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

Cancer Letters 388 (2017) 303e311

Contents lists available at ScienceDirect

Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Original Article

ARF6, induced by mutant Kras, promotes proliferation and Warburg effect in pancreatic cancer Chen Liang a, b, c, 1, Yi Qin a, b, c, 1, Bo Zhang a, b, c, 1, Shunrong Ji a, b, c, Si Shi a, b, c, Wenyan Xu a, b, c, Jiang Liu a, b, c, Jinfeng Xiang a, b, c, Dingkong Liang a, b, c, Qiangsheng Hu a, b, c, Quanxing Ni a, b, c, Xianjun Yu a, b, c, *, Jin Xu a, b, c, ** a b c

Department of Pancreatic Surgery, Fudan University Shanghai Cancer Center, Shanghai 200032, China Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China Pancreatic Cancer Institute, Fudan University, Shanghai 200032, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 October 2016 Received in revised form 12 December 2016 Accepted 12 December 2016

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. © 2016 Elsevier Ireland Ltd. All rights reserved.

Keywords: ADP-ribosylation factor 6 Kras/ERK axis Warburg effect c-Myc

Introduction Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with highly lethal rate, which is equal to incidence of the disease [1]. Only 15e20% 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 [3e5]. However, thirty years after its discovery, mutant Kras still poses a formidable challenge to researchers and clinicians

* Corresponding author. Department of Pancreatic Surgery, Fudan University Shanghai Cancer Center, Shanghai 200032, China. Fax: þ86 21 64031446. ** Corresponding author. Department of Pancreatic Surgery, Fudan University Shanghai Cancer Center, Shanghai 200032, China. Fax: þ86 21 64031446. E-mail addresses: [email protected] (X. Yu), [email protected] (J. Xu). 1 These authors contributed equally to this article. http://dx.doi.org/10.1016/j.canlet.2016.12.014 0304-3835/© 2016 Elsevier Ireland Ltd. All rights reserved.

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 a-subunit of the heterotrimeric G protein Gs in the middle of 1980s [8,9]. In

304

C. Liang et al. / Cancer Letters 388 (2017) 303e311

Fig. 1. ARF6 is a prognostic factor for pancreatic cancer. (AeE) KaplaneMeier analysis of the correlation between the ARF1, ARF3, ARF4, ARF5 and ARF6 levels and overall survival of pancreatic cancer patients with high (n ¼ 80) and low (n ¼ 80) ARF6 expression in the TCGA cohort. (F) Immunohistochemical analysis for the tissue ARF6 expression in the FUSCC cohort. (G) High level of tissue ARF6, as describe in (F), predicts a poor prognosis to pancreatic cancer in the FUSCC cohort.

mammals, the ARF family consists of 6 related gene products, ARF16, 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 [12e14]. 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 [15e18]. 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. Materials and method

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 medium, with FBS concentration of 10% and horse serum in a concentration of 2.5%. RNA isolation and quantitative real-time PCR Total RNA was prepared by using TRIzol reagent (Invitrogen, USA). Quantitative real-time PCR was performed as described previously [19]. Primers sequences are listed as Supplementary Table 1. Western blot analysis Western blot was performed as described previously [19]. b-Actin, c-Myc, GLUT1 (glucose transporter 1), HK2 (hexokinase 2) and LDHA (Lactate dehydrogenase A) and Kras antibodies were purchased from Proteintech. Antibodies against ARF6, p44/42 MAPK (ERK1/2), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), p44 MAP kinase (ERK1) and p42 MAP kinase (ERK2) were purchased from Cell Signaling Technology. Lentivirus production and stable cell line selection In order to generate shRNA expression constructs against ARF6, pLKO.1 TRC cloning vector (Addgene plamid: 10878) was employed [20]. 21bp targets against ARF6 were 50 -GCTCACATGGTTAACCTCTAA-30 and 50 -AGCTGCACCGCATTATCAATG30 , respectively. Lentiviral particles were produce by co-transfection of pLKO.1shARF6 constructs with psPAX2 and pMD2.G into HEK-293T cells in a ratio of 4:3:1. Cell lines were obtained by infection of PANC-1 and MiaPaCa-2 cells with lentiviral particles followed by puromycin selection.

Cell culture

CCK-8 proliferation assay

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

Cell proliferation was determined by CCK-8 assays by using CCK-8 reagents (Dojindo, Japan) and performed according to the manufacturer's protocol.

