Various types of protein kinase A (PKA) alterations have been observed in adrenocortical tumours and Carney complex (CNC). PKA is a heterotetramer of two regulatory and two catalytic subunits. The R1A and R2B proteins are the most abundant regulatory subunits in endocrine tissues. A decrease in R2B protein levels has been observed in adrenal adenoma, whereas tumours from patients with CNC display a decrease in R1A protein levels. Dysregulation of the balance between R1A and R2B may thus be involved in adrenal tumourigenesis. We investigated the impact of the differences in the balance of PKA subunits on cell growth using specific cAMP analogues. We assessed the effects of 8-chloroadenosine-cAMP (8Cl-cAMP), a site-selective activator of PKA R2B, in H295R adrenocortical cells. 8Cl-cAMP stimulated PKA activity, decreased R1A levels and increased R2B levels. It had no cytotoxic effects, initially stimulating DNA synthesis and then inducing apoptosis by disrupting G2/M progression. We observed an initial accumulation of cells in the S phase, translocation of cyclin A to the nucleus, CDK2 activation, sustained DNA synthesis and proliferating cell nuclear antigen accumulation. Cell cycle arrest in the G2 phase was parallel with the accumulation of cyclin B and the inactivation of CDC2 kinase. The 8CPT-cAMP, which activates the R2B subunit, had similar effects. R2B silencing reduced the apoptosis induced by tumour necrosis factor α and transforming growth factor β. Thus, R2B is a key regulator of proliferation/differentiation in H295R cell line along with the complex balance between the PKA subunits. Activation of PKA R2B and dysregulation of the R1A/R2B balance regulate cell cycle progression and apoptosis in adrenocortical cells by modulating cyclin production and cyclin-dependent kinase activities.
Zhor Bouizar, Bruno Ragazzon, Lucie Viou, Mariuccia Hortane, Jerôme Bertherat and Marthe Rizk-Rabin
A Picon, M Leblond-Francillard, M-L Raffin-Sanson, F Lenne, X Bertagna and Y de Keyzer
DMS-79 is a human cell line derived from a small cell lung carcinoma (SCLC), which expresses the pro-opiomelanocortin (POMC) gene. We took it as a model in which to study the mechanism of POMC gene expression in non-pituitary tumours. DMS-79 reproduces the usual characteristics of POMC expression in these tumours: precursor processing is altered and gene expression is essentially unresponsive to glucocorticoids. POMC gene structure appeared normal by Southern blot analysis, indicating that gene rearrangement was not responsible for its expression in DMS-79. Indeed, using transient expression of human POMC—luciferase fusion genes in DMS-79, we showed that (1) the normal human POMC promoter was functional in DMS-79, and (2) the same proximal promoter region (−417;+21) produced the full transcriptional activity in DMS-79 and in the mouse pituitary cell line AtT-20. Progressive 5′ deletion analysis revealed differences between AtT-20 and DMS-79: region (−161;−376) was active in AtT-20 and not in DMS-79, whereas region (−95;−161) was active in both cell lines and (−376;−417) was only active in DMS-79. The latter partially overlaps a motif homologous to the DE-2 rat element which confers the tissue-specific expression of POMC in AtT-20 cells; however, this motif had no effect in DMS-79. These data suggest that POMC gene transcription is achieved through a different set of transacting factors in DMS-79 and AtT-20. Altogether, our results provide evidence that DMS-79 is a valid model of tumours responsible for the ectopic ACTH syndrome and that the mechanism of POMC gene expression in these SCLC cells is different from that in pituitary cells.
N Hoggard, K Callaghan, A Levy and J R E Davis
Pit-1, a member of the POU family of homeodomain transcription factors, activates prolactin and GH gene expression but also has a role in pituitary cell differentiation and proliferation. Expression of Pit-1 may therefore be of central importance in the function and phenotype of human pituitary adenomas. We have found evidence that, in addition to Pit-1 mRNA, Pit-1-like immunoreactivity and DNA-binding activity are readily detectable in a series of human pituitary adenomas. Gel mobility shift assays using adenoma protein extracts with two Pit-1-binding sites from the human prolactin gene promoter demonstrated the formation of several DNA sequence-specific protein—DNA complexes; some of these could be accounted for by Oct-1-binding activity. Pit-1 activity was anticipated in prolactin- and GH-secreting adenomas, but was also detected in a proportion of endocrine-inactive (non-secreting) adenomas that did not express Pit-1 target genes.
