Giada Poli

Influence of JAK2V617F allele burden on phenotype in essential thrombocythemia.

Variable proportions of mutant alleles are found in patients with JAK2 (V617F)-positive myeloproliferative disorders. This study shows that this variable mutant allele burden influences the clinical phenotype of JAK2 (V617F)-positive essential thrombocythemia. Background Fifty to sixty percent of patients with essential thrombocythemia harbor the JAK2V617F mutation. The impact of this mutation on clinical phenotype is still debated. The aim of this study was to evaluate possible correlations between JAK2V617F mutant allele burden and both clinical presentation and hematologic abnormalities in essential thrombocythemia patients. Design and Methods In this single-center retrospective study, JAK2V617F allele load was measured by sensitive quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in the granulocytes of 260 patients diagnosed as having essential thrombocythemia according to WHO criteria. Results Median V617F allele burden in patients with the mutation (n=165, 63.4%) was 24%, ranging from 1% to 87%; an allele burden greater than 51% was found in 5% of the patients. Older patients presented progressively higher percentages of the V617F allele. Signs of stimulated erythropoiesis and myelopoiesis, as well as higher PRV-1 levels, were found in patients with the mutation, but no linear correlation with load of mutant allele could be ascertained; on the other hand, the frequency of patients with erythropoietin-independent erythroid colonies progressively increased depending on mutant allele load. Splenomegaly and microvessel symptoms were significantly more represented among patients with greater than 50% and 25% JAK2V617F allele burden, respectively. Increasing mutant allele load correlated with higher frequency of arterial thrombosis at diagnosis, as confirmed also in multivariate analysis; the relative risk was 3.0 (95% CI 1.3–6.8; p=0.01) in patients having a greater than 25% mutant allele burden. Conclusions The JAK2V617F mutant allele burden contributes to determining the clinical phenotype in patients with essential thrombocythemia.

Functional Differences in Visceral and Subcutaneous Fat Pads Originate from Differences in the Adipose Stem Cell

Metabolic pathologies mainly originate from adipose tissue (AT) dysfunctions. AT differences are associated with fat-depot anatomic distribution in subcutaneous (SAT) and visceral omental (VAT) pads. We address the question whether the functional differences between the two compartments may be present early in the adipose stem cell (ASC) instead of being restricted to the mature adipocytes. Using a specific human ASC model, we evaluated proliferation/differentiation of ASC from abdominal SAT-(S-ASC) and VAT-(V-ASC) paired biopsies in parallel as well as the electrophysiological properties and functional activity of ASC and their in vitro-derived adipocytes. A dramatic difference in proliferation and adipogenic potential was observed between the two ASC populations, S-ASC having a growth rate and adipogenic potential significantly higher than V-ASC and giving rise to more functional and better organized adipocytes. To our knowledge, this is the first comprehensive electrophysiological analysis of ASC and derived-adipocytes, showing electrophysiological properties, such as membrane potential, capacitance and K+-current parameters which confirm the better functionality of S-ASC and their derived adipocytes. We document the greater ability of S-ASC-derived adipocytes to secrete adiponectin and their reduced susceptibility to lipolysis. These features may account for the metabolic differences observed between the SAT and VAT. Our findings suggest that VAT and SAT functional differences originate at the level of the adult ASC which maintains a memory of its fat pad of origin. Such stem cell differences may account for differential adipose depot susceptibility to the development of metabolic dysfunction and may represent a suitable target for specific therapeutic approaches.

Morphofunctional effects of mitotane on mitochondria in human adrenocortical cancer cells

