| BackgroundWe previously reported that β6 integrin plays an important role in the progression of colon cancer. In this study, we demonstrated that β6 integrin induces the expression of MMP-3/MMP-9 and the invasion of colon cancer cells. Moreover, that function was abolished by the inhibition of ERK/MAPK pathways or knockdown of ETS1, an important transcription factor of MMP genes. Here, we showed that β6 induced phosphorylation of ETS1 via the ERK/MAPK pathways, through which the MMP-3/MMP-9 promoters were stimulated, lead to the up-regulation of MMP-3/MMP-9, and subsequent invasion of colon cancer cells.Colorectal cancer (CRC) is the third most common malignant neoplasm and the fourth leading cause of cancer related deaths worldwide. It is estimated that 20% of patients with CRC are found to have metastatic disease at the time of diagnosis, whereas many others will experience metastasis during the course of the disease. Therefore, a detailed understanding of the mechanism behind invasion and metastasis of CRC may be helpful in the development of a new therapeutic strategy and thereby reduce mortality rate.Matrix metalloproteinases (MMPs) have fundamental roles in pathological processes through degradation of basal membranes and extracellular matrix, and some MMPs contribute to tumor invasion and metastasis. Several experimental and clinical studies documented that CRC aggression significantly correlated with increased levels of MMP-2, MMP-3, and MMP-9. At the transcriptional level, the synthesis of MMPs is regulated by the surrounding stroma, ECM proteins, systemic hormones, and other factors.Integrins are major cell adhesion receptors that mediate cell migration via cell-cell and cell-matrix interactions. They belong to a cell surface adhesion molecular family, which is composed of α and β subunits associated non-covalently. Integrin αvβ6 is the only heterodimer that 06 subunit can form and is expressed exclusively in epithelial cells, and is absent or lowly expressed in healthy adult epithelia, but however highly expressed during embryogenesis, tissue repair and carcinogenesis. We have reported that β6 integrin plays an important role in invasion, metastasis and degradation of extracellular matrix of colorectal cancer, thyroid carcinoma, gastric carcinoma, and pancreatic carcinoma. We recently verified that αvβ6 could mediate colon cancer cell migration on fibronectin by internalization and recycling of avP6 integrin, indicating direct contribution of αvβ6 to colon cancer cell migration. In addition, we demonstrated a direct link between extracellular signal-regulated kinase (ERK2) and β6 integrin in colon cancer cells, and blockage of this direct binding markedly impaired cytosolic ERK/MAP kinase activity, cell density-dependent expression of β6 integrin and β6-mediated MMP-9 secretion. However, the precise mechanism underlying β6-MMP cross-talk, especially at the transcriptional level remains largely unknown.ETS transcription factors are helix-turn-helix proteins with a highly conserved ETS domain that binds to the core consensus sequence GGA(A/T). Many MMP genes have been reported to possess an ETS binding sequence (EBS) in their promoter region. The ETS family of transcription factors consists of over 200 target genes and is involved in several biological processes, such as angiogenesis, development, and apoptosis. Among the different types in their class, ETS1, ETS2, and ELK1 are reported to have prometastatic functions. Therefore, we aimed to study these factors in β6-mediated MMP gene regulation.ETS is co-expressed with integrins in different types of cancers. However, regarding the involvement of MAPK signaling pathways and transcription factors in P6-mediated MMP expression, a clear picture is still unavailable. In this study, we investigated the mechanism involved in β6-induced MMPs up-regulation as well as the invasion of colon cancer cells, and the role of ETS transcription factors during that process.Part 1 Role of Integrin αvβ6 in Invasion and Migration of Colon CarcinomaObjectiveThis study was to investigate the role of integrin αvβ6 in invasion and migration of SW480, HT-29 and WiDr colon cancer cells.Methods1. HT-29 and WiDr colon cancer cells were divided into three groups:wild-type, Antisence 06 and control of mock, SW480 colon cancer cells were divided into three groups:wild-type, mock transfected and 06 transfected, Flow cytometry and western blot were used to examine the expression of 06 integrin in these three groups in order to test the result of our transfection for the next step of experiments.2. Quantitative Real-time PCR was used to test the expression of MMPs in the mRNA level in each group.3. ELISA was used to test the expression of MMPs in the protein level in each group.Results1. Results of flow cytometry and western blotting showed tha β6 integrin was effectively up-regulated in 06-transfected SW480 cells, while