Gennaro Chiappetta

Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27(Kip1) by PKB/Akt-mediated phosphorylation in breast cancer.

The cyclin-dependent kinase inhibitor p27kip1 is a putative tumor suppressor for human cancer. The mechanism underlying p27kip1 deregulation in human cancer is, however, poorly understood. We demonstrate that the serine/threonine kinase Akt regulates cell proliferation in breast cancer cells by preventing p27kip1-mediated growth arrest. Threonine 157 (T157), which maps within the nuclear localization signal of p27kip1, is a predicted Akt-phosphorylation site. Akt-induced T157 phosphorylation causes retention of p27kip1 in the cytoplasm, precluding p27kip1-induced G1 arrest. Conversely, the p27kip1-T157A mutant accumulates in cell nuclei and Akt does not affect p27kip1–T157A-mediated cell cycle arrest. Lastly, T157-phosphorylated p27kip1 accumulates in the cytoplasm of primary human breast cancer cells coincident with Akt activation. Thus, cytoplasmic relocalization of p27kip1, secondary to Akt-mediated phosphorylation, is a novel mechanism whereby the growth inhibitory properties of p27kip1 are functionally inactivated and the proliferation of breast cancer cells is sustained.

An increased high-mobility group A2 expression level is associated with malignant phenotype in pancreatic exocrine tissue.

The altered form of the high-mobility group A2 (HMGA2) gene is somehow related to the generation of human benign and malignant tumours of mesenchymal origin. However, only a few data on the expression of HMGA2 in malignant tumour originating from epithelial tissue are available. In this study, we examined the HMGA2 expression level in pancreatic carcinoma, and investigated whether alterations in the HMGA2 expression level are associated with a malignant phenotype in pancreatic tissue. High-mobility group A2 mRNA and protein expression was determined in eight surgically resected specimens of non-neoplastic tissue (six specimens of normal pancreatic tissue and two of chronic pancreatitis tissue) and 27 pancreatic carcinomas by highly sensitive reverse transcriptase–polymerase chain reaction (RT–PCR) techniques and immunohistochemical staining, respectively. Reverse transcriptase–polymerase chain reaction analysis revealed the expression of the HMGA2 gene in non-neoplastic pancreatic tissue, although its expression level was significantly lower than that in carcinoma. Immunohistochemical analysis indicated that the presence of the HMGA2 gene in non-neoplastic pancreatic tissue observed in RT–PCR reflects its abundant expression in islet cells, together with its focal expression in duct epithelial cells. Intense and multifocal or diffuse HMGA2 immunoreactivity was noted in all the pancreatic carcinoma examined. A strong correlation between HMGA2 overexpression and the diagnosis of carcinoma was statistically verified. Based on these findings, we propose that an increased expression level of the HMGA2 protein is closely associated with the malignant phenotype in the pancreatic exocrine system, and accordingly, HMGA2 could serve as a potential diagnostic molecular marker for distinguishing pancreatic malignant cells from non-neoplastic pancreatic exocrine cells.

The neutrophil gelatinase-associated lipocalin (NGAL), a NF-B-regulated gene, is a survival factor for thyroid neoplastic cells

NF-κB is constitutively activated in primary human thyroid tumors, particularly in those of anaplastic type. The inhibition of NF-κB activity in the human anaplastic thyroid carcinoma cell line, FRO, leads to an increased susceptibility to chemotherapeutic drug-induced apoptosis and to the blockage of their ability to form tumors in nude mice. To identify NF-κB target genes involved in thyroid cancer, we analyzed the secretome of conditioned media from parental and NF-κB-null FRO cells. Proteomic analysis revealed that the neutrophil gelatinase-associated lipocalin (NGAL), a protein involved in inflammatory and immune responses, is secreted by FRO cells whereas its expression is strongly reduced in the NF-κB-null FRO cells. NGAL is highly expressed in human thyroid carcinomas, and knocking down its expression blocks the ability of FRO cells to grow in soft agar and form tumors in nude mice. These effects are reverted by the addition of either recombinant NGAL or FRO conditioned medium. In addition, we show that the prosurvival activity of NGAL is mediated by its ability to bind and transport iron inside the cells. Our data suggest that NF-κB contributes to thyroid tumor cell survival by controlling iron uptake via NGAL.

