Rosa Marina Melillo

PTC is a novel rearranged form of the ret proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas

We recently detected a novel activated oncogene by transfection analysis on NIH 3T3 cells in five out of 20 primary human thyroid papillary carcinomas and in the available lymph node metastases. We designated this transforming gene PTC (for papillary thyroid carcinoma). Here we describe the molecular cloning and sequencing of the gene. The new oncogene resulted from the rearrangement of an unknown amino-terminal sequence to the tyrosine kinase domain of the ret proto-oncogene. This gene rearrangement was detected in all of the transfectants and in all of the original tumor DNAs, but not in normal DNA of the same patients, thus indicating that this genetic lesion occurred in vivo and is specific to somatic tumors. Moreover, the transcript coded for by the fused gene was detected in an additional PTC-positive human papillary carcinoma for which mRNA was available.

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.

Ret oncogene activation in human thyroid neoplasms is restricted to the papillary cancer subtype.

We have recently reported the activation of a new oncogene in human papillary thyroid carcinomas. This oncogene, papillary thyroid carcinoma (PTC), is a novel rearranged version of the ret tyrosine-kinase protooncogene. Thyroid neoplasms include a broad spectrum of malignant tumors, ranging from well-differentiated tumors to undifferentiated anaplastic carcinomas. To determine the frequency of ret oncogene activation, we analyzed 286 cases of human thyroid tumors of diverse histologic types. We found the presence of an activated form of the ret oncogene in 33 (19%) of 177 papillary carcinomas. By contrast, none of the other 109 thyroid tumors, which included 37 follicular, 15 anaplastic, and 18 medullary carcinomas, and 34 benign lesions, showed ret activation.

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.

Molecular Mechanisms of RET Activation in Human Cancer

Abstract: Mutations that produce oncogenes with dominant gain of function target receptor protein tyrosine kinases (PTKs) in cancer and confer uncontrolled proliferation, impaired differentiation, or unrestrained survival to the cancer cell. However, insufficient PTK signaling may be responsible for developmental diseases. Gain of function of the RET receptor PTK is associated with human cancer. At the germline level, point mutations of RET are responsible for multiple endocrine neoplasia type 2 (MEN2A, MEN2B, and FMTC). Mutations of extracellular cysteines are found in MEN2A patients, and a Met918Thr mutation is responsible for most MEN2B cases. At the somatic level, gene rearrangements juxtaposing the tyrosine kinase domain of RET to heterologous gene partners are found in papillary carcinomas of the thyroid. These rearrangements generate the chimeric RET/PTC oncogenes. Both MEN2 mutations and PTC gene rearrangements potentiate the intrinsic tyrosine kinase activity of RET and, ultimately, the RET downstream signaling events. A multidocking site of the C‐tail of RET is essential for both mitogenic and survival RET signaling. Such a site is involved in the recruitment of several intracellular molecules, such as the Shc, FRS2, IRS1, Gab1/2, and Enigma. The different activating mutations not only potentiate the enzymatic activity of the RET kinase but also may alter qualitatively RET signaling properties by: (1) altering RET autophosphorylation (in the case of the MEN2B mutation), (2) modifying the subcellular distribution of the active kinase, and (3) providing the active kinase with a scaffold for novel protein‐protein interactions (as in the case of RET/PTC oncoproteins). This review describes the molecular mechanisms by which the different genetic alterations cause the conversion of RET into a dominant transforming oncogene.

Signalling of the Ret receptor tyrosine kinase through the c-Jun NH2-terminal protein kinases (JNKS): evidence for a divergence of the ERKs and JNKs pathways induced by Ret.

The RET proto-oncogene encodes a functional receptor tyrosine kinase (Ret) for the Glial cell line Derived Neurotrophic Factor (GDNF). RET is involved in several neoplastic and non-neoplastic human diseases. Oncogenic activation of RET is detected in human papillary thyroid tumours and in multiple endocrine neoplasia type 2 syndromes. Inactivating mutations of RET have been associated to the congenital megacolon, i.e. Hirschprungs disease. In order to identify pathways that are relevant for Ret signalling to the nucleus, we have investigated its ability to induce the c-Jun NH2-terminal protein kinases (JNK). Here we show that triggering the endogenous Ret, expressed in PC12 cells, induces JNK activity; moreover, Ret is able to activate JNK either when transiently transfected in COS-1 cells or when stably expressed in NIH3T3 fibroblasts or in PC Cl 3 epithelial thyroid cells. JNK activation is dependent on the Ret kinase function, as a kinase-deficient RET mutant, associated with Hirschsprungs disease, fails to activate JNK. The pathway leading to the activation of JNK by RET is clearly divergent from that leading to the activation of ERK: substitution of the tyrosine 1062 of Ret, the Shc binding site, for phenylalanine abrogates ERK but not JNK activation. Experiments conducted with dominant negative mutants or with negative regulators demonstrate that JNK activation by Ret is mediated by Rho/Rac related small GTPases and, particularly, by Cdc42.

