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The gene, collagen triple helix repeat containing 1 (CTHRC1), has been reported to increase in several kinds of human solid cancers and is associated with tumor invasion and metastasis. To date, the expression and function of CTHRC1 in gastric cancer (GC) have not been reported. The aim of this study was to investigate the expression levels and regulatory transcription mechanisms of CTHRC1 in GC. Immunohistochemical analysis revealed that CTHRC1 expression was markedly increased in carcinoma compared with normal gastric mucosa, chronic atrophic gastritis, and intestinal metaplasia (P < 0.05 for all), and this overexpression in tumor was related to depth of tumor invasion. Moreover, RNA interference-mediated knockdown and ectopic expression of CTHRC1 showed that CTHRC1 promoted tumor cell invasion in vitro. We then investigated the mechanisms underlying the aberrant expression of CTHRC1 in GC and found that CTHRC1 expression was restored after GC cell lines were treated with the demethylating agent, 5-aza-2'-deoxycytidine. Transforming growth factor-β1 led to an increase in levels of CTHRC1 mRNA and protein. Overall, our data revealed that the upregulated expression of CTHRC1 in gastric carcinogenesis contributes to tumor cell invasion and metastasis, and promoter demethylation and transforming growth factor-β1 may co-regulate the expression of CTHRC1. (Cancer Sci 2012; 103: 1327–1333)
Gastric cancer (GC) is one of most common cancers worldwide. It has been reported that multiple genetic and epigenetic alterations are implicated in the development of GC,[2, 3] but the molecular mechanisms of its pathogenesis remain unclear. More recently, a growing body of published reports has defined a class of genes that function, positively or negatively, in regulating the invasion and metastasis of malignant cancer cells.[4-6] As invasion and metastasis is the leading cause of GC-related death, identifying novel metastasis-related molecules may be useful for the prediction of prognosis and the treatment of advanced GC.
Collagen triple helix repeat containing 1 (CTHRC1) was first found in a screen for differentially expressed sequences in balloon-injured versus normal rat arteries. Tang et al. reported that CTHRC1 was increased in several kinds of human solid cancers and associated with invasion and metastasis. However, to date, no investigation has addressed the expression of CTHRC1 and the mechanisms that underlie the aberrant expression of CTHRC1 in GC.
In this study, we have found that CTHRC1 expression was increased in gastric carcinogenesis by immunohistochemical (IHC) analysis. This increase was associated with depth of tumor invasion. Moreover, RNA interference-mediated knockdown and re-expression of CTHRC1 showed that CTHRC1 promoted tumor cell invasion and metastasis in vitro. In addition, we investigated the mechanisms by which CTHRC1 expression was regulated.
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- Materials and Methods
- Disclosure Statement
Gastric carcinogenesis is a multistep and chronic process, where the gastric tissues undergo atrophy, intestinal metaplasia, dysplasia, and ultimately, cancer. CTHRC1 is a glycosylated protein that contains a NH2-terminal signal peptide for extracellular secretion, a short collagen triple helix repeat of 36 amino acids, and a COOH-terminal globular domain.[7, 17, 18] It has been found to be overexpressed in several malignant tumors, including breast cancer, GC, and malignant melanoma. However, CHTRC1 was simply screened out as being increased in GC, but an initial biochemical and functional characterization of this molecule in GC has not been undertaken.
In this study, using IHC, we observed that the level of CTHRC1 gradually increased with the development of GC. Furthermore, GC samples with deeper depths of invasion showed higher levels of CTHRC1 expression, indicating that CTHRC1 could have important implications in GC invasion and metastasis. Importantly, this result was further confirmed through in vitro experiments. Re-expression of CTHRC1 in GC cells led to a significant increase in their ability to invade, whereas the knockdown of CTHRC1 gene expression by siRNA had an adverse effect. Therefore, CTHRC1 is a potential metastasis-related gene in gastric carcinogenesis. In addition, it was interesting to note that CTHRC1 staining of GC was detected mainly in the cytoplasm and extracellular space, but CHTRC1 expression in normal gastric mucosa, CAG, and intestinal metaplasia tissues was mostly present in the nucleus. CTHRC1 is a secreted extracellular protein. Pyagay et al. found that CTHRC1 may undergo proteolytic processing and this could have implications for the activity of the molecule. Our ongoing studies showed that there was no difference in mRNA levels of collagen type I and III between control and CTHRC1 overexpressing cells (data not shown). Therefore, it is possible that the secreted form of CTHRC1 overproduced by GC cells may act on the surrounding microenvironment, such as the stromal cells and ECM, and thus promotes tumor invasion.
