Collagen Triple Helix Repeat Containing 1 Research Articles

Lentivirus-Mediated Knockdown of CTHRC1 Inhibits Osteosarcoma Cell Proliferation and Migration.

Collagen triple helix repeat containing-1 (CTHRC1), a secreted protein, is transiently expressed in the arterial wall in response to injury, indicating that it may contribute to vascular remodeling by limiting collagen matrix deposition and promoting cell migration. Recent studies showed that it is aberrantly upregulated in most human solid tumors, yet its role in osteosarcoma remains unclear. In this study, the authors investigated the role of CTHRC1 in human osteosarcoma tumorigenesis. The authors used lentivirus-mediated short hairpin RNA (shRNA) against CTHRC1 to limit its endogenous expression in U2OS and SW1353 cells. Interestingly, they found that depletion of CTHRC1 significantly inhibited cell proliferation and colony formation in U2OS and SW1353 cells. Flow cytometry assay showed that knockdown of CTHRC1 increased the cell percentage of G0/G1 phase, resulting in cell cycle arrest in U2OS cells. Moreover, CTHRC1 silencing induced the cell cycle arrest by a decrease in the cell percentage in G0/G1 phase and increased in G2/M phase in SW1353 cells. In addition, crystal violet staining suggested CTHRC1 silencing inhibited migration of U2OS and SW1353 cells. These results demonstrated that CTHRC1 might play an important role in osteosarcoma progression.

Gene expression analyses of primary melanomas reveal CTHRC1 as an important player in melanoma progression.

Melanoma is notorious for its high tendency to metastasize and its refractoriness to conventional treatments after metastasis, and the responses to most targeted therapies are short-lived. A better understanding of the molecular mechanisms behind melanoma development and progression is needed to develop more effective therapies and to identify new markers to predict disease behavior. Here, we compared the gene expression profiles of benign nevi, and non-metastatic and metastatic primary melanomas to identify any common changes in disease progression. We identified several genes associated with inflammation, angiogenesis, and extracellular matrix modification to be upregulated in metastatic melanomas. We selected one of these genes, collagen triple helix repeat containing 1 (CTHRC1), for detailed analysis, and found that CTHRC1 was expressed in both melanoma cells and the associated fibroblasts, as well as in the endothelium of tumor blood vessels. Knockdown of CTHRC1 expression by shRNAs in melanoma cells inhibited their migration in Transwell assays and their invasion in three-dimensional collagen and Matrigel matrices. We also elucidated the possible down-stream effectors of CTHRC1 by gene expression profiling of the CTHRC1-knockdown cells. Our analyses showed that CTHRC1 is regulated coordinately with fibronectin and integrin β3 by the pro-invasive and -angiogenic transcription factor NFATC2. We also found CTHRC1 to be a target of TFGβ and BRAF. These data highlight the importance of tumor stroma in melanoma progression. Furthermore, CTHRC1 was recognized as an important mediator of melanoma cell migration and invasion, providing together with its regulators—NFATC2, TGFβ, and BRAF—attractive therapeutic targets against metastatic melanomas.

MicroRNA-98 suppresses cell proliferation, migration and invasion by targeting collagen triple helix repeat containing 1 in hepatocellular carcinoma.

MicroRNAs (miRNAs) are emerging as critical regulators in carcinogenesis and tumor progression. miR-98 has previously been verified to be important in tumor progression, however, its function in hepatocellular carcinoma (HCC) remains to be elucidated. The expression levels of miR-98 in HCC tissues and cell lines were determined by reverse transcription quantitative polymerase chain reaction. Subsequently, the effect of miR‑98 on cell proliferation, migration and invasion was evaluated by MTT assay, transwell migration assay and transwell invasion assay. Furthermore, a luciferase reporter assay was conducted to confirm the action of miR‑98 on downstream target genes, including collagen triple helix repeat containing 1 (CTHRC1). In the present study, it was confirmed that miR‑98 was significantly downregulated in HCC tissues and cell lines. Overexpression of miR‑98 inhibited HCC cell proliferation, migration and invasion in vitro. In addition, at the molecular level, the tumor oncogene CTHRC1 was identified to be the direct target of miR-98. Our findings suggested that miR‑98 was significantly downregulated in HCC and suppressed HCC cell proliferation, migration and invasion partially via the downregulation of CTHRC1. Thus, these data demonstrated that miR-98 could be a potential therapeutic target in HCC.

