Sobral Lays M, Sousa Lucas O, Coletta Ricardo D, Cabral Hamilton, Greene Lewis J, Tajara Eloiza H, Gutkind J Silvio, Curti Carlos, Leopoldino Andréia M
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.
Mol Cancer. 2014 Feb 20;13:32. doi: 10.1186/1476-4598-13-32.
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.
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.
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.
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.
SET/I2PP2A是一种多功能蛋白，在头颈部鳞状细胞癌（HNSCC）中上调。SET在HNSCC致瘤性中的作用尚不清楚。
通过shRNA（shSET）在三种HNSCC细胞系（HN12、HN13和Cal27）中建立稳定的SET敲低。通过蛋白质印迹和免疫荧光测定蛋白质表达和磷酸化蛋白水平，通过功能分析测量细胞迁移和侵袭，并使用丝氨酸/苏氨酸磷酸酶测定法测定PP2A活性。使用实时PCR阵列定量84个与细胞运动相关的基因。通过酶谱法和荧光测定法评估金属蛋白酶（MMP）活性。在Balb/c裸鼠中建立HN12shSET异种移植瘤（侧翼和舌模型）；通过宏观、免疫组织化学和组织学分析证明异种移植瘤的特征和顺铂敏感性，以及通过组织学证明淋巴结转移。
与对照相比，HN12shSET细胞显示ERK1/2和p53磷酸化降低。ShSET降低了HN12细胞增殖，并增加了HN12和Cal27细胞的亚G1期群体。PP2A活性增加也与shSET相关。PCR阵列表明HN12细胞中三种mRNA上调：波形蛋白、基质金属蛋白酶-9（MMP9）和非肌肉肌球蛋白重链IIB。SET敲低的结果还显示E-钙黏蛋白和全细胞角蛋白减少，以及波形蛋白增加。这些变化伴随着MMP-9和MMP-2活性、迁移和侵袭的增加。HN12shSET皮下异种移植瘤表现出低分化表型、细胞增殖减少和顺铂敏感性。使用HN12shSET细胞的原位异种移植瘤模型显示转移潜能增加。
SET积累在HNSCC中具有重要作用。作为一种癌基因，SET促进体内细胞增殖、存活和顺铂诱导的细胞死亡抗性。作为一种转移抑制因子，SET调节侵袭、上皮间质转化和转移。
