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CYP24A1 is a potential biomarker for the progression and prognosis of human colorectal cancer
Human Pathology (2016) 50, 101–108
www.elsevier.com/locate/humpath
Original contribution
CYP24A1 is a potential biomarker for the progression and prognosis of human colorectal cancer☆,☆☆ Hongyan Sun MD a,b , Chuanwen Wang MD c , Miao Hao PhD d , Ran Sun MD d , Yuqian Wang PhD d , Tie Liu MD a , Xianling Cong MD a,⁎,1 , Ya Liu MD b,⁎⁎,1 a
Department of Pathology, China-Japan Union Hospital of Jilin University, Changchun, Jilin Province 130033, China Department of Nutrition and Food Hygiene, School of Public Health, Jilin University, Changchun, Jilin Province 130021, China c Department of Radiological Health, Occupation Disease Prevention and Control Center of Jilin Province, Changchun, Jilin Province 130021, China d Scientific Research Center, China-Japan Union Hospital of Jilin University, Changchun, Jilin Province 130033, China b
Received 12 September 2015; revised 15 November 2015; accepted 18 November 2015
Keywords: CYP24A1; Colorectal cancer; Immunohistochemistry; Prognosis; Oncogene; Tissue microarray
Summary Our study aims to fully evaluate clinicopathological and prognostic values of CYP24A1 in colorectal cancer (CRC) patients. Tissue microarrays of formalin-fixed and paraffin-embedded tumor samples and matched adjacent nontumor colorectal tissues from 99 CRC patients were studied for CYP24A1 protein expression by immunohistochemistry. Messenger RNA expression of CYP24A1 was further evaluated by quantitative real-time polymerase chain reaction in 12 pairs of fresh frozen CRC samples. CYP24A1 expression was significantly higher in CRC tissues compared to corresponding noncancerous tissues. The expression of CYP24A1 protein in CRC was correlated with the depth of tumor invasion (P = .000), lymph node metastasis (P = .030), venous permeation (P = .016), and overall survival (P = .008). A Kaplan-Meier analysis of the CRC patients with high CYP24A1 expression showed significantly reduced overall survival and disease-free survival compared to the patients with low expression (P = 0.026 and .009). A prognostic significance of CYP24A1 was also found in the subgroup of venous permeation condition classification. A multivariate Cox regression analysis showed that CYP24A1 expression was an independent prognostic factor for CRC recurrence (P = .032). In conclusion, CYP24A1 expression is closely associated with CRC progression, and it might be a novel prognostic biomarker for CRC. © 2016 Elsevier Inc. All rights reserved.
☆
Competing interests: The authors declare that they have no conflict of interest. Funding/Support: This study was funded by the National Nature Scientific Foundation of China (nos. 81472209 and 81401966) and the Scientific Research Foundation of Jilin Province (nos. 20130206001YY and 20140414061GH). ⁎ Correspondence to: X. Cong, MD, Department of Pathology, China-Japan Union Hospital of Jilin University, Xiantai Road 163, Changchun, Jilin 130033, PR China. ⁎⁎ Correspondence to: Y. Liu, MD, Department of Nutrition and Food Hygiene, School of Public Health, Jilin University, Xinmin Road 1163, Changchun, Jilin 130021, PR China. E-mail addresses: [email protected] (X. Cong), [email protected] (Y. Liu). 1 Ya Liu and Xianling Cong contributed equally to this article. ☆☆
http://dx.doi.org/10.1016/j.humpath.2015.11.008 0046-8177/© 2016 Elsevier Inc. All rights reserved.
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1. Introduction Colorectal cancer (CRC) is the third most common type of malignancy worldwide, with an estimated incidence of 1.2 million newly diagnosed cases and 600 000 cancer deaths every year [1]. It is the third leading cause of cancer-related mortality in both males and females in the United States [2]. Meanwhile, the incidence of CRC is rapidly increasing in several Eastern Asian and Eastern European countries, including China, Japan, and the Czech Republic [3]. Despite the development of therapeutic modalities for CRC treatment, including surgery and a combination of chemotherapy and adjuvant therapy, a large portion of CRC patients, especially advanced CRC patients, develop local recurrence or metastasis as the disease progresses, which ultimately leads to death. The overall prognosis of CRC is still unsatisfactory, and the 5-year survival rate of CRC patients remains low [4,5]. Consequently, it is critical to focus research on the detection of diagnostic and prognostic biomarkers that could inform therapeutic strategies and prolong the lives of CRC patients [6]. Accumulating data indicate that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays an important role in inhibiting proliferation, invasion, and metastasis in a variety of cancer cell types [7,8]. 24-Hydroxylase, encoded by the CYP24A1 gene, is the key enzyme that catabolizes 1,25(OH)2D3 to the less active calcitroic acid [9]. It has been reported that CYP24A1 is overexpressed in several human tumor types and that changes in CYP24A1 expression are associated with cancer development and progression [10-15]. Moreover, elevated tumor CYP24A1 expression is correlated with a poorer prognosis. Thus, CYP24A1 may be a candidate oncogene and an important biomarker for cancer diagnosis and prognosis [12,16]; however, the prognostic significance of CYP24A1 expression in CRC patients has not been examined. The purpose of the present study was to characterize the relationship between CYP24A1 expression and the clinicopathological features of CRC patients and to investigate whether CYP24A1 expression could be an independent prognostic factor for CRC. Our results show for the first time that CYP24A1 could be a novel predictor of poor prognosis in CRC patients.
