Reduced expression of let-7f activates TGF-ß/ALK5 pathway and leads to impaired ischaemia-induced neovascularization after cigarette smoke exposure.

This study sought to determine the potential role of microRNAs (miRNAs) in the detrimental effects of cigarette smoke on angiogenesis and neovascularization. Using large-scale miRNA profiling and qRT-PCR analyses, we identified let-7f as a pro-angiogenic miRNA which expression is significantly reduced in HUVECs treated with cigarette smoke extracts (CSE), and in the ischemic muscles of mice that are exposed to cigarette smoke (MES). In a mouse model of hindlimb ischaemia, intramuscular injection of let-7f mimic restored ischaemia-induced neovascularization in MES. Doppler flow ratios and capillary density in ischemic muscles were significantly improved in MES treated with let-7f mimic. Clinically, this was associated with reduced ambulatory impairment and hindlimb ischaemic damage. Treatment with let-7f mimic could also rescue pro-angiogenic cell (PAC) number and function (attachment, proliferation, migration) in MES. ALK5 (TGF-ßR1), an important modulator of angiogenesis, is a target of let-7f. Here we show that ALK5 is increased in HUVECs exposed to CSE and in the ischaemic muscles of MES. This is associated with a downstream activation of the anti-angiogenic factors SMAD2/3 and PAI-1. Importantly, treatment with let-7f mimic reduces the expression of ALK5, SMAD2/3 and PAI-1 both in vitro and in vivo. Moreover, let-7f overexpression or ALK5 inhibition can rescue angiogenesis in HUVECs exposed to CSE. Cigarette smoke exposure is associated with reduced expression of let-7f and activation of the anti-angiogenic TGF-ß/ALK5 pathway. Overexpression of let-7f using a miRNA mimic could constitute a novel therapeutic strategy to improve ischaemia-induced neovascularization in pathological conditions.

Low levels of IGFBP7 expression in high-grade serous ovarian carcinoma is associated with patient outcome.

BACKGROUND: Insulin-like growth factor binding protein 7 (IGFBP7) has been suggested to act as a tumour suppressor gene in various human cancers, yet its role in epithelial ovarian cancer (EOC) has not yet been investigated. We previously observed that IGFBP7 was one of several genes found significantly upregulated in an EOC cell line model rendered non-tumourigenic as consequence of genetic manipulation. The aim of the present study was to investigate the role of IGFBP7 in high-grade serous ovarian carcinomas (HGSC), the most common type of EOC. METHODS: We analysed IGFBP7 gene expression in 11 normal ovarian surface epithelial cells (NOSE), 79 high-grade serous ovarian carcinomas (HGSC), and seven EOC cell lines using a custom gene expression array platform. IGFBP7 mRNA expression profiles were also extracted from publicly available databases. Protein expression was assessed by immunohistochemistry of 175 HGSC and 10 normal fallopian tube samples using tissue microarray and related to disease outcome. We used EOC cells to investigate possible mechanisms of gene inactivation and describe various in vitro growth effects of exposing EOC cell lines to human recombinant IGFBP7 protein and conditioned media. RESULTS: All HGSCs exhibited IGFBP7 expression levels that were significantly (p = 0.001) lower than the mean of the expression value of NOSE samples and that of a whole ovary sample. IGFBP7 gene and protein expression were lower in tumourigenic EOC cell lines relative to a non-tumourigenic EOC cell line. None of the EOC cell lines harboured a somatic mutation in IGFBP7, although loss of heterozygosity (LOH) of the IGFBP7 locus and epigenetic methylation silencing of the IGFBP7 promoter was observed in two of the cell lines exhibiting loss of gene/protein expression. In vitro functional assays revealed an alteration of the EOC cell migration capacity. Protein expression analysis of HGSC samples revealed that the large majority of tumour cores (72.6%) showed low or absence of IGFBP7 staining and revealed a significant correlation between IGFBP7 protein expression and a prolonged overall survival (p = 0.044). CONCLUSION: The low levels of IGFPB7 in HGSC relative to normal tissues, and association with survival are consistent with a purported role in tumour suppressor pathways.

Genetic Characterization of GnRH/LH-Responsive Primary Aldosteronism.

