Gabriella De Vita

A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.

A Genetic Screen Implicates miRNA-372 and miRNA-373 As Oncogenes in Testicular Germ Cell Tumors

Endogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in cellular transformation, we identified miR-372 and miR-373, each permitting proliferation and tumorigenesis of primary human cells that harbor both oncogenic RAS and active wild-type p53. These miRNAs neutralize p53-mediated CDK inhibition, possibly through direct inhibition of the expression of the tumorsuppressor LATS2. We provide evidence that these miRNAs are potential novel oncogenes participating in the development of human testicular germ cell tumors by numbing the p53 pathway, thus allowing tumorigenic growth in the presence of wild-type p53.

Dual effect on the RET receptor of MEN 2 mutations affecting specific extracytoplasmic cysteines

The RET gene encodes a receptor tyrosine kinase whose function is essential during the development of kidney and the intestinal nervous system. Germline mutations affecting one of five cysteines (Cys609, 611, 618, 620 and 634) located in the juxtamembrane domain of the RET receptor are responsible for the vast majority of two cancer-prone disorders, multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC). These mutations lead to the replacement of a cysteine by an alternate amino acid. Mutations of the RET gene are also the underlying genetic cause of Hirschsprung disease (HSCR), a congenital aganglionosis of the hindgut. In a fraction of kindreds, MEN 2A cosegregate with HSCR and affected individuals carry a single mutation at codons 609, 618 or 620. To examine the consequences of cysteine substitution on RET function, we have introduced a Cys to Arg mutation into the wild-type RET at either codons 609, 618, 620, 630 or 634. We now report that each mutation induces a constitutive catalytic activity due to the aberrant disulfide homodimerization of RET. However, mutations 630 and 634 activate RET more strongly than mutations 609, 618 or 620 as demonstrated by quantitative assays in rodent fibroblasts and pheochromocytoma PC12 cells. Biochemical analysis revealed that mutations 618 and 620, and to a lesser extent mutation 609, result in a marked reduction of the level of RET at the cell surface and as a consequence decrease the amount of RET covalent dimer. These findings provide a molecular basis explaining the range of phenotype engendered by alterations of RET cysteines and suggest a novel mechanism whereby mutations of cysteines 609, 618 and 620 exert both activating and inactivating effects.

Replacement of K-Ras with H-Ras supports normal embryonic development despite inducing cardiovascular pathology in adult mice

Ras proteins are highly related GTPases that have key roles in regulating growth, differentiation and tumorigenesis. Gene‐targeting experiments have shown that, out of the three mammalian ras genes, only K‐ras is essential for normal mouse embryogenesis, and that mice deprived of H‐ras and/or N‐ras show no major phenotype. We generated mice (HrasKI) in which the K‐ras gene had been modified to encode H‐Ras protein. HrasKI mice produce undetectable amounts of K‐Ras but—in contrast to mice homozygous for a null K‐ras allele—they are born at the expected mendelian frequency, indicating that H‐Ras can be substituted for K‐Ras in embryonic development. However, adult HrasKI mice show dilated cardiomyopathy associated with arterial hypertension. Our results show that K‐Ras can be replaced by H‐Ras in its essential function in embryogenesis, and indicate that K‐Ras has a unique role in cardiovascular homeostasis.

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.

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.

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.

The microRNA-Processing Enzyme Dicer Is Essential for Thyroid Function

Dicer is a type III ribonuclease required for the biogenesis of microRNAs (miRNAs), a class of small non-coding RNAs regulating gene expression at the post-transcriptional level. To explore the functional role of miRNAs in thyroid gland function, we generated a thyrocyte-specific Dicer conditional knockout mouse. Here we show that development and early differentiation of the thyroid gland are not affected by the absence of Dicer, while severe hypothyroidism gradually develops after birth, leading to reduced body weight and shortened life span. Histological and molecular characterization of knockout mice reveals a dramatic loss of the thyroid gland follicular architecture associated with functional aberrations and down-regulation of several differentiation markers. The data presented in this study show for the first time that an intact miRNAs processing machinery is essential for thyroid physiology, suggesting that deregulation of specific miRNAs could be also involved in human thyroid dysfunctions.

Different mutations of the RET gene cause different human tumoral diseases.

The RET gene encodes a tyrosine kinase receptor for neurotrophic molecules. RET is a conceptually valuable example of how different mutations of a single gene may cause different diseases. Gene rearrangements activate the oncogenic potential of RET in human thyroid papillary carcinomas. On the other side, different point mutations activate RET in familial multiple endocrine neoplasia syndromes. Finally, inactivating mutations of RET can be present in Hirschsprungs disease patients. The detailed knowledge of the specific RET mutations responsible for human tumors provides relevant tools for the clinical management of these diseases. Moreover, the recent discovery of the growth factors which in vivo stimulate its signaling may shed new light on the role played by RET in the development and differentiation of the central and peripheral nervous system.

Ligand stimulation of a Ret chimeric receptor carrying the activating mutation responsible for the multiple endocrine neoplasia type 2B

Inherited activating mutations of Ret, a receptor tyrosine kinase, predispose to multiple endocrine neoplasia (MEN) types 2A and 2B and familial medullary thyroid carcinoma. To investigate the effects induced by acute stimulation of Ret, we transfected both PC12 and NIH 3T3 cells with a molecular construct in which the ligand-binding domain of the epidermal growth factor receptor was fused to the catalytic domain of Ret. Acute stimulation of the chimeric receptor induced PC12 cells to express a neuronal-like phenotype. Moreover, we introduced the dominant mutation, responsible for the multiple endocrine neoplasia type 2B, in the catalytic domain of the Ret chimera. Expression of the mutant chimera, in the absence of ligand stimulation, induces the PC12 cells to acquire a flat morphology with short neuritic processes and transforms the NIH 3T3 cells. Stimulation of the mutant chimera with epidermal growth factor causes a drastic overgrowth of long neuritic processes, with the induction of the suc1-associated protein tyrosine phosphorylation in PC12 cells and higher transforming efficiency in NIH 3T3 cells. These data indicate that the gain-of-function MEN2B mutation does not abrogate ligand responsiveness of Ret and suggest that the presence of Ret ligand could play a role in the pathogenesis of the MEN2B syndrome.