Biomedical subjects

Rosa Marina Melillo

Publications and source records attributed to Rosa Marina Melillo.

At least 19 recordsLinked to original sources

PubMed2007

RET/papillary thyroid carcinoma oncogenic signaling through the Rap1 small GTPase.

RET/papillary thyroid carcinoma (PTC) oncoproteins result from the in-frame fusion of the RET receptor tyrosine kinase with protein dimerization motifs encoded by heterologous genes. Here, we show that RET/PTC1 activates the Rap1 small GTPase. The activation of Rap1 was dependent on the phosphorylation of RET Tyr(1062). RET/PTC1 recruited a complex containing growth factor receptor binding protein 2-associated binding protein 1 (Gab1), CrkII (v-crk sarcoma virus CT10 oncogene homologue II), and C3G (Rap guanine nucleotide exchange factor 1). By using dominant-negative and small interfering duplex (small interfering RNA) oligonucleotides, we show that RET/PTC1-mediated Rap1 activation was dependent on CrkII, C3G, and Gab1. Activation of Rap1 was involved in the RET/PTC1-mediated stimulation of the BRAF kinase and the p42/p44 mitogen-activated protein kinases. Proliferation and stress fiber formation of RET/PTC1-expressing PC Cl 3 thyroid follicular cells were inhibited by the dominant-negative Rap1(N17) and by Rap1-specific GTPase-activating protein. Thus, Rap1 is a downstream effector of RET/PTC and may contribute to the transformed phenotype of RET/PTC-expressing thyrocytes.

Actins↗

PubMed2006

The receptor-type protein tyrosine phosphatase J antagonizes the biochemical and biological effects of RET-derived oncoproteins.

Thyroid cancer is frequently associated with the oncogenic conversion of the RET receptor tyrosine kinase. RET gene rearrangements, which lead to the generation of chimeric RET/papillary thyroid carcinoma (PTC) oncogenes, occur in PTC, whereas RET point mutations occur in familial multiple endocrine neoplasia type 2 (MEN2) and sporadic medullary thyroid carcinomas (MTC). We showed previously that the expression of the receptor-type protein tyrosine phosphatase J (PTPRJ) is suppressed in neoplastically transformed follicular thyroid cells. We now report that PTPRJ coimmunoprecipitates with wild-type RET and with the MEN2A-associated RET(C634R) oncoprotein but not with the RET/PTC1 and RET-MEN2B isoforms. Using mutated forms of PTPRJ and RET-MEN2A, we show that the integrity of the respective catalytic domains is required for the PTPRJ/RET-MEN2A interaction. PTPRJ expression induces dephosphorylation of the RET(C634R) and, probably via an indirect mechanism, RET/PTC1 oncoproteins on two key RET autophosphorylation sites (Tyr1062 and Tyr905). This results in a significant decrease of RET-induced Shc and extracellular signal-regulated kinase 1/2 phosphorylation levels. In line with this finding, adoptive PTPRJ expression reduced the oncogenic activity of RET(C634R) in an in vitro focus formation assay of NIH3T3 cells. As expected from the coimmunoprecipitation results, the RET(M918T) oncoprotein, which is associated to MEN2B and sporadic MTC, was resistant to the dephosphorylating activity of PTPRJ. Taken together, these findings identify RET as a novel substrate of PTPRJ and suggest that PTPRJ expression levels may affect tumor phenotype associated with RET/PTC1 and RET(C634R) mutants. On the other hand, resistance to PTPRJ may be part of the mechanism of RET oncogenic conversion secondary to the M918T mutation.

Cell Line, Tumor↗

PubMed2006

BRAF is a therapeutic target in aggressive thyroid carcinoma.

