Claudin-low Breast Cancer Subtype Research Articles

MRCK as a Potential Target for Claudin-Low Subtype of Breast Cancer.

To find new molecular targets for triple negative breast cancer (TNBC), we analyzed a large-scale drug screening dataset based on breast cancer subtypes. We discovered that BDP-9066, a specific MRCK inhibitor (MRCKi), may be an effective drug against TNBC. After confirming the efficacy and specificity of BDP-9066 against TNBC in vitro and in vivo, we further analyzed the underlying mechanism of specific activity of BDP-9066 against TNBC. Comparing the transcriptome of BDP-9066-sensitive and -resistant cells, the activation of the focal adhesion and YAP/TAZ pathway were found to play an important role in the sensitive cells. Furthermore, YAP/TAZ is indeed repressed by BDP-9066 in the sensitive cells, and active form of YAP suppresses the effects of BDP-9066. YAP/TAZ expression and activity are high in TNBC, especially the Claudin-low subtype, consistent with the expression of focal adhesion-related genes. Interestingly, NF-κB functions downstream of YAP/TAZ in TNBC cells and is suppressed by BDP-9066. Furthermore, the PI3 kinase pathway adversely affected the effects of BDP-9066 and that alpelisib, a PI3 kinase inhibitor, synergistically increased the effects of BDP-9066, in PIK3CA mutant TNBC cells. Taken together, we have shown for the first time that MRCKi can be new drugs against TNBC, particularly the Claudin-low subtype.

Cellular Plasticity in Mammary Gland Development and Breast Cancer

Cellular plasticity is a phenomenon where cells adopt different identities during development and tissue homeostasis as a response to physiological and pathological conditions. This review provides a general introduction to processes by which cells change their identity as well as the current definition of cellular plasticity in the field of mammary gland biology. Following a synopsis of the evolving model of the hierarchical development of mammary epithelial cell lineages, we discuss changes in cell identity during normal mammary gland development with particular emphasis on the effect of the gestation cycle on the emergence of new cellular states. Next, we summarize known mechanisms that promote the plasticity of epithelial lineages in the normal mammary gland and highlight the importance of the microenvironment and extracellular matrix. A discourse of cellular reprogramming during the early stages of mammary tumorigenesis that follows focuses on the origin of basal-like breast cancers from luminal progenitors and oncogenic signaling networks that orchestrate diverse developmental trajectories of transforming epithelial cells. In addition to the epithelial-to-mesenchymal transition, we highlight events of cellular reprogramming during breast cancer progression in the context of intrinsic molecular subtype switching and the genesis of the claudin-low breast cancer subtype, which represents the far end of the spectrum of epithelial cell plasticity. In the final section, we will discuss recent advances in the design of genetically engineered models to gain insight into the dynamic processes that promote cellular plasticity during mammary gland development and tumorigenesis in vivo.

Quantitative Proteomics Reveals Knockdown of CD44 Promotes Proliferation and Migration in Claudin-Low MDA-MB-231 and Hs 578T Breast Cancer Cell Lines.

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.

Comprehensive landscape of epigenetic-dysregulated lncRNAs reveals a profound role of enhancers in carcinogenesis in BC subtypes

Aberrant expression of long non-coding RNAs (lncRNA) is associated with altered DNA methylation and histone modifications during carcinogenesis. However, identifying epigenetically dysregulated lncRNAs and characterizing their functional mechanisms in different cancer subtypes are still major challenges for cancer studies. In this study, we systematically analyzed the epigenetic alterations of lncRNAs at important regulatory elements in three breast cancer subtypes. We identified 87, 691, and 1,197 epigenetically dysregulated lncRNAs in luminal, basal, and claudin-low subtypes of breast cancer, respectively. The landscape of epigenetically dysregulated lncRNAs at enhancer elements revealed that epigenetic changes of the majority of lncRNAs occurred in a subtype-specific manner and contributed to subtype-specific biological functions. We identified six acetylation of lysine 27 on histone H3 (H3K27ac)-dysregulated lncRNAs and three DNA methylation-dysregulated lncRNAs (CTC-303L1.2, RP11-738B7.1, and SLC26A4-AS1) as prognostic biomarkers of basal subtype. These lncRNAs were involved in immune response-related biological functions. Treatment of the basal breast cancer cell line MDA-MB-468 with CREBBP/EP300 bromodomain inhibitors downregulated H3K27 acetylation levels and caused a decrease in the expression of five H3K27ac-dysregulated lncRNAs (LINC00393, KB-1836B5.1, RP1-140K8.5, AC005162.1, and AC020916.2) and inhibition of the growth of breast cancer cells. One epigenetically dysregulated lncRNA (LINC01983) and four lncRNA regulators (UCA1, RP11-221J22.2, RP11-221J22.1, and RP1-212P9.3) were identified as prognostic biomarkers of the luminal molecular subtype of breast cancer by controlling the tumor necrosis factor (TNF) signaling pathway, T helper (Th)17 cell differentiation, and T cell migration. Finally, our results highlighted a profound role of enhancer-related H3K27ac-dysregulated lncRNAs, DNA methylation-dysregulated lncRNAs, and lncRNA regulators in breast cancer subtype carcinogenesis and their potential prognostic value.