C. Liang et al. / Cancer Letters 388 (2017) 303e311

305

Fig. 2. ARF6 contributes to proliferation of pancreatic cancer cells. (AeB) The shRNA against ARF6 were transfected into the PANC-1 and MiaPaCa-2 cells for 48 h to examine the decrease of ARF6 mRNA and protein. (CeD) Silencing ARF6 expression, as indicated in (AeB), decreased proliferation of PANC-1 and MiaPaCa-2 cells reflected by CCK-8 proliferation assay. (EeF) Decreased ARF6 expression inhibited cloning formation capacity of PANC-1 and MiaPaCa-2 cells.

Colony-formation assay

Analysis of promoter activity with dual luciferase assay

PANC-1 and MiaPaCa-2 cells (5  102) stably expressing shRNA targets against ARF6 and its relative control cells were seeded. After cultivating for 10 days, 4% paraformaldehyde was used to fix the cells followed by staining with 1% crystal violet. The colonies were counted subsequently.

Promoters of human ARF6 promoter regions, spanning from 2000 to 300 of the transcription starting site, were cloned into pGL3-Basic vector. Cells were plated on 96-well culture plates and transfected with pGL3 constructs and Renilla luciferase expression vectors by using Lipofectamine™ 2000 (Invitrogen). Next, the cells were assayed for both firefly and Renilla luciferase activities using a dual-luciferase system (Promega), as described in the manufacturer's protocol.

Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) Cellular glycolytic capacity and mitochondrial function were measured by using the Seahorse Bioscience XF96 Extracellular Flux Analyzer, all according to the manufacturer's instructions of seahorse XF Glycolysis Stress Test Kit and Cell Mito stress test kit [21]. TCGA dataset analysis TCGA-PAAD on RNA expression (Level 3) of pancreatic cancer patients in terms of RNA-seq by Expectation-Maximization was downloaded from the Cancer Genomics Brower of the University of California, Santa Cruz (UCSC) (https://genomecancer.ucsc.edu/). In total, 160 primary pancreatic cancer samples from patients with detailed expression data were chosen from the updated TCGA database according to parameters mentioned. Detailed demographics of these patients were characterized by the TCGA consortium. Immunohistochemical staining (IHC) Immunohistochemical staining of paraffin-embedded tissues with antibodies for c-Myc and ARF6 were performed and scored to determine the proteins expression according to standard procedures described previously [22].

Chromatin immunoprecipitation (ChIP) assay ChIP was performed according to the instructions of the Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (Merck Millipore Corporation). The nuclear DNA extracts were amplified using two pairs of primers that spanned the ARF6 promoter region.

Statistical analyses Statistical analyses were performed by SPSS software (version 17.0, IBM Corp., Armonk, NY, USA) using independent Student's t-test (two-tailed) or one-way analysis of variance (ANOVA). Logistic regression was used to determine the correlation between ARF6, Kras, GLUT1, HK2 and LDHA expression level and clinicopathological characteristics in the TCGA cohorts. Statistical significance was based on two-sided P values of <0.05.

306

C. Liang et al. / Cancer Letters 388 (2017) 303e311

Fig. 3. Kras/ERK axis induces ARF6 expression in the pancreatic cancer cells. (AeC) The 2 mg of siRNA against Kras were transfected into the PANC-1 and MiaPaCa-2 cells for 48 h to examine the decrease of ARF6 mRNA and protein. (D) ARF6 expression was decreased in a dose-dependent manner by treatment with UO126 in pancreatic cancer cells. (E) UO126 decreased the phosphorylation level of ERK and ARF6 protein expression. (FeG) KrasG12D induction by Doxy increased ARF6 mRNA and protein levels. (H) UO126 treatment reversed the Kras-induced upregulation of ARF6. (I) Silencing ERK1 or ERK2 expression reversed the Kras-induced upregulation of ARF6. (J) KaplaneMeier analysis of the correlation between the Kras level and overall survival of pancreatic cancer patients with high (n ¼ 80) and low (n ¼ 80) Kras expression in the TCGA cohort. (K) Pearson analysis of the correlation between the levels of ARF6 and Kras in the TCGA cohort.

