In epithelial cancers, aberrant COL17A1 promoter methylation predicts its misexpression and increased invasion

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; HR = 1.18, 95% CI = 1.11–1.25, p = 8.11 × 10; HR = 0.86, 95% CI = 0.81–0.92, p = 4.57 × 10; 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

Gene expression profiling of tumour epithelial and stromal compartments during breast cancer progression

The progression of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) marks a critical step in the evolution of breast cancer. There is some evidence to suggest that dynamic interactions between the neoplastic cells and the tumour microenvironment play an important role. Using the whole-genome cDNA-mediated annealing, selection, extension and ligation assay (WG-DASL, Illumina), we performed gene expression profiling on 87 formalin-fixed paraffin-embedded (FFPE) samples from 17 patients consisting of matched IDC, DCIS and three types of stroma: IDC-S ( 10 mm from IDC or DCIS). Differential gene expression analysis was validated by quantitative real time-PCR, immunohistochemistry and immunofluorescence. The expression of several genes was down-regulated in stroma from cancer patients relative to normal stroma from reduction mammoplasties. In contrast, neoplastic epithelium underwent more gene expression changes during progression, including down regulation of SFRP1. In particular, we observed that molecules related to extracellular matrix (ECM) remodelling (e.g. COL11A1, COL5A2 and MMP13) were differentially expressed between DCIS and IDC. COL11A1 was overexpressed in IDC relative to DCIS and was expressed by both the epithelial and stromal compartments but was enriched in invading neoplastic epithelial cells. The contributions of both the epithelial and stromal compartments to the clinically important scenario of progression from DCIS to IDC. Gene expression profiles, we identified differential expression of genes related to ECM remodelling, and specifically the elevated expression of genes such as COL11A1, COL5A2 and MMP13 in epithelial cells of IDC. We propose that these expression changes could be involved in facilitating the transition from in situ disease to invasive cancer and may thus mark a critical point in disease development

Genomics and proteomics approaches to the study of cancer-stroma interactions

<p>Abstract</p> <p>Background</p> <p>The development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression.</p> <p>Methods</p> <p>The study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells.</p> <p>Results</p> <p>We observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (<it>ARID4A</it>, <it>CALR</it>, <it>GNB2L1</it>, <it>RNF10</it>, <it>SQSTM1</it>, <it>USP9X</it>) were validated by real time PCR.</p> <p>Conclusions</p> <p>A significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.</p