Clinical Endocrinology (2014)
doi: 10.1111/cen.12396
ORIGINAL ARTICLE
Increased CYP24A1 expression is associated with BRAFV600E mutation and advanced stages in papillary thyroid carcinoma Minjing Zou*, Faisal S. BinHumaid*, Ali S. Alzahrani†, Essa Y. Baitei*, Futwan A. Al-Mohanna‡, Brian F. Meyer* and Yufei Shi* *Department of Genetics, King Faisal Specialist Hospital and Research Centre, †Department of Medicine, King Faisal Specialist Hospital and Research Centre and ‡Department of Cell Biology, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia
Abstract Objective 1a, 25(OH)2D3 (calcitriol), the active form of vitamin D, has been shown to exert antiproliferative effects in many cancers. Overexpression of CYP24A1, the primary vitamin D-inactivating enzyme, is also observed in a variety of human cancers, thus potentially neutralizing the antitumour effect of 1a, 25(OH)2D3. This study investigates the expression of CYP24A1 and the effect of BRAFV600E on its expression in thyroid cancer. Methods We investigated 60 papillary thyroid carcinoma (PTC) specimens for CYP24A1 expression and its association with BRAF mutation and disease progression. CYP24A1 expression was measured by real-time RT-PCR, and BRAFV600E mutation was detected by PCR-DNA sequencing analysis. The interaction between BRAFV600E and CYP24A1 expression was determined by Western blot analysis and real-time RT-PCR. Results CYP24A1 expression was increased in PTC as compared to benign multinodular goitre. The expression was further increased in stage III and IV tumours. There is a strong correlation between CYP24A1 overexpression and BRAFV600E mutation (P < 001). In thyroid cancer cell lines expressing BRAFV600E, CYP24A1 expression was significantly higher when compared to those without BRAFV600E expression. BRAFV600E transgene expression in CAL62 cell line can induce CYP24A1 expression. Furthermore, BRAFV600E inhibitor PLX4720 can significantly down-regulate CYP24A1 expression and enhance the antiproliferative effects of calcitriol in thyroid cancer cell lines. Conclusion CYP24A1 overexpression is a poor prognostic indicator for PTC and may reflect BRAFV600E mutation and MARK activation. The crosstalk between vitamin D and MAPK signalling pathways results in resistance to calcitriol-mediated antitumour effects, and the resistance can be reversed by BRAFV600E inhibitor PLX4720.
Correspondence: Yufei Shi, MBC 3, Department of Genetics, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia. Tel.: 966 1 442 4768; Fax: 966 1 442 4585; E-mail: [email protected] © 2014 John Wiley & Sons Ltd
(Received 28 August 2013; returned for revision 15 September 2013; finally revised 1 December 2013; accepted 20 December 2013)
Introduction Vitamin D exists in two major forms: ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3).1 Both forms need two-step hydroxylation at carbons 1 and 25 for activation. The first step occurs in the liver where vitamin D is hydroxylated to 25-hydroxyvitamin D [25(OH)D] by either mitochondrial vitamin D 25-hydroxylase (CYP27A1) or microsomal vitamin D 25-hydroxylase (CYP2R1).1 The second step occurs mainly in the kidney where 25(OH)D is hydroxylated by the mitochondrial vitamin D1a-hydroxylase (CYP27B1) to biologically active hormone 1a, 25(OH)2D (calcitriol), which binds to its nuclear receptor, the vitamin D receptor (VDR), to exert its biological activities.1 Although vitamin D is best recognized for its effects on bone and mineral metabolism, it also plays an important role in cancer prevention and treatment.2 It is known that vitamin D can exert significant antiproliferative effects (growth inhibition, increased apoptosis and cell cycle arrest) in many human cancer cells such as thyroid, prostate, breast, colorectal and lung cancers.1,3–5 VDR knockout mice display a higher incidence of carcinogen-induced breast and skin tumours,6 and vitamin D deficiency promotes human breast cancer growth.7 Clinical trials have shown potential therapeutic effects of calcitriol in prostate cancer patients.8 Vitamin D 24-hydroxylase (CYP24A1) is the primary vitamin D-inactivating enzyme, which catabolizes 1a, 25(OH)2D and to a lesser extent, 25(OH)2D by 24 hydroxylation into inactive 1a, 24,25(OH)3D and 24,25(OH)2D. The vitamin D-mediated antiproliferative effects can be disrupted by overexpression of CYP24A1 during tumour development.3 Increased expression of CYP24A1 has been observed in many human cancers including thyroid,9 lung,10 colon,11 oesophageal,12 and breast13 and has been linked to poor prognosis in patients with lung and oesophageal cancers.12,14 The diminished antitumour effects of calcitriol can be restored by inhibition of CYP24A1.15 In patients with 1
2 M. Zou et al. thyroid cancer, serum level of 1a, 25(OH)2D3 is significantly lower16 although there is no significant difference in serum 25 (OH) D3 level between thyroid nodule and thyroid cancer patients,16,17 indicating that 1a, 25(OH)2D3 might be converted into inactive 1a, 24,25(OH)3D3 by increased CYP24A1 expression. It has been reported that vitamin D can inhibit thyroid cancer cell growth with varying degrees of sensitivity.4,18,19 The sensitive thyroid cancer cell lines (TPC-1, C643 and HTh74) are associated with lower baseline CYP24A1 expression, whereas the resistant cell lines (8505C, SW1736, KTC-1, BCPAP and HTh7) have higher CYP24A1 expression and/or poor response to calcitriol stimulation. Interestingly, the resistant cell lines often carry BRAF (8505C, SW1736, BCPAP) mutation, which raise the question of whether BRAF mutation can cause elevated expression of CYP24A1 and result in resistance to calcitriol treatment. In this study, we investigated CYP24A1 expression and BRAF mutation in 60 PTC from Saudi Arabia to determine whether CYP24A1 expression is associated with disease progression and BRAF mutation is involved in inducing CYP24A1 expression. We found that CYP24A1 expression is increased and the level of expression is associated with disease progression and poor prognosis. The higher level of CYP24A1 expression is often correlated with BRAF mutation. Furthermore, BRAFV600E inhibitor PLX4720 can significantly enhance the antiproliferative effects of calcitriol in thyroid cancer cell lines.
Materials and methods Thyroid tumour specimens All tumour tissues were obtained at surgery with informed consent and were immediately frozen in liquid nitrogen and stored at 70 °C until processed. The clinical staging of thyroid cancer was based on the TNM classification.20 Sixty PTC diagnosed between 1987 and 2006 were selected randomly and included in the study: 55 CPTC (classic PTC), 3 tall cell variants and 2 FVPTC (follicular variant of PTC). Eleven normal thyroid tissues removed from tumour surrounding, and 11 multinodular goitres were also included in the study. The study was approved by the institutional review board (Office of Research Affairs at King Faisal Specialist Hospital and Research Centre).
