Sonia Moretti

Germline BRAF mutations in noonan, LEOPARD, and cardiofaciocutaneous Syndromes: Molecular diversity and associated phenotypic spectrum

Noonan, LEOPARD, and cardiofaciocutaneous syndromes (NS, LS, and CFCS) are developmental disorders with overlapping features including distinctive facial dysmorphia, reduced growth, cardiac defects, skeletal and ectodermal anomalies, and variable cognitive deficits. Dysregulated RAS–mitogen‐activated protein kinase (MAPK) signal traffic has been established to represent the molecular pathogenic cause underlying these conditions. To investigate the phenotypic spectrum and molecular diversity of germline mutations affecting BRAF, which encodes a serine/threonine kinase functioning as a RAS effector frequently mutated in CFCS, subjects with a diagnosis of NS (N=270), LS (N=6), and CFCS (N=33), and no mutation in PTPN11, SOS1, KRAS, RAF1, MEK1, or MEK2, were screened for the entire coding sequence of the gene. Besides the expected high prevalence of mutations observed among CFCS patients (52%), a de novo heterozygous missense change was identified in one subject with LS (17%) and five individuals with NS (1.9%). Mutations mapped to multiple protein domains and largely did not overlap with cancer‐associated defects. NS‐causing mutations had not been documented in CFCS, suggesting that the phenotypes arising from germline BRAF defects might be allele specific. Selected mutant BRAF proteins promoted variable gain of function of the kinase, but appeared less activating compared to the recurrent cancer‐associated p.Val600Glu mutant. Our findings provide evidence for a wide phenotypic diversity associated with mutations affecting BRAF, and occurrence of a clinical continuum associated with these molecular lesions. Hum Mutat 0:1–8, 2009.

Balancing inflammation and tolerance in vivo through dendritic cells by the commensal Candida albicans.

We analyzed the contribution of intracellular signaling to the functional plasticity of dendritic cells (DCs) presenting Candida albicans, a human commensal associated with severe diseases. Distinct intracellular pathways were activated by recognition of different fungal morphotypes in distinct DC subsets and in Peyers patches DCs. Inflammatory DCs initiated Th17/Th2 responses to yeasts through the adaptor myeloid differentiation factor-88 (MyD88), whereas tolerogenic DCs activate Th1/T regulatory cell (Treg) differentiation programs to hyphae involving Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF) as an intermediary of signaling. In addition, signal transducer and activator of transcription 3 (STAT3), affecting the balance between canonical and non-canonical activation of nuclear factor-kappaB (NF-κB) and 2,3 indoleamine dioxygenase (IDO), pivotally contributed to DC plasticity and functional specialization. As Candida-induced tolerogenic DCs ameliorated experimental colitis, our data qualify Candida as a commensal with immunoregulatory activity, resulting from the orchestrated usage of multiple, yet functionally distinct, receptor–signaling pathways in DCs. Ultimately, affecting the local Th17/Treg balance might likely be exploited by the fungus for either commensalism or pathogenicity.

Genotyping of an Italian papillary thyroid carcinoma cohort revealed high prevalence of BRAF mutations, absence of RAS mutations and allowed the detection of a new mutation of BRAF oncoprotein (BRAFV599Ins)

Objectivesu2002 The genes RET and RAS, and more recently BRAF, have been shown to be frequently mutated in human papillary thyroid carcinomas (PTC). The aim of this study was to genotype for these mutations a cohort of thyroid tumours collected at our institutions.

Intranasally delivered siRNA targeting PI3K/Akt/mTOR inflammatory pathways protects from aspergillosis

Innate responses combine with adaptive immunity to generate the most effective form of anti-Aspergillus immune resistance. Although some degree of inflammation is required for protection, progressive inflammation may worsen disease and ultimately prevents pathogen eradication. To define molecular pathways leading to or diverting from pathogenic inflammation in infection, we resorted to dendritic cells (DCs), known to activate distinct signaling pathways in response to pathogens. We found that distinct intracellular pathways mediated the sensing of conidia and hyphae by lung DCs in vitro, which translate in vivo in the activation of protective Th1/Treg responses by conidia or inflammatory Th2/Th17 responses by hyphae. In vivo targeting inflammatory (PI3K/Akt/mTOR) or anti-inflammatory (STAT3/IDO) DC pathways by intranasally delivered small interfering RNA (siRNA) accordingly modified inflammation and immunity to infection. Thus, the screening of signaling pathways in DCs through a systems biology approach may be exploited for the development of siRNA therapeutics to attenuate inflammation in respiratory fungal infections and diseases.