Activating E17K mutation in the gene encoding the protein kinase AKT1 in a subset of squamous cell carcinoma of the lung

Somatic mutation (E17K) that constitutively activates the protein kinase AKT1 has been found in human cancer patients. We determined the role of the E17K mutation of AKT1 in lung cancer, through sequencing of AKT1 exon 4 in 105 resected, clinically annotated non-small cell lung cancer specimens. We detected a missense mutations G->A transition at nucleotide 49 (that results in the E17K substitution) in two squamous cell carcinoma (2/36) but not in adenocarcinoma (0/53). The activity of the endogenous kinase carrying the E17K mutation immunoprecipitated by tumour tissue was significantly higher compared with the wild-type kinase immunoprecipitated by the adjacent normal tissue as determined both by in vitro kinase assay using a consensus peptide as substrate and by in vivo analysis of the phosphorylation status of AKT1 itself (pT308, pS473) or of known downstream substrates such as GSK3 (pS9/S22) and p27 (T198). Immunostaining or immunoblot analysis on membrane-enriched extracts indicated that the enhanced membrane localization exhibited by the endogenous E17K-AKT1 may account for the observed increased activity of mutant E17K kinase in comparison with the wild-type AKT1 from adjacent normal tissue. In conclusion, this is the first report of AKT1 mutation in lung cancer. Our data provide evidence that, although AKT1 mutations are apparently rare in lung cancer (1.9%), the oncogenic properties of E17K-AKT1 may contribute to the development of a fraction of lung carcinoma with squamous histotype (5.5%).

PTEN expression is reduced in a subset of sporadic thyroid carcinomas: evidence that PTEN-growth suppressing activity in thyroid cancer cells is mediated by p27 kip1

The dual-specificity phosphatase PTEN/MMAC1/TEP1 has recently been identified as the tumor suppressor gene most frequently mutated and/or deleted in human tumors. Germline mutations of PTEN give rise to Cowden Disease (CD), an autosomal dominantly-inherited cancer syndrome which predisposes to increased risk of developing breast and thyroid tumors. However, PTEN mutations have rarely been detected in sporadic thyroid carcinomas. In this study, we confirm that PTEN mutations in sporadic thyroid cancer are infrequent as we found one point mutation and one heterozygous deletion of PTEN gene in 26 tumors and eight cell lines screened. However, we report that PTEN expression is reduced – both at the mRNA and at the protein level – in five out of eight tumor-derived cell lines and in 24 out of 61 primary tumors. In most cases, decreased PTEN expression is correlated with increased phosphorylation of the PTEN-regulated protein kinase Akt/PKB. Moreover, we demonstrate that PTEN may act as a suppressor of thyroid cancerogenesis as the constitutive re-expression of PTEN into two different thyroid tumor cell lines markedly inhibits cell growth. PTEN-dependent inhibition of BrdU incorporation is accompanied by enhanced expression of the cyclin-dependent kinase inhibitor p27kip1 and can be overcome by simultaneous co-transfection of an excess p27kip1 antisense plasmid. Accordingly, in a subset of thyroid primary carcinomas and tumor-derived cell lines, a striking correlation between PTEN expression and the level of p27kip1 protein was observed. In conclusion, our findings demonstrate that inactivation of PTEN may play a role in the development of sporadic thyroid carcinomas and that one key target of PTEN suppressor activity is represented by the cyclin-dependent kinase inhibitor p27kip1.

Assessment of RET/PTC Oncogene Activation and Clonality in Thyroid Nodules with Incomplete Morphological Evidence of Papillary Carcinoma : A Search for the Early Precursors of Papillary Cancer

Noninvasive thyroid nodules that exhibit borderline morphological signs of papillary cancer are difficult to diagnose and we do not know if they represent papillary carcinoma precursor lesions. Forty-six such nodules were analyzed for RET activation by immunohistochemistry and, in selected cases, by reverse transcriptase-polymerase chain reaction performed on RNA extracted after laser capture microdissection (LCM) of the tumor foci with and without papillary carcinoma features and positive RET immunoreactivity. RET immunoreactivity was identified, at least focally, in 30 of 46 (65.2%) of the nodules where it closely paralleled the morphological changes. Enough RNA was obtained after LCM in seven samples. RET/PTC1 or RET/PTC3 were detected in microscopic foci with papillary carcinoma features in most of the thyroid nodules (five of seven cases). No RET/PTC1 or RET/PTC3 rearrangements were detected in areas of the same tumors that lacked the cytological alterations. Analysis of clonality in the same nodules selected for LCM demonstrated that two were monoclonal and six were polyclonal. We conclude that RET activation closely parallels the morphological changes, that it is restricted to those areas of the tumor with the cytological alterations and that it is detectable in both mono- and polyclonal tumors. Although the finding of microscopic foci indicative of papillary carcinoma in a hyperplastic or adenomatous nodule does not justify the interpretation of the entire lesion as papillary carcinoma, it is possible that such foci may precede the development of invasive papillary cancer.

RET/PTC activation in hyalinizing trabecular tumors of the thyroid.

