Calcitriol (1,25-dihydroxyvitamin D3), the hormonally active metabolite of vitamin D, exerts its anti-proliferative activity in breast cancer (BCa) cells by multiple mechanisms including the downregulation of the expression of estrogen receptor α (ER). We analyzed an ∼3.5 kb ER promoter sequence and demonstrated the presence of two potential negative vitamin D response elements (nVDREs), a newly identified putative nVDRE upstream at −2488 to −2473 bp (distal nVDRE) and a previously published sequence (proximal nVDRE) at −94 to −70 bp proximal to the P1 start site. Transactivation analysis using ER promoter deletion constructs and heterologous promoter–reporter constructs revealed that both nVDREs functioned to mediate calcitriol transrepression. In the electrophoretic mobility shift assay, the vitamin D receptor (VDR) showed strong binding to both nVDREs in the presence of calcitriol, and the chromatin immunoprecipitation assay demonstrated the recruitment of the VDR to the distal nVDRE site. Mutations in the 5′ hexameric DNA sequence of the distal nVDRE resulted in the loss of calcitriol-mediated transrepression and the inhibition of protein–DNA complex formation, demonstrating the importance of these nucleotides in VDR DNA binding and transrepression. A putative nuclear factor-Y (NFY) binding site, identified within the distal nVDRE, led to the findings that NFY bound to the distal nVDRE site interfered with the binding of the VDR at the site and reduced calcitriol-mediated transrepression. In conclusion, the ER promoter region contains two negative VDREs that act in concert to bind to the VDR and both nVDREs are required for the maximal inhibition of ER expression by calcitriol. The suppression of ER expression and estrogen-mediated signaling by calcitriol in BCa cells suggests that vitamin D may be useful in the treatment of ER+ BCa.
Srilatha Swami, Aruna V Krishnan, Lihong Peng, Johan Lundqvist, and David Feldman
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.
María Jesús Larriba, Noelia Valle, Héctor G Pálmer, Paloma Ordóñez-Morán, Silvia Álvarez-Díaz, Karl-Friedrich Becker, Carlos Gamallo, Antonio García de Herreros, José Manuel González-Sancho, and Alberto Muñoz
The Wnt/β-catenin signalling pathway is activated in 90% of human colon cancers by nuclear accumulation of β-catenin protein due to its own mutation or to that of adenomatous polyposis coli. In the nucleus, β-catenin regulates gene expression promoting cell proliferation, migration and invasiveness. 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits β-catenin signalling by inducing its binding to vitamin D receptor (VDR) and by promoting β-catenin nuclear export. The transcription factor Snail1 represses VDR expression and we demonstrate here that Snail1 also abolishes the nuclear export of β-catenin induced by 1,25(OH)2D3 in SW480-ADH cells. Accordingly, Snail1 relieves the inhibition exerted by 1,25(OH)2D3 on genes whose expression is driven by β-catenin, such as c-MYC, ectodermal-neural cortex-1 (ENC-1) or ephrin receptor B2 (EPHB2). In addition, Snail1 abrogates the inhibitory effect of 1,25(OH)2D3 on cell proliferation and migration. In xenografted mice, Snail1 impedes the nuclear export of β-catenin and the inhibition of ENC-1 expression induced by EB1089, a 1,25(OH)2D3 analogue. The elevation of endogenous SNAIL1 protein levels reproduces the effect of an ectopic Snail1 gene. Remarkably, the expression of exogenous VDR in cells with high levels of Snail1 normalizes the transcriptional responses to 1,25(OH)2D3. However, this exogenous VDR failed to fully restore the blockage of the Wnt/β-catenin pathway by 1,25(OH)2D3. This suggests that the effects of Snail1 on this pathway are not merely due to the repression of VDR gene. We conclude that Snail1 is a positive regulator of the Wnt/β-catenin signalling pathway in part through the abrogation of the inhibitory action of 1,25(OH)2D3.
Marc Sinotte, François Rousseau, Pierre Ayotte, Eric Dewailly, Caroline Diorio, Yves Giguère, Sylvie Bérubé, and Jacques Brisson
Vitamin D has been associated with reduced breast cancer risk. We studied the association of two vitamin D receptor (VDR) gene single nucleotide polymorphisms restriction enzyme detecting SNP of VDR (FokI and BsmI) with breast cancer risk in two independent case–control studies carried out in the same population. The modifying effect of family history of breast cancer on this relationship was also evaluated. The first and second studies included respectively 718 (255 cases/463 controls) and 1596 (622 cases/974 controls) women recruited in Quebec City, Canada. FokI and BsmI genotypes were assessed. Relative risks of breast cancer were estimated by multivariate logistic regression. Compared with homozygotes for the common F allele (FF genotype), FokI ff homozygotes had a higher breast cancer risk (study 1: odds ratio (OR)=1.22, 95% confidence interval (CI)=0.76–1.95; study 2: OR=1.44, 95% CI=1.05–1.99; and combined studies: OR=1.33, 95% CI=1.03–1.73). Significant interactions were observed between FokI and family history of breast cancer in the two studies as well as in the combined analysis (P interaction=0.031, 0.050 and 0.0059 respectively). Among women without family history, odds ratios were 1.00, 1.27 (95% CI=1.02–1.58) and 1.57 (95% CI=1.18–2.10) respectively for FF, Ff and ff carriers (P trend=0.0013). BsmI Bb+bb genotypes were associated with a weak non-significant increased risk in the two studies (combined OR=1.22, 95% CI=0.95–1.57) without interaction with family history. Results support the idea that vitamin D, through its signalling pathway, can affect breast cancer risk. They also suggest that variability in observed associations between VDR FokI and breast cancer from different studies may partly be explained by the proportion of study subjects with a family history of breast cancer.
Alyson Murray, Stephen F Madden, Naoise C Synnott, Rut Klinger, Darran O'Connor, Norma O'Donovan, William Gallagher, John Crown, and Michael J Duffy
Considerable epidemiological evidence suggests that high levels of circulating vitamin D (VD) are associated with a decreased incidence and increased survival from cancer, i.e., VD may possess anti-cancer properties. The aim of this investigation was therefore to investigate the anti-cancer potential of a low calcaemic vitamin D analogue, i.e., inecalcitol and compare it with the active form of vitamin D, i.e., calcitriol, in a panel of breast cancer cell lines (n = 15). Using the MTT assay, IC50 concentrations for response to calcitriol varied from 0.12 µM to >20 µM, whereas those for inecalcitol were significantly lower, ranging from 2.5 nM to 63 nM (P = 0.001). Sensitivity to calcitriol and inecalcitol was higher in VD receptor (VDR)-positive compared to VDR-negative cell lines (P = 0.0007 and 0.0080, respectively) and in ER-positive compared to ER-negative cell lines (P = 0.043 and 0.005, respectively). Using RNA-seq analysis, substantial but not complete overlap was found between genes differentially regulated by calcitriol and inecalcitol. In particular, significantly enriched gene ontology terms such as cell surface signalling and cell communication were found after treatment with inecalcitol but not with calcitriol. In contrast, ossification and bone morphogenesis were found significantly enriched after treatment with calcitriol but not with inecalcitol. Our preclinical results suggest that calcitriol and inecalcitol can inhibit breast cancer cell line growth, especially in cells expressing ER and VDR. As inecalcitol is significantly more potent than calcitriol and has low calcaemic potential, it should be further investigated for the treatment of breast cancer.
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.
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.