with RET/PTC-1 rearrangement. The cell lines were propagated in DMEM/Ham’s F12 medium containing 10% foetal bovine serum, 100 units/ml penicillin and 100 lg/ml streptomycin at 37 °C with 5% CO2 in a humidified environment. Quantitative real-time reverse transcriptase (RT)-PCR analysis for CYP24A1 expression Total RNAs from normal thyroid, thyroid tumour and cell lines were isolated by guanidinium thiocyanate-phenol-chloroform method. The integrity of RNA was verified by denaturing gel electrophoresis. Two lg of each total RNA was reverse-transcribed to cDNA using the Promega RT system (Promega, Madison, WI, USA). LightCycler DNA Master SYBR Green 1 kit was used for quantitative real-time PCR analysis.21 The cDNA mix was diluted 10-fold, and 2 ll of the dilution was used for realtime PCR analysis. PCR primers for the 154-bp CYP24A1 cDNA fragment were as follows: 5′-GGAAGTGATGAAGCTGGACAA CA-3′ (sense, located in exon 3) and 5′-CTCATACAACACG AGGCAGATAC-3′ (antisense, located in exon 5). The sense primer spans over intron 3 and 4 of 5 kb so that the contaminated genomic DNA will not be amplified. The CYP24A1 cDNA fragment was verified by DNA sequencing. The mRNA level of housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and a 300-bp PCR product was amplified using the following two primers: 5′-ACAG TCAGCCGCATCTTCTT-3′ (sense) and 5′-TTGATTTTGGAGG GATCTCG-3′ (antisense). The PCR conditions are 95 °C for 30 s followed by 30 cycles of amplification (95 °C for 10 s, 48 °C for 5 s and 72 °C for 10 s). The resulting concentration of CYP24A1 PCR products was normalized by comparison with GAPDH and was used to determine the relative mRNA level of CYP24A1 among thyroid tumour specimens. Detection of BRAF mutation Tumour tissues were obtained by standard sectioning, and DNA was extracted by standard proteinase-K treatment followed by phenol/chloroform extraction. BRAF exon 15 was amplified by PCR and directly sequenced as described previously.22 Induction of CYP24A1 expression by BRAFV600E
Cell lines and cell culture The following cell lines were studied for CYP24A1 expression: N-Thy, K1, BCPAP, 8505C, BHT101, CAL62, TPC-1 and FRO. N-thy is a normal thyroid follicular cell line immortalized by SV40 and was purchased from the European Collection of Animal Cell Culture (Salisbury, UK). K1, BCPAP, 8505C, BHT101 and CAL62 cell lines were purchased from DSMZ (Braunschweig, Germany). TPC-1 and FRO were kindly provided by Dr. James Fagin (Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York). K1 and BCPAP are PTC cell lines with BRAFV600E mutation. 8505C, BHT101 and FRO are anaplastic thyroid carcinoma (ATC) cell lines with BRAFV600E mutation, and CAL62 is an ATC cell line with KRASG12R mutation; TPC-1 is a PTC cell line
The BRAFV600E cDNA was cloned into pcDNA3.1 as described previously.22 HRASG12V cDNA was obtained from Biomyx, CA and subcloned into pcDNA3.1 vector as well. 20 lg of each construct and vector control were transfected into CAL62 cell line using Lipofectamine (Invitrogen, Carlsbad, CA, USA) and then selected for 4 weeks with 400 lg/ml G418. Stable clones were pooled and used to measure CYP24A1 expression and phosphoERK 1/2 activity. Cell proliferation assay Cell proliferation was measured by a nonradioactive MTT assay kit according to manufacturer’s procedure (Promega Corp, © 2014 John Wiley & Sons Ltd Clinical Endocrinology (2014), 0, 1–8
CYP24A1 activation in thyroid carcinoma 3 Madison, WI, USA). Briefly, cells were plated into triplicate wells of 96-well plates (103 cells/well) in growth medium containing vehicle (1% ethanol or DMSO or both), 100 nM calcitriol (Sigma-Aldrich, St. Louis, MO, USA), 100 nM PLX4720 (Selleck Chemicals, Houston, TX, USA) or a combination of calcitriol and PLX4720 for 72 h. The medium was changed, and cells were cultured for additional 24 h in the growth medium containing the same concentration of drugs. At the final 2 h of incubation, 20 ll of CellTiter 96â AQueous One Solution reagent was added into each well for the measurement of cell viability. Western blot analysis Protein extracts (40 lg/lane) from cells were loaded onto a 12% SDS-polyacrylamide gel. Proteins were transferred to a PVDF membrane and subject to Western blot analysis using antiphospho-ERK 1/2 antibody (1:1000, Cell Signaling Technology, Inc, Danvers, MA, USA) and anti-CYP24A1 antibody (1:3000, OriGene, Inc, Rockville, MD, USA). Statistical analysis The significant difference in CYP24A1 gene expression and cell proliferation among different groups was carried out using the unpaired Student’s t-test (two-tailed). Fisher’s exact test (twotailed) was used to compare BRAF mutation rate among different stages of cancer. A P-value of 005 or less was considered significant.
Results CYP24A1 expression in thyroid cancer CYP24A1 mRNA expression was investigated in 11 paired PTC, 11 multinodular goitres and 49 nonpaired PTC by real-time RTPCR. The same group of samples was also screened for BRAF mutation. As shown in Fig. 1(a) and Table S1, there is significant difference in CYP24A1 expression between normal thyroid and matched tumour tissues (32 06 vs 571 148, P < 001).There was no significant difference in CYP24A1 expression between normal thyroids and multinodular goitres (32 06 vs 28 05, P = 03, n = 11, Fig. 1b). Increased expression of CYP24A1 was observed in 41 PTC samples (68%) as compared to normal thyroids and multinodular goitres. The difference in CYP24A1 mRNA expression between 60 PTC vs normal thyroids and multinodular goitres was statistically significant (704 179 vs 30 04, P = 0025, Fig. 1b). BRAFV600E was detected in 28 samples (47%), and its frequency was significantly higher in advanced stage (stage III and IV, P < 002, Fig. 1c & d). No BRAFV600E was detected in normal controls and nodular goitres. As there was a large variation in CYP24A1 mRNA expression among PTC samples and CYP24A1 mRNA expression was higher in PTC with BRAFV600E, we stratified PTC samples into BRAFV600E-positive and BRAFV600E-negative groups to see whether increased CYP24A1 expression was associated with BRAFV600E. As shown in Fig. 2, the variation was © 2014 John Wiley & Sons Ltd Clinical Endocrinology (2014), 0, 1–8
significantly reduced and CYP24A1 expression was significantly higher in PTC with BRAFV600E (1356 345, n = 28) as compared to those without BRAFV600E (106 18, n = 29, P < 0001). CYP24A1 expression was still higher in BRAFV600Enegative PTC as compared to multinodular goitres (1327 23 vs 28 05, P < 005). We next examined 7 thyroid cancer cell lines to see whether CYP24A1 expression was higher in cell lines harbouring BRAFV600E. As shown in Fig. 3, CYP24A1 expression in mRNA and protein was indeed higher in BRAFV600E-positive cell lines (BHT101, FRO, 8305C, K1, and BCPAP) than BRAFV600E-negative cell lines (CAL62 and TPC-1). Interestingly, CAL62 and TPC-1 harbour KRASG12R and RET/PTC-1 rearrangement, respectively, which results in MAP kinase activation like BRAFV600E. CYP24A1 expression in different stages of thyroid cancer CYP24A1 overexpression was linked to poor prognosis in patients with lung and oesophageal cancers12,14, we sought to investigate whether its overexpression was associated with advanced stage of PTC. As shown in Fig. 4, the level of CYP24A1 mRNA expression is higher in stage III (739 362, n = 10) and IV (1969 872, n = 10) PTC as compared to stage I (171 37, n = 30) and II (437 216, n = 10) PTC. The difference in CYP24A1 mRNA expression between early stage (stage I and II) and late stage (stage III and IV) PTC was statistically significant (237 61, n = 40 vs 1354 481, n = 20, P < 001). When PTC samples were stratified into BRAFV600E-positive and BRAFV600E-negative groups and analysed separately, the difference in CYP24A1 mRNA expression was observed only in PTC with BRAFV600E: 519 119 in early stage (n = 14) and 1891 639 in late stage (n = 14, P < 005). There was no significant difference in CYP24A1 mRNA expression among PTC without BRAFV600E: 133 27 in early stage (n = 25) and 101 48 in late stage (n = 6, P = 0595). Induction of CYP24A1 expression by BRAFV600E The strong association between CYP24A1 expression and BRAFV600E suggests that BRAFV600E mutation may result in CYP24A1 overexpression. To confirm the hypothesis, we transfected BRAFV600E or HRASG12V into CAL62 cells and CYP24A1 expression was determined by Western blot analysis. As shown in Fig. 5, CYP24A1 expression was increased following BRAFV600E transfection. The induction of CYP24A1 expression by HRASG12V was much less than BRAFV600E although HRASG12V caused higher p-ERK expression than BRAFV600E (Fig. 5). Reduction of CYP24A1 expression by BRAFV600E inhibitor PLX4720 As BRAFV600E can induce CYP24A1 expression, we next investigated whether PLX4720 could reduce CYP24A1 expression and enhance or sensitize calcitriol-mediated antiproliferative effects in thyroid cancer cell lines. Thyroid cancer cell lines were treated
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Fig. 1 CYP24A1 gene expression and BRAF mutation among normal, multinodular goitre and PTC. (a) The CYP24A1 expression in 11 paired normal and PTC samples. CYP24A1 mRNA level was quantified by real-time RT-PCR. Data are expressed as relative expression of CYP24A1 after normalization to GAPDH expression in each sample.* indicate BRAFV600E mutation in the sample. (b) CYP24A1 expression among normal, multinodular goitre and PTC. A column scatter graph is used to show each data point in a group with a line at the mean. (c) Frequency of BRAF mutation in different stages of PTC. BRAFV600E mutation rate is significantly higher in advanced stage of PTC (stage III and IV). No mutation was detected in the goitres. (d) A representative sequence electropherogram is shown and BRAFV600E mutation from 3 patients is indicated by an arrow.
