Osteosarcoma (OS) is a primary malignant tumour of bone occurring predominantly in children and young adults. Despite chemotherapy, relapse is common and mortality remains high. Non-transformed osteoblasts are highly sensitive to glucocorticoids, which reduce proliferation and induce apoptosis. Previously, we observed that OS cells, but not normal osteoblasts, express 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). This enzyme inactivates cortisol (active) to cortisone (inactive) and expression of 11β-HSD2 renders OS cells resistant to glucocorticoids. By contrast, the related enzyme 11β-HSD1 converts cortisone to cortisol and reduces OS cell proliferation in vitro. Some synthetic glucocorticoids (e.g. dehydrodexamethasone (DHD), inactive counterpart of dexamethasone (DEX)) have been reported to be activated by 11β-HSD2. We therefore investigated expression and enzymatic activity of 11β-HSD isozymes in human OS tissue, determined whether 11β-HSD expression has prognostic value in the response to therapy, and evaluated the potential use of synthetic glucocorticoids to selectively target OS cells. OS samples expressed both 11β-HSD1 and 11β-HSD2. 11β-HSD1 expression in pretreatment biopsy specimens positively correlated with primary tumour size. Expression and activity of 11β-HSD1 in post-treatment biopsies were unrelated to the degree of tumour necrosis following chemotherapy. However, high 11β-HSD2 expression in post-treatment biopsies correlated with a poor response to therapy. OS cells that expressed 11β-HSD2 inactivated endogenous glucocorticoids; but these cells were also able to generate DEX from DHD. These results suggest that OS treatment response is related to 11β-HSD2 enzyme expression. Furthermore, OS cells expressing this enzyme could be targeted by treatment with synthetic glucocorticoids that are selectively reactivated by the enzyme.
Pushpa Patel, Rowan Hardy, Vaiyapuri Sumathi, Gillian Bartle, Lars-Gunnar Kindblom, Robert Grimer, Iwona Bujalska, Paul M Stewart, Elizabeth Rabbitt, Neil J L Gittoes, and Mark S Cooper
A Raitila, M Georgitsi, A Karhu, K Tuppurainen, M J Mäkinen, K Birkenkamp-Demtröder, K Salmenkivi, T F Ørntoft, J Arola, V Launonen, P Vahteristo, and L A Aaltonen
Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene were recently observed in patients with pituitary adenoma predisposition (PAP). Though AIP mutation-positive individuals with prolactin-, mixed growth hormone/prolactin-, and ACTH-producing pituitary adenomas as well as non-secreting pituitary adenomas have been reported, most mutation-positive patients have had growth hormone-producing adenomas diagnosed at relatively young age. Pituitary adenomas are also component tumors of some familial endocrine neoplasia syndromes such as multiple endocrine neoplasia type 1 (MEN1) and Carney complex (CNC). Genes underlying MEN1 and CNC are rarely mutated in sporadic pituitary adenomas, but more often in other lesions contributing to these two syndromes. Thus far, the occurrence of somatic AIP mutations has not been studied in endocrine tumors other than pituitary adenomas. Here, we have analyzed 32 pituitary adenomas and 79 other tumors of the endocrine system for somatic AIP mutations by direct sequencing. No somatic mutations were identified. However, two out of nine patients with prolactin-producing adenoma were shown to harbor a Finnish founder mutation (Q14X) with a complete loss of the wild-type allele in the tumors. These results are in agreement with previous studies in that prolactin-producing adenomas are component tumors in PAP. The data also support the previous finding that somatic AIP mutations are not common in pituitary adenomas and suggest that such mutations are rare in other endocrine tumors as well.
K Revill, K J Dudley, R N Clayton, A M McNicol, and W E Farrell
The imprinted gene, neuronatin (NNAT), is one of the most abundant transcripts in the pituitary and is thought to be involved in the development and maturation of this gland. In a recent whole-genome approach, exploiting a pituitary tumour cell line, we identified hypermethylation associated loss of NNAT. In this report, we determined the expression pattern of NNAT in individual cell types of the normal gland and within each of the different pituitary adenoma subtypes. In addition, we determined associations between expression and CpG island methylation and used colony forming efficiency assays (CFE) to gain further insight into the tumour-suppressor function of this gene. Immunohistochemical (IHC) co-localization studies of normal pituitaries showed that each of the hormone secreting cells (GH, PRL, ACTH, FSH and TSH) expressed NNAT. However, 33 out of 47 adenomas comprising, 11 somatotrophinomas, 10 prolactinomas, 12 corticotrophinomas and 14 non-functioning tumours, irrespective of subtype failed to express either NNAT transcript or protein as determined by quantitative real-time RT-PCR and IHC respectively. In normal pituitaries and adenomas that expressed NNAT the promoter-associated CpG island showed characteristics of an imprinted gene where ∼50% of molecules were densely methylated. However, in the majority of adenomas that showed loss or significantly reduced expression of NNAT, relative to normal pituitaries, the gene-associated CpG island showed significantly increased methylation. Induced expression of NNAT in transfected AtT-20 cells significantly reduced CFE. Collectively, these findings point to an important role for NNAT in the pituitary and perhaps tumour development in this gland.