2. Materials and methods 2.1. Patients and samples Formalin-fixed and paraffin-embedded primary CRC and matched adjacent nontumor colorectal tissues from 99 patients between 2009 and 2011 were collected for tissue microarray (TMA) construction. TMAs were constructed by ALPHELYS MiniCore series 3; 1-mm cores from donor blocks were transferred into a recipient block. None of the patients received preoperative radiation or chemotherapy. Among them, 12 pairs of fresh CRC tissues and matched normal tissues were selected for the detection of CYP24A1 messenger RNA (mRNA)
H. Sun et al. expression. The tissues were obtained directly after surgical resection, snap frozen in liquid nitrogen overnight, and then stored at −80°C until RNA extraction. The demographic and clinicopathological information for 99 CRC cases, including age, sex, tumor size, tumor location, tumor differentiation, depth of invasion, lymph node metastasis, venous permeation, perineural invasion, and Ki-67 expression, were simultaneously collected from each patient's medical records. Complete follow-up data were available until May 2015 for all CRC patients. Disease-free survival (DFS) was calculated from the date of tumor resection until the detection of tumor recurrence or last observation, and overall survival (OS) was calculated from the date of resection to the date of death or last follow-up. All tissue samples were obtained from the tissue bank of the China-Japan Union Hospital of Jilin University. The present study was approved by the Ethical Committee of the China-Japan Union Hospital of Jilin University and conducted with the informed consent of all patients.
2.2. Immunohistochemistry Immunohistochemistry (IHC) was performed using a standard avidin-biotin-peroxidase method. Four-micrometerthick sections of TMAs mentioned previously were deparaffinized in xylene, dehydrated in gradient concentrations of ethanol, and then subjected to high-pressure antigen retrieval in a pressure cooker for 3 minutes in preheated 10 mmol/L sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 10 minutes, and nonspecific staining was eliminated by incubating the sections with normal goat serum for 15 minutes at room temperature. The sections were incubated with the primary antibody (1:25 dilution) at 4°C overnight, then they were incubated overnight at 4°C with the primary antibody diluted at 1:25 (ab175976; Abcam, Cambridge, MA). After being washed with phosphate-buffered saline, sections were incubated with diluted biotinylated goat anti-rabbit secondary antibody for 10 minutes and then incubated with the avidin-biotin-peroxidase complex for another 10 minutes with repeated washing steps. Staining was visualized using 3,3′-diaminobenzidine solution (Maxim, Fuzhou, China). Sections were then counterstained with hematoxylin after dehydration and clearing with xylene. Coverslips were added to the slides and examined. Negative controls were obtained by omission of the primary antibody. CYP24A1 immunostaining was semiquantitatively assessed according to the intensity and percentage of positive cancer cells on the basis of an immunoreactive score [17]. Staining intensity was scored as follows: 0, none; 1, weak; 2, moderate; and 3, strong staining. The percentage of positively stained cells was scored as follows: 0, no staining; 1, less than 10% of cells; 2, 11% to 50% of cells; 3, 51% to 80% of cells; and 4, greater than 81% of cells stained. The product of the intensity and percentage scores was calculated as the final staining score, which ranged from 0 to 12; 0 indicated no staining, 1 indicated weak staining, 2 or 3 indicated moderate staining, and more than 3 indicated
CYP24A1 is a prognostic biomarker in colorectal cancer strong staining. In the final analysis, tissue samples with a score less than 4 were considered to have low CYP24A1 expression, whereas those with a score greater than or equal to 4 were considered to have high CYP24A1 expression [18,19]. IHC staining was scored by 2 independent pathologists.
2.3. Quantitative real-time polymerase chain reaction Total RNA was extracted from fresh frozen CRC tissues and the matched noncancerous tissues using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Extracted RNA samples were treated with RQ1 RNase-Free DNase (Promega, Madison, WI) to eliminate DNA contamination. Complementary DNA was prepared by reverse transcription using a Goscript Reverse Transcription System Kit (A5000; Promega) from 500 ng of total RNA. mRNA expression levels were quantified by quantitative real-time polymerase chain reaction (qPCR) with the SYBR Premix Ex Taq II (Takara Bio, Otsu, Shiga, Japan) with the following primers: CYP24A1: forward primer 5′-GCCGTATTTAAAAGCCTGTCTGAA-3′ and reverse primer 5′-ACCTGGGTATTTAGCATGAG CACTG-3′. The glyceraldehyde 3-phosphate dehydrogenase mRNA level was used to standardize the measurements of CYP24A1, and the primers for glyceraldehyde 3-phosphate dehydrogenase were as follows: forward primer 5′-AGAAGGCTGGGGCTCATTTG-3′ and reverse primer 5′-AGGGGCCATCCACAGTCTTC-3′. Relative mRNA levels were quantified by the comparative 2−ΔΔCt method.
2.4. Statistical analysis Statistical analysis was performed using SPSS 18.0 (SPSS, Chicago, IL). Correlations between CYP24A1 protein expression and clinicopathological factors were analyzed by Pearson χ2 test or Fisher exact probability test. The differences in CYP24A1 mRNA expression between carcinoma and normal samples were evaluated by the independent-sample t test. The correlation between mRNA expression and the IHC score was examined using Spearman correlation coefficients by rank test. Survival curves were calculated using the Kaplan-Meier method and compared using a log-rank test. Univariate and multivariate analyses were performed using Cox proportional hazards regression models. All of the tests were 2 sided, and P b .05 was considered statistically significant.
3. Results 3.1. Differential expression of CYP24A1 in normal colorectal tissues and CRC tissues IHC analysis was performed to investigate the role of CYP24A1 in CRC. Among the 99 paired tissues, high CYP24A1 expression was detected in 69 CRC tissues
103 (69.7%) and in only 20 tumor-adjacent nontumor tissues (20.2%). CYP24A1 expression was more frequently present in CRC tissues than in the adjacent nontumor tissues (P b .05). Expression of CYP24A1 was mainly localized to the cytoplasm of CRC cells. Typical IHC staining patterns for CYP24A1 in CRC are shown in Fig. 1. qPCR analysis further confirmed that CYP24A1 mRNA was significantly increased in the tumor tissues compared to matched nontumor tissues (7.27 ± 3.39 versus 1.21 ± 0.77; P b .05; Fig. 2A). Higher mRNA expression correlated with a higher IHC score (r = 0.89; P b .05; Fig. 2B).
