| BackgroundGastric cancer is one of the most common malignancies and is a leading cause of cancer death worldwide, especially in Eastern Asia. A recent study reported that 640,000 men and 350,000 women are diagnosed with gastric cancer, and 464,000 men and 273,000 women die from gastric cancer every year. Metastasis is the cause of 90% of all deaths from cancer and exhibits a unique set of clinical characteristics. Heparanase (HPA) as an endoglucuronidase that cleaves heparan sulfate chains of proteoglycans (HSPG) plays important roles in tumor metastasis. The cleavage of heparan sulfate proteoglycans can promote the shedding of heparin-binding growth factors, such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and hepatocyte growth factor (HGF), from the ECM to enhance endothelial cell migration and proliferation. However, few studies have examined the regulation of HPA expression. For example, epigenetic changes and/or mutational inactivation of p53 during cancer development may provide a molecular explanation for the induction of HPA expression observed in many human tumors. Other studies have also reported that SP1 and Ets transcription factor families are associated with the expression of HPA. Furthermore, our previous studies also demonstrated that multiple antigenic peptides vaccines based on cytotoxic T lymphocyte epitopes of human heparanase can be used as potent immunogenic target for tumor immunotherapy, which significantly decreased tumor growth and metastasis. So it was very significant to explore the expression of HPA in cancer tissues,c-met as an oncogene was significantly expressed by tumor cells. It has been demonstrated that c-met play important roles in many tumors progression, including breast cancer, hepatocellular carcinoma, osteosarcoma and brain tumors. In our previous study, it was found by 30 clinical tissues that the c-met expression was positive related to HPA expression. Furthermore, it was also reported that HGF/c-met can activate a series of signaling pathways, including PI3K/Akt, ERK, NF-κB, and STAT3. However, whether these signaling pathways can induce the expression of HPA remains unknown. So in this study we considered if HGF can regulate HPA expression to promote tumor metastasis.Methods:1. IHC was used to detect the expression of HGF and HPA in 58 cancer and para-cancer tissues. Furthermore it was also analyses about HGF or HPA expression and the poor survival, TNM stage, depth of invasion and Lymph node metastasis.2. qRT-PCR and Western blot were used to detected HPA expression after treated with different concentration of HGF in both MKN74 and MKN45 cells.3. c-met inhibitor Su 11274 and HGF together treated to MKN74 and MKN45 cells for 24h, then Western blot was used to detected the expression of HPA.4. MKN74 and MKN45 cells was first treated with HGF for 0,15,30,45,60 and 90 min, then the expression of p-AKT and p-p65 was detected by Western blot.PI3K/Akt signal inhibitor GSK and NF-κB signal inhibitor was treated to MKN74 cells, then the expression of p-AKT and p-p65 was detected by Western blot.5. c-met inhibitor Su11274, PI3K/Akt signal inhibitor GSK and NF-κB signal inhibitor was treated to MKN74 cells for 24h, then the expression of HPA was detected by Western blot.6. Luciferase assay was used to detect if HGF can promote HPA promoter luciferase assay.7. Bioinformatics technique was used to predict the potential bind sites for p65 to bind with HPA promoter.8. ChIP assay was used to detected the the detail binding cites of p65 to the HPA promoter.9. CCK-8 assay, wound healing assay, Transwell assay and peritoneal dissemination in nude mice experiments were applied to analyze the function of HGF and HPA in tumor metastasis.10. ELISA was used to analysis the expression of HGF in HPA-cDNA MKN74 cells and sh-HPA MKN45 cells.Results:1. HGF and HPA expression were significantly expressed and associated with poor prognosis in 58 patients with gastric cancer. HGF was strongly expressed in gastric cancer samples compared with normal gastric tissues. Similarly, HPA was also weakly expressed in the normal gastric tissues but was significantly increased in gastric cancer samples. In addition, we analyzed the association between HGF or HPA and the clinicopathological features of cancer patients. High levels of HGF positively correlated with TNM stage, lymph node metastasis and depth of invasion. Consistently, the strong expression of HPA was also associated with the TNM stage and depth of invasion. Notably, HGF overexpression was strongly associated with the poor survival of patients with gastric cancer. High expression of HPA was also positively correlated with poor survival. Furthermore, HGF and HPA were positively correlated.2. HGF can significantly up-regulate HPA expression in MKN74 cells, but