Giancarlo Vecchio

One- and two-step transformations of rat thyroid epithelial cells by retroviral oncogenes.

A system of epithelial cells is described in which it is possible to study the number and the nature of genes capable of conferring the malignant phenotype. Two fully differentiated, hormone-responsive cell lines from rat thyroid glands are presented which are susceptible to one-step or two-step transformation upon infection with several murine acute retroviruses. After infection, both cell lines became independent from their thyrotropic hormone requirement for growth. However, complete transformation was achieved with one of the cell lines (FRTL-5 Cl 2), whereas the other cell line (PC Cl 3) failed to grow in agar and to give rise to tumors in vivo. The latter cell line was susceptible to complete transformation upon cooperation of the v-ras-Ha and the human c-myc oncogenes.

Disease associated mutations at valine 804 in the RET receptor tyrosine kinase confer resistance to selective kinase inhibitors.

We have recently demonstrated that the pyrazolopyrimidines PP1 and PP2 and the 4-anilinoquinazoline ZD6474 display a strong inhibitory activity (IC50⩽100 nM) towards constitutively active oncogenic RET kinases. Here, we show that most oncogenic MEN2-associated RET kinase mutants are highly susceptible to PP1, PP2 and ZD6474 inhibition. In contrast, MEN2-associated swap of bulky hydrophobic leucine or methionine residues for valine 804 in the RET kinase domain causes resistance to the three compounds. Substitution of valine 804 with the small amino- acid glycine renders the RET kinase even more susceptible to inhibition (ZD6474 IC50: 20 nM) than the wild-type kinase. Our data identify valine 804 of RET as a structural determinant mediating resistance to pyrazolopyrimidines and 4-anilinoquinazolines.

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.

Elevated levels of a specific class of nuclear phosphoproteins in cells transformed with v-ras and v-mos oncogenes and by cotransfection with c-myc and polyoma middle T genes.

Transformation of a rat thyroid epithelial cell line (FRTL5‐C12) with Kirsten and Harvey murine sarcoma viruses (carrying the ras oncogenes) results in elevated levels of three perchloric acid‐soluble nuclear phosphoproteins. These three proteins are also induced to high levels in the PC‐C13 thyroid epithelial cell line when transformed by the myeloproliferative sarcoma virus (carrying the v‐mos oncogene) and when transformed by transfection with the c‐myc proto‐oncogene followed by infection with the polyoma leukaemia virus (PyMuLV) carry the polyoma middle T antigen gene. Neither c‐myc or PyMuLV alone induced high levels of the three nuclear proteins. Untransformed thyroid fibroblasts have high levels of two of the three proteins and can be transformed by PyMuLV alone resulting in the appearance of the third protein. Transformation with Harvey sarcoma virus also results in the induction of the third protein. The three phosphoproteins have been purified by h.p.l.c. and shown to be related to the HeLa protein HMGI already described. The results of these studies indicate that elevated levels of these HMGI‐like proteins are associated with neoplastic transformation and/or with an undifferentiated phenotype.

The insulin receptor substrate (IRS)-1 recruits phosphatidylinositol 3-kinase to Ret: evidence for a competition between Shc and IRS-1 for the binding to Ret.

Tyrosine 1062 of Ret, which represents an intracytoplasmic docking site for multiple signaling molecules, is essential for Ret-mediated activation of phosphatidylinositol 3-Kinase (PI3-K). PI3-K, in turn, has been implicated in inducing cell survival and neoplastic transformation mediated by Ret. We have examined the mechanisms by which Ret stimulates PI3-K. Here we show that the Insulin Receptor Substrate-1 (IRS-1) is tyrosine phosphorylated and associated with the p85 regulatory subunit of PI3-K in response to Ret activation. IRS-1 coimmunoprecipitates with Ret and co-expression of IRS-1 results in the potentiation of Ret-mediated activation of Akt(PKB), a bona fide effector of PI3-K. The association with the PTB domain of IRS-1 depends on the phosphorylation of tyrosine 1062 of Ret. The deletion of asparagine 1059 (delN1059) and the substitution of leucine 1061 (L1061P), two Ret mutations identified in families affected by congenital megacolon (Hirschsprungs disease), impair the binding of IRS-1 to Ret as well as Ret-mediated Akt(PKB) stimulation. Finally, we show that Shc, which was previously identified as another ligand of Y1062 of Ret, competes with IRS-1 for the binding to Ret pY1062. All together, these findings suggest that IRS-1 is a component of the signaling pathway which leads to Ret-mediated PI3-K activation, a pathway which can be targeted by Hirschsprung-associated Ret mutations. The alternative binding of Shc and IRS-1 to Ret pY1062 can be a system to modulate the activation of different intracellular signaling pathways and to elicit different biological responses following Ret activation.

Pathophysiology of ageing, longevity and age related diseases

On April 18, 2007 an international meeting on Pathophysiology of Ageing, Longevity and Age-Related Diseases was held in Palermo, Italy. Several interesting topics on Cancer, Immunosenescence, Age-related inflammatory diseases and longevity were discussed. In this report we summarize the most important issues. However, ageing must be considered an unavoidable end point of the life history of each individual, nevertheless the increasing knowledge on ageing mechanisms, allows envisaging many different strategies to cope with, and delay it. So, a better understanding of pathophysiology of ageing and age-related disease is essential for giving everybody a reasonable chance for living a long and enjoyable final part of the life.

