Aberrant methylation-mediated silencing of microRNAs contributes to HPV-induced anchorage independence

Abstract

Cervical cancer and a subset of anogenital and head-and-neck carcinomas are caused by high-risk types of the human papillomavirus (hrHPV). During hrHPV-induced malignant transformation keratinocytes become able to grow anchorage independently, a tumorigenic trait at least partly associated with inactivation of tumor suppressor genes. We used hrHPV-containing keratinocytes to investigate the role of DNA methylation-mediated silencing of microRNAs (miRNAs) in the acquisition of anchorage independence.Anchorage dependent (n=11) and independent passages (n=19) of 4 hrHPV-immortalized keratinocyte cell lines were treated with 2'-deoxy-5-azacytidine (DAC). Genome-wide miRNA expression profiles before and after treatment were compared to identify miRNAs silenced by methylation. Bisulfite sequencing and methylation-specific PCR showed increased methylation of hsa-mir-129-2/-137/-935/-3663/-3665 and -4281 in anchorage independent HPV-transformed keratinocytes and cervical cancer cell lines. Mature miRNAs derived from hsa-mir-129-2/-137/-3663 and -3665 showed functional relevance as they decreased anchorage independence in cervical cancer cell lines. Cervical (pre)cancerous lesions demonstrated increased methylation of hsa-mir-129-2/-935/-3663/-3665 and -4281, underlining the clinical relevance of our findings.In conclusion, methylation-mediated silencing of tumor suppressive miRNAs contributes to acquisition of an anchorage independent phenotype. This study further substantiates the importance of miRNAs during early stages of carcinogenesis and underlines their potential as both disease markers and therapeutic targets.

Keywords: CIN lesion; DNA methylation; anoikis; cervical cancer.

Figure 1. Characterization of our longitudinal in vitro model system of hrHPV-induced transformation

In A. anchorage dependent (grey) and independent (white) timepoints (T) of all 4 cell lines are shown in relation to the transformation process. Unsupervised hierarchical cluster results based on overall miRNA expression are shown for B. FK16A, C. FK16B, D. FK18A, and E. FK18B. Anchorage dependent timepoints are marked in grey, whereas anchorage independent timepoints are white.

Figure 2. Schematic overview of the analysis pipeline used to select the most promising potential methylation targets

Figure 3. Bisulfite sequencing results of selected genomic regions in primary keratinocytes (HFK), HPV-transformed keratinocytes and cervical cancer cells

Results are shown for A. hsa-mir-129-2, B. hsa-mir-137, C. hsa-mir-615, D. hsa-mir-675, E. hsa-mir-935, F. hsa-mir-2277, G. hsa-mir-3663, H. hsa-mir-3665, I. hsa-mir-4281, and J. hsa-mir-4323. Methylation-independent sequencing primers are indicated by black arrows and methylation-specific MSP primers are indicated by grey arrows. TSS; transcription start site.

Figure 4. MSP results for selected miRNA genes in 3 cervical cancer cell lines (SiHa, HeLa, CaSki), primary keratinocytes (HFK) of 3 independent donors, and anchorage independent passages of 4 HPV-transformed keratinocyte cell lines (FK16A, FK16B, FK18A, and FK18B)

Results are shown for A. hsa-mir-129-2, B. hsa-mir-137, C. hsa-mir-615, D. hsa-mir-935, E. hsa-mir-3663, F. hsa-mir-3665, G. hsa-mir-4281, and H. ACTB (reference gene). In vitro methylated DNA (IVD) and unmodified DNA were included as positive and negative controls, respectively.

Figure 5. Functional effects of miRNAs silenced by methylation on HPV-induced transformation

Effects of ectopic expression of mimics of all mature miRNAs derived from the methylated gene loci that showed a DAC effect (miR-129-5p, -129-2-3p, -137, -935, -;3663, -3665, and -4281) were determined on A. cell viability and B. anchorage independent growth. Dashed lines indicate the threshold of 30% we used to discriminate true effects from random variation. Results are representative of 2 independent experiments.

Figure 6. Methylation levels of selected miRNA genes relative to the reference gene (ACTB) in normal cervical tissue specimens (n=22), CIN3 lesions (n=23) and SCCs (n=28)

Results are shown for A. hsa-mir-129-2, B. hsa-mir-137, C. hsa-mir-935, D. hsa-mir-3663, E. hsa-mir-3665, and F. hsa-mir-4281. ** indicates a two-sided p-value <0.001 (non-parametric Wilcoxon rank test); * indicates a two-sided p-value <0.05.