had no significantly influence in MKN45 cells. At the mRNA levels, different concentrations of HGF increased the expression of HPA in MKN74 cells. However, HGF did not increase the expression of HPA in MKN45 cells. Consistently, similar results were observed at the protein level; HGF promoted the expression of HPA in MKN74 cells but had no role in MKN45 cells. These results indicate that exogenous HGF can significantly induce HPA expression in HGF-/c-met+ cells but has no role in HGF+/c-met+ cells. When the c-met inhibitor SU11274 was added, the expression of HPA in both MKN74 and MKN45 cells were decreased, indicating that HGF promoted the expression of HPA through c-met.3. HGF promoted the expression of HPA by activating PI3K/Akt and downstream NF-kB signaling. Western blotting revealed that p-AKT (Ser473) was significantly increased after 30 min, which indicated that HGF induced the activation of PI3K/Akt signaling. Furthermore, NF-κB was also activated after 45 min. PI3K/Akt inhibitor GSK and NF-κB inhibitor Bay were used in MKN74 cells. It was demonstrated that GSK can significantly inhibit the activation of PI3K/Akt signaling, whereas Bay had no effect on the activation of PI3K/Akt pathway, indicating that NF-κB signaling may be the downstream of PI3K/Akt pathway. Furthermore, it was also demonstrated that both GSK and Bay can inhibit the activity of NF-κB signaling, further suggesting that HGF-activated NF-κB signaling may be mediated downstreamPI3K/Akt signaling. It was demonstrated that all inhibitors significantly inhibited the expression of HPA.4. p65 binds to the promoter of HPA to induce its expression. Luciferase reporter assay revealed that HGF could enhance the promoter activity of HPA, whereas Bay could decrease HGF-enhanced promoter activity. This indicated that NF-κB may bind to the promoter of HPA. Moreover, bioinformatics analysis predicted that there are three binding sites (BS) for p65 in the promoter of HPA gene. Subsequently, ChIP assay was used to investigate the binding site for p65. Furthermore, it was revealed that p65 can only directly bind to BS1 elements (-769～-760 bps) in the promoter of HPA. Furthermore, we constructed a vector containing the HPA promoter with a deletion of the BS1 elements (HPA Promoter Mut). Luciferase reporter analysis revealed that HGF could not increase the activity of HPA Promoter Mut.5. It was demonstrated by in vivo and vitro experiments that HGF/c-met and HPA played important roles for gastric cancer metastasis. The scratch migration assay and Transwell assay revealed that when MKN74 cells were treated with HGF, cell migration was significantly increased. Furthermore, Bay significantly decreased migration. However, in MKN45 cells, exogenous HGF could not increase migration; when the cells were treated with Bay, the migration of MKN45 cells was significantly decreased. Transwell experiments revealed that the migration of MKN74-HPA cDNA cells was significantly greater than that of MKN74-NC cells. Furthermore, the migration of MKN45-sh-HPA cells was significantly decreased relative to MKN45-NC cells. These results indicate that HPA plays a positive role in cancer cell metastasis. In vivo, peritoneal dissemination of MKN45-sh-HPA cells was significantly low than MKN45-NC cells.6. HPA inversely regulated the expression of HGF. The concentration of HGF in MKN74-HPA cDNA supernatant was significantly higher than in MKN74-NC. Furthermore, HGF concentrations in MKN45-sh-HPA supernatant were significantly lower than in MKN45-NC. These data indicated that HPA can also regulate the level of HGF.Conclusion:1. HGF and HPA expression were significantly expressed in 58 patients of gastric cancer (p<0.001). High levels of HGF and HPA were positively correlated with poor survival, TNM stage, lymph node metastasis and depth of invasion. Furthermore, it was found HGF was positively correlated to HPA (R=0.7011, p<0.001). Exogenous HGF combining with the receptor c-met significantly increased the expression of HPA in MKN74 cells, but had no roles for MKN45 cells.2. HGF/c-met activated PT3K/Akt signal first and then activated the downstream NF-κB signal to promote HPA expression. p65 combined to HPA promoter-769～-760 bps to promote HPA expression in MKN74 cells.3. HGF and HPA played important roles for gastric cancer metastasis.4. HPA can reversely regulate the expression of HGF, which finally formed a positive feedback loop to promote tumor metastasis.In total, this study demonstrated that HGF regulates the expression of HPA by activating PI3K/Akt and downstream NF-κB signaling. Subsequently, p65 binds to the promoter of HPA to influence its expression. Furthermore, HPA can inversely regulate HGF expression to promote tumor metastasis... |
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