Results 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. 1AeE). The correlation of ARF6 expression with clinicopathological characteristics was shown in Supplementary Table 2. To further validate this observation, we performed IHC staining to

examine the ARF6 expression in PDAC tissue microarray from Fudan University Shanghai Cancer Center (FUSCC) as a validation cohort (Fig. 1F). Consistent with the results from the TCGA cohort, higher levels of ARF6 significantly correlated with poor survival (Fig. 1G). ARF6 contributes to proliferation of pancreatic cancer cells To further confirm the function of ARF6 in tumor proliferation, we firstly generated PANC-1 and MiaPaCa-2 cell lines that stably expressing shRNAs against ARF6, and the silencing effect was validated by using real time PCR and immunoblot (Fig. 2A and B). Then we performed the CCK-8 proliferation assays to validate the function of ARF6 in cell viability. As observed, silencing ARF6

C. Liang et al. / Cancer Letters 388 (2017) 303e311

307

Fig. 4. ARF6 maintains Warburg effect by ERK/c-Myc axis. (A) Silencing ARF6 expression inhibited the activation of ERK1/2 in PANC-1 and MiaPaCa-2 cells. (B) Silencing ARF6 expression decreased c-Myc levels in PANC-1 and MiaPaCa-2 cells. (CeD) Silencing ARF6 expression inhibited the glycolytic capacity of PANC-1 and MiaPaCa-2 cells, as reflected by ECAR analysis. (EeF) Silencing ARF6 expression increased mitochondrial respiration in PANC-1 and MiaPaCa-2 cells. (G) Silencing ARF6 expression inhibited mRNA expression of GLUT1. (H) Silencing ARF6 expression inhibited mRNA expression of HK2. (I) Silencing ARF6 expression inhibited mRNA expression of LDHA. (J) Silencing ARF6 expression decreased the protein levels of GLUT1, HK2 and LDHA in PANC-1 and MiaPaCa-2 cells.

significantly decreased cell viability, indicating its role in cell viability maintenance in PANC-1 and MiaPaCa-2 cells (Fig. 2C and D). Furthermore, we performed clone formation assay, and observed that silencing ARF6 expression significantly inhibited clone formation capacity of PANC-1 and MiaPaCa-2 cells (Fig. 2E and F). ARF6 is a downstream target of Kras Due to the decisive role of Kras mutation in pancreatic cancer oncogenesis and progression, we asked whether ARF6 was a target

and regulated by Kras in pancreatic cancer. In PANC-1 and MiaPaCa2 cells, we silenced Kras expression and observed that expression of ARF6 was decreased accordingly (Fig. 3AeC). Ras-Raf-MEK-ERK pathway is a decisive pathway that governs pancreatic cancer oncogenesis and progression. To test whether ARF6 expression was regulated by Kras-activated ERK, we inhibited ERK activity by treatment of cells with kinase inhibitor UO126. As observed, inhibition of ERK activity decreased ARF6 in transcriptional and protein levels (Fig. 3D and E). To further confirm the role of Kras in ARF6 expressional regulation, we used an inducible KrasG12D expression cell line, namely iKras cells. Upon KrasG12D expression, ARF6

308

C. Liang et al. / Cancer Letters 388 (2017) 303e311

Fig. 5. Clinical significance of glycolytic genes and their correlations with ARF6 in pancreatic cancer patients. (A) Pearson analysis of the correlation between the levels of ARF6 and GLUT1 expression in the TCGA cohort. (B) Pearson analysis of the correlation between the levels of ARF6 and HK2 expression in the TCGA cohort. (C) Pearson analysis of the correlation between the levels of ARF6 and LDHA expression in the TCGA cohort. (DeF) The expression data were divided into High expression group (n ¼ 80) and low expression group (n ¼ 80) based on the median. KaplaneMeier analysis of the correlation between the GLUT1, HK2 and LDHA levels and overall survival of pancreatic cancer patients in the TCGA cohort. (GeI) Survival analysis by combination of ARF6 with GLUT1, HK2 and LDHA.

