Role of Epigenetic Therapy in the Modulation of Tumor Growth and Migration in Human Castration-Resistant Prostate Cancer Cells with Neuroendocrine Differentiation.

INTRODUCTION: Neuroendocrine transdifferentiation (NED) of prostate cancer (PC) cells is associated with the development of resistance to antiandrogen therapy and poor prognosis in patients with castration-resistant PC (CRPC). Many of the molecular events, involved in NED, appear to be mediated by epigenetic mechanisms. In this study, we evaluated the antitumor activity and epigenetic modulation of 2 epigenetic drugs, such as the demethylating agent 5-aza-2'-deoxycytidine (AZA) and the methyl donor S-adenosylmethionine (SAM), in 2 human CRPC cell lines with NED (DU-145 and PC-3). METHODS: The effects of AZA and SAM on cell viability, cell cycle, apoptosis, migration, and genome-wide DNA methylation profiling have been evaluated. RESULTS: Both drugs showed a prominent antitumor activity in DU-145 and PC-3 cells, through perturbation of cell cycle progression, induction of apoptosis, and inhibition of cell migration. AZA and SAM reversed NED in DU-145 and PC-3, respectively. Moreover, AZA treatment modified DNA methylation pattern in DU-145 cells, sustaining a pervasive hypomethylation of the genome, with a relevant effect on several pathways involved in the regulation of cell proliferation, apoptosis, and cell migration, in particular Wnt/ß-catenin. CONCLUSIONS: A relevant antitumor activity of these epigenetic drugs on CRPC cell lines with NED opens a new scenario in the therapy of this lethal variant of PC.

A frequent oligogenic involvement in congenital hypothyroidism.

Congenital hypothyroidism (CH), the most frequent form of preventable mental retardation, is predicted to have a relevant genetic origin. However, CH is frequently reported to be sporadic and candidate gene variations were found in <10% of the investigated patients. Here, we characterize the involvement of 11 candidate genes through a systematic Next Generation Sequencing (NGS) analysis. The NGS was performed in 177 unrelated CH patients (94 gland-in-situ; 83 dysgenesis) and in 3,538 control subjects. Non-synonymous or splicing rare variants (MAF < 0.01) were accepted, and their functional impact was predicted by a comprehensive bioinformatic approach and co-segregation studies. The frequency of variations in cases and controls was extended to 18 CH-unrelated genes. At least one rare variant was accepted in 103/177 patients. Monogenic recessive forms of the disease were found in five cases, but oligogenic involvement was detected in 39 patients. The 167 variations were found to affect all genes independently of the CH phenotype. These findings were replicated in an independent cohort of additional 145 CH cases. When compared to 3,538 controls, the CH population was significantly enriched with disrupting variants in the candidate genes (P = 5.5 × 10-7), but not with rare variations in CH-unrelated genes. Co-segregation studies of the hypothyroid phenotype with multiple gene variants in several pedigrees confirmed the potential oligogenic origin of CH. The systematic NGS approach reveals the frequent combination of rare variations in morphogenetic or functional candidate genes in CH patients independently of phenotype. The oligogenic origin represents a suitable explanation for the frequent sporadic CH occurrence.

Mild TSH resistance: Clinical and hormonal features in childhood and adulthood.

OBJECTIVE: Mutations in TSH receptor (TSHR) are associated with TSH resistance, a genetic defect characterized by a heterogeneous phenotype ranging from severe hypothyroidism to subclinical hypothyroidism (SCH). We assessed the clinical and hormonal pattern of TSHR variants in a series of pediatric patients, and the long-term outcome of growth, biochemical measurements of metabolism, and neuropsychological functions in TSHR mutations carriers. DESIGN: Observational, retrospective study. PATIENTS: Thirty four children (age 7 days to 11 years) and 18 adult carriers of TSHR variants. MEASUREMENTS: The TSHR gene was sequenced by PCR-amplified direct sequencing in 111 pediatric patients with slight to moderate elevation of TSH and normal FT4 levels. The study focused on the: auxological and biochemical parameters, thyroid ultrasound, bone age, bone mineral density (BMD), and intellectual outcome (IQ) were collected during the long follow-up (1-15 years). RESULTS: Seventeen different TSHR variants (eight novel) were identified in 34 of the 111 pediatric patients, with a high prevalence of familial cases (27/34). Neonatal screening for congenital hypothyroidism was positive in half of the TSHR carriers. Growth, IQ, BMD, and biochemical parameters were normal in all subjects. Twenty patients received L-T4 replacement therapy, in all cases before genetic analysis. After re-evaluation, six patients resumed L-T4 therapy: they were compound heterozygous, or single heterozygous and with associated conditions at risk of thyroid impairment (SGA). No adults presented clinical features consistent with impaired thyroid function. CONCLUSIONS: Children carriers of TSHR variants, regardless of L-T4 treatment, show regular growth and neuropsychological development, with no evident biochemical and US alterations.

