- Email: [email protected]
Vitamin K epoxide reductase expression and prostate cancer risk
Urologic Oncology: Seminars and Original Investigations ] (2016) ∎∎∎–∎∎∎
Original article
Vitamin K epoxide reductase expression and prostate cancer risk Ben Yi Tew, Ph.D.a,1, Sumanta K. Pal, M.D.b,1, Miaoling He, B.S.a, Tommy Tong, M.D.c, Huiqing Wu, M.D.c, JoAnn Hsu, B.S.b, Xueli Liu, Ph.D.d, Susan L. Neuhausen, Ph.D.e, Jeremy O. Jones, Ph.D.a,* a
Department of Cancer Biology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA Department of Medical Oncology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA c Department of Pathology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA d Department of Biostatistics, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA e Department of Population Sciences, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA b
Received 3 May 2016; received in revised form 24 October 2016; accepted 26 October 2016
Abstract Purpose: Increasing evidence has demonstrated that men taking the anticoagulant warfarin have a lower risk of developing prostate cancer. This phenomenon is not observed in other cancers. We sought to determine if the target of warfarin, vitamin K epoxide reductase (VKOR), is expressed in benign and cancerous prostate tissues and if a functional single nucleotide polymorphism (SNP) in the VKOR gene is associated with prostate cancer risk. Materials and methods: The expression of VKOR was quantified by immunohistochemistry in an institutional series of 54 radical prostatectomy samples and metastatic biopsies, as well as in 40 other cancers and matched benign tissues on a tissue microarray. Genotyping of SNP rs2359612 was performed in a prospective series of 57 patients. Results: VKOR is highly expressed in benign human prostate epithelial cells but is not expressed or expressed at very low levels in cancerous cells. This expression pattern is unique to prostate cancer. Additionally, the proportion of the carrier C allele of rs2359612 in the patients with prostate cancer was significantly higher than in the population, suggesting an association between this allele and the risk of having a diagnosis of prostate cancer. Conclusions: The expression of VKOR in benign prostate epithelial cells, along with the association between a functional VKOR SNP and prostate cancer risk, suggests a possible role for VKOR in mediating the effect of warfarin on prostate cancer risk. Larger multiinstitutional cohort studies are warranted, as are molecular studies on the role of VKOR in prostate cancer development. r 2016 Elsevier Inc. All rights reserved.
Keywords: Prostate cancer; Vitamin K epoxide reductase; Warfarin
1. Introduction Despite multiple advances in detection and treatment, prostate cancer remains the second leading cause of cancer death in American men [1]. Retrospective studies have shown that prolonged use of the anticoagulant warfarin is associated with a reduced incidence of prostate cancer, an 1
Both authors contributed equally to this work. Corresponding author. Tel.: þ1-626-256-4673, ext: 80270; fax: þ1-626-471-3902. E-mail address: [email protected] (J.O. Jones). *
http://dx.doi.org/10.1016/j.urolonc.2016.10.020 1078-1439/r 2016 Elsevier Inc. All rights reserved.
effect that is not observed for other cancers. Specifically, Tagalakis et al. showed that among 135,882 men, those with 4 years of warfarin use had an adjusted prostate cancer incidence rate of 0.80 (95% CI: 0.65–0.99). No significant association was found between warfarin and risk of other urogenital cancers [2]. Pengo et al. [3], with 3,231 cases and 72,777 controls, found that patients with any use of vitamin K cycle antagonists (VKAs, mostly warfarin) had an adjusted prostate cancer incidence rate of 0.69 (95% CI: 0.50–0.97; P ¼ 0.008). The adjusted hazard ratio for any cancer-related mortality and overall mortality rate was 1.07 (95% CI: 0.92–1.24) and 1.12 (95% CI:
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B.Y. Tew et al. / Urologic Oncology: Seminars and Original Investigations ] (2016) 1–6
1.05–1.19), respectively. Pottegard et al. [4], with 238,196 cases and 1,713,176 controls, found that long-term VKA use (mostly warfarin) was associated with a decreased risk of prostate cancer (odds ratio ¼ 0.86, 95% CI: 0.78–0.95), but not other cancers. Schulman et al. [5] reported an overall decrease in the incidence of genitourinary cancers with 6 months compared with 6 weeks of warfarin use (odds ratio ¼ 0.40, 95% CI: 0.20–0.77), most of these tumors were prostate cancer, and follow-up studies showed no effect on bladder cancers alone [6]. These studies, from 3 different groups in 3 different countries, all of which used large, well-controlled cohorts, with medical record databases for warfarin usage, strongly suggest a specific association between warfarin usage and decreased prostate cancer risk. How warfarin usage reduces the risk of prostate cancer is unknown. Warfarin and other VKAs target the C1 subunit of vitamin K epoxide reductase (VKOR), the rate limiting enzyme in the vitamin K cycle [7]. VKOR controls the γcarboxylation of target proteins by γ-glutamyl carboxylase by limiting the availability of vitamin KH2 (Fig. 1). Thus, warfarin is able to inhibit coagulation by inhibiting the γcarboxylation of clotting factors. VKOR has also been linked to regulation of angiogenesis, cellular migration, and a wide array of other cellular processes that may foster the growth and invasion of metastasis [8]. It is, therefore, possible that the inhibition of VKOR by warfarin is related to its ability to reduce prostate cancer risk. To evaluate this possibility, we quantified the expression of VKOR in a series of radical prostatectomy (RP) samples and metastatic biopsies, as well as in other cancers and matched benign tissues.