PURPOSE: Oncogenic conversion of BRAF occurs in approximately 44% of papillary thyroid carcinomas and 24% of anaplastic thyroid carcinomas. In papillary thyroid carcinomas, this mutation is associated with an unfavorable clinicopathologic outcome. Our aim was to exploit BRAF as a potential therapeutic target for thyroid carcinoma. EXPERIMENTAL DESIGN: We used RNA interference to evaluate the effect of BRAF knockdown in the human anaplastic thyroid carcinoma cell lines FRO and ARO carrying the BRAF V600E (V600EBRAF) mutation. We also exploited the effect of BAY 43-9006 [N-(3-trifluoromethyl-4-chlorophenyl)-N'-(4-(2-methylcarbamoyl pyridin-4-yl)oxyphenyl)urea], a multikinase inhibitor able to inhibit RAF family kinases in a panel of six (V600E)BRAF-positive thyroid carcinoma cell lines and in nude mice bearing ARO cell xenografts. Statistical tests were two sided. RESULTS: Knockdown of BRAF by small inhibitory duplex RNA, but not control small inhibitory duplex RNA, inhibited the mitogen-activated protein kinase signaling cascade and the growth of ARO and FRO cells (P < 0.0001). These effects were mimicked by thyroid carcinoma cell treatment with BAY 43-9006 (IC50 = 0.5-1 micromol/L; P < 0.0001), whereas the compound had negligible effects in normal thyrocytes. ARO cell tumor xenografts were significantly (P < 0.0001) smaller in nude mice treated with BAY 43-9006 than in control mice. This inhibition was associated with suppression of phospho-mitogen-activated protein kinase levels. CONCLUSIONS: BRAF provides signals crucial for proliferation of thyroid carcinoma cells spontaneously harboring the (V600E)BRAF mutation and, therefore, BRAF suppression might have therapeutic potential in (V600E)BRAF-positive thyroid cancer.

Animals↗

PubMed2006

HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity.

HMGA2 gene amplification and overexpression in human prolactinomas and the development of pituitary adenomas in HMGA2 transgenic mice showed that HMGA2 plays a crucial role in pituitary tumorigenesis. We have explored the pRB/E2F1 pathway to investigate the mechanism by which HMGA2 acts. Here we show that HMGA2 interacts with pRB and induces E2F1 activity in mouse pituitary adenomas by displacing HDAC1 from the pRB/E2F1 complex-a process that results in E2F1 acetylation. We found that loss of E2F1 function (obtained by mating HMGA2 and E2F1(-/-) mice) suppressed pituitary tumorigenesis in HMGA2 mice. Thus, HMGA2-mediated E2F1 activation is a crucial event in the onset of these tumors in transgenic mice and probably also in human prolactinomas.

Acetylation↗

PubMed2006

RET/PTC activation in papillary thyroid carcinoma: European Journal of Endocrinology Prize Lecture.

Papillary thyroid carcinoma (PTC) is frequently associated with RET gene rearrangements that generate the so-called RET/PTC oncogenes. In this review, we examine the data about the mechanisms of thyroid cell transformation, activation of downstream signal transduction pathways and modulation of gene expression induced by RET/PTC. These findings have advanced our understanding of the processes underlying PTC formation and provide the basis for novel therapeutic approaches to this disease.

Animals↗

PubMed2005

Overexpression of the cytokine osteopontin identifies aggressive laryngeal squamous cell carcinomas and enhances carcinoma cell proliferation and invasiveness.

PURPOSE: Osteopontin is a secreted cytokine that binds to the cell surface CD44v6 receptor. We studied osteopontin and CD44v6 expression in laryngeal squamous cell carcinomas and correlated osteopontin expression levels with clinicopathologic tumor features. EXPERIMENTAL DESIGN: We used immunohistochemistry, immunoblotting, and reverse transcription-PCR to study osteopontin expression in 58 laryngeal squamous cell carcinomas. Cultured squamous carcinoma cells were treated with exogenous osteopontin or with RNA interference to knockdown osteopontin expression. RESULTS: Osteopontin expression was higher in all the invasive carcinomas than in patient-matched normal mucosa. Its expression levels were significantly correlated with tumor stage and grade and with the presence of lymph node and distant metastases. Osteopontin positivity was negatively correlated with overall survival (P = 0.03). Osteopontin expression was paralleled by intense cell surface reactivity for CD44v6. Treatment of squamous carcinoma cells with recombinant osteopontin sharply increased proliferation and Matrigel invasion in comparison with the untreated cells parallel to activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/mitogen-activated protein kinase signaling cascade. Osteopontin knockdown by RNA interference, anti-CD44 antibodies, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibition prevented these effects. CONCLUSIONS: These results identify osteopontin as a marker and a potential therapeutic target in cases of aggressive laryngeal squamous cell carcinomas.

