The clinical presentation of primary hyperparathyroidism (PHPT) varies widely, although the underlying mechanistic reasons for this disparity remain unknown. We recently reported that parathyroid tumors can be functionally segregated into two distinct groups on the basis of their relative responsiveness to ambient calcium, and that patients in these groups differ significantly in their likelihood of manifesting bone disability. To examine the molecular basis for this phenotypic variation in PHPT, we compared the global gene expression profiles of calcium-sensitive and calcium-resistant parathyroid tumors. RNAseq and proteomic analysis identified a candidate set of differentially expressed genes highly correlated with calcium-sensing capacity. Subsequent quantitative assessment of the expression levels of these genes in an independent cohort of parathyroid tumors confirmed that calcium-sensitive tumors cluster in a discrete transcriptional profile group. These data indicate that PHPT is not an etiologically monolithic disorder and suggest that divergent molecular mechanisms could drive the observed phenotypic differences in PHPT disease course, provenance, and outcome.
James Koh, Joyce A Hogue, Sanziana A Roman, Randall P Scheri, Hèléne Fradin, David L Corcoran, and Julie A Sosa
G R Mundy and T A Guise
Tumors cause multiple effects on the skeleton and on calcium homeostasis, but they do so in specific patterns which are becoming better defined as the mediators responsible become more fully characterized. Metastatic bone disease occurs in the majority of patients with advanced cancer, and is particularly frequent in breast, lung and prostate cancers, which are the most common of all human tumors. Approximately 1,000 000 people die each year in Western Europe and the United States from these three malignancies, and the majority of these people have bone metastases. Bone is the third most common site of metastatic disease in tumors of all types and the second most common in breast and prostate cancer. In this review, the role of the tumor peptide parathyroid hormone-related peptide (PTH-rP) in the effects of cancer on the skeleton will be discussed.
S Humez, M Monet, G Legrand, G Lepage, P Delcourt, and N Prevarskaya
Neuroendocrine differentiation (NED) has been implicated in prostate cancer progression and hormone-therapy failure. Neuroendocrine cells are non-proliferating and escape apoptotic cell death, although their origin and the causes of their apoptotic resistance have as yet been poorly elucidated. This study demonstrates a new mechanism involved in controlling NED. We report that epidermal growth factor (5–50 ng/ml) promotes neuroendocrine-like differentiation of androgen-independent DU145 prostate cancer cells. This differentiation is associated with an increase in the expression of Neuron Specific Enolase (NSE) and a reduction in cell proliferation and is blocked by inhibiting tyrosine kinase activity with genistein and with compound 56 (C56). An increase in the cAMP level, using dibutryl cAMP (db-cAMP) (1 mM) and isobutylmethylxanthine (100 μM), does not promote NED by itself, but does increase the effect of EGF on NED. In addition, EGF-induced NED protects cells from apoptosis induced with thapsigargin (1 μM) by reducing the thapsigargin-induced cytosolic calcium overload. In order to describe how EGF-induced NED protects cells against thapigargin-induced calcium overload we investigated the spatiotemporal calcium signalling linked to apoptosis. By using thapsigargin in various conditions on DU145 cells and using micro-fluorimetric calcium measurements, we show that depletion of intracellular calcium store induces apoptosis and that the amplitude and duration of the capacitive calcium entry are two apoptosis-modulating parameters. We show that protection against thapsigargin-induced apoptosis conferred by NED is achieved by reducing the amount and the speed of calcium that can be released from calcium pools, as well as modulating the amplitude of the subsequent calcium entry.
