Cancer Letters

Cancer Letters

Volume 374, Issue 1, 28 April 2016, Pages 127-135
Cancer Letters

Original Articles
ALDOA functions as an oncogene in the highly metastatic pancreatic cancer

https://doi.org/10.1016/j.canlet.2016.01.054Get rights and content

Highlights

Abstract

Pancreatic cancer is an aggressive and devastating disease that is characterized by uncontrolled progression, invasiveness and resistance to conventional treatment. In the past decades, much effort has been given to cancer genetics and pathological classification of this disease. Our previous study has uncovered a subgroup of patients with poor outcome, which is characterized by serum signature of CEA+/CA125+/CA19-9 ≥ 1000 U/mL; however, the underlying biology mechanism remains poorly understood. By using high-throughput screening analysis, we analyzed gene expression signature in highly malignant patients with serum markers of CEA+/CA125+/CA19-9 ≥ 1000 U/mL. Multiple differentially expressed genes were identified, many of which were closely related with cancer metabolic changes. Treatment of pancreatic cancer cell lines PANC-1 with transforming growth factor-β (TGF-β), which was commonly used to induce metastasis, has uncovered that the glycolytic process and antioxidant response was up-regulated upon TGF-β stimulation. These results were consistent with the high-throughput screening analysis. Subsequent analysis indicated that among glycolytic genes, aldolase A (ALDOA) increased the most significantly upon TGF-β treatment. Further in vitro and in vivo results demonstrated that ALDOA was associated with proliferation and metastasis of pancreatic cancer cells. Moreover, ALDOA predicted poor prognosis of pancreatic cancer, partially due to its role in E-cadherin expression regulation, and the results were further validated by analysis of the correlation between ALDOA and E-cadherin expression in pancreatic cancer tissue samples. Mechanistically, the role of ALDOA in pancreatic cancer might attribute to its regulation of c-Myc, HIF1α and NRF2 (Nuclear Factor, Erythroid 2-Like 2), which were key regulators of glycolysis and antioxidant response control.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States, and is projected to surpass breast cancers to become the second by 2030 [1]. Based on epidemiological studies, the incidence rate of pancreatic cancer is 17.28 per 100,000 for male and 14.04 per 100,000 for female in 2009 in Shanghai [2]. Patients who are diagnosed with pancreatic cancer have a poor prognosis, with an overall 5-year survival rate about 6% [3]. This poor prognosis is mainly caused by a lack of typical symptoms at the early stages of the disease, uncontrolled local recurrence and metastasis and resistance to existing chemotherapeutic agents [4]. Surgical resection remains the most effective modality to cure the disease. However, early distant metastasis after radical pancreatectomy is the major reason for dismal surgical outcome [5]. In order to identify whether a patient would likely to benefit from surgery preoperatively, we previously analyzed eight tumor markers from 1000 patients and found that a subgroup of patients with preoperative serum signature of CEA+/CA125+/CA19-9 ≥ 1000 U/mL was associated with poor surgical outcome. This was attributed to a higher prevalence of early distant metastasis after surgery in this subgroup [6]. Therefore, in order to improve the treatment of PDAC, it is necessary to explore molecular markers to elucidate the biological mechanism governing the processes of rapid proliferation and metastasis. Employing high-throughput gene expression analysis in our previously described subgroup patients with CEA+/CA125+/CA19-9 ≥ 1000 U/mL, we identified multiple differentially expressed genes, many of which were closely related with cancer metabolic adaptation to hypoxic stress and antioxidant stress response. TGF-β was reported to induce metastasis of many cancers by promoting the epithelial-mesenchymal transition (EMT) process [7]. The role of TGF-β in hypoxia stress and oxidant stress adaptation has seldom been reported. Thus we used TGF-β treatment in vitro to mimic the metastasis process to observe changes in hypoxia and antioxidant stress. And results demonstrated that TGF-β stimulation induced up-regulation of hypoxia and antioxidant stress response. It is well accepted that anabolic glycolysis process is connected to glucose metabolism transformation and antioxidant stress response, two phenomenona commonly observed in highly metastatic pancreatic cancer cells [8], [9], [10], [11]. And the anabolic process involves a series of glycolytic genes, which ensures abundant building blocks and energy supply in hypoxic cancer cells [12]. Furthermore, we examined the changes of glycolytic genes expressions upon TGF-β treatment, and observed that aldolase A (ALDOA) changed the most significantly, indicating that ALDOA might participate in the metastasis process.

