University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Spring 5-6-2017 Role of Endothelin Axis in Pancreatic Tumor Microenvironment Suprit Gupta University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Cancer Biology Commons Recommended Citation Gupta, Suprit, "Role of Endothelin Axis in Pancreatic Tumor Microenvironment" (2017). Theses & Dissertations. 203. https://digitalcommons.unmc.edu/etd/203 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. i Role of Endothelin Axis in Pancreatic Tumor Microenvironment By Suprit Gupta Presented to the Faculty of The Graduate College in the University of Nebraska In Partial fulfillment of Requirements For the degree of Doctor of Philosophy Department of Biochemistry and Molecular Biology Under the Supervision of Dr. Maneesh Jain University of Nebraska Medical Center Omaha, Nebraska April 2017 ii iii Role of Endothelin Axis in Pancreatic Tumor Microenvironment (TME) Suprit Gupta, PhD. University of Nebraska Medical Center 2017 Supervisor: Maneesh Jain, PhD. Endothelins (ETs) are a family of three 21 amino-acid vasoactive peptides ET-1, ET-2 and ET-3 that mediate their effects via two G-protein couple receptors ETAR and ETBR which are expressed on various cell types. Apart from their physiological role in vasoconstriction, there is emerging evidence supporting the role of endothelin axis (ET- axis) in cancer. Due to the expression of ET receptors on various cell-types, ET-axis can exert pleotropic effects and contribute to various aspects of cancer pathobiology. Several studies have provided a fragmented picture of the diverse roles or ET-axis in various tumors. However, the comprehensive picture of the pathobiological role of this axis in any given cancer is poorly understood. Given that PC epitomizes the complexity of tumor microenvironment (TME), which is an active player in disease progression and therapy resistance, the overarching goal of this dissertation was to define the role of ET-axis in this lethal malignancy. Specifically, the dissertation was aimed at defining the expression pattern of ET axis in PC TME and elucidating the pathobiological significance of ET axis in PC. Immunohistochemistry (IHC) analysis of surgically resected tumor tissues from PC patients indicated expression of ECE-1, ET-1, ETAR and ETBR expression in both primary and metastatic lesions. In addition to tumor cells, ETAR and ETBR expression was observed on blood vessels (BV), stromal cells including stellate cells and infiltrating immune cells. The expression of ETAR and ETBR in various cellular compartments was also analyzed using marker for tumor cell (CK19), blood vessel (CD31), stellate cell (alpha SMA) and macrophages (CD68 and F4/80). Importantly, analysis of survival data showed ETBR positivity on BV is correlated with poor prognosis of the PC patients. iv Bioinformatics analysis of TCGA database revealed high positive correlation of the pro- fibrotic gene signatures with both ETAR and ETBR particularly Collagen I (Col1A2, Col3A1, Col5A2, Col6A3), Platelet derived growth factor receptor beta (PDGFRβ), Fibroblast activation protein (FAP), suggesting a pro-fibrotic role of ET axis in PC. In the second part of the dissertation, we studied the impact of ET-axis inhibition in autochthonous tumors that develop in genetically engineered mouse model (KPC). Treatment with dual ET receptor antagonist Bosentan induced cell death in the autochthonous tumors, decreased IHC signal for extracellular matrix proteins (α-SMA, Collagen I, Fibronectin and CTGF). Transcriptomic analysis using fibrosis gene array indicated anti-fibrogenic effects of Bosentan in KPC tumors. Further, treatment of murine pancreatic stellate cells (PSCs) and human cancer associated fibroblasts (CAFs) with recombinant ET-1 in vitro induced the expression of pro-fibrotic genes was abrogated by selective inhibition of ETAR (BQ123) and ETBR (BQ788) signaling with synergistic effects observed with dual receptor inhibition. Further, ET-1 stimulation induced a significant increase in the p-ERK and p-AKT in a time dependent manner and dual receptor antagonist Bosentan significantly attenuated the ET-1 mediated induction. Our study also demonstrates that targeting ETAR with a specific inhibitor BQ123 enhances perfusion selectively in the tumor and reduces hypoxia in xenograft PC tumors. The third part of the dissertation describes a possible involvement of ET axis in inflammation associated pancreatic tumor progression in presence if mutated KrasG12D. The expression of ET axis components initially is restricted to pancreatic acinar and islet cell compartment