with calcitriol in the presence or absence of different concentrations of PLX4720, and CYP24A1mRNA level was measured by quantitative real-time RT-PCR. As shown in Fig. 6(a), PLX4720 could significantly reduce calcitriol-induced CYP24A1 mRNA expression (P < 001) in the cell lines, and it was dose-dependent. The calcitriol-mediated growth inhibition was also significantly increased as a result of PLX4720 treatment, especially in cell lines harbouring BRAFV600E (8505C, FRO and K1) (Fig. 6b, P < 001).
Discussion In the present study, we reported for the first time that CYP24A1 overexpression was associated with late stage of PTC and BRAFV600E could drive CYP24A1 overexpression, linking
vitamin D and MAPK signalling pathways in thyroid cancer progression. Inhibition of MAPK can reduce CYP24A1 expression and significantly enhance calcitriol-mediated antiproliferative effects. Overexpression of CYP24A1 has been reported in many cancers and can lead to abrogation of growth control mediated by vitamin D. It has thus been proposed as a candidate oncogene.23 Multiple mechanisms are involved in its overexpression: for example, gene amplification in breast and colon cancers,23,24 down-regulation of miR-125b in breast cancer25 and activation of protein kinase CK2 in human prostate cancer.26 miR-125b regulates CYP24A1 expression post-transcriptionally, and its expression is inversely correlated with CYP24A1 protein level.25 Interestingly, miR-125b is down-regulated in anaplastic thyroid carcinoma.27 CK2 is a multitask kinase and is overexpressed in © 2014 John Wiley & Sons Ltd Clinical Endocrinology (2014), 0, 1–8
CYP24A1 activation in thyroid carcinoma 5 (a)
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Fig. 2 CYP24A1 gene expression among normal, multinodular goitre, PTC with BRAFV600E and PTC without BRAFV600E. The CYP24A1 mRNA level was quantified by real-time RT-PCR. Data are expressed as relative expression of CYP24A1 after normalization to GAPDH expression in each sample. A column scatter graph is used to show each data point in a group with a line at the mean.
many solid tumours.28 CK2 is involved in many cellular processes related to cell growth and survival.28 In the present study, we have demonstrated that BRAFV600E, a key component in the MAPK signalling, can cause CYP24A1 overexpression. It is not clear why similar effect was not observed in cells with KRASG12R (CAL62) or RET/TPC-1 (TPC-1) mutation. BRAFV600E may cause more severe disruption of normal vitamin D signalling than KRASG12R or RET/TPC-1. Early studies4,18 have shown that basal level of CYP24A1 expression in TPC-1 is low and can be significantly induced by calcitriol as compared to other thyroid cancer cell lines harbouring BRAFV600E such as 8505C, BCPAP, SW1736 and KTC, indicating relative normal vitamin D signalling. TPC-1 is also sensitive to calcitriol-mediated growth inhibition. C643 (ATC with HRASG13R) and HTh74 (ATC with no BRAF or RAS mutation) are two other cell lines responsive to calcitriol-mediated antiproliferative effects. Again, in these two cell lines, the basal level of CYP24A1 is low and its expression can be significantly induced by calcitriol.4,18 Although the basal levels of CYP24A1 are low in CAL62 (current study) and HTh7 [ATC with NRASQ61K, previous studies],4,18 they are not responsive to calcitriol stimulation and resistance to calcitriol-mediated growth inhibition. This may reflect the loss of vitamin D signalling resulting from de-differentiation of ATC. These data suggest that the sensitivity of calcitriol antitumour activity is dependent on the level of CYP24A1 expression and/or the integrity of vitamin D signalling pathway in tumour cells. © 2014 John Wiley & Sons Ltd Clinical Endocrinology (2014), 0, 1–8
Fig. 3 CYP24A1 gene expression in thyroid cancer cell lines. (a) The CYP24A1 mRNA level in each cell line was quantified by real-time RT-PCR. Data are expressed as relative expression of CYP24A1 after normalization to GAPDH expression in each sample. (b) The CYP24A1 protein level in each cell line was analysed by Western blot analysis. Protein extracts (40 lg/lane) were loaded onto a 12% SDSpolyacrylamide gel. The blot was probed with anti-CYP24A1 antibody (upper panel) and reprobed with anti-GAPDH antibody (lower panel). Lane 1: BHI101; lane 2: TPC-1; lane 3: BCPAP; lane 4: CAL62; lane 5: K1; lane 6:8505; lane 7: FRO; lane 8: N-thy.
Vitamin D signalling pathway is compromised during tumorigenesis as evidenced by down-regulation of VDR and vitamin D independent up-regulation of CYP24A1 in many cancers. Constitutive activation of MAPK pathway has been shown to cause impairment of vitamin D signalling in human prostate epithelial cells.29 In our current study, we have demonstrated strong correlation of CYP24A1 expression with advanced disease stage and BRAFV600E. It is known that BRAFV600E is associated with poor prognosis in PTC.30 The association of CYP24A1 expression with advanced PTC is likely due to BRAFV600E, because BRAFV600E can induce higher CYP24A1 expression and BRAFV600E inhibitor PLX4720 results in down-regulation of CYP24A1. Indeed, MAP kinases have been shown to participate in the calcitriol-induced transactivation of CYP24A1 promoter,31 and the functional consequence of the crosstalk between vitamin D and MAPK pathways is cell type specific.32 In PTC, we have shown that the crosstalk results in increased CYP24A1 expression and resistance to calcitriol-mediated growth inhibition.
