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Gynecologic carcinosarcomas (CS) are biphasic neoplasms composed of carcinomatous (C) and sarcomatous (S) malignant components. Because of their rarity and histologic complexity, genetic and functional studies on CS are scarce and the mechanisms of initiation and development remain largely unknown. Whole-genome analysis of the C and S components reveals shared genomic alterations, thus emphasizing the clonal evolution of CS. Reconstructions of the evolutionary history of each tumor further reveal that C and S samples are composed of both ancestral cell populations and component-specific subclones, supporting a common origin followed by distinct evolutionary trajectories. However, while we do not find any recurrent genomic features associated with phenotypic divergence, transcriptomic and methylome analyses identify a common mechanism across the cohort, the epithelial-to-mesenchymal transition (EMT), suggesting a role for nongenetic factors in inflicting changes to cellular fate. Altogether, these data accredit the hypothesis that CS tumors are driven by both clonal evolution and transcriptomic reprogramming, essential for susceptibility to transdifferentiation upon encountering environmental cues, thus linking CS heterogeneity to genetic, transcriptomic, and epigenetic influences. Significance: We have provided a detailed characterization of the genomic landscape of CS and identified EMT as a common mechanism associated with phenotypic divergence, linking CS heterogeneity to genetic, transcriptomic, and epigenetic influences.

Key gene alterations in uterine and ovarian CS. Oncoprint of alterations identified in the 50 most altered genes from TCGA cancer pathways and custom lists of CRG. The type of genomic alteration (deletion, amplification, fusion, broken by SV, missense, truncated) is described in the legend. Number and proportions of alteration types are summarized by gene (horizontal bars on right) and by sample (vertical bars on top). The samples are classified into MSI and CN-high according to TCGA endometrial carcinoma classification.

Complete clonal evolution of P12 tumor.

Evolutionary histories of CS. Clonal lineage histories of P02 MSI phenotype tumor (A), P12 BRCAness-phenotype tumor (B), and P15 ovarian bilateral tumor (C). Left, Clonal lineage inference for the whole tumor: subclonal populations (Ci), main cancer gene alterations, genome doubling events, and representative mutational signature proportions are represented. Right, Subclonal population frequencies projection in each tumor sample. Clones colored in gray are found in both derived samples, pink (or green) clones are specific to sarcoma- (or carcinoma-) derived samples, respectively.

Differential expression and methylation analysis (C vs. S). A, Heatmap and pathway overrepresentation analysis of differentially expressed genes between C and S (or undifferentiated) components of the 10 samples derived from the five selected CS tumors. Gene clustering method: Ward's; distance: Spearman. FDR: false discovery rate. B, Heatmaps of EMT and stemness pathways scores (left) and methylation level of EMT-related miRNAs (right). mRNA level: scaled TPM. Pathway scores: scaled ssGSEA scores. Methylation level: scaled beta-values.

Curated list of chromatin remodeling genes.

Genomic landscape of CS. A, Tissue origin and histological subtypes for each tumor (from P01 to P15). B, Main component and detailed percentage content for both samples (a and b) derived from the same tumor. C, CNA analysis: tumor purity (%), ploidy, FGA, and CNA breakpoints number. D, SV number, classified as deletion, duplication, inversion, and interchromosomal translocation. E, SNV and small indel number. F, Proportion of each of four mutational signatures deciphered in the whole cohort. G, MSI phenotype: MSI score (representing the percentage of unstable microsatellite loci), MLH1 promoter methylation (beta-value), and mRNA expression level (TPM – transcripts per million). H, Kataegis number illustrating APOBEC error-prone DNA repair process. I, BRCAness phenotype: number of LST, (white stars highlight samples with LST number ≥20), BRCA1 promoter methylation level (beta-value), BRCA1/2 genes alterations and TDP score, collectively summarized by the HRD status track.

List of differentially methylated gene promoters (paired analysis of 5 selected tumors).

List of differentially expressed genes (paired analysis of 5 selected tumors).

Breast cancer is one of the most prominent types of cancers, in which therapeutic resistance is still a major clinical hurdle. Specific subtypes like Claudin-low (CL) and metaplastic breast cancers (MpBC) have been associated with high non-genetic plasticity, which can facilitate resistance. The overlaps and differences between these orthogonal subtypes, respectively identified by molecular and histopathological analyses, are however still insufficiently characterised. Adequate methods to identify high-plasticity tumours to better anticipate resistance are furthermore still lacking. Here we analysed 11 triple negative breast tumours, including 3 CL and 4 MpBC samples,viahigh-resolution spatial transcriptomics. We combined pathological annotations and deconvolution approaches to precisely identify tumour spots, on which we performed signature enrichment, differential expression and copy-number analyses. We used the TCGA and CCLE public databases for external validation of expression markers. By levying spatial transcriptomics to focus analyses only to tumour cells in MpBC samples, and therefore bypassing the negative impact of stromal contamination, we could identify specific markers that are not expressed in other subtypes nor stromal cells. Three markers (BMPER, POPDC3andSH3RF3) could furthermore be validated in external expression databases encompassing bulk tumour material and stroma-free cell lines. We find that existing bulk expression signatures of high-plasticity breast cancers are relevant in mesenchymal transdifferentiated compartments but can be hindered by stromal cell prevalence in tumour samples, negatively impacting their clinical applicability. Spatial transcriptomics analyses can however help identify more specific expression markers, and could thus enhance diagnosis and clinical care of rare high-plasticity breast cancers.

Supplementary Table 1: List of primers used for quantitative real time PCR. Supplementary Table. 2: List of primers used for construction of pGL2 and pMIR reporter plasmids. Supplementary Table. 3: List of primers used to generate PCR amplicons for pyrosequencing analysis. Supplementary Table. 4: Sequencing primers used for pyrosequencing. Supplementary Table. 5: Primers used for qPCR analysis in ChIP assay.

A subset of primary breast tumors expresses a 308-gene signature that correlated with gene signatures specifically expressed in immune cells.