Correlation of Collagen Triple Helix Repeat Containing 1 Overexpression With Lymph Node and Peritoneal Metastasis in Epithelial Ovarian Cancer.

This study aimed to reveal whether collagen triple helix repeat containing 1 (CTHRC1) was a predictor of peritoneal and lymph node metastases in epithelial ovarian cancer, which had not been researched by others previously. Western blot analysis showed that dramatic overexpression of CTHRC1 could be seen in most metastatic tissues. Univariate and multivariate logistic regression analyses demonstrated that overexpression of CTHRC1 was linked with peritoneal dissemination and lymph node metastasis in epithelial ovarian cancer. The negative and sensitivity-predictive values of CTHRC1 staining were excellent for both lymph node and peritoneal metastases. The odds ratio of high versus low staining for peritoneal dissemination was 2.250 (95% confidence interval, 1.126-4.496), and that for lymph node metastasis was 13.102 (95% confidence interval, 6.036-28.439). Collagen triple helix repeat containing 1 may potentially be used as a predictive marker of clinical progression in ovarian cancer either alone or in combination with other markers.

High expression of CTHRC1 promotes EMT of epithelial ovarian cancer (EOC) and is associated with poor prognosis.

Collagen triple helix repeat-containing 1 (CTHRC1) is aberrantly overexpressed in multiple malignant tumors. However, the expression characteristics and function of CTHRC1 in epithelial ovarian cancer (EOC) remain unclear. We found that CTHRC1 expression was up-regulated in the paraffin-embedded EOC tissues compared to borderline or benign tumor tissues. CTHRC1 expression was positively correlated with tumor size (p = 0.008), menopause (p = 0.037), clinical stage (p = 0.002) and lymph node metastasis (p < 0.001) and was also an important prognostic factor for the overall survival of EOC patients, as revealed by Kaplan-Meier analysis. CTHRC1 increased the invasive capabilities of EOC cells in vitro by activating the Wnt/β-catenin signaling pathway. We showed that ectopic transfection of CTHRC1 in EOC cells up-regulated the expression of EMT markers such as N-cadherin and vimentin, and EMT-associated transcriptional factor Snail. Knockdown of CTHRC1 expression in EOC cells resulted in down-regulation of N-cadherin, vimentin, Snail and translocation of β-catenin. Collectively, CTHRC1 may promote EOC metastasis through the induction of EMT process and serve as a potential biomarker for prognosis as well as a target for therapy.

Treatment of cholestatic fibrosis by altering gene expression of Cthrc1: Implications for autoimmune and non-autoimmune liver disease

Collagen triple helix repeat containing-1 (Cthrc1) is a documented specific inhibitor of TGF-β signaling. Based on this observation, we developed the hypothesis that knocking in/knocking out the Cthrc1 gene in murine models of cholestasis would alter the natural history of cholestatic fibrosis. To study this thesis, we studied two murine models of fibrosis, first, common bile duct ligation (CBDL) and second, feeding of 3, 5-diethoxy-carbonyl-1, 4-dihydrocollidine (DDC). In both models, we administered well-defined adenoviral vectors that expressed either Cthrc1 or, alternatively, a short hairpin RNA (shRNA)-targeting Cthrc1 either before or after establishment of fibrosis. Importantly, when Cthrc1 gene expression was enhanced, we noted a significant improvement of hepatic fibrosis, both microscopically and by analysis of fibrotic gene expression. In contrast, when Cthrc1 gene expression was deleted, there was a significant exacerbation of fibrosis. To identify the mechanism of action of these significant effects produced by knocking in/knocking out Cthrc gene expression, we thence studied the interaction of Cthrc1 gene expression using hepatic stellate cells (HSCs) and human LX-2 cells. Importantly, we demonstrate that Cthrc1 is induced by TGF-β1 via phospho-Smad3 binding to the promoter with subsequent transcription activation. In addition, we demonstrate that Cthrc1 inhibits TGF-β signaling by accelerating degradation of phospho-Smad3 through a proteosomal pathway. Importantly, the anti-fibrotic effects can be recapitulated with a truncated fragment of Cthrc1. In conclusion, our findings uncover a critical negative feedback regulatory loop in which TGF-β1 induces Cthrc1, which can attenuate fibrosis by accelerating degradation of phospho-Smad3.