3.2. Correlations between CYP24A1 protein expression and clinicopathological features The correlations between high CYP24A1 protein expression and clinicopathological features in 99 CRC patients are shown in Table 1. CYP24A1 protein expression was significantly associated with the depth of invasion (P = .000), lymph node metastasis (P = .030), lymphatic or venous permeation (P = .016), and OS (P = .008).
3.3. Survival analysis A Kaplan-Meier analysis of OS showed reduced survival in patients with high CYP24A1 protein expression compared to patients with low CYP24A1 protein expression (log-rank, P = .026) (Fig. 3A). The mean OS was 44.4 months in the low CYP24A1 expression group, whereas it was only 33.9 months in the high CYP24A1 expression group. Moreover, higher CYP24A1 expression was correlated with shorter DFS (log-rank, P = .009). The mean DFS was 40.1 months for patients with low CYP24A1 expression compared to 28.9 months for patients with high CYP24A1 protein expression (Fig. 3B). Univariate analysis showed that 7 factors, including tumor size (P = .015), tumor location (P = .025), tumor differentiation (P = .013), lymph node metastasis (P = .010), venous permeation (P = .000), perineural invasion (P = .009), and CYP24A1 expression (P = .036), were associated with OS and 5 factors showed a significant correlation with DFS (Table 2). These included histology differentiation (P = .023), lymph node metastasis (P = .001), venous permeation (P = .000), perineural invasion (P = .000), and CYP24A1 expression (P = .015). Multivariate analysis further confirmed that high CYP24A1 expression was an independent prognostic factor for DFS (P = .032), but not for OS currently (Table 3). We further analyzed the prognostic value of CYP24A1 expression in selective patient subgroups stratified according to the venous permeation condition. The 2-year DFS rate of the patients with high CYP24A1 expression was 76.7% in the subgroup without venous permeation, whereas it was only 19.9% in the venous permeation subgroup; CRC patients with higher CYP24A1 expression had shorter DFS than those with low expression when there was venous permeation (log-rank, P = .007, Fig. 3C). However,
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Fig. 1 Representative images of CYP24A1 expression in CRC tissues and corresponding noncancerous tissues. A-C, High immunohistochemical staining of CYP24A1 expression in CRC tissue samples. D-F, Low immunohistochemical staining of CYP24A1 expression in CRC tissues. G-I, High immunohistochemical staining of CYP24A1 expression in noncancerous tissues. J-L, Low immunohistochemical staining of CYP24A1 expression in noncancerous tissues. Original magnification ×40 (A, D, G, and J), ×200 (B, E, H, and K), and ×400 (C, F, I, and L).
Kaplan-Meier analysis of the subgroup without venous permeation did not show statistically significant differences between patients with different CYP24A1 expression levels (log-rank, P = .481; Fig. 3D).
4. Discussion Calcitriol, which is also known as 1,25(OH)2D3, is the active form of vitamin D. It not only plays an important role
in regulating calcium homeostasis and bone mineralization but also modulates cellular proliferation and differentiation in a variety of cell types, including cancer cells [20]. The level and biological activity of 1,25(OH)2D3 in tissues depends on the rates of its synthesis by CYP27B1 and inactivation by CYP24A1. CYP24A1 is a member of the cytochrome P450 enzyme family and the key enzyme that catabolizes 1,25(OH)2D3 into less active calcitroic acid through 24-hydroxylation, which is the rate-limiting step in vitamin D3 metabolism [21]. The expression level of the catabolic enzyme CYP24A1 is aberrantly increased in
CYP24A1 is a prognostic biomarker in colorectal cancer
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Fig. 2 CYP24A1 mRNA expression in CRC tissues and corresponding noncancerous tissues. A, The expression of CYP24A1 mRNA in CRC tissues was significantly higher than that in matched normal tissues (P b .05). B, Correlation between mRNA and protein expression of CYP24A1. Higher IHC score corresponded to higher mRNA level (r = 0.89; P b .05).
Table 1 Correlation between CYP24A1 expression and clinicopathological features of colorectal carcinoma Variable
Age (y) ≥60 b60 Sex Male Female Tumor size (cm) ≥5 b5 Tumor location Colon Rectum Tumor differentiation Well and moderate Poor Depth of invasion Tis-T2 T3-T4 Lymph node metastasis Yes No Venous permeation Yes No Perineural invasion Yes No Survival status Death Survival Ki-67 b50% ≥50%
No. of cases
CYP24A1 expression
P
High (n = 69)
Low (n = 30)
55 44
41 28
14 16
64 35
46 23
18 12
62 37
47 22
15 15
51 48
40 29
11 19
83 16
55 14
28 2
16 83
2 67
14 16
59 40
46 23
13 17
41 58
34 35
7 23
29 70
24 45
5 25
32 67
28 41
4 26
22 77
18 51
4 26
.241
.524
.087
.051
.137
.000
.030
.016
.069
.008
.161
several cancer cells. CYP24A1 was identified as a candidate oncogene first in breast cancer by the array comparative genomic hybridization method [22], and studies that have focused on CYP24A1 have revealed that up-regulation of CYP24A1 was associated with a poorer prognosis in some cancers [12,16]. For CRC, Horváth et al [23] compared CYP24A1 expression in normal human colon mucosa, colorectal adenomas, and adenocarcinomas, and the results showed that CYP24A1 was a potential biomarker for colorectal tumorigenesis; however, no studies have investigated the prognostic value of the expression of CYP24A1 in CRC patients. In this study, we found that CRC tissues demonstrated a higher expression of CYP24A1 compared with normal noncancerous tissues by IHC. Although Horváth et al [23] observed discordance between mRNA and protein expression levels, our qPCR results confirmed that higher CYP24A1 mRNA expression was detected in CRC tissues than in matched noncancerous tissues. These data were consistent with previous publications and suggested an oncogenic role of CYP24A1 in colorectal tumorigenesis [24,25]. Currently, the role of CYP24A1 cellular localization is controversial. Matusiak et al [25] for the first time noted that CYP24A1 was found exclusively in the nucleus of normal colonic epithelial cells but was mainly detected in the cytoplasm of CRC cells, suggesting that translocation of this protein from the nucleus to the cytoplasm is a function of malignant progression. However, in the study of Horváth et al [23], expression of CYP24A1 was only detected in the cytoplasm of cells. In the present study, we found that CYP24A1 was predominantly located in the cytoplasm both in normal colonic epithelial cells and CRC cells, which was not consistent with the study of Horváth et al [23]. This discrepancy might be due to antibody specificity. CYP24A1 is located in the inner mitochondrial membrane, where it is a member of the mini-electron transport chain consisting of ferredoxin, NADPH-ferredoxin reductase, and the terminal cytochrome CYP24A1. Thus, whether CYP24A1 is
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Fig. 3 The prognostic significance of CYP24A1 evaluated by Kaplan-Meier analysis. The OS (A) and DFS (B) of patients with high CYP24A1 expression were significantly shorter than those of patients with low expression. CRC patients with high CYP24A1 expression had shorter DFS than those with low expression in venous permeation subgroup (C); no statistically significant difference in DFS rate between patients with different expression CYP24A1 level was found in the subgroup without venous permeation (D).