Jessica Svedlund, Elham Barazeghi, Peter Stålberg, Per Hellman, Göran Åkerström, Peyman Björklund, and Gunnar Westin
Primary hyperparathyroidism (pHPT) resulting from parathyroid tumors is a common endocrine disorder with incompletely understood etiology. In renal failure, secondary hyperparathyroidism (sHPT) occurs with multiple tumor development as a result of calcium and vitamin D regulatory disturbance. The aim of this study was to investigate a potential role of the histone 3 lysine 27 methyltransferase EZH2 in parathyroid tumorigenesis. Parathyroid tumors from patients with pHPT included adenomas and carcinomas. Hyperplastic parathyroid glands from patients with HPT secondary to uremia and normal parathyroid tissue specimens were included in this study. Quantitative RT-PCR, western blotting, bisulfite pyrosequencing, colony formation assay, and RNA interference were used. EZH2 was overexpressed in a subset of the benign and in all malignant parathyroid tumors as determined by quantitative RT-PCR and western blotting analyses. Overexpression was explained by EZH2 gene amplification in a large fraction of tumors. EZH2 depletion by RNA interference inhibited sHPT-1 parathyroid cell line proliferation as determined by tritium–thymidine incorporation and colony formation assays. EZH2 depletion also interfered with the Wnt/β-catenin signaling pathway by increased expression of growth-suppressive AXIN2, a negative regulator of β-catenin stability. Indeed, EZH2 contributed to the total level of aberrantly accumulated transcriptionally active (nonphosphoylated) β-catenin in the parathyroid tumor cells. To our knowledge EZH2 gene amplification presents the first genetic aberration common to parathyroid adenomas, secondary hyperplastic parathyroid glands, and parathyroid carcinomas. This supports the possibility of a common pathway in parathyroid tumor development.
Luqman Sulaiman, Inga-Lena Nilsson, C Christofer Juhlin, Felix Haglund, Anders Höög, Catharina Larsson, and Jamileh Hashemi
In this study, we genetically characterized parathyroid adenomas with large glandular weights, for which independent observations suggest pronounced clinical manifestations. Large parathyroid adenomas (LPTAs) were defined as the 5% largest sporadic parathyroid adenomas identified among the 590 cases operated in our institution during 2005–2009. The LPTA group showed a higher relative number of male cases and significantly higher levels of total plasma and ionized serum calcium (P<0.001). Further analysis of 21 LPTAs revealed low MIB1 proliferation index (0.1–1.5%), MEN1 mutations in five cases, and one HRPT2 (CDC73) mutation. Total or partial loss of parafibromin expression was observed in ten tumors, two of which also showed loss of APC expression. Using array CGH, we demonstrated recurrent copy number alterations most frequently involving loss in 1p (29%), gain in 5 (38%), and loss in 11q (33%). Totally, 21 minimal overlapping regions were defined for losses in 1p, 7q, 9p, 11, and 15q and gains in 3q, 5, 7p, 8p, 16q, 17p, and 19q. In addition, 12 tumors showed gross alterations of entire or almost entire chromosomes most frequently gain of 5 and loss of chromosome 11. While gain of 5 was the most frequent alteration observed in LPTAs, it was only detected in a small proportion (4/58 cases, 7%) of parathyroid adenomas. A significant positive correlation was observed between parathyroid hormone level and total copy number gain (r=0.48, P=0.031). These results support that LPTAs represent a group of patients with pronounced parathyroid hyperfunction and associated with specific genomic features.
G A Clines and T A Guise
Calcium homeostasis is a tightly regulated process involving the co-ordinated efforts of the skeleton, kidney, parathyroid glands and intestine. Neoplasms can alter this homeostasis indirectly through the production of endocrine factors resulting in humoral hypercalcaemia of malignancy. Relatively common with breast and lung cancer, this paraneoplastic condition is most often due to tumour production of parathyroid hormone-related protein and ensuing increased osteoclastic bone resorption. Although control of hypercalcaemia is generally successful, the development of this complication is associated with a poor prognosis. The metastasis of tumour cells to bone represents another skeletal complication of malignancy. As explained in the ‘seed and soil’ hypothesis, bone represents a fertile ground for cancer cells to flourish. The molecular mechanisms of this mutually beneficial relationship between bone and cancer cells are beginning to be understood. In the case of osteolytic bone disease, tumour-produced parathyroid hormone-related protein stimulates osteoclasts that in turn secrete tumour-activating transforming growth factor-β that further stimulates local cancer cells. This ‘vicious cycle’ of bone metastases represents reciprocal bone/cancer cellular signals that likely modulate osteoblastic bone metastatic lesions as well. The development of targeted therapies to either block initial cancer cell chemotaxis, invasion and adhesion or to break the ‘vicious cycle’ is dependent on a more complete understanding of bone metastases. Although bisphosphonates delay progression of skeletal metastases, it is clear that more effective therapies are needed. Cancer-associated bone morbidity remains a major public health problem, and to improve therapy and prevention it is important to understand the pathophysiology of the effects of cancer on bone. This review will detail scientific advances regarding this area.
