Tatiana A. Karakasheva

Hypoxia-Dependent Modification of Collagen Networks Promotes Sarcoma Metastasis

UNLABELLED Intratumoral hypoxia and expression of hypoxia-inducible factor-1α (HIF-1α) correlate with metastasis and poor survival in patients with sarcoma. We show here that hypoxia controls sarcoma metastasis through a novel mechanism wherein HIF-1α enhances expression of the intracellular enzyme procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2). We show that loss of HIF-1α or PLOD2 expression disrupts collagen modification, cell migration, and pulmonary metastasis (but not primary tumor growth) in allograft and autochthonous LSL-Kras(G12D/+); Trp53(fl/fl) murine sarcoma models. Furthermore, ectopic PLOD2 expression restores migration and metastatic potential in HIF-1α-deficient tumors, and analysis of human sarcomas reveals elevated HIF1A and PLOD2 expression in metastatic primary lesions. Pharmacologic inhibition of PLOD enzymatic activity suppresses metastases. Collectively, these data indicate that HIF-1α controls sarcoma metastasis through PLOD2-dependent collagen modification and organization in primary tumors. We conclude that PLOD2 is a novel therapeutic target in sarcomas and successful inhibition of this enzyme may reduce tumor cell dissemination. SIGNIFICANCE Undifferentiated pleomorphic sarcoma (UPS) is a commonly diagnosed and particularly aggressive sarcoma subtype in adults, which frequently and fatally metastasizes to the lung. Here, we show the potential use of a novel therapeutic target for the treatment of metastatic UPS, specifi cally the collagen-modifying enzyme PLOD2.

Myeloid derived suppressor cells: Targets for therapy

The goal of achieving measurable response with cancer immunotherapy requires counteracting the immunosuppressive characteristics of tumors. One of the mechanisms that tumors utilize to escape immunosurveillance is the activation of myeloid derived suppressor cells (MDSCs). Upon activation by tumor-derived signals, MDSCs inhibit the ability of the host to mount an anti-tumor immune response via their capacity to suppress both the innate and adaptive immune systems. Despite their relatively recent discovery and characterization, anti-MDSC agents have been identified, which may improve immunotherapy efficacy.

CD38-Expressing Myeloid-Derived Suppressor Cells Promote Tumor Growth in a Murine Model of Esophageal Cancer

Myeloid-derived suppressor cells (MDSC) are an immunosuppressive population of immature myeloid cells found in advanced-stage cancer patients and mouse tumor models. Production of inducible nitric oxide synthase (iNOS) and arginase, as well as other suppressive mechanisms, allows MDSCs to suppress T-cell-mediated tumor clearance and foster tumor progression. Using an unbiased global gene expression approach in conditional p120-catenin knockout mice (L2-cre;p120ctn(f/f)), a model of oral-esophageal cancer, we have identified CD38 as playing a vital role in MDSC biology, previously unknown. CD38 belongs to the ADP-ribosyl cyclase family and possesses both ectoenzyme and receptor functions. It has been described to function in lymphoid and early myeloid cell differentiation, cell activation, and neutrophil chemotaxis. We find that CD38 expression in MDSCs is evident in other mouse tumor models of esophageal carcinogenesis, and CD38(high) MDSCs are more immature than MDSCs lacking CD38 expression, suggesting a potential role for CD38 in the maturation halt found in MDSC populations. CD38(high) MDSCs also possess a greater capacity to suppress activated T cells, and promote tumor growth to a greater degree than CD38(low) MDSCs, likely as a result of increased iNOS production. In addition, we have identified novel tumor-derived factors, specifically IL6, IGFBP3, and CXCL16, which induce CD38 expression by MDSCs ex vivo. Finally, we have detected an expansion of CD38(+) MDSCs in peripheral blood of advanced-stage cancer patients and validated targeting CD38 in vivo as a novel approach to cancer therapy.

The tumor microenvironment in esophageal cancer

Esophageal cancer is a deadly disease, ranking sixth among all cancers in mortality. Despite incremental advances in diagnostics and therapeutics, esophageal cancer still carries a poor prognosis, and thus, there remains a need to elucidate the molecular mechanisms underlying this disease. There is accumulating evidence that a comprehensive understanding of the molecular composition of esophageal cancer requires attention to not only tumor cells but also the tumor microenvironment (TME), which contains diverse cell populations, signaling factors and structural molecules that interact with tumor cells and support all stages of tumorigenesis. In esophageal cancer, environmental exposures can trigger chronic inflammation, which leads to constitutive activation of pro-inflammatory signaling pathways that promote survival and proliferation. Antitumor immunity is attenuated by cell populations such as myeloid-derived suppressor cells and regulatory T cells, as well as immune checkpoints like programmed death-1. Other immune cells such as tumor-associated macrophages can have other pro-tumorigenic functions, including the induction of angiogenesis and tumor cell invasion. Cancer-associated fibroblasts secrete growth factors and alter the extracellular matrix to create a tumor niche and enhance tumor cell migration and metastasis. Further study of how these TME components relate to the different stages of tumor progression in each esophageal cancer subtype will lead to development of novel and specific TME-targeting therapeutic strategies, which offer considerable potential especially in the setting of combination therapy.

