TPH1 and 5-HT7 Receptor Overexpression Leading to Gemcitabine-Resistance Requires Non-Canonical Permissive Action of EZH2 in Pancreatic Ductal Adenocarcinoma.

Prakash Chaudhary,Jae-Hoon Chang,Jung-Ae Kim,Diwakar Guragain

doi:10.3390/cancers13215305

Abstract

Simple SummaryMost patients with pancreatic cancer initially respond to the first-choice drug gemcitabine, but the cancer cells rapidly acquire drug resistance, resulting in poor survival. In this study, we investigated whether the serotonin (5-hydroxytryptamine, 5-HT) system plays an important role in gemcitabine resistance and the maintenance of pancreatic cancer stem cells (CSCs) in association with an Enhancer of zeste homolog 2 (EZH2), an epigenetic regulator of transcription. Herein, we demonstrate that long-term exposure of PDAC cells to 5-HT leads to enhanced EZH2 expression which, in turn, allows upregulation of TPH1 and 5-HT7, resulting in EZH2-TPH1-5-HT7 axis operating in a feed-forward manner. The results suggest that the EZH2-TPH1-5-HT7 axis may be a highly efficient therapeutic target against drug-resistant pancreatic ductal adenocarcinoma (PDAC).In the present study, we investigated the regulatory mechanisms underlying overexpression of EZH2, tryptophan hydroxylase 1 (TPH1), and 5-HT7, in relation to gemcitabine resistance and CSC survival in PDAC cells. In aggressive PANC-1 and MIA PaCa-2 cells, knock-down (KD) of EZH2, TPH1, or HTR7 induced a decrease in CSCs and recovery from gemcitabine resistance, while preconditioning of less aggressive Capan-1 cells with 5-HT induced gemcitabine resistance with increased expression of EZH2, TPH1, and 5-HT7. Such effects of the gene KD and 5-HT treatment were mediated through PI3K/Akt and JAK2/STAT3 signaling pathways. EZH2 KD or GSK-126 (an EZH2 inhibitor) inhibited activities of these signaling pathways which altered nuclear level of NF-kB, Sp1, and p-STAT3, accompanied by downregulation of TPH1 and 5-HT7. Co-immunoprecipation with EZH2 and pan-methyl lysine antibodies revealed that auto-methylated EZH2 served as a scaffold for binding with methylated NF-kB and Sp1 as well as unmethylated p-STAT3. Furthermore, the inhibitor of EZH2, TPH1, or 5-HT7 effectively regressed pancreatic tumor growth in a xenografted mouse tumor model. Overall, the results revealed that long-term exposure to 5-HT upregulated EZH2, and the noncanonical action of EZH2 allowed the expression of TPH1-5-HT7 axis leading to gemcitabine resistance and CSC population in PDAC.

Highlights

Pancreatic ductal adenocarcinoma (PDAC) accounts for over 90% of pancreatic cancer cases worldwide and is one of the most aggressive human malignancies
Antibodies against p-phosphoinositide 3kinases (PI3Ks), PI3K, p-Akt, Akt, p-JAK2, JAK2, p-STAT3, STAT3, Enhancer of zeste homolog 2 (EZH2), and NF-κB P65 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA); 5-HT1A, 5-HT1B, Nanog, CD44, Sp1, and EZH1 were from Abcam (Cambridge, MA, USA); tryptophan hydroxylase 1 (TPH1) from Invitrogen (Carlsbad, CA, USA); β-Actin and Lamin B from Santa Cruz Biotechnology (Dallas, Texas, USA)
Corresponding to gemcitabine resistance (GemR) levels, the level of EZH2, TPH1 and 5-HT7 receptor expressions was the highest in PANC-1, followed by MIA PaCa-2, Capan-1, and Capan-2 (Figure 1B and Figure S1A), whereas EZH1, 5-HT1A, and 5-HT1B levels did not correlate with GemR levels, and they were higher in Capan-1 and Capan-2 cells than PANC-1 and MIA PaCa-2 cells (Figure 1B)

Summary

Introduction

Pancreatic ductal adenocarcinoma (PDAC) accounts for over 90% of pancreatic cancer cases worldwide and is one of the most aggressive human malignancies. The activating mutation in KRAS is present in 80–90% of pancreatic cancers [4], and this mutation results in overactivation of various signaling molecules, such as Raf/MEK/extracellular signal-regulated kinase (ERK) and phosphoinositide 3kinases (PI3Ks)/Akt [5]. These signaling molecules mediate early distant metastasis, resistance to conventional chemotherapy [6,7], and development of pancreatic cancer stem cell (CSC) characteristics [8]. The epigenetic alterations are responsible for over-activation of the growth signaling pathway and the silencing of tumor suppressor and cell cycle checkpoint genes in PDAC [10,11]

Methods

Results

Discussion

Conclusion