Angela Greco

An oncogene isolated by transfection of Kaposi's sarcoma DNA encodes a growth factor that is a member of the FGF family

We recently reported the cloning of a rearranged human oncogene following transfection of DNA from Kaposis sarcoma into NIH 3T3 cells. To identify the protein(s) encoded in two novel mRNAs of 3.5 and 1.2 kb expressed in NIH 3T3 transformants, we constructed a cDNA library. One of the cDNA clones isolated (KS3) corresponded to the 1.2 kb mRNA and transformed NIH 3T3 cell when inserted into a mammalian expression vector. The 1152 nucleotide KS3 cDNA encodes a protein of 206 amino acids with significant homology to the growth factors basic FGF and acidic FGF. Expression of the KS3 product as a bacterial fusion protein or in COS cells allowed us to determine that both proteins had significant growth-promoting activity and that the COS cell protein was glycosylated. Thus one of the mRNAs transcribed from the KS oncogene encodes a growth factor that could transform cells by an autocrine mechanism and appears to represent a new member of the FGF family.

Alternative mutations of BRAF , RET and NTRK1 are associated with similar but distinct gene expression patterns in papillary thyroid cancer

Papillary thyroid carcinoma (PTC) is associated with RET and NTRK1 rearrangements and BRAF mutations. A series of 60 PTCs collected in a single center from Italian patients were histologically re-examined and subclassified as well differentiated or tall cell variant. The sample collection was analysed for the presence of all the reported PTC-associated genetic alterations through DNA or cDNA amplification, followed by automated sequencing. The analysis of exons 11 and 15 of BRAF gene revealed the T1796A (V599E) mutation in 32% of cases, and this alteration is significantly associated with PTC tall cell variant. Oncogenic rearrangements of RET and NTRK1 receptors were found in 33 and 5% of cases, respectively. No Ras mutations were detected. Overall, genetic alterations were detected in two-thirds of samples, and in no single case more than one mutational event was found simultaneously. Gene expression profiling of a subset of 31 tumors performed using cDNA microarray chips showed no strong differences in global gene expression among the different cases. However, a supervised analysis of the obtained data identified a subset of genes differentially expressed in tumors carrying BRAF mutation or RTK rearrangement.

Cytogenetics and molecular genetics of carcinomas arising from thyroid epithelial follicular cells

Cytogenetic and molecular analyses of thyroid tumors have indicated that these neoplasms represent a good model for analyzing human epithelial cell multistep carcinogenesis. They comprise, in fact, a broad spectrum of lesions with different phenotypes and variable biological and clinical behavior. Molecular analysis has detected specific genetic alterations in the different types of thyroid tumors. In particular, the well‐differentiated carcinomas of the papillary type are characterized by activation of the receptor tyrosine kinases (RTKs), RET and NTRKI proto‐oncogenes. Cytogenetic analysis of these tumors has contributed to defining the chromosomal mechanisms leading to RTK oncogenic activation. In the majority of cases, intrachromosomal inversions of chromosome 10 and chromosome I led to the formation of RET‐derived and NTRKI‐derived oncogenes, respectively. Interestingly, molecular analysis of these oncogenes revealed their nature of chimeric fusion proteins all sharing the tyrosine kinase (TK) domains of the respective proto‐oncogenes. Moreover, the sequencing of the oncogenic rearrangements led to the identification of a breakpoint cluster region in both RTK proto‐oncogenes. Exposure to ionizing radiation is associated with papillary carcinomas and RET activation has been suggested to be related to this event. Conversely, RAS point mutations are frequently observed in tumor with follicular histology and have been associated with metastatic dissemination. Iodide‐deficient areas seem to provide a higher‐frequency of RAS positive follicular carcinomas. Finally, a high prevalence of TP53 point mutations has been detected only in undifferentiated or anaplastic carcinomas and found to correlate inversely with BCL2 expression. All of these findings are contributing to the definition of genetic and environmental factors relevant for the pathogenesis of thyroid tumors. Moreover, the characterization of specific genetic lesions could provide significant molecular tools for a better differential diagnosis and for the development of novel therapeutic avenues for thyroid cancer. Genes Chromosom Cancer 16:1–14 (1996).

Transforming activity of the chimeric sequence formed by the fusion of collagen gene COL1A1 and the platelet derived growth factor b-chain gene in dermatofibrosarcoma protuberans.