ALDOA is one of the three aldolase isozymes (A, B, and C), encoded by three different genes [13]. These aldolases are differentially expressed during development. The ALDOA gene is expressed extensively in muscle tissues, whereas the aldolase B gene is expressed strongly in kidney, liver, stomach and intestine and the aldolase C gene is expressed in brain, heart and ovary. ALDOA contributes to various cellular functions and biological process related to muscle maintenance, regulation of cell shape and mobility, striated muscle contraction, actin filament organization and ATP biosynthetic process [14], [15], [16]. ALDOA deficiency is associated with myopathy and hemolytic anemia [17]. Notably, ALDOA has been found highly expressed in a variety of cancers, including human oral squamous cell carcinomas, lung squamous cell carcinomas, renal cell carcinomas, colorectal cancer, hepatocellular carcinomas and osteosarcoma [18], [19], [20]. However, none of these reports examined the involvement of ALDOA in PDAC progression and metastasis. In this study, we reported that ALDOA is highly expressed in patients with serum CEA+/CA125+/CA19-9 ≥ 1000 U/mL. Moreover, high ALDOA expression level was correlated with poor prognosis of PDAC patients. Functional assays showed that down-regulation of ALDOA in pancreatic cancer cells reduced cell viability, colony formation and metastasis capacity. Mechanistically, these functions in part due to its association with HIF1α and c-Myc mediated hypoxia and antioxidant stress response pathway. These observations suggest that ALDOA is a potential biomarker of PDAC metastasis and plays an oncogenic role in PDAC.

Section snippets

Cell culture

The human pancreatic cancer cell lines PANC-1 was obtained from the American Type Culture Collection (ATCC) and was cultured at 37 °C and 5% CO2 in DMEM (high Glucose) plus 10% heat-inactivated FBS plus 1 mM Na-Pyruvate and Penicillin/Streptomycin.

RNA extraction, ss-cDNA synthesis and microarray analysis

Whole tumor cell RNA was extracted by Trizol/Chloroform, and then purified with magnetic beads of Agencourt Ampure (APN 000132, Beckman Coulter). Target preparation for microarray processing was carried out according to the GeneChip® WT PLUS Reagent

High throughput screening of gene signatures and the related signaling pathway in highly metastatic pancreatic cancer subgroup

To understand the molecular mechanism underlying the highly metastatic pancreatic cancer patients, we analyzed gene expression profiles in patients with serum CEA+/CA125+/CA19-9 ≥ 1000 U/mL and non-CEA+/CA125+/CA19-9 ≥ 1000 U/mL samples. This analysis identified a series of genes that significantly varied between CEA+/CA125+/CA19-9 ≥ 1000 U/mL and non-CEA+/CA125+/CA19-9 ≥ 1000 U/mL groups (Supplementary Table S4). Unsupervised hierarchical clustering of the 68 differentially expressed genes

Discussion

The identification of signature gene mutations in pancreatic adenocarcinoma was recognized as a valuable starting point, which were translated into classical biological features and signaling networks that control cellular growth, survival, differentiation and metastasis of the disease [21]. Despite substantial progress in the understanding and therapy of pancreatic cancer, the overall 5-year survival rate remains steadily at about 6%. The underlying reason for this unsatisfying progress is

Conflict of interest

The authors declare no conflict interest.

Acknowledgments

This work was supported by National Natural Science Foundation of China [81372651, 81201900, 81172276 and 81101565], Sino-German Center [GZ857], Ph.D. Programs Foundation of Ministry of Education of the People's Republic of China [20120071120104], Program of Science and Technology Commission of Shanghai Municipality [13431900105, 13DZ1942802].

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