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Although synthesis and degradation of calcitriol occur mainly in the kidney, they are also present in extra renal tissues. It is known that the genes (VDR, CYP27A1, CYP2R1, CYP27B1 and CYP24A1) involved in vitamin D metabolism and signalling are expressed in thyroid epithelial cells and thyroid cancer cells.4,18 The major role of 1a, 25(OH)2D derived from autocrine/paracrine mechanism is to inhibit cell proliferation and induce cell differentiation. However, overexpression of CYP24A1 in cancer cells may affect bioavailability and antiproliferative activity of 1a, 25(OH)2D. Inhibition of CYP24A1 could increase 1a, 25 (OH)2D-mediated antitumour activity by reducing its degradation. Indeed, the therapeutic effects of CYP24A1 inhibitors such as ketoconazole and CTA091 have been seen both in vitro and
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Fig. 5 Induction of CYP24A1 expression by BRAFV600E in CAL62 cells. CAL62 cells expressing BRAFV600E or HRASG12V plasmid were subjected to Western blot analysis for CYP24A1 expression (top). The same blot was reprobed with p-ERK (middle) and GAPDH (bottom). Increased CYP24A1 expression was observed in BRAFV600E transfected cells, but not in HRASG12V transfected cells, although p-ERK level was higher in HRASG12V transfected cells. Lane 1: CAL62 cells expressing BRAFV600E; lane 2: CAL62 cells expressing HRASG12V; lane 3: CAL62 cells transfected with vector only.
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Fig. 4 CYP24A1 expression among different stages of thyroid tumours. CYP24A1mRNA levels were quantified by real-time RT-PCR and normalized using the expression of GAPDH. Data are expressed as relative CYP24A1 expression among different tumour stages. A column scatter graph is used to show each data point in a group with a line at the mean.
Calcitriol Calcitriol+1 µM PLX Calcitriol+0·1 µMPLX
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Fig. 6 Effects of calcitriol and PLX4720 on CYP24A1 expression and cell proliferation in thyroid cancer cell lines. (a) Induction of CYP24A1 expression by calcitriol and attenuation of its effect by PLX4720. Cells were plated into 12-well plates (2 9 104 cells/well) in growth medium for 24 h and then treated with vehicle, 100 nM calcitriol, 100 nM calcitriol + 01 lM PLX4720 or 100 nM calcitriol + 1 lM PLX4720 for 4 h. RNA was extracted from the cells, and CYP24A1 expression was determined by real-time RT-PCR. CYP24A1 expression in cells treated with vehicle was used as baseline control. Data are expressed as fold change in CYP24A1 expression compared to baseline. Each data point represents the mean SEM of three independent experiments. (b) Inhibition of cell proliferation by calcitriol and PLX4720. Cells were treated with 100 nM calcitriol, 100 nM PLX4720 or a combination of 100 nM calcitriol and 100 nM PLX4720 for 4 days. Cells treated with vehicle (1% ethanol or DMSO) were used as control. Cell proliferation was measured by a modified MTT assay. Data are expressed as % of reduction in cell proliferation compared to vehicle treatment. Each data point represents the mean SEM of three independent experiments.
in vivo.15,33 Our data show that BRAFV600E inhibitor PLX4720 can also down-regulate CYP24A1 expression and enhance calcitriol-mediated antitumour activity. PLX4720 is effective against thyroid cancer harbouring BRAFV600E in preclinical studies.34,35 Given that PLX4720 can inhibit both CYP24A1 and BRAFV600E, combination of PLX4720 and calcitriol may offer better treatment outcome than combined CYP24A1 inhibitors and calcitriol. © 2014 John Wiley & Sons Ltd Clinical Endocrinology (2014), 0, 1–8
CYP24A1 activation in thyroid carcinoma 7
Limitations of the study To study CYP24A1 expression in thyroid cancer, the best control is paired samples taken from both cancerous and normal tissues of the same thyroid gland. Due to the limitation to obtain normal thyroid tissue, we only studied 11 paired samples. The sample size is relatively small especially in the late stage of thyroid cancer.
Disclosure of conflict of interests All authors have nothing to declare.
Acknowledgement This study was supported by the King Abdulaziz City of Science and Technology (KACST) Biotechnology grant.
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12 Mimori, K., Tanaka, Y., Yoshinaga, K. et al. (2004) Clinical significance of the overexpression of the candidate oncogene CYP24 in esophageal cancer. Annals of Oncology, 15, 236–241. 13 Lopes, N., Sousa, B., Martins, D. et al. (2010) Alterations in Vitamin D signalling and metabolic pathways in breast cancer progression: a study of VDR, CYP27B1 and CYP24A1 expression in benign and malignant breast lesions. BMC Cancer, 10, 483. 14 Chen, G., Kim, S.H., King, A.N. et al. (2011) CYP24A1 is an independent prognostic marker of survival in patients with lung adenocarcinoma. Clinical Cancer Research, 17, 817–826. 15 Muindi, J.R., Yu, W.D., Ma, Y. et al. (2010) CYP24A1 inhibition enhances the antitumor activity of calcitriol. Endocrinology, 151, 4301–4312. 16 Stepien, T., Krupinski, R., Sopinski, J. et al. (2010) Decreased 1–25 dihydroxyvitamin D3 concentration in peripheral blood serum of patients with thyroid cancer. Archives of Medical Research, 41, 190–194. 17 Laney, N., Meza, J., Lyden, E. et al. (2010) The prevalence of vitamin D deficiency is similar between thyroid nodule and thyroid cancer patients. International Journal of Endocrinology, 2010, 805716. 18 Sharma, V., Fretwell, D., Crees, Z. et al. (2010) Thyroid cancer resistance to vitamin D receptor activation is associated with 24-hydroxylase levels but not the ff FokI polymorphism. Thyroid, 20, 1103–1111. 19 Clinckspoor, I., Verlinden, L., Overbergh, L. et al. (2011) 1,25dihydroxyvitamin D3 and a superagonistic analog in combination with paclitaxel or suberoylanilide hydroxamic acid have potent antiproliferative effects on anaplastic thyroid cancer. Journal of Steroid Biochemistry and Molecular Biology, 124, 1–9. 20 Thyroid (2010) In: S.B. Edge, D.R. Byrd, C.C. Compton, et al., eds. AJCC Cancer Staging Manual. 7th ed. pp. 87–96. Springer, New York, NY. 21 Zou, M., Famulski, K.S., Parhar, R.S. et al. (2004) Microarray analysis of metastasis-associated gene expression profiling in a murine model of thyroid carcinoma pulmonary metastasis: identification of S100A4 (Mts1) gene overexpression as a poor prognostic marker for thyroid carcinoma. Journal of Clinical Endocrinology & Metabolism, 89, 6146–6154. 22 Baitei, E.Y., Zou, M., Al-Mohanna, F. et al. (2009) Aberrant BRAF splicing as an alternative mechanism for oncogenic B-Raf activation in thyroid carcinoma. Journal of Pathology, 217, 707– 715. 23 Albertson, D.G., Ylstra, B., Segraves, R. et al. (2000) Quantitative mapping of amplicon structure by array CGH identifies CYP24 as a candidate oncogene. Nature Genetics, 25, 144–146. 24 Hobaus, J., Hummel, D.M., Thiem, U. et al. (2013) Increased copy-number and not DNA hypomethylation causes overexpression of the candidate proto-oncogene CYP24A1 in colorectal cancer. International Journal of Cancer 133, 1380–1388. 25 Komagata, S., Nakajima, M., Takagi, S. et al. (2009) Human CYP24 catalyzing the inactivation of calcitriol is post-transcriptionally regulated by miR-125b. Molecular Pharmacology, 76, 702–709. 26 Luo, W., Yu, W.D., Ma, Y. et al. (2013) Inhibition of protein kinase CK2 reduces cyp24a1 expression and enhances 1,25dihydroxyvitamin d3 antitumor activity in human prostate cancer cells. Cancer Research, 73, 2289–2297. 27 Visone, R., Pallante, P., Vecchione, A. et al. (2007) Specific microRNAs are downregulated in human thyroid anaplastic carcinomas. Oncogene, 26, 7590–7595.