Hepatitis B virus hijacks CTHRC1 to evade host immunity and maintain replication.

Hepatitis B virus (HBV) infection causes acute and chronic liver diseases, but is not directly cytopathic. Liver injury results from repeated attempts of the cellular immune response system to control the viral infection. Here, we investigate the roles of cellular factors and signaling pathways involved in the regulation of HBV replication to reveal the mechanism underlying HBV infection and pathogenesis. We show that collagen triple helix repeat containing 1 (CTHRC1) expression is elevated in HBV-infected patients and in HBV-transfected cells through epigenetic modification and transcriptional regulation. CTHRC1 facilitates HBV replication in cultured cells and BALB/c mice by activating the PKCα/ERK/JNK/c-Jun cascade to repress the IFN/JAK/STAT pathway. HBV-activated CTHRC1 downregulates the activity of type I interferon (IFN), the production of IFN-stimulated genes (ISGs), and the phosphorylation of signal transducer and activator of transcription 1/2 (STAT1/2), whereas it upregulates the phosphorylation and ubiquitination of type I IFN receptors (IFNARα/β). Thus, our results show that HBV uses a novel mechanism to hijack cellular factors and signal cascades in order to evade host antiviral immunity and maintain persistent infection. We also demonstrate that CTHRC1 has a novel role in viral infection.

Abstract A85: Collagen triple helix repeat containing-1 enhances the aggressiveness of pancreatic tumor by increased cancer cell motility and adhesiveness

Abstract Collagen triple helix repeat containing-1 (CTHRC1) is a secreted protein involved in vascular remodeling, bone formation and developmental morphogenesis. CTHRC1 is expressed in human cancers such as breast cancer and melanoma. In this study, we show that CTHRC1 is highly expressed in human pancreatic cancer tissues and plays a role in the progression and metastasis of the disease. CTHRC1 promoted primary tumor growth and metastatic spread of cancer cells to distant organs in orthotopic xenograft tumor mouse models. Overexpression of CTHRC1 in cancer cells resulted in increased motility and adhesiveness. CTHRC1 activated several key signaling molecules, including Src, focal adhesion kinase, paxillin, mitogen-activated protein kinase kinase (MEK), extracellular signal-regulated kinase and Rac1. Treatment of xenograft mice of pancreatic cancer cells with a CTHRC1-specific monoclonal antibody led to significantly smaller tumor sizes compared with those of untreated control mice. Collectively, our results suggest that CTHRC1 has a role in pancreatic cancer progression and metastasis by regulating migration and adhesion activities of cancer cells. Citation Format: Leem Sun-Hee, Kang Tae-Hong, Chung Jin Woong, Hwang Yeonsil, Kim Seokho, Koh Sang Seok. Collagen triple helix repeat containing-1 enhances the aggressiveness of pancreatic tumor by increased cancer cell motility and adhesiveness. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A85.

