Role Of Integrin ανβ6in Colon And Thyroid Carcinomas

Part ⅠRole of Integrin αvβ6in Chemo Resistance of Colon CarcinomaBackgroundColorectal cancer (CRC) is the third most prevalent cancer and the fourth leading cause of cancer deaths worldwide. With the increasingly serious environmental pollution and the change of people’s diet in recent years, the incidence and mortality rates of colorectal cancer have gradually increased. Currently, surgical resection is still the main treatment for colorectal cancer. However, tumor recurrence was found in about50%of resectable colorectal cancers, and most patients lose the opportunity of cure. Present study suggests that, CRC patients with stage III need systemic adjuvant chemotherapy to destroy the body’s tiny metastases, reducing tumor recurrence and prolonging life. Multiple chemotherapy regimens based on5-fluorouracil (5-FU) have been applied to the treatment of CRC patients for30years. Due to the generation of the primary or acquired drug resistance, chemotherapy often did not show good results, even failed. In recent years, a number of novel chemotherapy drugs are emerging, such as irinotecan and oxaliplatin, however, the generation of drug resistance is still the urgent problem in chemotherapy for colorectal cancer.Integrins belong to cell surface adhesion molecular family, which are composed of a and β subunits associated noncovalently. On the one hand, integrins combine with extracellular matrix proteins or adjacent cells, thus connecting extracellular matrix to cell skeletal system, and finally constituting the physical bracket of the tumor microenvironment. On the other hand, integrins combine with a variety of cytokines, and transduct cellular signals in a bidirectionally way, thus constituting the information transduction center for tumor microenvironment. Evidence has implicated that integrins may be involved in chemo resistance of cancers for increased expression of some integrin subunits and/or heterodimers have been found in drug resistant cells. In addition, integrins may participate in apoptosis of cancer cells induced by chemotherapeutic drugs. Blocking the link between integrins and extracellular matrix or adjacent cells, by molecular biology techniques, such as blocking antibodies and siRNA interference, can effectively inhibit drug resistance of tumors to chemotherapy. Integrins have become extremely potential targets for solving the problem of drug resistance of tumors. On this basis, doctors can analyze integrin phenotype of tumors by microarray technology and determine the most effective chemotherapy regimens. Especially, individualized treatments against specific integrin phenotype will have good prospects for clinical application.Integrin avβ6is a subtype of integrins, which expresses strictly in epithelia and is up-regulated in parallel with embryo formation, oncogenesis and epithelial repair,whereas it ismostly undetectable in normal tissue. We have reported that avβ6plays an important role in invasion, metastasis and degradation of extracellular matrix of colon cancer. In addition, we demonstrated a direct link between avβ36and the extracellular signal-regulated kinase (ERK2), and this direct link plays a key role in regulatory function of avβ6. However, it was not known about the role of avβ6in chemo resistance of colonic or other cancers.In the present study, we used two colon cancer cell lines SW480and HT-29and used5-FU, the representative of colorectal cancer chemotherapeutic drugs, to investigate whether avβ6participated in5-FU resistance and explored the mechanisms involved.Section1Role of Integrin avP6in Proliferation and Apoptosis of Colon Cancer Cells with5-FU Treatment Objective This study was to investigate the role of integrin avβ6in proliferation and apoptosis of SW480and HT-29colon cancer cells with5-FU treatment. Methods1. SW480colon cancer cells were divided into three groups:wild-type,β6-transfectant and Mock-transfectant, Flow cytometry and western blotting were used to examine the expression of β6-integrin in three groups.2. We generated the β6-integrin-specific shRNA expression vectors and control vectors by using pSIREN-DNR-DsRed-Express Vector plasmid, then transfected them into HT-29colon cancer cells in liposome way. HT-29colon cancer cells were divided into three groups: wild-type,β6-shRNA and control-shRNA. Flow cytometry and western blotting were used to examine the expression of β6-integrin in three groups.3. Both SW480and HT-29colon cancer cells were exposed to5-FU under various concentrations for48h, then cell proliferation were examined by MTT assay, and IC50values at48h were also determined and compared.4. Both SW480and HT-29colon cancer cells were exposed to5-FU under IC50concentrations for48h, and subjected to Hoechst33258nuclear staining assay and AnnexinV-FITC/PI cell apoptosis assay combined with a flow cytometer.Results1. Results of flow cytometry and western blotting showed that, in SW480cells, β6-transfected cells showed obvious expression of β6-integrin, while mock-transfected or wild type cells did not show obvious expression. In HT-29cells, plasmid vector based shRNA effectively down-regulated β6-integrin expression. As to av-integrin, the transfection did not result in obvious effects.2. Results of MTT assay showed that, the survival rate was higher in SW480β6-transfected cells, compared to the wild type and mock-transfected cells in all dose points. Besides, suppression of β6-integrin by the specific siRNA resulted in markedly lower survival rate in HT-29cells.3. Results of Hoechst33258assay and AnnexinV-FITC/PI cell apoptosis