The Roles Of KLF9 In Gastric Cancer Metastasis

Gastric cancer is one of the most lethal human malignancies.According to the latest statistics,the incidence and lethality of gastric cancer rank second among all types of cancer in China.The major reason for poor treatment and prognosis of gastric cancer is lack of early detection.Most of the patients who are diagnosed with gastric cancer have been in the advanced stage,and some have already developed distant metastasis.Metastatic disease accounts for most of gastric cancer related mortality due to its systemic nature.However,the precise mechanism of gastric cancer metastasis remains unclear.Therefore,developing new strategies for early diagnosis and exploring the precise mechanism of metastasis are crucial for better gastric cancer management.Transcription factors function in a coordinated pattern to direct numerous biological processes throughout life,including cell proliferation,differentiation,migration and apoptosis.As previously reported,transcription factors dysregulation is essential for carcinogenesis and metastasis.Kriippel-like factors(KLFs)are a family of transcription factors with three carboxyl-terminal C2H2-type zinc finger structure motif.The KLF family contains 17 family members which regulate various biological processes.Alterations in KLFs function have been associated with numerous human diseases,including cardiovascular disease,metabolic disorders,and cancer.Kriippel-like factor 9(KLF9)is previously known as basic transcription element binding protein(BTEB)due to its specific binding to the basic transcription element(BTE),a GC box of CYP1A1 gene promoter region.Mice lacking KLF9 have normal life span,but show subfertility,uterine hypoplasia and partial progesterone resistance.More recently,KLF9 has been indicated to be associated with colorectal cancer,endometrial carcinoma,breast cancer,prostate cancer and hepatocellular carcinoma.However,the function of KLF9 in gastric cancer remains undefined.To further investigate the mechanism of gastric cancer metastasis,we applied single-sample gene set enrichment analysis(ssGSEA)to divide the gastric cancer patients of The Cancer Genome Atlas(TCGA)into high or low metastatic potential group,and then identified 1770 differential expressed genes between the two groups.Considering its essential character in carcinogenesis and metastasis,we further identified 143 transcription factors among these differential expressed genes.After validating these transcription factors in other four gastric cancer microarrays,we finally identified five gastric cancer metastasis associated genes,including KLF9.Collectively,these results indicate that KLF9 might play an important role in the process of gastric cancer metastasis.Furthermore,we explored the expression pattern of KLF9 between patients with or without distant metastasis,and results showed that KLF9 mRNA level was evidently lower in gastric cancer tissues of patients with distant metastasis compared to the matched tissues from non-metastasis patients,suggesting it might act as a suppressor for gastric cancer metastasis.Further gene set enrichment analysis(GSEA)also revealed that multiple metastasis-related gene sets were enriched in low KLF9 expressed gastric cancer patients.We further investigated the effect of KLF9 on gastric cancer cells by ectopic expression or RNA interference.Downregulation of KLF9 endows gastric cancer cells with significantly stronger migration and invasion capability.Consistently,ectopic expression of KLF9 dramatically inhibits the migration and invasion capability of gastric cancer cells.Notably,altering the expression level of KLF9 does not affect the proliferation and colony formation capability of gastric cancer cells.Furthermore,we assessed the invasion and metastasis capability in vivo by tail intravenous injection.Results showed that ectopic KLF9 expression strongly inhibits the lung metastasis capability of gastric cancer cells.Taken together,these results indicate that KLF9 is a potent suppressor for gastric cancer metastasis.Next,we aim to figure out how KLF9 inhibits gastric cancer metastasis.Transcription factor is a set of proteins that controls the rate of transcription by binding to a specific DNA sequence,thus to make sure the target genes are expressed in the right cell with the right amount at the right time.To explore the downstream targets for KLF9,we screened the entire human ENCODE(Encyclopedia of DNA Elements)database for cell lines which conducted KLF9 chromatin immunoprecipitation coupled with high-throughput sequencing(ChIP-seq)experiments,and identified 823 potential target genes.Next,we combined these potential target genes with those which had been previous reported to be involved in the process of metastasis,and then tested their expression pattern by qRT-PCR.Finally,we found that mRNA and protein level of matrix metalloproteinase 28(MMP28)was severly suppressed by KLF9.Furthermore,we confirmed that there was specific binding of KLF9 on the promoter of MMP28 via dual luciferase reporter assay.These results indicate that KLF9 specifically inhibits MMP28 expression in gastric cancer cells.To investigate whether KLF9 suppressed gastric cancer metastasis in a MMP28-dependent manner,we conducted rescue experiment.Importantly,reintroduction of MMP28 to KLF9 overexpressed cells endows them with stronger invasiveness.Furthermore,reintroduction of MMP28 recovered KLF9 overexpressed gastric cancer cells to similar lung metastasis capability of negative control group.Collectively,reinforcement of MMP28 rescues gastric cancer metastasis defect caused by KLF9,which indicate that KLF9 suppresses gastric cancer metastasis through transcriptional inhibition of MMP28.Overall,our research indicates that transcription factor KLF9 is a potent suppressor for gastric cancer metastasis,and it works through directly inhibiting MMP28 transcription.Our research sheds new lights on the mechanism of gastric cancer metastasis,and we also provide promising targets for gastric cancer treatment.