expression was upregulated, and the process could be reversed by treatment with UO126 (Fig. 3FeH). Moreover, we silenced ERK1 and ERK2 expression in iKras cells (Supplementary Fig. 1A and 1B) and demonstrated that ERK1 or ERK2 silencing attenuated Krasinduced ARF6 expression (Fig. 3I). In the end, we discussed the expressional correlation between Kras and ARF6 by analysis of their expression status in the TCGA dataset. In consistent with previous studies, higher Kras expression predicted worse prognosis of pancreatic cancer patients (Fig. 3J). Moreover, there was a strong correlation between Kras and ARF6 expression, and these results further supported our in vitro observations that ARF6 was correlated with Kras (Fig. 3K). ARF6 maintains Warburg effect via ERK/c-Myc axis To test whether ARF6 is required for activation of ERK activation, we examined the status of activated ERK1/2 in PANC-1 and MiaPaCa-2 cells with ARF6 knockdown. As observed, silencing ARF6 inhibited activation of ERK1/2, suggesting its role in Ras-RafMEK-ERK signaling activation (Fig. 4A). c-Myc is a well-established downstream effector of the Ras-Raf-MEK-ERK signaling pathway, and as indicated in our results, the protein level of c-Myc decreased in ARF6 knockdown PANC-1 and MiaPaCa-2 cells (Fig. 4B). Some most recent studies validated that c-Myc is primarily responsible for the metabolic reprogramming. Thus, we examined the change

in aerobic glycolysis in ARF6-silenced PANC-1 and MiaPaCa-2 cells by using the Seahorse XF analyzers. The extracellular acidification rate (ECAR) significantly decreased in ARF6 knockdown cells, indicating that silencing ARF6 inhibited glycolytic process in pancreatic cancer cells (Fig. 4C and D). Oxygen consumption rate (OCR) value reflects glucose mitochondrial oxidation. In consistent with the ECAR results, silencing ARF6 promoted OCR in pancreatic cancer cells, suggesting its role in mitochondrial respiration inhibition (Fig. 4E and F). The Warburg effect, or aerobic glycolysis, is accompanied by the activation of a series of glycolytic genes and among them, GLUT1, HK2 and LDHA plays important roles for metabolize glucose into lactate. The oncogene c-Myc is a transcription factor that regulated the expression of GLUT1, HK2 and LDHA. As expected, silencing ARF6 expression decreased GLUT1, HK2 and LDHA expression in mRNA level in PANC-1 and MiaPaCa-2 cells (Fig. 4GeI). The results were further validated by detecting the protein level, reinforcing the contribution of ARF6 to Warburg effect maintenance (Fig. 4J). Clinical significance of GLUT1, HK2 and LDHA and their correlations with ARF6 in pancreatic cancer patients Based on the above observations, we examined the clinical significance of GLUT1, HK2 and LDHA in prognosis in pancreatic cancer by analysis of the TCGA dataset. The clinicopathological

C. Liang et al. / Cancer Letters 388 (2017) 303e311

309

Fig. 6. c-Myc was involved in the ARF6 transcriptional expression in PDAC. (A) Silencing c-Myc decreased the ARF6 mRNA expression in PANC-1 and MiaPaCa-2 cells. (B) Silencing c-Myc decreased the ARF6 protein expression in PANC-1 and MiaPaCa-2 cells. (C) The position of the c-Mycebinding site in the human ARF6 promoter. (D) c-Myc occupies the binding sites of the ARF6 promoter region in HEK-293T cell, as measured by ChIP assay. (E) c-Myc affected ARF6 promoter activity in HEK-293T cells. (F) The correlation between ARF6 and c-Myc expression derived from clinical specimen.

characteristics with expression of GLUT1, HK2 and LDHA were shown in Supplementary Table 3, 4 and 5. Moreover, ARF6 showed a strong correlation between GLUT1, HK2 and LDHA in pancreatic cancer patients, respectively, further supported our in vitro observations (Fig. 5AeC). Furthermore, higher expressions of GLUT1, HK2 and LDHA predicted worse prognosis of pancreatic cancer (Fig. 5DeF). In the end, to effectively predict prognosis of pancreatic cancer, we combined expression status of ARF6 with GLUT1, HK2 and LDHA, respectively. As illustrated, patient groups of ARF6High/ GLUT1High, ARF6High/HK2High and ARF6High/LDHAHigh exhibited shorter survivals (Fig. 5GeI).

c-Myc activates the ARF6 transcription in pancreatic cancer cells To explore the role of c-Myc in ARF6 expression, we silenced cMyc and found ARF6 was significantly lowered expression in PANC1 and MiaPaCa-2 cells (Fig. 6A and B). Furthermore, we analyzed the promoter region of ARF6, and found two putative c-Myc binding sites (Fig. 6C), and performed ChIP assay to demonstrate that c-Myc was enriched in the two binding sites in the promoter

region (Fig. 6D). Moreover, manipulation of c-Myc expression altered ARF6 promoter activity by using luciferase assays (Fig. 6E). In supporting the clues obtained from in vitro experiments with cell lines, we analyzed the expressional correlation between tissue cMyc and ARF6 levels in pancreatic cancer samples. We observed that c-Myc level strongly correlated with ARF6 levels in pancreatic cancer tissue samples (Fig. 6F).

