| BackgroundThe chromosomes consist of an end of TTAGGG repeat, which was named telomere. With the increased number of cell division in homeostasis, the length of telomere will become shorter and undergo cell senescence. Telomerase is the key enzyme maintaining the extension and stability of telomere, and the overexpression and re-activation of telomerase resulted in infinite proliferous and immortalized cells. Telomerase is a ribonucleoprotein, consisting of RNA and telomerase reverse transcriptase that was able to transcribe the specific TTAGGG repeat using the RNA template to extend the telomere. Human telomerase reverse transcriptase(hTERT) is the catalytic subunit of telomerase, which plays a key role in activation of telomerase, and the expression of hTERT could represent the telomerase activation. hTERT is overexpressed in over ninety percent of human malignancies, which can promote unlimited tumor proliferation through the canonical enzyme activation. Moreover, increasing research demonstrated that hTERT could also modulate gene expression independent of its enzyme activation.As a high incidence in China, the morbidity of gastric cancer in men and women is the second and third, respectively, and the mortality has reached the second among the malignancies. Early invasion and metastasis is the key factor for death of gastric cancer, while the molecular mechanisms are not fully understood. As we know, tumor invasion and metastasis is a complicated process, mainly including adhesion, degradation of extracellular matrix(ECM), epithelial-mesenchymal transition(EMT) and angiogenesis. Heparanase is the unique endoglycosidase that can degrade the heparin sulfate proteoglycan in ECM. Heparanase degrade the ECM, destroy the barrier, and release various cellular growth factors anchored in ECM, and finally promotes the tumor invasion and metastasis. Heparanase is overexpressed in tumor tissues and serum, closely correlating with tumor metastasis and tumor blood vessels density. Inhibiting heparanase expression could significantly inhibit tumor metastasis, suggesting that heparanase could be a potential target for cancer therapy.Previous studies mainly focused on the role of hTERT in tumor proliferation, increasing evidence revealed that hTERT might also play a key role in tumor invasion and metastasis. Our research group found that overexpression of hTERT could enhance the cell invasion in vitro, and hTERT expression in gastric cancer was closely related to tumor size, lymph node and distant metastasis, suggesting that hTERT might be involved in regulation of gastric cancer invasion and metastasis. However, the specific mechanisms are still unknown. This study will elucidate the role and molecular mechanism of hTERT upregulating heparanase and promoting gastric cancer invasion and metastasis, and provide the novel theoretical basis for hTERT-targeted diagnosis and treatment in gastric cancer.Methods1. The role of hTERT in gastric cancer invasion and metastasis was evaluated using transwell invasion assay, lung metastasis and peritoneal dissemination assay.2. Luciferase assay, quantitative PCR and western blot assay were used to detect the hTERT-induced promoter activity, mRNA and protein expression of heparanase.3. Bioinformatics and quantitative PCR were applied to screen and identify the candidate involved in hTERT-induced heparanase expression.4. Luciferase and western blot assay were used to test the role of overexpression or inhibition of c-Myc on promoter activity and protein expression of heparanase. ChIP analysis was used to detect the direct binding of c-Myc to heparanase promoter.5. After mutation of c-Myc binding site in heparanase promoter and inhibition of c-Myc expression, luciferase and western blot assay were used to examine the promoter activation and protein expression of heparanase.6. Co-immunoprecipitation, immunofluorescence and western blot were applied to evaluate the formation of complex of hTERT and c-Myc and role of binding to heparanase promoter.7. After overexpression or inhibition of c-Myc or hTERT, luciferase and western blot assay were used to test the promoter activation and protein expression of heparanase to further investigate the essential role of hTERT and c-Myc in heparanase expression.8. TOP/FOP flash assay, immunofluorescence and western blot were used to examine the role of hTERT in β-catenin activation and nuclear translocation, and to test the feedback regulation between hTERT and c-Myc.9. After inhibition of heparanase expression, in vitro invasion and in vivo metastasis assay were applied to examine the role of heparanase in hTERT-induced gastric cancer invasion and metastasis.10. Immunohistochemistry was used to test the expression levels of hTERT, c-Myc and heparanase in gastric cancer tissues and adjacent normal tissues. The correlation between the expression and prognosis of gastric cancer patients were further analyzed.Results1. The invasion and metastasis assay showed that overexpression or inhibition of hTERT could promote or inhibit gastric cancer invasion and metastasis.2. Overexpression or inhibition of hTERT upregulated or suppressed promoter activity, mRNA and protein expression of heparanase, suggesting hTERT is able to regulate heparanase expression and is closely associated with transcriptional activity of heparanase.3. Bioinformatics, luciferase and qPCR assay found that inhibition of c-Myc expression could attenuate hTERT-induced upregulation of heparanase promoter activity and expression, suggesting that c-Myc might be involved in modulation of heparanase transcription.4. ChIP analysis revealed that c-Myc could directly bind to heparanase promoter, while mutation of c-Myc binding cite significantly blocked the upregulation of heparanase promoter activity induced by hTERT, further confirming that hTERT regulation of the heparanase promoter activity was c-Myc-dependent.5. Co-immunoprecipitation and immunofluorescence co-localization analysis confirmed that hTERT interacted with c-Myc in nucleus. ChIP and reChIP assay showed that the complex formed by hTERT and c-Myc could bind to heparanase promoter through c-Myc, and overexpression of hTERT increased the formation of complex and the binding to heparanase promoter to eventually transactivate heparanase transcription.6. The elevated promoter activity and protein expression induced by overexpression of hTERT or c-Myc could be abolished by the c-Myc shRNA or hTERT shRNA. Either hTERT or c-Myc overexpression could increase the promoter activity and protein expression of heparanase. Moreover, overexpression of both hTERT and c-Myc could stimulate much higher promoter activity and protein expression. Taken together, these findings demonstrated that the intact complex is essential for heparanase transcription and expression.7. Overexpression of hTERT could increase the activity of β-catenin and promote the nuclear translocation of β-catenin. Moreover, overexpression of hTERT significantly increased the expression of c-Myc through activation of Wnt/β-catenin signaling pathway. Previous studies demonstrated that c-Myc could bind to the hTERT promoter and transcriptionally regulate hTERT expression, suggesting that a feedback regulation mechanism existed in hTERT and c-Myc to maintain the high expression of hTERT, c-Myc and heparanase in gastric cancer.8. Inhibition of c-Myc or heparanase significantly suppressed hTERT-induced gastric cancer invasion and metastasis, suggesting that hTERT cooperated with c-Myc to upregulate heparanase expression and promoted gastric cancer invasion and metastasis.9. The immunohistochemistry analysis showed that the expression levels of hTERT, c-Myc and heparanase were much higher in gastric cancer tissues compared to adjacent normal tissues, and the expression was positively correlated with each other. Survival analysis revealed that the high expression levels of hTERT, c-Myc and heparanase were closely associated with poor prognosis of gastric cancer.ConclusionTaken together, we confirmed the important role of hTERT in gastric cancer invasion and metastasis, and found that hTERT could promote gastric cancer invasion and metastasis through upregulating heparanase expression. hTERT could act as co-activator to interact with c-Myc and form a transcriptional complex that promoted the transcriptional expression through binding to heparanase promoter. Moreover, hTERT activated Wnt/β-catenin signaling pathway to upregulate c-Myc, and c-Myc could bind to the hTERT promoter and transcriptionally upregulate hTERT, suggesting a feedback regulation existed in hTERT and c-Myc to upregulate heparanase expression and promote gastric cancer invasion and metastasis. |
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