Abstract

Simple SummaryPancreatic cancer is one of the deadliest solid malignancies. Pancreatic ductal adenocarcinoma accounts for 90% of pancreatic cancer cases with minimal response to traditional chemotherapies. Protein tyrosine kinases have been shown to be hyperactivated in cancers and thus can serve as therapeutic targets. Patient-derived tumor xenografts (PDXs) in animal models such as mice are an appropriate resource to identify such activated kinases. PDXs models are excellent for the identification of therapeutic targets as compared to cell line models as they better reflect an in vivo environment. We identified ephrin type-B receptor 4 (EphB4) as hyperactivated in PDXs derived from pancreatic ductal adenocarcinoma.Pancreatic ductal adenocarcinoma is a recalcitrant tumor with minimal response to conventional chemotherapeutic approaches. Oncogenic signaling by activated tyrosine kinases has been implicated in cancers resulting in activation of diverse effector signaling pathways. Thus, the discovery of aberrantly activated tyrosine kinases is of great interest in developing novel therapeutic strategies in the treatment and management of pancreatic cancer. Patient-derived tumor xenografts (PDXs) in mice serve as potentially valuable preclinical models as they maintain the histological and molecular heterogeneity of the original human tumor. Here, we employed high-resolution mass spectrometry combined with immunoaffinity purification using anti-phosphotyrosine antibodies to profile tyrosine phosphoproteome across 13 pancreatic ductal adenocarcinoma PDX models. This analysis resulted in the identification of 1199 tyrosine-phosphorylated sites mapping to 704 proteins. The mass spectrometric analysis revealed widespread and heterogeneous activation of both receptor and non-receptor tyrosine kinases. Preclinical studies confirmed ephrin type-B receptor 4 (EphB4) as a potential therapeutic target based on the efficacy of human serum albumin-conjugated soluble EphB4 in mice bearing orthotopic xenografts. Immunohistochemistry-based validation using tissue microarrays from 346 patients with PDAC showed significant expression of EphB4 in >70% of patients. In summary, we present a comprehensive landscape of tyrosine phosphoproteome with EphB4 as a promising therapeutic target in pancreatic ductal adenocarcinoma.

Highlights

  • Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of death from cancer in the USA and is predicted to be the second leading cause by the year2030 [1,2]

  • It is anticipated that tyrosine kinome would be heterogeneously activated as well and was confirmed by Western blot analysis of global tyrosine phosphorylation in a panel of 13 Patient-derived tumor xenografts (PDXs) (Figure 1A)

  • We identified 1199 unique tyrosine phosphorylation sites at least in one PDX sample corresponding to 704 proteins (Figure 2A)

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Summary

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of death from cancer in the USA and is predicted to be the second leading cause by the year2030 [1,2]. Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of death from cancer in the USA and is predicted to be the second leading cause by the year. The overall 5-year survival rate of PDAC patients from all stages of the disease combined is less than 10% and has not increased in the past five decades [3]. There are no effective therapies for PDAC that can cure or substantially increase survival, and only about 10%–20% of patients are considered candidates for surgery as most patients present with metastatic cancer. Gemcitabine chemotherapy in combination with the EGFR inhibitor erlotinib showed modest improvements in the overall survival rate [4]. Characterization of the underlying molecular mechanisms responsible for the development of PDAC may lead to the identification of novel therapeutic targets. Several genetic alterations, including KRAS, CDKN2A, TP53, SMAD4, and BRCA2, have been implicated in the pathogenesis of PDAC [5,6]

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