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 PI3K-Akt-mTOR pathways. In the past decades, continuous efforts have been given to target Kras and exploring possible strategies to inhibit this

310

C. Liang et al. / Cancer Letters 388 (2017) 303e311

Fig. 7. Schematic representation of the model, indicating the mechanism of ARF6mediated regulation of glucose metabolism via the ERK/c-Myc axis in pancreatic cancer cells.

decisive oncogene, however, no effective targets has been obtained and Kras is still considered to be an undruggable target. To improve the poor overall survival rate of pancreatic cancer, it is urgent to explore the underlying mechanism and downstream effectors of the constitutive activation of the Kras proto-oncogene. We explore The Cancer Genome Atlas with the aim to find novel Kras effectors and uncover the possible mechanism. The ADP-ribosylation factor (ARF) family of proteins belongs to the Ras superfamily of small GTPases. Like other Ras-related GTPbinding proteins, the ARF proteins cycle between their active-GTPbound and inactive-GDP-bound conformations. Hydrolysis of bound GTP is mediated by GTPase-activating proteins (GAPs), whereas the exchange of GDP for tri-phosphate nucleotide is mediated by guanine nucleotide-exchange factors (GEFs) [11,25]. ARF family consists of 6 members that could be divided into 3 classes. Among them, ARF6 received more attention due to its subcellular distribution and diverse cellular functions. Moreover, the co-localization between ARF6 and Ras evoked us to explore the contribution of ARF6 to Kras induced oncogenesis and progression in pancreatic cancer [26]. As exhibited in our results, ARF6 was a downstream target of Kras and maintains Kras-induced ERK activation. Moreover, silencing ARF6 reduced the plasma membrane presence of Ras and inhibited the phosphorylation of ERK [26], which is consistent with our results. Thus, ARF6 might be involved in the Kras/ERK signaling, which prompted us to further uncover the underlying molecular mechanism of ARF6 in pancreatic cancer. Previous study has identified the integrin trafficking pathway in which endocytosed integrins are transported from recycling endosomes to trans-Golgi network before being recycled to the plasma membrane [27]. This integrin trafficking played key roles in various cellular processes including migration, proliferation and survival [27]. Since ARF6 is an important protein involved in the regulation of trafficking [10,11], ARF6 could promote the proliferation of cancer cells by endocytosis and recycling of some membrane receptors. Moreover, it is evident that in order to fuel Kras-

induced proliferation, cancer cells require both sufficient ATP supply and biosynthetic precursors as cellular building blocks. Thus Kras-induced metabolism reprogramming is a requirement for uncontrolled proliferation and malignant properties maintenance [28,29]. In our study, we explored the function of ARF6 in pancreatic cancer cell metabolism. As reported in our work, silencing ARF6 inhibited the Warburg effect in Kras mutated PANC-1 and MiaPaCa-2 cells, indicating that ARF6 is an important regulator of Kras induced aerobic glycolysis. The contribution of ARF6 to Warburg effect maintenance has never been reported before, thus we sought to uncover the molecular mechanism underlying ARF6 in glycolysis regulation. In our work, we demonstrated that ARF6 is important for ERK activation. Our previous studies demonstrated that ERK was an important player in proto-oncogene c-Myc activation, and in Kras mutated pancreatic cancer cells, inhibition of ERK kinase resulted in change of c-Myc protein level [22]. In consistent with this observation, we observed a downregulation of c-Myc in ARF6 knockdown pancreatic cancer cells. This may account for ARF6 in aerobic glycolysis control, as c-Myc is an important regulator in cancer cell metabolism [30,31]. c-Myc regulated the aerobic glycolysis via upregulation of many key glycolytic genes GLUT1, HK2 and LDHA [32e34]. The similar conclusions were also validated in our observation, since the expression status of GLUT1, HK2 and LDHA were also decreased in pancreatic cancer cells with ARF6 knockdown. Thus, in line with these in vitro observations, the clinical significance of ARF6 in pancreatic cancer became more affirmative. However, there are still some questions that need to be answered. For example, how RasRaf-MEK-ERK signaling pathway regulates the expression of ARF6, what is the transcriptional factor that accounted for ARF6 expression, these questions need to be explored in future research. Moreover, ARF6 is a small GTPase, and whether its enzymatic activity is required for the maintenance of Warburg effect and the underlying molecular mechanism needed further investigations. Answers to these unresolved questions will help to develop novel target and strategies in intervention of ARF6. Taken together, ARF6 was robustly upregulated in pancreatic cancer by oncogenic Kras activation. Mechanistically, ARF6 is responsible for constitutive activation of ERK1/2 and downstream effector c-Myc, which regulated the Warburg effect that meets the continuous energy and nutrient demand for uncontrolled proliferation (Fig. 7). We, moreover, provided evidence supporting that high expression levels of ARF6, as well as glycolytic genes, including GLUT1, HK2 and LDHA, are tightly correlated with the poor overall survival of patients. These results will help to uncover new predictive and treatment targets for pancreatic cancer.