Adult↗

PubMed2005

RAI(ShcC/N-Shc)-dependent recruitment of GAB 1 to RET oncoproteins potentiates PI 3-K signalling in thyroid tumors.

RAI, also named ShcC/N-Shc, one of the members of the Shc proteins family, is a substrate of the RET receptor tyrosine kinase. Here, we show that RAI forms a protein complex with both RET/MEN 2 A and RET/PTC oncoproteins. By co-immunoprecipitation, we found that RAI associates with the Grb 2-associated binder 1 (GAB 1) adapter. This association is constitutive, but, in the presence of RET oncoproteins, both RAI and GAB 1 are tyrosine-phosphorylated, and the stoichiometry of this interaction remarkably increases. Consequently, the p 85 regulatory subunit of phosphatidylinositol-3 kinase (PI-3 K) is recruited to the complex, and its downstream effector Akt is activated. We show that human thyroid cancer cell lines derived from papillary or medullary thyroid carcinoma (PTC or MTC) carrying, respectively, RET/PTC and RET/MEN 2 A oncoproteins express RAI proteins. We also show that human PTC samples express higher levels of RAI, when compared to normal thyroid tissue. In thyroid cells expressing RET/PTC 1, ectopic expression of RAI protects cells from apoptosis; on the other hand, the silencing of endogenous RAI by small inhibitory duplex RNAs in a PTC cell line that expresses endogenous RET/PTC 1, increases the rate of spontaneous apoptosis. These data suggest that RAI is a critical substrate for RET oncoproteins in thyroid carcinomas.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Osteopontin is overexpressed in human papillary thyroid carcinomas and enhances thyroid carcinoma cell invasiveness.

UNLABELLED: The transmembrane glycoprotein CD44v6 is overexpressed in most papillary thyroid carcinomas (PTC). We previously reported that osteopontin (OPN), a secreted glycoprotein that functions as a ligand for CD44v6, is overexpressed in thyrocytes transformed by the RET/PTC oncogene. OBJECTIVE: In this study we asked whether OPN is overexpressed in human PTC samples, and whether its expression correlates with clinical and histological features of the tumors. Furthermore, we wanted to establish the functional role of the CD44-OPN axis in thyroid tumorigenesis. DESIGN: Thyroid samples from 117 patients who had undergone surgical resection of the thyroid gland for benign or malignant lesions were collected. OPN and CD44 expressions were evaluated by immunohistochemistry with specific monoclonal antibodies. OPN expression was correlated with different PTC histological variants, lymph node metastasis, and PTC size. RESULTS: In this study we show that OPN is overexpressed in human PTCs with respect to normal thyroid tissue, follicular adenomas, and multinodular goiters (P < 0.05). The prevalence and intensity of OPN staining were significantly correlated with the presence of lymph node metastases (P = 0.0091) and tumor size (P = 0.0001). We also show that treatment of human PTC cells with recombinant exogenous OPN stimulated Matrigel invasion and activated the ERK and V-AKT murine thymoma viral oncogene homolog 1/protein kinase B; signaling pathways. Blockage of anti-CD44 antibodies prevented these effects. CONCLUSIONS: Given its prevalence and its correlation with aggressive features of human PTCs, we suggest that OPN might be used as a diagnostic and prognostic marker for these tumors. Furthermore, given the role of the OPN-CD44v6 axis in PTC cells, we suggest that CD44 and/or OPN may be molecular targets for therapeutic intervention in aggressive PTCs.

Carcinoma, Papillary↗

PubMed2005

The RET/PTC-RAS-BRAF linear signaling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells.