Hyalinizing trabecular tumor (HTT) of the thyroid is a neoplasm of follicular derivation with a histogenesis that is still the subject of debate. Morphologic affinities between HTT and papillary carcinoma, including nuclear pseudoinclusions and grooves, suggest that these tumors may be of similar origin. The authors investigated the relationship between these two types of tumors by assessing HTT for the presence of rearrangements of the proto-oncogene rearranged during transfection (RET) that, in thyroid tumors, are specific for papillary carcinoma. A series of 14 HTTs, including two cases associated with classic papillary carcinoma, was studied by means of immunohistochemistry and reverse transcription–polymerase chain reaction. Seven follicular adenomas with focal hyalinized trabecular areas served as control cases. Three of the 14 HTT cases under consideration displayed rearrangements of RET generating the RET/papillary thyroid carcinoma type 1 (PTC1) oncogene. In another case, RET expression was detected focally by immunohistochemistry alone. Finally, in one mixed HTT–papillary carcinoma sample, RET/PTC1 expression was detected, but only in the papillary component. None of the control follicular adenomas contained rearrangements of RET/PTC. These findings demonstrate that a comparable percentage (28.6%) of HTTs and papillary carcinomas exhibit the same RET proto-oncogene alterations. Thus, HTT may represent the “hyalinizing trabecular” variant of papillary carcinoma rather than a separate entity.

Overexpressed cyclin D3 contributes to retaining the growth inhibitor p27 in the cytoplasm of thyroid tumor cells

The majority of thyroid carcinomas maintain the expression of the cell growth suppressor p27, an inhibitor of cyclin-dependent kinase-2 (Cdk2). However, we find that 80% of p27-expressing tumors show an uncommon cytoplasmic localization of p27 protein, associated with high Cdk2 activity. To reproduce such a situation, a mutant p27 devoid of its COOH-terminal nuclear-localization signal was generated (p27-NLS). p27-NLS accumulates in the cytoplasm and fails to induce growth arrest in 2 different cell lines, indicating that cytoplasm-residing p27 is inactive as a growth inhibitor, presumably because it does not interact with nuclear Cdk2. Overexpression of cyclin D3 may account in part for p27 cytoplasmic localization. In thyroid tumors and cell lines, cyclin D3 expression was associated with cytoplasmic localization of p27. Moreover, expression of cyclin D3 in thyroid carcinoma cells induced cytoplasmic retention of cotransfected p27 and rescued p27-imposed growth arrest. Endogenous p27 also localized prevalently to the cytoplasm in normal thyrocytes engineered to stably overexpress cyclin D3 (PC-D3 cells). In these cells, cyclin D3 induced the formation of cytoplasmic p27-cyclin D3-Cdk complexes, which titrated p27 away from intranuclear complexes that contain cyclins A-E and Cdk2. Our results demonstrate a novel mechanism that may contribute to overcoming the p27 inhibitory threshold in transformed thyroid cells.

Gene rearrangement and Chernobyl related thyroid cancers.

The increase in thyroid carcinoma post-Chernobyl has been largely confined to a specific subtype of papillary carcinoma (solid/follicular). This subtype is observed predominantly in children under 10 in unirradiated populations, but maintains a high frequency in those aged 10–15 from those areas exposed to fallout from the Chernobyl accident. The aim of this study was to link morphology with molecular biology. We examined 106 papillary carcinomas from children under the age of 15 at operation. All were examined for rearrangements of the RET oncogene by reverse transcription polymerase chain reaction (RT-PCR); a subset of these cases were also examined for mutations of the three ras oncogenes, exon 10 of the thyroid stimulating hormone receptor, associated more usually with a follicular rather than papillary morphology, and exons 5, 6, 7 and 8 of the p53 gene, commonly involved in undifferentiated thyroid carcinoma. Rearrangements of the RET oncogene were found in 44% of papillary carcinomas in which we studied fresh material; none of the tumours examined showed mutation in any of the other genes. The two rearrangements resulting from inversion of part of chromosome 10 (PTC1 and PTC3) accounted for the majority of RET rearrangements identified, with PTC1 being associated with papillary carcinomas of the classic and diffuse sclerosing variants and PTC3 with the solid/follicular variant.

A microRNA signature defines chemoresistance in ovarian cancer through modulation of angiogenesis

Epithelial ovarian cancer is the most lethal gynecologic malignancy; it is highly aggressive and causes almost 125,000 deaths yearly. Despite advances in detection and cytotoxic therapies, a low percentage of patients with advanced stage disease survive 5 y after the initial diagnosis. The high mortality of this disease is mainly caused by resistance to the available therapies. Here, we profiled microRNA (miR) expression in serous epithelial ovarian carcinomas to assess the possibility of a miR signature associated with chemoresistance. We analyzed tumor samples from 198 patients (86 patients as a training set and 112 patients as a validation set) for human miRs. A signature of 23 miRs associated with chemoresistance was generated by array analysis in the training set. Quantitative RT-PCR in the validation set confirmed that three miRs (miR-484, -642, and -217) were able to predict chemoresistance of these tumors. Additional analysis of miR-484 revealed that the sensitive phenotype is caused by a modulation of tumor vasculature through the regulation of the VEGFB and VEGFR2 pathways. We present compelling evidence that three miRs can classify the response to chemotherapy of ovarian cancer patients in a large multicenter cohort and that one of these three miRs is involved in the control of tumor angiogenesis, indicating an option in the treatment of these patients. Our results suggest, in fact, that blockage of VEGF through the use of an anti-VEGFA antibody may not be sufficient to improve survival in ovarian cancer patients unless VEGFB signaling is also blocked.