8 M. Zou et al. 28 Trembley, J.H., Chen, Z., Unger, G. et al. (2010) Emergence of protein kinase CK2 as a key target in cancer therapy. BioFactors, 36, 187–195. 29 Zhang, Z., Kovalenko, P., Cui, M. et al. (2010) Constitutive activation of the mitogen-activated protein kinase pathway impairs vitamin D signaling in human prostate epithelial cells. Journal of Cellular Physiology, 224, 433–442. 30 Xing, M., Alzahrani, A.S., Carson, K.A. et al. (2013) Association between BRAF V600E mutation and mortality in patients with papillary thyroid cancer. The Journal of the American Medical Association, 309, 1493–1501. 31 Dwivedi, P.P., Hii, C.S., Ferrante, A. et al. (2002) Role of MAP kinases in the 1,25-dihydroxyvitamin D3-induced transactivation of the rat cytochrome P450C24 (CYP24) promoter. Specific functions for ERK1/ERK2 and ERK5. Journal of Biological Chemistry, 277, 29643–29653. 32 Narayanan, R., Sepulveda, V.A., Falzon, M. et al. (2004) The functional consequences of cross-talk between the vitamin D receptor and ERK signaling pathways are cell-specific. Journal of Biological Chemistry, 279, 47298–47310.
33 Zhang, Q., Kanterewicz, B., Buch, S. et al. (2012) CYP24 inhibition preserves 1alpha, 25-dihydroxyvitamin D(3) anti-proliferative signaling in lung cancer cells. Molecular and Cellular Endocrinology, 355, 153–161. 34 Nucera, C., Nehs, M.A., Nagarkatti, S.S. et al. (2011) Targeting BRAFV600E with PLX4720 displays potent antimigratory and anti-invasive activity in preclinical models of human thyroid cancer. Oncologist, 16, 296–309. 35 Salerno, P., De Falco, V., Tamburrino, A. et al. (2010) Cytostatic activity of adenosine triphosphate-competitive kinase inhibitors in BRAF mutant thyroid carcinoma cells. Journal of Clinical Endocrinology & Metabolism, 95, 450–455.
Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. CYP24A1 Expression and BRAF mutation in PTC.
© 2014 John Wiley & Sons Ltd Clinical Endocrinology (2014), 0, 1–8
doi: 10.1111/cen.12396
ORIGINAL ARTICLE
Increased CYP24A1 expression is associated with BRAFV600E mutation and advanced stages in papillary thyroid carcinoma Minjing Zou*, Faisal S. BinHumaid*, Ali S. Alzahrani†, Essa Y. Baitei*, Futwan A. Al-Mohanna‡, Brian F. Meyer* and Yufei Shi* *Department of Genetics, King Faisal Specialist Hospital and Research Centre, †Department of Medicine, King Faisal Specialist Hospital and Research Centre and ‡Department of Cell Biology, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia
Abstract Objective 1a, 25(OH)2D3 (calcitriol), the active form of vitamin D, has been shown to exert antiproliferative effects in many cancers. Overexpression of CYP24A1, the primary vitamin D-inactivating enzyme, is also observed in a variety of human cancers, thus potentially neutralizing the antitumour effect of 1a, 25(OH)2D3. This study investigates the expression of CYP24A1 and the effect of BRAFV600E on its expression in thyroid cancer. Methods We investigated 60 papillary thyroid carcinoma (PTC) specimens for CYP24A1 expression and its association with BRAF mutation and disease progression. CYP24A1 expression was measured by real-time RT-PCR, and BRAFV600E mutation was detected by PCR-DNA sequencing analysis. The interaction between BRAFV600E and CYP24A1 expression was determined by Western blot analysis and real-time RT-PCR. Results CYP24A1 expression was increased in PTC as compared to benign multinodular goitre. The expression was further increased in stage III and IV tumours. There is a strong correlation between CYP24A1 overexpression and BRAFV600E mutation (P < 001). In thyroid cancer cell lines expressing BRAFV600E, CYP24A1 expression was significantly higher when compared to those without BRAFV600E expression. BRAFV600E transgene expression in CAL62 cell line can induce CYP24A1 expression. Furthermore, BRAFV600E inhibitor PLX4720 can significantly down-regulate CYP24A1 expression and enhance the antiproliferative effects of calcitriol in thyroid cancer cell lines. Conclusion CYP24A1 overexpression is a poor prognostic indicator for PTC and may reflect BRAFV600E mutation and MARK activation. The crosstalk between vitamin D and MAPK signalling pathways results in resistance to calcitriol-mediated antitumour effects, and the resistance can be reversed by BRAFV600E inhibitor PLX4720.
Correspondence: Yufei Shi, MBC 3, Department of Genetics, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia. Tel.: 966 1 442 4768; Fax: 966 1 442 4585; E-mail: [email protected] © 2014 John Wiley & Sons Ltd
(Received 28 August 2013; returned for revision 15 September 2013; finally revised 1 December 2013; accepted 20 December 2013)
Introduction Vitamin D exists in two major forms: ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3).1 Both forms need two-step hydroxylation at carbons 1 and 25 for activation. The first step occurs in the liver where vitamin D is hydroxylated to 25-hydroxyvitamin D [25(OH)D] by either mitochondrial vitamin D 25-hydroxylase (CYP27A1) or microsomal vitamin D 25-hydroxylase (CYP2R1).1 The second step occurs mainly in the kidney where 25(OH)D is hydroxylated by the mitochondrial vitamin D1a-hydroxylase (CYP27B1) to biologically active hormone 1a, 25(OH)2D (calcitriol), which binds to its nuclear receptor, the vitamin D receptor (VDR), to exert its biological activities.1 Although vitamin D is best recognized for its effects on bone and mineral metabolism, it also plays an important role in cancer prevention and treatment.2 It is known that vitamin D can exert significant antiproliferative effects (growth inhibition, increased apoptosis and cell cycle arrest) in many human cancer cells such as thyroid, prostate, breast, colorectal and lung cancers.1,3–5 VDR knockout mice display a higher incidence of carcinogen-induced breast and skin tumours,6 and vitamin D deficiency promotes human breast cancer growth.7 Clinical trials have shown potential therapeutic effects of calcitriol in prostate cancer patients.8 Vitamin D 24-hydroxylase (CYP24A1) is the primary vitamin D-inactivating enzyme, which catabolizes 1a, 25(OH)2D and to a lesser extent, 25(OH)2D by 24 hydroxylation into inactive 1a, 24,25(OH)3D and 24,25(OH)2D. The vitamin D-mediated antiproliferative effects can be disrupted by overexpression of CYP24A1 during tumour development.3 Increased expression of CYP24A1 has been observed in many human cancers including thyroid,9 lung,10 colon,11 oesophageal,12 and breast13 and has been linked to poor prognosis in patients with lung and oesophageal cancers.12,14 The diminished antitumour effects of calcitriol can be restored by inhibition of CYP24A1.15 In patients with 1
2 M. Zou et al. thyroid cancer, serum level of 1a, 25(OH)2D3 is significantly lower16 although there is no significant difference in serum 25 (OH) D3 level between thyroid nodule and thyroid cancer patients,16,17 indicating that 1a, 25(OH)2D3 might be converted into inactive 1a, 24,25(OH)3D3 by increased CYP24A1 expression. It has been reported that vitamin D can inhibit thyroid cancer cell growth with varying degrees of sensitivity.4,18,19 The sensitive thyroid cancer cell lines (TPC-1, C643 and HTh74) are associated with lower baseline CYP24A1 expression, whereas the resistant cell lines (8505C, SW1736, KTC-1, BCPAP and HTh7) have higher CYP24A1 expression and/or poor response to calcitriol stimulation. Interestingly, the resistant cell lines often carry BRAF (8505C, SW1736, BCPAP) mutation, which raise the question of whether BRAF mutation can cause elevated expression of CYP24A1 and result in resistance to calcitriol treatment. In this study, we investigated CYP24A1 expression and BRAF mutation in 60 PTC from Saudi Arabia to determine whether CYP24A1 expression is associated with disease progression and BRAF mutation is involved in inducing CYP24A1 expression. We found that CYP24A1 expression is increased and the level of expression is associated with disease progression and poor prognosis. The higher level of CYP24A1 expression is often correlated with BRAF mutation. Furthermore, BRAFV600E inhibitor PLX4720 can significantly enhance the antiproliferative effects of calcitriol in thyroid cancer cell lines.
