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

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.

Lymph node or perineural invasion is associated with low miR-15a, miR-34c and miR-199b levels in head and neck squamous cell carcinoma

“…These specific miRs were chosen because they participate in the regulation of pathways considered to be “hallmarks of cancers”, such as cell death, proliferation and migration/invasion [10], [11], [12], [13], [14]. We also assessed by immunohistochemistry in six selected HNSCC samples and their matched resection margins the SET (I2PP2A) protein, involved in the promotion of cancer cell proliferation [15], and PTEN protein, a classic tumor suppressor [16]. In HNSCC samples, lower levels of miR-15a, miR-34c and miR-199b were associated with lymph node invasion, while lower levels of miR-199b were associated with perineural invasion.…”

Chemical Characterization and in Vitro Antitumor Activity of the Essential Oils from the Leaves and Flowers of Callistemon viminalis

“…Thus, several studies have reported the potency of the constituents of EOs in the prevention and treatment of cancer [21]. Sobral et al (2014) [22] showed, through studies performed in vitro and in vivo, that the antitumor [23]. Moderate cytotoxicity of 1,8-cineole and α-terpineol, major constituents also found in this study, was observed in the HepG2 (liver carcinoma) and K-562 (leukemia) lines [24].…”

Enrichment of Cells with Cancer Stem Cell-Like Markers in Relapses of Chemoresistant Patients with Locally Advanced Head and Neck Squamous Cell Carcinoma

“…We hypothesize that residual or recurrent tumor tissue has a high content of chemotherapy-resistant cancer cells which can cause tumor regeneration. Several lines of research support this hypothesis [2,3,4]. …”

“…A similar loss of HLA-I expression could be expected in tumors with high numbers of CSCs. Also, a loss of pancytokeratin (pan-CK) expression by immunohistochemical analysis has been used as an indicator of the poor cell differentiation typically observed in tumors with CSC enrichment [4]. …”