Neonatal Screening for Congenital Hypothyroidism: What Can We Learn From Discordant Twins?

CONTEXT: Newborn screening program for congenital hypothyroidism (CH) adopting rescreening in at-risk neonates. OBJECTIVES: To estimate the concordance rate for CH in twin pairs discordant at the first screening; to verify whether long-term follow-up of healthy cotwins belonging to CH discordant pairs may be useful to diagnose thyroid hypofunction during development; to evaluate the importance of genetic and environmental influences on liability to permanent and transient CH. DESIGN AND PATIENTS: Forty-seven screening discordant twin pairs were investigated. Proband was defined as the twin in the pair with a positive test at the first screening and a confirmed diagnosis of CH. RESULTS: Seven screening discordant twin pairs became concordant for CH within the first month of life (pairwise concordance of 14.9%) because seven screening negative cotwins showed high TSH values when retested. During long-term follow-up (range, 3 to 21 years), hypothyroidism was diagnosed in two monozygotic screening negative cotwins at the age of 9 months and 12 years, respectively. Furthermore, the twin analysis showed that 95% of liability to transient CH was explained by genetic factors and 5% by environmental (unshared) factors, whereas 64% of phenotypic variance of permanent CH was explained by common environmental factors (shared during the fetal life) and 36% by unshared environmental factors. CONCLUSIONS: This study showed that the introduction of rescreening permits the diagnosis of CH in a greater number of twins. It also showed the importance of long-term follow-up in both twins in the pair, and the role of nongenetic factors in the etiology of permanent CH.

Synergistic activity of everolimus and 5-aza-2'-deoxycytidine in medullary thyroid carcinoma cell lines.

Medullary thyroid cancer (MTC) is a tumor highly resistant to chemo- and radiotherapy. Drug resistance can be induced by epigenetic changes such as aberrant DNA methylation. To overcome drug resistance, we explored a promising approach based on the use of 5-aza-2'-deoxycytidine (AZA), a demethylating agent, in combination with the mTOR inhibitor everolimus in MTC cells (MZ-CRC-1 and TT). This combined treatment showed a strong synergistic antiproliferative activity through the induction of apoptosis. The effect of everolimus and/or AZA on genome-wide expression profiling was evaluated by Illumina BeadChip in MZ-CRC-1 cells. An innovative bioinformatic pipeline identified four potential molecular pathways implicated in the synergy between AZA and everolimus: PI3K-Akt signaling, the neurotrophin pathway, ECM/receptor interaction, and focal adhesion. Among these, the neurotrophin signaling pathway was most directly involved in apoptosis, through the overexpression of NGFR and Bax genes. The increased expression of genes involved in the NGFR-MAPK10-TP53-Bax/Bcl2 pathway during incubation with AZA plus everolimus was validated by western blotting in MZ-CRC-1 cells. Interestingly, addition of a neutralizing anti-NGFR antibody inhibited the synergistic cytotoxic activity between AZA and everolimus. These results open a new therapeutic scenario for MTC and potentially other neuroendocrine tumors, where therapy with mTOR inhibitors is currently approved.

Loss-of-Function Variants in a Hungarian Cohort Reveal Structural Insights on TSH Receptor Maturation and Signaling.