At present, mitotane (MTT) represents the first-line pharmacological approach for the treatment of advanced adrenocortical carcinoma (ACC). Despite clear evidence that the drug can reduce the clinical signs of steroid excess in secreting ACC, the mechanism mediating the possible toxic effect of MTT on tumor cells still remains obscure. This study investigated the intracellular events underlying the toxic effect of MTT by studying qualitative and quantitative alterations in mitochondrial morphology and functions in human adrenocortical cancer cell lines, H295R and SW13. Increasing concentrations of MTT resulted in rapid intracellular accumulation and conversion of the drug. Cytostatic and cytotoxic effects were evident at doses corresponding to the therapeutic window (30-50 μM) through an apoptotic mechanism involving caspase 3/7. Electron microscopic analysis of cell mitochondria displayed MTT-induced dose- and time-dependent alterations in the morphology of the organelle. These alterations were characterized by a marked swelling and a decrease in the number of respiratory cristae, accompanied by a significant depolarization of the mitochondrial membrane potential, finally leading to the disruption of the organelle. A drastic reduction of oxygen consumption was observed due to mitochondrial membrane damage, which was accompanied by a decrease in the levels of VDAC1 integral membrane channel. These findings contribute to better understand the intracellular mechanism of action of MTT in ACC cells, showing that its cytotoxic effect seems to be mainly mediated by an apoptotic process activated by the disruption of mitochondria.

Rosiglitazone Inhibits Adrenocortical Cancer Cell Proliferation by Interfering with the IGF-IR Intracellular Signaling

Rosiglitazone (RGZ), a thiazolidinedione ligand of the peroxisome proliferator-activated receptor (PPAR)-γ, has been recently described as possessing antitumoral properties. We investigated RGZ effect on cell proliferation in two cell line models (SW13 and H295R) of human adrenocortical carcinoma (ACC) and its interaction with the signaling pathways of the activated IGF-I receptor (IGF-IR). We demonstrate a high expression of IGF-IR in the two cell lines and in ACC. Cell proliferation is stimulated by IGF-I in a dose- and time-dependent manner and is inhibited by RGZ. The analysis of the main intracellular signaling pathways downstream of the activated IGF-IR, phosphatidyl inositol 3-kinase (PI3K)-Akt, and extracellular signal-regulated kinase (ERK1/2) cascades reveals that RGZ rapidly interferes with the Akt and ERK1/2 phosphorylation/activation which mediates IGF-I stimulated proliferation. In conclusion, our results suggest that RGZ exerts an inhibitory effect on human ACC cell proliferation by interfering with the PI3K/Akt and ERK1/2 signaling pathways downstream of the activated IGF-IR.

Rosiglitazone impairs proliferation of human adrenocortical cancer: preclinical study in a xenograft mouse model

Adrenocortical carcinoma (ACC) is a rare aggressive tumor with a poor prognosis. The lack of a specific and effective medical treatment is due to the poor knowledge of the mechanisms underlying tumor growth. Research on potential drugs able to specifically interfere with tumor proliferation is essential to develop more efficacious therapies. We evaluated for the first time the in vivo effect of rosiglitazone (RGZ), an anti-diabetic drug with in vitro anti-tumor properties, on ACC proliferation in a xenograft model obtained by s.c. injection of human ACC H295R cells in athymic mice. When the tumor size reached 5 mm, animals were allocated to 5 mg/kg RGZ- or water-treated groups. Tumor volume was measured twice a week. A significant reduction of tumor growth in RGZ versus control (control) group was observed and was already maximal following 17 day treatment (1-T/C=75.4% (43.7-93.8%)). After 31 days of treatment, mice were killed and tumor analyzed. Tumor histological evaluation revealed characteristics of invasiveness, richness in small vessels and mitotic figures in control group, while RGZ group tumors presented non infiltrating borders, few vessels, and many apoptotic bodies. Tumor immunohistochemistry showed that Ki-67 was reduced in RGZ versus control group. Quantitative real-time RT-PCR demonstrated a significant reduction in the expression of angiogenic (VEGF), vascular (CD31), proliferation (BMI-1), and anti-apoptotic (Bcl-2) genes in RGZ versus control group tumors. The same inhibitory effects were confirmed in in vitro RGZ-treated H295R. Our findings support and expand the role of RGZ in controlling ACC proliferation and angiogenesis in vivo and in vitro.