functional outside the mitochondrion and a result of malignant progression needs further investigation. In this study, the expression of CYP24A1 in CRC was correlated with several clinical features, including the depth of tumor invasion, lymph node metastasis, venous permeation, and OS, which further confirm a prooncogenic effect of CYP24A1. The potential mechanism might be that the up-regulation of CYP24A1 leads to rapid inactivation of 1,25(OH)2D3, thus diminishing the effect of 1,25(OH)2D3 in modulating the proliferation and angiogenesis of CRC cells [26-29]. However, our finding contradicts the results of
Table 2
Horváth et al [23], who reported that CYP24A1 expression was not related to TNM stage and vessel invasion. A possible reason for this discrepancy is the different sample sizes. We detected CYP24A1 expression in 99 CRC patients, whereas only 48 cases were included in the study of Horváth et al. There was no correlation between the proliferation marker Ki-67 and CYP24A1 protein expression in CRC patients in our study. Horváth et al [23] reported the same result in carcinomas but a strong correlation between CYP24A1 and Ki-67 expression in the normal and benign samples, which suggested that the vitamin D system might play a more
Univariate analysis of prognostic factors associated with survival and recurrence
Variables Age (y), ≥60 vs b60 Sex, male vs female Tumor size (cm), ≥5 vs b5 Tumor location, colon vs rectum Tumor differentiation, well and moderate vs poor Depth of invasion, Tis-T2 vs T3-T4 Lymph node metastasis, yes vs no Venous permeation, yes vs no Perineural invasion, yes vs no Ki-67, b50% vs ≥50% CYP24A1 expression, high vs low
OS
DFS
Hazard ratio (95% CI)
P
Hazard ratio (95% CI)
P
1.172 (0.578-2.374) 1.546 (0.713-3.351) 3.014 (1.240-7.328) 2.359 (1.117-4.983) 0.388 (0.183-0.822) 0.038 (0.001-2.632) 3.226 (1.327-7.839) 4.358 (2.013-9.437) 2.528 (1.260-5.073) 1.229 (0.552-2.737) 3.072 (1.077-8.764)
.660 .270 .015 a .025 a .013 a .131 .010 a .000 a .009 a .614 .036 a
1.561 (0.794-3.071) 0.871 (0.451-1.685) 1.433 (0.720-2.854) 1.279 (0.669-2.443) 0.429 (0.207-0.889) 0.037 (0.001-1.678) 4.513 (1.872-10.879) 5.149 (2.525-10.499) 3.23 (1.685-6.192) 0.981 (0.448-2.147) 3.221 (1.225-8.270)
.197 .682 .306 .457 .023 a .090 .001 a .000 a .000 a .962 .015 a
Abbreviations: OS, overall survival; DFS, disease-free survival; CI, confidence interval. a Statistically significant.
CYP24A1 is a prognostic biomarker in colorectal cancer
the China-Japan Union Hospital of Jilin University and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Table 3 Multivariate Cox regression analysis of prognostic factors associated with OS and DFS Variables
Multivariate analysis Hazard ratio (95% CI)
OS Tumor size (cm), ≥5 vs b5 Tumor location, colon vs rectum Venous permeation, yes vs no CYP24A1 expression, high vs low DFS Venous permeation, yes vs no CYP24A1 expression, high vs low
107
P
References 2.493 (1.012-6.141) 2.210 (1.036-4.715) 4.348 (2.005-9.431) 1.875 (0.639-5.500)
a
.047 .040 a .000 a .252
4.923 (2.384-10.168) .000 a 2.829 (1.092-7.330) .032 a
Abbreviations: OS, overall survival; DFS, disease-free survival; CI, confidence interval. a Statistically significant.
important role in the prevention of cancer and early tumorigenesis. Therefore, inhibition of CYP24A1 would be a reasonable option when using vitamin D3 for CRC prevention and treatment. Kaplan-Meier analysis and the log-rank test suggested that the expression level of CYP24A1 in CRC was significantly associated with patients' OS and DFS. The OS and DFS of patients with high CYP24A1 expression were shorter than those of patients with low expression. A multivariate Cox regression analysis further confirmed that CYP24A1 protein expression was an independent negative prognostic factor for DFS. This finding indicated that CYP24A1 was sufficient to predict early recurrence and metastasis in CRC patients after surgery. The venous permeation condition was also an independent prognostic factor for DFS, and we further analyzed the prognostic value of CYP24A1 expression in selective patient subgroups stratified according to the venous permeation condition. The results of Kaplan-Meier analysis showed that CRC patients with higher CYP24A1 expression had shorter DFS than those with low expression when there is venous permeation, which suggested CYP24A1 expression can be combined with venous permeation status to predict early recurrence in CRC patients more accurately. In conclusion, our results demonstrate for the first time that elevated CYP24A1 protein expression is associated with significantly reduced DFS and OS in CRC patients. CYP24A1 might be a novel independent prognostic biomarker for CRC, which could serve as a powerful new molecular target in the prevention and treatment of CRC in the future.
