Multiple genetic alterations within the PI3K pathway are responsible for AKT activation in patients with ovarian carcinoma.

The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is activated in multiple cancers including ovarian carcinoma (OC). However, the relative contribution of the single components within the PI3K pathway to AKT activation in OC is still unclear. We examined 98 tumor samples from Italian OC patients for alterations in the members of the PI3K pathway. We report that AKT is significantly hyperactive in OC compared to normal tissue (n = 93; p<0.0001) and that AKT activation is preferentially observed in the elderly (>58 years old; n = 93; p<0.05). The most frequent alteration is the overexpression of the p110α catalytic subunit of PI3K (63/93, ∼68%); less frequent alterations comprise the loss of PTEN (24/89, 27%) and the overexpression of AKT1 (18/96, 19%) or AKT2 (11/88,12.5%). Mutations in the PIK3CA or KRAS genes were detected at lower frequency (12% and 10%, respectively) whereas mutations in AKT1 or AKT2 genes were absent. Although many tumors presented a single lesion (28/93, of which 23 overexpressed PIK3CA, 1 overexpressed AKT and 4 had lost PTEN), many OC (35/93) presented multiple alterations within the PI3K pathway. Apparently, aberrant PI3K signalling was mediated by activation of the canonical downstream AKT-dependent mTOR/S6K1/4EBP1 pathway and by regulation of expression of oncogenic transcription factors that include HMGA1, JUN-B, FOS and MYC but not by AKT-independent activation of SGK3. FISH analysis indicated that gene amplification of PIK3CA, AKT1 and AKT2 (but not of PI3KR1) and the loss of PTEN are common and may account for changes in the expression of the corresponding proteins. In conclusion, our results indicate that p110α overexpression represents the most frequent alteration within the PI3K/AKT pathway in OC. However, p110α overexpression may not be sufficient to activate AKT signalling and drive ovarian tumorigenesis since many tumors overexpressing PI3K presented at least one additional alteration.

Oncogenic role of the E3 ubiquitin ligase NEDD4-1, a PTEN negative regulator, in non-small-cell lung carcinomas.

Loss of the PTEN tumor suppressor gene occurs frequently in non-small-cell lung carcinoma (NSCLC), although neither genetic alterations nor epigenetic silencing are significant predictors of PTEN protein levels. Since recent reports implicated neural precursor cell expressed, developmentally down-regulated 4-1 (NEDD4-1) as the E3 ubiquitin ligase that regulates PTEN stability, we investigated the role of NEDD4-1 in the regulation of PTEN expression in cases of NSCLC. Our findings indicate that NEDD4-1 plays a critical role in the development of NSCLC and provides novel insight on the mechanisms that contribute to inactivate PTEN in lung cancer. Immunohistochemical analysis on tissue microarrays containing 103 NSCLC resections revealed NEDD4-1 overexpression in 80% of tumors, which correlated with the loss of PTEN protein (n=98; P<0.001). Accordingly, adoptive NEDD4-1 expression in NSCLC cells decreased PTEN protein stability, whereas knock-down of NEDD4-1 expression decreased PTEN ubiquitylation and increased PTEN protein levels. In 25% of cases, NEDD4-1 overexpression was due to gene amplification at 15q21. In addition, manipulation of NEDD4-1 expression in different lung cell systems demonstrated that suppression of NEDD4-1 expression significantly reduced proliferation of NSCLC cells in vitro and tumor growth in vivo, whereas NEDD4-1 overexpression facilitated anchorage-dependent and independent growth in vitro of nontransformed lung epithelial cells that lack pRB and TP53 (BEAS-2B). NEDD4-1 overexpression also augmented the tumorigenicity of lung cancer cells that have an intact PTEN gene (NCI-H460 cells).

The eighth fibronectin type III domain of protein tyrosine phosphatase receptor J influences the formation of protein complexes and cell localization.

Regulation of receptor-type phosphatases can involve the formation of higher-order structures, but the exact role played in this process by protein domains is not well understood. In this study we show the formation of different higher-order structures of the receptor-type phosphatase PTPRJ, detected in HEK293A cells transfected with different PTPRJ expression constructs. In the plasma membrane PTPRJ forms dimers detectable by treatment with the cross-linking reagent BS(3) (bis[sulfosuccinimidyl]suberate). However, other PTPRJ complexes, dependent on the formation of disulfide bonds, are detected by treatment with the oxidant agent H(2)O(2) or by a mutation Asp872Cys, located in the eighth fibronectin type III domain of PTPRJ. A deletion in the eighth fibronectin domain of PTPRJ impairs its dimerization in the plasma membrane and increases the formation of PTPRJ complexes dependent on disulfide bonds that remain trapped in the cytoplasm. The deletion mutant maintains the catalytic activity but is unable to carry out inhibition of proliferation on HeLa cells, achieved by the wild type form, since it does not reach the plasma membrane. Therefore, the intact structure of the eighth fibronectin domain of PTPRJ is critical for its localization in plasma membrane and biological function.

Overexpression of the S-phase kinase-associated protein 2 in thyroid cancer.

Loss of expression of the cyclin-dependent kinase inhibitor p27 through enhanced protein degradation frequently occurs in human cancer. Degradation of p27 requires ubiquitination by the S-phase kinase-associated protein 2 (Skp2), a member of the F-box family of Skp1-Cullin-F-box protein ubiquitin ligases. In the present study, we have investigated the role of Skp2 in human thyroid tumours. Immunohistochemistry analysis showed that Skp2 was overexpressed significantly in thyroid carcinomas (26 out of 51) compared with goitres (0 out of 12, P<0.001) or adenomas (1 out of 10, P<0.05), and that high Skp2 expression was detected more often in anaplastic thyroid (ATC; 83%, n=12) than follicular thyroid (FTC; 40%, n=20) or papillary thyroid (PTC; 42%, n=19) carcinomas (P<0.05). Thyroid cancer cell lines and tissues with high levels of Skp2 protein presented high p27 degradation activity and there was an inverse correlation between Skp2 and p27 expression in thyroid cancer tissues (n=68; P<0.05). In most cases, the observed overexpression of Skp2 protein was paralleled by an increase in the levels of Skp2 mRNA, and we observed Skp2 gene amplification at 5p13 in 2 out of 6 cell lines and in 9 out of 23 primary tumours (six out of eight ATCs, two out of nine PTCs and one out of six FTCs) using Q-PCR and/or fluorescence in situ hybridization analysis. Finally, in vitro experiments demonstrated that suppression of Skp2 expression drastically reduced proliferation of thyroid cancer cells and, conversely, forced expression of Skp2 circumvented serum dependency and contact inhibition in Skp2-negative cells by promoting p27 degradation. These findings indicate that Skp2 plays an important role for the development of thyroid cancer.