assay both showed that, apoptotic cells were fewer in SW480β6-transfected cells than the wild type or mock-transfected cells. In HT-29cells, more apoptosis occurred when β6-integrin was suppressed. ConclusionsThis part of the study revealed that, integrin avβ6protected SW480and HT-29colon cancer cells from5-FU-induced growth inhibition and apoptosis. The result provides precise theory for the study of targeted therapy or adjuvant chemotherapy for colon cancer. Section2Effects of integrin avβ6on5-FU-related mitochondrial apoptotic pathway in colon carcinomaObjectiveThis study was to investigate the effects of integrin avβ6on5-FU-related mitochondrial apoptotic pathway in colon carcinoma.Methods1. Cells were grouped in the same way as part I.2. Both SW480and HT-29colon cancer cells were exposed to5-FU under IC50concentrations for48h, the expression of cytoplasmic cytochrome C, Bcl-2and Bax were examined by western blotting, the activity of caspase-3and caspase-9were examined by caspase activity detection kit.Results1. In SW480cells with5-FU treatment, cytochrome C released from mitochondria was evidently lower in β6-transfected cells compared to the wild type or mock-transfected cells. Both the caspase-3and caspase-9activities were lower in β6-transfected cells. In HT-29cells with5-FU treatment, suppression of β6-integrin induced higher expression of cytosolic cytochrome C and higher activities of caspase-3and caspase-9.2. Western blotting analysis demonstrated that in SW480β6-transfected cells, theexpression of Bcl-2was higher and Bax was lower compared withthose in mock-transfected and wild type cells. Upon treatmentwith5-FU, Bcl-2expression also increased in P6-transfected cellsand remained low in mock-transfected and wild type cells. Meanwhile, Bax expression decreased in SW480β6-transfected cells andalmost remained unchanged in mock-transfected and wild typecells. This Bcl-2up-regulation and Bax down-regulation both, preand post5-FU treatment, which associated with β6-integrinexpression, was also found in HT-29cells.ConclusionsThis part of the study revealed that, integrin avβ6effectively inhibited cytochrome C releasing from the mitochondria and activating of caspase-3and caspase-9. Moreover, β6-integrin resulted in up-regulation of Bcl-2and down-regulation of Bax. Thus integrin avβ6effectively inhibited5-FU-induced mitochondrial apoptotic pathway in colon carcinoma. This part of the study identified the specific mechanism of integrin avβ6inhibition of5-FU-induced apoptosis in colon cancer, and provided precise theory for the study of targeted therapy or adjuvant chemotherapy for colon cancer. Section3Cell signaling pathway of integrin avβ6-mediated chemoresistance of colon carcinomaObjectiveThis study was to investigate the cell signaling pathway of integrin avβ6-mediated chemoresistance of colon carcinoma.Methods1. Cells were grouped in the same way as part Ⅰ. Expression of MAPK signal transduction pathway critical factors, such as phosphorylated/total ERK (p/t-ERK), phosphorylated/total JNK (p/t-JNK) and phosphorylated/total p-p38(p/t-p38) were examined by western blotting. 2. Cells were grouped in the same way as part I. After ERK/MAPK signal transduction pathway was blocked, both SW480and HT-29colon cancer cells were exposed to5-FU under IC50concentrations for48h, then cell proliferation were examined by MTT assay, cell apoptosis were examined by AnnexinV-FITC/PI cell apoptosis assay combined with a flow cytometer.3. SW480colon cancer cells were divided into three groups:β6-transfectant, Mock-transfectant and β6Mutant-transfectant(in which β6-integrin lost the ERK2binding site). Cells were exposed to5-FU under IC50concentrations for48h, then cell proliferation were examined by MTT assay, cell apoptosis were examined by AnnexinV-FITC/PI cell apoptosis assay combined with a flow cytometer, and expression of Bcl-2and Bax were examined by western blotting.Results1. Western blotting analysis demonstrated that higher expression of p-ERK1/2was associated with β6-integrin expression in both SW480and HT-29cells. The expression of t-ERK1/2, same as p/t-JNK and p/t-p38, were not obviously affected by β6-integrin expression.2. Results of MTT assay and AnnexinV-FITC/PI cell apoptosis assay showed that, PD98059effectively reverted β6-integrin-mediated chemo resistance to5-FU.3. When β6-integrin lost the ERK2binding site, β6-integrin-mediated chemoresistance to5-FU was markedly inhibited. In addition, β6-integrin-mediated Bcl-2up-regulation and Bax down-regulation both pre and post5-FU treatment, were also inhibited.ConclusionsThis part of the study revealed that, integrin avβ6induced5-FU resistance in colon cancer cells through the ERK/MAPK pathway and largely depending on the b6-ERK2direct binding. This part of the study identified the specific cell signaling pathway of integrin avβ6-mediated5-FU resistance in colon cancer, and provided precise theory for the study of targeted therapy or adjuvant chemotherapy for colon cancer. Part ⅡExpression of Integrin αvβ6in Different Thyroid Tissues and its Relationship with Clinicopathological Features of Thyroid Carcinoma PatientsBackgroundThyroid carcinoma accounts for no more than2%of all human carcinomas, but the incidence rate over the past decades has consistently increased. Although most thyroid carcinomas have a low malignancy potential, are curable with surgery, and have a good responsiveness to radioiodine, diagnostic difficulties still remain. Fine needle aspiration (FNA) biopsy is currently the most accurate diagnostic method for thyroid carcinoma, but doctors or pathologists are unable