Background: Recently, somatic ß-catenin mutations (CTNNB1) identified in aldosterone-producing adenomas (APAs) from three women were suggested to be responsible for the aberrant overexpression of luteinizing hormone/choriogonadotropin receptor and gonadotropin-releasing hormone receptor in the APA. Objective: To genetically characterize patients with primary aldosteronism (PA) evaluated in vivo for gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH)-responsive aldosterone secretion. Method: Patients with PA were evaluated in vivo to determine the possible regulation of aldosterone secretion by GnRH or LH. Genetic analysis of the CTNNB1, KCNJ5, ATP1A1, ATP2B3, CACNA1D, and GNAS genes were performed in this cohort and a control cohort of PA not tested in vivo for GnRH response. Results: We studied 50 patients with confirmed PA, including 36 APAs, 12 bilateral macronodular adrenal hyperplasias, 1 oncocytoma, and 1 bilateral hyperplasia with cosecretion of cortisol. Among 23 patients tested in vivo for GnRH response of aldosterone, 7 (30.4%) had a positive response, 4 (17.4%) a partial response, and 12 (52.2%) no response. No somatic CTNNB1 mutations were identified, but the disease-causing c.451G>C KCNJ5 mutation was found in two individuals with partial and no GnRH responses and an individual showing a positive response to LH. Two additional somatic pathogenic mutations, CACNA1D c.776T>A and ATP1A1 c.311T>G, were identified in two patients with no GnRH responses. In the 26 patients not tested for GnRH response, we identified 2 CTNNB1 (7.7%), 13 KCNJ5 (50%), and 1 CACNA1D (3.8%) mutations. Conclusion: Aberrant regulation of aldosterone by GnRH is frequent in PA, but is not often associated with somatic CTNNB1, although it may be found with somatic KCNJ5 mutations.

ARMC5 mutations in a large French-Canadian family with cortisol-secreting ß-adrenergic/vasopressin responsive bilateral macronodular adrenal hyperplasia.

BACKGROUND: Bilateral macronodular adrenal hyperplasia (BMAH) is a rare cause of Cushing's syndrome (CS) and its familial clustering has been described previously. Recent studies identified that ARMC5 mutations occur frequently in BMAH, but the relation between ARMC5 mutation and the expression of aberrant G-protein-coupled receptor has not been examined in detail yet. METHODS: We studied a large French-Canadian family with BMAH and sub-clinical or overt CS. Screening was performed using the 1-mg dexamethasone suppression test (DST) in 28 family members. Screening for aberrant regulation of cortisol by various hormone receptors were examined in vivo in nine individuals. Sequencing of the coding regions of ARMC5 gene was carried out. RESULTS: Morning ambulating cortisol post 1 mg DST were >50 nmol/l in 5/8 members in generation II (57-68 years old), 9/22 in generation III (26-46 years old). Adrenal size was enlarged at different degrees. All affected patients increased cortisol following upright posture, insulin-induced hypoglycemia and/or isoproterenol infusion. ß-blockers led to the reduction of cortisol secretion in all patients with the exception of two who had adrenalectomies because of ß-blockers intolerance. We identified a heterozygous germline variant in the ARMC5 gene c.327_328insC, (p.Ala110Argfs*9) in nine individuals with clinical or subclinical CS, in four out of six individuals with abnormal suppression to dexamethasone at initial investigation and one out of six individuals with current normal clinical screening tests. CONCLUSIONS: Systematic screening of members of the same family with hereditary BMAH allows the diagnosis of unsuspected subclinical CS associated with early BMAH. The relation between the causative ARMC5 mutation and the reproducible pattern of aberrant ß-adrenergic and V1-vasopressin receptors identified in this family remains to be elucidated.

RAN nucleo-cytoplasmic transport and mitotic spindle assembly partners XPO7 and TPX2 are new prognostic biomarkers in serous epithelial ovarian cancer.

PURPOSE: Epithelial ovarian cancer has the highest mortality rate of all gynecological malignancies. We have shown that high RAN expression strongly correlates with high-grade and poor patient survival in epithelial ovarian cancer. However, as RAN is a small GTPase involved in two main biological functions, nucleo-cytoplasmic transport and mitosis, it is still unknown which of these functions associate with poor prognosis. METHODS: To examine the biomarker value of RAN network components in serous epithelial ovarian cancer, protein expression of six specific RAN partners was analyzed by immunohistochemistry using a tissue microarray representing 143 patients associated with clinical parameters. The RAN GDP/GTP cycle was evaluated by the expression of RANBP1 and RCC1, the mitotic function by TPX2 and IMPß, and the nucleo-cytoplasmic trafficking function by XPO7, XPOT and IMPß. RESULTS: Based on Kaplan-Meier analyses, RAN, cytoplasmic XPO7 and TPX2 were significantly associated with poor overall patient survival, and RAN and TPX2 were associated with lower disease free survival in patients with high-grade serous carcinoma. Cox regression analysis revealed that RAN and TPX2 expression were independent prognostic factors for both overall and disease free survival, and that cytoplasmic XPO7 expression was a prognostic factor for overall patient survival. CONCLUSIONS: In this systematic study, we show that RAN and two protein partners involved in its nucleo-cytoplasmic and mitotic functions (XPO7 and TPX2, respectively) can be used as biomarkers to stratify patients based on prognosis. In particular, we reported for the first time the clinical relevance of the exportin XPO7 and showed that TPX2 expression had the strongest prognostic value. These findings suggest that protein partners in each of RAN's functions can discriminate between different outcomes in high-grade serous epithelial ovarian cancer patients. Furthermore, these proteins point to cellular processes that may ultimately be targeted to improve the survival in serous epithelial ovarian cancer.