Proteomic identification of tumor- and metastasis-associated galectin-1 in claudin-low breast cancer

BackgroundMetastasis 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. MethodsWe 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. ResultsGalectin-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. ConclusionsProteomics and genomics reveal high expression of galectin-1 protein and RNA in primary and metastatic claudin-low breast cancer. General significanceThis 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.

Abstract 5041: Inhibition of RUNX-CBFβ binding reduces RSPO3 expression and EMT features in breast cancer cells

Abstract We have recently identified R-spondin3 (RSPO3) as a novel key modulator of breast cancer development that may become a potential target for treatment of the basal subtype, which lacks efficient therapeutic options to date. RSPO3 is expressed in the basal stem cell-enriched compartment of mouse mammary glands whereas it is absent in the mature luminal cells. RSPO3 knockdown in mouse mammary tumor cells reduces canonical Wnt signaling pathway, epithelial-to-mesenchymal transition (EMT)-like features, migration capacity, and tumor formation in vivo. To evaluate the relevance of RSPO3 in human breast cancer, we analyzed samples from 75 patients with ductal infiltrating breast carcinomas, where we found a high prevalence of positive immunoreactivity for RSPO3 (70%). Expression data of The Cancer Genome Atlas Network from 1985 human breast carcinoma samples showed that RSPO3 is primarily expressed in Claudin-low breast cancer subtype, which is characterized for having stem cells features. In culture assays revealed that knockdown of RSPO3 expression reduced SOX2 expression and impaired mammary tumor cell ability to form mammospheres. Previous reports suggested the potential involvement of RUNX1-CBFβ on RSPO3 transcription in mammary tumor cells. Therefore, we evaluated if pharmacologic inhibition of RUNX1 activity would reduce RSPO3 expression and activity in the cancer stem cell compartment. Treatment of breast cancer cells with AI-10-104, a small molecule that inhibits CBFβ-RUNX interaction, reduced RSPO3 mRNA and protein levels, and reduced the migration ability of MDA-MB231 breast cancer cells, ability that was recovered upon treatment with recombinant RSPO3. In addition, AI-10-104 inhibited the expression of canonical Wnt pathway targets, EMT and stem-cell markers. Therefore, our results demonstrate that inhibition of RUNX-CBFβ transcriptional activity leads to a down-regulation of RSPO3 expression, constituting a new molecular target for therapeutic modulation in breast cancer treatment. Citation Format: Carla M. Felcher, Johanna M. Tocci, Martin E. Garcia Sola, John H. Bushweller, Lucio H. Castilla, Edith C. Kordon. Inhibition of RUNX-CBFβ binding reduces RSPO3 expression and EMT features in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5041.

A dual role of Irf1 in maintaining epithelial identity but also enabling EMT and metastasis formation of breast cancer cells.

An epithelial to mesenchymal transition (EMT) is an embryonic dedifferentiation program which is aberrantly activated in cancer cells to acquire cellular plasticity. This plasticity increases the ability of breast cancer cells to invade into surrounding tissue, to seed metastasis at distant sites and to resist to chemotherapy. In this study, we have observed a higher expression of interferon-related factors in basal-like and claudin-low subtypes of breast cancer in patients, known to be associated with EMT. Notably, Irf1 exerts essential functions during the EMT process, yet it is also required for the maintenance of an epithelial differentiation status of mammary gland epithelial cells: RNAi-mediated ablation of Irf1 in mammary epithelial cells results in the expression of mesenchymal factors and Smad transcriptional activity. Conversely, ablation of Irf1 during TGFβ-induced EMT prevents a mesenchymal transition and stabilizes the expression of E-cadherin. In the basal-like murine breast cancer cell line 4T1, RNAi-mediated ablation of Irf1 reduces colony formation and cell migration in vitro and shedding of circulating tumor cells and metastasis formation in vivo. This context-dependent dual role of Irf1 in the regulation of epithelial-mesenchymal plasticity provides important new insights into the functional contribution and therapeutic potential of interferon-regulated factors in breast cancer.