Biochemical and molecular characterization of the novel BRAF V599Ins mutation detected in a classic papillary thyroid carcinoma

Activating mutations of the BRAF gene are the most common genetic alterations in papillary thyroid carcinomas (PTCs) and the T1799A transversion, resulting in BRAFV600E, appeared virtually unique in this cancer type. Here, we report on the identification in a classic PTC of a novel BRAF mutation, namely a 1795GTT insertion, resulting in BRAFV599Ins, and describe its biochemical and molecular characterization. Kinase assays carried out on BRAFV599Ins and BRAFV600E revealed a three- to five-fold increase in the enzymatic activity of both mutants with respect to BRAFWT. Similarly, evaluation of BRAF-induced phosphorylation of MEK, MAPK and RSK revealed a significant MAPK cascade activation in cells expressing BRAFV599Ins or BRAFV600E, but not in cells expressing BRAFWT. Molecular dynamic simulations showed a destabilization of the inactive conformation of the enzyme in both BRAFV599Ins and BRAFV600E mutants, but not in BRAFWT. The analysis of the interaction energies inside the catalytic site allowed to demonstrate the presence of repulsive electrostatic forces acting on the activation loop and moving from inward to outward of the mutant enzymes. Finally, focus assays in NIH-3T3 cells confirmed a high transformation rate in the cells transfected either with BRAFV599Ins or BRAFV600E. In conclusion, this study demonstrated that BRAFV599Ins, as BRAFV600E, is a ‘gain of function’ mutation, characterized by a constitutive catalytic activation, which accounts for its causative role in the studied PTC.

Modifications in the Papillary Thyroid Cancer Gene Profile Over the Last 15 Years

BACKGROUNDnEvidence for an increased prevalence of BRAF(V600E) mutations has been documented in recent decades. The aim of this study was to evaluate the prevalence of both RET/PTC rearrangements and BRAF(V600E) mutations in an Italian cohort of papillary thyroid carcinoma (PTC) patients followed at the Endocrine Units of Pisa, Milano, and Perugia from 1996-2010.nnnPATIENTS AND METHODSnIn total, 401 PTC patients were examined and grouped according to the time of surgery: group 1, 1996-2000; group 2, 2001-2005; and group 3, 2006-2010. Patients were analyzed for clinical, pathological, and molecular features. In parallel, the molecular characteristics of 459 PTC from Sicily were studied.nnnRESULTSnThe genetic profiles of the three groups were significantly different (P < 0.0001). In particular, the frequency of RET/PTC rearrangements decreased from 1996-2010, occurring in 33 of 100 (33%) of the patients in group 1, 26 of 148 (17%) in group 2, and 15 of 153 (9.8%) in group 3. The incidence of BRAF(V600E) mutations increased over the same period, from 28% in group 1 (28 of 100) to 48.9% in group 2 (73 of 148) and 58.1% in group 3 (89 of 153). A consistent increase in BRAF(V600E) prevalence was observed in the Sicilian group (P < 0.0001). Moreover, a statistically significant increase in the mean age at diagnosis and decrease in tumor size over the study period was observed.nnnCONCLUSIONnThe genetic profile of PTC changed over the last 15 yr, with a significant decrease in the prevalence of RET/PTC rearrangements and an increase in BRAF(V600E) mutations. In addition, the mean age at diagnosis increased and tumor size decreased over the study period.

Analysis of human MDM4 variants in papillary thyroid carcinomas reveals new potential markers of cancer properties

A wild-type (wt) p53 gene characterizes thyroid tumors, except for the rare anaplastic histotype. Because p53 inactivation is a prerequisite for tumor development, alterations of p53 regulators represent an alternative way to impair p53 function. Indeed, murine double minute 2 (MDM2), the main p53 negative regulator, is overexpressed in many tumor histotypes including those of the thyroid. A new p53 regulator, MDM4 (a.k.a. MDMX or HDMX) an analog of MDM2, represents a new oncogene although its impact on tumor properties remains largely unexplored. We estimated levels of MDM2, MDM4, and its variants, MDM4-S (originally HDMX-S) and MDM4-211 (originally HDMX211), in a group of 57 papillary thyroid carcinomas (PTC), characterized by wt tumor protein 53, in comparison to matched contra-lateral lobe normal tissue. Further, we evaluated the association between expression levels of these genes and the histopathological features of tumors. Quantitative real-time polymerase chain reaction revealed a highly significant downregulation of MDM4 mRNA in tumor tissue compared to control tissue (Pu2009<u20090.0001), a finding confirmed by western blot on a subset of 20 tissue pairs. Moreover, the tumor-to-normal ratio of MDM4 levels for each individual was significantly lower in late tumor stages, suggesting a specific downregulation of MDM4 expression with tumor progression. In comparison, MDM2 messenger RNA (mRNA) and protein levels were frequently upregulated with no correlation with MDM4 levels. Lastly, we frequently detected overexpression of MDM4-S mRNA and presence of the aberrant form, MDM4-211 in this tumor group. These findings indicate that MDM4 alterations are a frequent event in PTC. It is worthy to note that the significant downregulation of full-length MDM4 in PTC reveals a novel status of this factor in human cancer that counsels careful evaluation of its role in human tumorigenesis and of its potential as therapeutic target.