Acknowledgment All procedures performed in studies involving human participants were in accordance with the ethical standards of
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Original contribution
CYP24A1 is a potential biomarker for the progression and prognosis of human colorectal cancer☆,☆☆ Hongyan Sun MD a,b , Chuanwen Wang MD c , Miao Hao PhD d , Ran Sun MD d , Yuqian Wang PhD d , Tie Liu MD a , Xianling Cong MD a,⁎,1 , Ya Liu MD b,⁎⁎,1 a
Department of Pathology, China-Japan Union Hospital of Jilin University, Changchun, Jilin Province 130033, China Department of Nutrition and Food Hygiene, School of Public Health, Jilin University, Changchun, Jilin Province 130021, China c Department of Radiological Health, Occupation Disease Prevention and Control Center of Jilin Province, Changchun, Jilin Province 130021, China d Scientific Research Center, China-Japan Union Hospital of Jilin University, Changchun, Jilin Province 130033, China b
Received 12 September 2015; revised 15 November 2015; accepted 18 November 2015
Keywords: CYP24A1; Colorectal cancer; Immunohistochemistry; Prognosis; Oncogene; Tissue microarray
Summary Our study aims to fully evaluate clinicopathological and prognostic values of CYP24A1 in colorectal cancer (CRC) patients. Tissue microarrays of formalin-fixed and paraffin-embedded tumor samples and matched adjacent nontumor colorectal tissues from 99 CRC patients were studied for CYP24A1 protein expression by immunohistochemistry. Messenger RNA expression of CYP24A1 was further evaluated by quantitative real-time polymerase chain reaction in 12 pairs of fresh frozen CRC samples. CYP24A1 expression was significantly higher in CRC tissues compared to corresponding noncancerous tissues. The expression of CYP24A1 protein in CRC was correlated with the depth of tumor invasion (P = .000), lymph node metastasis (P = .030), venous permeation (P = .016), and overall survival (P = .008). A Kaplan-Meier analysis of the CRC patients with high CYP24A1 expression showed significantly reduced overall survival and disease-free survival compared to the patients with low expression (P = 0.026 and .009). A prognostic significance of CYP24A1 was also found in the subgroup of venous permeation condition classification. A multivariate Cox regression analysis showed that CYP24A1 expression was an independent prognostic factor for CRC recurrence (P = .032). In conclusion, CYP24A1 expression is closely associated with CRC progression, and it might be a novel prognostic biomarker for CRC. © 2016 Elsevier Inc. All rights reserved.
☆
Competing interests: The authors declare that they have no conflict of interest. Funding/Support: This study was funded by the National Nature Scientific Foundation of China (nos. 81472209 and 81401966) and the Scientific Research Foundation of Jilin Province (nos. 20130206001YY and 20140414061GH). ⁎ Correspondence to: X. Cong, MD, Department of Pathology, China-Japan Union Hospital of Jilin University, Xiantai Road 163, Changchun, Jilin 130033, PR China. ⁎⁎ Correspondence to: Y. Liu, MD, Department of Nutrition and Food Hygiene, School of Public Health, Jilin University, Xinmin Road 1163, Changchun, Jilin 130021, PR China. E-mail addresses: [email protected] (X. Cong), [email protected] (Y. Liu). 1 Ya Liu and Xianling Cong contributed equally to this article. ☆☆
http://dx.doi.org/10.1016/j.humpath.2015.11.008 0046-8177/© 2016 Elsevier Inc. All rights reserved.
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1. Introduction Colorectal cancer (CRC) is the third most common type of malignancy worldwide, with an estimated incidence of 1.2 million newly diagnosed cases and 600 000 cancer deaths every year [1]. It is the third leading cause of cancer-related mortality in both males and females in the United States [2]. Meanwhile, the incidence of CRC is rapidly increasing in several Eastern Asian and Eastern European countries, including China, Japan, and the Czech Republic [3]. Despite the development of therapeutic modalities for CRC treatment, including surgery and a combination of chemotherapy and adjuvant therapy, a large portion of CRC patients, especially advanced CRC patients, develop local recurrence or metastasis as the disease progresses, which ultimately leads to death. The overall prognosis of CRC is still unsatisfactory, and the 5-year survival rate of CRC patients remains low [4,5]. Consequently, it is critical to focus research on the detection of diagnostic and prognostic biomarkers that could inform therapeutic strategies and prolong the lives of CRC patients [6]. Accumulating data indicate that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays an important role in inhibiting proliferation, invasion, and metastasis in a variety of cancer cell types [7,8]. 24-Hydroxylase, encoded by the CYP24A1 gene, is the key enzyme that catabolizes 1,25(OH)2D3 to the less active calcitroic acid [9]. It has been reported that CYP24A1 is overexpressed in several human tumor types and that changes in CYP24A1 expression are associated with cancer development and progression [10-15]. Moreover, elevated tumor CYP24A1 expression is correlated with a poorer prognosis. Thus, CYP24A1 may be a candidate oncogene and an important biomarker for cancer diagnosis and prognosis [12,16]; however, the prognostic significance of CYP24A1 expression in CRC patients has not been examined. The purpose of the present study was to characterize the relationship between CYP24A1 expression and the clinicopathological features of CRC patients and to investigate whether CYP24A1 expression could be an independent prognostic factor for CRC. Our results show for the first time that CYP24A1 could be a novel predictor of poor prognosis in CRC patients.