Re-definition of claudin-low as a breast cancer phenotype

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.

Abstract B13: Immune cells, tumor-initiating cells, and drug sensitivity in claudin-low TNBC: A delicate balance

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

Claudin-low-like mouse mammary tumors show distinct transcriptomic patterns uncoupled from genomic drivers

BackgroundClaudin-low breast cancer is a molecular subtype associated with poor prognosis and without targeted treatment options. The claudin-low subtype is defined by certain biological characteristics, some of which may be clinically actionable, such as high immunogenicity. In mice, the medroxyprogesterone acetate (MPA) and 7,12-dimethylbenzanthracene (DMBA)-induced mammary tumor model yields a heterogeneous set of tumors, a subset of which display claudin-low features. Neither the genomic characteristics of MPA/DMBA-induced claudin-low tumors nor those of human claudin-low breast tumors have been thoroughly explored.MethodsThe transcriptomic characteristics and subtypes of MPA/DMBA-induced mouse mammary tumors were determined using gene expression microarrays. Somatic mutations and copy number aberrations in MPA/DMBA-induced tumors were identified from whole exome sequencing data. A publicly available dataset was queried to explore the genomic characteristics of human claudin-low breast cancer and to validate findings in the murine tumors.ResultsHalf of MPA/DMBA-induced tumors showed a claudin-low-like subtype. All tumors carried mutations in known driver genes. While the specific genes carrying mutations varied between tumors, there was a consistent mutational signature with an overweight of T>A transversions in TG dinucleotides. Most tumors carried copy number aberrations with a potential oncogenic driver effect. Overall, several genomic events were observed recurrently; however, none accurately delineated claudin-low-like tumors. Human claudin-low breast cancers carried a distinct set of genomic characteristics, in particular a relatively low burden of mutations and copy number aberrations. The gene expression characteristics of claudin-low-like MPA/DMBA-induced tumors accurately reflected those of human claudin-low tumors, including epithelial-mesenchymal transition phenotype, high level of immune activation, and low degree of differentiation. There was an elevated expression of the immunosuppressive genes PTGS2 (encoding COX-2) and CD274 (encoding PD-L1) in human and murine claudin-low tumors.ConclusionsOur findings show that the claudin-low breast cancer subtype is not demarcated by specific genomic aberrations, but carries potentially targetable characteristics warranting further research.

Stem cell-like breast cancer cells with acquired resistance to metformin are sensitive to inhibitors of NADH-dependent CtBP dimerization.

Altered flux through major metabolic pathways is a hallmark of cancer cells and provides opportunities for therapy. Stem cell-like cancer (SCLC) cells can cause metastasis and therapy resistance. They possess metabolic plasticity, theoretically enabling resistance to therapies targeting a specific metabolic state. The C-terminal binding protein (CtBP) transcriptional regulators are potential therapeutic targets in highly glycolytic cancer cells, as they are activated by the glycolytic coenzyme nicotinamide adenine dinucleotide (NADH). However, SCLC cells commonly exist in an oxidative state with low rates of glycolysis. Metformin inhibits complex I of the mitochondrial electron transport chain; it can kill oxidative SCLC cells and has anti-cancer activity in patients. SCLC cells can acquire resistance to metformin through increased glycolysis. Given the potential for long-term metformin therapy, we have studied acquired metformin resistance in cells from the claudin-low subtype of breast cancer. Cells cultured for 8 weeks in sub-IC50 metformin concentration proliferated comparably to untreated cells and exhibited higher rates of glucose uptake. SCLC cells were enriched in metformin-adapted cultures. These SCLC cells acquired sensitivity to multiple methods of inhibition of CtBP function, including a cyclic peptide inhibitor of NADH-induced CtBP dimerization. Single-cell mRNA sequencing identified a reprogramming of epithelial-mesenchymal and stem cell gene expression in the metformin-adapted SCLC cells. These SCLC cells demonstrated an acquired dependency on one of these genes, Tenascin C. Thus, in addition to acquisition of sensitivity to glycolysis-targeting therapeutic strategies, the reprograming of gene expression in the metformin-adapted SCLC cells renders them sensitive to potential therapeutic approaches not directly linked to cell metabolism.

