Philip D. Hicks

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.

Prrx1 isoform switching regulates pancreatic cancer invasion and metastatic colonization

The two major isoforms of the paired-related homeodomain transcription factor 1 (Prrx1), Prrx1a and Prrx1b, are involved in pancreatic development, pancreatitis, and carcinogenesis, although the biological role that these isoforms serve in the systemic dissemination of pancreatic ductal adenocarcinoma (PDAC) has not been investigated. An epithelial-mesenchymal transition (EMT) is believed to be important for primary tumor progression and dissemination, whereas a mesenchymal-epithelial transition (MET) appears crucial for metastatic colonization. Here, we describe novel roles for both isoforms in the metastatic cascade using complementary in vitro and in vivo models. Prrx1b promotes invasion, tumor dedifferentiation, and EMT. In contrast, Prrx1a stimulates metastatic outgrowth in the liver, tumor differentiation, and MET. We further demonstrate that the switch from Prrx1b to Prrx1a governs EMT plasticity in both mouse models of PDAC and human PDAC. Last, we identify hepatocyte growth factor ( HGF) as a novel transcriptional target of Prrx1b. Targeted therapy of HGF in combination with gemcitabine in a preclinical model of PDAC reduces primary tumor volume and eliminates metastatic disease. Overall, we provide new insights into the isoform-specific roles of Prrx1a and Prrx1b in primary PDAC formation, dissemination, and metastatic colonization, allowing for novel therapeutic strategies targeting EMT plasticity.

Loss of Stromal IMP1 Promotes a Tumorigenic Microenvironment in the Colon.

The colon tumor microenvironment is becoming increasingly recognized as a complex but central player in the development of many cancers. Previously, we identified an oncogenic role for the mRNA-binding protein IMP1 (IGF2BP1) in the epithelium during colon tumorigenesis. In the current study, we reveal the contribution of stromal IMP1 in the context of colitis-associated colon tumorigenesis. Interestingly, stromal deletion of Imp1 (Dermo1Cre;Imp1LoxP/LoxP, or Imp1ΔMes) in the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colitis-associated cancer resulted in increased tumor numbers of larger size and more advanced histologic grade than controls. In addition, Imp1ΔMes mice exhibited a global increase in protumorigenic microenvironment factors, including enhanced inflammation and stromal components. Evaluation of purified mesenchyme from AOM/DSS-treated Imp1ΔMes mice demonstrated an increase in hepatocyte growth factor (HGF), which has not been associated with regulation via IMP1. Genetic knockdown of Imp1 in human primary fibroblasts confirmed an increase in HGF with Imp1 loss, demonstrating a specific, cell-autonomous role for Imp1 loss to increase HGF expression. Taken together, these data demonstrate a novel tumor-suppressive role for IMP1 in colon stromal cells and underscore an exquisite, context-specific function for mRNA-binding proteins, such as IMP1, in disease states. Implications: The tumor-suppressive role of stromal IMP1 and its ability to modulate protumorigenic factors suggest that IMP1 status is important for the initiation and growth of epithelial tumors. Mol Cancer Res; 13(11); 1478–86. ©2015 AACR. See related article by Koltsova and Grivennikov, p. 1452

The LIN28B–IMP1 post-transcriptional regulon has opposing effects on oncogenic signaling in the intestine

RNA-binding proteins (RBPs) are expressed broadly during both development and malignant transformation, yet their mechanistic roles in epithelial homeostasis or as drivers of tumor initiation and progression are incompletely understood. Here we describe a novel interplay between RBPs LIN28B and IMP1 in intestinal epithelial cells. Ribosome profiling and RNA sequencing identified IMP1 as a principle node for gene expression regulation downstream from LIN28B In vitro and in vivo data demonstrate that epithelial IMP1 loss increases expression of WNT target genes and enhances LIN28B-mediated intestinal tumorigenesis, which was reversed when we overexpressed IMP1 independently in vivo. Furthermore, IMP1 loss in wild-type or LIN28B-overexpressing mice enhances the regenerative response to irradiation. Together, our data provide new evidence for the opposing effects of the LIN28B-IMP1 axis on post-transcriptional regulation of canonical WNT signaling, with implications in intestinal homeostasis, regeneration and tumorigenesis.

ETV5 regulates ductal morphogenesis with Sox9 and is critical for regeneration from pancreatitis: ETV5 in Pancreatic Regeneration

Background: The plasticity of pancreatic acinar cells to undergo acinar to ductal metaplasia (ADM) has been demonstrated to contribute to the regeneration of the pancreas in response to injury. Sox9 is critical for ductal cell fate and important in the formation of ADM, most likely in concert with a complex hierarchy of, as yet, not fully elucidated transcription factors. Results: By using a mouse model of acute pancreatitis and three dimensional organoid culture of primary pancreatic ductal cells, we herein characterize the Ets‐transcription factor Etv5 as a pivotal regulator of ductal cell identity and ADM that acts upstream of Sox9 and is essential for Sox9 expression in ADM. Loss of Etv5 is associated with increased severity of acute pancreatitis and impaired ADM formation leading to delayed tissue regeneration and recovery in response to injury. Conclusions: Our data provide new insights in the regulation of ADM with implications in our understanding of pancreatic homeostasis, pancreatitis and epithelial plasticity. Developmental Dynamics 247:854–866, 2018.

