Mechanism Regulation By Kai1 In Lymphatic Metastasis Of Pancreatic Cancer

[Background]Pancreatic cancer is characterized by poor prognosis, early metastasis and resistance to chemoradiotherapy. The potentially curative resection is possible in only 10-15% of the pancreatic cancer patients, overall 5 year survival with 1-5%. Metastasis of this neoplasm through lymphatic system is one of the most important factor which affects the treatment and prognosis. Preventing and controling the lymphatic metastasis may make great sense in effective integrated treament of pancreatic carcinoma. With the development of the lymphatic tracer, the specific markers of lymphatic vessels and the improved understanding about cancer cell migration, invasion, lymphangiogenesis, lymphatic invasion, and other parts of the tumor lymphatic metastasis, the research about lymphatic metastasis of cancer has made a great progress. However, the accurate mechanism of lymphatic metastasis in pancreatic cancer is still unknow and the targeted therapies were less reported.The research has become a hotspot. Kangai 1(KAI1) gene is a newly identified tumor suppressor gene. It encoded transmembrane protein belonging to the transmembrane 4 superfamily (TM4SF). Currently, the research in a variety of tumors, including pancreatic cancer, showed that KAI1 may regulate tumor cell adhesion and movement, inhibite tumor cell migration and invasion. The studies about KAI1 related mechanism of action are further carried out. In addition, the clinicopathological study found that KAI1 gene expression is negative with tumor lymphatic metastasis. We firstly found that KAI1 gene is low expression in the event of lymphatic metastasis in pancreatic cancer. Thus, KAI1 gene may play an important role in lymphatic metastasis of pancreatic cancer.[Aims]Investigate the regulation of function and mechanisms of KAI1 gene in lymphatic metastasis of pancreatic cancer.[Methods]1. The pcMV-KAIl vector which contained the full length of KAI1 cDNA was transfected into pancreatic cancer cells MIA PaCa-2, followed by screening and verifying.2. The proliferation of pancreatic cancer cells transfected with KAI1 vector was evaluated by MTT method.3.Wound-healing assay and cell invasion assay were used to detect the influence of KAI1 on the migration and invasion of pancreatic cancer cells.4.The migration and invasion related molecules were dectede by Western blot.5. The effect of KAI1 on tumorigenicity and lymphatic metastasis of pancreatic cancer cells was revealed by tumorigenesis in nude mice. 6. The expression of LYVE-1 related antigen in xenograft tumor was detected by immunohistochemistry and the LVD was compared among different xenograft tumors.7. The lymphangiogenesis related molecules were dectede by Western blot. The concentration of VEGF-C in the supernatant of transfectants was determined by ELISA.8. The signal transduction is evaluated by Western blot analysis. 【Results】1. The plasmid pcMV-KAIl and pcMC were transfected into MIA PaCa-2 cell lines respectively. The cell lines with high expression of KAI1 were established and identified with the name of MIA PaCa-2-K. The empty vector transfected cells named as MIA PaCa-2-p.2. There were not significantly different in cell growth between MiaPaCa2, MiaPaCa2-p and MiaPaCa2-K, showed by MTT assay. The wound- healing assay showed that the migration ability were cut down in MIA PaCa-2-K at 24h, 48h,72h time point. The Transwell assay indicated that the number of the invasive cells through the membrane is 48±15.4,50±12.4,12±3.8 respectivly, in MIA PaCa-2, MIA PaCa-2-p and MIA PaCa-2-K cell lines (p<0.05). There is statistical significance within different groups.3. Western blot revealed that the E-cadherin ratio increased, while the MMP2 and MMP9 ratio decreased in MIA PaCa-2-K.4. The subcutaneous tumor formation rate of MiaPaCa2-K was decreased. But the growth curve of subcutaneous tumor from different cells showed that KAIldoes not affect primary tumor growth.5. The weights of subcutaneous tumor from MIA PaCa-2, MIA PaCa-2-p and MIA PaCa-2-K was 1364.40±211.30 mg,1426.13±175.10 mg and 1294.37±195.40 mg respectivly, and there were no significant different (p> 0.05)6.42.9%(3/7) nude mice occurred lymph node metastasis, and the average number of lymph node metastasis is 1.33 in MIA PaCa-2-K group, which is significantly decreased than 80%(8/10) and 2.47,70%(7/10) and 2.14 from MIA PaCa-2 and MIA PaCa-2-p groups.7. The stain of LYVE-1-related antigen was positive in the lymphatic vessels of nude mice subcutaneous tumors. The density of lymphatic vessels was significantly lower in MIA PaCa-2-K group compared to MIA PaCa-2 and MIA PaCa-2-p groups (p<0.05).8. Western blot revealed that the VEGF-A ratio is similar in MIA PaCa-2-K, MIA PaCa-2 and MIA PaCa-2-p cells, whereas the VEGF-C ratio decreased in MIA PaCa-2-K. The concentration of VEGF-C in the supernatant of MIA PaCa-2-K were decreased when compared to the control cells.9. Western blot analysis showed that high expression of KAI1 may inhibit the phosphorylation Src and STAT3. The expression of VEGF-C can be partely inhibited by PP2 and AG490, the special inhibitors of Src and STAT3, respectively. On the other hand, PP2 can inhibited the phosphorylation of STAT3, while AG490 did not effect Src phosphorylation.[Conclusions]1. Our results suggest that up-regulation of KAI1 may induce the expression of E-cadherin and reduce the expression of MMP2 and MMP9 to inhibit migration of pancreatic cancer cells in vitro.2. KAI1 can decrease the lymphangiogenesis in the animal subcutaneous tumor models, and reduce lymphatic vessel density to suppress lymphatic metastases of pancreatic cancer. KAI1 may regulate the phosphorylation of Src/STAT3 signaling pathway to inhibit VEGF-C synthesis of pancreatic cancer cell.3. All these data revealed that KAI1 may play an important role in regulating the process of lymphatic metastasis of pancreatic cancer, and it may be considered as a valuable therapy target for the pancreatic cancer in the future.