Cancer Letters

Cancer Letters

Volume 388, 1 March 2017, Pages 303-311
Cancer Letters

Original Article
ARF6, induced by mutant Kras, promotes proliferation and Warburg effect in pancreatic cancer

https://doi.org/10.1016/j.canlet.2016.12.014Get rights and content

Highlights

  • ARF6 predicts poor prognosis and plays oncogenic roles in pancreatic cancer cells.

  • ARF6 is responsible for maintaining the Kras-induced activation of ERK1/2.

  • ARF6 leads to enhancement of c-Myc expression and concomitant aerobic glycolysis.

  • ARF6 cooperates with Kras/ERK axis to sustain growth of pancreatic cancer cells.

  • ARF6 is a downstream target of c-Myc and forms a positive loop with c-Myc.

Abstract

Though significant progress has been made in the availability of diagnostic techniques and treatment strategies, pancreatic cancer remains a disease of high mortality rates. Therefore, there is an urgent need for a better understanding of the molecular mechanisms that governs the oncogenesis and metastasis process of pancreatic cancer. In our study, by using the Cancer Genome Atlas (TCGA) dataset analysis, we demonstrated that the small guanosine triphosphatase (GTPase) ADP-ribosylation factor 6 (ARF6) serves as a biomarker for predicting prognosis of pancreatic cancer. In vitro studies demonstrated that silencing ARF6 expression reduced cell proliferation and attenuated the Warburg effect. Moreover, we observed that ARF6 was a downstream target of Kras/ERK signaling pathway, and the strong correlation of expression between Kras and ARF6 in the TCGA dataset further confirmed this observation. Taken together, our novel findings suggest ARF6, a target of mutant Kras, may promote pancreatic cancer development by enhancing the Warburg effect.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with highly lethal rate, which is equal to incidence of the disease [1]. Only 15–20% of the patients with pancreatic cancer are able to perform surgery and the 5-year survival rate is disappointedly around 6% [2]. Thus there is an urgent need for a better understanding of the molecular mechanisms that underlying the oncogenesis and metastasis of pancreatic cancer. Over 90% of PDAC patients bear activation mutations in the Kras oncogene, and silencing Kras expression inhibits pancreatic cancer cell proliferation in vitro, these results suggest that Kras may be an ideal target for PDAC [3], [4], [5]. However, thirty years after its discovery, mutant Kras still poses a formidable challenge to researchers and clinicians alike, and attempts to directly target this protein have, so far, failed [6]. Until now, mutant Kras is considered to be an undruggable target, thus much efforts have been focused on the discovery of downstream signaling pathways with the hope of identifying novel targets for pancreatic cancer therapy.

One recent progress in Kras contribution to pancreatic cancer oncogenesis and progression is that Kras-induced metabolism reprogramming. It is well-accepted that metabolic reprogramming meets the demands of proliferative cancerous cells for the requirement of building blocks for macromolecule synthesis and energy production. Oncogenic Kras activity promoted upregulation of a series of key enzymes involved in glucose metabolism, including glycolysis, hexosamine biosynthesis and the pentose phosphate pathway. Thus it is generalized that Kras feeds uncontrolled proliferation of pancreatic cancer, and this presents a great challenge to directly target metabolic pathways as treatment targets [7].

The small GTPase ADP-ribosylation factor (ARF), which belongs to the Ras superfamily, was originally identified as a cofactor that promotes cholera toxin-catylyzed ADP-ribosylation of α-subunit of the heterotrimeric G protein Gs in the middle of 1980s [8], [9]. In mammals, the ARF family consists of 6 related gene products, ARF1-6, that fall into 3 classes based on their sequence homology, including Class I (ARF1-3), Class II (ARF4-5) and Class III (ARF6). Classes I and II ARFs are mainly localized at the Golgi and endoplasmic reticulum, and participated in the regulation of vesicular trafficking between intracellular organelles [10], [11]. However, ARF6 is localized to the plasma membrane and several endosomes. Besides its important roles in membrane trafficking, ARF6 also regulates membrane associated pathological processes such as membrane ruffle formation, neurite outgrowth and cell migration and invasion [12], [13], [14]. Clinically, robust expression of ARF6 and activation of its downstream signaling pathways have been observed in several tumor types and are related to poor overall survival, such as breast cancer, lung adenocarcinoma and head and neck cancers [15], [16], [17], [18]. However, the function of ARF in pancreatic cancer and its correlation of expression with Kras have seldom been discussed before.

In the present study, we sought to uncover the function of ARF6 in pancreatic cancer and explain the possible underlying mechanisms. Our study provided novel findings of Kras/ARF6 axis in contributing to pancreatic cancer tumorigenesis, shedding light on the molecular mechanism underlying its function and provided novel predictive and treatment targets for pancreatic cancer.

Section snippets

Cell culture

The human pancreatic cancer cell lines PANC-1 and MiaPaCa-2 were obtained from ATCC and cultured according to standard ATCC protocols. iKras cell lines, which expresses KrasG12D upon doxycycline (Doxy) treatment was generously provided by Professor Paul J.Chiao from MD Aderson Cancer Center. In brief, PANC-1 and iKras cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM), containing fetal bovine serum (FBS) in a final concentration of 10%. MiaPaCa-2 cells were cultured in DMEM

ARF6 is a prognostic factor for pancreatic cancer

In order to assess the function of ARF family members in pancreatic cancer, we firstly analyzed the prognostic values of ARF1, ARF3, ARF4, ARF5 and ARF6 in pancreatic cancer by using the TCGA dataset. Our results demonstrated among these ARF family proteins, only ARF6 was a predictive marker for pancreatic cancer and higher expression of ARF6 predicted worse overall survival (Fig. 1A–E). The correlation of ARF6 expression with clinicopathological characteristics was shown in Supplementary

Discussion

Malignant progression from pancreatic intraepithelial neoplasia (PanINs) to invasive and metastatic disease is accompanied by the early acquisition of activating mutations in the Kras oncogene, which occurs in >90% of cases, and subsequent loss of tumor suppressors including INK4A/ARF, TP53 and SMAD4 [23], [24]. Constitutive KrasG12D activation drives uncontrolled proliferation and enhances survival of cancer cells through its downstream signaling pathways, such as the Ras-Raf-MEK-ERK and

Acknowledgements

This work was supported by the National Science Fund for Distinguished Young Scholars [grant number 81625016]. This work was also supported by the National Natural Science Foundation [grant numbers 81372651, 81502031 and 81602085]; the Sino-German Center [grant number GZ857], Ph.D. Programs of the Foundation of the Ministry of Education of China [grant number 20120071120104], the Program of Science and Technology Commission of Shanghai [grant numbers 13431900105 and 13DZ1942802] and Shanghai

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