A Longitudinal Study of the Association between Mammographic Density and Gene Expression in Normal Breast Tissue

High mammographic density (MD) is associated with a 4–6 times increase in breast cancer risk. For post-menopausal women, MD often decreases over time, but little is known about the underlying biological mechanisms. MD reflects breast tissue composition, and may be associated with microenvironment subtypes previously identified in tumor-adjacent normal tissue. Currently, these subtypes have not been explored in normal breast tissue. We obtained biopsies from breasts of healthy women at two different time points several years apart and performed microarray gene expression analysis. At time point 1, 65 samples with both MD and gene expression were available. At time point 2, gene expression and MD data were available from 17 women, of which 11 also had gene expression data available from the first time point. We validated findings from our previous study; negative correlation between RBL1 and MD in post-menopausal women, indicating involvement of the TGFβ pathway. We also found that breast tissue samples from women with a large decrease in MD sustained higher expression of genes in the histone family H4. In addition, we explored the previously defined active and inactive microenvironment subtypes and demonstrated that normal breast samples of the active subtype had characteristics similar to the claudin-low breast cancer subtype. Breast biopsies from healthy women are challenging to obtain, but despite a limited sample size, we have identified possible mechanisms relevant for changes in breast biology and MD over time that may be of importance for breast cancer risk and tumor initiation.

PO-287 ’Annex’in A1 to the breast tumour microenvironment by aiding in macrophage polarisation

IntroductionTumor-associated macrophages (TAMs) choreograph various aspects of the tumour microenvironment. Macrophages exhibit cellular plasticity and can be polarised to M1 or M2 subtypes in the presence of different microenvironment ’signals’. Annexin A1 (ANXA1), an anti-inflammatory protein is highly expressed in the metastatic breast cancer. Many studies have found that the TAMs exhibit a higher pro-tumorigenic and anti-inflammatory, M2 phenotype.Material and methodsGene Expression Omnibus (GEO) and ArrayExpress were used to assess the association between TAMs and breast cancer in the patients. Percentage of M2 macrophages was evaluated using flow cytometry in the breast tumours from the MMTV mice and the mammary fat pad of the control mice. Macrophage education by breast cancer was assessed by ex vivo differentiation of bone-marrow derived macrophages (BMDMs) in the presence or absence of breast cancer conditioned media by flow cytometry, ELISA, and mRNA expression.Results and discussionsClinically, we found that the M2 TAMs were highly enriched in the claudin-low breast cancer subtype and was strongly associated with ANXA1 gene expression. In the MMTV mouse model, M2 TAMs were higher in the breast tumours as compared to the mammary tissues along with higher expression of ANXA1 and other M2 markers. Additionally, BMDMs were skewed to a more M2 TAM-like phenotype upon co-culture with the breast cancer cells, with enhanced migratory and invasive properties and phagocytic potential, which was reduced in the ANXAI-deficient BMDMs. In another in vivo mouse model employed in our study, we found that the pro-tumorigenic M2 macrophages were significantly reduced in the breast tumours isolated from the ANXA1-deficient mice as compared to the wild type, when 4 T1 had been injected orthotopically in them.ConclusionCollectively, our data describe a novel regulatory role of Annexin A1 in promoting macrophage polarisation to a more M2 phenotype in the breast tumour microenvironment both ex vivo and in vivo, thus presenting itself and its associated receptor as a potential drug target.Furthermore, our secretome data has also revealed some interesting results and Affymetrix microarray profiling and RNA sequencing studies are underway to explore the unique signature molecules and the signalling mechanism involved in governing this dynamic process.