The data demonstrate the presence of Pit-1 in a range of pituitary adenomas. Different adenomas generated slightly differing patterns of DNA-binding activity, though Pit-1 mRNA and protein size appeared normal in all tumours so far examined.
S. Tejura, G. R. Rodgers, M. H. Dunion, M. A. Parsons, J. C. E. Underwood and P. M. Ingleton
The results of this study confirm our previous report of increased androgen receptor expression in livers of female SUAH Wistar rats during development of liver tumours induced by diethylnitrosamine (DENA). In adult female rats not treated with DENA, removal of the ovary increased liver androgen receptor levels but testosterone did not further enhance the androgen receptor status of ovariectomized rats. In normal adult males the testis and/or testosterone maintained high levels of androgen receptors but oestrogen reduced them in castrated rats. Oestrogen receptor levels were not significantly changed in either males or females by gonadectomy. Treatment of female rats with DENA for 10 and 16 weeks increased liver androgen receptors but oestrogen receptors were only reduced by 16 weeks of DENA treatment, whether the rats were intact or ovariectomized. Concentrations of liver androgen receptors were increased in intact and castrated male rats by 10 and 16 weeks of DENA treatment, an increase not seen in the previous experiments. Oestrogen appeared to inhibit both the increases in liver androgen receptor expression and liver tumour development in rats treated with the weakly carcinogenic dose of 10 weeks of DENA. However, the full carcinogenic dose of 16 weeks of DENA increased liver androgen receptors and decreased oestrogen receptors in female rats regardless of sex-steroid status. Development of malignant hepatocellular carcinoma (HCC) was associated with both an increase in liver androgen receptors and a decrease in oestrogen receptors. Maintenance of relatively high levels of liver oestrogen receptors appeared to protect the liver against development of HCC.
M Delhase, P Vergani, A Malur, B Velkeniers, E Teugels, J Trouillas and E L Hooghe-Peters
Adenomas can develop from each cell type of the anterior pituitary. In the normal pituitary, three of these cell types, the GH-, prolactin- and TSH-secreting cells, express the transcription factor Pit-1/GHF-1 which is responsible for prolactin and GH (and probably TSH) cell commitment, differentiation, probably proliferation and gene expression. We have analysed the expression of Pit-1/GHF-1 in a panel of human pituitary adenomas. All GH-, prolactin- and TSH-expressing adenomas studied expressed the Pit-1/GHF-1 factor, as demonstrated by in-situ hybridization and immunocytochemistry. The expression was higher in adenomas than in normal human pituitary. In contrast, ACTH- and LH—FSH-secreting and non-secreting adenomas were negative. Seven transplants of the spontaneous rat prolactinoma SMtTW were also investigated and all were found to be positive.
This further stresses the analogy between these tumours and human prolactinomas. Taken together, the data confirm that Pit-1/GHF-1 expression is restricted to GH-, prolactin- and TSH-expressing cells, and the increased expression in adenomas is compatible with a role of Pit-1/GHF-1 in cell proliferation.
JM Rey, P Pujol, P Callier, V Cavailles, G Freiss, T Maudelonde and JP Brouillet
The increasing number of factors to be taken into account in the oestrogen transcriptional process has created a need to develop a rapid screening method to evaluate their role in physiology and pathology. Molecular biology techniques enable gene expression studies at the mRNA level with small amounts of tissues. We therefore developed a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique using fluorescent oligonucleotides to analyse simultaneously a large panel of interrelated genes involved in the oestrogen transcriptional pathway using a moderately expressed housekeeping gene, the hypoxanthine phosphoribosyltransferase gene (HPRT), as the reference gene. Expression levels of oestrogen receptors (ERalpha, ERbeta), cofactors AIB1, RIP140, SMRT and the Fas-associated protein-tyrosine phophatase-1 (FAP-1) genes were evaluated in breast, endometrial and ovarian cancer cell lines and in three ERalpha-positive and three ERalpha-negative breast cancer tumours. This technique provides a rapid and reliable way to quantitate simultaneously numerous mRNAs of genes involved in the oestrogen pathway from small amounts of tissues.
