Ursula Thiem

Role of Calcium, Vitamin D, and the Extrarenal Vitamin D Hydroxylases in Carcinogenesis

Vitamin D deficiency and low calcium intake are considered risk factors for several cancers. Vitamin D, synthesized in the skin or ingested through the diet, is transformed through two hydroxylation steps to the active metabolite, 1α,25-dihydroxyvitamin D3 (1,25-D3). 25-hydroxylases in the liver are responsible for the first hydroxylation step. The ultimate activation is performed by the renal 25-hydroxyvitamin D 1α-hydroxylase (CYP27B1), while the 1,25-dihydroxyvitamin D 24-hydroxylase (CYP24A1) in the kidneys degrades the active metabolite. These two renal vitamin D hydroxylases control the endocrine serum 1,25-D3 levels, and are responsible for maintaining mineral homeostasis. In addition, the active vitamin D hormone 1,25-D3 regulates cellular proliferation, differentiation, and apoptosis in multiple tissues in a paracrine/autocrine manner. Interestingly, it is the low serum level of the precursor 25- hydroxyvitamin D3 (25-D3) that predisposes to numerous cancers and other chronic diseases, and not the serum concentration of the active vitamin D hormone. The extra-renal autocrine/paracrine vitamin D system is able to synthesize and degrade locally the active 1,25- D3 necessary to maintain normal cell growth and to counteract mitogenic stimuli. Thus, vitamin D hydroxylases play a prominent role in this process. The present review describes the role of the vitamin D hydroxylases in cancer pathogenesis and the cross-talk between the extra-renal autocrine/paracrine vitamin D system and calcium in cancer prevention.

Increased copy-number and not DNA hypomethylation causes overexpression of the candidate proto-oncogene CYP24A1 in colorectal cancer

In colorectal cancer (CRC) the vitamin D catabolizing enzyme 1,25‐dihydroxyvitamin D 24‐hydroxylase (CYP24A1) is overexpressed with a potentially significant, positive impact on the catabolism of 1,25‐dihydroxyvitamin D3 (1,25‐D3). However, the underlying mechanism of CYP24A1 overexpression is poorly understood. In the present study, we investigated possible causes including hypomethylation of the CYP24A1 promoter, amplification of the CYP24A1 gene locus (20q13.2), and altered expression of CYP24A1‐specific transcription factors. We quantified CYP24A1 gene copy‐number, performed bisulfite sequencing of the CYP24A1 promoter to assess DNA methylation, and measured mRNA expression of CYP24A1, 25‐hydroxyvitamin D 1α‐hydroxylase (CYP27B1), vitamin D receptor (VDR) and retinoid X receptor (RXR). We found that 77 (60%) out of 127 colorectal tumors showed increased CYP24A1 gene copy‐number and that more than 6 copies of CYP24A1 correlated positively with CYP24A1 mRNA expression suggestive of a causal relationship. No differences in CYP24A1 promoter methylation were found between tumor tissue and adjacent mucosa from the same patient or between tissues with high or low mRNA expression, thus excluding DNA hypomethylation as a possible cause of CYP24A1 overexpression in CRC. Furthermore, mRNA expression of several factors involved in replication licensing positively correlated with CYP24A1 mRNA expression, raising the possibility that CYP24A1 overexpression might favor increased proliferation in tumors by suppressing local 1,25‐D3 levels. We conclude that high copy‐number gain is a key determinant of CYP24A1 overexpression in CRC. Other postulated causes of CYP24A1 overexpression including promoter hypomethylation and enhanced VDR and/or RXR expression do not appear to be involved.

VITA-D: cholecalciferol substitution in vitamin D deficient kidney transplant recipients: a randomized, placebo-controlled study to evaluate the post-transplant outcome.