O-010 Novel Regulation of Autophagy and Intestinal Homeostasis Via mRNA Binding Protein IMP1.

Background:IMP1 (Insulin-like growth factor-2 mRNA binding protein 1) is essential for normal gut development and aberrant overexpression promotes colorectal tumors; however, the role of IMP1 in epithelial homeostasis in the adult intestine remains unclear. Our preliminary findings suggest that Imp1 loss may alter autophagy in intestinal epithelium during homeostasis and response to injury. Recent studies have linked aberrations in autophagy to Crohns disease. We therefore sought to determine if Imp1-mediated changes in autophagy may affect response to injury in the intestine epithelium and whether Imp1 expression is altered in Crohns disease patients. Methods:Mice with intestine-epithelial specific Imp1 deletion (VillinCre;Imp1fl/fl) were used to evaluate autophagy flux and response to irradiation or Heligmosomoides polygyrus infection via gene expression analyses, flow cytometry, and IHC/IF. Crypt enteroid assays were utilized to evaluate stem cell growth. Imp1 expression in Crohns disease patients was evaluated via qRT-PCR. Results:Imp1 expression is enriched in the crypt region of the small intestine, and VillinCre;Imp1fl/fl mice exhibit an increase in autophagy flux and enhanced expression of Paneth and stem cell markers in isolated crypts. Following challenge with irradiation or Heligmosomoides polygyrus infection, VillinCre;Imp1fl/fl mice exhibit robust crypt enteroid growth and improved clinical parameters consistent with Imp1 loss being protective in these contexts. Analysis of tissue biopsies from Crohns disease patients reveal a significant upregulation of Imp1 compared to unaffected patients, suggesting the possibility that overexpression of Imp1 may contribute to autophagy-related pathogenesis in Crohns disease. Conclusions:Our data demonstrate in 2 independent models that intestinal epithelial deletion of Imp1 promotes enhanced recovery from injury, possibly due to upregulation of stem cell gene expression and autophagy. Furthermore, our data reveal for the first time that Imp1 expression is increased in tissue from Crohns disease patients. Taken together, our findings may suggest a novel mechanism for IMP1 to promote pathogenesis of Crohns disease via negative regulation of autophagy.

Abstract 1136: Cooperative functional roles of RNA binding proteins LIN28B and IMP1 in the pathogenesis of colorectal cancer

Introduction: RNA binding proteins and miRNAs have emerged as crucial regulators of intestinal homeostasis by controlling the stability and translation of target mRNAs. LIN28B, an mRNA binding protein, plays a critical role in regulating growth and proliferation in the intestinal epithelium. Previous work in our lab revealed that LIN28B promotes growth and tumorigenesis of the intestinal epithelium via suppression of mature let-7 miRNAs.LIN28B suppression of let-7 promotes upregulation of let-7 targets, including IMP1 (Insulin-like growth factor II mRNA-binding protein 1). Indeed, previous studies from our lab have shown that transgenic mice expressing LIN28B from the mouse Vil1 promoter (Vil-Lin28b mice) have an increase in IMP1 protein levels that is increased further with conditional knockout of let-7. Mechanistically, Let-7 isoforms have been known to physically and functionally interact with IMP1; however, the specific role of IMP1 in Lin28b-let 7-mediated tumorigenesis remains unknown. The current study tested the hypothesis that IMP1 maybe required for LIN28B-mediated tumorigenesis and that LIN28B and IMP1 may cooperatively promote a tumor-initiating phenotype. Methods: We evaluated LIN28B and IMP1 expression and localization in colorectal cancer patient samples using tissue microarrays and clinical outcomes. We used intestinal epithelial cell lines with LIN28B overexpression and CRISPR-Cas9 mediated knockout of IMP1 to study the functional consequences on migration, invasion and proliferation. Results: LIN28B expression correlates with expression of IMP1 in colorectal cancer patient samples. Individually, LIN28B and IMP1 expression intensity each was associated with worse prognosis in stage II colon cancer. Knock-down of IMP1 in Lin28B overexpressing cells decreased migration of colorectal cancer cell lines, suggesting a relationship of IMP1 for the tumorigenic effects of LIN28B. Furthermore, our RNA target analyses show that Lin28b and IMP1 both bind to target mRNAs in the WNT and adherens junction pathways. Conclusions: These data implicate a novel role for the Lin28b-let7-IMP1 axis in colorectal cancer and support an emerging paradigm for a critical and cooperative role of RNA-binding proteins in intestinal homeostasis and cancer. We are using the cell lines generated for orthotopic xenograft studies to see the effects of IMP1 loss and Lin28b overexpression on tumor growth and dissemination. Furthermore, we are generating mice with intestinal epithelium specific Lin28b overexpression-IMP1 loss to study the effects in vivo. Citation Format: Priya Chatterji, Kathryn Hamilton, Sarah Andres, Rei Mizuno, Philip Hicks, Arjun Jeganathan, Monte M. Winslow, Antoni Castells, Miriam Cuatrecasas, Blair Madison, Anil Rustgi. Cooperative functional roles of RNA binding proteins LIN28B and IMP1 in the pathogenesis of colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1136.