A Cell Proliferation and Chromosomal Instability Signature in Anaplastic Thyroid Carcinoma

Here, we show that the anaplastic thyroid carcinoma (ATC) features the up-regulation of a set of genes involved in the control of cell cycle progression and chromosome segregation. This phenotype differentiates ATC from normal tissue and from well-differentiated papillary thyroid carcinoma. Transcriptional promoters of the ATC up-regulated genes are characterized by a modular organization featuring binding sites for E2F and NF-Y transcription factors and cell cycle-dependent element (CDE)/cell cycle gene homology region (CHR) cis-regulatory elements. Two protein kinases involved in cell cycle regulation, namely, Polo-like kinase 1 (PLK1) and T cell tyrosine kinase (TTK), are part of the gene set that is up-regulated in ATC. Adoptive overexpression of p53, p21 (CIP1/WAF1), and E2F4 down-regulated transcription from the PLK1 and TTK promoters in ATC cells, suggesting that these genes might be under the negative control of tumor suppressors of the p53 and pRB families. ATC, but not normal thyroid, cells depended on PLK1 for survival. RNAi-mediated PLK1 knockdown caused cell cycle arrest associated with 4N DNA content and massive mitotic cell death. Thus, thyroid cell anaplastic transformation is accompanied by the overexpression of a cell proliferation/genetic instability-related gene cluster that includes PLK1 kinase, which is a potential molecular target for ATC treatment.

Molecular heterogeneity of RET loss of function in Hirschsprung's disease.

The RET proto‐oncogene encodes a receptor with tyrosine kinase activity (RET) that is involved in several neoplastic and non‐neoplastic diseases. Oncogenic activation of RET, achieved by different mechanisms, is detected in a sizeable fraction of human thyroid tumors, as well as in multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) and familial medullary thyroid carcinoma tumoral syndromes. Germline mutations of RET have also been associated with a non‐neoplastic disease, the congenital colonic aganglionosis, i.e. Hirschsprungs disease (HSCR). To analyse the impact of HSCR mutations on RET function, we have introduced into wild‐type RET and activated RET(MEN2A) and RET(MEN2B) alleles three missense mutations associated with HSCR. Here we show that the three mutations caused a loss of function of RET when assayed in two model cell systems, NIH 3T3 and PC12 cells. The effect of different HSCR mutations was due to different molecular mechanisms. The HSCR972 (Arg972–>Gly) mutation, mapping in the intracytoplasmic region of RET, impaired its tyrosine kinase activity, while two extracellular mutations, HSCR32 (Ser32–>Leu) and HSCR393 (Phe393–>Leu), inhibited the biological activity of RET by impairing the correct maturation of the RET protein and its transport to the cell surface.

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-1α/β. 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-1α was able to induce a biological response in thyroid cells. We observed that SDF-1α induced S-phase entry and survival of thyroid cells. Invasion through a reconstituted extracellular matrix was also supported by SDF-1α 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-1α receptor–ligand pathway to proliferate, survive and migrate.

Thyroid cancer and inflammation

Some cancer types are strongly associated with chronic inflammatory or infectious diseases whereas others are not, but an inflammatory component is present in most human neoplastic lesions. This review focuses on various aspects of thyroid cancer and inflammation. The incidence of thyroid cancer, in particular of well-differentiated papillary thyroid carcinomas (PTCs), is increased in autoimmune thyroid diseases such as Hashimotos thyroiditis. Thyroid cancer often has an inflammatory cell infiltrate, which includes lymphocytes, macrophages, dendritic cells and mast cells, whose role in thyroid cancer is still not completely understood. However, most experimental evidence suggests these cells exert a protumorigenic function. Moreover, oncoproteins typically expressed in human PTCs, such as RET/PTC, RAS, and BRAF, trigger a proinflammatory programme in thyreocytes. These data suggest that inflammatory molecules are promising targets for thyroid cancer therapy.