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 SinoGerman 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 Sailing Program [grant number 16YF1401800].

Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.

C. Liang et al. / Cancer Letters 388 (2017) 303e311

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.canlet.2016.12.014. References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2015, CA Cancer J. Clin. 65 (2015) 5e29. [2] D.P. Ryan, T.S. Hong, N. Bardeesy, Pancreatic adenocarcinoma, N. Engl. J. Med. 371 (2014) 1039e1049. [3] H. Ying, P. Dey, W. Yao, A.C. Kimmelman, G.F. Draetta, A. Maitra, et al., Genetics and biology of pancreatic ductal adenocarcinoma, Genes Dev. 30 (2016) 355e385. [4] A.F. Hezel, A.C. Kimmelman, B.Z. Stanger, N. Bardeesy, R.A. Depinho, Genetics and biology of pancreatic ductal adenocarcinoma, Genes Dev. 20 (2006) 1218e1249. [5] J.J. Yeh, C.J. Der, Targeting signal transduction in pancreatic cancer treatment, Expert Opin. Ther. Targets 11 (2007) 673e694. [6] M. Russo, F. Di Nicolantonio, A. Bardelli, Climbing RAS, the everest of oncogenes, Cancer Discov. 4 (2014) 19e21. [7] C. Liang, Y. Qin, B. Zhang, S. Ji, S. Shi, W. Xu, et al., Energy Sources Identify Metabolic Phenotypes in Pancreatic Cancer, Acta Biochim Biophys Sin, Shanghai, 2016. [8] R.A. Kahn, A.G. Gilman, The protein cofactor necessary for ADP-ribosylation of Gs by cholera toxin is itself a GTP binding protein, J. Biol. Chem. 261 (1986) 7906e7911. [9] A.K. Gillingham, S. Munro, The small G proteins of the Arf family and their regulators, Annu. Rev. Cell Dev. Biol. 23 (2007) 579e611. [10] T. Hongu, Y. Yamauchi, Y. Funakoshi, N. Katagiri, N. Ohbayashi, Y. Kanaho, Pathological functions of the small GTPase Arf6 in cancer progression: tumor angiogenesis and metastasis, Small GTPases 7 (2016) 47e53. [11] C. D'Souza-Schorey, P. Chavrier, ARF proteins: roles in membrane traffic and beyond, Nat. Rev. Mol. Cell Biol. 7 (2006) 347e358. [12] A. Honda, M. Nogami, T. Yokozeki, M. Yamazaki, H. Nakamura, H. Watanabe, et al., Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation, Cell. 99 (1999) 521e532. [13] J. Jaworski, ARF6 in the nervous system, Eur. J. Cell Biol. 86 (2007) 513e524. [14] H. Sabe, Requirement for Arf6 in cell adhesion, migration, and cancer cell invasion, J. Biochem. 134 (2003) 485e489. [15] Y. Onodera, S. Hashimoto, A. Hashimoto, M. Morishige, Y. Mazaki, A. Yamada, et al., Expression of AMAP1, an ArfGAP, provides novel targets to inhibit breast cancer invasive activities, EMBO J. 24 (2005) 963e973. [16] M. Morishige, S. Hashimoto, E. Ogawa, Y. Toda, H. Kotani, M. Hirose, et al., GEP100 links epidermal growth factor receptor signalling to Arf6 activation to induce breast cancer invasion, Nat. Cell Biol. 10 (2008) 85e92. [17] T. Menju, S. Hashimoto, A. Hashimoto, Y. Otsuka, H. Handa, E. Ogawa, et al., Engagement of overexpressed Her2 with GEP100 induces autonomous