2. Materials and methods 2.1. Patients and samples Formalin-fixed and paraffin-embedded primary CRC and matched adjacent nontumor colorectal tissues from 99 patients between 2009 and 2011 were collected for tissue microarray (TMA) construction. TMAs were constructed by ALPHELYS MiniCore series 3; 1-mm cores from donor blocks were transferred into a recipient block. None of the patients received preoperative radiation or chemotherapy. Among them, 12 pairs of fresh CRC tissues and matched normal tissues were selected for the detection of CYP24A1 messenger RNA (mRNA)
H. Sun et al. expression. The tissues were obtained directly after surgical resection, snap frozen in liquid nitrogen overnight, and then stored at −80°C until RNA extraction. The demographic and clinicopathological information for 99 CRC cases, including age, sex, tumor size, tumor location, tumor differentiation, depth of invasion, lymph node metastasis, venous permeation, perineural invasion, and Ki-67 expression, were simultaneously collected from each patient's medical records. Complete follow-up data were available until May 2015 for all CRC patients. Disease-free survival (DFS) was calculated from the date of tumor resection until the detection of tumor recurrence or last observation, and overall survival (OS) was calculated from the date of resection to the date of death or last follow-up. All tissue samples were obtained from the tissue bank of the China-Japan Union Hospital of Jilin University. The present study was approved by the Ethical Committee of the China-Japan Union Hospital of Jilin University and conducted with the informed consent of all patients.
2.2. Immunohistochemistry Immunohistochemistry (IHC) was performed using a standard avidin-biotin-peroxidase method. Four-micrometerthick sections of TMAs mentioned previously were deparaffinized in xylene, dehydrated in gradient concentrations of ethanol, and then subjected to high-pressure antigen retrieval in a pressure cooker for 3 minutes in preheated 10 mmol/L sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 10 minutes, and nonspecific staining was eliminated by incubating the sections with normal goat serum for 15 minutes at room temperature. The sections were incubated with the primary antibody (1:25 dilution) at 4°C overnight, then they were incubated overnight at 4°C with the primary antibody diluted at 1:25 (ab175976; Abcam, Cambridge, MA). After being washed with phosphate-buffered saline, sections were incubated with diluted biotinylated goat anti-rabbit secondary antibody for 10 minutes and then incubated with the avidin-biotin-peroxidase complex for another 10 minutes with repeated washing steps. Staining was visualized using 3,3′-diaminobenzidine solution (Maxim, Fuzhou, China). Sections were then counterstained with hematoxylin after dehydration and clearing with xylene. Coverslips were added to the slides and examined. Negative controls were obtained by omission of the primary antibody. CYP24A1 immunostaining was semiquantitatively assessed according to the intensity and percentage of positive cancer cells on the basis of an immunoreactive score [17]. Staining intensity was scored as follows: 0, none; 1, weak; 2, moderate; and 3, strong staining. The percentage of positively stained cells was scored as follows: 0, no staining; 1, less than 10% of cells; 2, 11% to 50% of cells; 3, 51% to 80% of cells; and 4, greater than 81% of cells stained. The product of the intensity and percentage scores was calculated as the final staining score, which ranged from 0 to 12; 0 indicated no staining, 1 indicated weak staining, 2 or 3 indicated moderate staining, and more than 3 indicated
CYP24A1 is a prognostic biomarker in colorectal cancer strong staining. In the final analysis, tissue samples with a score less than 4 were considered to have low CYP24A1 expression, whereas those with a score greater than or equal to 4 were considered to have high CYP24A1 expression [18,19]. IHC staining was scored by 2 independent pathologists.
2.3. Quantitative real-time polymerase chain reaction Total RNA was extracted from fresh frozen CRC tissues and the matched noncancerous tissues using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Extracted RNA samples were treated with RQ1 RNase-Free DNase (Promega, Madison, WI) to eliminate DNA contamination. Complementary DNA was prepared by reverse transcription using a Goscript Reverse Transcription System Kit (A5000; Promega) from 500 ng of total RNA. mRNA expression levels were quantified by quantitative real-time polymerase chain reaction (qPCR) with the SYBR Premix Ex Taq II (Takara Bio, Otsu, Shiga, Japan) with the following primers: CYP24A1: forward primer 5′-GCCGTATTTAAAAGCCTGTCTGAA-3′ and reverse primer 5′-ACCTGGGTATTTAGCATGAG CACTG-3′. The glyceraldehyde 3-phosphate dehydrogenase mRNA level was used to standardize the measurements of CYP24A1, and the primers for glyceraldehyde 3-phosphate dehydrogenase were as follows: forward primer 5′-AGAAGGCTGGGGCTCATTTG-3′ and reverse primer 5′-AGGGGCCATCCACAGTCTTC-3′. Relative mRNA levels were quantified by the comparative 2−ΔΔCt method.
2.4. Statistical analysis Statistical analysis was performed using SPSS 18.0 (SPSS, Chicago, IL). Correlations between CYP24A1 protein expression and clinicopathological factors were analyzed by Pearson χ2 test or Fisher exact probability test. The differences in CYP24A1 mRNA expression between carcinoma and normal samples were evaluated by the independent-sample t test. The correlation between mRNA expression and the IHC score was examined using Spearman correlation coefficients by rank test. Survival curves were calculated using the Kaplan-Meier method and compared using a log-rank test. Univariate and multivariate analyses were performed using Cox proportional hazards regression models. All of the tests were 2 sided, and P b .05 was considered statistically significant.
3. Results 3.1. Differential expression of CYP24A1 in normal colorectal tissues and CRC tissues IHC analysis was performed to investigate the role of CYP24A1 in CRC. Among the 99 paired tissues, high CYP24A1 expression was detected in 69 CRC tissues
103 (69.7%) and in only 20 tumor-adjacent nontumor tissues (20.2%). CYP24A1 expression was more frequently present in CRC tissues than in the adjacent nontumor tissues (P b .05). Expression of CYP24A1 was mainly localized to the cytoplasm of CRC cells. Typical IHC staining patterns for CYP24A1 in CRC are shown in Fig. 1. qPCR analysis further confirmed that CYP24A1 mRNA was significantly increased in the tumor tissues compared to matched nontumor tissues (7.27 ± 3.39 versus 1.21 ± 0.77; P b .05; Fig. 2A). Higher mRNA expression correlated with a higher IHC score (r = 0.89; P b .05; Fig. 2B).
3.2. Correlations between CYP24A1 protein expression and clinicopathological features The correlations between high CYP24A1 protein expression and clinicopathological features in 99 CRC patients are shown in Table 1. CYP24A1 protein expression was significantly associated with the depth of invasion (P = .000), lymph node metastasis (P = .030), lymphatic or venous permeation (P = .016), and OS (P = .008).