Materials and methods Thyroid tumour specimens All tumour tissues were obtained at surgery with informed consent and were immediately frozen in liquid nitrogen and stored at 70 °C until processed. The clinical staging of thyroid cancer was based on the TNM classification.20 Sixty PTC diagnosed between 1987 and 2006 were selected randomly and included in the study: 55 CPTC (classic PTC), 3 tall cell variants and 2 FVPTC (follicular variant of PTC). Eleven normal thyroid tissues removed from tumour surrounding, and 11 multinodular goitres were also included in the study. The study was approved by the institutional review board (Office of Research Affairs at King Faisal Specialist Hospital and Research Centre).
with RET/PTC-1 rearrangement. The cell lines were propagated in DMEM/Ham’s F12 medium containing 10% foetal bovine serum, 100 units/ml penicillin and 100 lg/ml streptomycin at 37 °C with 5% CO2 in a humidified environment. Quantitative real-time reverse transcriptase (RT)-PCR analysis for CYP24A1 expression Total RNAs from normal thyroid, thyroid tumour and cell lines were isolated by guanidinium thiocyanate-phenol-chloroform method. The integrity of RNA was verified by denaturing gel electrophoresis. Two lg of each total RNA was reverse-transcribed to cDNA using the Promega RT system (Promega, Madison, WI, USA). LightCycler DNA Master SYBR Green 1 kit was used for quantitative real-time PCR analysis.21 The cDNA mix was diluted 10-fold, and 2 ll of the dilution was used for realtime PCR analysis. PCR primers for the 154-bp CYP24A1 cDNA fragment were as follows: 5′-GGAAGTGATGAAGCTGGACAA CA-3′ (sense, located in exon 3) and 5′-CTCATACAACACG AGGCAGATAC-3′ (antisense, located in exon 5). The sense primer spans over intron 3 and 4 of 5 kb so that the contaminated genomic DNA will not be amplified. The CYP24A1 cDNA fragment was verified by DNA sequencing. The mRNA level of housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and a 300-bp PCR product was amplified using the following two primers: 5′-ACAG TCAGCCGCATCTTCTT-3′ (sense) and 5′-TTGATTTTGGAGG GATCTCG-3′ (antisense). The PCR conditions are 95 °C for 30 s followed by 30 cycles of amplification (95 °C for 10 s, 48 °C for 5 s and 72 °C for 10 s). The resulting concentration of CYP24A1 PCR products was normalized by comparison with GAPDH and was used to determine the relative mRNA level of CYP24A1 among thyroid tumour specimens. Detection of BRAF mutation Tumour tissues were obtained by standard sectioning, and DNA was extracted by standard proteinase-K treatment followed by phenol/chloroform extraction. BRAF exon 15 was amplified by PCR and directly sequenced as described previously.22 Induction of CYP24A1 expression by BRAFV600E
Cell lines and cell culture The following cell lines were studied for CYP24A1 expression: N-Thy, K1, BCPAP, 8505C, BHT101, CAL62, TPC-1 and FRO. N-thy is a normal thyroid follicular cell line immortalized by SV40 and was purchased from the European Collection of Animal Cell Culture (Salisbury, UK). K1, BCPAP, 8505C, BHT101 and CAL62 cell lines were purchased from DSMZ (Braunschweig, Germany). TPC-1 and FRO were kindly provided by Dr. James Fagin (Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York). K1 and BCPAP are PTC cell lines with BRAFV600E mutation. 8505C, BHT101 and FRO are anaplastic thyroid carcinoma (ATC) cell lines with BRAFV600E mutation, and CAL62 is an ATC cell line with KRASG12R mutation; TPC-1 is a PTC cell line
The BRAFV600E cDNA was cloned into pcDNA3.1 as described previously.22 HRASG12V cDNA was obtained from Biomyx, CA and subcloned into pcDNA3.1 vector as well. 20 lg of each construct and vector control were transfected into CAL62 cell line using Lipofectamine (Invitrogen, Carlsbad, CA, USA) and then selected for 4 weeks with 400 lg/ml G418. Stable clones were pooled and used to measure CYP24A1 expression and phosphoERK 1/2 activity. Cell proliferation assay Cell proliferation was measured by a nonradioactive MTT assay kit according to manufacturer’s procedure (Promega Corp, © 2014 John Wiley & Sons Ltd Clinical Endocrinology (2014), 0, 1–8
CYP24A1 activation in thyroid carcinoma 3 Madison, WI, USA). Briefly, cells were plated into triplicate wells of 96-well plates (103 cells/well) in growth medium containing vehicle (1% ethanol or DMSO or both), 100 nM calcitriol (Sigma-Aldrich, St. Louis, MO, USA), 100 nM PLX4720 (Selleck Chemicals, Houston, TX, USA) or a combination of calcitriol and PLX4720 for 72 h. The medium was changed, and cells were cultured for additional 24 h in the growth medium containing the same concentration of drugs. At the final 2 h of incubation, 20 ll of CellTiter 96â AQueous One Solution reagent was added into each well for the measurement of cell viability. Western blot analysis Protein extracts (40 lg/lane) from cells were loaded onto a 12% SDS-polyacrylamide gel. Proteins were transferred to a PVDF membrane and subject to Western blot analysis using antiphospho-ERK 1/2 antibody (1:1000, Cell Signaling Technology, Inc, Danvers, MA, USA) and anti-CYP24A1 antibody (1:3000, OriGene, Inc, Rockville, MD, USA). Statistical analysis The significant difference in CYP24A1 gene expression and cell proliferation among different groups was carried out using the unpaired Student’s t-test (two-tailed). Fisher’s exact test (twotailed) was used to compare BRAF mutation rate among different stages of cancer. A P-value of 005 or less was considered significant.
