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 modelsReport as inadecuate




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 - Download this document for free, or read online. Document in PDF available to download.

Molecular Cancer

, 13:32

First Online: 20 February 2014Received: 09 December 2013Accepted: 13 February 2014DOI: 10.1186-1476-4598-13-32

Cite this article as: Sobral, L.M., Sousa, L.O., Coletta, R.D. et al. Mol Cancer 2014 13: 32. doi:10.1186-1476-4598-13-32

Abstract

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.

KeywordsSET Head and neck cancer MMP ERK EMT p53 Invasion Cisplatin Metastasis AbbreviationsAktProtein kinase B

APMA4-aminophenylmercuric acetate

BrdUBromodeoxyuridine

DMEMDulbecco’s modified medium

EMTEpithelial-mesenchymal transition

ERKExtracellular signal-regulated kinase

FBSFetal bovine serum

HandEHematoxylin-eosin

HNSCCsHead and neck squamous cell carcinomas

JNKc-Jun NH2-terminal kinase

LIMK1LIM kinase

MAPKsMitogen-activated protein kinases

MMPMatrix metalloproteinase

OSCCsOral squamous cell carcinomas

PAN-CTKRPan-cytokeratin

PBSPhosphate-buffered saline

PIPropidium iodide

PI3KPhosphatidylinositol 3-kinase

PP2AProtein phosphatase 2A

SDS-PAGESodium dodecyl sulfate-polyacrylamide gel electrophoresis

shRNAShort-hairpin RNA

SRCc-Src

TBSTTris-buffered saline with 10% Tween 20

WASLWiskott-Aldrich syndrome-like.

Electronic supplementary materialThe online version of this article doi:10.1186-1476-4598-13-32 contains supplementary material, which is available to authorized users.

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Author: Lays M Sobral - Lucas O Sousa - Ricardo D Coletta - Hamilton Cabral - Lewis J Greene - Eloiza H Tajara - J Silvio Gut

Source: https://link.springer.com/







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