Triple-negative breast cancer (TNBC) cells are modulated in reaction to tumor-infiltrating lymphocytes. However, their specific responses to this immune pressure are unknown. In order to address this question, we first used mRNA sequencing to compare the immunophenotype of the TNBC cell line MDA-MB-231 and the luminal breast cancer cell line MCF7 after both were cocultured with activated human T cells. Despite similarities in the cytokine-induced immune signatures of the two cell lines, MDA-MD-231 cells were able to transcribe more IDO1 than MCF7 cells. The two cell lines had similar upstream JAK/STAT1 signaling and IDO1 mRNA stability. However, using a series of breast cancer cell lines, IFNγ stimulated IDO1 protein expression and enzymatic activity only in ER−, not ER+, cell lines. Treatment with 5-aza-deoxycytidine reversed the suppression of IDO1 expression in MCF7 cells, suggesting that DNA methylation was potentially involved in IDO1 induction. By analyzing several breast cancer datasets, we discovered subtype-specific mRNA and promoter methylation differences in IDO1, with TNBC/basal subtypes exhibiting lower methylation/higher expression and ER+/luminal subtypes exhibiting higher methylation/lower expression. We confirmed this trend of IDO1 methylation by bisulfite pyrosequencing breast cancer cell lines and an independent cohort of primary breast tumors. Taken together, these findings suggest that IDO1 promoter methylation regulates anti-immune responses in breast cancer subtypes and could be used as a predictive biomarker for IDO1 inhibitor–based immunotherapy. Cancer Immunol Res; 5(4); 330–44. ©2017 AACR.

Supplemental Figure 1. Notch4 mRNA expression correlates with Notch ligands and downstream effectors; Supplemental Figure 2. A schematic outline of experimental procedures; Supplemental Figure 3. A single Notch+ cell has the capacity to generate tumors that represents the initial heterogeneity; Supplemental Figure 4. Evaluation of tumor growth following the treatment of animals with GSI, Docetaxel or combination treatments.

Figure Legends for Supplementary Figures 1-6

Elevated levels of the proinflammatory cytokine IL6 are associated with poor survival outcomes in many cancers. Antibodies targeting IL6 and its receptor have been developed for chronic inflammatory disease, but they have not yet been shown to clearly benefit cancer patients, possibly due to antibody potency or the settings in which they have been tested. In this study, we describe the development of a novel high-affinity anti-IL6 antibody, MEDI5117, which features an extended half-life and potent inhibitory effects on IL6 biologic activity. MEDI5117 inhibited IL6-mediated activation of STAT3, suppressing the growth of several tumor types driven by IL6 autocrine signaling. In the same models, MEDI5117 displayed superior preclinical activity relative to a previously developed anti-IL6 antibody. Consistent with roles for IL6 in promoting tumor angiogenesis, we found that MEDI5117 inhibited the growth of endothelial cells, which can produce IL6 and support tumorigenesis. Notably, in tumor xenograft assays in mice, we documented the ability of MEDI5117 to enhance the antitumor activities of chemotherapy or gefitinib in combination treatment regimens. MEDI5117 also displayed robust activity on its own against trastuzumab-resistant HER2+ tumor cells by targeting the CD44+CD24− cancer stem cell population. Collectively, our findings extend the evidence of important pleiotropic roles of IL6 in tumorigenesis and drug resistance, and offer a preclinical proof of concept for the use of IL6 antibodies in combination regimens to heighten therapeutic responses and overcome drug resistance. Cancer Res; 76(2); 480–90. ©2016 AACR.

Supplemental Figures

Metastatic potential in basal-like breast cancers typically correspond with increased enrichment of EpCAM-/CD49f- cancer stem cells (CSC). With this premise in mind, it is important to better understand the mechanistic driver of these cell populations and their distinctive potential to interact with the tumor microenvironment (TME) for cancer promotion. Previous work from our lab has compared the 450K DNA methylation profile of EpCAM-/CD49f- poor breast cancer cell lines to that of EpCAM-/CD49f- enriched breast cancer cell lines and found the IL32 promoter to be hypomethylated in EpCAM-/CD49f- enriched cell lines, a result which corresponded basal-like patient samples in TCGA. By identifying IL32 being differentially regulated in CSC-enriched cell lines, we further sought to characterize IL32’s role in breast cancer aggressiveness. We first were able to identify several overarching mechanisms altered in siIL32 treated SUM15PT cells by RNAseq differential expression analysis (FDR p-value Citation Format: Megan A. Wilson, Elayne M. Benson, Emma Gray, Paige Cairns, Maria Ouzounova, Hasan Korkaya, Austin Y. Shull. Epigenetic driven IL32 expression contributes to a JNK related cell stress response in breast cancer stem cells to promote cellular invasion. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3595.

SummaryWhile uncommon in breast cancers, oncogenic activation of the RAS/MAPK signalling pathway is frequent in claudin-low (CL) tumours, a subtype of breast malignancies enriched in features of epithelial-mesenchymal transition (EMT), suggesting an interplay between RAS activation and EMT. Using inducible models of human mammary epithelial cells, we show that RAS-mediated transformation relies on cellular reprogramming governed by the EMT-inducing transcription factor ZEB1. The path to ZEB1 induction involves a paracrine process: cells entering a senescent state following RAS activation release proinflammatory cytokines, notably IL-6 and IL-1α which promote ZEB1 expression and activity in neighbouring cells, thereby fostering their malignant transformation. Collectively, our findings unveil a previously unprecedented role for senescence in bridging RAS activation and EMT over the course of malignant transformation of human mammary epithelial cells.

Simple Summary Senescence is a form of cell cycle arrest induced by stresses such as DNA damage and oncogenes and therefore constitutes a crucial barrier against cancer. Nevertheless, senescent cells can escape or bypass this tumor suppressor mechanism and evolve towards an altered, pre-cancerous genotype. Furthermore, accumulated senescent cells that are not cleared by the immune system secrete pro-inflammatory factors, promoting malignant phenotypes. This pro-tumor activity of senescence is associated with genetic reprogramming and the acquisition of cellular plasticity. In this review, we aim to unravel the interconnection between senescence, senescence-associated pro-inflammatory cytokines and the induction of cellular plasticity, which enables the adaptability of tumor cells at different stages of carcinogenesis. Abstract Senescence is a dynamic, multistep program that results in permanent cell cycle arrest and is triggered by developmental or environmental, oncogenic or therapy-induced stress signals. Senescence is considered as a tumor suppressor mechanism that prevents the risk of neoplastic transformation by restricting the proliferation of damaged cells. Cells undergoing senescence sustain important morphological changes, chromatin remodeling and metabolic reprogramming, and secrete pro-inflammatory factors termed senescence-associated secretory phenotype (SASP). SASP activation is required for the clearance of senescent cells by innate immunity. Therefore, escape from senescence and the associated immune editing would be a prerequisite for tumor initiation and progression as well as therapeutic resistance. One of the possible mechanisms for overcoming senescence could be the acquisition of cellular plasticity resulting from the accumulation of genomic alterations and genetic and epigenetic reprogramming. The modified composition of the SASP produced by these reprogrammed cancer cells would create a permissive environment, allowing their immune evasion. Additionally, the SASP produced by cancer cells could enhance the cellular plasticity of neighboring cells, thus hindering their recognition by the immune system. Here, we propose a comprehensive review of the literature, highlighting the role of cellular plasticity in the pro-tumoral activity of senescence in normal cells and in the cancer context.

