Investigation Of EphA2on The Invasiveness And Metastasis In Squamous Cell Carcinoma Of The Head And Neck In Vitro And In Vivo

Charpter1Clinical significance of EphA2expression in squamous cell carcinoma of the head and neckObjective:To evaluate the expression of EphA2in tissue specimens and cell lines of squamous cell carcinoma of the head and neck (SCCHN), and further correlate EphA2expression with clinicopathological parameters and prognosis in SCCHN.Methods:EphA2expression in98primary SCCHN tissue specimens was analyzed by immunohistochemistry and correlated with clinicopathological parameters. Additionally,13paired SCCHN tissues and6SCCHN cell lines were evaluated for EphA2expression by RT-PCR and immunoblotting.Results:Both of the mRNA and protein of EphA2overexpressed in SCCHN tissues and SCCHN cell lines. More importantly, high EphA2expression was signifcantly associated with tumor site (P=0.018), T classifcation (P=0.016), clinical stage (P=0.001), recurrence (P=0.002), and lymph node metastasis (P=0.002), respectively. Kaplan-Meier survival analysis demonstrated that patients with high EphA2expression had both poorer disease-free survival and overall survival than patients with low EphA2expression (64.1%vs27.8%;82.1%vs38.9%; both P<0.05). Multivariate Cox regression analysis revealed that EphA2overexpression was an independent prognostic factor for patients with SCCHN (P=0.006).Conclusion:This study demonstrated that EphA2protein expression was obviously increased in SCCHN tissue samples and cell line, and EphA2protein overexpression was associated with tumor recurrence, metastasis and poorer prognosis in SCCHN patients. These results suggested that EphA2might play a critical role in the initiation and progression of SCCHN, implicating EphA2as a valuable marker for the prediction of recurrence, metastasis and prognosis in SCCHN. Charpter2Investigation of the regulation of EphA2on the proliferation, migration and ivasion of the squamous cell carcinoma of the head and neck in vitroObjective:EphA2, a member of the receptor tyrosine kinases, is overexpressed in a variety of solid tumors and cancer cell lines. Our previous study has demonstrated that EphA2is overexpressed in SCCHN, and EphA2protein overexpression was associated with tumor recurrence, metastasis and poorer prognosis in SCCHN patients, suggesting the biological significance of EphA2in SCCHN progression. However, the mechanisms of EphA2and the consequence of its down-regulation in SCCHN have not been fully elucidated in vitro.Methods:EphA2shRNA lentiviral particles were used to knockdown EphA2gene expression in SCCHN cell lines Tu686, RT-PCR and Western blotting were used to validate the gene silencing efficiency of EphA2in SCCHN cell lines. Stable clones were obtained by puromycin screening. CCK-8assay, fluorescence-activated cell sorting analysis, invasion and migration assay were carried out to observe the effects of EphA2inhibition on the proliferation, cell cycle, apoptosis, migration and invasion in vitro.Results:EphA2shRNA lentiviral particles efficiently decreased the mRNA and protein expression of EphA2in SCCHN cell lines Tu686. Tu686EphA2RNAi+cell grew slowly than both control groups (Tu686and Tu686EphA2RNAi-). The percentage of Tu686EphA2RNAi+cells in G0/G1phase was significantly increased (75.04±1.08vs56.09±1.58,55.04±1.38)(P<0.01), whereas the percentage of cells in S phase (18.33±0.77vs32.54±1.70,32.66±0.99) and G2/M phase (6.64±1.00vs12.30±0.76,11.37±0.87) were obviously decreased (both P<0.01). However, there was no significant difference in apoptotic rate between differently treated groups(P>0.05). Moreover, inhibited EphA2expression led to significantly decreased migration [Tu686EphA2RNAi+group, wound closure rate (46.7±7.6)%vs Tu686, Tu686EphA2RNAi-control groups, wound closure rate (88.7±4.5)%and (86.0±2.6)%), P<0.01] and invasion (123±13vs288±20,303±11; P<0.01) of SCCHN Tu686cells. Conclusion:Knockdown the expression of EphA2led to the inhibition of tumor growth, migration and invasion in vitro, suggetting that EphA2contributes to the aggressive behavior of SCCHN, indicating inhibition of EphA2may be a promising targeted approach to block SCCHN progression. Charpter3Investigation of the regulation of EphA2on the ivasion and metastasis of the squamous cell carcinoma of the head and neck in vivoObjective:To further confirm the above results we obtained in vivo, and initially explore the mechanisms of EphA2mediating cervical lymph nodes metastasis of SCCHN in xenografted tumors from nude mouse.Methods:EphA2shRNA lentiviral particles were used to knockdown EphA2gene expression in SCCHN cell lines M2with high lymph nodes metastasis rate, RT-PCR and Western blotting were used to validate the gene silencing efficiency of EphA2in SCCHN cell lines. Stable clones, obtained by puromycin screening, were used to establish SCCHN metastatic xenograft mouse model. Hematoxylin-eosin staining were applied to identify cervical lymph nodes metastasis of SCCHN in xenografted tumors. Western blot was used to investigate the protein expression of EphA2and VEGF. Immunohistochemistry was used to observe microvessel density (CD31).Results:EphA2shRNA lentiviral particles efficiently decreased the mRNA and protein expression of EphA2in SCCHN cell lines M2, which were further successfully utilized to establish SCCHN metastatic xenograft mouse model. Xenografted tumors in group of M2EphA2RNAi+decreased63.5%～69.4%in volume and51.8%～60.0%in weight compared with xenografted tumors in groups of M2and M2EphA2RNA1-. More importantly, the cervical lymph nodes metastasis rate in M2EphA2RNAi+was also declined (21.4%vs66.7%,58.3%)(P<0.05). Decreased protein expression of EphA2and VEGF and microvessel density were observed in group of M2EphA2RNAi+Conclusion:Knockdown the expression of EphA2led to the inhibition of tumor growth and metastasis in SCCHN nude mouse model. More importantly, SCCHN angiogenesis was also impeded, which might be associated with the decreased expression of VEGF.