Fig. 1. Schematic of the vitamin K cycle and involvement of VKOR. Color version of figure is available online.
2. Materials and methods 2.1. Patient population All studies were conducted with approval from the City of Hope Institutional Review Board under protocols 11058 and 11020. For inclusion in the study, patients must have had cytologically or pathologically verified diagnoses of prostate cancer. The median age at diagnosis was 65 years (range: 50–74 years). Of the 57 patients genotyped, 44 were Non-Hispanic White, 6 were Hispanic, 4 were AfricanAmerican, and 3 were Asian. RP ( n ¼ 45) and metastatic biopsy (n ¼ 9) tissues were accessed from the City of Hope tissue repository. RP sections were chosen to represent varying Gleason grade cancers as well as surrounding benign tissue (Grade 6: n ¼ 8, Grade 7: n ¼ 29, Grade 8: n ¼ 3, and Grade 9: n ¼ 5). Gleason grades 3, 4, and 5 lesions were represented on at least 25% of all RP specimens. Metastatic biopsy tissues came from bone (n ¼ 3), distant lymph node (n ¼ 3), brain (n ¼ 2), and lung (n ¼ 1). 2.2. Immunohistochemistry and scoring Immunohistochemistry and H&E staining were performed using standard protocols. Antigen retrieval was performed on paraffin-embedded sections using citratebased antigen unmasking solution (Vector Labs, Burlingame, CA). Slides were blocked with 10% normal goal serum, and then stained with VKOR antibody developed by Berkner [9] (diluted 1:200 in TBST) overnight at 41C. Slides were then incubated in biotinylated antirabbit secondary antibody (Vector Labs), followed by Vectastain Elite ABC reagent (Vector Labs) and developed using DAB substrate (Vector Labs). Sections were counterstained with Harris hematoxylin (Poly Scientific, Bay Shore, NY). Scoring was accomplished by 2 complementary methods. Sections were graded and marked for areas of benign, prostatic intraepithelial neoplasia (PIN), and adenocarcinomous tissue by a board-certified pathologist (T.T.). The same pathologist also scored VKOR staining. An estimate of the average percentage of positively staining cells (0%–100%) and their staining intensity (0–3) in the benign, PIN, and adenocarcinoma sections of each slide was recorded; the extent and intensity values were multiplied for a final staining score as we have done previously [10]. As very little VKOR expression was observed in adenocarcinoma of any grade, all grades were grouped together for this analysis. Analysis of variance methods were used to determine significant differences in expression among groups. We also quantified the intensity of staining using image analysis software (Image J). One or two 4 images were obtained from each slide; each image contained adenocarcinoma and adjacent benign tissue. Regions of interest were identified within benign sections and cancerous sections with efforts made to minimize unstained
B.Y. Tew et al. / Urologic Oncology: Seminars and Original Investigations ] (2016) 1–6
luminal areas. The mean staining within the regions of interest was recorded, and VKOR staining was calculated relative to staining in primary cancer areas for each image. PIN sections were very small and very few sections could be found with benign epithelial cells in the same area and as there was no surrounding benign prostate tissue in metastatic samples, this analysis focused only on primary adenocarcinomas. 2.3. Tissue microarray A tissue microarray (TMA) (Pantomics MTU951) containing 95 cases of 40 types of cancer and matched benign tissue from 27 organs/sites (1.5 mm) was used for staining with VKOR. A second tissue array (Pantomics PRO01P) containing 5 benign prostate samples, derived from men with no history of prostate cancer, was also stained for VKOR. Staining and scoring was performed by the study pathologist as described earlier for RP samples. 