Rap1GAP inhibits tumor progression in endometrial cancer

Abstract

Objective

Endometrioid adenocarcinoma (EAC) is a common endometrial cancer with recent dramatic increases in incidence. Previous findings indicate that Rap1GAP acts as a tumor suppressor inhibiting Ras superfamily protein Rap1 in multiple aggressive carcinomas; however, Rap1GAP expression in EAC has not been investigated. In this study, the tumor suppressing activity of Rap1GAP in EAC was explored.

Methods

EAC cell lines were used to examine Rap1GAP levels by real-time RT-PCR and western blotting and the effects of Rap1GAP on cancer cell invasion and migration. Rap1GAP expression was analyzed by immunohistochemical staining for Rap1GAP, E-cadherin in surgically resected tumors of 114 EAC patients scored according to EAC differentiation grade. Prognostic variables such as age, stage, grade, tumor size, and immunostaining for Rap1GAP, E-cadherin were evaluated using Cox regression multivariate analysis.

Results

Low Rap1GAP expression was detected in poorly differentiated EAC cells. Rap1GAP deficiency significantly accelerated while Rap1 deficiency decreased cancer cell migration and invasion. Patients with higher Rap1GAP, E-cadherin, and especially combined Rap1GAP/E-cadherin levels had better overall survival than EAC patients with no or weak expression. In addition, Rap1GAP expression was an independent prognostic factor in EAC.

Conclusions

Inhibition of Rap1GAP expression increases EAC cell migration and invasion through upregulation of Rap1. Low expression of Rap1GAP correlates with poor EAC differentiation. Our findings suggest that Rap1GAP is an important tumor suppressor with high prognostic value in EAC.

Introduction

Endometrial carcinoma (EC) is the seventh most common malignancy in the world. Annually, EC is detected in about 142,000 women, causing 42,000 deaths [1]. In Japan, EC incidence grew from 3722 EC patients in 2003 to 7273 in 2011. Endometrioid adenocarcinoma (EAC), the most common histological type of EC, is classified into three histologic grades according to differentiation. Early-stage patients have a high cure rate; however, older women or patients with low differentiation, lymphatic metastasis, or vascular and myometrial EC invasion have poor prognosis [2], [3].

In EC, inactivating mutations in the PTEN gene and activating mutations in the KRAS (encoding GTPase KRas) and PIK3CA genes are detected in 30–50%, 10–30%, and 30–40% cases, respectively [4]. Furthermore, mutations or overexpression of the genes involved in these pathways have been associated with metastatic invasion, and used as prognostic factors for EC and other carcinomas [5].

Rap proteins Rap1A/B and Rap2A/B/C are members of the RAS superfamily of small GTPases. Rap regulates the balance in cell/matrix and cell/cell adhesion via integrin activation [6], [7], [8] and cadherin-mediated cell/cell junctions [9]. Furthermore, Rap proteins are active when bound to GTP, and inactive when bound to GDP and are regulated by the same guanine nucleotide exchange factors (RapGEFs) and GTPase-activating proteins (RapGAPs) [10], [11]. The RAP1GAP gene mapped to chromosome 1p36.1-p35 encodes a 663-amino acid protein with a molecular weight of 73 kDa, which has GAP activity specific for Rap1 [12].

Rap1GAP has been identified as a tumor suppressor gene; many human malignant tumors, including thyroid [13], [14], pancreas [15], colon [16], melanomas [17], prostate [11], and head and neck carcinomas [18] are either Rap1GAP-deficient or have decreased Rap1GAP expression. The mechanisms of Rap1GAP tumor suppressing activity have recently been reported. Thus, it has been shown that in pancreatic carcinoma, high-frequency loss of Rap1GAP heterozygosity followed by Rap1GAP deficiency is likely due to the loss of the second allele [14]. In squamous cell carcinoma of the head and neck, Rap1GAP inhibits tumor growth by delaying the G1/S transition of the cell cycle [19]. Rap1GAP is also identified as a novel Ret-binding protein [20]. However, the significance of Rap1GAP in gynecologic carcinomas is unknown. In this study, we identified Rap1GAP, a suppressor of RAS superfamily Rap1 activity, as a putative tumor invasion inhibitor in EAC.