PIK3CA-mediated PI3-kinase signalling is essential for HPV-induced transformation in vitro.
PIK3CA-mediated PI3-kinase signalling is essential for HPV-induced transformation in vitro.
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DOI：
10.1186/1476-4598-10-71
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发表时间：
2011-06-10
期刊：
影响因子：
37.3
通讯作者：
Steenbergen RD
中科院分区：
文献类型：
--
作者：
Henken FE;Banerjee NS;Snijders PJ;Meijer CJ;De-Castro Arce J;Rösl F;Broker TR;Chow LT;Steenbergen RD
高危型人乳头瘤病毒（hrHPV）感染与宫颈癌的发生有因果关系。然而，驱动hrHPV感染细胞恶性转化的额外（epi）遗传改变尚未完全阐明。在这项研究中，我们实验性地评估了PI 3激酶通路及其调节因子PIK 3CA在HPV诱导的转化中的作用，PIK 3CA在宫颈癌中经常改变。分别通过定量RT-PCR和免疫组化染色评估宫颈癌和宫颈外段对照的PIK 3CA mRNA和蛋白表达。使用化学途径抑制即LY 294002处理和PIK 3CA RNA干扰，测定代表永生和锚定独立表型的hrHPV转染的角质形成细胞的纵向体外模型系统的PI 3激酶活化和功能。检查的表型包括细胞活力、迁移、锚定独立生长和分化。采用定量RT-PCR和北方印迹法检测hTERT和HPV 16 E6 E7 mRNA的表达。与对照相比，宫颈癌显示PIK 3CA的显著过表达。在体外HPV诱导的转化过程中，催化亚基PIK 3CA的表达以及下游效应子PKB/AKT的激活平行地逐渐增加。通过化学干扰或siRNA介导的PIK 3CA沉默抑制HPV 16转染的角质形成细胞中的PI 3激酶信号传导导致PKB/AKT磷酸化降低。此外，PI 3-激酶的阻断导致细胞活力、迁移和锚定非依赖性生长降低。HPV 16 E7和hTERTmRNA表达下调。在HPV 16和HPV 18永生化细胞的器官型筏培养中，磷酸化PKB/AKT主要见于细胞角蛋白10（CK 10）染色阳性的分化细胞。在PI 3-激酶信号传导抑制后，上皮组织发育严重受损，p-PKB/AKT和CK 10显著降低。目前的数据表明，通过PIK 3CA激活PI 3-激酶/PKB/AKT途径调节HPV永生化细胞的各种转化表型以及生长和分化，因此可能在HPV诱导的致癌作用中发挥关键作用。
High-risk human papillomavirus (hrHPV) infections are causally related to cervical cancer development. The additional (epi)genetic alterations driving malignant transformation of hrHPV-infected cells however, are not yet fully elucidated. In this study we experimentally assessed the role of the PI3-kinase pathway and its regulator PIK3CA, which is frequently altered in cervical cancer, in HPV-induced transformation. Cervical carcinomas and ectocervical controls were assessed for PIK3CA mRNA and protein expression by quantitative RT-PCR and immunohistochemical staining, respectively. A longitudinal in vitro model system of hrHPV-transfected keratinocytes, representing the immortal and anchorage independent phenotype, was assayed for PI3-kinase activation and function using chemical pathway inhibition i.e. LY294002 treatment, and PIK3CA RNA interference. Phenotypes examined included cellular viability, migration, anchorage independent growth and differentiation. mRNA expression of hTERT and HPV16 E6E7 were studied using quantitative RT-PCR and Northern blotting. Cervical carcinomas showed significant overexpression of PIK3CA compared to controls. During HPV-induced transformation in vitro, expression of the catalytic subunit PIK3CA as well as activation of downstream effector PKB/AKT progressively increased in parallel. Inhibition of PI3-kinase signalling in HPV16-transfected keratinocytes by chemical interference or siRNA-mediated silencing of PIK3CA resulted in a decreased phosphorylation of PKB/AKT. Moreover, blockage of PI3-kinase resulted in reduced cellular viability, migration, and anchorage independent growth. These properties were accompanied with a downregulation of HPV16E7 and hTERT mRNA expression. In organotypic raft cultures of HPV16- and HPV18-immortalized cells, phosphorylated PKB/AKT was primarily seen in differentiated cells staining positive for cytokeratin 10 (CK10). Upon PI3-kinase signalling inhibition, there was a severe impairment in epithelial tissue development as well as a dramatic reduction in p-PKB/AKT and CK10. The present data indicate that activation of the PI3-kinase/PKB/AKT pathway through PIK3CA regulates various transformed phenotypes as well as growth and differentiation of HPV-immortalized cells and may therefore play a pivotal role in HPV-induced carcinogenesis.
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