in physiological conditions. However, during inflammation or injury the acinar expression is abrogated and is seen in early pre-cancerous lesions and neoplastic cells. The reprogramming of acinar phenotype into early pre-neoplastic lesions indicates an essential role of ET axis in pancreatic acinar to ductal metaplasia. This trans-differentiation is followed by excessive accumulation of ECM proteins and v inflammatory reaction in the pancreas, indicating further involvement of ET axis in influencing micro-environmental factors in initiation and progression of pancreatic cancer. The fourth part of the dissertation describes the generation of the mouse model aimed at delineating the role of ET-1 in PC progression. Genetically engineered mouse model of PC (K-rasG12D; Trp53R172H/+; Pdx-1-Cre) that harbors a Kras and p53 mutation in the pancreas were crossed with the ET-1 flox/flox mice. Taken together, studies in this dissertation demonstrate that ET axis plays a pleotropic role in the TME, and targeting ET axis can modulate the obstructive and immunosuppressive TME and make it potentially more amenable for chemotherapy and immunotherapy. vi TABLE OF CONTENTS 1A:Introduction................................................................................................................1 1. Synopsis……………………………………………………………………………………2 2. Introduction to Endothelins (ETs)………………………………………………………..3 3. The Endothelin system……………………………………………………………………5 4. Role of Endothelin(s) in inflammation………………………………………………….10 4.1 ETs in fibroblasts……………………………………………….……………..12 4.2 ETs in Immune system……………………………………………………….16 4.3 ETs in endothelial cells……………………………………………………….19 5. Endothelin in normal pancreas physiology……………………………………………22 5.1 Role in pancreatic islets………………………………………………………22 5.2 Role in acinar cells……………………………………………………………25 5.3 Role in pancreatic microcirculation………………………………………….26 6. Endothelin in pancreas pathophysiology………………………………………………27 6.1 Role in pancreatitis and inflammation………………………………………27 6.2 Role in pancreatic stellate cells and pancreas cancer……………………34 7. Conclusions and Future perspective…………………………………………………..36 1B: General Hypothesis and Objectives………………………………………………….71 1. Background and Rationale……………………………………………………………72 2. Hypothesis……………………………………………………………………………...73 3. Objectives………………………………………………………………………………73 Chapter 2: Materials and Methods…………………………………………………………76 1. Cell Culture……………………………………………………………………………..77 2. Tissue specimens……………………………………………………………………...77 vii 3. Immunohistochemistry and Immunofluorescence………………………………….78 4. RNA isolation, reverse transcription and RT- PCR analysis……………………...80 5. In vitro assays of cell migration………………………………………………………80 6. In vitro 2D co-culture…………………………………………………………………..81 7. In vitro assays of cell proliferation……………………………………………………82 8. Cell cycle analysis……………………………………………………………………..82 9. Annexin V staining and flow cytometry……………………………………………...82 10. Cytotoxicity assay using MTT………………………………………………………...83 11. Western blotting………………………………………………………………………..83 12. In vivo ET axis antagonism using Bosentan………………………………………..84 13. RNA extraction from mouse tissues, reverse transcription and real time PCR analysis…………………………………………………………………………………85 14. Microarray analysis……………………………………………………………………86 15. Perfusion analysis using BQ123……………………………………………………..86 16. Procurement of animals………………………………………………………………87 17. DNA isolation, genotyping and maintenance of animals…………………………..88 18. Statistical analysis……………………………………………………………………..88 Chapter 3: Expression of endothelin converting enzyme (ECE-1), endothelin-1 (ET- 1), endothelin A receptor (ETAR) and endothelin B receptor (ETBR) in pancreatic cancer and its microenvironment………………………………………………………….90 1. Synopsis………………………………………………………………………………..91 2. Background and Rationale……………………………………………………………92 3. Results………………………………………………………………………………….95 A. Endothelin-1 and its receptors are expressed in human pancreatic cancer cells…………………………………………………………………………………95 viii B. ET axis components are overexpressed in pancreatic cancer (PC) tissues..95 C. Elevated expression of ETBR on tumor blood vessels is correlated with patient survival……………………………………………………………………..97 D. Expression of ET axis in mouse progression model of PC…………………...98 E. Expression of ET axis in tumor microenvironment of human PC and mice KPC tissues………………………………………………………………………100 F. ET-1 and receptors correlate with tumor grade and stage in human TCGA database…………………………………………………………………………..101 4. Discussion…………………………………………………………………………….102 Chapter 4: Targeting Endothelin axis in pancreatic cancer using selective