As a consequence of a reciprocal translocation t(17;22)(q22;q13) and of supernumerary ring chromosomes derived from the t(17;22), a fusion between the platelet-derived growth factor b-chain (PDGF, c-sis proto-oncogene) and the collagen type 1A1 (COL1A1) genes has been recently described in dermatofibrosarcoma protuberans (DP), an infiltrating skin tumor (). Although PDGFB has been implicated in transforming processes via autocrine and paracrine pathways, by the activation of the cognate receptor, no direct evidence of its involvement in neoplastic transformation of human tumours has been so far provided. In this report, we have tested the DNA from four DPs in the classical DNA transfection assay onto NIH3T3 fibroblast cell line. All the DNAs induced the formation of transformed foci in the transfected cultures whose derived cell lines were shown to contain a fused sequence comprising the human COL1A1 and PDGF genes. The relative breakpoint regions have been sequenced revealing that this gene fusion deleted exon 1 of PDGF and released the growth factor from its normal regulation. All the biochemical and biological assays were consistent with the model of an autocrine mechanism for NIH3T3 transformation by the human rearranged PDGFB gene involving the activation of the endogeneous PDGF receptor.

Chromosome I rearrangements involving the genes TPR and NTRK1 produce structurally different thyroid‐specific TRK oncogenes

The NTRK1 gene in the q arm of chromosome I encodes one of the receptors for the nerve growth factor and is frequently activated as an oncogene in papillary thyroid carcinomas. The activation is due to chromosomal rearrangements juxtaposing the NTRK1 tyrosine kinase domain to 5′‐end sequences from different genes. The thyroid TRK oncogenes are activated by recombination with at least three different genes: the gene coding for tropomyosin and TPR, both on chromosome I, and TFG on chromosome 3. In a previous study, we showed that two tumors carrying the TPR/NTRK1 rearrangement contained structurally different oncogenes named TRK‐T1 and TRK‐T2. In this paper, we report (1) the cDNA structure of TRK‐T2. (2) evidence that TRK‐T2 is generated by different rearrangements in two thyroid tumors, and (3) a detailed analysis of the three different TPR/NTRK1 rearrangements. With molecular studies based on Southern blot hybridization, cloning, and sequencing, we show that all the rearrangements are nearly balanced, involving deletion, insertion, or duplication of only few nucleotides. In one case, an additional rearrangement involving sequences derived from chromosome 17 was detected. Genes Chromosom. Cancer 19:112–123, 1997.

Role of the TFG N-terminus and coiled-coil domain in the transforming activity of the thyroid TRK-T3 oncogene

The thyroid TRK-T3 oncogene results from the fusion of the tyrosine kinase (TK) domain of NTRK1 (one of the receptors for the Nerve Growth Factor) on chromosome 1 to sequences of a novel gene, TFG, on chromosome 3. The 68 kDa TRK-T3 fusion oncoprotein displays a constitutive tyrosine kinase activity resulting in its capability to transform mouse NIH3T3 cells. The TFG portion of TRK-T3 contains a coiled-coil domain most likely responsible for the constitutive, ligand-independent activation of the receptor tyrosine kinase activity. We have previously shown that TRK-T3 oncoprotein forms, in vivo, complexes of three or four molecules. By mean of different experimental approaches, we show here that TRK-T3 activity depends on oligomers formation. In addition, the analysis of different TRK-T3 mutants indicates that the TFG coiled-coil domain and its N-terminal region are both required for the activation and the fully transforming activity of the TRK-T3 oncoprotein, although, most likely, they play a role in different steps of the transforming process. The deletion of the coiled-coil domain abrogates the oligomers formation leading to a constitutive activation; the deletion of the N-terminal region, although not affecting phosphorylation and complexes formation, abrogates transformation, thus suggesting a role in cellular localization and/or interaction with substrata.

Expression of ligand-activated KIT and platelet-derived growth factor receptor β tyrosine kinase receptors in synovial sarcoma