2.4. SNP sequencing rs2359612 was prospectively genotyped from 57 patients in institutional review board studies 11058 and 11020 using published primers [11]. Expected allele frequencies were derived from the 1,000 genomes project [12] and weighted by the proportion of each race. 2.5. Statistical considerations For VKOR expression scoring on a scale of 0–3, and a standard deviation from the mean of o0.2 for all groups, there was a 95% power to detect a 0.152 difference between means using 45 RP specimens. Too few metastatic samples were available for analysis for appropriate power to detect this difference. For single nucleotide polymorphism (SNP) genotyping, as we had no a priori expectations of differences between patient groups, no power calculations were performed; however, the analysis was post hoc. 3. Results 3.1. VKOR expression in prostate and other tissues RP tissue and metastatic biopsies from 45 to 9 patients, respectively, were stained for expression of VKOR (Fig. 2). VKOR expression was observed in both basal and luminal epithelial cells. There was a significant difference in VKOR expression between benign and cancerous or precancerous PIN areas of patient samples. VKOR expression was nearly always strong in benign areas and was nearly always very weak or nonexistent in PIN or cancerous areas, as determined by pathologist scoring (Fig. 2B) or image quantitation software (Fig. 2C). VKOR expression often provided a stark demarcation between benign and cancerous sections of
3
the RP specimen (Fig. 2A). There were no significant differences found by Gleason grade. Samples of 5 normal prostate from men with no history of prostate cancer were also stained for VKOR expression (Fig. 2A). Strong epithelial expression was observed in these tissues as well, indicating that VKOR is expressed in benign tissue of prostate from both healthy and diseased men. Although progression to adenocarcinoma paralleled decreased VKOR expression, expression in metastatic biopsies was variable, with 3 of 9 having strong staining, with no apparent correlation to the area of the body from which the biopsy was taken. It is possible that VKOR is re-expressed in some metastatic cancers. VKOR is strongly expressed in a panel of prostate cancer cell lines derived from metastatic lesions (Fig. 2D). As warfarin usage has not been associated with a reduced risk of other cancers, the expression of VKOR in benign and cancerous sections of other tissues was quantified on a TMA (Fig. 2E). The TMA contained multiple cancers from 27 tissues, including prostate, most with matched benign tissue. Only prostate cancer demonstrated a pattern of high expression in benign tissue and low to no expression in cancerous tissue. Thus, the pattern of VKOR expression, like the sensitivity to warfarin, is unique to prostate cancer. Interestingly, lung tissue shows the opposite pattern to prostate, where VKOR expression increases in malignant tissue. The relevance of this observation is unknown, but it represents an important area of future investigation.
3.2. VKOR SNP analysis There are several known SNPs in the VKORC1 gene, which have been associated with different sensitivities to warfarin. rs2359612, located þ2255 of VKORC1, has been found to be associated with VKOR activity and warfarin dosage, as well as with risk of vascular disease [11,13]. The “C” allele was found to be dominant regarding risk for vascular disease. This SNP was evaluated in germline DNA from 57 COH patients with prostate cancer (Fig. 3A). The population carrier rate of a C (C/C or C/T), taking into account the ethnicities of the patients, was 0.67. In our patients, the frequency was 0.88, which was significantly higher than expected (P ¼ 0.0003). Thus, the proportion of the carrier C allele in our patients is significantly higher than the population carrier rate, suggesting that this SNP in VKOR is associated with development of prostate cancer, further implicating VKOR in the mechanism of warfarin-mediated prostate cancer prevention. Interestingly, this SNP is highly divergent across ethnicities (Fig. 3B). The C allele is highest in African-American men, who have the highest risk of prostate cancer, and lowest in East-Asian men, who have the lowest risk of prostate cancer [14]. Two other SNPs in the VKOR gene, rs7294 and rs9934438, have similar ethnic distributions. Although there are many such SNPs that vary in this way, this information, combined with our other data, strongly implicates VKORC1 in the development of prostate cancer.