to reach a consensus on the pathological diagnosis for as many as30%of these FNA biopsies. This low yield consensus is due to many papillary and follicular tumors appearing structurally similar to normal follicular tissue. In many cases, an unnecessary thyroidectomy was performed that in retrospect was a benign tumor. Additionally, some patients develop more aggressive forms of the disease including lymphatic and distant metastasis. Papillary carcinoma, which accounts for the majority of thyroid carcinomas, has a characteristic early stage lymph node metastasis. Studies have shown that patients over the age of45with lateral or mediastinal cervical lymph node metastasis have an increased risk of death from this disease. Thus, it is needed to identify new molecular markers to increase diagnostic accuracy and predict the metastatic potential for thyroid carcinoma.As members of the cell adhesion molecular family, integrins are composed of a and β subunits associated noncovalently. Integrins are not only receptors for extracellular matrix protein cell adhesion but also transmembrane connections for bidirectional cellular signal pathway transduction.αvβ6is a subtype of integrins, expressed strictly in epithelia, up-regulated in parallel with embryo formation, oncogenesis and epithelial repair, and rarely expressed in normal tissue. Previously studies showed that integrin αvβ6played an important role in invasion, prolifration, apoptosis, tumor immunity and EMT of malignant tumors. It is worth noting that avP6expression was closely related with clinicopathological features of many carcinomas, as well as patients prognosis. In colon cancer, the median survival time of patients with high expression of avβ6, compared with patients with no or low expression of avβ6, was significantly shorter. In stomach carcinoma, avP6expression is closely associated with Lauren type, differentiation, N stage and TNM stage. More importantly, avP6expression is linked to significantly reduced survival times. In addition, in oral squamous cell carcinoma, breast carcinoma and ovarian carcinoma, the relationship between avP6expression and the clinicopathological features of carcinomas, as well as patients prognosis, has also been confirmed by studies.There is still mystery about avβ6expression in the thyroid. In this study, we examined avP6expression in a series of malignant and non-malignant thyroid tissue. Furthermore, we investigated the relationship between avβ6expression and the clinicopathological features of patients with thyroid carcinoma.ObjectiveThis study was to examine integrin avP6expression in different thyroid tissues by immunohistochemistry and investigated the relationship between avβ6and clinicopathological features of thyroid carcinoma patients.Methods1. Purchased microarray containing a variety of thyroid tissues, and collectted thyroid cancer tissue samples and patient information from the Department of Pathology, QiLu Hospital. Before the study, all cases were reviewed for diagnostic confirmation.2. avβ6expression in different thyroid tissues was examined by immunohistochemistry, and the results were evaluated according to the criteria established.3. Compared avβ6expression in different thyroid tissues, and explored the relationship between avβ6expression and clinicopathological characteristics of patients with thyroid cancer, including age, gender, histological type, TNM stage, etc.Results 1. avβ6expression was significantly different among tissues with different histopathological types. In normal thyroid tissue, carcinoma adjacent normal tissue, and thyroid adenomas, avP6expression was generally negative or weak. Among them,10cases of normal thyroid tissue were consistently avβ6-negative. Only1out of10cases in carcinoma adjacent normal tissue and3out of10cases in thyroid adenomas were avβ6-positive. Thyroid goiters and thyroiditis tissue both showed wide and strong avβ6expression with7out of10cases in thyroid goiters and8out of10cases in thyroiditis tissue as avβ6-positive. In thyroid carcinomas, expression was positive and strong in71out of90cases. In addition,10cases of metastatic lymph node tissue from papillary thyroid carcinoma showed consistent and strong avP6expression2. avβ6expression distinguished thyroid carcinoma from non-malignant tissues with a sensitivity of78.89%and specificity of62%. The predictive positive value was78.89%and the predictive negative value was62%. avβ6expression differentiated thyroid carcinoma from normal thyroid tissue, carcinoma adjacent normal tissue, and thyroid adenoma. According to the level of avβ6expression, it is impossible to differentiate thyroid carcinoma from thyroid nodular goiter and thyroiditis tissue. There was no significant difference in avβ6expression between papillary and follicular thyroid carcinoma.3. Analysis showed that there was no statistical difference between avβ6-positive and avP6-negative tumors regarding gender, histopathological patterns, T stage and TNM stage, but statistical differences existed regarding patient age and N stage.ConclusionsThis study showed that avβ6expression was significantly different among tissues with different histopathological types. avβ6expression distinguished thyroid carcinoma from non-malignant thyroid tissues with a good sensitivity and specificity. Additionally, avP6expression was closely related to lymph node metastasis from thyroid carcinoma. A potentially consequence of our work is that detection of avβ6expression may help supplement FNA biopsy to increase diagnostic accuracy and help predict the potential lymph node metastasis of thyroid carcinoma.