C Schaaf, B Shan, M Buchfelder, M Losa, J Kreutzer, W Rachinger, G K Stalla, T Schilling, E Arzt, M J Perone, and U Renner
Curcumin (diferuloylmethane) is the active ingredient of the spice plant Curcuma longa and has been shown to act anti-tumorigenic in different types of tumours. Therefore, we have studied its effect in pituitary tumour cell lines and adenomas. Proliferation of lactosomatotroph GH3 and somatotroph MtT/S rat pituitary cells as well as of corticotroph AtT20 mouse pituitary cells was inhibited by curcumin in monolayer cell culture and in colony formation assay in soft agar. Fluorescence-activated cell sorting (FACS) analysis demonstrated curcumin-induced cell cycle arrest at G2/M. Analysis of cell cycle proteins by immunoblotting showed reduction in cyclin D1, cyclin-dependent kinase 4 and no change in p27kip. FACS analysis with Annexin V-FITC/7-aminoactinomycin D staining demonstrated curcumin-induced early apoptosis after 3, 6, 12 and 24 h treatment and nearly no necrosis. Induction of DNA fragmentation, reduction of Bcl-2 and enhancement of cleaved caspase-3 further confirmed induction of apoptosis by curcumin. Growth of GH3 tumours in athymic nude mice was suppressed by curcumin in vivo. In endocrine pituitary tumour cell lines, GH, ACTH and prolactin production were inhibited by curcumin. Studies in 25 human pituitary adenoma cell cultures have confirmed the anti-tumorigenic and hormone-suppressive effects of curcumin. Altogether, the results described in this report suggest this natural compound as a good candidate for therapeutic use on pituitary tumours.
Masanori Murakami, Takanobu Yoshimoto, Kazuhiko Nakabayashi, Yujiro Nakano, Takahiro Fukaishi, Kyoichiro Tsuchiya, Isao Minami, Ryotaro Bouchi, Kohji Okamura, Yasuhisa Fujii, Koshi Hashimoto, Ken-ichiro Hata, Kazunori Kihara, and Yoshihiro Ogawa
The pathophysiology of aldosterone-producing adenomas (APAs) has been investigated via genetic approaches and the pathogenic significance of a series of somatic mutations, including KCNJ5, has been uncovered. However, how the mutational status of an APA is associated with its molecular characteristics, including its transcriptome and methylome, has not been fully understood. This study was undertaken to explore the molecular characteristics of APAs, specifically focusing on APAs with KCNJ5 mutations as opposed to those without KCNJ5 mutations, by comparing their transcriptome and methylome status. Cortisol-producing adenomas (CPAs) were used as reference. We conducted transcriptome and methylome analyses of 29 APAs with KCNJ5 mutations, 8 APAs without KCNJ5 mutations and 5 CPAs. Genome-wide gene expression and CpG methylation profiles were obtained from RNA and DNA samples extracted from these 42 adrenal tumors. Cluster analysis of the transcriptome and methylome revealed molecular heterogeneity in APAs depending on their mutational status. DNA hypomethylation and gene expression changes in Wnt signaling and inflammatory response pathways were characteristic of APAs with KCNJ5 mutations. Comparisons between transcriptome data from our APAs and that from normal adrenal cortex obtained from the Gene Expression Omnibus suggested similarities between APAs with KCNJ5 mutations and zona glomerulosa. The present study, which is based on transcriptome and methylome analyses, indicates the molecular heterogeneity of APAs depends on their mutational status. Here, we report the unique characteristics of APAs with KCNJ5 mutations.
