High RAB25 expression is associated with good clinical outcome in patients with locally advanced head and neck squamous cell carcinoma

Abstract

Currently there are no molecular markers able to predict clinical outcome in locally advanced head and neck squamous cell carcinoma (HNSCC). In a previous microarray study, RAB25 was identified as a potential prognostic marker. The aim of this study was to analyze the association between RAB25 expression and clinical outcome in patients with locally advanced HNSCC treated with standard therapy. In a retrospective immunohistochemical study (n = 97), we observed that RAB25-negative tumors had lower survival (log-rank, P = 0.01) than patients bearing positive tumors. In an independent prospective mRNA study (n = 117), low RAB25 mRNA expression was associated with poor prognosis. Using classification and regression tree analysis (CART) we established two groups of patients according to their RAB25 mRNA level and their risk of death. Low mRNA level was associated with poor local recurrence-free (log-rank, P = 0.005), progression-free (log-rank, P = 0.002) and cancer-specific (log-rank, P < 0.001) survival. Multivariate Cox model analysis showed that low expression of RAB25 was an independent poor prognostic factor for survival (hazard ratio: 3.84, 95% confidence interval: 1.93-7.62, P < 0.001). Patients whose tumors showed high RAB25 expression had a low probability of death after treatment. We also found lower RAB25 expression in tumors than in normal tissue (Mann-Whitney U, P < 0.001). Moreover, overexpression of RAB25 in the UM-SCC-74B HNSCC cell line increased cisplatin sensitivity, and reduced cell migration and invasion. Our findings support a tumor suppressor role for RAB25 in HNSCC and its potential use to identify locally advanced patients with a high probability of survival after genotoxic treatment.

Keywords: HNSCC; RAB25; prognosis; tumor suppressor.

Figure 1

RAB25 protein expression analysis in pretreatment tumor biopsies of 97 patients included in the retrospective study. (A) Representative images of positive and negative RAB25 tumors at 200× and 400× magnifications. Images show a cytoplasmic and membrane staining pattern of RAB25. (B) Kaplan–Meier curves and log rank test showed that patients with RAB25 negative tumors have a lower cancer-specific survival (CSS) than patients bearing positive tumors.

Figure 2

Patients included in the prospective study (n = 117) were stratified in two groups according to the RAB25 mRNA level. Kaplan–Meier curves and log-rank test showed significant differences in local recurrence-free survival (A), progression-free survival (B), and cancer-specific survival (C) between patients with high expression (>1.75) and patients with low expression (<1.75).

Figure 3

(A) Box plots showed a lower RAB25 expression in tumor tissue (n = 117) than in normal tissue (n = 19). We confirmed significant differences using three independent microarrays datasets (B) Thurlow et al. (C) GSE23558 and (D) GSE25099 (Mann–Whitney U test).

Figure 4

(A) RAB25 mRNA levels analyzed in HNSCC cell lines by quantitative RT-PCR. (B) UM-SCC-74B clones overexpressing RAB25 detected by qRT-PCR. (C) Proliferation assays performed seeding cells at a density of 30,000 cells/well in 6 well/plates, during 48, 96, and 144 h. (D) XTT cytoxicity assays. Cells were exposed during 48 h to cisplatin (2.5–40 μmol/L). (E) Scattering, migration, and invasion assays. (Mann–Whitney U test). HNSCC, head and neck squamous cell carcinoma; RT-PCR, real-time polymerase chain reaction.