Abstract 676: Microrna-30 and Cthrc1: Translating Aging into Vascular Stiffness and Abdominal Aortic Aneurysm

Abdominal aortic aneurysm (AAA) is a major source of vascular morbidity and mortality, with increasing age representing one of the strongest risk factors. While increases in aortic extra-cellular matrix deposition may be protective, changes in vessel architecture with age result in heterogeneous stiffness and vascular calcification, which appear to pre-dispose to AAA development. MicroRNAs (miRs) are key regulators of vascular homeostasis and pathobiology. Array profiling and qRT-PCR of aneurysmal aortic tissue in a murine AAA model (elastase-infused C57/B6) showed significant down-regulation of the miR-30 family, which is believed to have a role in vascular calcification. Increased age augmented this response, particularly for miR-30a, 30b and 30c. RNASeq profiling of a related AAA model (Ang-II in ApoE-/- KO) showed similar aortic down-regulation. Further, of differentially down-regulated miRs, miR-30 had the highest inverse correlation with mRNA gene targets. One predicted target - Cthrc1 (collagen triple helix repeat containing-1) - was the most consistently and significantly up-regulated gene across all time points in the AAA models. The protein resides within vascular smooth muscle cells (SMCs) and fibroblasts, responds to injury, and may regulate collagen expression and deposition. Immunofluorescence staining of AAA vs. normal aortas revealed increased expression of CTHRC1. We further found that miR-30 family members bind to the CTHRC1 3’ UTR and regulate gene expression in vitro, and that antagomir suppression of miR-30 upregulates CTHRC1 in SMCs. We also demonstrated that signaling pathways known to increase in activity with aging within the aorta, and which are associated with vascular calcification and fibrosis (e.g. IL6-based inflammatory signaling, TGF-β signaling, and BMP-2 signaling) down-regulate miR-30 family expression in SMCs, and inversely up-regulate CTHRC1 expression. Forced overexpression of miR-30 in SMC in vitro down-regulates RUNX2, a key promoter of vascular stiffness and calcification. Taken together, these results suggest a significant role for miR-30 in pathways related to matrix deposition and calcification, regulating aortic pathobiology and age-related susceptibility to AAA.

Collagen Triple Helix Repeat Containing-1 (CTHRC1) Expression in Oral Squamous Cell Carcinoma (OSCC): Prognostic Value and Clinico-Pathological Implications.

BACKGROUND: Collagen Triple Helix Repeat Containing 1 (CTHRC1) is a protein often found to be over-expressed in various types of human cancers. However, correlation between CTHRC1 expression level with clinico-pathological characteristics and prognosis in oral cancer remains unclear. Therefore, this study aimed to determine mRNA and protein expression of CTHRC1 in oral squamous cell carcinoma (OSCC) and to evaluate the clinical and prognostic impact of CTHRC1 in OSCC.METHODS: In this study, mRNA and protein expression of CTHRC1 in OSCCs were determined by quantitative PCR and immunohistochemistry, respectively. The association between CTHRC1 and clinico-pathological parameters were evaluated by univariate and multivariate binary logistic regression analyses. Correlation between CTHRC1 protein expressions with survival were analysed using Kaplan-Meier and Cox regression models.RESULTS: Current study demonstrated CTHRC1 was significantly overexpressed at the mRNA level in OSCC. Univariate analyses indicated a high-expression of CTHRC1 that was significantly associated with advanced stage pTNM staging, tumour size ≥ 4 cm and positive lymph node metastasis (LNM). However, only positive LNM remained significant after adjusting with other confounder factors in multivariate logistic regression analyses. Kaplan-Meier survival analyses and Cox model demonstrated that patients with high-expression of CTHRC1 protein were associated with poor prognosis and is an independent prognostic factor in OSCC.CONCLUSION: This study indicated that over-expression of CTHRC1 potentially as an independent predictor for positive LNM and poor prognosis in OSCC.

Overexpression of collagen triple helix repeat containing 1 (CTHRC1) is associated with tumour aggressiveness and poor prognosis in human non-small cell lung cancer.

