To determine whether P16 methylation directly increased the sensitivity of cancer cells to palbociclib, we induced P16 methylation in the lung cancer cell lines H661 and HCC827 and the gastric cancer cell line BGC823 via an engineered P16-specific DNA methyltransferase (P16-Dnmt) and found that the sensitivity of these cells to palbociclib was significantly increased.
Multiple logistic regression analysis showed that the odds ratio for having lung cancer was 10.204 for individuals with p16 methylation (P = .013) and 9.952 for individuals with RASSFIA methylation (P = .019).
Our data show that a combination of cytological analysis of sputum and examination of p16 hypermethylation in sputum and plasma identified 92.0% (46/50) of the lung cancer patients studied, offering an effective means of early detection of lung cancer.
Polymerase chain reaction amplification and direct sequencing of p16 exons 1 and 2 revealed no mutations, indicating that p16-altered expression in lung cancer is not necessarily linked to mutational events of these genes.
Based on smoking status, the promoter methylation ratios of both RASSF1A and p16 was significantly higher in lung cancer patients with smoking history compared to nonsmokers.
These results provide the first link between germ-line functional deficits in pathways that protect the cell from tobacco- and radon-induced DNA damage, and the development of aberrant promoter methylation of the p16 and MGMT genes in the respiratory epithelium of individuals at high risk for lung cancer.
Knockdown of DeltaDNMT3B4 expression by small interfering RNA resulted in a rapid demethylation of RASSF1A promoter and reexpression of RASSF1A mRNA but had no effect on p16 promoter in the lung cancer cells.
All of these findings are consistent with loss of Fhit protein expression being as frequent an abnormality in lung cancer pathogenesis as are p53 and p16 protein abnormalities and that such loss occurs independently of the commitment to the metastatic state and of most other molecular abnormalities.
Furthermore, the inactivation of the p16 gene by these carcinogenic exposures supports a possible role for oxidative stress and inflammation in the etiology of human lung cancer.
Evaluation of p16 and ESR1 promoter methylation in blood using real-time PCR appears to be very useful for lung cancer diagnosis and there is some possibility that these methylated genes might come to represent useful biomarkers for the early detection of lung cancer.
In contrast, cancer cell lines which were wild-type for p16 but mutant or null for pRb (Saos-2, C33a and H358) were <threefold more sensitive to Adp16 when compared to a control virus.
In contrast, p16 expression was lost in moderate dysplasia (12%) and in CIS (30%) in patients with lung cancer. p16 loss occurred exclusively in patients who displayed loss of p16 expression in their related invasive carcinoma.
Furthermore, rat lung cancer appears to be an excellent model in which to investigate the mechanisms of de novo gene methylation and the role of p16 dysfunction in the progression of neoplasia.
In summary, our data suggest that targeted DNA methylation silencing of ECAD and DAPK occurs in the early stages and that of p16 and MGMT in the later stages of lung cancer progression.
The strong association seen between p16 methylation in the bronchial epithelium and corresponding primary tumor substantiates that inactivation of this gene, although not transforming by itself, is likely permissive for the acquisition of additional genetic and epigenetic changes leading to lung cancer.
Since somatic mutations of the p53, RB and p16 genes occur frequently in lung cancer and the replication error phenotype is seen in a subset of lung cancer, it is possible that germ-line mutations of the p53, RB, p16 and mismatch repair genes influence the susceptibility to lung cancer.