Detection of Circulating Tumor Cells in Patients With Adrenocortical Carcinoma: A Monocentric Preliminary Study

CONTEXT Adrenocortical carcinoma (ACC) is a rare malignancy, the prognosis of which is mainly dependent on stage at diagnosis. The identification of disease-associated markers for early diagnosis and drug monitoring is mandatory. Circulating tumor cells (CTCs) are released into the bloodstream from primary tumor/metastasis. CTC detection in blood samples may have enormous potential for assisting in the diagnosis of malignancy, estimating prognosis, and monitoring the disease. OBJECTIVE The aim of the study was to investigate the presence of CTCs in blood samples of patients with ACC or benign adrenocortical adenoma (ACA). SETTING We conducted the study at a university hospital. INTERVENTION CTC analysis was performed in blood samples from 14 ACC patients and 10 ACA patients. CTCs were isolated on the basis of cell size by filtration through ScreenCell devices, followed by identification according to validated morphometric criteria and immunocytochemistry. MAIN OUTCOME MEASURE We measured the difference in CTC detection between ACC and ACA. RESULTS CTCs were detected in all ACC samples, but not in ACA samples. Immunocytochemistry confirmed the adrenocortical origin. When ACC patients were stratified according to the median value of tumor diameter and metastatic condition, a statistically significant difference was found in the number of CTCs detected after surgery. A significant correlation between the number of CTCs in postsurgical samples and clinical parameters was found for tumor diameter alone. CONCLUSIONS Our findings provide the first evidence for adrenocortical tumors that CTCs may represent a useful marker to support differential diagnosis between ACC and ACA. The correlation with some clinical parameters suggests a possible relevance of CTC analysis for prognosis and noninvasive monitoring of disease progression and drug response.

Role of the PPAR-γ system in normal and tumoral pituitary corticotropic cells and adrenal cells.

PPAR-γ is a member of the nuclear hormone receptor superfamily of transcription factors, whose thiazolidinedione ligands (TZD) have been recently demonstrated to also possess anticancer properties in addition to their well-known insulin-sensitizer and glucose/lipid regulation activity. In this minireview, we summarize the current knowledge on PPAR-γ in normal and tumoral corticotropic pituitary and adrenal cells. The receptor expression has been shown in ACTH-secreting cells in both normal and adenomal pituitary as well as in normal and tumor adrenal cortex. Preclinical studies conducted both in vitro on tumor cells and in vivo on xenograft tumor models obtained by subcutaneous injection of cancer cells have evidenced the anticancer properties of TZD, in particular rosiglitazone (RGZ) and pioglitazone (PIO). In both pituitary and adrenocortical cancer, RGZ treatment results in inhibition of cell proliferation, through G0/G1 cell-cycle arrest and induction of cell apoptosis, leading to significant inhibition of tumor growth in the xenograft tumor models. In addition, since RGZ can reduce ACTH and corticosterone secretion in mouse corticotropic pituitary tumors, both RGZ and PIO have been used in the treatment of Cushing’s disease with variable but generally unsatisfactory results. Discrepancies in the antitumor effects of TZD observed between successful preclinical and unsuccessful clinical studies may be particularly due to differences in treatment duration and doses used.

Metformin as a new anti-cancer drug in adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is a rare heterogeneous malignancy with poor prognosis. Since radical surgery is the only available treatment, more specific and effective drugs are urgently required. The anti-diabetic drug metformin has been associated with a decreased cancer prevalence and mortality in several solid tumors, prompting its possible use for ACC treatment. This paper evaluates the in vitro and in vivo anti-cancer effects of metformin using the ACC cell model H295R. Metformin treatment significantly reduces cell viability and proliferation in a dose- and time-dependent manner and associates with a significant inhibition of ERK1/2 and mTOR phosphorylation/activation, as well as with stimulation of AMPK activity. Metformin also triggers the apoptotic pathway, shown by the decreased expression of Bcl-2 and HSP27, HSP60 and HSP70, and enhanced membrane exposure of annexin V, resulting in activation of caspase-3 apoptotic effector. Metformin interferes with the proliferative autocrine loop of IGF2/IGF-1R, which supports adrenal cancer growth. Finally, in the ACC xenograft mouse model, obtained by subcutaneous injection of H295R cells, metformin intraperitoneal administration inhibits tumor growth, confirmed by the significant reduction of Ki67%. Our data suggest that metformin inhibits H295R cell growth both in vitro and in vivo. Further preclinical studies are necessary to validate the potential anti-cancer effect of metformin in patients affected by ACC.