wild type of SW480 cells and mock transfected cells did not show obvious expression.2. Results of flow cytometry and western blotting showed that HT-29 and WiDr cells showed obvious expression of β6 integrin compared with HT-29 and WiDr Antisence β6 transfectants. The transfection did not result in obvious effects in the expression of av integrin.3. Results of quantitative real-time PCR showed the expression of MMP-2, MMP-3 and MMP-9 mRNA was highly decreased in the HT-29 and WiDr Antisence β6 transfectants compared with the wild type cells.4. Results of quantitative real-time PCR showed in SW480 cells, the MMP-2, MMP-3 and MMP-9 mRNA showed obvious augment after transfected with β6 integrin while other MMPs did not show obvious change.5. ELISA data suggested that the protein expression of MMP-2, MMP-3 and MMP-9 were significantly decreased after suppression of β6 expression in HT-29 and WiDr.6. ELISA data showed the expression of MMP-2, MMP-3 and MMP-9 was higher in SW480 β6-transfected cells, compared to the wild type and mock-transfected SW480 cells.ConclusionsThis part of the study is to investigate the role of integrin αvβ6 on the expression of MMPs at the level of mRNA and protein. Flow cytometry and western blot were used to examine the expression of β6 integrin after overexpression or knockdown of β6 integrin. Results of quantitative real-time PCR and ELISA showed 06 integrin increased the expression of MMP-2, MMP-3 and MMP-9. The result of this part provides theory for the study of ntegrin αvβ6 promoting invasion and migration of colon carcinoma.Part 2 ETS1 involves integrin αvβ6 regulate MMPs expression in colon carcinomaObjectiveThis study was to investigate the effects of transcription factor ETS1 on the integrin αvβ6 promoting expression of MMPs in colon carcinoma.Methods1. Division of cell groups were the same way as section 1.2. Quantitative Real-time PCR and western blot were used to test the expression of αvβ6 and some transcription factors such as ETS1, ETS2 and ELK1 from ETS family.3. Luciferase kit was used to examined whether transcription factor ETS1 was involved during the process integrin αvβ6 induced MMP-3 and MMP-9.4. MEK inhibitor PD98059 was used to examined whether ERK/MAPK signal pathway was involved during the process integrin αvβ6 activate ETS1.5. Primaquine, which inhibits receptor recycling and vesicular transport, was used to examined whether internalization of integrin αvβ6 was involved during the process integrin αvβ6 activate ETS1.6. Role of the β6-ERK2 direct binding in β6-induced ETS1 activation, stably transfected SW480 cells with a deletion mutant β6 (which lost the ERK2 binding site:746EAERSKAKWOTGTNPLYRG764) were used to investigate the role of the β6-ERK2 direct binding in β6-induced ETS1 activation.Results1. Western blot showed that the ETS1, ETS2, ELK1 gene expression unchanged. However, the ETS1 activation (phosphorylation of ETS1) was significantly increased in SW480 β6-transfected cells compared with the wild type of SW480 cells, while phosphorylated ETS2 and ELK1 expression remained unchanged.2. For HT-29 and WiDr cells, Western blot showed that the phosphorylation of ETS1 decreased significantly after suppression of β integrin.3. Luciferase assay result indicates that the promoter activity of MMP-3 and MMP-9 was reduced in ETS1 siRNA-transfected cells, while the MMP-2 promoter activity unchanged in HT-29 and WiDr cells.4. For the HT-29 and WiDr β6 integrin Antisence cells, Luciferase assay result indicates the promoter activity of MMP-3 and MMP-9 were significantly decreased compared with the wild type groups.5. For SW480 cells, the promoter activity of MMP-3 and MMP-9 was increased in SW480 β6-transfected cells, and again red Luciferase assay result indicates uced in ETS1 siRNA-transfected cells.6. The western blot showed that, ERK inhibitor PD98059 decreased the expression of p-ETS1, while t-ETS1 remained unchanged. In SW480 cells, PD98059 effectively reverted 06 integrin-mediated ETS1 activation.7. The western blot showed that primaquine decreased the expression of p-ETS1, while t-ETS1 remained unchanged. In SW480 cells, β6-induced ETS1 activation was again reduced after primaquine was added.8. The result of western blot data showed that the ERK activation decreased in deletion mutant β6-transfected cells compared with full length β6-transfected cells. The loss of the ERK2 binding site in β6-integrin largely reverted β6-induced ETS1 activities in SW480 cells.ConclusionsThis part of the study revealed that ETS 1 plays a critical role in β6 integrin induced CRC progression, and expression of MMP-3/MMP-9 can be up-regulated by β6 integrin through increased phosphorylation of ERK and subsequent activation of ETS 1. Our present study presents a novel mechanism for β6 integrin-induced CRC progression. Our data confirms avP6 trafficking has a large contribution to cell migration and suggests a