Despite the advances in early diagnostics and therapeutics, women with metastatic breast cancer have limited treatment options. Women with TNBC, who constitute 15-20% of breast cancer patients, are often diagnosed with aggressive/metastatic disease. Advanced studies implicated immunosuppressive tumor microenvironment (TME) in aggressive/metastatic properties of TNBC subtype. Alternatively activated immature myeloid cells including tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), tumor-associated dendritic cells (TADC) and myeloid derived suppressor cells (MDSC) constitute a major component of TME. However, anti-tumorigenic microenvironment is also reported and that may have clinical relevance in early TNBC patients. Therefore, our hypothesis is that myeloid cells polarize to become immunosuppressive and infiltrate tumors and pre-metastatic niches in patients with advanced disease, while patients with early TNBCs may elicit anti-tumor immune response eliminating disseminated tumor cells (DTC). The utilization of syngeneic immunocompetent mouse models has contributed to our current understanding of immunosuppressive or immunomodulatory TME. Using these models, we have demonstrated that tumor dissemination and growth at metastatic sites is facilitated by MDSC’s. Emerging technologies; single cell RNA sequencing (scRNAseq), mass cytometry (CyTOF) or cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) has been powerful platforms for detailed characterization of tumors and TME compartments. Our bulk gene expression data of the myeloid cell populations of tumor microenvironment, lung, spleen and BM from 4T1 tumor-bearing mice showed distinct MDSC gene signatures. When applied to publicly available scRNAseq data, lung gMDSCs from 4T1 metastatic tumor model appeared to show different trajectory of polarization than the tumor gMDSCs. Consistent with previous findings by Hedrick Lab, lung gMDSCs from 4T1 mice also express higher levels of NeP markers compared to BM and tumor gMDSCs as well as lung gMDSCs from EMT6 mice. However, analyses of immune cells from EMT6 tumor bearing mice exhibited an anti-tumor immune signature which is consistent with the clearance of the DTCs following complete resection of the primary tumors. Using the murine TNBC models in syngeneic mice, we provide evidence that early TNBC tumors may elicit anti-tumor immune responses and thus the survival outcome in those patients is substantially increased after complete surgical resection of the primary tumors. Whereas immunosuppressive tumor microenvironment contributes to the poor overall survival in patients with advanced TNBCs. Therefore, identifying an anti-tumor immune signature in early TNBC patients may be utilized as a clinical biomarker before surgical intervention as well as improve the survival outcome. Citation Format: Fulya Alkan, Raziye Piranlioglu, Eunmi Lee, Maria Ouzounova, Catherine C Hedrick, Huidong Shi, Hasan Korkaya. Understanding the pro- and anti-tumorigenic microenvironments in syngeneic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2223.

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Suppressor of cytokine signaling (SOCS) family of proteins plays critical role in the regulation of immune responses controlling JAK/STAT mediated inflammatory cytokines. Among the members, SOCS1 and SOCS3 contain a kinase inhibitory region (KIR) and SOCS3 binds to JAK/STAT/gp130 complex by inhibiting the downstream signaling and suppressing inflammatory cytokines. Loss or reduced levels of SOCS3 have been linked to cancer-associated inflammation and suppressive immunity leading to enhanced tumor growth and metastasis. In line with these reports, we previously demonstrated that proteolytic degradation of SOCS3 in triple negative breast cancer (TNBC) subtype drives the expression of inflammatory cytokines. Therefore, we postulated that SOCS3 mimetics might suppress the inflammatory cytokine production in TNBC subtype and inhibit tumor growth and metastasis. Here we designed and characterized five linear peptides derived from the N-terminal region of SOCS3 encompassing regions that interface with the JAK2/gp130 complex using the Circular Dichroism and Surface Plasmon Resonance spectroscopies. The KIRESS peptide resulted the sequence containing the most part of the hot-spots required for binding to JAK2 and was further investigated in vivo in mouse xenografts of MDA-MB-231-luci tumors as models of human TNBC subtype. Expectedly, this peptide showed a significant inhibition of primary tumor growth and pulmonary metastasis. Our studies suggest that SOCS3 peptidomimetics may possess a therapeutic potential in aggressive cancers, such as TNBC subtype, with activated inflammatory cytokines.

