Stable SET knockdown in head and neck squamous cell carcinoma promotes cell invasion and the mesenchymal-like phenotype in vitro, as well as necrosis, cisplatin sensitivity and lymph node metastasis in xenograft tumor models

Molecular Cancer

Research

Lewis J Greene¹  Lucas O Sousa¹  Andréia M Leopoldino²  Lays M Sobral²  Eloiza H Tajara³  Ricardo D Coletta⁴  Hamilton Cabral⁵  Carlos Curti⁶  J Silvio Gutkind⁷ 

[1] Department of Cellular and Molecular Biology, School of Medicine, University of São Paulo, Ribeirão Preto, SP, Brazil and Center for Cell Therapy and Hemotherapy of Ribeirão Preto, INCT, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil;Department of Clinical Analysis, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Riberião Preto, University of São Paulo, Av. Café, s/n, 14040-903, Ribeirão Preto, SP, Brazil;Department of Molecular Biology, School of Medicine of São José do Rio Preto, Ribeirão Preto, SP, Brazil;Department of Oral Diagnosis, Piracicaba Dental School, University of Campinas, Piracicaba, SP, Brazil;Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil;Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil;Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA;

关键词: SET;    Head and neck cancer;    MMP;    ERK;    EMT;    p53;    Invasion;    Cisplatin;    Metastasis;   

DOI  :  10.1186/1476-4598-13-32

 received in 2013-12-09, accepted in 2014-02-13,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundSET/I2PP2A is a multifunctional protein that is up-regulated in head and neck squamous cell carcinoma (HNSCC). The action of SET in HNSCC tumorigenicity is unknown.MethodsStable SET knockdown by shRNA (shSET) was established in three HNSCC cell lines: HN12, HN13, and Cal27. Protein expression and phosphorylated protein levels were determined by Western blotting and immunofluorescence, cell migration and invasion were measured by functional analysis, and PP2A activity was determined using a serine/threonine phosphatase assay. A real-time PCR array was used to quantify 84 genes associated with cell motility. Metalloproteinase (MMP) activity was assessed by zymographic and fluorometric assays. HN12shSET xenograft tumors (flank and tongue models) were established in Balb/c nude mice; the xenograft characteristics and cisplatin sensitivity were demonstrated by macroscopic, immunohistochemical, and histological analyses, as well as lymph node metastasis by histology.ResultsThe HN12shSET cells displayed reduced ERK1/2 and p53 phosphorylation compared with control. ShSET reduced HN12 cell proliferation and increased the sub-G1 population of HN12 and Cal27 cells. Increased PP2A activity was also associated with shSET. The PCR array indicated up-regulation of three mRNAs in HN12 cells: vimentin, matrix metalloproteinase-9 (MMP9) and non-muscle myosin heavy chain IIB. Reduced E-cadherin and pan-cytokeratin, as well as increased vimentin, were also demonstrated as the result of SET knockdown. These changes were accompanied by an increase in MMP-9 and MMP-2 activities, migration and invasion. The HN12shSET subcutaneous xenograft tumors presented a poorly differentiated phenotype, reduced cell proliferation, and cisplatin sensitivity. An orthotopic xenograft tumor model using the HN12shSET cells displayed increased metastatic potential.ConclusionsSET accumulation has important actions in HNSCC. As an oncogene, SET promotes cell proliferation, survival, and resistance to cell death by cisplatin in vivo. As a metastasis suppressor, SET regulates invasion, the epithelial mesenchymal transition, and metastasis.

【 授权许可】

Unknown   
© Sobral et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.