C Verdelli, L Avagliano, P Creo, V Guarnieri, A Scillitani, L Vicentini, G B Steffano, E Beretta, L Soldati, E Costa, A Spada, G P Bulfamante, and S Corbetta
Components of the tumour microenvironment initiate and promote cancer development. In this study, we investigated the stromal component of parathyroid neoplasia. Immunohistochemistry for alpha-smooth muscle actin (α-SMA) showed an abundant periacinar distribution of α-SMA+ cells in normal parathyroid glands (n=3). This pattern was progressively lost in parathyroid adenomas (PAds; n=6) where α-SMA+cells were found to surround new microvessels, as observed in foetal parathyroid glands (n=2). Moreover, in atypical adenomas (n=5) and carcinomas (n=4), α-SMA+ cells disappeared from the parenchyma and accumulated in the capsula and fibrous bands. At variance with normal glands, parathyroid tumours (n=37) expressed high levels of fibroblast-activation protein (FAP) transcripts, a marker of tumour-associated fibroblasts. We analysed the ability of PAd-derived cells to activate fibroblasts using human bone-marrow mesenchymal stem cells (hBM-MSCs). PAd-derived cells induced a significant increase in FAP and vascular endothelial growth factor A (VEGFA) mRNA levels in co-cultured hBM-MSCs. Furthermore, the role of the calcium-sensing receptor (CASR) and of the CXCL12/CXCR4 pathway in the PAd-induced activation of hBM-MSCs was investigated. Treatment of co-cultures of hBM-MSCs and PAd-derived cells with the CXCR4 inhibitor AMD3100 reduced the stimulated VEGFA levels, while CASR activation by the R568 agonist was ineffective. PAd-derived cells co-expressing parathyroid hormone (PTH)/CXCR4 and PTH/CXCL12 were identified by FACS, suggesting a paracrine/autocrine signalling. Finally, CXCR4 blockade by AMD3100 reduced PTH gene expression levels in PAd-derived cells. In conclusion, i) PAd-derived cells activated cells of mesenchymal origin; ii) PAd-associated fibroblasts were involved in tumuor neoangiogenesis and iii) CXCL12/CXCR4 pathway was expressed and active in PAd cells, likely contributing to parathyroid tumour neoangiogenesis and PTH synthesis modulation.
S Corbetta, L Vicentini, S Ferrero, A Lania, G Mantovani, D Cordella, P Beck-Peccoz, and A Spada
Previous studies indicate that nuclear factor kappaB (NF-κB) transcription factor is deregulated and overexpressed in several human neoplasias. The aim of this study was to test the hypothesis that the NF-κB pathway may be involved in parathyroid tumorigenesis. For this purpose, we determined the level of NF-κB activity, evaluated as phosphorylation of the transcription subunit p65, its modulation by specific and non-specific agents and its impact on cyclin D1 expression. Phosphorylated p65 levels present in parathyroid neoplasias (n = 13) were significantly lower than those found in normal tissues (n = 3; mean optical density (OD) 0.19 ± 0.1 vs 0.4 ± 0.1, P = 0.007), but there was no significant difference between adenomas and secondary and multiple endocrine neoplasia type 1 (MEN1)-related hyperplasia. Conversely, MEN2A (Cys634Arg)-related parathyroid samples showed extremely high levels of phosphorylated p65 that exhibited a nuclear localization at immunohistochemistry (n = 3). Phosphorylated p65 levels negatively correlated with menin expression (r 2 = 0.42, P = 0.05). Tumor necrosis factor-α (TNFα) caused a significant increase in phosphorylated p65 levels (183 ± 13.8% of basal) while calcium sensing receptor (CaR) agonists exerted a significant inhibition (19.2 ± 3.3% of basal). Although TNFα was poorly effective in increasing cyclin D1 expression, NF-κB blockade by the specific inhibitor BAY11-7082 reduced FCS-stimulated cyclin D1 by about 60%. Finally, the inhibitory effects of CaR and BAY11-7082 on cyclin D1 expression were not additive – by blocking NF-κB CaR activation did not induce a further reduction in cyclin D1 levels. In conclusion, the study demonstrated that in parathyroid tumors: (1) p65 phosphorylation was dramatically increased by RET constitutive activation and was negatively correlated with menin expression, (2) p65 phosphorylation was increased and reduced by TNFα and CaR agonists respectively, and (3) blockade of the NF-κB pathway caused a significant decrease in cyclin D1 expression.
