Increased RhoA Activity Predicts Worse Overall Survival in Patients Undergoing Surgical Resection for Lauren Diffuse-Type Gastric Adenocarcinoma

Abstract

Background: Several studies have reported a high rate of RHOA mutations in the Lauren diffuse-type gastric adenocarcinoma (GA) but not in intestinal-type GA. The aim of this study was to determine if RhoA activity is prognostic for overall survival (OS) in patients with resectable GA.

Methods: Retrospective review was performed on a prospective database of GA patients who underwent potentially curative resection between 2003 and 2012 at a single institution. Tissue microarrays were constructed from surgical specimens and analyzed for phosphorylated RhoA, a marker of inactive RhoA signaling. OS was estimated by the Kaplan-Meier method, and multivariate analysis was performed by Cox proportional hazards regression modeling.

Results: One hundred thirty-six patients with diffuse-type GA and 129 patients with intestinal-type GA were examined. Compared to intestinal-type GA, diffuse-type GA tumors were significantly associated with increased tumor size and advanced tumor, node, metastasis (TNM) classification system stage. In patients with diffuse-type GA, high RhoA activity was associated with significantly worse OS when compared to low RhoA activity (5-year OS 52.5 vs. 81.0 %, p = 0.017). This difference in OS was not observed in patients with intestinal-type GA (5-year OS 83.9 vs. 81.6 %, p = 0.766). On multivariate analysis of diffuse-type GA patients, high RhoA activity was an independent negative prognostic factor for OS (hazard ratio 2.38, 95 % confidence interval 1.07-5.28).

Conclusions: Increased RhoA activity is predictive of worse OS in patients with diffuse-type GA who undergo potentially curative surgical resection. Along with findings from genomic studies, these results suggest RhoA may be a novel therapeutic target in diffuse-type GA.

FIG. 1

(a) Schematic overview of the RhoA signaling pathway. RhoA and its downstream effector Rho-associated protein kinase (ROCK) have key regulatory roles in cell cycle regulation, actin-myosin-dependent cell contractility, cell motility, and cell proliferation. RhoA cycles between a GDP-bound inactive form and a GTP-bound active form. This process is mediated by guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP). Protein kinase A (PKA) phosphorylation of active Rho-GTP induces a tight association with Rho-GDP-dissociation inhibitor (GDI), thereby sequestering RhoA in the cytosol in an inactive state. (b) Immunohistochemistry analysis of phosphorylated(Ser188)-RhoA (phospho-RhoA) in Lauren intestinal-type and diffuse-type gastric adenocarcinoma tumor samples. Tissue incubated without primary antibody served as a negative control. Representative fields corresponding to variable staining intensities are shown. Phospho-RhoA staining was predominantly localized to the cytosol. Scale bars represent 20μm.

FIG. 2

Kaplan-Meier overall survival curves stratified by RhoA activity level in (a) all patients with gastric adenocarcinoma (GA), (b) patients with intestinal-type GA, and (c) patients with diffuse-type GA.