Graduation Date

Spring 5-6-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Dr. Maneesh Jain

Abstract

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. 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 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.

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