Abstract A32: Il-6 initiates a self-sustaining signaling loop to promote nurturing tumor microenvironment

Desmoplasia, or deposition of connective tissue proper in the stroma, is a key feature of esophageal squamous cell carcinoma (ESCC), a common malignancy worldwide. The cellular component of connective tissue proper is comprised of fibroblasts, and cancer-associated fibroblasts (CAFs) are widely recognized as a major constituent of the tumor microenvironment (TME). The purpose of this study was to investigate the cross-talk between CAFs and tumor cells in ESCC, as well as identify factors that mediate this cross-talk and evaluate these factors as therapeutic targets in ESCC and potentially other squamous cell carcinomas. We have observed that co-culture with CAFs prompts ESCC cells to acquire a more mesenchymal phenotype and express potent mediators of cell migration and invasion, such as matricellular proteins (periostin, fibrillin, osteonectin) and growth factors (EGF, HGF, VEGF). Furthermore, co-culture of ESCC cells with CAFs promotes tumor cell migration in our 3D organotypic culture, while co-transplantation of ESCC cells with fibroblasts leads to enhanced tumor growth in vivo , compared to tumors transplanted without fibroblasts. In order to identify potential mediators of ESCC-CAF cross-talk, we have performed a comprehensive cytokine array and found that interleukin 6 (IL-6) is significantly overexpressed in conditioned media from co-culture of esophageal CAFs and ESCC cells, compared to mono-culture. IL-6 is known to play important roles in the development of multiple types of cancer, mostly via activation of the STAT3 signaling pathway. To confirm relevance of our in vitro findings, we have performed immunohistochemical staining of tissue samples from ESCC patients and found that, compared to normal esophagus, expression of IL-6 is enhanced in ESCC in both epithelial cells and fibroblasts. Interestingly, knockdown of IL-6 resulted in altered morphology of ESCC cells in 3D organotypic culture. This was accompanied by restored membrane localization of E-cadherin and reduced activation of STAT3 signaling. In order to investigate the importance of STAT3 and MEK/ERK signaling (the latter being another pathway activated by IL-6 in cancer) on ESCC cell biology, we have treated 3D organotypic cultures with stattic and trametinib (inhibitors of STAT3 and MEK, respectively). This treatment resulted in impaired invasion, reduced proliferation and enhanced apoptosis of ESCC cells, accompanied by decreased expression of the basal cell marker p63. Furthermore, treatment with stattic and trametinib leads to reduced secretion of IL-6 by these cultures. These effects were more pronounced upon treatment with combination of stattic and trametinib. Since we found IL-6 to be important for ESCC progression in vitro , we have conducted a therapeutic study utilizing tocilizumab (an FDA-approved anti-human IL-6R neutralizing antibody). Subcutaneous xenograft tumors comprised of human ESCC and CAF cell lines, were treated with tocilizumab, which resulted in suppressed tumor growth, accompanied by decreased activation of STAT3 and ERK within the tumor, compared to control IgG-treated tumors. Furthermore, tocilizumab-treated animals had reduced numbers of splenic monocytic immature myeloid cells (CD11b + Ly-6C + ), which is in agreement with previously published findings regarding MDSCs in murine models of ESCC. In summary, our findings indicate that IL-6, at least in part, is responsible for migration, proliferation, survival and poor differentiation of ESCC cells, promoted by CAFs in the TME. We also report that these effects of IL-6 are mediated by STAT3 and ERK signaling. Finally, we demonstrate that inhibition of IL-6 signaling suppresses ESCC tumor growth in vivo . This is the first report of anti-tumorigenic activity of tocilizumab in ESCC, which makes it a promising candidate for ESCC therapy. Supported by NCI P01 CA098101. Citation Format: Tatiana A. Karakasheva, Monica Soni, Todd J. Waldron, Eric W. Lin, Philip D. Hicks, Andres Klein-Szanto, Kwok K. Wong, Adam J. Bass, Anil K. Rustgi. Il-6 initiates a self-sustaining signaling loop to promote nurturing 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 A32.

756 ETV5, an ETS-Transcription Factor, Regulates Ductal Morphogenesis in Association With SOX9 and Is Critical for Regeneration From Acute Pancreatitis

NA not assessed, INT intensive therapy, NPDR non proliferative diabetic retinopathy, a Values are numbers (%) or mean (SEM) unless stated otherwise. b CAN prevalence is defined as any of: R-R variation 300 mg/day, renal transplant or dialysis. Table 2. Multiple Variable Predictor Models for Delayed Gastric Emptying in Type 1 Diabetes Mellitus