Previous studies have revealed the effect of 1,25(OH)2D in suppressing proliferation, invasion, angiogenesis, and metastasis of cancer cells, and in promoting differentiation and apoptosis of these cells in vitro (Giovannucci, 2005). Results of epidemiological studies suggested a weak effect of vitamin D in reducing the risk for cancers (Giovannucci, 2005, 2006; Holick, 2006; Bertone-Johnson, 2009; Feldman et al., 2014). However, there are studies that failed to confirm the antineoplastic effect of vitamin D (Baron et al., 2015). To explain these inconsistent findings, several issues may be considered. First, the antineoplastic effect of vitamin D is likely weak. Second, cancer development is influenced by multiple factors, some of which may mask the effect of vitamin D. Furthermore, cancer incidence in the general population is low and the majority of clinical studies cover only short periods of time and therefore may not be suitable for detecting subtle effects.
We considered an alternative strategy by means of using conditions with increased incidence of tumors, in particular, the tumor suppressor gene disorder neurofibromatosis type 1 (NF1), which has the hallmark of multiple cutaneous neurofibromas (Anderson and Gutmann, 2015). In our previous study, we could indeed show that 25-hydroxyvitamin D serum concentration is inversely correlated with the number of neurofibromas (P<0.0001) in NF1 patients (Lammert et al., 2006).
The effect of 1,25(OH)2D is transmitted via its binding to and the subsequent activation of its receptor: the vitamin D receptor (VDR), coded by the VDR gene on chromosome 12. High VDR expression in breast and prostate tumors was reported to be associated with favorite prognosis (Hendrickson et al., 2011; Ditsch et al., 2012). In a mouse model, VDR was found to function as a master regulator for genes that are involved in tumorigenesis, and low VDR expression was associated with higher tumor susceptibility (Quigley et al., 2009). Lack of VDR in the epidermis leads to predisposition of tumor formation (Bikle, 2015).
In the present study, we examined VDR mRNA in blood of 87 NF1 patients and evaluated its correlation with burden of neurofibromas and other NF1-related tumors including plexiform neurofibromas and malignant peripheral nerve sheath tumors.
Patients and methods
Patients and samples
A total of 141 patients aged between 12 and 75 years were recruited for the study. Most patients were adults aged between 20 and 60 years (Fig. 1b). For 101 patients, data of serum vitamin 25(OH)D (VitD) were available (Schnabel et al., 2014). All patients were informed about the purpose and protocol of the study, and signed written consent. The study protocol was approved by the local ethical review board (no. WF-042/10).
Vitamin D receptor mRNA level in peripheral leukocytes
From 87 patients, fresh blood was available as a nontumor tissue for studying VDR mRNA. From each patient, 2×5 ml blood was taken in PaxGene tubes (Qiagen, Hilden, Germany) and stored at −80°C until RNA preparation using the corresponding kit (Qiagen). Reagents in the PaxGene tube immediately stabilize intracellular RNA and prevent degradation. From these total RNA, cDNA was synthesized using random primers and standard protocols.
mRNA of VDR in each sample was measured in four replicates using dual-probe real-time PCR. Each assay contained components of two TaqMan (Thermo Fisher Scientific, Waltham, Massachusetts, USA) assay kits, one for the target VDR mRNA and the other for mRNA of a reference housekeeping gene GAPDH. Cycle numbers at a defined threshold for VDR (Ct VDR) and GAPDH (Ct GAPDH) were read and the difference between the two was calculated as [INCREMENT]Ct=Ct VDR−Ct GAPDH. Subsequently, the four [INCREMENT]Ct values in the four replicates for one sample were used to calculate the mean [INCREMENT]C for each sample. Relative copy number of VDR mRNA to fictive 1000 copies of GAPDH-mRNA was calculated as 1000/
Correlation with clinical data
Evaluation of VDR mRNA level and serum VitD with clinical data was carried out using the statistic program R by a statistician (G.S., one of the coauthors). Hypothesis was two-tailed and statistical significance was set at P value less than 0.05. Regarding neurofibromas, patients were divided into five groups according to the order of the number of their neurofibromas: 0, 10, 100, 1000, and more than 1000. Nonparametric Spearman’s analysis was used for examination of possible correlations. Regarding plexiform neurofibromas, patients were divided into four groups according to the burden of this kind of tumor: no, small, medium, and large.
Immunohistochemistry for vitamin D receptor in tumors
Sections were made from neurofibroma, plexiform neurofibrom, and malignant peripheral nerve sheath tumors from NF1 patients. Immunohistochemistry was performed in an automated stainer (Ventana Benchmark XT; Roche Diagnostics, Mannheim, Germany) using an antibody against vitamin D1 receptor (sc-13133, dilution 1 : 100; Santa Cruz, San Jose, California, USA). A healthy skin sample was included as a control.
Results and discussion
Vitamin D receptor mRNA and lack of correlation with VitD serum concentration
VDR mRNA was measured in total RNA from blood of 87 NF1 patients. The VDR mRNA level is normally distributed (Fig. 1a). No trend regarding age and no difference between male and female patients were observed (Fig. 1b and c). VDR mRNA did not correlate with VitD serum concentration (Fig. 1d).
Correlation of VitD and vitamin D receptor mRNA with number of cutaneous neurofibromas
For a total of 75 patients, data regarding both number of neurofibromas and VDR mRNA were available. When the patients were divided into five groups according to the number of tumors, an inverse correlation was found between the tumor burden and VDR mRNA level (Fig. 2a). Nonparametric Spearman’s analysis revealed that this correlation was slightly significant (ρ=−0.23, P=0.045).