CONTEXT: Congenital hypothyroidism (CH) is one of the most common inborn endocrine disorders with genetic background. Despite the well-established newborn CH screening program in Hungary, no systematic examination of the underlying genetic alterations has been performed as yet. OBJECTIVE: We aimed to explore TSH receptor (TSHR) mutations in a cohort of Hungarian patients with CH. PATIENTS: Eighty-five unrelated patients with permanent primary CH, all diagnosed at newborn screening, were selected. MAIN OUTCOME MEASURES: Coding exons of the TSHR gene were sequenced and evaluated together with the thyroid-specific clinical parameters. Functional features of the novel mutations were experimentally examined, and their comparative molecular models were built. RESULTS: In four patients (one heterozygous and three compound heterozygous), seven TSHR mutations were identified. Among these, N432(1.50)D and P449(2.39)L are novel missense alterations. Importantly, the N432(1.50) residue is highly conserved among G protein-coupled receptors, and its function has not been examined yet in human glycoprotein hormone receptors. Our results indicate that the N432(1.50)D mutation disrupts important, architecture-stabilizing intramolecular interactions and ultimately leads to the complete intracellular retention of the receptor. On the other hand, P449(2.39) is located in the intracellular part of the receptor, which is important in G protein coupling. The P449(2.39)L mutation results in signaling impairment, with a more profound effect on the Gq/11 pathway. CONCLUSION: TSHR mutations are common among Hungarian patients with CH. The novel genetic alterations revealed an important structural role of the N432(1.50) and the P449(2.39) residues in receptor expression and signaling, respectively.

Frequent TSH receptor genetic alterations with variable signaling impairment in a large series of children with nonautoimmune isolated hyperthyrotropinemia.

CONTEXT: Heterozygous mutations in the TSH receptor gene (TSHR) are associated with partial TSH resistance, characterized by isolated nonautoimmune hyperthyrotropinemia (NAHT). The prevalence and management of this condition is controversial. OBJECTIVE: Our objective was to investigate the prevalence and clinical impact of TSHR alterations in a large series of pediatric patients with NAHT and to dissect their mechanism of action. DESIGN AND SETTING: For this prospective multicenter study, clinical data and samples were collected in the clinical units and conveyed to a centralized laboratory for analysis. PATIENTS: Subjects included 153 unrelated patients with NAHT aged <18 yr. Exclusion criteria included thyroid dysgenesis or major associated congenital defects. MAIN OUTCOME MEASURES: Parameters of thyroid function, TSHR gene analysis, and TSHR functional assays were evaluated. RESULTS: The frequency of heterozygous nonpolymorphic TSHR variations was 11.8%. We identified seven previously unknown variations: a frameshift (p.Q33PfsX46), one intronic (g.IVS4+2A→G), and five novel missense (p.P162L, p.Y466C, p.I583T, p.I607T, and p.R609Q) variations. The missense variations variably affected TSHR membrane expression and G(s) and/or G(q/11) signaling. Several variations cosegregated with NAHT in the affected families. Parameters of thyroid function were similar between affected and unaffected family members. CONCLUSIONS: Nonpolymorphic alterations in the TSHR gene are commonly associated with isolated NAHT in young patients, thus configuring partial TSH resistance as the most frequent inheritable cause of isolated NAHT. The identification of TSHR defects may thus be helpful for a tailored management of subclinical hypothyroidism. We provide further evidence that besides the well-known defects in G(s) signaling, TSHR genetic alternations found in NAHT may frequently impair the G(q/11) pathway.

Genetics and phenomics of hypothyroidism due to TSH resistance.

The resistance to thyrotropin (TSH) action is the disease associated with molecular defects hampering the adequate transmission of TSH stimulatory signal into thyroid cells. The defect may in principle affect every step along the cascade of events following the binding of TSH to its receptor (TSHR) on thyroid cell membranes. After the description of the first family affected with loss-of-function (LOF) TSHR mutations in 1995, there is now evidence that TSH resistance is a disease with a broad range of expressivity going from severe congenital hypothyroidism (CH) with thyroid hypoplasia to mild hyperthyrotropinemia (hyperTSH) associated with an apparent euthyroid state. More severe forms occur in patients with disrupting biallelic TSHR mutations and follow a recessive pattern of inheritance. Differential diagnosis in these cases includes the exclusion of other causes of thyroid dysgenesis, such as mutations in thyroid transcription factors. More mild forms may instead occur in patients with monoallelic TSHR defects following a dominant mode of inheritance. In these cases we described the dominant negative effect exerted by some LOF mutants on the activity of the wild-type TSHR. Differential diagnosis involves the exclusion of mild hypothyroidism in autoimmune thyroid disease or pseudohypoparathyroidism associated with genetic or epigenetic defects at the GNAS locus. This review will focus on the prevalence of TSHR mutations, on the molecular mechanisms leading to TSH resistance and on the variable clinical expression of this disease.