Collagen triple helix repeat-containing 1 (CTHRC1), a novel oncogene, was identified to be aberrantly overexpressed in several malignant tumors. However, the expression profile of CTHRC1 and its clinical significance in non-small cell lung cancer (NSCLC) remain unknown. In this study, we showed that CTHRC1 was evidently overexpressed in human NSCLC tissues and NSCLC cell lines at the protein and mRNA level. Ectopic up-regulation of CTHRC1 in cancer cells resulted in elevated invasive and proliferative abilities, which were attenuated by the specific CTHRC1 siRNA. The biological effect of CTHRC1 on metastasis and proliferation was mediated by the activation of the Wnt/β-catenin pathway. Furthermore, CTHRC1 immunoreactivity was evidently overexpressed in paraffin-embedded NSCLC tissues (212/292, 72.60%) in comparison to corresponding adjacent non-cancerous tissues (6/66, 9.09%) (p<0.001). Clinicopathologic analysis showed that CTHRC1 expression was significantly correlated with differentiation degree (p<0.001), clinical stage (p<0.001), T classification (p<0.001), lymph node metastasis (p=0.013) and distant metastasis (p<0.001). Kaplan-Meier analysis revealed that patients with high CTHRC1 expression had poorer overall survival rates than those with low CTHRC1 expression. Multivariate analysis indicated that CTHRC1 expression was an independent prognostic factor for the overall survival of NSCLC patients. Collectively, CTHRC1 plays important roles in NSCLC progression, and the evaluation of CTHRC1 expression could serve as a potential marker for metastasis progression and prognosis in NSCLC patients.

Collagen triple helix repeat containing 1 is overexpressed in hepatocellular carcinoma and promotes cell proliferation and motility.

Although several therapeutic options are available for hepatocellular carcinoma (HCC), the outcome is still very poor. One reason is the complexity of signal transduction in the pathogenesis of HCC. The aim of this study was to identify new HCC-related genes and to investigate the functions of these genes in the pathogenesis and progression of HCC. Whole genomes of 15 surgically resected HCC specimens were examined for copy number alterations with comparative genomic hybridization. Gene expression was compared between HCC and normal liver tissues. The roles of the new genes in the progression of HCC were studied using cultured cell lines. Copy number gain in chromosome 8q was detected in 53% of HCC tissues examined. The gene that coded for collagen triple helix repeat containing 1 (CTHRC1), located at chromosome 8q22.3, was overexpressed in HCC compared with normal or liver cirrhosis tissues and identified as a new HCC-related gene. CTHRC1 deletion with short hairpin RNA significantly reduced proliferation, migration and invasion of HepG2 and Huh7 cells. In addition, mRNA of integrins β-2 and β-3 was downregulated, with deletion of CTHRC1 in these cells. Immunohistochemical staining on resected HCC tissues showing positive staining areas for CTHRC1 was significantly greater in poorly-differentiated HCC compared with well-differentiated HCC. Moreover, some cases showed strong staining for CTHRC1 in invasive areas of HCC. CTHRC1 has the potential to be a new biomarker for the aggressive HCC, and to be a new therapeutic target in treating HCC.

CTHRC1 Acts as a Prognostic Factor and Promotes Invasiveness of Gastrointestinal Stromal Tumors by Activating Wnt/PCP-Rho Signaling

Gastrointestinal stromal tumors (GISTs) are the major gastrointestinal mesenchymal tumors with a variable malignancy ranging from a curable disorder to highly malignant sarcomas. Metastasis and recurrence are the main causes of death in GIST patients. To further explore the mechanism of metastasis and to more accurately estimate the recurrence risk of GISTs after surgery, the clinical significance and functional role of collagen triple helix repeat containing-1 (CTHRC1) in GIST were investigated. We found that CTHRC1 expression was gradually elevated as the risk grade of NIH classification increased, and was closely correlated with disease-free survival and overall survival in 412 GIST patients. In vitro experiments showed that recombinant CTHRC1 protein promoted the migration and invasion capacities of primary GIST cells. A luciferase reporter assay and pull down assay demonstrated that recombinant CTHRC1 protein activated noncanonical Wnt/PCP-Rho signaling but inhibited canonical Wnt signaling. The pro-motility effect of CTHRC1 on GIST cells was reversed by using a Wnt5a neutralizing antibody and inhibitors of Rac1 or ROCK. Taken together, these data indicate that CTHRC1 may serve as a new predictor of recurrence risk and prognosis in post-operative GIST patients and may play an important role in facilitating GIST progression. Furthermore, CTHRC1 promotes GIST cell migration and invasion by activating Wnt/PCP-Rho signaling, suggesting that the CTHRC1-Wnt/PCP-Rho axis may be a new therapeutic target for interventions against GIST invasion and metastasis.