B.Y. Tew et al. / Urologic Oncology: Seminars and Original Investigations ] (2016) 1–6
4
A
H&E
VKOR
4x
B
VKOR - benign
4x
C
20x
Quantitation software Relative VKOR staining intensity
Pathologist scoring
20x
1.5
1
*
0.5
0 Benign Primary
VKOR B-actin
DU-145
PC-3
22Rv1
LAPC-4
E LNCaP
D
Tissue
Normal
Cancer
Adrenal Gland None Bladder Strong Breast None Bone No data Brain None Esophagus Strong Stomach None Small Intestine Weak Colon Weak Rectum Weak Kidney None Liver None
None Strong None None None Strong None Weak Weak Weak None None
Tissue
Normal
Cancer
Lung None Lymphoma, Lymph Node No data Head & Neck, Oral Cavity No data Head & Neck, Salivary Gland None Ovary None Pancreas Weak Prostate Strong Skin Weak Testis None Thyroid None Uterus, Cervix Weak Uterus, Endometrium None
Strong None Strong None None Weak None Weak None None Weak None
Fig. 2. VKOR expression in prostate and other cancers. (A) Representative staining of a radical prostatectomy slide (left, with H&E comparison) and benign prostate slide (right) with the VKOR antibody. Areas of benign tissue have strong VKOR staining, which is strikingly lost in areas of cancerous tissue. (B and C) Quantification of VKOR staining. (B) A trained pathologist scored the intensity of staining (0–3) in benign, PIN, and primary adenocarcinomous areas of RP slides (n ¼ 45) and in prostate cancer metastases (n ¼ 9). VKOR staining was significantly higher in benign cells than in PIN or carcinoma areas (standard error is shown, * P o 0.05). (C) Image J was used to quantify VKOR staining in primary cancer and adjacent benign areas of RP slides. Staining is expressed relative to primary cancer areas (standard error is shown, * P o 0.001). (D) Expression of VKOR in prostate cancer cell lines. VKOR expression is strong in all cell lines, all of which were developed from metastatic lesions. (E) Summary of results of VKOR staining of a tissue microarray. A trained pathologist scored the intensity of staining as none, weak, or strong. Prostate cancer was the only cancer in which VKOR staining was strong in normal cells but absent in cancer cells. Color version of figure is available online.
4. Discussion Overwhelming evidence indicates that long-term warfarin usage is associated with a decreased risk of prostate
cancer diagnosis. Although these data are necessarily retrospective in nature, as a prospective study testing the effects of the anticoagulant warfarin is not feasible owing to safety concerns, the 4 largest experiments contain more than
B.Y. Tew et al. / Urologic Oncology: Seminars and Original Investigations ] (2016) 1–6 Allele frequencies of rs2359612 expected in general population and in patient cohort (n=57) SNP Genotype T/T T/C or C/C
A
Population
0.331
0.669
Patient Sample
0.123
0.877
p-value
0.0003
B Allele frequencies of rs2359612 and prostate cancer risk across ethnicities SNP Genotype
T/T
T/C or C/C
Prostate Cancer Incidence (per 100,000)
African American
0.033
0.967
236.0
Hispanic
0.199
0.801
125.9
White/European
0.166
0.834
146.9
East Asian
0.836
0.164
85.4
Fig. 3. SNPs in the VKOR gene are associated with prostate cancer risk. (A) VKOR SNP rs2359612 was prospectively sequenced in an institutional series of patients with prostate cancer (n ¼ 57). Expected population allele frequencies based on the ethnicities of patients in the cohort were derived from values in the 1,000 genomes dataset. The carrier “C” allele was found at a significantly higher frequency in patients with prostate cancer than would be expected in the population. (B) rs2359612 allele frequencies have a similar trend as prostate cancer incidence across ethnicities. Color version of figure is available online.
