Deregulated Expression of SRC, LYN and CKB Kinases by DNA Methylation and Its Potential Role in Gastric Cancer Invasiveness and Metastasis

<div><p>Kinases are downstream modulators and effectors of several cellular signaling cascades and play key roles in the development of neoplastic disease. In this study, we aimed to evaluate SRC, LYN and CKB protein and mRNA expression, as well as their promoter methylation, in gastric cancer. We found elevated expression of SRC and LYN kinase mRNA and protein but decreased levels of CKB kinase, alterations that may have a role in the invasiveness and metastasis of gastric tumors. Expression of the three studied kinases was also associated with MYC oncogene expression, a possible biomarker for gastric cancer. To understand the mechanisms that regulate the expression of these genes, we evaluated the DNA promoter methylation of the three kinases. We found that reduced <i>SRC</i> and <i>LYN</i> methylation and increased <i>CKB</i> methylation was associated with gastric cancer. The reduced <i>SRC</i> and <i>LYN</i> methylation was associated with increased levels of mRNA and protein expression, suggesting that DNA methylation is involved in regulating the expression of these kinases. Conversely, reduced <i>CKB</i> methylation was observed in samples with reduced mRNA and protein expression, suggesting CKB expression was found to be only partly regulated by DNA methylation. Additionally, we found that alterations in the DNA methylation pattern of the three studied kinases were also associated with the gastric cancer onset, advanced gastric cancer, deeper tumor invasion and the presence of metastasis. Therefore, SRC, LYN and CKB expression or DNA methylation could be useful markers for predicting tumor progression and targeting in anti-cancer strategies.</p></div

Kinases expression in gastric cancer.

<p>A) Association between SRC immunoreactivity and its protein expression; B) SRC protein expression; C) Association between SRC immunoreactivity and its mRNA expression; D) <i>SRC</i> mRNA expression. E) Association between LYN immunoreactivity and its protein expression; F) LYN protein expression; G) Association between LYN immunoreactivity and its mRNA expression; H) <i>LYN</i> mRNA expression; I) CKB protein expression; J) <i>CKB</i> mRNA expression; K) representative image of Western-blot, in each TNM of each sample is show. Protein and mRNA expression were determined by Western-blot and RT-qPCR analysis, respectively. In all graphs, the expression in gastric tumors was normalized by matched non-neoplastic gastric tissue. *Significant difference between groups by Mann-Whitney (p < 0.05). IHC+: cases presenting protein immunoreactivity; IHC–: cases without protein immunoreactivity; NM: normal mucosa sample.</p

Kinases expression regulation by DNA methylation.

<p>A) <i>SRC</i> mRNA expression; B) SRC protein expression; C) SRC immunoreactivity; D) <i>LYN</i> mRNA expression; E) LYN protein expression; F) LYN protein immunoreactivity; G) <i>CKB</i> mRNA expression; H) CKB protein expression. Protein and mRNA expression were determined by Western-blot and RT-qPCR analysis, respectively. In these analyses, the expression in gastric tumors was normalized by matched non-neoplastic gastric tissue. *Significant difference between groups by χ² (for analysis involving IHC data) or Mann-Whitney tests (p < 0.05). IHC–: cases without protein immunoreactivity; IHC+: cases presenting protein immunoreactivity.</p

Percentage of methylation in gastric samples.

<p>A) <i>SRC</i> methylation in gastric tumors and non-tumor samples; B) <i>LYN</i> methylation in gastric tumors and non-tumor samples; C) <i>CKB</i> methylation in gastric tumors and non-tumor samples; D) Correlation between the percentage of <i>SRC</i> methylation in gastric tumors and paired non-tumor samples; E) Correlation between the percentage of <i>LYN</i> methylation in gastric tumors and paired non-tumor samples; F) Correlation between the percentage of <i>CKB</i> methylation in gastric tumors and paired non-tumor samples. *Significant difference between groups by Mann-Whitney test (p < 0.05).</p

Primer sequences (5’-3’) for methylation analysis.

<p>MSP: primers for methyl-specific PCR; BSP: primers for Bisulfite Sequencing PCR; M: primer for methylated sequence by MSP; U: primer for unmethylated sequences by MSP; bp: base pair.</p><p>Primer sequences (5’-3’) for methylation analysis.</p

Correlation between <i>MYC</i> and kinases mRNA expression.

<p>A) <i>MYC</i> and <i>SRC</i>; B) <i>MYC</i> and <i>LYN</i>; C) <i>MYC</i> and <i>CKB</i>.</p

Methylation analysis of the kinases promoters showing methylated and unmethylated bands.

<p>A) <i>SRC</i> promoter methylation analysis, in which samples 1 and 2 presented hypomethylated promoter, sample 3 presented partial methylation and sample 4 presented hypermethylated promoter; B) <i>LYN</i> promoter methylation analysis, in which samples 1 presented hypomethylated promoter, sample 2 presented partial methylation and samples 3 and 4 presented hypermethylated promoter; C) <i>CKB</i> promoter methylation analysis, in which samples 1 and 2 presented hypermethylated promoter, and samples 3 and 4 presented hypomethylated promoter. C–: blank; C+: positive control, gDNA sample completely methylated; U: PCR with unmethylated primer set; M: PCR with methylated primer set; MW: molecular weight marker; bp: base pairs.</p

Protein kinases methylation pattern in gastric samples.

<p>^ap-value of χ² test</p><p>^bp-value of the post-hoc comparison between tissue samples hypermethylated and partial-methylated</p><p>^cp-value of the post-hoc comparison between tissue samples partial-methylated and hypomethylated</p><p>^dp-value of the post-hoc comparison between tissue samples hypermethylated and hypomethylated</p><p>*Statistically significant difference between neoplastic and non-neoplastic samples by χ² test (p < 0.05)</p><p>**Statistically significant difference between groups by χ² test followed Bonferroni adjustment (p < 0.016)</p><p>Protein kinases methylation pattern in gastric samples.</p

Kinases immunoreactivity in gastric tissue samples.

<p>A) gastric mucosa without SRC immunoreactivity; B) diffuse-type gastric cancer presenting cell membrane and cytoplasmic immunoreactivity of SRC; C) non-neoplastic gastric tissue without LYN immunoreactivity; D) intestinal-type gastric cancer presenting LYN immunoreactivity; E) non-neoplastic gastric mucosa showing weak cytoplasmic CKB staining in glandular cells; F) diffuse-type gastric cancer cells without CKB immunoreactivity.</p