Indoleamine 2,3-Dioxygenase 1 (IDO1) Is Up-Regulated in Thyroid Carcinoma and Drives the Development of an Immunosuppressant Tumor Microenvironment

CONTEXTnIndoleamine 2,3-dioxygenase 1 (IDO1) is a single chain oxidoreductase that catalyzes tryptophan degradation to kynurenine. In cancer, it appears to exert an immunosuppressive function as part of an acquired mechanism of immune escape mediated by the inhibition of lymphocyte proliferation and survival and by the induction of FoxP3+ T regulatory cells.nnnOBJECTIVEnThe objective of the study was to evaluate IDO1 expression in thyroid carcinoma and demonstrate its immunosuppressive function in the context of thyroid tumors.nnnSETTINGnIDO1 expression was evaluated by quantitative PCR in 105 papillary thyroid carcinomas (PTCs), 11 medullary thyroid carcinomas, six anaplastic thyroid carcinomas, and five thyroid carcinoma cell lines (TCCLs), by immunohistochemistry in 55 PTCs and by Western blotting in five TCCLs. FoxP3+ Treg lymphocyte density was evaluated by immunohistochemistry in 29 PTCs. IDO1 inhibitory effect on lymphocyte proliferation was tested in coculture experiments of TCCLs and activated lymphocytes.nnnRESULTSnIDO1 mRNA expression resulted significantly higher in all the analyzed thyroid carcinoma histotypes compared with normal thyroid. Interestingly, an increase of IDO1 mRNA expression magnitude could be observed with gain of aggressiveness (PTCs and medullary thyroid carcinomas ≪ anaplastic thyroid carcinomas). In PTCs, IDO1 mRNA expression magnitude correlated with IDO1 immunostaining intensity in cancer cells and with FoxP3+ Treg lymphocyte density in the tumor microenvironment. IDO1 was expressed in human thyroid cancer cell lines in vitro, and FTC-133 cells showed high kynurenine concentration in the conditioned medium and a strong suppressive action on the proliferation of activated lymphocytes in coculture experiments.nnnCONCLUSIONSnFor the first time, this study demonstrates a pivotal role of IDO1 in the suppression of lymphocyte function in thyroid carcinoma microenvironment.

Gemcitabine-loaded biocompatible nanocapsules for the effective treatment of human cancer

AIMnTo encapsulate the nucleoside gemcitabine (GEM) in novel PEGylated polymeric nanocapsules (NanoGEM).nnnMATERIALS & METHODSnThe biological activity of NanoGEM was tested both in vitro and in vivo in comparison with the free drug. The NanoGEM was made of polylactic acid and allowed the entrapment of a great amount of GEM.nnnRESULTS & DISCUSSIONnThe NanoGEM showed mean sizes of approximately 200 nm, a polydispersity index of approximately 0.1 and a ζ-potential of -30 mV. It exerted a stronger, quicker effect on the reduction of HEK293 cell growth in vitro in comparison with free GEM and had an in vivo antitumoral effect on the proliferation of xenograft tumors at a drug dosage tenfold less than its saline solution. The employment of nanocapsules increased the plasmatic half-life of the drug and allowed a great accumulation of GEM inside the tumors.nnnCONCLUSIONnNanoGEM represents a promising new tool for the treatment of cancer.

Microsomal Prostaglandin E2 Synthase-1 Is Induced by Conditional Expression of RET/PTC in Thyroid PCCL3 Cells through the Activation of the MEK-ERK Pathway

RET/PTC rearrangements are believed to be tumor-initiating events in papillary thyroid carcinomas. We identified microsomal prostaglandin E2 synthase-1 (mPGES-1) as a RET/PTC-inducible gene through subtraction hybridization cloning and expression profiling with custom microarrays. The inducible prostaglandin E2 (PGE2) biosynthetic enzymes cyclooxygenase-2 (COX-2) and mPGES-1 are up-regulated in many cancers. COX-2 is overexpressed in thyroid malignancies compared with benign nodules and normal thyroid tissues. Eicosanoids may promote tumorigenesis through effects on tumor cell growth, immune surveillance, and angiogenesis. Conditional RET/PTC1 or RET/PTC3 expression in PCCL3 thyroid cells markedly induced mPGES-1 and COX-2. PGE2 was the principal prostanoid and up-regulated (by ∼60-fold), whereas hydroxyeicosatetraenoic acid metabolites were decreased, consistent with shunting of prostanoid biosynthesis toward PGE2 by coactivation of the two enzymes. RET/PTC activated mPGES-1 gene transcription. Based on experiments with kinase inhibitors, with PCCL3 cell lines with doxycycline-inducible expression of RET/PTC mutants with substitutions of critical tyrosine residues in the kinase domain, and lines with inducible expression of activated mutants of H-RAS and MEK1, RET/PTC was found to regulate mPGES-1 through Shc-RAS-MEK-ERK. These data show a direct relationship between activation of a tyrosine kinase receptor oncogene and regulation of PGE2 biosynthesis. As enzymes involved in prostanoid biosynthesis can be targeted with pharmacological inhibitors, these findings may have therapeutic implications.