3.3. Survival analysis A Kaplan-Meier analysis of OS showed reduced survival in patients with high CYP24A1 protein expression compared to patients with low CYP24A1 protein expression (log-rank, P = .026) (Fig. 3A). The mean OS was 44.4 months in the low CYP24A1 expression group, whereas it was only 33.9 months in the high CYP24A1 expression group. Moreover, higher CYP24A1 expression was correlated with shorter DFS (log-rank, P = .009). The mean DFS was 40.1 months for patients with low CYP24A1 expression compared to 28.9 months for patients with high CYP24A1 protein expression (Fig. 3B). Univariate analysis showed that 7 factors, including tumor size (P = .015), tumor location (P = .025), tumor differentiation (P = .013), lymph node metastasis (P = .010), venous permeation (P = .000), perineural invasion (P = .009), and CYP24A1 expression (P = .036), were associated with OS and 5 factors showed a significant correlation with DFS (Table 2). These included histology differentiation (P = .023), lymph node metastasis (P = .001), venous permeation (P = .000), perineural invasion (P = .000), and CYP24A1 expression (P = .015). Multivariate analysis further confirmed that high CYP24A1 expression was an independent prognostic factor for DFS (P = .032), but not for OS currently (Table 3). We further analyzed the prognostic value of CYP24A1 expression in selective patient subgroups stratified according to the venous permeation condition. The 2-year DFS rate of the patients with high CYP24A1 expression was 76.7% in the subgroup without venous permeation, whereas it was only 19.9% in the venous permeation subgroup; CRC patients with higher CYP24A1 expression had shorter DFS than those with low expression when there was venous permeation (log-rank, P = .007, Fig. 3C). However,
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Fig. 1 Representative images of CYP24A1 expression in CRC tissues and corresponding noncancerous tissues. A-C, High immunohistochemical staining of CYP24A1 expression in CRC tissue samples. D-F, Low immunohistochemical staining of CYP24A1 expression in CRC tissues. G-I, High immunohistochemical staining of CYP24A1 expression in noncancerous tissues. J-L, Low immunohistochemical staining of CYP24A1 expression in noncancerous tissues. Original magnification ×40 (A, D, G, and J), ×200 (B, E, H, and K), and ×400 (C, F, I, and L).
Kaplan-Meier analysis of the subgroup without venous permeation did not show statistically significant differences between patients with different CYP24A1 expression levels (log-rank, P = .481; Fig. 3D).
4. Discussion Calcitriol, which is also known as 1,25(OH)2D3, is the active form of vitamin D. It not only plays an important role
in regulating calcium homeostasis and bone mineralization but also modulates cellular proliferation and differentiation in a variety of cell types, including cancer cells [20]. The level and biological activity of 1,25(OH)2D3 in tissues depends on the rates of its synthesis by CYP27B1 and inactivation by CYP24A1. CYP24A1 is a member of the cytochrome P450 enzyme family and the key enzyme that catabolizes 1,25(OH)2D3 into less active calcitroic acid through 24-hydroxylation, which is the rate-limiting step in vitamin D3 metabolism [21]. The expression level of the catabolic enzyme CYP24A1 is aberrantly increased in
CYP24A1 is a prognostic biomarker in colorectal cancer
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Fig. 2 CYP24A1 mRNA expression in CRC tissues and corresponding noncancerous tissues. A, The expression of CYP24A1 mRNA in CRC tissues was significantly higher than that in matched normal tissues (P b .05). B, Correlation between mRNA and protein expression of CYP24A1. Higher IHC score corresponded to higher mRNA level (r = 0.89; P b .05).
Table 1 Correlation between CYP24A1 expression and clinicopathological features of colorectal carcinoma Variable
Age (y) ≥60 b60 Sex Male Female Tumor size (cm) ≥5 b5 Tumor location Colon Rectum Tumor differentiation Well and moderate Poor Depth of invasion Tis-T2 T3-T4 Lymph node metastasis Yes No Venous permeation Yes No Perineural invasion Yes No Survival status Death Survival Ki-67 b50% ≥50%
No. of cases
CYP24A1 expression
P
High (n = 69)
Low (n = 30)
55 44
41 28
14 16
64 35
46 23
18 12
62 37
47 22
15 15
51 48
40 29
11 19
83 16
55 14
28 2
16 83
2 67
14 16
59 40
46 23
13 17
41 58
34 35
7 23
29 70
24 45
5 25
32 67
28 41
4 26
22 77
18 51
4 26
.241
.524
.087
.051
.137
.000
.030
.016
.069
.008
.161
several cancer cells. CYP24A1 was identified as a candidate oncogene first in breast cancer by the array comparative genomic hybridization method [22], and studies that have focused on CYP24A1 have revealed that up-regulation of CYP24A1 was associated with a poorer prognosis in some cancers [12,16]. For CRC, Horváth et al [23] compared CYP24A1 expression in normal human colon mucosa, colorectal adenomas, and adenocarcinomas, and the results showed that CYP24A1 was a potential biomarker for colorectal tumorigenesis; however, no studies have investigated the prognostic value of the expression of CYP24A1 in CRC patients. In this study, we found that CRC tissues demonstrated a higher expression of CYP24A1 compared with normal noncancerous tissues by IHC. Although Horváth et al [23] observed discordance between mRNA and protein expression levels, our qPCR results confirmed that higher CYP24A1 mRNA expression was detected in CRC tissues than in matched noncancerous tissues. These data were consistent with previous publications and suggested an oncogenic role of CYP24A1 in colorectal tumorigenesis [24,25]. Currently, the role of CYP24A1 cellular localization is controversial. Matusiak et al [25] for the first time noted that CYP24A1 was found exclusively in the nucleus of normal colonic epithelial cells but was mainly detected in the cytoplasm of CRC cells, suggesting that translocation of this protein from the nucleus to the cytoplasm is a function of malignant progression. However, in the study of Horváth et al [23], expression of CYP24A1 was only detected in the cytoplasm of cells. In the present study, we found that CYP24A1 was predominantly located in the cytoplasm both in normal colonic epithelial cells and CRC cells, which was not consistent with the study of Horváth et al [23]. This discrepancy might be due to antibody specificity. CYP24A1 is located in the inner mitochondrial membrane, where it is a member of the mini-electron transport chain consisting of ferredoxin, NADPH-ferredoxin reductase, and the terminal cytochrome CYP24A1. Thus, whether CYP24A1 is
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Fig. 3 The prognostic significance of CYP24A1 evaluated by Kaplan-Meier analysis. The OS (A) and DFS (B) of patients with high CYP24A1 expression were significantly shorter than those of patients with low expression. CRC patients with high CYP24A1 expression had shorter DFS than those with low expression in venous permeation subgroup (C); no statistically significant difference in DFS rate between patients with different expression CYP24A1 level was found in the subgroup without venous permeation (D).