Purpose: The use of tyrosine kinase receptor inhibitors is increasingly becoming a valuable therapeutic alternative in tumors carrying activated tyrosine kinase receptors. In a previous study, we described a coexpression of KIT and stem cell factor (SCF) mRNA in Synovial sarcomas, (SS) and in a limited number of cases, we demonstrated the presence of an activated receptor. Here, in a wider number of cases, we investigated the expression level and phosphorylation status of two structurally related tyrosine kinase receptors, KIT and platelet-derived growth factor receptor β (PDGFRβ), at the light of their role as possible targets of tyrosine kinase receptors inhibitor molecules. Experimental Design: Forty-three SS cases were analyzed for KIT and PDGFRβ expression/activation by immunoprecipitation/Western blotting experiments. The cognate ligands, SCF and PDGFB, were detected by reverse transcription-PCR. Results: KIT was observed in 48 and 41% (45% total) whereas PDGFRβ in 54 and 33% (45% total) of monophasic and biphasic SS cases, respectively. With respect to the fusion transcript type SYTSSX1 and SYTSSX2, KIT was more expressed in SYTSSX1 carrying cases (48 versus 38%), whereas PDGFRβ resulted more frequently expressed in SYTSSX2 ones (54 versus 37%). When expressed, the receptors were phosphorylated. Their ligands were detected in all of the activated cases. Conclusions: About 70% of the cases express one of the two activated tyrosine kinase receptors with a mutually exclusive expression trend. Coexpression is not frequent and seems to be restricted to monophasic subtype. These data indicate that a consistent fraction of this tumor type could represent a good candidate for kinase inhibitor molecules effective on KIT and PDGFRβ where their activation is sustained by an autocrine loop.

9p21 locus analysis in high‐risk gastrointestinal stromal tumors characterized for c‐kit and platelet‐derived growth factor receptor α gene alterations

Gastrointestinal stromal tumors (GISTs) are noncomplex sarcomas that often are due to c‐kit‐activating and platelet‐derived growth factor receptor α gene (PDGFRα)‐activating mutations and perturbations of their related signaling pathways. Molecular and cytogenetic findings have indicated correlations between tumor progression and high‐risk GISTs with c‐kit mutations, the overexpression of genes such as ezrin, and losses at 9p. In particular, it was reported recently that malignant GISTs showed alterations in the p16INK4a gene located at the 9p21 locus.

Detection of Overexpressed and Phosphorylated Wild-Type Kit Receptor in Surgical Specimens of Small Cell Lung Cancer

Purpose: The combinations of various chemotherapeutic drugs currently used to treat advanced small cell lung cancer (SCLC) led to similarly poor survival outcomes, which is why new molecular biology approaches are needed to design and select targeted therapies. Experimental Design: Thirteen stage I SCLC surgical specimens were screened for c-Kit gene mutations by sequencing whole cDNA and for KIT receptor expression/activation by immunoprecipitation and Western blotting. Both the paraffin-embedded and frozen materials were analyzed by immunocytochemistry, and the stem cell factor cognate ligand was assessed by retrotranscription PCR. Results: In all cases, we showed the presence of wild-type KIT receptors by analyzing the entire coding sequence, which together with the detection of the cognate ligand stem cell factor, supports the establishment of an autocrine loop. In addition, the KIT receptor was activated/phosphorylated. The immunoprecipitation/Western blotting data fit the observed immunophenotype. Interestingly, comparison of the level of KIT expression was at least 10 times higher in the tumoral specimens than the normal reference lungs. Conclusions: The KIT molecular profile derived from the analysis of SCLC surgical specimens shows that wild-type KIT is overexpressed and phosphorylated in the presence of stem cell factor. This finding, which is consistent with pathological KIT activation driven by an autocrine loop, is particularly interesting in the light of the recent development of new tyrosine kinase inhibitory drugs, which are highly effective in blocking wild-type KIT receptors.

Proximity of TPR and NTRK1 rearranging loci in human thyrocytes

Chromosomal rearrangements are frequently associated with cancer; the mechanisms underlying their cell-type specificity are poorly understood. Papillary thyroid carcinomas are marked by a high frequency of chromosome rearrangements involving the RET and NTRK1 tyrosine kinase receptor genes and producing RET and TRK oncogenes. An explanation for the propensity of thyrocytes to undergo gene rearrangements has been recently proposed by Nikiforova and colleagues, who showed that the recombination between RET and H4 is favored by the loci proximity in interphase nuclei. We investigated whether the spatial proximity is a contributing factor also in the generation of the thyroid-specific TRK oncogenes. The distance between NTRK1 and its oncogenic partner TPR was determined by two-color fluorescence in situ hybridization and two-dimensional microscopy. A three-dimensional reconstruction of the data was also done. We show that the two loci in thyrocytes nuclei display a distance reduced with respect to peripheral blood lymphocytes, thus supporting the notion that spatial proximity of translocation-prone gene loci may favor gene rearrangements.