Overexpression of <i>MEOX2</i> and <i>TWIST1</i> Is Associated with H3K27me3 Levels and Determines Lung Cancer Chemoresistance and Prognosis

<div><p>Lung cancer is the leading cause of death from malignant diseases worldwide, with the non-small cell (NSCLC) subtype accounting for the majority of cases. NSCLC is characterized by frequent genomic imbalances and copy number variations (CNVs), but the epigenetic aberrations that are associated with clinical prognosis and therapeutic failure remain not completely identify. In the present study, a total of 55 lung cancer patients were included and we conducted genomic and genetic expression analyses, immunohistochemical protein detection, DNA methylation and chromatin immunoprecipitation assays to obtain genetic and epigenetic profiles associated to prognosis and chemoresponse of NSCLC patients. Finally, siRNA transfection-mediated genetic silencing and cisplatinum cellular cytotoxicity assays in NSCLC cell lines A-427 and INER-37 were assessed to describe chemoresistance mechanisms involved. Our results identified high frequencies of CNVs (66–51% of cases) in the 7p22.3–p21.1 and 7p15.3–p15.2 cytogenetic regions. However, overexpression of genes, such as <i>MEOX2</i>, <i>HDAC9</i>, <i>TWIST1</i> and <i>AhR</i>, at 7p21.2–p21.1 locus occurred despite the absence of CNVs and little changes in DNA methylation. In contrast, the promoter sequences of <i>MEOX2</i> and <i>TWIST1</i> displayed significantly lower/decrease in the repressive histone mark H3K27me3 and increased in the active histone mark H3K4me3 levels. Finally these results correlate with poor survival in NSCLC patients and cellular chemoresistance to oncologic drugs in NSCLC cell lines in a <i>MEOX2</i> and <i>TWIST1</i> overexpression dependent-manner. In conclusion, we report for the first time that <i>MEOX2</i> participates in chemoresistance irrespective of high CNV, but it is significantly dependent upon H3K27me3 enrichment probably associated with aggressiveness and chemotherapy failure in NSCLC patients, however additional clinical studies must be performed to confirm our findings as new probable clinical markers in NSCLC patients.</p></div

Outcomes clinical data of NSCLC patients.

<p>(AD) Adenocarcinoma Patients; (N.D.) Not Detected; and (N.A.) Not Applied.</p><p>Outcomes clinical data of NSCLC patients.</p

Genome-wide analysis of normal lung and paired tumors.

<p>Focal analysis of the whole genome and chromosome 7. (A) A whole-genome amplification and a bioinformatics segmental analysis of three lung cancer patients was conducted using matched, histologically adjacent non-involved lung tissue (LNAT) and lung carcinomas (LT). (B) Whole-genome and focal analyses of normal lung, lung precursor lesions and lung carcinomas (18–14 of 27 lung lesions with high CNV). (C) A focal analysis of chromosome 7 revealed a high frequency of CNV at region 7p21 (arrow) in precursor lung lesions and lung carcinomas.</p

mRNA expression, DNA methylation and protein expression analyses in lung carcinomas.

<p>(A) <i>MEOX2</i>, <i>HDAC9</i> and <i>TWIST1</i> mRNA expression. Error bars represent 95% of confidence interval of the mean. (B) Protein expression in adjacent non-involved lung tissue (LNAT), lung precursor lesions (LP) and lung carcinomas (LT) (microphotographs at 200X and 400X), as well as formalin-fixed and paraffin-embedded (FFPE) and fresh frozen (FF) tissues, were compared. (C) Promoter sequence methylation analysis. The differences were statistically significant with respect to the LNAT and were detected using Fisher's exact test, an unpaired <i>t</i>-test and a Mann-Whitney U test, <b>*</b><i>p</i>≤0.05; <b>**</b><i>p</i>≤0.005; <b>***</b><i>p</i>≤0.001. Error bars indicating min to max rank with 95% confidence interval, and box plots represent standard deviations of the mean.</p

Histone modification profile at the <i>MEOX2</i> and <i>TWIST1</i> promoters under baseline conditions and after cisplatinum treatment in NSCLC cell lines (A-427 and INER-37).

<p>(A) <i>MEOX2</i> histone profile under baseline conditions. (B) <i>MEOX2</i> histone modification profile changes after cisplatinum treatment. (C) <i>TWIST1</i> histone profile under baseline conditions. (D) <i>TWIST1</i> histone modification profile changes after cisplatinum treatment. (E) <i>MEOX2</i> mRNA expression induction by cisplatinum stimulation as compared to the baseline conditions from both NSCLC cell lines. (F) <i>TWIST1</i> mRNA expression induction by cisplatinum stimulation as compared to the baseline conditions from both NSCLC cell lines. (G) Chemoresistance curves evaluating cellular viability for both NSCLC cell lines. (H) <i>MEOX2</i> and <i>TWIST1</i> siRNA silencing assays and cellular viability analyses. Statistically significant differences with respect to the baseline control conditions as assessed using an unpaired t-test, *<i>p</i>≤0.05 and **<i>p</i>≤0.001. Error bars represent standard errors of the mean of three technical replicates.</p