2.5 million subjects combined, and each reaches a very similar conclusion: warfarin usage of more than a half of a year reduces the risk of a prostate cancer diagnosis 25%. Although men on warfarin tend to be less healthy, and therefore less likely to undergo aggressive screening for prostate cancer, the association between warfarin use and decreased risk of prostate cancer is not likely attributable to ascertainment bias. Several of the studies control for age of screening and most importantly, the Schulman et al. [5], study compared men with 6 months warfarin usage with 6 weeks of warfarin usage, thus mitigating any potential ascertainment bias. Recall bias is also not an issue with most of these studies, as prerecorded prescription histories were prospectively collected through patient registries. Having established the association between warfarin use and the decreased risk of prostate cancer diagnosis, it is now essential to elucidate the mechanism by which warfarin acts. It is possible that warfarin could prevent the development of new lesions or the growth of existing lesions or both. Although this cannot be accurately determined from existing literature or our study, here we demonstrate that the molecular target of warfarin, VKOR, is expressed in benign prostate cells but not in most cancerous cells in a series of 54 patient samples. The expression of VKOR in benign prostate tissue suggests that it has the potential to mediate the chemoprotective effects of warfarin. In contrast, the near absence of VKOR expression in PIN and primary cancers regardless of grade suggests that warfarin is unlikely to be useful to control the growth of cancers once they develop, at least using a VKOR-mediated mechanism. Interestingly, Tagalakis et al. [15] demonstrated that contrary to warfarin’s association with the reduced risk of prostate cancer diagnosis, men treated with warfarin after diagnosis with
5
prostate cancer have worse prostate cancer–specific and overall survival. A larger, more recent study reached similar conclusions [16]. Our VKOR expression data fit with a model, wherein VKOR mediates the chemoprotective effects of warfarin but does not influence the effect of warfarin on the growth of established cancers. We also demonstrated that the C allele of rs2359612 SNP in the VKOR gene is found at a higher rate in our prospective series of patient with prostate cancer than would be expected in the general population. Unfortunately, rs2359612 was not included in the collaborative ovarian, prostate, and breast gene-environment study (GOGS) dataset [17], which has been used to detect associations between polymorphisms and prostate cancer risk. A detailed analysis of rs2359612 in this dataset and others like, it is warranted. Although no direct connection can be assumed, the ethnic distribution of rs2359612 is intriguing as it is parallel when compared with prostate cancer risk across ethnicities. It is challenging to envision warfarin as a suitable strategy for preventing prostate cancer as, in the aged population that develops prostate cancer, the risks of bleeding associated with warfarin would likely outweigh any preventative benefit. However, understanding the mechanistic connection between warfarin usage and prostate cancer risk may allow us to separate the effects on cancer from the effects on coagulation. Based on the data presented herein, our laboratory is actively investigating potential mechanisms of VKOR action in prostate cells and identifying targets downstream of VKOR inactivation that may represent more reasonable drug targets. As noted previously, warfarin regulates the gamma-carboxylation of target proteins through its effects on VKOR. For instance, GAS6, a ligand for the AXL tyrosine kinase, is a gammacarboxylated protein that is potently inhibited by warfarin [18]. It has been shown that activation of AXL may promote growth of prostate cancer in preclinical models [19,20]. Although cabozantinib, a potent inhibitor of AXL [21], failed in phase III trials in prostate cancer, there was substantial enthusiasm surrounding phase II data suggesting potent anticancer activity with disappearance of bony lesions on nuclear medicine scans [22]. Thus, it is possible that GAS6/AXL signaling contributes to the effect of VKOR inhibition on prostate cancer development. Several limitations of this work should be noted. First, validation in larger series would be necessary to more convincingly demonstrate the presence and absence of VKOR in benign and malignant prostate tissue, respectively. A relatively limited number of metastatic samples were explored, and we are currently developing a much larger repository of paired primary and metastatic samples through a prospective study. A second limitation is the inherent challenge of characterizing VKOR expression by immunohistochemistry, which is subject to interpreter bias and potential intrapatient heterogeneity. This is mitigated by the use of a second quantitation method, which reached the same conclusion that VKOR expression is significantly