Periostin and its emerging role in systemic carcinogenesis

Dear Editor, I read with great interest the recent article by Merle et al. [1] periostin may also play a major role in carcinogenesis and tumor progression in a number of systemic malignancies besides its role in bone metabolism. For instance, periostin augments lymph angiogenesis in head and neck malignancies. It mediates this effect directly by enhancing tube formation of lymphatic endothelial cells as well as by upregulating the expression of vascular endothelial growth factor C [2]. Akt is responsible in part for periostininduced lymph angiogenesis in head and neck squamous cell carcinomas. Similarly, increased upregulation of periostin expression is seen in nasopharyngeal carcinomas, and it significantly influences the stage of the tumor. Reduced overall survival is typically seen in nasopharyngeal carcinomas with accentuated periostin expression, and periostin in fact is an independent prognostic indicator [3]. Similarly, accentuated periostin expression is seen in nearly 31 % of breast carcinomas [4]. Patients with increased periostin expression tend to show a poor clinical outcome and poor disease-specific survival. Triple negative breast carcinomas typically tend to show higher periostin levels. In fact, periostin is an independent parameter influencing tumor prognosis in mammary malignancies as it is closely related to tumor stage as well as grade [2]. Interestingly, the risk of tumor metastasis in patients with high periostin expression is augmented by accentuated collagen triple helix repeat containing 1 (CTHRC1) expression [5]. Recently, a periostin neutralizing antibody, PN1-Ab, has been developed, and it attenuates tumor growth of the primary breast carcinoma in animal models [6]. The antibody also increases survival rates by decreasing bone destruction as well as distant metastasis. Similarly, upregulation of periostin is seen in non-small cell lung carcinomas [7]. Typically, higher levels are seen in pulmonary adenocarcinomas in contrast to comparatively lower levels in squamous cell pulmonary carcinomas [3]. Periostin expression correlates closely with tumor differentiation and grade [8]. Larger tumors tend to have higher periostin levels indicating its role in tumor progression and invasiveness. Similarly, accentuated expression of periostin is seen in 34% of prostate malignancies. The periostin expression closely correlates with the tumor stage [9]. Similarly, a close association has been seen between high periostin expression and highGleason scores as well as decreased PSA relapse-free survival times [4, 6]. This makes periostin a possible therapeutic target in the management of prostate carcinomas. Similarly, periostin plays a role in enhancing tumor growth in cholangiocarcinomas. It has modulatory effect on a PI3K/AKT-dependent pathway resulting in the ultimate activation of the integrin ITGα5β1 [10]. Increased expression of intracellular pAKT is seen as a result, which facilitates increased invasiveness of the primary tumor. An increased periostin serum level is one emerging way to distinguish cholangiocarcinomas from liver malignancies [11]. The above examples clearly illustrate the significant role that periostin plays in systemic carcinogenesis and the urgent need to identify treatment modalities to modulate periostin expression.

Cthrc1 overexpression is an independent prognostic marker in gastric cancer.

Collagen triple helix repeat containing 1 (CTHRC1) was identified as a novel gene expressed in the adventitia and neointima on arterial injury and was found to be overexpressed in several malignant tumors, such as breast cancer and malignant melanoma. However, the expression of Cthrc1 and its role in gastric cancer progression remain unknown. We investigated the expression of the Cthrc1 protein by immunohistochemistry in 30 normal tissues from the control subjects and 166 gastric carcinomas and analyzed its correlation with various clinicopathological features, including patient outcome. Cthrc1 immunoreactivity was overexpressed in gastric carcinoma cases compared with normal tissues (P < .001). High Cthrc1 expression was found in 108 (65.06%) of these 166 carcinomas and was positively correlated with the American Joint Committee on Cancer stage classification, depth of gastric wall invasion, lymph node metastasis, lymphovascular space involvement, and recurrence but not with age, tumor site, and carcinoembryonic antigen level. Patients with high Cthrc1 expression had significantly poorer overall survival and disease-free survival compared with patients with low expression of Cthrc1 (P = .001 and P = .002, respectively). Multivariate analysis showed that high Cthrc1 expression was an independent prognostic factor for both overall survival and disease-free survival of patients with gastric carcinoma (both P = .005). These results showed that high Cthrc1 expression was associated with progression and prognosis of gastric carcinoma.