V V Vax, M Gueorguiev, I I Dedov, A B Grossman, and M Korbonits
The oncogenes and/or tumour suppressor genes which may be involved in the transformation process for the vast majority of pituitary tumours remain unknown. There is substantial evidence for derangement of cell cycle control in such tumours, but cell cycle protein mutations identified in other human malignancies are restricted to only a very small subset of sporadic pituitary neoplasms. Kr√ºppel-like factors are DNA-binding transcriptional regulators with diverse effects including the upregulation of the cell cycle protein p21(WAF1/CIP1). It has been reported that the Kr√ºppel-like transcription factor 6 (KLF6) gene is mutated in a proportion (15-55%) of human prostate cancers, and more recent data are emerging regarding mutated KLF6 in nasopharyngeal carcinomas, astrocytoid gliomas and colorectal cancer. We therefore speculated that other tumours such as pituitary adenomas might also harbour such mutations that may be involved in the control of cell proliferation in the pituitary. The aim of the current study was thus to analyse the KLF6 gene for mutations in sporadic pituitary tumours. We analysed 60 pituitary adenomas (15 GH-, four ACTH-, two PRL-secreting and 39 non-functioning) with direct sequence analysis of exons 2 and 3 of the KLF6 gene, the region where most of the previously described mutations are located. Three non-functioning pituitary adenomas of the 60 pituitary tumours (5%) had two identical sequence changes in exon 2 (missense mutation Val165Met, 523G-->A and a silent substitution in Ser77Ser codon 261C-->T). Analysis of genomic DNA extracted from peripheral lymphocytes in one patient confirmed these changes to be present in the germline and they therefore probably represent polymorphisms, although we cannot exclude the possibility that these are predisposing germline mutations. We conclude that mutations of the KLF6 gene are unlikely to play an important role in sporadic pituitary tumorigenesis.
D Dworakowska, E Wlodek, C A Leontiou, S Igreja, M Cakir, M Teng, N Prodromou, M I Góth, S Grozinsky-Glasberg, M Gueorguiev, B Kola, M Korbonits, and A B Grossman
Raf/MEK/ERK and phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) cascades are key signalling pathways interacting with each other to regulate cell growth and tumourigenesis. We have previously shown B-Raf and Akt overexpression and/or overactivation in pituitary adenomas. The aim of this study is to assess the expression of their downstream components (MEK1/2, ERK1/2, mTOR, TSC2, p70S6K) and effectors (c-MYC and CYCLIN D1). We studied tissue from 16 non-functioning pituitary adenomas (NFPAs), six GH-omas, six prolactinomas and six ACTH-omas, all collected at transsphenoidal surgery; 16 normal autopsy pituitaries were used as controls. The expression of phospho and total protein was assessed with western immunoblotting, and the mRNA expression with quantitative RT-PCR. The expression of pSer217/221 MEK1/2 and pThr183 ERK1/2 (but not total MEK1/2 or ERK1/2) was significantly higher in all tumour subtypes in comparison to normal pituitaries. There was no difference in the expression of phosphorylated/total mTOR, TSC2 or p70S6K between pituitary adenomas and controls. Neither c-MYC phosphorylation at Ser 62 nor total c-MYC was changed in the tumours. However, c-MYC phosphorylation at Thr58/Ser62 (a response target for Akt) was decreased in all tumour types. CYCLIN D1 expression was higher only in NFPAs. The mRNA expression of MEK1, MEK2, ERK1, ERK2, c-MYC and CCND1 was similar in all groups. Our data indicate that in pituitary adenomas both the Raf/MEK/ERK and PI3K/Akt/mTOR pathways are upregulated in their initial cascade, implicating a pro-proliferative signal derangement upstream to their point of convergence. However, we speculate that other processes, such as senescence, attenuate the changes downstream in these benign tumours.
I Bossis, A Voutetakis, T Bei, F Sandrini, K J Griffin, and C A Stratakis
The type 1 alpha regulatory subunit (R1alpha) of cAMP-dependent protein kinase A (PKA) (PRKAR1A) is an important regulator of the serine-threonine kinase activity catalyzed by the PKA holoenzyme. Carney complex (CNC) describes the association 'of spotty skin pigmentation, myxomas, and endocrine overactivity'; CNC is in essence the latest form of multiple endocrine neoplasia to be described and affects the pituitary, thyroid, adrenal and gonadal glands. Primary pigmented nodular adrenocortical disease (PPNAD), a micronodular form of bilateral adrenal hyperplasia that causes a unique, inherited form of Cushing syndrome, is also the most common endocrine manifestation of CNC. CNC and PPNAD are genetically heterogeneous but one of the responsible genes is PRKAR1A, at least for those families that map to 17q22-24 (the chromosomal region that harbors PRKAR1A). CNC and/or PPNAD are the first human diseases to be caused by mutations in one of the subunits of the PKA holoenzyme. Despite the extensive literature on R1alpha and PKA, little is known about their potential involvement in cell cycle regulation, growth and/or proliferation. The presence of inactivating germline mutations and the loss of its wild-type allele in CNC lesions indicated that PRKAR1A could function as a tumor-suppressor gene in these tissues. However, there are conflicting data in the literature about PRKAR1A's role in human neoplasms, cancer cell lines and animal models. In this report, we review briefly the genetics of CNC and focus on the involvement of PRKAR1A in human tumorigenesis in an effort to reconcile the often diametrically opposite reports on R1alpha.
