Methylation of RASSF1A, RASSF2A, and HIN-1 is associated with poor outcome after radiotherapy, but not surgery, in oral squamous cell carcinoma.
OBJECTIVE
Radiotherapy is the standard adjuvant treatment for oral squamous cell carcinoma (OSCC). The Ras/PI3K/AKT pathway is the major mechanism associated with radioresistance. To evaluate the potential significance on the outcome of radiotherapy in OSCC of the Ras/PI3K/AKT pathway with respect to methylation of negative regulators, we examined the methylation status of genes known to be involved in Ras/PI3K/AKT pathway and aberrantly methylated in human cancers together with the mutation status of K-ras/H-ras.


METHODS
PCR--denaturing high-performance liquid chromatography was used to examine the methylation status of the RASSF1A, RASSF2A, PTEN, and HIN-1 genes, and PCR-RFLP was used to determine the mutation status of K-ras/H-ras in 482 OSCCs. Associations between mutation, methylation, clinicopathologic parameters, and outcome were evaluated.


RESULTS
The frequencies of K-ras/H-ras mutation and promoter methylation of the RASSF1A, RASSF2A, PTEN, and HIN-1 genes were 6.6%, 22.4%, 27.8%, 1.2%, and 7.3%, respectively. A combination of RASSF1A and RASSF2A methylation was found to be significantly associated with poor disease-free survival (DFS). Furthermore, a gene dosage effect of the activated Ras/PI3K/AKT signal on DFS was observed in patients treated with radiotherapy after surgery but not in patients treated with surgery alone. The Ras/PI3K/AKT pathway was activated in 140 primary OSCCs among 286 patients treated with radiotherapy after surgery and methylation of RASSF1A/RASSF2A (75.7%) was the most common mechanism.


CONCLUSIONS
Our study indicates that epigenetic silencing of tumor suppressor genes involved in the Ras/PI3K/AKT pathway plays an important role in OSCC radioresistance and this provides a rationale for exploring novel treatment strategies.
Huang
Kuo-Hao
KH
Graduate Institute of Basic Medical Science, Chang Gung University, Tao-Yuan, Taiwan.
Huang
Shiang-Fu
SF
Chen
I-How
IH
Liao
Chun-Ta
CT
Wang
Hung-Ming
HM
Hsieh
Ling-Ling
LL
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000532:radiotherapy* / Q000601:surgery; D016207:Cytokines / Q000235:genetics*; D019175:DNA Methylation; D018572:Disease-Free Survival; D044127:Epigenesis, Genetic; D005500:Follow-Up Studies; D015972:Gene Expression Regulation, Neoplastic; D005787:Gene Frequency; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D053208:Kaplan-Meier Estimate; D008875:Middle Aged; D009062:Mouth Neoplasms / Q000235:genetics / Q000532:radiotherapy* / Q000601:surgery; D009154:Mutation / Q000235:genetics; D051059:PTEN Phosphohydrolase / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011401:Promoter Regions, Genetic; D016016:Proportional Hazards Models; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011836:Radiation Tolerance / Q000235:genetics*; D012044:Regression Analysis; D016896:Treatment Outcome; D025521:Tumor Suppressor Proteins / Q000235:genetics*
D016207:Cytokines; C411762:RASSF1 protein, human; C425300:RASSF2 protein, human; C434732:SCGB3A1 protein, human; D025521:Tumor Suppressor Proteins; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human