Results CYP24A1 expression in thyroid cancer CYP24A1 mRNA expression was investigated in 11 paired PTC, 11 multinodular goitres and 49 nonpaired PTC by real-time RTPCR. The same group of samples was also screened for BRAF mutation. As shown in Fig. 1(a) and Table S1, there is significant difference in CYP24A1 expression between normal thyroid and matched tumour tissues (32 06 vs 571 148, P < 001).There was no significant difference in CYP24A1 expression between normal thyroids and multinodular goitres (32 06 vs 28 05, P = 03, n = 11, Fig. 1b). Increased expression of CYP24A1 was observed in 41 PTC samples (68%) as compared to normal thyroids and multinodular goitres. The difference in CYP24A1 mRNA expression between 60 PTC vs normal thyroids and multinodular goitres was statistically significant (704 179 vs 30 04, P = 0025, Fig. 1b). BRAFV600E was detected in 28 samples (47%), and its frequency was significantly higher in advanced stage (stage III and IV, P < 002, Fig. 1c & d). No BRAFV600E was detected in normal controls and nodular goitres. As there was a large variation in CYP24A1 mRNA expression among PTC samples and CYP24A1 mRNA expression was higher in PTC with BRAFV600E, we stratified PTC samples into BRAFV600E-positive and BRAFV600E-negative groups to see whether increased CYP24A1 expression was associated with BRAFV600E. As shown in Fig. 2, the variation was © 2014 John Wiley & Sons Ltd Clinical Endocrinology (2014), 0, 1–8
significantly reduced and CYP24A1 expression was significantly higher in PTC with BRAFV600E (1356 345, n = 28) as compared to those without BRAFV600E (106 18, n = 29, P < 0001). CYP24A1 expression was still higher in BRAFV600Enegative PTC as compared to multinodular goitres (1327 23 vs 28 05, P < 005). We next examined 7 thyroid cancer cell lines to see whether CYP24A1 expression was higher in cell lines harbouring BRAFV600E. As shown in Fig. 3, CYP24A1 expression in mRNA and protein was indeed higher in BRAFV600E-positive cell lines (BHT101, FRO, 8305C, K1, and BCPAP) than BRAFV600E-negative cell lines (CAL62 and TPC-1). Interestingly, CAL62 and TPC-1 harbour KRASG12R and RET/PTC-1 rearrangement, respectively, which results in MAP kinase activation like BRAFV600E. CYP24A1 expression in different stages of thyroid cancer CYP24A1 overexpression was linked to poor prognosis in patients with lung and oesophageal cancers12,14, we sought to investigate whether its overexpression was associated with advanced stage of PTC. As shown in Fig. 4, the level of CYP24A1 mRNA expression is higher in stage III (739 362, n = 10) and IV (1969 872, n = 10) PTC as compared to stage I (171 37, n = 30) and II (437 216, n = 10) PTC. The difference in CYP24A1 mRNA expression between early stage (stage I and II) and late stage (stage III and IV) PTC was statistically significant (237 61, n = 40 vs 1354 481, n = 20, P < 001). When PTC samples were stratified into BRAFV600E-positive and BRAFV600E-negative groups and analysed separately, the difference in CYP24A1 mRNA expression was observed only in PTC with BRAFV600E: 519 119 in early stage (n = 14) and 1891 639 in late stage (n = 14, P < 005). There was no significant difference in CYP24A1 mRNA expression among PTC without BRAFV600E: 133 27 in early stage (n = 25) and 101 48 in late stage (n = 6, P = 0595). Induction of CYP24A1 expression by BRAFV600E The strong association between CYP24A1 expression and BRAFV600E suggests that BRAFV600E mutation may result in CYP24A1 overexpression. To confirm the hypothesis, we transfected BRAFV600E or HRASG12V into CAL62 cells and CYP24A1 expression was determined by Western blot analysis. As shown in Fig. 5, CYP24A1 expression was increased following BRAFV600E transfection. The induction of CYP24A1 expression by HRASG12V was much less than BRAFV600E although HRASG12V caused higher p-ERK expression than BRAFV600E (Fig. 5). Reduction of CYP24A1 expression by BRAFV600E inhibitor PLX4720 As BRAFV600E can induce CYP24A1 expression, we next investigated whether PLX4720 could reduce CYP24A1 expression and enhance or sensitize calcitriol-mediated antiproliferative effects in thyroid cancer cell lines. Thyroid cancer cell lines were treated
4 M. Zou et al. (a)
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Fig. 1 CYP24A1 gene expression and BRAF mutation among normal, multinodular goitre and PTC. (a) The CYP24A1 expression in 11 paired normal and PTC samples. CYP24A1 mRNA level was quantified by real-time RT-PCR. Data are expressed as relative expression of CYP24A1 after normalization to GAPDH expression in each sample.* indicate BRAFV600E mutation in the sample. (b) CYP24A1 expression among normal, multinodular goitre and PTC. A column scatter graph is used to show each data point in a group with a line at the mean. (c) Frequency of BRAF mutation in different stages of PTC. BRAFV600E mutation rate is significantly higher in advanced stage of PTC (stage III and IV). No mutation was detected in the goitres. (d) A representative sequence electropherogram is shown and BRAFV600E mutation from 3 patients is indicated by an arrow.
with calcitriol in the presence or absence of different concentrations of PLX4720, and CYP24A1mRNA level was measured by quantitative real-time RT-PCR. As shown in Fig. 6(a), PLX4720 could significantly reduce calcitriol-induced CYP24A1 mRNA expression (P < 001) in the cell lines, and it was dose-dependent. The calcitriol-mediated growth inhibition was also significantly increased as a result of PLX4720 treatment, especially in cell lines harbouring BRAFV600E (8505C, FRO and K1) (Fig. 6b, P < 001).
