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

Author(s)

Sobral, Lays M. ^[1] ; Sousa, Lucas O. ^{[2, 3]} ; Coletta, Ricardo D. ^[4] ; Cabral, Hamilton ^[5] ; Greene, Lewis J. ^{[2, 3]} ; Tajara, Eloiza H. ^[6] ; Gutkind, J. Silvio ^[7] ; Curti, Carlos ^[8] ; Leopoldino, Andreia M. ^[1]

Affiliation

^[1] Univ Sao Paulo, Sch Pharmaceut Sci Riberiao Preto, Dept Clin Anal Toxicol & Food Sci, BR-14040903 Ribeirao Preto, SP - Brazil

^[2] Univ Sao Paulo, Sch Med, Dept Cellular & Mol Biol, BR-14040903 Ribeirao Preto, SP - Brazil

^[3] Univ Sao Paulo, Sch Med Ribeirao Preto, INCT, Ctr Cell Therapy & Hemotherapy Ribeirao Preto, BR-14040903 Ribeirao Preto, SP - Brazil

^[4] Univ Estadual Campinas, Piracicaba Dent Sch, Dept Oral Diag, Piracicaba, SP - Brazil

^[5] Univ Sao Paulo, Sch Pharmaceut Sci Riberiao Preto, Dept Pharmaceut Sci, BR-14040903 Ribeirao Preto, SP - Brazil

^[6] Sch Med Sao Jose do Rio Preto, Dept Mol Biol, Ribeirao Preto, SP - Brazil

^[7] Natl Inst Dent & Craniofacial Res, Oral & Pharyngeal Canc Branch, NIH, Bethesda, MD - USA

^[8] Univ Sao Paulo, Sch Pharmaceut Sci Riberiao Preto, Dept Chem & Phys, BR-14040903 Ribeirao Preto, SP - Brazil

• Document type: Journal article
• Source: Molecular Cancer; v. 13, FEB 20 2014.
• Web of Science Citations: 26

Abstract

Background: SET/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. Methods: Stable 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. Results: The 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. Conclusions: SET 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. (AU)

• FAPESP's process: 10/18544-9 - Profile analysis of SET-associated miRNAS and its effects on proliferation, invasion and migration in an oral squamous cell carcinoma model - in vivo and in vitro.
• Grantee: Lays Martin Sobral
• Support type: Scholarships in Brazil - Post-Doctorate
• FAPESP's process: 13/10898-4 - Study of the molecular mechanisms by protein SET with impact on tumorigenesis and progression of oral cancer
• Grantee: Carlos Curti
• Support type: Regular Research Grants
• FAPESP's process: 09/52228-0 - Studies on resistance to apoptosis in cancer, head/neck model: signaling via with emphasis on PIP3-Akt/SET, oxidative stress, mitochondria and relationships
• Grantee: Carlos Curti
• Support type: Research Projects - Thematic Grants
• FAPESP's process: 10/20384-0 - Identification of genes, miRNAs and proteins regulated by set oncoprotein and associated on malignization and tumor progression in HNSCC using in vitro and in vivo models
• Grantee: Andréia Machado Leopoldino
• Support type: Regular Research Grants
• FAPESP's process: 13/08135-2 - CTC - Center for Cell-Based Therapy
• Grantee: Dimas Tadeu Covas
• Support type: Research Grants - Research, Innovation and Dissemination Centers - RIDC