Comparative transcriptomes of adenocarcinomas and squamous cell carcinomas reveal molecular similarities that span classical anatomic boundaries

Advances in genomics in recent years have provided key insights into defining cancer subtypes “within-a-tissue”—that is, respecting traditional anatomically driven divisions of medicine. However, there remains a dearth of data regarding molecular profiles that are shared across tissues, an understanding of which could lead to the development of highly versatile, broadly applicable therapies. Using data acquired from The Cancer Genome Atlas (TCGA), we performed a transcriptomics-centered analysis on 1494 patient samples, comparing the two major histological subtypes of solid tumors (adenocarcinomas and squamous cell carcinomas) across organs, with a focus on tissues in which both subtypes arise: esophagus, lung, and uterine cervix. Via principal component and hierarchical clustering analysis, we discovered that histology-driven differences accounted for a greater degree of inherent molecular variation in the tumors than did tissue of origin. We then analyzed differential gene expression, DNA methylation, and non-coding RNA expression between adenocarcinomas and squamous cell carcinomas and found 1733 genes, 346 CpG sites, and 42 microRNAs in common between organ sites, indicating specific adenocarcinoma-associated and squamous cell carcinoma-associated molecular patterns that were conserved across tissues. We then identified specific pathways that may be critical to the development of adenocarcinomas and squamous cell carcinomas, including Liver X receptor activation, which was upregulated in adenocarcinomas but downregulated in squamous cell carcinomas, possibly indicating important differences in cancer cell metabolism between these two histological subtypes of cancer. In addition, we highlighted genes that may be common drivers of adenocarcinomas specifically, such as IGF2BP1, which suggests a possible link between embryonic development and tumor subtype. Altogether, we demonstrate the need to consider biological similarities that transcend anatomical boundaries to inform the development of novel therapeutic strategies. All data sets from our analysis are available as a resource for further investigation.

Esophageal Expression of Active IκB Kinase-β in Mice Up-Regulates Tumor Necrosis Factor and Granulocyte-Macrophage Colony-Stimulating Factor, Promoting Inflammation and Angiogenesis

BACKGROUND & AIMS IκB kinase-β (IKKβ) mediates activation of the nuclear factor-κB, which regulates immune and inflammatory responses. Although nuclear factor-κB is activated in cells from patients with inflammatory diseases or cancer, little is known about its roles in the development and progression of esophageal diseases. We investigated whether mice that express an activated form of IKKβ in the esophageal epithelia develop esophageal disorders. METHODS We generated ED-L2-Cre/Rosa26-IKK2caSFL mice, in which the ED-L2 promoter activates expression of Cre in the esophageal epithelia, leading to expression of a constitutively active form of IKKβ (IKKβca) in epithelial cells but not in inflammatory cells or the surrounding stroma (IKKβca mice). Mice lacking the Cre transgene served as controls. Some mice were given intraperitoneal injections of neutralizing antibodies against granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumor necrosis factor (TNF), or immunoglobulin G1 (control), starting at 1 month of age. Epithelial tissues were collected and analyzed by immunofluorescence, immunohistochemical, and quantitative real-time polymerase chain reaction assays. Transgenes were overexpressed from retroviral vectors in primary human keratinocytes. RESULTS IKKβca mice developed esophagitis and had increased numbers of blood vessels in the esophageal stroma, compared with controls. Esophageal tissues from IKKβca mice had increased levels of GM-CSF. Expression of IKKβca in primary human esophageal keratinocytes led to 11-fold overexpression of GM-CSF and 200-fold overexpression of TNF. Incubation of human umbilical vein endothelial cells with conditioned media from these keratinocytes increased endothelial cell migration by 42% and promoted formation of capillary tubes; these effects were blocked by a neutralizing antibody against GM-CSF. Injections of anti-GM-CSF reduced angiogenesis and numbers of CD31+ blood vessels in esophageal tissues of IKKβca mice, but did not alter the esophageal vasculature of control mice and did not alter recruitment of intraepithelial leukocytes to esophageal tissues of IKKβca mice. Injections of anti-TNF prevented the development of esophagitis in IKKβca mice. CONCLUSIONS Constitutive activation of IKKβ in the esophageal epithelia of mice leads to inflammation and angiogenesis, mediated by TNF and GM-CSF, respectively.