potential role for ETS1 in CRC progression, especially in patients with 06 integrin-induced CRC progression. Part 3 Integrin αvβ6 induces the Invasion and Migration of Colon Carcinoma via ERK-ETS1 pathwayObjectiveThis study was mainly to investigate the primary cell signal pathway of integrin αvβ6-mediated Invasion and Migration of colon carcinoma.Methods1. Cells were grouped in the same way as section 1.2. Quantitative Real-time PCR and ELISA were used to test the expression of MMPs in the mRNA level and protein level in both HT-29 and WiDr cells after inhibition of integrin αaβ6 trafficking by Primaquine, inhibition of ERK phosphorylation by PD98059 or inhibition of ETS1 activity by ETS1 siRNA.3. Quantitative Real-time PCR and ELISA were used to test the expression of MMPs in the mRNA level and protein level in integrin αvβ6 knocked down HT-29 and WiDr cells after ETS1overexpressed.4. Quantitative Real-time PCR and ELISA were used to test the expression of MMPs in the mRNA level and protein level in integrin αvβ6 overexpressed SW480 cells after inhibition of integrin αvβ6 trafficking by Primaquine, inhibition of ERK phosphorylation by PD98059 or inhibition of ETS1 activity by ETS1 siRNA.5. Serum-stimulated matrigel invasion assay and wound healing assay were used to examine the change of the invasion and migration of colon cancer cells after inhibition of avP6 by neutralizing antibody 10D5, inhibition of ERK phosphorylation by PD98059 or inhibition of ETS1 activity by ETS1 siRNA.6. SW480 colon cancer cells were divided into three groups:mock transfected, β6 transfected, and cells lost of the ERK2 binding site in β6-integrin. Serum-stimulated matrigel invasion assay and wound healing assay were used to examine the change of the invasion and migration of cells in each groups after inhibition of αvβ6 by neutralizing antibody 10D5, inhibition of ERK phosphorylation by PD98059 or inhibition of ETS1 activity by ETS1 siRNA.Results1. Real-time PCR analysis demonstrated that PD98059, primaquine or ETS1 siRNA decreased mRNA levels of MMP-3/MMP-9 in HT-29 and WiDr wild type cells. This result was confirmed by the ELISA data.2. The β6 Antisence stable HT-29 and WiDr cells were transfected with the expression plasmid pcDNA-ETS1, which contains the gene for transcription factor ETS1 (with the pcDNA3.1 plasmid as a negative control). The down-regulation of MMP-3/MMP-9 by knockdown of β6 integrin in HT-29 and WiDr cells was rescued after the overexpression of ETS1 both in Real-time PCR and the ELISA results.3. In SW480 P6 integrin overexpression cells, Real-time PCR analysis and the ELISA data showed that β6 integrin-mediated MMP-3/MMP-9 was again reduced at mRNA levels and protein levels after pretreated with a specific concentration of PD98059, primaquine or ETS1 siRNA.4. The serum-stimulated matrigel invasion assay shows inhibition of αvβ6 by neutralizing antibody 10D5, inhibition of ERK phosphorylation by PD98059 or inhibition of ETS1 activity by ETS1 siRNA greatly decreased the invasive ability of HT29 cells. The wound healing assay showed at 24 h after wounding, HT29 cells pretreated with 10D5, PD98059, or ETS1 siRNA presented a significantly slower closing of scratch wound compared with control groups cells.5. The invasive ability was detected in mock vector and β6-integrin transfected SW480 cells separately.β6 integrin dramatically increased the invasive ability of SW480 cells. Moreover, inhibition of ETS1 activation by ETS1 siRNA significantly alleviated invasion of β6-integrin transfected SW480 cells. On the contrary, β6-integrin transfected SW480 cells exhibited an elevated capacity of wound healing, but this capacity was again abolished after inhibition of αvβ6 by neutralizing antibody 10D5, inhibition of ERK phosphorylation by PD98059 or inhibition of ETS1 activity by ETS1 siRNA, which is consistent with our invasion assay. Moreover, both the invasion assay and the wound healing assay showed that the loss of the ERK2 binding site in β6-integrin (Del. Mutant) largely reverted β6-induced invasion and migration of colon cancer cells.ConclusionsWe firstly report that ETS1 plays a critical role in β6 integrin induced CRC progression, and expression of MMP-3/MMP-9 can be up-regulated by β6 integrin through increased phosphorylation of ERK and subsequent activation of ETS1 (Fig.8). Our present study presents a novel mechanism for P6 integrin-induced CRC progression. Our data confirms avP6 trafficking has a large contribution to cell migration and suggests a potential role for ETS1 in CRC progression, especially in patients with β6 integrin-induced CRC progression. This finding advances our understanding of the mechanisms of CRC progression, and it may lead to the development of ETS1-based strategies for treating the disease and sheds light on effective therapeutic approaches in the progression of CRC. |
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