Epigenetic Deregulation of Protein Tyrosine Kinase 6 Promotes Carcinogenesis of Oral Squamous Cell Carcinoma

Abstract

Oral squamous cell carcinoma (OSCC) accounts for over 90% of oral cancers and causes considerable morbidity and mortality. Epigenetic deregulation is a common mechanism underlying carcinogenesis. DNA methylation deregulation is the epigenetic change observed during the transformation of normal cells to precancerous and eventually cancer cells. This study investigated the DNA methylation patterns of PTK6 during the development of OSCC. Bisulfite genomic DNA sequencing was performed to determine the PTK6 methylation level. OSCC animal models were established to examine changes in PTK6 expression in the different stages of OSCC development. The DNA methylation of PTK6 was decreased during the development of OSCC. The mRNA and protein expression of PTK6 was increased in OSCC cell lines compared with human normal oral keratinocytes. In mice, the methylation level of PTK6 decreased after treatment with 4-nitroquinoline 1-oxide and arecoline, and the mRNA and protein expression of PTK6 was increased. PTK6 hypomethylation can be a diagnostic marker of OSCC. Upregulation of PTK6 promoted the proliferation, migration, and invasion of OSCC cells. PTK6 promoted carcinogenesis and metastasis by increasing STAT3 phosphorylation and ZEB1 expression. The epigenetic deregulation of PTK6 can serve as a biomarker for the early detection of OSCC and as a treatment target.

Keywords: epigenetic regulation; oral squamous cell carcinoma; oral squamous cell carcinoma carcinogenesis; protein tyrosine kinase 6 (PTK6).

Figure 1

PTK6 was hypomethylated with OSCC development. (a) CpG sites on the PTK6 exon 1 and two specific regions were designed for pyrosequencing. (b) The methylation levels of PTK6 in 35 normal tissues, 18 precancerous lesions, and 51 OSCC patient samples were measured through bisulfite pyrosequencing. (c) The relative DNA methylation of PTK6 in tumor tissues and their corresponding normal tissues from the same patient. (d) The methylation levels of PTK6 in 10 normal and 10 OSCC patients’ saliva samples were measured through bisulfite pyrosequencing. * p < 0.05. *** p < 0.005.

Figure 2

PTK6 hypomethylation induces OSCC carcinogenesis by increasing PTK6 transcriptional expression in the animal model. (a) Flowchart of exposure of mice to carcinogens (200 µg/mL of 4-NQO and 500 µg/mL of arecoline) in drinking water. The mice treated with 4-NQO and arecoline and corresponding control mice were sacrificed at 8, 16, 20, and 29 weeks for analysis. (b) Methylation analysis of PTK6 in the tongue tissues of OSCC mice at different time points. (c) PTK6 transcriptional expression in the tongue tissues of the OSCC mice at different time points. (d) The protein expression of PTK6 in the tongue tissues of the OSCC mice at different time points. * p < 0.05. ** p < 0.01; *** p < 0.005.

Figure 3

Downregulation of PTK6 suppressed OSCC cell migration and invasion. (a) OCMC and OECM1 cell lines were used to knock down PTK6 by using shRNA. The mRNA and protein expression were examined through real-time PCR and Western blotting in PTK6 knockdown cells. (b) Cell viability of control and PTK6 knockdown cells was evaluated using a WST-1 assay. Absorbance was measured at 450 nm at seven time points (0, 24, 48, 72, 96, 120, and 144 h) to calculate the proliferation rate. (c) The migration abilities of control and PTK6 knockdown cells were examined using a transwell assay. Migrated cells were quantified using ImageJ. (d) The invasive abilities of control and PTK6 knockdown cells were measured using a transwell Matrigel assay. Invasive cells were quantified using ImageJ. ns means not significant. **** p < 0.001.

Figure 4

Upregulation of PTK6 facilitated OSCC cell migration and invasion. (a) PTK6 overexpression was induced in OML3 and OCSL cell lines by transfecting a PTK6 overexpression plasmid. The mRNA and protein expression were examined through real-time PCR and Western blotting in PTK6 overexpression cells. (b) Cell viabilities of control and PTK6 overexpression cells were examined using a WST-1 assay. Absorbance was measured at 450 nm at seven time points (0, 24, 48, 72, 96, 120, and 144 h) to calculate the proliferation rate. (c) The migration abilities of control and PTK6 overexpression cells were examined using a transwell assay. Migrated cells were quantified using ImageJ. (d) The invasive abilities of control and PTK6 overexpression cells were measured using a transwell Matrigel assay. Invasive cells were quantified using ImageJ. ns means not significant. ** p < 0.01; *** p < 0.005; **** p < 0.001.

Figure 5

PTK6 promoted tumorigenesis in vivo. (a) Images show the sizes of tumors collected from control and PTK6 knockdown xenograft tumor models. (b,c) Tumor volumes and weights at 4 weeks post injection were measured. (d) Images show the sizes of tumors received from control and PTK6 overexpression xenograft tumor models. (e,f) Tumor volumes and weights at 8 weeks post injection were measured. Tumor volume was calculated using the following formula: TV (mm³) = (L × W²)/2. * p < 0.05. ** p < 0.01.

Figure 6

Analysis of candidate genes downstream of PTK6. (a,b) The protein levels of candidate genes downstream of PTK6 downstream were detected through Western blotting. (c,d)The intensities of Western blotting data were measured with ImageJ and normalized to GAPDH. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.001; ns: not significant.