BackgroundVitamin D does not only regulate calcium homeostasis but also plays an important role as an immune modulator. It influences the immune system through the induction of immune shifts and regulatory cells resulting in immunologic tolerance. As such, vitamin D is thought to exert beneficial effects within the transplant setting, especially in kidney transplant recipients, considering the high prevalence of vitamin D deficiency in kidney transplant recipients.Methods/DesignThe VITA-D study, a randomized, placebo-controlled, double-blind study with two parallel groups including a total of 200 kidney transplant recipients, is designed to investigate the immunomodulatory and renoprotective effects of cholecalciferol (vitamin D3) within the transplant setting. Kidney transplant recipients found to have vitamin D deficiency defined as 25-hydroxyvitamin D3 < 50 nmol per liter will be randomly assigned to receive either oral cholecalciferol therapy or placebo and will be followed for one year. Cholecalciferol will be administered at a dose of 6800 International Units daily over a time period of one year.The objective is to evaluate the influence of vitamin D3 substitution in vitamin D deficient kidney transplant recipients on the post-transplant outcome. As a primary endpoint glomerular filtration rate calculated with the MDRD formula (modification of diet in renal disease) one year after kidney transplantation will be evaluated. Incidence of acute rejection episodes, and the number and severity of infections (analyzed by means of C-reactive protein) within the first year after transplantation will be monitored as well. As a secondary endpoint the influence of vitamin D3 on bone mineral density within the first year post-transplant will be assessed. Three DXA analyses will be performed, one within the first four weeks post-transplant, one five months and one twelve months after kidney transplantation.Trial RegistrationClinicalTrials.gov NCT00752401

Prevention of preneoplastic lesions by dietary vitamin D in a mouse model of colorectal carcinogenesis

Highlights ► High dietary vitamin D was able to prevent premalignant lesions caused by AOM/DSS. ► Increasing vitamin D intake raised serum 25-D3 levels reaching a plateau ≥1000 IU/kg. ► Serum 25-D3 levels over 30 ng/ml are needed to prevent tumorigenesis.

Impact of CYP24A1 overexpression on growth of colorectal tumour xenografts in mice fed with vitamin D and soy

Our previous studies showed that the 1,25‐dihydroxyvitamin D (1,25‐D3) catabolizing enzyme, 1,25‐dihydoxyvitamin D 24 hydroxylase (CYP24A1) was overexpressed in colorectal tumours and its level correlated with increased proliferation. We hypothesised that cells overexpressing CYP24A1 have growth advantage and a diet rich in vitamin D and soy would restore sensitivity to the anti‐tumourigenic effects of vitamin D. Soy contains genistein, a natural CYP24A1 inhibitor. To determine causality between CYP24A1 and tumour growth, we established xenografts in male SCID mice with HT29 cells stably overexpressing either GFP‐tagged CYP24A1 or GFP. Mice were fed with either high (2500 IU D3/kg) or low vitamin D (100 IU D3/kg) diet in the presence or absence of soy (20% diet). In vitro, cells overexpressing CYP24A1 grew faster than controls. 1,25‐D3, the active vitamin D metabolite, reduced cell number only in the presence of the CYP24A1 inhibitor VID400. Regardless of the amount of vitamin D in the diet, xenografts overexpressing CYP24A1 grew faster, were heavier and more aggressive. Soy reduced tumour volume only in the control xenografts, while the tumours overexpressing CYP24A1 were larger in the presence of dietary soy. In conclusion, we demonstrate that CYP24A1 overexpression results in increased aggressiveness and proliferative potential of colorectal tumours. Irrespective of the dietary vitamin D3, dietary soy is able to increase tumour volume when tumours overexpress CYP24A1, suggesting that combination of vitamin D3 and soy could have an anti‐tumourigenic effect only if CYP24A1 levels are normal.

Overexpression of a functional calcium-sensing receptor dramatically increases osteolytic potential of MDA-MB-231 cells in a mouse model of bone metastasis through epiregulin-mediated osteoprotegerin downregulation