Despite an enhanced interest, the origin of cancer stem cells (CSCs) remains equivocal. A strong body of evidence suggests that, in some adult carcinomas, CSC may originate from normal mature differentiated cells through a dedifferentiation process and the acquisition of stem-like properties. At the crux of this concept is the epithelial-mesenchymal transition (EMT), an embryonic transdifferentiation process that can be abnormally reactivated over the course of tumor development as a result of oncogenic or microenvironmental cues. High expression of EMT genes and stemness features are hallmarks of claudin-low (CL) breast cancers, a rare subtype of breast malignancies, generally considered as the most primitive breast cancers. We have recently reported that this molecular subtype of breast cancers exhibits a significant diversity, comprising three main subgroups (CL1, CL2 and CL3), that emerge from unique evolutionary processes. Genetic and epigenetic analyses support the hypothesis that CL1 tumors generate from the transformation of a normal mammary stem cells, whereas CL2 and CL3 arise from the reactivation of an EMT process over the course of tumor progression. Interestingly, CL tumors share the frequent activation of the RAS-MAPK pathway, suggesting a potent role for the RAS pathway in the development of CL tumors. Based on these findings, we wondered whether RAS pathway activation could induce the reactivation of EMT-TFs allowing the emergence of CSCs displaying CL2 and CL3 characteristics. By expressing an oncogenic form of RAS in differentiated epithelial cells (HME), we observed a phenotypic switch towards a CD24-/low/CD44+ phenotype, associated with CSC properties, and suggesting an acquisition of cellular plasticity after RAS activation. By using CRISPR/Cas9 knock-down experiments we have shown i) the emergence of CD24-/low/CD44+ cells, ii) the acquisition of transformation and stemness features, and iii) that the cellular plasticity acquired after RAS activation in differentiated HME strongly rely on ZEB1. Single-cell RNA sequencing analyses after RAS activation in HME-RAS cells suggested two different outcomes: senescence or EMT. Treatment with a senolytic drug resulted in reduction of RAS-induced senescent cells associated with a decrease in the emergence of CD24-/low/CD44+ cells and ZEB1 expression. Finally, a co-culture reporter system and a depletive approach with cytokine-neutralizing antibodies highlighted a paracrine effect of IL-6 and IL-1α, produced by senescent RAS-expressing cells on their neighboring differentiated cells, leading to the acquisition of ZEB1-dependent cellular plasticity. Hence, we unveiled a new paracrine process, in which cytokines secreted by cells undergoing oncogene-induced senescence drive ZEB1 expression, thus promoting cellular plasticity correlated with the emergence of CSCs. Citation Format: ALAIN PUISIEUX, Hadrien De Blander, Laurie Tonon, Frédérique Fauvet, Roxane Pommier, Christelle Lamblot, Rahma Benhassoun, Benjamin Gibert, Maria Ouzounova, Anne-Pierre Morel. RAS-induced transformation of mammary epithelial cells relies upon a ZEB1-dependent cellular reprogramming through a paracrine process [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 2667.

One of the ground-breaking discoveries of the international cancer genome sequencing endeavours is the high frequency of mutational and non-mutational changes in epigenetic regulator genes (ERGs), which constitute a “genetic smoking gun” that epigenetic mechanisms lie in the very heart of cancer biology. However, functional importance of disruption of ERGs in tumorigenesis and cancer phenotype is poorly understood. We hypothesise that these genes are candidates to be drivers («epidrivers») of cancer onset and progression, thus regulating mechanisms underpinning cancer development. Moreover, deregulation of epidrivers may play a role in epithelial-to-mesenchymal transition (EMT) and emergence of cancer resilience. The overarching aim of this study was to identify and functionally characterize “Epidrivers” in tumorigenesis and cancer cell plasticity. To this end, we have set up novel epigenome-wide functional screens in human cultured cells (and organoids) combined with state-of-the-art genome-editing approach (based on CRISPR/Cas9 screen) and multiparametric phenotyping. We designed a custom-made lentiviral CRISPR library that consists of 1,649 gRNAs targeting all known ERGs (426 genes). Lentiviral CRISPR library was used to deliver the ERG gRNAs to target cells expressing the RNA-guided DNA endonuclease Cas9. Independent clones (derived from transduced breast and lung cancer cell lines constitutively expressing Cas9) are screened for acquisition of distinct features of transformed cells. The results of the identification of Epidriver genes based on the analysis of deregulation of core cellular processes, epigenome (ChIP-seq) as well as EMT and multiparametric phenotyping, will be presented. Citation Format: Andrea Halaburkova, Vincent Cahais, Cyrille Cuenin, Rita Khoueiry, Maria Ouzounova, Akram Ghantous, Zdenko Herceg. Identifying and characterizing epigenetic «driver» genes («epidrivers») in regulatory pathways involved in tumorigenesis and tumour cell plasticity [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 4313.

Although clinically apparent metastasis is associated with late stages of cancer development, micro-metastatic dissemination may be an early event. However, the fate of these early disseminated tumor cells (DTC) remains elusive. We show that despite their capacity to disseminate into secondary organs, 4T1 tumor models develop overt metastasis while EMT6-tumor bearing mice clear DTCs shed from primary tumors as well as those introduced by intravenous (IV) injection. Following the surgical resection of primary EMT6 tumors, mice do not develop detectable metastasis and reject IV-injected tumor cells. In contrast, these cells readily grow and metastasize in immuno-deficient athymic or Rag2−/− mice, an effect mimicked by CD8+ T-cell depletion in immunocompetent mice. Furthermore, recombinant G-CSF or adoptive transfer of granulocytic-MDSCs isolated from 4T1 tumor-bearing mice, induce metastasis by suppressing CD8+ T-cells in EMT6-primed mice. Our studies support the concept of immune surveillance providing molecular insights into the immune mechanisms during tumor progression., Dissemination of tumor cells from the primary site is an early event. Here, the authors show that the early disseminated tumor cells are actively cleared by the host cytotoxic T lymphocytes induced by the primary tumor and that infiltration of granulocytic myeloid-derived suppressor cells counteracts such immune protection and allow metastasis development.

WOS: 000456589800002 PubMed ID: 30166590 TNF alpha is a pleiotropic cytokine which fuels tumor cell growth, invasion, and metastasis in some malignancies, while in others it induces cytotoxic cell death. However, the molecular mechanism by which TNF alpha exerts its diverse effects on breast cancer subtypes remains elusive. Using in vitro assays and mouse xenografts, we show here that TNF alpha contributes to the aggressive properties of triple negative breast cancer (TNBC) cell lines via upregulation of TNFAIP3(A20). In a striking contrast, TNF alpha induces a potent cytotoxic cell death in luminal (ER+) breast cancer cell lines which fail to upregulate A20 expression. Overexpression of A20 not only protects luminal breast cancer cell lines from TNF alpha-induced cell death via inducing HSP70-mediated anti-apoptotic pathway but also promotes a robust EMT/CSC phenotype by activating the pStat3-mediated inflammatory signaling. Furthermore, A20 overexpression in luminal breast cancer cells induces aggressive metastatic properties in mouse xenografts via generating a permissive inflammatory microenvironment constituted by granulocytic-MDSCs. Collectively, our results reveal a mechanism by which A20 mediates pleiotropic effects of TNF alpha playing role in aggressive behaviors of TNBC subtype while its deficiency results in TNF alpha-induced apoptotic cell death in luminal breast cancer subtype. NCI NIH HHS [R35 CA197585]