IL-6 Mediates Cross-Talk between Tumor Cells and Activated Fibroblasts in the Tumor Microenvironment

The tumor microenvironment (TME) plays a major role in the pathogenesis of multiple cancer types, including upper-gastrointestinal (GI) cancers that currently lack effective therapeutic options. Cancer-associated fibroblasts (CAF) are an essential component of the TME, contributing to tumorigenesis by secreting growth factors, modifying the extracellular matrix, supporting angiogenesis, and suppressing antitumor immune responses. Through an unbiased approach, we have established that IL-6 mediates cross-talk between tumor cells and CAF not only by supporting tumor cell growth, but also by promoting fibroblast activation. As a result, IL-6 receptor (IL6Rα) and downstream effectors offer opportunities for targeted therapy in upper-GI cancers. IL-6 loss suppressed tumorigenesis in physiologically relevant three-dimensional (3D) organotypic and 3D tumoroid models and murine models of esophageal cancer. Tocilizumab, an anti-IL6Rα antibody, suppressed tumor growth in vivo in part via inhibition of STAT3 and MEK/ERK signaling. Analysis of a pan-cancer TCGA dataset revealed an inverse correlation between IL-6 and IL6Rα overexpression and patient survival. Therefore, we expanded evaluation of tocilizumab to head and neck squamous cell carcinoma patient-derived xenografts and gastric adenocarcinoma xenografts, demonstrating suppression of tumor growth and altered STAT3 and ERK1/2 gene signatures. We used small-molecule inhibitors of STAT3 and MEK1/2 signaling to suppress tumorigenesis in the 3D organotypic model of esophageal cancer. We demonstrate that IL6 is a major contributor to the dynamic cross-talk between tumor cells and CAF in the TME. Our findings provide a translational rationale for inhibition of IL6Rα and downstream signaling pathways as a novel targeted therapy in oral-upper-GI cancers.Significance: These findings demonstrate the interaction of esophageal cancer and cancer-associated fibroblasts through IL-6 signaling, providing rationale for a novel therapeutic approach to target these cancers. Cancer Res; 78(17); 4957-70. ©2018 AACR.

CD38+ M-MDSC expansion characterizes a subset of advanced colorectal cancer patients

BACKGROUND Myeloid-derived suppressor cells (MDSCs) are a population of immature immune cells with several protumorigenic functions. CD38 is a transmembrane receptor-ectoenzyme expressed by MDSCs in murine models of esophageal cancer. We hypothesized that CD38 could be expressed on MDSCs in human colorectal cancer (CRC), which might allow for a new perspective on therapeutic targeting of human MDSCs with anti-CD38 monoclonal antibodies in this cancer. METHODS Blood samples were collected from 41 CRC patients and 8 healthy donors, followed by peripheral blood mononuclear cell (PBMC) separation. Polymorphonuclear (PMN-) and monocytic (M-) MDSCs and CD38 expression levels were quantified by flow cytometry. The immunosuppressive capacity of M-MDSCs from 10 CRC patients was validated in a mixed lymphocyte reaction (MLR) assay. RESULTS A significant expansion of CD38+ M-MDSCs and a trend of expansion of CD38+ PMN-MDSCs (accompanied by a trend of increased CD38 expression on both M- and PMN-MDSCs) were observed in PBMCs of CRC patients when compared with healthy donors. The CD38+ M-MDSCs from CRC patients were found to be immunosuppressive when compared with mature monocytes. CD38+ M- and PMN-MDSC frequencies were significantly higher in CRC patients who previously received treatment when compared with treatment-naive patients. CONCLUSIONS This study provides a rationale for an attempt to target M-MDSCs with an anti-CD38 monoclonal antibody in metastatic CRC patients. FUNDING NCI P01-CA14305603, the American Cancer Society, Scott and Suzi Lustgarten Family Colon Cancer Research Fund, Hansen Foundation, and Janssen Research and Development.