Introduction and Aims Osteolytic bone metastases are observed in advanced cases of breast cancer. In vitro data suggest that the activity of the calcium-sensing receptor (CaSR) expressed by metastatic cells could potentiate their osteolytic potential. This study aimed to demonstrate in vivo the involvement of the CaSR in breast cancer cells osteolytic potential and to identify potential targets linked to CaSR activity. Methods and Results MDA-MB-231 stably transfected with plasmids containing either a full-length wild-type CaSR (CaSR-WT), or a functionally inactive dominant negative mutant (CaSR-DN) or an empty vector (EV) were intratibially injected into Balb/c-Nude mice. X-ray analysis performed 19 days after injection showed a dramatic increase of osteolytic lesions in mice injected with CaSR-WT-transfected cells as compared to mice injected with EV- or CaSR-DN-transfected cells. This was associated with decreased BV/TV ratio and increased tumor burden. Epiregulin, an EGF-like ligand, was identified by a DNA microarray as a possible candidate involved in CaSR-mediated osteolysis. Indeed, in vitro, CaSR overexpression increased both epiregulin expression and secretion as compared to EV- or CaSR-DN-transfected cells. Increased epiregulin expression was also detected in osteolytic bone lesions from mice injected with CaSR-WT-transfected MDA-MB-231. In vitro, exposure of osteoblastic cells (HOB and SaOS2) to exogenous epiregulin significantly decreased OPG mRNA expression. Exposure of osteoblastic cells to conditioned media prepared from CaSR-WT-transfected cells also decreased OPG expression. This effect was partially blocked after addition of an anti-epiregulin antibody. Conclusions Overexpression of a functional CaSR in metastatic breast cancer cells dramatically amplifies their osteolytic potential through epiregulin-mediated OPG downregulation.INTRODUCTION AND AIMS Osteolytic bone metastases are observed in advanced cases of breast cancer. In vitro data suggest that the activity of the calcium-sensing receptor (CaSR) expressed by metastatic cells could potentiate their osteolytic potential. This study aimed to demonstrate in vivo the involvement of the CaSR in breast cancer cells osteolytic potential and to identify potential targets linked to CaSR activity. METHODS AND RESULTS MDA-MB-231 stably transfected with plasmids containing either a full-length wild-type CaSR (CaSR-WT), or a functionally inactive dominant negative mutant (CaSR-DN) or an empty vector (EV) were intratibially injected into Balb/c-Nude mice. X-ray analysis performed 19 days after injection showed a dramatic increase of osteolytic lesions in mice injected with CaSR-WT-transfected cells as compared to mice injected with EV- or CaSR-DN-transfected cells. This was associated with decreased BV/TV ratio and increased tumor burden. Epiregulin, an EGF-like ligand, was identified by a DNA microarray as a possible candidate involved in CaSR-mediated osteolysis. Indeed, in vitro, CaSR overexpression increased both epiregulin expression and secretion as compared to EV- or CaSR-DN-transfected cells. Increased epiregulin expression was also detected in osteolytic bone lesions from mice injected with CaSR-WT-transfected MDA-MB-231. In vitro, exposure of osteoblastic cells (HOB and SaOS2) to exogenous epiregulin significantly decreased OPG mRNA expression. Exposure of osteoblastic cells to conditioned media prepared from CaSR-WT-transfected cells also decreased OPG expression. This effect was partially blocked after addition of an anti-epiregulin antibody. CONCLUSIONS Overexpression of a functional CaSR in metastatic breast cancer cells dramatically amplifies their osteolytic potential through epiregulin-mediated OPG downregulation.

Long-term clinical practice experience with cinacalcet for treatment of hypercalcemic hyperparathyroidism after kidney transplantation.

Within this prospective, open-label, self-controlled study, we evaluated the long-term effects of the calcimimetic cinacalcet on calcium and phosphate homeostasis in 44 kidney transplant recipients (KTRs) with hypercalcemic hyperparathyroidism by comparing biochemical parameters of mineral metabolism between pre- and posttreatment periods. Results are described as mean differences (95% CIs) between pre- and posttreatment medians that summarize all repeated measurements of a parameter of interest between the date of initial hypercalcemia and cinacalcet initiation (median of 1.6 (IQR: 0.6–3.8) years) and up to four years after treatment start, respectively. Cinacalcet was initiated after 1.8 (0.8–4.7) years posttransplant and maintained for 6.2 (3.9–7.6) years. It significantly decreased total serum calcium (−0.30 (−0.34 to −0.26) mmol/L, P < 0.001) and parathyroid hormone levels (−79 (−103 to −55) pg/mL, P < 0.001). Serum levels of inorganic phosphate (Pi) and renal tubular reabsorption of phosphate to glomerular filtration rate (TmP/GFR) increased simultaneously (Pi: 0.19 (0.15–0.23) mmol/L, P < 0.001, TmP/GFR: 0.20 (0.16–0.23) mmol/L, P < 0.001). In summary, cinacalcet effectively controlled hypercalcemic hyperparathyroidism in KTRs in the long-term and increased low Pi levels without causing hyperphosphatemia, pointing towards a novel indication for the use of cinacalcet in KTRs.