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higher in benign prostate glands vs. cancerous areas. Although the difference is not as great as the pathologist scoring system, this is likely owing to the difficulties of removing the white space of the lumen in the image analysis, which leads to underestimations of staining intensity. Because VKOR is expressed in so few lesions, it was not possible to derive any associations between VKOR staining and National Comprehensive Cancer Network or other risk categories or any specific clinical outcome. Despite these limitations, our immunohistochemistry data provide a compelling suggestion that the association between warfarin use and reduced prostate cancer incidence may be tied to expression of VKOR. Supporting this is the strong association observed between a functional VKOR SNP and prostate cancer incidence. If validated in larger multi-institutional series, drugs that target proteins downstream of VKOR and that lack the deleterious anticoagulant effects of warfarin could be explored as a possible means of chemoprevention. Acknowledgments S.L.N. is the Morris and Horowitz Families Endowed Professor and B.Y.T. was supported by the Dr. Norman and Melinda Payson Graduate School Fellowship. References [1] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin 2016;66:7. [2] Tagalakis V, Tamim H, Blostein M, Collet JP, Hanley JA, Kahn SR. Use of warfarin and risk of urogenital cancer: a population-based, nested case-control study. Lancet Oncol 2007;8:395. [3] Pengo V, Noventa F, Denas G, Pengo MF, Gallo U, Grion AM, et al. Long-term use of vitamin K antagonists and incidence of cancer: a population-based study. Blood 2011;117:1707. [4] Pottegard A, Friis S, Hallas J. Cancer risk in long-term users of vitamin K antagonists: a population-based case-control study. Int J Cancer 2013;132:2606. [5] Schulman S, Rhedin AS, Lindmarker P, Carlsson A, Larfars G, Nicol P, et al. A comparison of six weeks with six months of oral anticoagulant therapy after a first episode of venous thromboembolism. Duration of Anticoagulation Trial Study Group. N Engl J Med 1995;332:1661. [6] Blumentals WA, Foulis PR, Schwartz SW, Mason TJ. Does warfarin therapy influence the risk of bladder cancer? Thromb Haemost 2004;91:801. [7] Oldenburg J, Watzka M, Rost S, Muller CR. VKORC1: molecular target of coumarins. J Thromb Haemost 2007;5(Suppl. 1):S1.
[8] Wang Y, Zhen Y, Shi Y, Chen J, Zhang C, Wang X, et al. Vitamin k epoxide reductase: a protein involved in angiogenesis. Mol Cancer Res 2005;3:317. [9] Berkner KL. Expression of recombinant vitamin K-dependent proteins in mammalian cells: factors IX and VII. Methods Enzymol 1993;222:450. [10] Pal SK, He M, Tong T, Wu H, Liu X, Lau C, et al. RNA-seq reveals aurora kinase-driven mTOR pathway activation in patients with sarcomatoid metastatic renal cell carcinoma. Mol Cancer Res 2015;13:130. [11] Wang Y, Zhang W, Zhang Y, Yang Y, Sun L, Hu S, et al. VKORC1 haplotypes are associated with arterial vascular diseases (stroke, coronary heart disease, and aortic dissection). Circulation 2006; 113:1615. [12] Genomes Project C, Abecasis GR, Auton A, Brooks LD, DePristo MA, Durbin RM, et al. An integrated map of genetic variation from 1,092 human genomes. Nature 2012;491:56. [13] Wadelius M, Chen LY, Downes K, Ghori J, Hunt S, Eriksson N, et al. Common VKORC1 and GGCX polymorphisms associated with warfarin dose. Pharmacogenomics J 2005;5:262. [14] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin 2015;65:5. [15] Tagalakis V, Tamim H, Blostein M, Hanley JA, Kahn SR. Risk of prostate cancer death in long-term users of warfarin: a populationbased case-control study. Cancer Causes Control 2013;24:1079. [16] O’Rorke MA, Murray LJ, Hughes CM, Cantwell MM, Cardwell CR. The effect of warfarin therapy on breast, colorectal, lung, and prostate cancer survival: a population-based cohort study using the Clinical Practice Research Datalink. Cancer Causes Control 2015;26:355. [17] Eeles RA, Olama AA, Benlloch S, Saunders EJ, Leongamornlert DA, Tymrakiewicz M, et al. Identification of 23 new prostate cancer susceptibility loci using the iCOGS custom genotyping array. Nat Genet 2013;45:385. [18] Nakano T, Kawamoto K, Kishino J, Nomura K, Higashino K, Arita H. Requirement of gamma-carboxyglutamic acid residues for the biological activity of Gas6: contribution of endogenous Gas6 to the proliferation of vascular smooth muscle cells. Biochem J 1997;323(Pt 2):387. [19] Sainaghi PP, Castello L, Bergamasco L, Galletti M, Bellosta P, Avanzi GC. Gas6 induces proliferation in prostate carcinoma cell lines expressing the Axl receptor. J Cell Physiol 2005;204:36. [20] Shiozawa Y, Pedersen EA, Patel LR, Ziegler AM, Havens AM, Jung Y, et al. GAS6/AXL axis regulates prostate cancer invasion, proliferation, and survival in the bone marrow niche. Neoplasia 2010;12:116. [21] Zhou L, Liu XD, Sun M, Zhang X, German P, Bai S, et al. Targeting MET and AXL overcomes resistance to sunitinib therapy in renal cell carcinoma. Oncogene 2016;35:2687. [22] Smith MR, Sweeney CJ, Corn PG, Rathkopf DE, Smith DC, Hussain M, et al. Cabozantinib in chemotherapy-pretreated metastatic castration-resistant prostate cancer: results of a phase II nonrandomized expansion study. J Clin Oncol 2014;32:3391.