Synergistic effect of pyrazoles derivatives and doxorubicin in claudin-low breast cancer subtype

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

EMT programs promote basal mammary stem cell and tumor-initiating cell stemness by inducing primary ciliogenesis and Hedgehog signaling

Tissue regeneration relies on adult stem cells (SCs) that possess the ability to self-renew and produce differentiating progeny. In an analogous manner, the development of certain carcinomas depends on a small subset of tumor cells, called "tumor-initiating cells" (TICs), with SC-like properties. Mammary SCs (MaSCs) reside in the basal compartment of the mammary epithelium, and their neoplastic counterparts, mammary TICs (MaTICs), are thought to serve as the TICs for the claudin-low subtype of breast cancer. MaSCs and MaTICs both use epithelial-mesenchymal transition (EMT) programs to acquire SC properties, but the mechanism(s) connecting EMT programs to stemness remain unclear. Here we show that this depends on primary cilia, which are nonmotile, cell-surface structures that serve as platforms for receiving cues and enable activation of various signaling pathways. We show that MaSC and MaTIC EMT programs induce primary cilia formation and Hedgehog (Hh) signaling, which has previously been implicated in both MaSC and MaTIC function. Moreover, ablation of these primary cilia is sufficient to repress Hh signaling, the stemness of MaSCs, and the tumor-forming potential of MaTICs. Together, our findings establish primary ciliogenesis and consequent Hh signaling as a key mechanism by which MaSC and MaTIC EMT programs promote stemness and thereby support mammary tissue outgrowth and tumors of basal origin.

PPARγ Ligand-induced Annexin A1 Expression Determines Chemotherapy Response via Deubiquitination of Death Domain Kinase RIP in Triple-negative Breast Cancers.

Metastatic breast cancer is still incurable so far; new specifically targeted and more effective therapies for triple-negative breast cancer (TNBC) are required in the clinic. In this study, our clinical data have established that basal and claudin-low subtypes of breast cancer (TNBC types) express significantly higher levels of Annexin A1 (ANXA1) with poor survival outcomes. Using human cancer cell lines that model the TNBC subtype, we observed a strong positive correlation between expression of ANXA1 and PPARγ. A similar correlation between these two markers was also established in our clinical breast cancer patients' specimens. To establish a link between these two markers in TNBC, we show de novo expression of ANXA1 is induced by activation of PPARγ both in vitro and in vivo and it has a predictive value in determining chemosensitivity to PPARγ ligands. Mechanistically, we show for the first time PPARγ-induced ANXA1 protein directly interacts with receptor interacting protein-1 (RIP1), promoting its deubiquitination and thereby activating the caspase-8-dependent death pathway. We further identified this underlying mechanism also involved a PPARγ-induced ANXA1-dependent autoubiquitination of cIAP1, the direct E3 ligase of RIP1, shifting cIAP1 toward proteosomal degradation. Collectively, our study provides first insight for the suitability of using drug-induced expression of ANXA1 as a new player in RIP1-induced death machinery in TNBCs, presenting itself both as an inclusion criterion for patient selection and surrogate marker for drug response in future PPARγ chemotherapy trials. Mol Cancer Ther; 16(11); 2528-42. ©2017 AACR.

Abstract P1-07-01: A pan-cancer perspective of functional proteomics provides novel information content for uncommon breast cancer subtypes

Abstract Cancer cell lines (CCLs) serve as models to study the functional consequences of the genomic lesions in patients and as screening platforms for prediction of drug response. While genomic and transcriptomic data have proven to be useful predictors, the ability of these omics platforms to predict protein level and function is limited. Furthermore, since proteins are the targets of the majority of the targeted therapies, protein levels and importantly protein function would be expected to provide more powerful predictions than DNA or RNA data. While large scale genomic and transcriptomic data linked to drug sensitivity are available for over a thousand CCLs, proteomic data is available for only a small subset of lines. Here we performed proteomic profiling of 736 cell lines using reverse-phase protein arrays (RPPAs) with approximately 300 antibodies providing an unbiased sparse representation of the majority of signaling pathways. The functional proteomic analysis revealed 10 protein-based clusters across all cell lines. Similar to human tumors, the breast cell lines fell into three major clusters representing basal-like, luminal/Her2-amplified and claudin-low breast cancer subtypes. The basal-like and claudin-low clusters contained all of the representative breast cancer cell lines as well as a much larger number of other CCLs. For example, the 6 claudin-low breast cancers analyzed reside in an EMT cluster, in which only 8/126 are breast cell lines. However, the complete cluster including multiple non-breast cancer cell lines recapitulated mRNA and protein features of claudin-low breast tumors, including a high EMT signature and low level of hormone receptor pathway activity. We thus explored whether we could gain power for linking the limited number of basal and claudin-low breast cancer cell lines to therapeutic sensitivity by assessing patterns of drug sensitivity in each cluster for both the breast and non-breast cancer cell lines in the cluster. We explored drug sensitivity of 481 therapeutic compounds from the Cancer Therapeutic Response Portal (CTRP v2) and demonstrated that the non- breast cancer and breast cancer cell lines in each cluster provided similar patterns of drug sensitivity. For example, Claudin-low/EMT cell lines of both breast cancer and non-breast cancer origin showed decreased sensitivity to PI3K/mTOR inhibitors compared to luminal breast cancers (p<0.05 for 4 mTOR inhibitors) and drugs targeting EGFR family compared to basal cell lines (p<0.05 for 7 EGFR/ERBB2 inhibitors). Thus it is possible to gain information by characterizing cell lines with similar patterns of protein expression and provide important information related to drug sensitivity of uncommon breast cancer lineages. The functional proteomic analysis provides a wealth of information that complements the genomic and transciptomic studies of cancer cell lines, and demonstrates the opportunity to leverage cell line 'pan-cancer' proteomic patterns to improve characterization of specific breast cancer subtypes. To facilitate broad access to these data, we developed a user-friendly data portal, the MD Anderson Cell Lines Project (MCLP), that provides both data analysis and download (http://ibl.mdanderson.org/mclp/). Citation Format: Zhao W, Li J, Lu Y, Akbani R, Liang H, Mills GB. A pan-cancer perspective of functional proteomics provides novel information content for uncommon breast cancer subtypes [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-07-01.