Low-dose calcium versus pentagastrin for stimulation of calcitonin in chronic hemodialysis patients: a pilot study.

CONTEXT Elevated calcitonin levels occur in up to 46% of patients with chronic hemodialysis (CHD) and frequently reflect benign C-cell hyperplasia rather than medullary thyroid carcinoma. For the differential diagnosis of hypercalcitoninemia, the pentagastrin-stimulated calcitonin test was used until its availability became restricted. OBJECTIVE This study sought to compare calcium and pentagastrin in terms of their ability to stimulate calcitonin secretion and their side effects in patients with CHD. SETTING AND DESIGN This prospective pilot study was conducted at the chronic hemodialysis unit of the Medical University of Vienna between December 2012 and September 2013. PATIENTS We studied six male patients with CHD with elevated basal calcitonin levels. INTERVENTION The stimulation test was performed first with 0.5 μg/kg pentagastrin and then with 1 mg/kg calcium after a median washout period of 7 (6-9) months. MAIN OUTCOME MEASURES We measured calcitonin, serum ionized calcium, intact PTH (iPTH), and C-terminal fibroblast growth factor 23 levels before and 2, 5, and 10 minutes after iv infusion of the stimulant and assessed the tolerability of the two substances by a questionnaire. RESULTS Both pentagastrin and calcium significantly stimulated calcitonin secretion at 2 and 5 minutes. Partial correlation analysis revealed a strong association between calcium- and pentagastrin-stimulated calcitonin levels (r=0.875, P < .0001). Only after calcium infusion serum ionized calcium levels increased from 1.09 (0.91-1.16) mmol/l to 1.4 (1.14-1.65) mmol/l at 2 minutes (P < .01) but returned to baseline levels at 5 minutes. Moreover, calcium infusion led to a significant decrease in iPTH levels from 315 (203-723) pg/ml to 182 (121-415) pg/ml at 5 minutes (P < .05) and 171 (91-346) pg/ml at 10 minutes (P < .001). In general, calcium caused fewer and less severe side effects than pentagastrin. CONCLUSIONS In patients with CHD, the response of calcitonin to calcium and pentagastrin was comparable, making calcium a potential substitute for pentagastrin in these patients.

Calcidiol deficiency in end-stage organ failure and after solid organ transplantation: status quo.

Among patients with organ failure, vitamin D deficiency is extremely common and frequently does not resolve after transplantation. This review crystallizes and summarizes existing data on the status quo of vitamin D deficiency in patients with organ failure and in solid organ transplant recipients. Interventional studies evaluating different treatment strategies, as well as current clinical practice guidelines and recommendations on the management of low vitamin D status in these patients are also discussed.

Vitamin D in solid organ transplantation with special emphasis on kidney transplantation.

Within the past decades, vitamin D was identified as having additional physiological functions far beyond calcium homeostasis and bone metabolism. Stimulated by the discovery of the vitamin D receptor in a broad range of tissues as well as the expression of 1α-hydroxylase, the enzyme responsible for the activation of vitamin D, it became evident that the actions of vitamin D are not restricted to cells involved in mineral and bone metabolism. In fact, it affects proliferation, differentiation, and function of a large number of different cell types including cells of the immune system. Vitamin D receptor agonists were found to exert immunosuppressive effects on the adaptive immune system, thus being able to mediate immunologic tolerance. However, they promote the innate immune system and thereby improve the ability of the host to combat invading pathogens. This review summarizes our current understanding of vitamin D as an immunomodulatory agent with special emphasis on its clinical implications in the transplant setting.