In papillary thyroid carcinomas (PTCs), rearrangements of the RET receptor (RET/PTC) and activating mutations in the BRAF or RAS oncogenes are mutually exclusive. Here we show that the 3 proteins function along a linear oncogenic signaling cascade in which RET/PTC induces RAS-dependent BRAF activation and RAS- and BRAF-dependent ERK activation. Adoptive activation of the RET/PTC-RAS-BRAF axis induced cell proliferation and Matrigel invasion of thyroid follicular cells. Gene expression profiling revealed that the 3 oncogenes activate a common transcriptional program in thyroid cells that includes upregulation of the CXCL1 and CXCL10 chemokines, which in turn stimulate proliferation and invasion. Thus, motile and mitogenic properties are intrinsic to transformed thyroid cells and are governed by an epistatic oncogenic signaling cascade.

Animals↗

PubMed2004

Minireview: RET: normal and abnormal functions.

The RET gene encodes a single-pass transmembrane receptor tyrosine kinase. RET is the oncogene that causes papillary thyroid carcinoma and medullary thyroid carcinoma. The latter may arise as a component of multiple endocrine neoplasia type 2 syndromes; germline mutations in RET are responsible for multiple endocrine neoplasia type 2 inheritance. In this report we review data on the mechanisms leading to RET oncogenic conversion and on RET targeting as a strategy in thyroid cancer treatment.

Gene Expression Regulation, Neoplastic↗

PubMed2004

Functional expression of the CXCR4 chemokine receptor is induced by RET/PTC oncogenes and is a common event in human papillary thyroid carcinomas.

To identify genes involved in the transformation of thyroid follicular cells, we explored, using DNA oligonucleotide microarrays, the transcriptional response of PC Cl3 rat thyroid epithelial cells to the ectopic expression of the RET/PTC oncogenes. We found that RET/PTC was able to induce the expression of CXCR4, the receptor for the chemokine CXCL12/SDF-1alpha/beta. We observed that CXCR4 expression correlated with the transforming ability of the oncoprotein and depended on the integrity of the RET/PTC-RAS/ERK signaling pathway. We found that CXCR4 was expressed in RET/PTC-positive human thyroid cancer cell lines, but not in normal thyroid cells. Furthermore, we found CXCR4 expression in human thyroid carcinomas, but not in normal thyroid samples by immunohistochemistry. Since CXCR4 has been recently implicated in tumor proliferation, motility and invasiveness, we asked whether treatment with SDF-1alpha was able to induce a biological response in thyroid cells. We observed that SDF-1alpha induced S-phase entry and survival of thyroid cells. Invasion through a reconstituted extracellular matrix was also supported by SDF-1alpha and inhibited by a blocking antibody to CXCR4. Taken together, these results suggest that human thyroid cancers bearing RET/PTC rearrangements may use the CXCR4/SDF-1alpha receptor-ligand pathway to proliferate, survive and migrate.

Carcinoma, Papillary↗

PubMed2004

Regulation of p27Kip1 protein levels contributes to mitogenic effects of the RET/PTC kinase in thyroid carcinoma cells.

We show that treatment of a panel of thyroid carcinoma cell lines naturally harboring the RET/PTC1 oncogene, with the RET kinase inhibitors PP1 and ZD6474, results in reversible G(1) arrest. This is accompanied by interruption of Shc and mitogen-activated protein kinase (MAPK) phosphorylation, reduced levels of G(1) cyclins, and increased levels of the cyclin-dependent kinase inhibitor p27Kip1 because of a reduced protein turnover. MAP/extracellular signal-regulated kinase 1/2 inhibition by U0126 caused G(1) cyclins down-regulation and p27Kip1 up-regulation as well. Forced expression of RET/PTC in normal thyroid follicular cells caused a MAPK- and proteasome-dependent down-regulation of p27Kip1. Reduction of p27Kip1 protein levels by antisense oligonucleotides abrogated the G(1) arrest induced by RET/PTC blockade. Therefore, in thyroid cancer, RET/PTC-mediated MAPK activation contributes to p27Kip1 deregulation. This pathway is implicated in cell cycle progression and in response to small molecule kinase inhibitors.