Collagen Triple Helix Repeat Containing 1 (CTHRC1) acts via ERK-dependent induction of MMP9 to promote invasion of colorectal cancer cells

Collagen triple helix repeat-containing 1 (CTHRC1) is known to be aberrantly upregulated in most human solid tumors, although the functional roles of CTHRC1 in colorectal cancer remain unclear. In this study, we investigated the occurrence of CTHRC1 upregulation and its role in vivo and in vitro. The expression profile and clinical importance of CTHRC1 were examined by reverse transcription-polymerase chain reaction and immunohistochemical analyses in normal and tumor patient samples. CTHRC1 was detectable in normal tissues, but also was highly expressed in tumor specimens. CTHRC1 upregulation was significantly associated with demethylation of the CTHRC1 promoter in colon cancer cell lines and tumor tissues. Clinicopathologic analyses showed that nodal status and expression of CTHRC1 (95% CI 0.999-3.984, p=0.05) were significant prognostic factors for disease-free survival. Promoter CpG methylation and hypermethylation status were measured by bisulfite sequencing and pyrosequencing analysis. Furthermore, we showed that overexpression of CTHRC1 in the SW480 and HT-29 cell lines increased invasiveness, an effect mediated by extracellular signal-regulated kinase (ERK)-dependent upregulation of matrix metalloproteinase 9 (MMP9). Consistent with this, we found that knockdown of CTHRC1 attenuated ERK activation and cancer cell invasivity. These results demonstrate that CTHRC1 expression is elevated in human colon cancer cell lines and clinical specimens, and promotes cancer cell invasivity through ERK-dependent induction of MMP9 expression. Our results further suggest that high levels of CTHRC1 expression are associated with poor clinical outcomes.

Overexpression of CTHRC1 in Hepatocellular Carcinoma Promotes Tumor Invasion and Predicts Poor Prognosis

Collagen triple helix repeat containing-1 (CTHRC1) is a secreted glycoprotein that activates the planar cell polarity pathway of Wnt signaling. Using microarray analysis, we found that the CTHRC1 gene is overexpressed in hepatocellular carcinoma (HCC). The level of CTHRC1 mRNA was measured in 201 surgically resected HCCs using real time reverse transcription-polymerase chain reaction. Overexpression of CTHRC1 in HCC was associated with large tumor size and advanced tumor stage. Furthermore, expression of CTHRC1 as was identified as an independent prognostic factors in the multivariate analysis. Suppression of CTHRC1 expression inhibited tumor migration and invasion whereas overexpression of CTHRC1 promoted tumor invasion. Activation of RhoA, but not Rac1 or Cdc42, was found to play a crucial role in CTHRC1-induced cell migration. CTHRC1 promoted adhesion of cancer cells to extracellular matrix through induction of integrin β1 expression and activation of focal adhesion kinase. These results suggest CTHRC1 promotes tumor invasion and metastasis by enhancing the adhesion and migratory abilities of tumor cells. It is also a promising biomarker for predicting the prognosis of patients with HCC.

Protein expressions of collagen triple helix repeat containing-1, β-catenin and Wnt5a in lesions from patients with different stages of melanoma and their relationship

