ) and led to the elevation of TGF-β receptor dimerization and activity. Importantly, a peptide from TβR Ⅱ was identified as a naked peptide substrate of ppGalNAc-T4 with a higher affinity than ppGalNAc-T2. Further, Ser31, corresponding to the extracellular domain of TβR Ⅱ, was identified as the O-GalNAcylation site upon in vitro glycosylation by ppGalNAc-T4. The O-GalNAc-deficient S31A mutation enhanced TGF-β signaling activity and EMT in breast cancer cells. Together, these results identified a novel mechanism of ppGalNAc-T4-catalyzed TGF-β receptors O-GalNAcylation that suppresses breast cancer cell migration and invasion via the EMT process. Targeting ppGalNAc-T4 may be a potential therapeutic strategy for breast cancer treatment.

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

Breast cancer is the leading cause of cancer death in women. Particularly one of the triple negative breast cancer subtypes, very aggressive claudin-low tumors exhibit the expression of factors involved in the epithelial to mesenchymal transition. This complex mechanism of cellular transdifferentiation is controlled by embryonic transcription factors such as ZEB1. In our team, it has been shown that some claudin-low tumors have evolved on the basis of low genetic instability, probably attributed to faulty repair of DNA lesions that arose during the process of tumor progression. The results, obtained during my thesis, show that claudin-low cells limit the use of a mutagenic junction repair pathway, called TMEJ, dependent in particular on the POLQ and PARP1 genes. Indeed, claudin-low cells expressing very strongly ZEB1, conversely, exhibit a very low level of expression of POLQ and a decrease in PARylation activity, leading to a reduction in the use of the TMEJ pathway. Thus, this original mechanism of TMEJ regulation highlights ZEB1 as a key player in genome stability during cancer progression, via its repression of POLQ. All of this thesis work enabled by characterizing the role of ZEB1 on the relative use of DNA repair pathways to exploit this modulation in therapeutic terms for the treatment of these claudin-low tumors currently resistant to conventional chemotherapy, Les cancers du sein constituent la première cause de mortalité par cancer chez la femme. En particulier un des sous-types de cancers du sein triples négatifs, les tumeurs claudin-low très agressives présentent l’expression de facteurs impliqués dans la transition épithélio-mésenchymateuse. Ce mécanisme complexe de transdifférenciation cellulaire est contrôlé par des facteurs de transcription embryonnaires comme ZEB1. Au sein de notre équipe, il a été montré que certaines tumeurs claudin-low ont évolué sur la base d’une faible instabilité génétique, probablement imputable à la réparation fautive de lésions de l’ADN apparues au cours du processus de progression tumorale. Les résultats, obtenus au cours de ma thèse, montrent que les cellules claudin-low limitent l’utilisation d’une voie de réparation par jonction très mutagène, appelée TMEJ dépendante notamment du gène POLQ et de PARP1. En effet, les cellules claudin-low, exprimant très fortement ZEB1, présentent à l’inverse de très faible niveau d’expression de POLQ et une diminution de l’activité de PARylation, conduisant à une réduction de l’utilisation de la voie du TMEJ. Ainsi ce mécanisme original de régulation TMEJ met en évidence ZEB1 comme un acteur clé de la stabilité du génome lors de la progression du cancer, via sa répression de POLQ. L’ensemble de ces travaux de thèse a permis en caractérisant le rôle de ZEB1 sur l’utilisation relative des voies de réparation de l’ADN d’exploiter cette modulation en terme thérapeutique pour le traitement ces tumeurs claudin-low actuellement résistantes aux chimiothérapies conventionnelles

