Quantitative Methylation Profiles for Multiple Tumor Suppressor Gene Promoters in Salivary Gland Tumors

Abstract

BackgroundMethylation profiling of tumor suppressor gene (TSGs) promoters is quickly becoming a powerful diagnostic tool for the early detection, prognosis, and even prediction of clinical response to treatment. Few studies address this in salivary gland tumors (SGTs); hence the promoter methylation profile of various TSGs was quantitatively assessed in primary SGT tissue to determine if tumor-specific alterations could be detected.MethodologyDNA isolated from 78 tumor and 17 normal parotid gland specimens was assayed for promoter methylation status of 19 TSGs by fluorescence-based, quantitative methylation-specific PCR (qMSP). The data were utilized in a binary fashion as well as quantitatively (using a methylation quotient) allowing for better profiling and interpretation of results.Principal FindingsThe average number of methylation events across the studied genes was highest in salivary duct carcinoma (SDC), with a methylation value of 9.6, compared to the normal 4.5 (p<0.0003). There was a variable frequency and individual methylation quotient detected, depending on the TSG and the tumor type. When comparing normal, benign, and malignant SGTs, there was a statistically significant trend for increasing methylation in APC, Mint 1, PGP9.5, RAR-β, and Timp3.Conclusions/SignificanceScreening promoter methylation profiles in SGTs showed considerable heterogeneity. The methylation status of certain markers was surprisingly high in even normal salivary tissue, confirming the need for such controls. Several TSGs were found to be associated with malignant SGTs, especially SDC. Further study is needed to evaluate the potential use of these associations in the detection, prognosis, and therapeutic outcome of these rare tumors.

Aquaporin‐1 Promoter Hypermethylation Is Associated with Improved Prognosis in Salivary Gland Adenoid Cystic Carcinoma

“…14 Each reaction was carried out in a reaction volume of 10 μL, with components as previously described. 4 Calibration curves were constructed for each plate with serial dilutions of bisulfite-converted universal methylated human DNA (Zymo Research, Irvine, California). Each plate included samples, water blanks, and calibration curves in triplicate.…”

Molecular biology of adenoid cystic carcinoma

“…Interestingly a promoter methylation study performed on several TSG’s including FHIT showed that FHIT promoter methylation rates were actually much higher in normal tissue than in benign or malignant salivary gland tumors (100) . The reason for this inverse relationship is unclear.…”

“…Published reports on methylation in ACC mostly focus on candidate genes known to play important roles in non-ACC tumors (100, 118–123) . In some studies, differentially methylated genes were further correlated with available clinical and pathological parameters of the given tumor samples (121, 122) , to identify their biological function and clinical importance ( see Table 3).…”

Death Receptor Pathway Activation and Increase of ROS Production by the Triple Epigenetic Inhibitor UVI5008

“…UVI5008 treatment of HCT116-DKO did not further enhance transcription as compared with the parental HCT116 suggesting that its DNMT but not HDAC and/or SIRTi caused the low but significant activation of the UCHL1 locus in wild-type cells. TIMP3, a TSG known both to be repressed by DNMT (39,40) and to be SIRT1 target (41) was reactivated by UVI5008 only in HCT116-DKO cells and not in the untreated or wild-type cells (Fig. 5C).…”