Original article
Vitamin K epoxide reductase expression and prostate cancer risk Ben Yi Tew, Ph.D.a,1, Sumanta K. Pal, M.D.b,1, Miaoling He, B.S.a, Tommy Tong, M.D.c, Huiqing Wu, M.D.c, JoAnn Hsu, B.S.b, Xueli Liu, Ph.D.d, Susan L. Neuhausen, Ph.D.e, Jeremy O. Jones, Ph.D.a,* a
Department of Cancer Biology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA Department of Medical Oncology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA c Department of Pathology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA d Department of Biostatistics, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA e Department of Population Sciences, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA b
Received 3 May 2016; received in revised form 24 October 2016; accepted 26 October 2016
Abstract Purpose: Increasing evidence has demonstrated that men taking the anticoagulant warfarin have a lower risk of developing prostate cancer. This phenomenon is not observed in other cancers. We sought to determine if the target of warfarin, vitamin K epoxide reductase (VKOR), is expressed in benign and cancerous prostate tissues and if a functional single nucleotide polymorphism (SNP) in the VKOR gene is associated with prostate cancer risk. Materials and methods: The expression of VKOR was quantified by immunohistochemistry in an institutional series of 54 radical prostatectomy samples and metastatic biopsies, as well as in 40 other cancers and matched benign tissues on a tissue microarray. Genotyping of SNP rs2359612 was performed in a prospective series of 57 patients. Results: VKOR is highly expressed in benign human prostate epithelial cells but is not expressed or expressed at very low levels in cancerous cells. This expression pattern is unique to prostate cancer. Additionally, the proportion of the carrier C allele of rs2359612 in the patients with prostate cancer was significantly higher than in the population, suggesting an association between this allele and the risk of having a diagnosis of prostate cancer. Conclusions: The expression of VKOR in benign prostate epithelial cells, along with the association between a functional VKOR SNP and prostate cancer risk, suggests a possible role for VKOR in mediating the effect of warfarin on prostate cancer risk. Larger multiinstitutional cohort studies are warranted, as are molecular studies on the role of VKOR in prostate cancer development. r 2016 Elsevier Inc. All rights reserved.
Keywords: Prostate cancer; Vitamin K epoxide reductase; Warfarin
1. Introduction Despite multiple advances in detection and treatment, prostate cancer remains the second leading cause of cancer death in American men [1]. Retrospective studies have shown that prolonged use of the anticoagulant warfarin is associated with a reduced incidence of prostate cancer, an 1
Both authors contributed equally to this work. Corresponding author. Tel.: þ1-626-256-4673, ext: 80270; fax: þ1-626-471-3902. E-mail address: [email protected] (J.O. Jones). *
http://dx.doi.org/10.1016/j.urolonc.2016.10.020 1078-1439/r 2016 Elsevier Inc. All rights reserved.