To date, the regulating mechanism by which CTHRC1 is upregulated in human cancers is not known. There are varied ways to regulate gene expression, including genetic variation, epigenetic variation, and cytokine induction, amongst others. Changes of gene expression are usually caused by mutation in exons and regulatory region (between nucleotides within 1 kb upstream of the transcription start site). In this study, we did not find any mutations in the above regions to be associated with the aberrant expression of CTHRC1. However, the samples are relatively small and further studies are needed to validate this result.
Promoter methylation, which can be a mechanism for the inactivation of tumor suppressor genes, has been associated with cancer-specific expression differences in human malignancies.[20-22] To date, few examples of promoter hypomethylation of putative oncogenes have been reported.[23, 24] Here, our data on 5-aza-dC treatment and methylation analyses strongly indicate that CTHRC1 silencing is related to promoter hypermethylation. Hence, promoter demethylation may be one cause of upregulation of CHTRC1 in GC cell lines. Additional studies are needed to investigate the methylation status of CTHRC1 in GC tissues. Thus, CHTRC1 methylation might also function as a prognostic biomarker and therapeutic target.
Sequence analysis of the CTHRC1 promoter region reveals a binding site of Smad, which is responsive to regulation by TGF-β. It has been reported that dysregulation of TGF-β signaling is implicated in tumor growth, angiogenesis, invasion, and metastasis,[25-27] and the upregulation of TGF-β1 is present in a variety of human cancers, including GC.[28, 29] CTHRC1 is induced by TGF-β1 and bone morphogenetic protein-4 in murine NIH3T3 cells. Thus, we hypothesized that TGF-β might also induce CTHRC1 expression during gastric carcinogenesis. Our own in vitro studies showed that both CTHRC1 mRNA and protein levels increased gradually in response to TGF-β1. In addition, we observed that TGF-β-mediated Smad signaling activation was also increased, but this change did not coincide with the upregulation of CTHRC1. These results differed from those of LeClair et al., who reported that Cthrc1 is a cell-type-specific inhibitor of TGF-β, which in turn impacts collagen type I and III deposition, neointimal formation, and the dedifferentiation of smooth muscle cells. There are potential confounders for these findings: (i) compared with the function of CTHRC1 in tissue repair, TGF-β may play a reverse role in tumorigenesis; and (ii) the signaling pathways are very complex. CHTRC1 may participate in the TGF-β-mediated Smad signaling pathway, or may have no relation with this pathway and is only regulated by TGF-β1. Thus, we provide novel mechanistic evidence that TGF-β1 may upregulate CTHRC1 expression in GC. Further studies will be needed to address the relationship between CTHRC1 and TGF-β-mediated Smad signaling or other pathways. In addition, as epigenetic alterations in promoter methylation are associated with downregulation of gene expression, and the CTHRC1 promoter region shows a binding site of Smad, it is possible that promoter demethylation and TGF-β1 may co-regulate the expression of CTHRC1. Understanding the mechanism by which CTHRC1 is regulated requires further investigation in GC tissues.
In summary, CTHRC1 is a potential metastasis-related gene in gastric carcinogenesis. Promoter demethylation and TGF-β1 may coregulate the expression of CTHRC1. Understanding the mechanism by which CTHRC1 increases cell invasion requires further investigation. Thus, it will not only provide new insights into the metastasis mechanism but provide a new therapeutic strategy for the treatment of GC in the future.