R. K. Iles and T. Chard
Material with the immunochemical characteristics of human chorionic gonadotrophin (hCG) is produced by bladder tumour cells in vitro and in vivo. In order to characterize this material further, media were collected from 17 cell cultures (three choriocarcinomas, seven bladder carcinomas and seven 'normal' urothelium). The hCG-like material was compared with pregnancy hCG and purified α- and β-subunits by specific radioimmunoassays. Media were also submitted to affinity chromatography and the fractions further analysed by SDS-PAGE and Western blotting. It was shown that both the neoplastic and normal urothelium produced only free β-subunit-like material. This urothelial 'β-hCG' has the same molecular weight and electrophoretic mobility as that present in the intact hCG of pregnancy.
James F H Pittaway and Leonardo Guasti
Adrenocortical carcinoma (ACC) is a rare malignancy with an incidence worldwide of 0.7–2.0 cases/million/year. Initial staging is the most important factor in determining prognosis. If diagnosed early, complete surgical resection +/− adjuvant treatment can lead to 5-year survival of up to 80%. However, often it is diagnosed late and in advanced disease, 5-year survival is <15% with a high recurrence rate even after radical surgery. The mainstay of adjuvant treatment is with the drug mitotane. Mitotane has a specific cytotoxic effect on steroidogenic cells of the adrenal cortex, but despite this, progression through treatment is common. Developments in genetic analysis in the form of next-generation sequencing, aided by bioinformatics, have enabled high-throughput molecular characterisation of these tumours. This, in addition to a better appreciation of the processes of physiological, homeostatic self-renewal of the adrenal cortex, has furthered our understanding of the pathogenesis of this malignancy. In this review, we have detailed the pathobiology and genetic alterations in adrenocortical carcinoma by integrating current understanding of homeostasis and self-renewal in the normal adrenal cortex with molecular profiling of tumours from recent genetic analyses. Improved understanding of the mechanisms involved in self-renewal and stem cell hierarchy in normal human adrenal cortices, together with the identification of cell populations likely to be co-opted by oncogenic mutations, will enable further progress in the definition of the molecular pathways involved in the pathogenesis of ACC. The combination of these advances eventually will lead to the development of novel, effective and personalised strategies to eradicate molecularly annotated ACCs.
Barry V L Potter
Steroid sulphatase is an emerging drug target for the endocrine therapy of hormone-dependent diseases, catalysing oestrogen sulphate hydrolysis to oestrogen. Drug discovery, developing the core aryl O-sulphamate pharmacophore, has led to steroidal and non-steroidal drugs entering numerous clinical trials, with promising results in oncology and women’s health. Steroidal oestrogen sulphamate derivatives were the first irreversible active-site-directed inhibitors and one was developed clinically as an oral oestradiol pro-drug and for endometriosis applications. This review summarizes work leading to the therapeutic concept of sulphatase inhibition, clinical trials executed to date and new insights into the mechanism of inhibition of steroid sulphatase. To date, the non-steroidal sulphatase inhibitor Irosustat has been evaluated clinically in breast cancer, alone and in combination, in endometrial cancer and in prostate cancer. The versatile core pharmacophore both imbues attractive pharmaceutical properties and functions via three distinct mechanisms of action, as a pro-drug, an enzyme active-site-modifying motif, likely through direct sulphamoyl group transfer, and as a structural component augmenting activity, for example by enhancing interactions at the colchicine binding site of tubulin. Preliminary new structural data on the Pseudomonas aeruginosa arylsulphatase enzyme suggest two possible sulphamate-based adducts with the active site formylglycine as candidates for the inhibition end product via sulphamoyl or sulphonylamine transfer, and a speculative choice is suggested. The clinical status of sulphatase inhibition is surveyed and how it might develop in the future. Also discussed are dual-targeting approaches, development of 2-substituted steroidal sulphamates and non-steroidal derivatives as multi-targeting agents for hormone-independent tumours, with other emerging directions.
H. M. Docherty and D. A. Heath
The extensive chromatographic characterization of four parathyroid hormone (PTH)-like proteins in a human bronchial carcinoid tumour associated with humoral hypercalcaemia and severe osteitis fibrosa is described. PTH-like bioactivity was detected in acetic acid extracts of the tumour using an in-vitro osteosarcoma cell bioassay. The active tumour proteins were positively charged at physiological pH and had apparent M r of approximately 29 000, 16 000, 4000–9000 and < 4000. The proteins were immunologically distinct from PTH, but each stimulated PTH-sensitive adenylate cyclase in cultured osteoblastic cells. There was no evidence of PTH gene expression by the tumour. These proteins represent different molecular forms of PTH-related protein.