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It is widely accepted that dynamic and reversible tumour cell plasticity is required for metastasis, however, in vivo steps and molecular mechanisms are poorly elucidated. We demonstrate here that monocytic (mMDSC) and granulocytic (gMDSC) subsets of myeloid-derived suppressor cells infiltrate in the primary tumour and distant organs with different time kinetics and regulate spatiotemporal tumour plasticity. Using co-culture experiments and mouse transcriptome analyses in syngeneic mouse models, we provide evidence that tumour-infiltrated mMDSCs facilitate tumour cell dissemination from the primary site by inducing EMT/CSC phenotype. In contrast, pulmonary gMDSC infiltrates support the metastatic growth by reverting EMT/CSC phenotype and promoting tumour cell proliferation. Furthermore, lung-derived gMDSCs isolated from tumour-bearing animals enhance metastatic growth of already disseminated tumour cells. MDSC-induced ‘metastatic gene signature' derived from murine syngeneic model predicts poor patient survival in the majority of human solid tumours. Thus spatiotemporal MDSC infiltration may have clinical implications in tumour progression., Myeloid-derived suppressive cells (MDSCs) promote metastasis. Here, the authors show that the monocytic MDSCs subset promotes epithelial to mesenchymal transition at the primary site while the granulocytic subset promotes the reverse transition at the metastatic site enabling dynamic tumour cells plasticity.

Triple-negative breast cancer (TNBC) cells are modulated in reaction to tumor-infiltrating lymphocytes. However, their specific responses to this immune pressure are unknown. In order to address this question, we first used mRNA sequencing to compare the immunophenotype of the TNBC cell line MDA-MB-231 and the luminal breast cancer cell line MCF7 after both were cocultured with activated human T cells. Despite similarities in the cytokine-induced immune signatures of the two cell lines, MDA-MD-231 cells were able to transcribe more IDO1 than MCF7 cells. The two cell lines had similar upstream JAK/STAT1 signaling and IDO1 mRNA stability. However, using a series of breast cancer cell lines, IFNγ stimulated IDO1 protein expression and enzymatic activity only in ER−, not ER+, cell lines. Treatment with 5-aza-deoxycytidine reversed the suppression of IDO1 expression in MCF7 cells, suggesting that DNA methylation was potentially involved in IDO1 induction. By analyzing several breast cancer datasets, we discovered subtype-specific mRNA and promoter methylation differences in IDO1, with TNBC/basal subtypes exhibiting lower methylation/higher expression and ER+/luminal subtypes exhibiting higher methylation/lower expression. We confirmed this trend of IDO1 methylation by bisulfite pyrosequencing breast cancer cell lines and an independent cohort of primary breast tumors. Taken together, these findings suggest that IDO1 promoter methylation regulates anti-immune responses in breast cancer subtypes and could be used as a predictive biomarker for IDO1 inhibitor–based immunotherapy. Cancer Immunol Res; 5(4); 330–44. ©2017 AACR.

Somatic mutations or deletions of TP53 and PTEN in ductal carcinoma in situ lesions have been implicated in progression to invasive ductal carcinomas. A recent molecular and mutational analysis of breast cancers revealed that inactivation of tumor suppressors, p53 and PTEN, are strongly associated with triple negative breast cancer. In addition, these tumor suppressors have important roles in regulating self-renewal in normal and malignant stem cells. To investigate their role in breast carcinogenesis, we knocked down these genes in human mammary cells and in non-transformed MCF10A cells. p53 and PTEN knockdown synergized to activate pro-inflammatory interleukin-6 (IL6)/Stat3/nuclear factor κB signaling. This resulted in generation of highly metastatic epithelial-to-mesenchymal transition-like cancer stem cells resulting in tumors whose gene expression profile mimicked that found in basal/claudin-low molecular subtype within the triple negative breast tumors. Constitutive activation of this loop in transformed cells was dependent on proteolytic degradation of suppressor of cytokine signaling 3 (SOCS3) resulting in low levels of this protein in basal/claudin-low cell lines and primary tumors. In non-transformed cells, transient activation of the IL6 inflammatory loop induced SOCS3 expression leading to pathway inactivation. In transformed cells, enforced expression of SOCS3 or interfering with IL6 pathway via IL6R blockade inhibited tumor growth and metastasis in mouse xenograft models. Furthermore, circulating tumor cells were significantly reduced in tumor-bearing animals when treated with anti-IL6R antibodies. These studies uncover important connections between inflammation and carcinogenesis and suggest that blocking pro-inflammatory cytokines may be utilized as an attractive strategy to target triple negative breast tumors, which currently lacks molecularly targeted therapies.

Large cohorts of recent clinical studies have firmly established that increased levels of tumor-infiltrating lymphocytes (TILs) in TNBC and HER2+ subtypes predicted better clinical outcome compared to the luminal subtype. These observations led to the hypothesis that women with TNBC or HER2+ subtypes may respond to a checkpoint blockade. However, early results from these trials using check point inhibitors alone or in combination with chemotherapy have shown very little promise in breast cancer patients, despite the remarkable long-lasting responses in other hard to treat malignancies such as non-small cell lung and melanoma. Although the outcome falls short of the expectation, it has suggested that the combinations of check point blockade with therapeutics that target immunosuppression may potentiate its efficacy. TNFα exhibits paradoxical roles; it may fuel tumor cell growth, invasion and metastasis in some tumor types, while in others it induces cytotoxic cell death. We recently demonstrated that TNFα distinctly induces A20 in TNBC subtype and protects these cells from TNFα-induced cytotoxic cell death by upregulating HSP70 protein and maintaining EMT/CSC phenotype. In contrast, luminal MCF7 or ZR75-1 cells display approximately 70% apoptosis when treated with TNFα. Overexpression of A20 in luminal cells not only protected them from TNFα-induced cytotoxicity by upregulating HSP70 and EMT/CSC phenotype, but also exhibited aggressive metastatic properties in mouse xenograft models. Furthermore, we show that A20/HSP70 pathway attracts tumor-infiltrating lymphocytes (TILs) while inducing the accumulation of immunosuppressive MDSCs in syngeneic mouse models. Interestingly, pulmonary DTCs as well as the immune infiltrates from 4T1 tumor-bearing mice exhibited significantly higher HSP70 expression. Therefore, we proposed that targeting HSP70 may potentiate the efficacy of immunotherapy in preclinical models of breast cancer. As previously reported, murine 4T1 tumors do respond to check point inhibitors. We reasoned that this may be an appropriate model to test the efficacy of HSP70 inhibitor, JG-231. Expectedly, there was no difference in tumor growth and metastasis between control and anti-PDL1 treated animals, however, combination of anti-PDL1 antibody ed with JG-231 and chemotherapy (cyclophosphamide-CTX) significantly reduced primary tumor growth (>10 fold) and eliminated metastasis. Collectively, our pilot experiments provide a strong rationale for testing our hypothesis and may lead to a rapid translation into the clinical utility. Citation Format: Hasan Korkaya, Eunmi Lee, Raziye Piranioglu, Maria Ouzounova, Ahmet Korkaya, Jason Gestwicki, Max S. Wicha, Esteban Celis. Improving the effectiveness of immunotherapy in breast cancer by targeting the tumor microenvironment [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 2245.