Overview of Genetically Engineered Mouse Models of Distinct Breast Cancer Subtypes.

Advances in the screening of new therapeutic options have significantly reduced the breast cancer death rate over the last decade. Despite these advances, breast cancer remains the second leading cause of cancer death among women. This is due in part to the complexity of the disease, which is characterized by multiple subtypes that are driven by different genetic mechanisms and that likely arise from different cell types of origin. Because these differences often drive treatment options and outcomes, it is important to select relevant preclinical model systems to study new therapeutic interventions and tumor biology. Described in this unit are the characteristics and applications of validated genetically engineered mouse models (GEMMs) of basal-like, luminal, and claudin-low human subtypes of breast cancer. These different subtypes have different clinical outcomes and require different treatment strategies. These GEMMs can be considered faithful surrogates of their human disease counterparts. They represent alternative preclinical tumor models to cell line and patient-derived xenografts for preclinical drug discovery and tumor biology studies.

BRCA Mutation-Related and Claudin-Low Breast Cancer: Blood Relatives or Stepsisters?

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

Abstract 2326: Sox10 promotes both stem-like and EMT properties in mammary cells

Abstract Molecular pathways that guide cell proliferation and programming events during embryonic development may be aberrantly re-activated in the cancer state to drive tumor progression. To identify mechanisms that drive progression of aggressive breast cancers, we identified molecules that are present in the fetal mammary stem cells (fMaSCs) that drive early development of the mammary gland. These analyses revealed that Sox10 is one of the most highly expressed transcription factors within fMaSCs, and prompted us to investigate the function of Sox10 in the mammary gland and in subtypes of cancer originating from it. Our studies described here indicate that Sox10 is specifically expressed in mammary cells that exhibit higher levels of stem or progenitor cell functions using in vitro or in vivo assays. This includes fetal and adult mammary cells in vivo, as well as mammary cells cultured in vitro as organoids, and for the first time, male fetal mammary stem cells. Sox10 appears to contribute to these stem/progenitor behaviors, as the genetic deletion of Sox10 limits stem/progenitor activities, while the overexpression of Sox10 significantly expands clonogenic behavior. Intriguingly, ectopic Sox10 expression is also able to elicit an EMT-like response in mammary organoids. Sox10 thus represents a single molecule capable of directly mediating both stem-like and EMT-like properties in mammary cells. Furthermore, by modulating Sox10 levels in 3D mammary organoids, we are able to induce cells into sequential motile and stem/progenitor states. This provides a striking in vitro surrogate for metastatic behavior. Consistent with these findings, we find that Sox10 is highly expressed in basal-like and claudin-low subtypes of breast cancer, which are the most stem-like and EMT-like manifestations of the disease, respectively. Finally, we show that Sox10 expression is regulated through a feedback loop involving FGF signaling, and that inhibition of FGF signaling can block EMT-like cell behaviors mediated by Sox10. These findings have important implications in how stem-like and metastatic properties may be specified in mammary cells. Citation Format: Christopher Dravis, Geoffrey M. Wahl. Sox10 promotes both stem-like and EMT properties in mammary cells. [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 2326. doi:10.1158/1538-7445.AM2015-2326