Discussion In the present study, we reported for the first time that CYP24A1 overexpression was associated with late stage of PTC and BRAFV600E could drive CYP24A1 overexpression, linking
vitamin D and MAPK signalling pathways in thyroid cancer progression. Inhibition of MAPK can reduce CYP24A1 expression and significantly enhance calcitriol-mediated antiproliferative effects. Overexpression of CYP24A1 has been reported in many cancers and can lead to abrogation of growth control mediated by vitamin D. It has thus been proposed as a candidate oncogene.23 Multiple mechanisms are involved in its overexpression: for example, gene amplification in breast and colon cancers,23,24 down-regulation of miR-125b in breast cancer25 and activation of protein kinase CK2 in human prostate cancer.26 miR-125b regulates CYP24A1 expression post-transcriptionally, and its expression is inversely correlated with CYP24A1 protein level.25 Interestingly, miR-125b is down-regulated in anaplastic thyroid carcinoma.27 CK2 is a multitask kinase and is overexpressed in © 2014 John Wiley & Sons Ltd Clinical Endocrinology (2014), 0, 1–8
CYP24A1 activation in thyroid carcinoma 5 (a)
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many solid tumours.28 CK2 is involved in many cellular processes related to cell growth and survival.28 In the present study, we have demonstrated that BRAFV600E, a key component in the MAPK signalling, can cause CYP24A1 overexpression. It is not clear why similar effect was not observed in cells with KRASG12R (CAL62) or RET/TPC-1 (TPC-1) mutation. BRAFV600E may cause more severe disruption of normal vitamin D signalling than KRASG12R or RET/TPC-1. Early studies4,18 have shown that basal level of CYP24A1 expression in TPC-1 is low and can be significantly induced by calcitriol as compared to other thyroid cancer cell lines harbouring BRAFV600E such as 8505C, BCPAP, SW1736 and KTC, indicating relative normal vitamin D signalling. TPC-1 is also sensitive to calcitriol-mediated growth inhibition. C643 (ATC with HRASG13R) and HTh74 (ATC with no BRAF or RAS mutation) are two other cell lines responsive to calcitriol-mediated antiproliferative effects. Again, in these two cell lines, the basal level of CYP24A1 is low and its expression can be significantly induced by calcitriol.4,18 Although the basal levels of CYP24A1 are low in CAL62 (current study) and HTh7 [ATC with NRASQ61K, previous studies],4,18 they are not responsive to calcitriol stimulation and resistance to calcitriol-mediated growth inhibition. This may reflect the loss of vitamin D signalling resulting from de-differentiation of ATC. These data suggest that the sensitivity of calcitriol antitumour activity is dependent on the level of CYP24A1 expression and/or the integrity of vitamin D signalling pathway in tumour cells. © 2014 John Wiley & Sons Ltd Clinical Endocrinology (2014), 0, 1–8
Fig. 3 CYP24A1 gene expression in thyroid cancer cell lines. (a) The CYP24A1 mRNA level in each cell line was quantified by real-time RT-PCR. Data are expressed as relative expression of CYP24A1 after normalization to GAPDH expression in each sample. (b) The CYP24A1 protein level in each cell line was analysed by Western blot analysis. Protein extracts (40 lg/lane) were loaded onto a 12% SDSpolyacrylamide gel. The blot was probed with anti-CYP24A1 antibody (upper panel) and reprobed with anti-GAPDH antibody (lower panel). Lane 1: BHI101; lane 2: TPC-1; lane 3: BCPAP; lane 4: CAL62; lane 5: K1; lane 6:8505; lane 7: FRO; lane 8: N-thy.
Vitamin D signalling pathway is compromised during tumorigenesis as evidenced by down-regulation of VDR and vitamin D independent up-regulation of CYP24A1 in many cancers. Constitutive activation of MAPK pathway has been shown to cause impairment of vitamin D signalling in human prostate epithelial cells.29 In our current study, we have demonstrated strong correlation of CYP24A1 expression with advanced disease stage and BRAFV600E. It is known that BRAFV600E is associated with poor prognosis in PTC.30 The association of CYP24A1 expression with advanced PTC is likely due to BRAFV600E, because BRAFV600E can induce higher CYP24A1 expression and BRAFV600E inhibitor PLX4720 results in down-regulation of CYP24A1. Indeed, MAP kinases have been shown to participate in the calcitriol-induced transactivation of CYP24A1 promoter,31 and the functional consequence of the crosstalk between vitamin D and MAPK pathways is cell type specific.32 In PTC, we have shown that the crosstalk results in increased CYP24A1 expression and resistance to calcitriol-mediated growth inhibition.
6 M. Zou et al. 6
CYP24A1 mRNA (fold change compared to baseline)
(a)
Although synthesis and degradation of calcitriol occur mainly in the kidney, they are also present in extra renal tissues. It is known that the genes (VDR, CYP27A1, CYP2R1, CYP27B1 and CYP24A1) involved in vitamin D metabolism and signalling are expressed in thyroid epithelial cells and thyroid cancer cells.4,18 The major role of 1a, 25(OH)2D derived from autocrine/paracrine mechanism is to inhibit cell proliferation and induce cell differentiation. However, overexpression of CYP24A1 in cancer cells may affect bioavailability and antiproliferative activity of 1a, 25(OH)2D. Inhibition of CYP24A1 could increase 1a, 25 (OH)2D-mediated antitumour activity by reducing its degradation. Indeed, the therapeutic effects of CYP24A1 inhibitors such as ketoconazole and CTA091 have been seen both in vitro and
3 2 1
8505C
FRO
K1
CAL62 Calcitriol PLX4720 PLX4720+Calcitriol
(b) 40
30
20
10
0
Fig. 5 Induction of CYP24A1 expression by BRAFV600E in CAL62 cells. CAL62 cells expressing BRAFV600E or HRASG12V plasmid were subjected to Western blot analysis for CYP24A1 expression (top). The same blot was reprobed with p-ERK (middle) and GAPDH (bottom). Increased CYP24A1 expression was observed in BRAFV600E transfected cells, but not in HRASG12V transfected cells, although p-ERK level was higher in HRASG12V transfected cells. Lane 1: CAL62 cells expressing BRAFV600E; lane 2: CAL62 cells expressing HRASG12V; lane 3: CAL62 cells transfected with vector only.
4
0
Growth inhibition (% of control)
Fig. 4 CYP24A1 expression among different stages of thyroid tumours. CYP24A1mRNA levels were quantified by real-time RT-PCR and normalized using the expression of GAPDH. Data are expressed as relative CYP24A1 expression among different tumour stages. A column scatter graph is used to show each data point in a group with a line at the mean.
Calcitriol Calcitriol+1 µM PLX Calcitriol+0·1 µMPLX
5
8505C
FRO
K1
CAL62
Fig. 6 Effects of calcitriol and PLX4720 on CYP24A1 expression and cell proliferation in thyroid cancer cell lines. (a) Induction of CYP24A1 expression by calcitriol and attenuation of its effect by PLX4720. Cells were plated into 12-well plates (2 9 104 cells/well) in growth medium for 24 h and then treated with vehicle, 100 nM calcitriol, 100 nM calcitriol + 01 lM PLX4720 or 100 nM calcitriol + 1 lM PLX4720 for 4 h. RNA was extracted from the cells, and CYP24A1 expression was determined by real-time RT-PCR. CYP24A1 expression in cells treated with vehicle was used as baseline control. Data are expressed as fold change in CYP24A1 expression compared to baseline. Each data point represents the mean SEM of three independent experiments. (b) Inhibition of cell proliferation by calcitriol and PLX4720. Cells were treated with 100 nM calcitriol, 100 nM PLX4720 or a combination of 100 nM calcitriol and 100 nM PLX4720 for 4 days. Cells treated with vehicle (1% ethanol or DMSO) were used as control. Cell proliferation was measured by a modified MTT assay. Data are expressed as % of reduction in cell proliferation compared to vehicle treatment. Each data point represents the mean SEM of three independent experiments.
in vivo.15,33 Our data show that BRAFV600E inhibitor PLX4720 can also down-regulate CYP24A1 expression and enhance calcitriol-mediated antitumour activity. PLX4720 is effective against thyroid cancer harbouring BRAFV600E in preclinical studies.34,35 Given that PLX4720 can inhibit both CYP24A1 and BRAFV600E, combination of PLX4720 and calcitriol may offer better treatment outcome than combined CYP24A1 inhibitors and calcitriol. © 2014 John Wiley & Sons Ltd Clinical Endocrinology (2014), 0, 1–8
CYP24A1 activation in thyroid carcinoma 7
Limitations of the study To study CYP24A1 expression in thyroid cancer, the best control is paired samples taken from both cancerous and normal tissues of the same thyroid gland. Due to the limitation to obtain normal thyroid tissue, we only studied 11 paired samples. The sample size is relatively small especially in the late stage of thyroid cancer.
Disclosure of conflict of interests All authors have nothing to declare.
Acknowledgement This study was supported by the King Abdulaziz City of Science and Technology (KACST) Biotechnology grant.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. CYP24A1 Expression and BRAF mutation in PTC.
© 2014 John Wiley & Sons Ltd Clinical Endocrinology (2014), 0, 1–8