Metastatic basal-like breast cancers are believed to correspond with EpCAM-/Cd49f- cancer stem cell (CSC) enrichment. As well, basal-like breast cancers typically correspond with tumor inflammation and immunoediting phenotypes. However, the exact interplay between CSCs and the inflammatory signature of basal-like breast cancers is not well understood. To provide insight regarding the clinical overlap between breast cancer stem cells and tumor inflammation, we compared the 450K DNA methylation profile of EpCAM-/CD49f- CSCs from the isogenic MCF10A p53-/PTEN- breast cell line against the corresponding EpCAM+/CD49f+ and EpCAM-/CD49f+ subpopulations to determine whether differential DNA methylation occurred within the promoters of immune-related genes in CSCs. In addition, we also overlapped the 450K DNA methylation profile from 16 established breast cancer cell lines of varying EpCAM-/CD49f- concentrations to compare against the isolated CSCs. Based on our results, we identified 1432 differentially methylated promoter regions overall (ANOVA FDR p-value Citation Format: Emma V. Gray, Caroline E. Dyar, Maria Ouzounova, Max S. Wicha, Hasan Korkaya, Austin Y. Shull. IL32 expression is epigenetically regulated in EpCAM-/Cd49f- basal-like breast cancers and can be suppressed by the bromodomain inhibitor JQ1 [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 3683.

Background Naturally occurring polyphenolic compounds from fruits, particularly from blueberries, have been reported to be significantly involved in cancer chemoprevention and chemotherapy. Biotransformation of blueberry juice by Serratia vaccinii increases its polyphenolic content and endows it with anti-inflammatory properties. Methods This study evaluated the effect of a polyphenol-enriched blueberry preparation (PEBP) and its non-fermented counterpart (NBJ), on mammary cancer stem cell (CSC) development in in vitro, in vivo and ex vivo settings. Effects of PEBP on cell proliferation, mobility, invasion, and mammosphere formation were measured in vitro in three cell lines: murine 4T1 and human MCF7 and MDA-MB-231. Ex vivo mammosphere formation, tumor growth and metastasis observations were carried out in a BALB/c mouse model. Results Our research revealed that PEBP influence cellular signaling cascades of breast CSCs, regulating the activity of transcription factors and, consequently, inhibiting tumor growth in vivo by decreasing metastasis and controlling PI3K/AKT, MAPK/ERK, and STAT3 pathways, central nodes in CSC inflammatory signaling. PEBP significantly inhibited cell proliferation of 4T1, MCF-7 and MDA-MB-231. In all cell lines, PEBP reduced mammosphere formation, cell mobility and cell migration. In vivo, PEBP significantly reduced tumor development, inhibited the formation of ex vivo mammospheres, and significantly reduced lung metastasis. Conclusions This study showed that polyphenol enrichment of a blueberry preparation by fermentation increases its chemopreventive potential by protecting mice against tumor development, inhibiting the formation of cancer stem cells and reducing lung metastasis. Thus, PEBP may represent a novel complementary alternative medicine therapy and a source for novel therapeutic agents against breast cancer.

International audience; Growing evidence supports the existence of a subpopulation of cancer cells with stem cell characteristics within breast tumors. In spite of its potential clinical implications, an understanding of the mechanisms responsible for retaining the stem cell characteristics in these cells is still lacking. Here, we used the mammosphere model combined with DNA methylation bead arrays and quantitative gene expression to characterize the epigenetic mechanisms involved in the regulation of developmental pathways in putative breast cancer stem cells. Our results revealed that MCF7-derived mammospheres exhibit distinct CpG promoter methylation profiles in a specific set of genes, including those involved in Jak-STAT signaling pathway. Hypomethylation of several gene components of the Jak-STAT pathway was correlated with an increased expression in mammospheres relative to parental cells. Remarkably, cell sorting of the cells with a putative cancer stem cell phenotype (CD44+/CD24 low) suggests a constitutive activation of Jak-STAT pathway in these cells. These results show that Jak-STAT activation may represent a characteristic of putative breast cancer stem cells. In addition, they favor the concept that the expression of cancer stem-like pathways and the establishment and maintenance of defining properties of cancer stem cells are orchestrated by epigenetic mechanisms.

