PIK3CA, encoding the catalytic subunit p110alpha of phosphatidylinositol 3-kinase (PI3K), is activated in malignant diseases. However, the role of the PIK3CA gene aberrations for tumourigenesis of head and neck squamous cell carcinoma (HNSCC) is to date unclear. The present study was designed to determine the genomic aberration of PIK3CA in invasive HNSCC and dysplastic precursor lesions by fluorescence in situ hybridization (FISH) with a YAC probe, containing the PIK3CA gene, on isolated interphase nuclei from histomorphologically well-defined regions of formalin-fixed tissue sections and to compare these data with protein and mRNA expression of p110alpha. The mRNA and protein levels of p110alpha were assessed, respectively, by in situ hybridization and immunohistochemistry on consecutive tissue sections. Copy number gains at 3q26 were observed in one of six low-to-moderate dysplasias (17%) and in seven of nine high-grade dysplasias (78%), as well as in 11 carcinomas (100%). In addition, one of seven high-grade dysplasias (14%) and 6 of 11 carcinomas (55%) had amplifications of 3q26. The majority of cases with copy number gain in more than 50% of the cells and/or amplification in more than 10% of cells showed increased p110alpha mRNA and protein expression, whereas only two cases (18%) (one high-grade dysplasia and one carcinoma) with no gain or low-level gain displayed increased p110alpha protein expression. These data suggest that 3q26 copy number gain and amplification represent early genomic aberrations in HNSCC carcinogenesis. In addition, p110alpha mRNA and protein expression in HNSCC may be regulated by these genomic aberrations as well as by epigenetic events.

ABSTRACT:The acetylation state of core histones is controlled by two classes of enzymes, histone acetyl transferases (HATs) and histone deacetylases (HDACs). HDAC inhibitors, such as trichostatin-A (TSA), are able to induce cell cycle arrest by stimulating transcription of genes that negatively regulate cell growth and survival. However, little is known about the effect of HDAC inhibitors on spermatogenesis. TSA treatment of cultured murine germ cells from whole testes resulted in an increase of histone H4 acetylation in round spermatids, suggesting that a hypoacetylated state of these cells is important for their normal differentiation. In the present study, the in vivo effects of TSA on murine spermatogenesis were investigated. Subcutaneously applied TSA resulted in a dose-dependent decrease in relative testis weight due to impaired spermatogenesis. No obvious toxic effects of TSA treatment could be found. A second animal experiment confirmed that male mice receiving TSA under the same conditions as in the first experiment became infertile. This phenomenon was completely reversible. No evidence of histone H4 hyperacetylation in round spermatids could be found; however, the number of spermatids significantly decreased with increasing TSA concentrations. Additionally, a dramatic loss of pachytene-diplotene spermatocytes due to increased apoptosis was observed. This suggests that TSA was mainly effective at the level of meiosis. The other male reproductive organs showed no morphological changes compared to controls, suggesting that TSA action on the testis was not mediated by sex hormones.

Disrupted phosphatidylinositol 3-kinase (PI3K) activity and its effect on the downstream target AKT plays an important role in malignant diseases. Gain and/or amplification of PIK3CA gene, encoding the catalytic subunit of phosphatidylinositol 3-kinase (p110 alpha) and its increased expression are associated with enhanced PI3K activity in ovarian cancer cell lines. In this study, ovarian carcinomas with documented clinical outcome were assessed for genetic aberrations at the 3q26.3 locus, including PIK3CA, by fluorescence in situ hybridization. PIK3CA amplification was evaluated by quantitative real-time PCR with respect to a control gene situated at 3q13. The expression of p110 alpha, phosphorylated AKT (pAKT) and the proliferation marker Ki-67 were immunohistochemically investigated. PIK3CA amplification and Ki-67 index were strong predictors for an early tumour-associated death. p110 alpha expression correlated with 3q26.3 gain and Ki-67 index but not with the patient outcome. No relationship could be observed between p110 alpha and pAKT or between pAKT and disease outcome. It is interesting to note that cases with a nuclear pAKT immunoreactivity showed a trend of improved overall survival. Our results underline the prognostic significance of PIK3CA in ovarian carcinoma and argue against a simple linear model of PIK3CA gain/amplification followed by PI3K activation and consecutive AKT phosphorylation in ovarian carcinoma.

PIK3CA, which encodes the catalytic subunit, p110alpha, of phosphatidylinositol 3-kinase (PI3K), is implicated in the development and progression of numerous neoplasias including head and neck squamous cell carcinoma (HNSCC). In the present study, we investigated the occurrence of PIK3CA hot-spot mutations in exons 9 and 20, the genomic gain and amplification of PIK3CA, the expression of PIK3CA mRNA and the p110α protein, as well as the expression of phosphorylated-Akt (pAkt) in 33 cases of HNSCC and compared the results with the clinicopathological data. No non-synonymous mutations were detected. PIK3CA copy number gain and amplification were found in 36.4 and 9% of the cases, respectively, whereas mRNA overexpression was observed in 48.5% of the cases. No correlations could be stated between DNA gain, DNA amplification and mRNA expression, either between DNA or mRNA status and p110α expression. Direct associations were found between PIK3CA gain and lymph node metastases (p=0.025) and between mRNA expression and tumour stage (p=0.015). A strong correlation was detected between p110α and pAkt expression (p<0.001). Concluding, PIK3CA could be an oncogene implicated in HNSCC development. However, our data suggest that amplifications or mutations of this gene seldom occur in HNSCC and that epigenetic events can play an important role in advanced tumour stages.