Breast cancer is the leading cause of cancer death in women. Particularly one of the triple negative breast cancer subtypes, very aggressive claudin-low tumors exhibit the expression of factors involved in the epithelial to mesenchymal transition. This complex mechanism of cellular transdifferentiation is controlled by embryonic transcription factors such as ZEB1. In our team, it has been shown that some claudin-low tumors have evolved on the basis of low genetic instability, probably attributed to faulty repair of DNA lesions that arose during the process of tumor progression. The results, obtained during my thesis, show that claudin-low cells limit the use of a mutagenic junction repair pathway, called TMEJ, dependent in particular on the POLQ and PARP1 genes. Indeed, claudin-low cells expressing very strongly ZEB1, conversely, exhibit a very low level of expression of POLQ and a decrease in PARylation activity, leading to a reduction in the use of the TMEJ pathway. Thus, this original mechanism of TMEJ regulation highlights ZEB1 as a key player in genome stability during cancer progression, via its repression of POLQ. All of this thesis work enabled by characterizing the role of ZEB1 on the relative use of DNA repair pathways to exploit this modulation in therapeutic terms for the treatment of these claudin-low tumors currently resistant to conventional chemotherapy; Les cancers du sein constituent la première cause de mortalité par cancer chez la femme. En particulier un des sous-types de cancers du sein triples négatifs, les tumeurs claudin-low très agressives présentent l’expression de facteurs impliqués dans la transition épithélio-mésenchymateuse. Ce mécanisme complexe de transdifférenciation cellulaire est contrôlé par des facteurs de transcription embryonnaires comme ZEB1. Au sein de notre équipe, il a été montré que certaines tumeurs claudin-low ont évolué sur la base d’une faible instabilité génétique, probablement imputable à la réparation fautive de lésions de l’ADN apparues au cours du processus de progression tumorale. Les résultats, obtenus au cours de ma thèse, montrent que les cellules claudin-low limitent l’utilisation d’une voie de réparation par jonction très mutagène, appelée TMEJ dépendante notamment du gène POLQ et de PARP1. En effet, les cellules claudin-low, exprimant très fortement ZEB1, présentent à l’inverse de très faible niveau d’expression de POLQ et une diminution de l’activité de PARylation, conduisant à une réduction de l’utilisation de la voie du TMEJ. Ainsi ce mécanisme original de régulation TMEJ met en évidence ZEB1 comme un acteur clé de la stabilité du génome lors de la progression du cancer, via sa répression de POLQ. L’ensemble de ces travaux de thèse a permis en caractérisant le rôle de ZEB1 sur l’utilisation relative des voies de réparation de l’ADN d’exploiter cette modulation en terme thérapeutique pour le traitement ces tumeurs claudin-low actuellement résistantes aux chimiothérapies conventionnelles

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

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

Significance Mass spectrometry-based proteogenomics of patient-derived xenografts (PDXs) identified dihydropyrimidinase-like-3 (DPYSL3) as a multilevel (RNA/protein/phosphoprotein) expression outlier specific to a claudin-low (CLOW) PDX. DPYSL3 has established functions in neural cell migration and axon outgrowth but is understudied in breast cancer. Here, we demonstrate that loss of DPYSL3 promotes cell-cycle arrest, multinucleation, and collapse of the vimentin microfilament network associated with increased phospho-vimentin. DPYSL3 is also a negative regulator of p21-activated kinase (PAK) and suppresses epithelial-to-mesenchymal transition (EMT). In turn, EMT regulators induce DPYSL3, suggesting that DPYSL3 provides negative feedback on EMT. DPYSL3 therefore serves as a biomarker for CLOW tumors that exhibit PAK-dependent motility and EMT and is also susceptible to therapeutic approaches that promote vimentin phosphorylation during mitosis., A Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteogenomic analysis prioritized dihydropyrimidinase-like-3 (DPYSL3) as a multilevel (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 knockdown in DPYSL3-positive (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 multifunctional signaling modulator. Slower proliferation in DPYSL3-negative (DPYSL3−) CLOW cells was associated with accumulation of multinucleated cells, indicating a mitotic defect that was associated with a collapse of the vimentin microfilament network and increased vimentin phosphorylation. DPYSL3 also suppressed the expression of EMT regulators SNAIL and TWIST and opposed p21 activated kinase 2 (PAK2)-dependent migration. However, these EMT regulators in turn induce DPYSL3 expression, suggesting that DPYSL3 participates in negative feedback on EMT. In conclusion, DPYSL3 expression identifies CLOW tumors that will be sensitive to approaches that promote vimentin phosphorylation during mitosis and inhibitors of PAK signaling during migration and EMT.

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

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