We recently demonstrated that infiltration of granulocytic MDSCs in distant organs is a critical step in metastatic process. The mouse transcriptome analysis of in vitro co-cultures and samples from syngeneic mouse model revealed that granulocytic subset of myeloid-derived suppressor cells (gMDSCs) from metastatic 4T1 tumor bearing mice are regulated a distinct set of genes. Two genes, S100A8 and S100A9 were highly upregulated in gMDSCs isolated from mice with metastatic 4T1 tumors compared to mice with non-invasive tumors. Using the recombinant single S100A8, S100A9 proteins or the S100A8/S100A9 heterotetramer (calprotectin), we demonstrated that calprotectin is a critical player in gMDSC induction and infiltration in distant organs particularly in lungs. Furthermore, injection of recombinant calprotectin but not single S100A8 or S100A9 proteins via tail vein enhanced the metastatic ability of nan-invasive EMT6 tumors in mice. We therefore performed computational screen of NCI compounds against the crystal structure of calprotectin and identified over 100 lead compounds. In vitro gMDSC differentiation assay identified 3 compounds with strong gMDSC inhibitory activity. We therefore designed a pilot experiment to test these compounds in preclinical mouse models. We also compared the activity of these compounds against tasquinimod, a small molecule targeting S100A9 only. However, tasquinimod treatment of 4T1 tumor-bearing mice had a moderate anti-tumor activity which may be due to a limited activity on granulocytic MDSC accumulation. In addition, we determined that there was a significant upregulation of S100A8 in MDSCs from tasquinimod treated mice. This data suggested that inhibiting only S100A9 leads to activation of S100A8 and thus may be ineffective targeting of MDSCs. In line with these data, one of the compounds (#10) showed a potent activity against gMDSCs in 4T1 tumor-bearing mice and significantly inhibited pulmonary metastasis. Moreover, this compound eliminated the cytotoxicity caused by cyclophosphamide and animals were free of cancer. In summary, we have identified a molecular target which regulates gMDSC induction and play critical role in metastatic process and our findings from in vitro screens and preclinical testing provide a strong rationale for targeting MDSCs. Citation Format: Hasan Korkaya, Eunmi Lee, Raziye Piranioglu, Thomas Albers, Maria Ouzounova, Charlie Weeks, Riley Rodier, Ahmet K. Korkaya, Khaled A. Hassan. Screening the chemical library against granulocytic MDSCs: Critical player in breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1100.

The metastatic potential of breast cancer cells is believed to correspond with the preferential enrichment of a tumor population known as cancer stem cells (CSCs). Thus, evaluating the unique molecular characteristics of breast CSCs is of great interest from a prognostic and therapeutic standpoint. In order to provide a more detailed profile of breast cancer stem cells, we compared the 450K DNA methylation landscape of the EpCAM-/CD49f- cancer stem cell subpopulation from the isogenic MCF10A p53-/PTEN- breast cell line against the corresponding EpCAM+/CD49f+ and EpCAM-/CD49f+ subpopulations to determine how DNA methylation varies within the different genomic regions of CSCs. In addition, we also overlapped the 450K DNA methylation profiles from 16 established breast cancer cell lines of varying subtypes and aggressiveness to determine how these cell lines relate epigenetically with the isolated CSCs. Based on unsupervised PCA and matrix dissimilarity clustering, we were able to identify 3 distinct groups that appeared to independently cluster based on EpCAM-/CD49f- enrichment status. It is also interesting to note that the aggressive cell lines SUM149 and MDA-MB-231 would cluster away from the EpCAM-/CD49f- subset when utilizing strictly promoter probes for clustering, whereas SUM149 and MDA-MB-231 would cluster more closely with the EpCAM-/CD49f- subset when utilizing gene body probes, potentially indicating a more sensitive correlation between gene body methylation and CSC-associated aggressiveness. To further investigate the differing promoter and gene body DNA methylation patterns in CSCs, we performed differential methylation analysis between the 3 previously defined groups. Based on our results, we discovered 1432 differentially methylated promoter probes and 7243 differentially methylated gene body probes (ANOVA FDR p-value Citation Format: Austin Y. Shull, Caroline E. Dyar, Maria Ouzounova, Nicole L. Hudson, Max S. Wicha, Hasan Korkaya. Deciphering the DNA methylation signature of EpCAM-/CD49f- breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5321.

The Wnt pathway is a key regulator of embryonic development and stem cell self-renewal, and hyperactivation of the Wnt signalling is associated with many human cancers. The central player in the Wnt pathway is beta-catenin, a cytoplasmic protein whose function is tightly controlled by ubiquitination and degradation, however the precise regulation of beta-catenin stability/degradation remains elusive. Here, we report a new mechanism of beta-catenin ubiquitination acting in the context of chromatin. This mechanism is mediated by the histone acetyltransferase (HAT) complex component TRRAP and Skp1, an invariable component of the Skp-Cullin-F-box (SCF) ubiquitin ligase complex. TRRAP interacts with Skp1/SCF and mediates its recruitment to beta-catenin target promoter in chromatin. TRRAP deletion leads to a reduced level of beta-catenin ubiquitination, lower degradation rate and accumulation of beta-catenin protein. Furthermore, recruitment of Skp1 to chromatin and ubiquitination of chromatin-bound beta-catenin are abolished upon TRRAP knock-down, leading to an abnormal retention of beta-catenin at the chromatin and concomitant hyperactivation of the canonical Wnt pathway. These results demonstrate that there is a distinct regulatory mechanism for beta-catenin ubiquitination/ destruction acting in the nucleus which functionally complements cytoplasmic destruction of beta-catenin and prevents its oncogenic stabilization and chronic activation of the canonical Wnt pathway.

Developmental pathways such as Notch play a pivotal role in tissue-specific stem cell self-renewal as well as in tumor development. However, the role of Notch signaling in breast cancer stem cells (CSC) remains to be determined. We utilized a lentiviral Notch reporter system to identify a subset of cells with a higher Notch activity (Notch+) or reduced activity (Notch−) in multiple breast cancer cell lines. Using in vitro and mouse xenotransplantation assays, we investigated the role of the Notch pathway in breast CSC regulation. Breast cancer cells with increased Notch activity displayed increased sphere formation as well as expression of breast CSC markers. Interestingly Notch+ cells displayed higher Notch4 expression in both basal and luminal breast cancer cell lines. Moreover, Notch+ cells demonstrated tumor initiation capacity at serial dilutions in mouse xenografts, whereas Notch− cells failed to generate tumors. γ-Secretase inhibitor (GSI), a Notch blocker but not a chemotherapeutic agent, effectively targets these Notch+ cells in vitro and in mouse xenografts. Furthermore, elevated Notch4 and Hey1 expression in primary patient samples correlated with poor patient survival. Our study revealed a molecular mechanism for the role of Notch-mediated regulation of breast CSCs and provided a compelling rationale for CSC-targeted therapeutics. Mol Cancer Ther; 14(3); 779–87. ©2015 AACR.

