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In Epithelial Cancers, Aberrant COL17A1 Promoter Methylation Predicts Its Misexpression and Increased Invasion

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In Epithelial Cancers, Aberrant COL17A1 Promoter Methylation Predicts Its Misexpression and Increased Invasion

Pulari U Thangavelu et al. Clin Epigenetics.

Abstract

Background: Metastasis is a leading cause of death among cancer patients. In the tumor microenvironment, altered levels of extracellular matrix proteins, such as collagens, can facilitate the first steps of cancer cell metastasis, including invasion into surrounding tissue and intravasation into the blood stream. However, the degree of misexpression of collagen genes in tumors remains understudied, even though this knowledge could greatly facilitate the development of cancer treatment options aimed at preventing metastasis.

Methods: We systematically evaluate the expression of all 44 collagen genes in breast cancer and assess whether their misexpression provides clinical prognostic significance. We use immunohistochemistry on 150 ductal breast cancers and 361 cervical cancers and study DNA methylation in various epithelial cancers.

Results: In breast cancer, various tests show that COL4A1 and COL4A2 overexpression and COL17A1 (BP180, BPAG2) underexpression provide independent prognostic strength (HR = 1.25, 95% CI = 1.17-1.34, p = 3.03 × 10-10; HR = 1.18, 95% CI = 1.11-1.25, p = 8.11 × 10-10; HR = 0.86, 95% CI = 0.81-0.92, p = 4.57 × 10-6; respectively). Immunohistochemistry on ductal breast cancers confirmed that the COL17A1 protein product, collagen XVII, is underexpressed. This strongly correlates with advanced stage, increased invasion, and postmenopausal status. In contrast, immunohistochemistry on cervical tumors showed that collagen XVII is overexpressed in cervical cancer and this is associated with increased local dissemination. Interestingly, consistent with the opposed direction of misexpression in these cancers, the COL17A1 promoter is hypermethylated in breast cancer and hypomethylated in cervical cancer. We also find that the COL17A1 promoter is hypomethylated in head and neck squamous cell carcinoma, lung squamous cell carcinoma, and lung adenocarcinoma, in all of which collagen XVII overexpression has previously been shown.

Conclusions: Paradoxically, collagen XVII is underexpressed in breast cancer and overexpressed in cervical and other epithelial cancers. However, the COL17A1 promoter methylation status accurately predicts both the direction of misexpression and the increased invasive nature for five out of five epithelial cancers. This implies that aberrant epigenetic control is a key driver of COL17A1 gene misexpression and tumor cell invasion. These findings have significant clinical implications, suggesting that the COL17A1 promoter methylation status can be used to predict patient outcome. Moreover, epigenetic targeting of COL17A1 could represent a novel strategy to prevent metastasis in patients.

Keywords: Breast cancer; Cervical cancer; Collagen XVII; Epigenetics; Invasion; Prognosis.

Figures

Fig. 1
Fig. 1
Reduced COL17A1 expression correlates with poor breast cancer patient prognosis. a Fold change in normalized COL4A1, COL4A2 (4A2), and COL17A1 (17A1) expression using 28 combined previously published datasets. p values: t test. b Fold change in normalized COL17A1 expression using the TCGA breast cancer RNAseq dataset. p value: Mann-Whitney U test. ce Distant metastasis-free survival (c), recurrence-free survival (d), and overall survival (e) for breast cancer patients whose tumors express high (green) or low (red) COL17A1 levels. Patients were split in low and high expression groups using the median expression level as the cut-off [23]. p values: log-rank test. p value summaries: n/s not significant; ****p < 0.0001
Fig. 2
Fig. 2
Collagen XVII is underexpressed in breast cancer. Tissue microarrays with a total of 57 normal breast tissues, 20 hyperplastic breast tissues, and 150 breast ductal carcinomas were stained with an anti-collagen XVII antibody [15, 16]. Numbers of samples in each category, as well as the total number of samples, are indicated. Percentages indicate frequencies of observations per row. Scale bar, 50 μm
Fig. 3
Fig. 3
Collagen XVII is overexpressed in cervical cancer. a COL17A1 is overexpressed twofold in the TCGA cervical cancer dataset. p values: t test. b, c Tissue microarrays with 31 normal control cervical tissue samples (b) and 331 cervical squamous cell carcinomas, 27 cervical adenocarcinomas, and 3 cervical adenosquamous carcinomas (c) were stained with an anti-collagen XVII antibody. Numbers of samples in each category, as well as the total number of samples, are indicated. Percentages indicate frequencies of observations per row. Scale bar, 50 μm
Fig. 4
Fig. 4
The COL17A1 promoter methylation status predicts the direction of misexpression in breast and cervical cancer. a COL17A1 allelic copy number gains and losses in relation to normalized COL17A1 expression level. Data are extracted from TCGA breast cancer RNAseq V2 RSEM and SNP6 array GISTIC copy number datasets. Error bars represent standard error of the mean. p values: Mann-Whitney U test. b Heat map of the degree of promoter methylation based on β values for each indicated probe. Each three-probe column corresponds to a sample. Data were extracted from the TCGA Illumina Infinium HumanMethylation450 breast cancer dataset [21, 22]. c Box plot for COL17A1 promoter methylation comparing normal samples to all samples, as well as to samples with indicated allelic copy numbers. Whiskers represent 10–90 percentiles of the data. p values: Mann-Whitney U test. d Scatter plot of COL17A1 promoter methylation compared to normalized COL17A1 gene expression. p value for linear regression line: Spearman correlation. eh Graphs as in (ad), respectively, for cervical cancer. Data were derived from the TCGA cervical dataset. p value summaries: n/s not significant; *p < 0.05; **p < 0.01; ****p < 0.0001
Fig. 5
Fig. 5
The COL17A1 promoter is hypomethylated in head and neck and lung cancers. ac Methylation status of the COL17A1 promoter in head and neck squamous cell carcinoma (SCC), lung adenocarcinoma, and lung SCC using the respective TCGA datasets [–43], as in Fig. 4c. p values: Mann-Whitney U test. df Scatter plots of COL17A1 promoter methylation compared to normalized gene expression for indicated cancer types, as in Fig. 4d. p values: Spearman correlation for linear regression. g Box plot comparing the absolute COL17A1 mRNA levels in five epithelial tumor types and their respective matched normal control tissues. Data were extracted from TCGA RNAseq datasets [, , –43]. N normal tissues, T tumor tissues. Sample numbers are indicated below each box. p values: Mann-Whitney U tests. p value summaries: **p < 0.01; ***p < 0.001; ****p < 0.0001

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