[18]

[19]

[20]

[21]

[22]

[23]

[24] [25] [26] [27]

[28] [29]

[30] [31] [32]

[33]

[34]

311

invasive activities and provides a biomarker for metastases of lung adenocarcinoma, PLoS One 6 (2011) e25301. H. Sato, K.C. Hatanaka, Y. Hatanaka, H. Hatakeyama, A. Hashimoto, Y. Matsuno, et al., High level expression of AMAP1 protein correlates with poor prognosis and survival after surgery of head and neck squamous cell carcinoma patients, Cell Commun. Signal. 12 (2014) 17. S. Ji, Y. Qin, C. Liang, R. Huang, S. Shi, J. Liu, et al., FBW7 (F-box and WD repeat domain-containing 7) negatively regulates glucose metabolism by targeting the c-Myc/TXNIP (thioredoxin-binding protein) axis in pancreatic cancer, Clin. Cancer Res. (2016). J. Moffat, D.A. Grueneberg, X. Yang, S.Y. Kim, A.M. Kloepfer, G. Hinkle, et al., A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen, Cell. 124 (2006) 1283e1298. T.N. Pathiraja, K.N. Thakkar, S. Jiang, S. Stratton, Z. Liu, M. Gagea, et al., TRIM24 links glucose metabolism with transformation of human mammary epithelial cells, Oncogene 34 (2015) 2836e2845. S. Ji, Y. Qin, S. Shi, X. Liu, H. Hu, H. Zhou, et al., ERK kinase phosphorylates and destabilizes the tumor suppressor FBW7 in pancreatic cancer, Cell Res. 25 (2015) 561e573. H. Ying, A.C. Kimmelman, C.A. Lyssiotis, S. Hua, G.C. Chu, E. Fletcher-Sananikone, et al., Oncogenic Kras maintains pancreatic tumors through regulation of anabolic glucose metabolism, Cell. 149 (2012) 656e670. M. Hidalgo, New insights into pancreatic cancer biology, Ann. Oncol. 23 (Suppl. 10) (2012) x135e138. P. Chavrier, B. Goud, The role of ARF and Rab GTPases in membrane transport, Curr. Opin. Cell Biol. 11 (1999) 466e475. C.G. Xie, S.M. Wei, J.T. Cai, K-Ras resides on the Arf6-mediated CIE system and its active type interacted with Arf6T27N, Cell. Signal. 24 (2012) 524e531. K.A. Riggs, N. Hasan, D. Humphrey, C. Raleigh, C. Nevitt, D. Corbin, et al., Regulation of integrin endocytic recycling and chemotactic cell migration by syntaxin 6 and VAMP3 interaction, J. Cell Sci. 125 (2012) 3827e3839. R.A. Cairns, I.S. Harris, T.W. Mak, Regulation of cancer cell metabolism, Nat. Rev. Cancer 11 (2011) 85e95. Y. Hu, W. Lu, G. Chen, P. Wang, Z. Chen, Y. Zhou, et al., K-ras(G12V) transformation leads to mitochondrial dysfunction and a metabolic switch from oxidative phosphorylation to glycolysis, Cell Res. 22 (2012) 399e412. Z.E. Stine, Z.E. Walton, B.J. Altman, A.L. Hsieh, C.V. Dang, MYC, metabolism, and cancer, Cancer Discov. 5 (2015) 1024e1039. D.M. Miller, S.D. Thomas, A. Islam, D. Muench, K. Sedoris, c-Myc and cancer metabolism, Clin. Cancer Res. 18 (2012) 5546e5553. J.D. Gordan, C.B. Thompson, M.C. Simon, HIF and c-Myc: sibling rivals for control of cancer cell metabolism and proliferation, Cancer Cell. 12 (2007) 108e113. H. Shim, C. Dolde, B.C. Lewis, C.S. Wu, G. Dang, R.A. Jungmann, et al., c-Myc transactivation of LDH-A: implications for tumor metabolism and growth, Proc. Natl. Acad. Sci. U S A. 94 (1997) 6658e6663. R.C. Osthus, H. Shim, S. Kim, Q. Li, R. Reddy, M. Mukherjee, et al., Deregulation of glucose transporter 1 and glycolytic gene expression by c-Myc, J. Biol. Chem. 275 (2000) 21797e21800.