Cell Cycle Proteins↗

PubMed2004

Autocrine stimulation by osteopontin plays a pivotal role in the expression of the mitogenic and invasive phenotype of RET/PTC-transformed thyroid cells.

Papillary thyroid carcinomas are characterized by rearrangements of the RET receptor tyrosine kinase generating RET/PTC oncogenes. Here we show that osteopontin (OPN), a secreted glycoprotein, is a major RET/PTC-induced transcriptional target in PC Cl 3 thyroid follicular cells. OPN upregulation depended on the integrity of the RET/PTC kinase and tyrosines Y1015 and Y1062, two major RET/PTC autophosphorylation sites. RET/PTC also induced a strong overexpression of CD44, a cell surface signalling receptor for OPN. Upregulation of CD44 was dependent on RET/PTC Y1062, as well. Constitutive OPN overexpression or treatment with exogenous recombinant OPN sharply increased proliferation, Matrigel invasion and spreading in collagen gels of RET/PTC-transformed PC Cl 3 cells. These effects were impaired by the treatment of PC Cl 3-RET/PTC cells with OPN- and CD44-locking antibodies. Thus, RET/PTC signalling triggers an autocrine loop involving OPN and CD44 that sustains proliferation and invasion of transfomed PC Cl 3 thyrocytes.

Carcinoma, Papillary↗

PubMed2004

The oncogenic activity of RET point mutants for follicular thyroid cells may account for the occurrence of papillary thyroid carcinoma in patients affected by familial medullary thyroid carcinoma.

Activating germ-line point mutations in the RET receptor are responsible for multiple endocrine neoplasia type 2-associated medullary thyroid carcinoma (MTC), whereas somatic RET rearrangements are prevalent in papillary thyroid carcinomas (PTCs). Some rare kindreds, carrying point mutations in RET, are affected by both cancer types, suggesting that, under specific circumstances, point mutations in RET can drive the generation of PTC. Here we describe a family whose siblings, affected by both PTC and MTC, carried a germ-line point mutation in the RET extracellular domain, converting cysteine 634 into serine. We tested on thyroid follicular cells the transforming activity of RET(C634S), RET(K603Q), another mutant identified in a kindred with both PTC and MTC, RET(C634R) a commonly isolated allele in MEN2A, RET(M918T) responsible for MEN2B and also identified in kindreds with both PTC and MTC, and RET/PTC1 the rearranged oncogene that characterizes bona fide PTC in patients without MTC. We show that the various RET point mutants, but not wild-type RET, scored constitutive kinase activity and exerted mitogenic effects for thyroid PC Cl 3 cells, albeit at significantly lower levels compared to RET/PTC1. The low mitogenic activity of RET point mutants paralleled their reduced kinase activity compared to RET/PTC. Furthermore, RET point mutants maintained a protein domain, the intracellular juxtamembrane domain, that exerted negative effects on the mitogenic activity. In conclusion, RET point mutants can behave as dominant oncogenes for thyroid follicular cells. Their transforming activity, however, is rather modest, providing a possible explanation for the rare association of MTC with PTC.

Adenocarcinoma, Follicular↗

PubMed2003

Protein kinase Calpha activation by RET: evidence for a negative feedback mechanism controlling RET tyrosine kinase.

We have studied the role of protein kinase C (PKC) in signaling of the RET tyrosine kinase receptor. By using a chimeric receptor (E/R) in which RET kinase can be tightly controlled by the addition of epidermal growth factor (EGF), we have found that RET triggering induces a strong increase of PKCalpha, PKCdelta and PKCzeta activity and that PKCalpha, not PKCdelta and PKCzeta, forms a ligand-dependent protein complex with E/R. We have identified tyrosine 1062 in the RET carboxyl-terminal tail as the docking site for PKCalpha. Block of PKC activity by bisindolylmaleimide or chronic phorbol esters treatment decreased EGF-induced serine/threonine phosphorylation of E/R, while it caused a similarly sized increase of EGF-induced E/R tyrosine kinase activity and mitogenic signaling. Conversely, acute phorbol esters treatment, which promotes PKC activity, increased the levels of E/R serine/threonine phosphorylation and significantly decreased its phosphotyrosine content. A threefold reduction of tyrosine phosphorylation levels of the constitutively active RET/MEN2A oncoprotein was observed upon coexpression with PKCalpha. We conclude that RET binds to and activates PKCalpha. PKCalpha, in turn, causes RET phosphorylation and downregulates RET tyrosine kinase and downstream signaling, thus functioning as a negative feedback loop to modulate RET activity.