Inga Mertens-Walker, Christine Bolitho, Robert C Baxter, and Deborah J Marsh
The gonadotropin hypothesis proposes that elevated serum gonadotropin levels may increase the risk of epithelial ovarian cancer (EOC). We have studied the effect of treating EOC cell lines (OV207 and OVCAR-3) with FSH or LH. Both gonadotropins activated the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and increased cell migration that was inhibited by the MAPK 1 inhibitor PD98059. Both extra- and intracellular calcium ion signalling were implicated in gonadotropin-induced ERK1/2 activation as treatment with either the calcium chelator EGTA or an inhibitor of intracellular calcium release, dantrolene, inhibited gonadotropin-induced ERK1/2 activation. Verapamil was also inhibitory, indicating that gonadotropins activate calcium influx via L-type voltage-dependent calcium channels. The cAMP/protein kinase A (PKA) pathway was not involved in the mediation of gonadotropin action in these cells as gonadotropins did not increase intracellular cAMP formation and inhibition of PKA did not affect gonadotropin-induced phosphorylation of ERK1/2. Activation of ERK1/2 was inhibited by the protein kinase C (PKC) inhibitor GF 109203X as well as by the PKCδ inhibitor rottlerin, and downregulation of PKCδ was inhibited by small interfering RNA (siRNA), highlighting the importance of PKCδ in the gonadotropin signalling cascade. Furthermore, in addition to inhibition by PD98059, gonadotropin-induced ovarian cancer cell migration was also inhibited by verapamil, GF 109203X and rottlerin. Similarly, gonadotropin-induced proliferation was inhibited by PD98059, verapamil, GF 109203X and PKCδ siRNA. Taken together, these results demonstrate that gonadotropins induce both ovarian cancer cell migration and proliferation by activation of ERK1/2 signalling in a calcium- and PKCδ-dependent manner.
A G Mackay, E A Ofori-Kuragu, A Lansdown, R C Coombes, L Binderup, and K W Colston
The anti-tumour effect of EB 1089, a novel vitamin D analogue with reduced calcaemic activity, was examined in vivo using the N-methyl-nitrosourea-induced rat mammary tumour model. The vitamin D compound was given orally at a dose of 1 pg/kg body weight alone and in combination with tamoxifen (1 mg/kg). Effects were compared with oral tamoxifen treatment alone. EB 1089 significantly inhibited tumour progression compared with controls with a response rate of 58% and a regression rate of 92% As expected, tamoxifen at the dose given also caused significant inhibition of tumour progression with a response rate of 73%. Combination of these two compounds did not lead to a marked increase in their effectiveness. Histological examination of tumours from EB 1089-treated rats showed a marked reduction in cellularity and mitotic activity.
At the dose given, EB 1089 produced a significant rise in serum calcium concentration and urinary calcium excretion. Tamoxifen treatment alone did not significantly alter serum calcium levels. However, combined treatment with tamoxifen and EB 1089 led to a significant reduction in hypercalcaemia compared with EB 1089 alone. It is suggested that vitamin D analogues with reduced calcaemic activity may provide a new therapeutic strategy for certain malignancies, either alone or in combination with established treatment regimens.
Endocrine-Related Cancer (1996) 3 327-335