Objective To compare the protein expressions of collagen triple helix repeat containing-1 (CTHRC1),β-catenin and WntSa in lesions of nevus versus melanoma in different stages,and to assess their possible roles and relationship in the initiation and progression of melanoma.Methods Totally,76 tissue specimens were studied in this study,including 23 cases of nevus,14 cases of melanoma in situ,21 cases of invasive melanoma and 18 cases of metastatic melanoma.Immunohistochemical SP method was used to detect the protein expressions of CTHRC1,β-catenin and Wnt5a in these tissue specimens.The clinical and pathological implications of these protein expressions were assessed.Results The expression rate in nevus and melanoma lesions was 34.8％ and 62.3％ respectively for CTHRC1 (P ＜0.05),21.7％ and 62.3％ respectively for β-catenin (P ＜0.05),34.8％ and 69.8％ respectively for Wnt5a (P ＜0.05).The expressions of CTHRC1 and β-catenin were associated with the invasion depth and lymph node metastasis of melanoma (both P ＜0.05),and the expression of Wnt5a was only related to the lymph node metastasis of melanoma (P ＜0.05).There was a positive correlation between the protein expression of CTHRC1 and β-catenin as well as between that of CTHRC1 and Wnt5a (r =0.798,0.623,respectively,both P ＜0.01).Conclusions CTHRC1,β-catenin and Wnt5a appear to play a certain role in the development of melanoma,and CTHRC 1 may serve as a cofactor of the Wnt signaling pathway in promoting the initiation and progression of melanoma. Key words: Melanoma; Nevus; CTHRC1; β-catenin; Wnt5a

N-Glycosylation Induces the CTHRC1 Protein and Drives Oral Cancer Cell Migration

Oral squamous cell carcinoma (OSCC) is one of the most pernicious malignancies, but the mechanisms underlying its development and progression are poorly understood. One of the key pathways implicated in OSCC is the canonical Wnt/β-catenin signaling pathway. Previously, we reported that canonical Wnt signaling functions in a positive feedback loop with the DPAGT1 gene, a principal regulator of the metabolic pathway of protein N-glycosylation, to hyperglycosylate E-cadherin and reduce intercellular adhesion. Here, we show that in OSCC, DPAGT1 and canonical Wnt signaling converge to up-regulate CTHRC1 (collagen triple helix repeat containing 1), an N-glycoprotein implicated in tumor invasion and metastasis. We found that in human OSCC specimens, amplification of the levels of CTHRC1 was associated with its hyperglycosylation. Partial inhibition of DPAGT1 expression in OSCC CAL27 cells reduced CTHRC1 abundance by increasing protein turnover, indicating that N-glycosylation stabilizes CTHRC1. Additionally, canonical Wnt signaling promoted β-catenin/T-cell factor transcriptional activity at the CTHRC1 promoter to further elevate CTHRC1 levels. We demonstrate that DPAGT1 promotes cell migration and drives the localization of CTHRC1 to cells at the leading edge of a wound front coincident with drastic changes in cell morphology. We propose that in OSCC, dysregulation of canonical Wnt signaling and DPAGT1-dependent N-glycosylation induces CTHRC1, thereby driving OSCC cell migration and tumor spread.

Effect of vascular endothelial growth factor C and collagen triple helix repeat containing 1 expression on prognosis of rectal carcinoma patients

To explore the relationship of vascular endothelial growth factor C (VEGF-C) and collagen triple helix repeat containing 1 (CTHRC1) expression with the carcinogenesis and prognosis of rectal cancer. Cancer tissue samples from 120 rectal cancer patients confirmed by pathology in the People's Hospital of Yichun City from September 2005 to September 2010 were included in the study. Expressions of CTHRC1 and VEGF-C were examined by immunohistochemistry and their correlations with clinicopathological features and prognosis were analyzed. The expression of VEGF-C was positively correlated with tumor size (r=0.943), TNM stages (r=0.784) and tumor differentiation (r=0.773) (all P<0.05). Similarly, the expression of CTRHC1 was also positively correlated with tumor size (r=0.829), TNM stages (r=0.632) and tumor differentiation (r=0.532) (all P<0.05). Rectal cancer patients with low expression of VEGF-C and CTHRC1 had significantly longer survival than those with high expression of VEGF-C and CTHRC1 [(40.0±1.3) vs. (35.4±0.5) months, P<0.01, (39.0±0.5) vs. (35.0±0.5) months, P=0.014]. VEGF-C and CTHRC1 may synergistically promote the invasion and metastasis of human rectal cancer, and provide evidence in predicting the prognosis of patients.