For 88 patients, data regarding both the number of neurofibromas and serum VitD concentration were available. In addition, a statistically significant inverse correlation was found (Fig. 2b, ρ=−0.29, P=0.006).
These results suggest that both VitD and its receptor VDR may play roles in the development of cutaneous neurofibromas. Furthermore, VitD and VDR may compensate each other. This would mean that for patients with lower VDR expression, keeping their VitD at adequate level may be beneficial in suppressing neurofibroma development.
No correlation of VitD and vitamin D receptor with other NF1-related tumors
By contrast, no correlation was evident for size of plexiform neurofibromas with neither VDR mRNA level (Fig. 3a) nor serum VitD (Fig. 3b). Neither the presence of malignant peripheral nerve sheath tumour was correlated with VDR mRNA level (Fig. 3c) nor serum VitD (Fig. 3d).
This finding may suggest that while VDR and VitD may play a role in tumor initialization, they may not be directly involved in tumor growth and progression. However, it has to be noted that the sample size for these two NF1-related tumors is smaller than the sample size for neurofibromas. For example, VDR mRNA could only be measured in eight malignant peripheral nerve sheath tumour cases. Subtle effect of VDR and VitD may therefore not be detected. Since NF1 is a rare disease, prospective collection of samples and data strictly following standardized protocol is challenging. For studies on larger sample size, multicenter collaboration is therefore necessary.
Lack of detectable vitamin D receptor in neurofibromatosis type 1-related tumors
Immunohistochemistry did not detect VDR in neurofibroma, plexiform neurofibroma, and in malignant peripheral nerve sheath tumor from NF1 patients. For comparison, VDR was clearly detected in normal skin, in particular in epithelial cells in the epidermal layer using the same antibody and staining procedure (Fig. 4).
Lack of VDR in NF1-tumors may indicate that (a) the tumors selectively developed in cells that do not contain VDR or express only very low levels of VDR or (b) VDR expression is suppressed during the tumor development.
Remaining issues and future prospective
In the present study, the VDR mRNA level was measured only in blood of the patients. Using blood samples has the advantage of standardizing the protocol. This is especially critical for studies involving RNA, which degrades quickly once the specimen is resected. In our study, RNA was immediately stabilized upon blood being taken. Considering ethical aspect, collecting nontumor tissues other than blood, for example, skin biopsy, is difficult to be justified. However, a critical issue is whether or not the VDR mRNA level in blood correlate to the expression and biological activity of VDR in cells that are involved in or regulate tumor development. Furthermore, the VDR mRNA level in our study was measured in patients who have already developed the tumors and may differ from those before or when the tumor were developing. One interesting issue for future studies could be to follow vitamin D serum concentration, VDR mRNA level, and disease-related tumors in same patients over long period starting in young age.
Both VitD and its receptor may play roles in suppressing the development of neurofibromas. Sustaining VitD at an adequate level may contribute to controlling neurofibromas and possibly also other tumors. This is especially important for individuals with lower expression of VDR.
The authors thank Ina Alster for managing and preparing samples, and carrying out the RT-PCR assays.
This study was financially supported by Deutsche Forschungsgemeinschaft, no. KL2379.
Conflicts of interest
There are no conflicts of interest.
Anderson JL, Gutmann DH (2015). Neurofibromatosis type 1
. Handb Clin Neurol 132:75–86.
Baron JA, Barry EL, Mott LA, Rees JR, Sandler RS, Snover DC, et al (2015). A trial of calcium and vitamin D
for the prevention of colorectal adenomas. N Engl J Med 373:1519–1530.
Bertone-Johnson ER (2009). Vitamin D
and breast cancer. Ann Epidemiol 19:462–467.
Bikle DD (2015). Vitamin D receptor
, a tumor
suppressor in skin. Can J Physiol Pharmacol 93:349–354.
Ditsch N, Toth B, Mayr D, Lenhard M, Gallwas J, Weissenbacher T, et al (2012). The association between vitamin D receptor
expression and prolonged overall survival in breast cancer. J Histochem Cytochem 60:121–129.
Feldman D, Krishnan AV, Swami S, Giovannucci E, Feldman BJ (2014). The role of vitamin D
in reducing cancer risk and progression. Nat Rev Cancer 14:342–357.
Giovannucci E (2005). The epidemiology of vitamin D
and cancer incidence and mortality: a review (United States). Cancer Causes Control 16:83–95.
Giovannucci E (2006). The epidemiology of vitamin D
and colorectal cancer: recent findings. Curr Opin Gastroenterol 22:24–29.
Hendrickson WK, Flavin R, Kasperzyk JL, Fiorentino M, Fang F, Lis R, et al (2011). Vitamin D receptor
protein expression in tumor
tissue and prostate cancer progression. J Clin Oncol 29:2378–2385.
Holick MF (2006). Vitamin D
: its role in cancer prevention and treatment. Prog Biophys Mol Biol 92:49–59.
Lammert M, Friedman JM, Roth HJ, Friedrich RE, Kluwe L, Atkins D, et al (2006). Vitamin D
deficiency associated with number of neurofibromas in neurofibromatosis 1. J Med Genet 43:810–813.
Quigley DA, To MD, Pérez-Losada J, Pelorosso FG, Mao JH, Nagase H, et al (2009). Genetic architecture of mouse skin inflammation and tumour susceptibility. Nature 458:505–508.
Schnabel C, Dahm S, Streichert T, Thierfelder W, Kluwe L, Mautner VF (2014). Differences of 25-hydroxyvitamin D3 concentrations in children and adults with neurofibromatosis type 1
. Clin Biochem 47:560–563.