Sara Jung, Zoltan Nagy, Martin Fassnacht, Gerard Zambetti, Max Weiss, Martin Reincke, Peter Igaz, Felix Beuschlein, and Constanze Hantel
Systemic therapy of adrenocortical carcinoma (ACC) is limited by heterogeneous tumor response and adverse effects. Recently, we demonstrated anti-tumor activity of LEDP-M (etoposide, liposomal doxorubicin, liposomal cisplatin, mitotane), a liposomal variant of EDP-M (etoposide, doxorubicin, cisplatin, mitotane). To improve the therapeutic efficacy and off-target profiles of the clinical gold standard EDP-M, we investigated liposomal EDP-M regimens in different preclinical settings and in a small number of ACC patients with very advanced disease. Short- and long-term experiments were performed on two ACC models (SW-13 and SJ-ACC3) in vivo. We evaluated the anti-tumoral effects and off-target profiles of EDP-M, LEDP-M and a novel regimen L(l)EDP-M including liposomal etoposide. Furthermore, the role of plasma microRNA-210 as a therapeutic biomarker and first clinical data were assessed. Classical and liposomal protocols revealed anti-proliferative efficacy against SW-13 (EDP-M P < 0.01; LEDP-M: P < 0.001; L(l)EDP-M: P < 0.001 vs controls), whereas in SJ-ACC3, only EDP-M (P < 0.05 vs controls) was slightly effective. Long-term experiments in SW-13 demonstrated anti-tumor efficacy for all treatment schemes (EDP-M: P < 0.01, LEDP-M: P < 0.05, L(l)EDP-M P < 0.001 vs controls). The analysis of pre-defined criteria leading to study termination revealed significant differences for control (P < 0.0001) and EDP-M (P = 0.003) compared to L(l)EDP-M treatment. Raising its potential for therapy monitoring, we detected elevated levels of circulating microRNA-210 in SW-13 after LEDP-M treatment (P < 0.05). In contrast, no comparable effects were detectable for SJ-ACC3. However, overall histological evaluation demonstrated improved off-target profiles following liposomal regimens. The first clinical data indicate improved tolerability of liposomal EDP-M, thus confirming our results. In summary, liposomal EDP-M regimens represent promising treatment options to improve clinical treatment of ACC.
Anand Pathak, Douglas R Stewart, Fabio R Faucz, Paraskevi Xekouki, Sara Bass, Aurelie Vogt, Xijun Zhang, Joseph Boland, Meredith Yeager, Jennifer T Loud, Katherine L Nathanson, Katherine A McGlynn, Constantine A Stratakis, Mark H Greene, and Lisa Mirabello
Germline inactivating mutations of isoform 4 of phosphodiesterase (PDE) 11A (coded by the PDE11A gene) have been associated with familial adrenocortical tumors and familial testicular cancer. Testicular tissue is unique in expressing all four isoforms of PDE11A. In a prior candidate gene study of 94 familial testicular germ cell tumor (TGCT) subjects, we identified a significant association between the presence of functionally abnormal variants in PDE11A and familial TGCT risk. To validate this novel observation, we sequenced the PDE11A coding region in 259 additional TGCT patients (both familial and sporadic) and 363 controls. We identified 55 PDE11A variants: 20 missense, four splice-site, two nonsense, seven synonymous, and 22 intronic. Ten missense variants were novel; nine occurred in transcript variant 4 and one in transcript variant 3. Five rare mutations (p.F258Y, p.G291R, p.V820M, p.R545X, and p.K568R) were present only in cases and were significantly more common in cases vs controls (P=0.0037). The latter two novel variants were functionally characterized and shown to be functionally inactivating, resulting in reduced PDE activity and increased cAMP levels. In further analysis of this cohort, we focused on white participants only to minimize confounding due to population stratification. This study builds upon our prior reports implicating PDE11A variants in familial TGCT, provides the first independent validation of those findings, extends that work to sporadic testicular cancer, demonstrates that these variants are uncommonly but reproducibly associated with TGCT, and refines our understanding regarding which specific inactivating PDE11A variants are most likely to be associated with TGCT risk.