CCL17-CCR4 Axis Promotes Metastasis Via ERK/MMP13 Pathway In Bladder Cancer

BackgroundBladder cancer is the most common cancer.The latest epidemiological studies suggest that bladder cancer is the ninth most common tumor in the world,with mortality ranking 13th,and developed countries showing a downward trend.According to China's epidemiological study,the incidence of bladder cancer in China is 7.49/100,000,ranking eighth in the incidence of all malignant tumors,accounting for 2.5%of the incidence of malignant tumors in China.And the mortality rate of bladder cancer in China is still high,and there is still a gap with developed countries.According to pathological classification,the vast majority of bladder tumors are bladder urothelial carcinoma.Clinically,especially high-grade myometrial invasive bladder cancer(MIBC,including T2 and above),it is characterized by high degree of malignancy,easy metastasis,rapid progress,and high mortality.As a family of small molecules,the role of chemokines is mainly achieved by binding to G protein-coupled receptors.In the process of tumor development,they mainly aggregate with specific receptors on the surface of tumor cells,thereby accumulating specific sites of chemotaxis,and achieving tumor metastasis and infiltration.A number of studies have demonstrated the pivotal role of chemokines and their receptors in the treatment of cancer.For example,studies have found that CXCR4 is involved in the distant metastasis of more than 20 different cancers.As an important chemokine receptor,CCR4 is expressed on a variety of T cells,particularly Treg cells and Th2 cells.A number of reports have reported the function of CCR4 in hematological malignancies and solid tumors such as gastric cancer,breast cancer and lung cancer.Studies have shown that CCR4 may be involved in CCL17/CCL22-induced tumor cell migration.However,little is known about the role of CCR4 in the development of bladder cancer.Therefore,the purpose of this study is to explore the expression of CCR4 in bladder cancer tissues,its correlationwith various clinicopathological factors and the role of CCL17-CCR4 in cell invasion and metastasis,and to explore its molecular mechanism.MethodsThe expression of CCR4 in bladder cancer and corresponding paracancerous tissues was detected by Western Blot.And we also investigated the expression of CCR4 in bladder cancer tissues by imunohistochemical staining(IHC),and the staining results of 122 specimens were scored.Recurrence rate curves were calculated by Kaplan-Meier curves and statistical single factor or multivariate analysis was performed by Cox risk regression analysis.The expression of CCR4 in bladder cancer cell lines T24 and 253J was detected by Western Blot,PCR and immunofluorescence.Subsequently,the effects of CCL17 on the invasion and migration ability of bladder cancer cells were examined by cell scratch test and Transwell test.Stable CCR4 low-expression cell lines were constructed using plasmids,and changes in migration ability were examined,and Western Blot was used to detect changes in related pathways.Using ERK pathway inhibitor U0126,the relationship between ERK-MMP13 pathway and CCL17-CCR4 was explored,and the corresponding changes of bladder cancer cells under U0126 were detected by cell scratch and Transwell assay.ResultsWestern Blot results showed that the expression of CCR4 in bladder cancer tissues was higher than that in adjacent tissues.At the same time,IHC results showed that among the 122 specimens,68 cases were highly expressed in CCR4,accounting for 55.7%.Kaplan-Meier curve statistical analysis The expression of CCR4 was significantly correlated with OS(P=0.002).In addition,multivariate Cox risk regression analysis showed that CCR4 expression was an independent prognostic factor(P=0.007).In order to detect the expression of CCR4 on bladder cancer cells,we used Western Blot,RT-qPCR and cellular immunofluorescence techniques respectively.The results showed that CCR4 was expressed in bladder cancer cell lines T24 and 253J.Subsequently,when the synthetic CCL17 chemokine was added to the cell supernatant,both T24 and 253J cells showed an increase in invasion and migration ability,while Western Blot also showed an increase in the expression of CCR4 in the cells,indicating that CCL17 can promote CCR4 expression.And in order to explore the role of CCR4 in the above process,we constructed a low-expression CCR4-expressing bladder cancer cell line(sh-T24 and sh-253J)by plasmid stabilization,and under the same dose of CCL17,low expression of bladder cancer cell lines.The ability to invade and migrate has decreased significantly.In order to explore its related molecular mechanism,we performed Western Blot experiments and found that the expression of P-ERK and MMP13 in bladder cancercell lines with low expression of CCR4 also decreased.By adding the ERK pathway inhibitor U0126,we found that the invasive migration ability of T24 and 253J showed a significant decrease,and there was no obvious recovery after the addition of CCL17,indicating that CCL17-CCR4 regulates bladder cancer cells through ERK-MMP13 signal path is implemented.ConclusionThis study shows that there is a correlation between the expression of CCR4 and the clinical prognosis of patients with bladder cancer,and CCL17-CCR4 can affect the invasion and metastasis of bladder cancer cells through ERK-MMP13 signaling pathway.Therefore,CCR4 can be a potential target for the treatment of bladder cancer and has more in-depth research value.