Abstract A37: The role of IL-6 in the interaction between fibroblasts and tumor cells in esophageal adenocarcinoma

Often described as wounds that do not heal, cancer cells depend on interactions with the surrounding stroma to develop and progress. Among the various stromal components, cancer-associated fibroblasts (CAFs) play a critical role in promoting tumor growth and invasion, leading to treatment resistance and poor survival in a number of cancers, including esophageal adenocarcinoma. Mechanistically, CAFs communicate with tumor cells in large part via secreted signaling factors. One such factor, interleukin-6 (IL-6), has been shown to be secreted by CAFs to promote angiogenesis, epithelial-to-mesenchymal transition (EMT), tumor cell stemness, and treatment resistance in adenocarcinomas of the colon, pancreas, stomach, and breast. In EAC, the upregulation of IL-6 signaling among tumor cells, particularly as a result of exposure to reflux, is well characterized. However, the role of IL-6 as a potential mediator of the CAF-tumor cell interaction in EAC remains poorly understood. To confirm that IL-6 is indeed involved in this interaction, we used ELISA to study the dynamics of IL-6 secretion by activated fibroblasts (FEF3303, FEF3 and PDF.G.P) and EAC cells (OE-19 and OE-33) in the setting of mono- and co-culture. We found that the interaction of CAFs and EAC cells in co-culture dramatically increased IL-6 levels compared to either EAC cells or fibroblasts alone. Next, we considered the relevance of these findings to human disease by examining human EAC biopsy specimens for patterns of IL-6 expression via immunohistochemical staining. IL-6 was strongly expressed in the tumor-associated stroma of 10/10 EAC biopsy specimens, with 5/10 showing tumor expression as well, which in several cases was localized to the invasive edge near the tumor-stromal interface. In addition, to assess for IL-6 signaling, we stained the sections for downstream mediators of IL-6 signaling, namely STAT3 and ERK1/2. We found nuclear localization of STAT3 and ERK in 10/10 and 7/10 samples, respectively, indicating activation of these pathways. In summary, our findings indicate that IL-6 is involved in the communication between CAFs and tumor cells in EAC. Furthermore, we have reason to believe, especially in cases where both tumor cells and stroma have strong IL-6 expression, that IL-6 may be a mediator of a bidirectional “crosstalk” between these two cell types. Additional investigation, for instance with the use of a 3-D organotypic model of EAC, will help to further characterize the functional role of IL-6 in this context. This is the first report describing a potential role for IL-6 in mediating crosstalk between CAFs and tumor cells in EAC. Citation Format: Eric W. Lin, Tatiana A. Karakasheva, Sarah Derks, Kwok K. Wong, Adam J. Bass, Anil K. Rustgi. The role of IL-6 in the interaction between fibroblasts and tumor cells in esophageal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A37.

Abstract IA13: The interplay between tumor cells, cancer associated fibroblasts, and immature myeloid cells in the esophageal tumor microenvironment

Esophageal cancer has two major subtypes: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). ESCC is more common worldwide and illustrative of other SCCs (e.g. head/neck, lung) from a molecular pathogenesis viewpoint. EAC is rising in incidence in the United States and Western Europe. Unfortunately, 5-year survival remains dismal for both types, at about 15%. Interrogation of the esophageal tumor microenvironment, replete with desmoplasia, CAFs and diverse immune cells, may afford opportunities to reveal new therapeutic targets, especially in a combinatorial fashion. We have generated previously a conditional knockout model of p120-catenin whereby mice develop preneoplastic and neoplastic lesions in the esophagus. Tumor-derived cells secrete granulocyte macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNFα). The tumors contain significant desmoplasia and immune cell infiltration. Immature myeloid cells (myeloid deriver suppressor cells or MDSCs) comprise a significant percentage of the immune cells present and likely participate in fostering a favorable tumor microenvironment, including the activation of fibroblasts. We have identified CD38 as playing a vital role in MDSC biology. CD38 belongs to the ADP-ribosyl cyclase family and possesses both ectoenzyme and receptor functions. It has been described to function in lymphoid and early myeloid cell differentiation, cell activation, and neutrophil chemotaxis. CD38high MDSCs are more immature than MDSCs lacking CD38 expression, suggesting a potential role for CD38 in the maturation halt found in MDSC populations. CD38high MDSCs also possess a greater capacity to suppress activated T cells, and promote tumor growth to a greater degree than CD38low MDSCs, likely as a result of increased iNOS production. In addition, we have identified novel tumor–derived factors, specifically IL-6, IGFBP3, and CXCL16, which induce CD38 expression by MDSCs ex vivo. Finally, we have detected an expansion of CD38+ MDSCs in peripheral blood of advanced-stage cancer patients. Additional work reveals cross-talk between CAFs and tumor cells through the induction of IL-6 and RANTES, suggesting there is interaction between tumor cells, CAFs and MDSCs. In aggregate, we find that CD38 (on MDSCs), IL-6 and RANTES (secreted by tumor cells and CAFs) serve to provide novel approaches in therapy. Citation Format: Tatiana Karakasheva, Eric Lin, Phil Hicks, Todd Waldron, Anil K. Rustgi. The interplay between tumor cells, cancer associated fibroblasts, and immature myeloid cells in the esophageal tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr IA13.