3T3 Cells↗

PubMed2003

Ras-mediated apoptosis of PC CL 3 rat thyroid cells induced by RET/PTC oncogenes.

RET gene rearrangements, which generate chimeric RET/PTC oncogenes, are early events in the evolution of thyroid papillary carcinomas. Expression of RET/PTC oncogenes promotes neoplastic transformation of cultured thyroid cells and of thyroid glands in transgenic mice. Notwithstanding these oncogenic effects, we have found that the expression of two RET/PTC oncogenes (H4-RET and RFG-RET) induces apoptosis of rat thyroid PC CL 3 cells. Promotion of thyroid cell death depends on the kinase activity of RET/PTC and on the phosphorylation of a tyrosine residue (tyrosine 1062) that maps in the carboxy-terminus of the RET protein. Tyrosine 1062 is essential for RET/PTC-mediated activation of the Ras/ERK pathway. Inhibition of Ras/ERK by a dominant negative Ras or by the MEKI inhibitor, PD98059, obstructed RET/PTC-mediated apoptosis. We also show that signals transmitted by tyrosine 1062 mediate proapoptotic events like Bcl-2 down regulation and Bax upregulation, and that adoptive overexpression of Bcl-2 overcomes RET/PTC-induced apoptosis. Thus, gene rearrangements that generate RET/PTC oncogenes subvert RET function by converting it into a chronically active kinase that is constitutively phosphorylated on tyrosine 1062. In turn, Y1062 phosphorylation transmits not only mitogenic but also proapoptotic signals to thyroid cells.

Animals↗

PubMed2003

Efficient inhibition of RET/papillary thyroid carcinoma oncogenic kinases by 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2).

Inappropriate activation of the RET receptor tyrosine kinase causes development of papillary and medullary thyroid cancer. We have previously shown that pyrazolopyrimidine is a potent inhibitor of the RET kinase. Here, we show that 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) (PP2), another pyrazolopyrimidine, blocks the enzymatic activity of the isolated RET kinase and RET/PTC1 oncoprotein at IC(50) in the nanomolar range. PP2 blocked in vivo phosphorylation and signaling of the RET/PTC1 oncoprotein. PP2 prevented serum-independent growth of RET/PTC1-transformed NIH3T3 fibroblasts and of TPC1 and FB2, two human papillary thyroid carcinoma cell lines that carry spontaneous RET/PTC1 rearrangements. Finally, PP2 blocked invasion of type I collagen matrix by TPC1 cells. Thus, pyrazolopirimidines hold promise for the treatment of human cancers sustaining oncogenic activation of RET.

3T3 Cells↗

PubMed2002

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 p27(kip1) is a putative tumor suppressor for human cancer. The mechanism underlying p27(kip1) 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 p27(kip1)-mediated growth arrest. Threonine 157 (T157), which maps within the nuclear localization signal of p27(kip1), is a predicted Akt-phosphorylation site. Akt-induced T157 phosphorylation causes retention of p27(kip1) in the cytoplasm, precluding p27(kip1)-induced G1 arrest. Conversely, the p27(kip1)-T157A mutant accumulates in cell nuclei and Akt does not affect p27(kip1)-T157A-mediated cell cycle arrest. Lastly, T157-phosphorylated p27(kip1) accumulates in the cytoplasm of primary human breast cancer cells coincident with Akt activation. Thus, cytoplasmic relocalization of p27(kip1), secondary to Akt-mediated phosphorylation, is a novel mechanism whereby the growth inhibitory properties of p27(kip1) are functionally inactivated and the proliferation of breast cancer cells is sustained.

Breast Neoplasms↗