functional outside the mitochondrion and a result of malignant progression needs further investigation. In this study, the expression of CYP24A1 in CRC was correlated with several clinical features, including the depth of tumor invasion, lymph node metastasis, venous permeation, and OS, which further confirm a prooncogenic effect of CYP24A1. The potential mechanism might be that the up-regulation of CYP24A1 leads to rapid inactivation of 1,25(OH)2D3, thus diminishing the effect of 1,25(OH)2D3 in modulating the proliferation and angiogenesis of CRC cells [26-29]. However, our finding contradicts the results of
Table 2
Horváth et al [23], who reported that CYP24A1 expression was not related to TNM stage and vessel invasion. A possible reason for this discrepancy is the different sample sizes. We detected CYP24A1 expression in 99 CRC patients, whereas only 48 cases were included in the study of Horváth et al. There was no correlation between the proliferation marker Ki-67 and CYP24A1 protein expression in CRC patients in our study. Horváth et al [23] reported the same result in carcinomas but a strong correlation between CYP24A1 and Ki-67 expression in the normal and benign samples, which suggested that the vitamin D system might play a more
Univariate analysis of prognostic factors associated with survival and recurrence
Variables Age (y), ≥60 vs b60 Sex, male vs female Tumor size (cm), ≥5 vs b5 Tumor location, colon vs rectum Tumor differentiation, well and moderate vs poor Depth of invasion, Tis-T2 vs T3-T4 Lymph node metastasis, yes vs no Venous permeation, yes vs no Perineural invasion, yes vs no Ki-67, b50% vs ≥50% CYP24A1 expression, high vs low
OS
DFS
Hazard ratio (95% CI)
P
Hazard ratio (95% CI)
P
1.172 (0.578-2.374) 1.546 (0.713-3.351) 3.014 (1.240-7.328) 2.359 (1.117-4.983) 0.388 (0.183-0.822) 0.038 (0.001-2.632) 3.226 (1.327-7.839) 4.358 (2.013-9.437) 2.528 (1.260-5.073) 1.229 (0.552-2.737) 3.072 (1.077-8.764)
.660 .270 .015 a .025 a .013 a .131 .010 a .000 a .009 a .614 .036 a
1.561 (0.794-3.071) 0.871 (0.451-1.685) 1.433 (0.720-2.854) 1.279 (0.669-2.443) 0.429 (0.207-0.889) 0.037 (0.001-1.678) 4.513 (1.872-10.879) 5.149 (2.525-10.499) 3.23 (1.685-6.192) 0.981 (0.448-2.147) 3.221 (1.225-8.270)
.197 .682 .306 .457 .023 a .090 .001 a .000 a .000 a .962 .015 a
Abbreviations: OS, overall survival; DFS, disease-free survival; CI, confidence interval. a Statistically significant.
CYP24A1 is a prognostic biomarker in colorectal cancer
the China-Japan Union Hospital of Jilin University and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Table 3 Multivariate Cox regression analysis of prognostic factors associated with OS and DFS Variables
Multivariate analysis Hazard ratio (95% CI)
OS Tumor size (cm), ≥5 vs b5 Tumor location, colon vs rectum Venous permeation, yes vs no CYP24A1 expression, high vs low DFS Venous permeation, yes vs no CYP24A1 expression, high vs low
107
P
References 2.493 (1.012-6.141) 2.210 (1.036-4.715) 4.348 (2.005-9.431) 1.875 (0.639-5.500)
a
.047 .040 a .000 a .252
4.923 (2.384-10.168) .000 a 2.829 (1.092-7.330) .032 a
Abbreviations: OS, overall survival; DFS, disease-free survival; CI, confidence interval. a Statistically significant.
important role in the prevention of cancer and early tumorigenesis. Therefore, inhibition of CYP24A1 would be a reasonable option when using vitamin D3 for CRC prevention and treatment. Kaplan-Meier analysis and the log-rank test suggested that the expression level of CYP24A1 in CRC was significantly associated with patients' OS and DFS. The OS and DFS of patients with high CYP24A1 expression were shorter than those of patients with low expression. A multivariate Cox regression analysis further confirmed that CYP24A1 protein expression was an independent negative prognostic factor for DFS. This finding indicated that CYP24A1 was sufficient to predict early recurrence and metastasis in CRC patients after surgery. The venous permeation condition was also an independent prognostic factor for DFS, and we further analyzed the prognostic value of CYP24A1 expression in selective patient subgroups stratified according to the venous permeation condition. The results of Kaplan-Meier analysis showed that CRC patients with higher CYP24A1 expression had shorter DFS than those with low expression when there is venous permeation, which suggested CYP24A1 expression can be combined with venous permeation status to predict early recurrence in CRC patients more accurately. In conclusion, our results demonstrate for the first time that elevated CYP24A1 protein expression is associated with significantly reduced DFS and OS in CRC patients. CYP24A1 might be a novel independent prognostic biomarker for CRC, which could serve as a powerful new molecular target in the prevention and treatment of CRC in the future.
Acknowledgment All procedures performed in studies involving human participants were in accordance with the ethical standards of
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