HPV16 E7 Promotes Proliferation, Invasion And Metastasis Of Cutaneous Squamous Cell Carcinoma By Upregulating Smad7 Expression

Background and Objective:Human papillomavirus (HPV) is the etiological agent in cervical cancer and also is implicated in other cancers of anogenital and oropharyngeal origins, and the most common genotype is HPV16. HPV16 E7 oncogene is an important transforming gene which appears to be essential motivation for the majority of related cancers. Transforming growth factor-beta 1 (TGF-beta 1) is a cytokine which is a potent growth inhibitor of most epithelial and endothelial cells. Smad proteins are downstream signal proteins of the TGF-beta superfamily, which are intimately related to the genesis of many human carcinomas. This study also provides a better understanding of the relationship between HPV16 E7 , Smad7 and cutaneous squamous cell carcinoma.HPV16 E7 expression is closely related to poor prognosis and higher tumor grade in patients with breast, oral and gastric carcinomas. Smad7 proteins have been identified as major inhibitor in the intracellular signaling of TGF-beta 1. High-level expression of Smad7 in gastric carcinomas is significantly associated with lymph node metastasis, and poor prognosis. It is reported that Smad7 plays a critical role in carcinogenesis and tumor metastasis through blocking TGF-beta signaling and upregulating the proliferation-related genes. Therefore, we investigated the relation of HPV16 E7 and Smad7 in invasion and metastasis of cutaneous squamous cell carcinoma.Methods and Result:Firstly, the levels of the expression of HPV16 E7 and Smad7 in 37 cutaneous squamous cell carcinoma tissues and 30 control tissues were detected by PCR and immunohistochemistry method respectively. In cutaneous squamous cell carcinoma tissues, positive rate of HPV16 E7 and Smad7 was 16.2% (6/37) and 86.5% (32/37), respectively. There was a significant difference in positive expression of HPV16 E7 or Smad7 between cutaneous squamous cell carcinoma tissues and normal control tissues (P < 0.05). There was a significant association between expression of HPV16 E7 and lymph node metastasis (P < 0.01), differentiation stages(P < 0.05). The expression of Smad7 was closely related to lymph node metastasis and differentiation (P < 0.05) too.Secondly, A vector ovexpressing HPV16 E7 named pEGFP-E7 was constructed and transfected into A431 cells. The expressions of Smad7 proteins and mRNAs in A431 cells were increased after HPV16 E7 overexpression,which was confirmed by quantitative RT-PCR assay and Western blotting. Western blotting showed that the expression level of Smad4 protein in HPV16 E7-overexpressed A431 cells was lower than that in normal controls, and the level of EGFR protein was opposite. The level of the expression of Smad1/2/3 and p-Smad2/3 proteins in A431 cells were unchanged after the transfection of HPV16 E7. Untreated A431 cells contained moderate Smad4 protein levels in the cytoplasm and nuclear, and with the weak fluorescence in the perinuclear region observed by confocal microscopy in the present of HPV16 E7. ELISA showed that HPV16 E7 contribute to the enhancement of TGF-beta 1 level secreted by A431 cells. HPV16 E7 overexpression promoted proliferation, migration and invasion of A431 cells by WST-1 assay and Tranwell experiment. HPV16 E7 overexpression also contributes to the acceleration of cell cycle in A431 cells analyzed by flow cytometry.Finally, an interference plasmid named pcDNA6.2-miR-Smad7 for inhibiting Smad7 expression was constructed using the pcDNA6.2-miR vectors. The effect of Smad7 knockdown was confirmed through transfecting the pcDNA6.2-miR-Smad7 into A431 cells. Expression of Smad7 mRNAs and proteins were examined by Western blotting and quantitative RT-PCR. Silencing of Smad7 reduced cell migration and inhibited cell proliferation of A431 cells observed by Tranwell experiment and WST-1 assay. Western blotting showed that the expression level of TGF-beta RI, Smad4 and Bax protein in Smad7-hypoexpressed A431 cells were higher than that in control A431 cells. Silencing of Smad7 contribute to decreasing the expression levels of PCNA, C-myc, EGFR proteins. The cytoplasmic-nucleo shuttling of Smad4 in A431 cells with silencing of Smad7 detected by immunofluorence assay and confocal microscopy. Cell cycle arrest and apoptosis of A431 cells were observed after Smad7 be silenced. Scanning electron microscope and transmission electron microscope micrographs showed that the apoptotic morphology of in A431 cells and tumor after treated. Smad7 silences suppress the growth of cutaneous squamous cell carcinoma of tumor-bearing nude mice.ConclusionsHPV16 E7 promotes invasion and metastasis of cutaneous squamous cell carcinoma by upregulating Smad7 expression. Smad7 plays an important role in invasion and metastasis of squamous cell carcinoma induced by HPV16 E7. These effects mentioned above may be coupled with elevated Smad7 expression through interfering TGF-beta signaling pathway and interacting with other apoptotic regulators.