Metastatic disease is the end stage of extremely inefficient processes that entail overcoming multiple barriers including anti-tumor immunity. Although evidences from preclinical and clinical settings suggest that dissemination of malignant cells is an early process, majority of disseminated cells either eliminated or remain dormant in distant organs, while very few cells eventually develop successful metastasis. Therefore, it is widely accepted that dynamic and reversible tumor cell plasticity is required for metastasis, however, in vivo steps and molecular mechanisms are poorly elucidated. We provide evidence that monocytic and granulocytic subsets of myeloid derived suppressor cells (m-MDS, g-MDSC) infiltrated in primary 4T1 tumor and distant organs with different time kinetics regulate spatiotemporal tumor plasticity. Using co-culture experiments and syngeneic mouse models of murine 4T1 (metastatic) tumor, we demonstrated that tumor infiltrated m-MDSCs facilitate tumor cell dissemination from the primary site by inducing the EMT/CSC phenotype. In contrast, g-MDSCs infiltrated in the lungs support metastatic growth by reverting the EMT/CSC phenotype and thus promoting tumor cell proliferation. In contrast, less invasive EMT6 tumors fail to induce efficient pulmonary infiltration of g-MDSCs and results in clearance of disseminated tumor cells in the lungs. Gene expression analyses of tumors and MDCS subsets in primary tumor site and distant organs at different time points reveal mechanistic temporal regulation of in vivo tumor plasticity by m-MDSC and g-MDSC subsets in 4T1 tumor-bearing mice. However, the lung microenvironment of EMT6 tumor-bearing mice display a gene expression signature of anti-tumor immunity which predict better survival in breast cancer patients. In our functional studies, we demonstrate that EMT6 tumor-bearing animals efficiently eliminate disseminated tumor cells in the lungs. Furthermore, g-MDSCs isolated from 4T1-tumor bearing animals significantly enhance metastatic growth of already disseminated tumor cells. Consistent with the “seed and soil” hypothesis, our studies provide a molecular mechanism by which the immune system regulate spatiotemporal tumor plasticity and generation of permissive or anti-tumorigenic microenvironment in distant organs determining the fate of disseminated tumor cells. Citation Format: Hasan Korkaya, Eunmi Lee, Raziye Piranioglu, Maria Ouzounova, Abdeljabar El-Andaloussi, Iskander Asm, Ahmet K. Korkaya, Gang Zhou, Ali Arbab, Paulo Rodriguez. Immune regulation of disseminated tumor cell clearance versus metastatic growth [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 3992. doi:10.1158/1538-7445.AM2017-3992

Parity and breastfeeding influence a woman’s risk of developing breast cancer. Understanding the underlying molecular changes in the breast will facilitate identification of additional factors during pregnancy and postpartum that modify breast cancer risk. Most prior studies in humans on this topic, however, have been cross-sectional studies comparing breast tissue samples from nulliparous versus post-pregnant women, and are limited in their ability to characterize longitudinal changes and within-person variations. Breast milk contains cells of the mature gland, and may have a potential as a noninvasive source for studying molecular characteristics of the pregnancy-associated changes in the breast and their impact on breast cancer risk. The objectives of this study were to (1) determine normative patterns of gene expression levels in longitudinally collected breast milk samples and (2) correlate gene expression data in relation to self-reported measures of psychosocial stress during pregnancy and postpartum period. Our preliminary results showed that the post-pregnancy gene expression levels in breast milk samples can be measured reproducibly over a short period of time. Whole-genome transcriptome analysis revealed that expression levels of the post-pregnancy genes are different in early (less than 3 month postpartum) and late lactation period (more than 6 month postpartum). Moreover cytokine expression profiling correlated expression of inflammatory cytokines with early versus late postpartum period. The unique contribution of this study is to capture longitudinal gene expression data from breast milk samples collected over the course of lactation, hence providing a valuable basis for future studies to identify differential dynamics associated with breast cancer susceptibility. Note: This abstract was not presented at the meeting. Citation Format: Maria Ouzounova, Georgina Lewis, Raziye Piranlioglu, Tiana Curry-McCoy, Wonsuk Yoo, Nita Maihle, Sangmi Kim, Hasan Korkaya. Determination of normative patterns of gene expression levels and breast cancer risk biomarkers in human breast milk [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 4240. doi:10.1158/1538-7445.AM2017-4240

We previously demonstrated that MDSC subsets accumulated in primary tumor and distant organs regulate tumor plasticity. The mouse transcriptome analysis of in vitro co-cultures and samples from syngeneic mouse model revealed that granulocytic subset of myeloid-derived suppressor cells (gMDSCs) from metastatic 4T1 tumor bearing mice regulate several hundred genes in tumor cells upon co-culture. The top genes are S100A8, S100A9, MMP8, FPR1, CCL3, and TGFβ2 which also predicted poor survival in human solid tumors including breast cancer. Therefore, we called these 6 genes as “metastatic gene signature”. It has been reported that S100A8/S100A9 heterotetramer, called calprotectin, play a key role in inflammation-associated cancer progression. To investigate the role of calprotectin (S100A8/A9) in tumor-mediated immunosuppression and metastasis, we first utilized Tasquinimod, a small molecule targeting S100A9 only. However, Tasquinimod treatment of 4T1 tumor-bearing mice had a moderate anti-tumor activity which may be due to a limited activity on granulocytic MDSC accumulation. In addition, we determined that there was a significant upregulation of S100A8 in MDSCs from Tasquinimod treated mice. This data suggested that inhibiting only S100A9 leads to activation of S100A8 and thus may be ineffective targeting of MDSCs. We therefore run a computational screen of the NCI chemical library against the crystal structure of calprotectin (S100A8/S100A9) and identified 40 lead compounds. We then performed in vitro screening assay to identify compounds that inhibit MDSC induction. We identified 3 compounds that significantly suppressed gMDSC differentiation. We are currently performing in vivo studies with these 3 candidate drugs in our murine breast cancer model and will present our findings at the AACR meeting. We believe that this study will provide a novel drug targeting S100A8/S100A9 heterotetramer, a key molecule in MDSC induction and its regulatory functions during tumor progression. Citation Format: Eunmi Lee, Maria Ouzounova, Raziye Piranlioglu, Abdeljabar El Andaloussi, Sena Arbag, Gang Zhou, Hasan Korkaya. Chemical library screen identifies compounds that target S100A8/S100A9 complex and MDSC accumulation [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 2956. doi:10.1158/1538-7445.AM2017-2956