The Role And Molecular Mechanism Of Fibroblast Growth Factor Receptor 3 In Progression Of Malignant Melanoma

BackgroundMalignant melanoma is one of the most invasive tumors worldwide with a higher incidence in developed countries in Europe and America. According to the statistical results by World Health Organization (WHO), the standardized incidence rate of melanoma in developed countries of Europe and America is more than 9/100 000 while this rate is less than 1/100 000 in less developed countries. In United States, malignant melanoma is the fifth most common tumors of male and the sixth most common tumors of female. As to Asian countries, the incidence of malignant melanoma is far less than the United States with an incidence rate less than 1/100000 in most areas. As to china, the incidence rate of malignant melanoma exhibits a continuous upward trend in the last decade, in addition, the total number of melanoma patients remains worrisome due to a huge population base. The poor prognosis of melanoma patients is mainly caused by early metastasis, additionally, recent studies about melanoma have made much progression and revealed that the risk factors of malignant melanoma including genetic risk, immune, and environmental factors and multiple therapeutic measures. However, the prognosis of metastatic melanoma patients remains poor with a 5-year survival of 15%. Thus, investigating for novel diagnostic and therapeutic targets for malignant melanoma is of great value.Fibroblast growth factor (FGF) and its receptor FGFR family consist a relatively conservative signaling pathway.22 kinds of FGFs and 4 kinds of FGFRs have been identified to now. FGFR3 was initially identified as a modulator of angiogenesis and embryonic development. FGFR3 knockout mice exhibited overgrowth of the long bones, while activated FGFR3 mutations were confirmed to be associated with dwarfism, indicating the negative role of FGFR3 in bone development. In recent years, overactivation of FGFR signaling had been identified to play critical rules in myeloma, cervical cancer, bladder cancer and other malignancies. Activated FGFR3 mutations could significantly enhance the malignant behaviors of tumor cells. Thus, FGFR3 had been considered as a potential therapeutic target for a variety of malignant tumors. Recently, the relationship between FGFRs and malignant melanoma had drawn much attention. FGFR1 could regulate the proliferation and angiogenesis ability of melanoma cells and silencing FGFR1 could significantly inhibit proliferation and poorly differentiated of melanoma cells. These studies indicated FGFR1 might be an oncogene of malignant melanoma. Considering the homology of FGFR family proteins, we wanted to explore the clinical significance of FGFR3 expression in melanoma tissues and the biological function of FGFR3 in melanoma cells.Objective1. The detection of FGFR3 expression in malignant melanoma tissues and non-tumor tissues.2. Evaluation of the relationship between FGFR3 expression and clinicopathological parameters of melanoma patients.3. Knocking down or overexpressing FGFR3 in melanoma cells to evaluate the role of FGFR3 on cell proliferation.4. Evaluating the role of FGFR3 on cell migration, invasion and EMT behaviors in vitro.5. Evaluating the role of FGFR3 on cell proliferation and migration in vivo.6. Detecting the change of signaling pathways after FGFR3 down-regulation.Methods1.42 paired melanoma samples were collected and the expression levels of FGFR3 in melanoma tissues and corresponding non-tumor tissues were evaluated by Quantitative Real-time PCR (qRT-PCR) and western blot respectively.2. The relationship between FGFR3 expression and the clinicopathological parameters of melanoma patients was evaluated by immunohistochemical staining, which was scored according to staining intensity and number of positive cells. Staining intensity was scored as 0-3 representing negative(-), weak(+), moderate(++), and strong (+++) staining, respectively. The stained cells were counted and scored as 0 (<10%),1 (10%-25%),2 (25%-50%),3(>50%). The final score was determined by two independent researchers. After multiplying score of staining intensity and stained cell numbers. The final score range was 0-9, and scores of 0-3 represent low expression level of LIFR and 4-9 represent high level.3. Establishment of cell lines expressing shFGFR3 or shNC by corresponding lentivirus product and cell lines expressing pcDNA3.0-FGFR3 or pcDNA3.0 by corresponding plasmids. The down-regulation or up-regulation efficiency was confirmed by qRT-PCR and western blot, respectively.4. The role of FGFR3 on cell proliferation was evaluated by CCK-8 assay. Cells at logarithmic growth phase were trypsinized, resuspended and plated into 96 well plate at a density of 1.0×104/ml with a total volume of 100ul. lOul CCK-8 was added into each plate at the same time of each day for 6 days.5. The role of FGFR3 on cell apoptosis was examined by flow cytometry. After stained by Annexin V and PI, the apoptosis rate of A375/LV-shFGFR and A375/LV-shNC cells were detected by flow cytometer respectively.6. The role of FGFR3 on cell migration and invasion was evaluated by transwell and matrigel invasion chambers according to the manufacturer's protocol.7. A375/LV-shFGFR3 and A375/LV-shNC cells were subcutaneously injected into the right flank of 4-week-old male nude mice (5 mice per group). The tumor volume was examined every week. The subcutaneous tumors were removed for evaluating the volume and weight. As to lung metastasis assay, A375/LV-shFGFR3 and A375/LV-shNC cells were injected into tail vein of nude mice and the metastatic nodes were calculated respectively.8. Western blot assay was employed to examine the expression levels of ERK, AKT, EGFR and the corresponding phosphorylation proteins after FGFR3 down-regulation.Results1. The expression level of FGFR3 was significantly higher in melanoma tissues compared with adjacent non-tumor tissues. FGFR3 expression in malignant melanoma tissues was correlated with Breslow thickness and lymph node metastasis (p<0.05).2. Silencing FGFR3 inhibited the proliferation ability of melanoma cells while FGFR3 restoration promoted the proliferation ability.3. Silencing FGFR3 promoted the apoptosis rate of melanoma cells while FGFR3 restoration inhibited apoptosis.4. Silencing FGFR3 inhibited the migration and invasion ability of melanoma cells in vitro while FGFR3 restoration promoted the migration and invasion ability in vitro.5. Silencing FGFR3 inhibited the proliferation and migration ability of melanoma cells in vivo.6. The levels of epithelial marker E-cadherin was up-regulated while the levels of mesenchymal markers N-cadherin and vimentin were down-regulated after silencing FGFR3.7. The expression level of ERK, AKT and EGFR didn't show much change after FGFR3 down-regulation while the phosphorylation levels of these proteins were significantly decreased.ConclusionsFGFR3 was overexpressed in malignant melanoma tissues and correlated with Breslow thickness and lymph node metastasis. FGFR3 regulated malignant melanoma cell proliferation, migration and EMT behaviors via influencing the phosphorylation level of ERK, AKT and EGFR.