The RFG oligomerization domain mediates kinase activation and re-localization of the RET/PTC3 oncoprotein to the plasma membrane.

The RET/PTC3 oncogene arises from the fusion between the N-terminal encoding domain of the RFG gene and the tyrosine kinase encoding domain of RET receptor. RET/PTC3 is very frequent in papillary thyroid carcinomas, especially in children exposed to the Chernobyl accident. We have studied the functional consequences of the RFG–RET fusion. Here we show that the N-terminal coiled-coil domain of RGF mediates oligomerization and activation of the kinase and of the transforming capability of RET/PTC3. In addition, the RFG coiled-coil domain mediates a physical association between RET/PTC3 and RGF proteins, rendering RFG a bona fide substrate of RET/PTC3 kinase. Finally, we show that the coiled-coil domain of RGF is essential for the distribution of the RET/PTC3 protein at the membrane/particulate cell compartment level, where also most of the RFG protein is localized. We propose that fusion to the RFG coiled-coil domain provides RET kinase with a scaffold that mediates oligomerization and re-localization of the RET/PTC3 protein, a process that may be crucial for the signalling of this specific RET/PTC variant.

Dissociation between transformed and differentiated phenotype in rat thyroid epithelial cells after transformation with a temperature-sensitive mutant of the Kirsten murine sarcoma virus.

Differentiated rat thyroid epithelial cells, infected in vitro with a temperature-sensitive mutant of the Kirsten murine sarcoma virus, expressed at the permissive temperature (33 degrees C) some phenotypic properties typical of transformed cells, including morphological features, colony formation in agar, and induction of tumors in newborn animals. Specific functional markers of these differentiated cells, i.e., synthesis/secretion of thyroglobulin, synthesis of thyroglobulin mRNA and iodide uptake, were blocked during growth at 33 degrees C. Normal morphology, failure to grow in agar, and the requirement of hormones for optimal growth were all restored after shifting to the temperature nonpermissive for transformation (39 degrees C), though the typical differentiated functions remained blocked. Infection with a leukemia helper virus clone (Moloney or Kirsten murine leukemia virus) did not lead to the loss of the differentiated phenotype of rat epithelial thyroid cells, thus demonstrating that the loss of the differentiated phenotype is caused by the sarcoma virus component. These results indicate that the expression of some of the phenotypic properties of transformed differentiated rat thyroid epithelial cells is under the direct control of the p21 thermosensitive activity, whereas the block in the expression of two typical differentiation markers of thyroid epithelial cells is irreversible and probably controlled by different mechanisms.

H4(D10S170), a gene frequently rearranged with RET in papillary thyroid carcinomas: functional characterization

Human thyroid papillary carcinomas are characterized by rearrangements of the RET protooncogene with a number of heterologous genes, which generate the RET/papillary thyroid carcinoma (PTC) oncogenes. One of the most frequent variants of these recombination events is the fusion of the intracellular kinase-encoding domain of RET to the first 101 amino acids of a gene named H4(D10S170). We have characterized the H4(D10S170) gene product, showing that it is a ubiquitously expressed 55 KDa nuclear and cytosolic protein that is phosphorylated following serum stimulation. This phosphorylation was found to depend on mitogen-activated protein kinase (MAPK) Erk1/2 activity and to be associated to the relocation of H4(D10S170) from the nucleus to the cytosol. Overexpression of the H4(D10S170) gene was able to induce apoptosis of thyroid follicular epithelial cells; conversely a carboxy-terminal truncated H4(D10S170) mutant H4(1–101), corresponding to the portion included in the RET/PTC1 oncoprotein, behaved as dominant negative on the proapoptotic function and nuclear localization of H4(D10S170). Furthermore, conditional expression of the H4(D10S170)-dominant negative truncated mutant protected cells from stress-induced apoptosis. The substitution of serine 244 with alanine abrogated the apoptotic function of H4(D10S170). These data suggest that loss of the H4(D10S170) gene function might have a role in thyroid carcinogenesis by impairing apoptosis.

Molecular Mechanisms of RET Activation in Human Neoplasia

Mutations that produce oncogenes with dominant gain of function may target receptor protein tyrosine kinases (PTK) in cancer and confer uncontrolled proliferation, impaired differentiation or unrestrained survival to the cancer cell. On the other hand, insufficient PTKs’ signaling may be responsible for developmental diseases. Gain of function of the RET receptor PTK is associated to human cancer. At the germ line 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 MEN2B. At the somatic level, gene rearrangements juxtaposing the TK 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-associated point mutations and PTC-associated gene rearrangements potentiate the intrinsic TK 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 signalling. Such a site is involved in the recruitment of several intracellular molecules, like the She, FRS2 and IRS1 docking proteins and Enigma. The different activating mutations may also alter qualitatively the RET signaling properties either by altering RET autophosphorylation (in the case of the MEN2B mutation) or the subcellular distribution of the active kinase or 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.