Osteocalcin increase after bariatric surgery predicts androgen recovery in hypogonadal obese males

OBJECTIVE:Bone modulates testis function through osteocalcin (OCN) production. This paper assesses the association between serum OCN and androgen production recovery in morbidly obese males at 9 months after bariatric surgery.SUBJECTS:A cohort of n=103 obese males with mean±s.d. body mass index (BMI) 47.7±8.2 kg m–2, age 42±11 years, consisting of n=76 patients undergoing gastric bypass and n=27 in the waiting list for surgery.RESULTS:At 9 months from surgery, a significant increase was observed in mean±s.d. total OCN (tOCN=10.4±10.3 ng ml–1, P<0.001) and undercarboxylated OCN (ucOCN=5.4±3.7 ng ml–1, P<0.001), total testosterone (TT, 5.6±6.5 nM, P<0.001) and calculated free testosterone (cFT, 0.035±0.133 nM, P<0.006), sex hormone binding globulin (SHBG, 21.2±16.7 nM, P<0.001) and decrease in estradiol (E2, −30.1±51.9 pM, P<0.001) levels only in operated patients, with a significant reduction in BMI (24%) and waist (20%). A positive correlation existed between tOCN and ucOCN (age-adjustment (age-adj.): β=0.692, P<0.001) and their variations (age-adj.: β=0.629, P<0.001) after surgery. Multivariate analysis in operated patients showed a significant positive association between variations in tOCN and TT (age-adj.: β=0.289, P=0.012), SHBG (age-adj.: β=0.326, P=0.005) but not with cFT variation. tOCN, but not luteinizing hormone (LH) variation was the only significant predictive factor of cFT recovery in the hypogonadal (TT<12 nM) operated subjects even after age- and BMI-adjustment (adj.: β=0.582, P<0.05). cFT improvement was significantly higher when considering operated patients with tOCN increase (0.045±0.123 vs −0.02±0.118 nM, P=0.015), hypogonadism (0.059±0.111 vs –0.059±0.138 nM, P=0.002) and younger than 35 years (0.102±0.108 vs –0.019±0.123 nM, P=0.009).CONCLUSION:OCN recovery observed after bariatric surgery is significantly associated with cFT improvement independently of BMI variation and age in hypogonadal morbidly obese males.

Peroxisome-proliferator-activated receptor gamma (PPARγ) is required for modulating endothelial inflammatory response through a nongenomic mechanism

Besides their well-known anti-diabetic effects, the peroxisome-proliferator-activated receptor gamma (PPARgamma) thiazolidinedione ligands (TZD) have been suggested to also display anti-inflammatory properties. The receptor role in mediating such effects is far from being elucidated. Here, we demonstrated that PPARgamma is necessary for TZD to interfere with TNFalpha and IFNgamma inflammatory activity in human endothelial cells. Different PPARgamma ligands similarly inhibit cytokinic stimulation of IFNgamma-inducible-protein-of-10-kDa (IP10) secretion in a dose-dependent manner and prevent the induced phosphorylation/activation of extracellular-signaling-regulated-kinases (ERK1/2). To further confirm the PPARgamma role in mediating both rapid and long term anti-inflammatory effects of its ligands, we evaluated RGZ inhibitory action in PPARgamma-silenced and -overexpressing cells. PPARgamma-silencing results in a reversion of RGZ inhibitory activity on cyto/chemokine secretions and rapid ERK phosphorylation. Conversely, receptor overexpression significantly increases RGZ inhibitory activity. Finally, PPARgamma-overexpression results in a reduction of ERK1/2 phosphorylation and inflammatory secretions in response to TNFalpha and IFNgamma even in the absence of RGZ, suggesting a restraining effect controlled by endogenous ligands. In conclusion, our data provide the first evidence that PPARgamma is involved in the anti-inflammatory action of TZD in endothelial cells, not only by modulating cyto/chemokine secretions but also by restraining ERK activation through a novel rapid nongenomic mechanism.