effect that is not observed for other cancers. Specifically, Tagalakis et al. showed that among 135,882 men, those with 4 years of warfarin use had an adjusted prostate cancer incidence rate of 0.80 (95% CI: 0.65–0.99). No significant association was found between warfarin and risk of other urogenital cancers [2]. Pengo et al. [3], with 3,231 cases and 72,777 controls, found that patients with any use of vitamin K cycle antagonists (VKAs, mostly warfarin) had an adjusted prostate cancer incidence rate of 0.69 (95% CI: 0.50–0.97; P ¼ 0.008). The adjusted hazard ratio for any cancer-related mortality and overall mortality rate was 1.07 (95% CI: 0.92–1.24) and 1.12 (95% CI:
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B.Y. Tew et al. / Urologic Oncology: Seminars and Original Investigations ] (2016) 1–6
1.05–1.19), respectively. Pottegard et al. [4], with 238,196 cases and 1,713,176 controls, found that long-term VKA use (mostly warfarin) was associated with a decreased risk of prostate cancer (odds ratio ¼ 0.86, 95% CI: 0.78–0.95), but not other cancers. Schulman et al. [5] reported an overall decrease in the incidence of genitourinary cancers with 6 months compared with 6 weeks of warfarin use (odds ratio ¼ 0.40, 95% CI: 0.20–0.77), most of these tumors were prostate cancer, and follow-up studies showed no effect on bladder cancers alone [6]. These studies, from 3 different groups in 3 different countries, all of which used large, well-controlled cohorts, with medical record databases for warfarin usage, strongly suggest a specific association between warfarin usage and decreased prostate cancer risk. How warfarin usage reduces the risk of prostate cancer is unknown. Warfarin and other VKAs target the C1 subunit of vitamin K epoxide reductase (VKOR), the rate limiting enzyme in the vitamin K cycle [7]. VKOR controls the γcarboxylation of target proteins by γ-glutamyl carboxylase by limiting the availability of vitamin KH2 (Fig. 1). Thus, warfarin is able to inhibit coagulation by inhibiting the γcarboxylation of clotting factors. VKOR has also been linked to regulation of angiogenesis, cellular migration, and a wide array of other cellular processes that may foster the growth and invasion of metastasis [8]. It is, therefore, possible that the inhibition of VKOR by warfarin is related to its ability to reduce prostate cancer risk. To evaluate this possibility, we quantified the expression of VKOR in a series of radical prostatectomy (RP) samples and metastatic biopsies, as well as in other cancers and matched benign tissues.
Fig. 1. Schematic of the vitamin K cycle and involvement of VKOR. Color version of figure is available online.
2. Materials and methods 2.1. Patient population All studies were conducted with approval from the City of Hope Institutional Review Board under protocols 11058 and 11020. For inclusion in the study, patients must have had cytologically or pathologically verified diagnoses of prostate cancer. The median age at diagnosis was 65 years (range: 50–74 years). Of the 57 patients genotyped, 44 were Non-Hispanic White, 6 were Hispanic, 4 were AfricanAmerican, and 3 were Asian. RP ( n ¼ 45) and metastatic biopsy (n ¼ 9) tissues were accessed from the City of Hope tissue repository. RP sections were chosen to represent varying Gleason grade cancers as well as surrounding benign tissue (Grade 6: n ¼ 8, Grade 7: n ¼ 29, Grade 8: n ¼ 3, and Grade 9: n ¼ 5). Gleason grades 3, 4, and 5 lesions were represented on at least 25% of all RP specimens. Metastatic biopsy tissues came from bone (n ¼ 3), distant lymph node (n ¼ 3), brain (n ¼ 2), and lung (n ¼ 1). 2.2. Immunohistochemistry and scoring Immunohistochemistry and H&E staining were performed using standard protocols. Antigen retrieval was performed on paraffin-embedded sections using citratebased antigen unmasking solution (Vector Labs, Burlingame, CA). Slides were blocked with 10% normal goal serum, and then stained with VKOR antibody developed by Berkner [9] (diluted 1:200 in TBST) overnight at 41C. Slides were then incubated in biotinylated antirabbit secondary antibody (Vector Labs), followed by Vectastain Elite ABC reagent (Vector Labs) and developed using DAB substrate (Vector Labs). Sections were counterstained with Harris hematoxylin (Poly Scientific, Bay Shore, NY). Scoring was accomplished by 2 complementary methods. Sections were graded and marked for areas of benign, prostatic intraepithelial neoplasia (PIN), and adenocarcinomous tissue by a board-certified pathologist (T.T.). The same pathologist also scored VKOR staining. An estimate of the average percentage of positively staining cells (0%–100%) and their staining intensity (0–3) in the benign, PIN, and adenocarcinoma sections of each slide was recorded; the extent and intensity values were multiplied for a final staining score as we have done previously [10]. As very little VKOR expression was observed in adenocarcinoma of any grade, all grades were grouped together for this analysis. Analysis of variance methods were used to determine significant differences in expression among groups. We also quantified the intensity of staining using image analysis software (Image J). One or two 4 images were obtained from each slide; each image contained adenocarcinoma and adjacent benign tissue. Regions of interest were identified within benign sections and cancerous sections with efforts made to minimize unstained
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luminal areas. The mean staining within the regions of interest was recorded, and VKOR staining was calculated relative to staining in primary cancer areas for each image. PIN sections were very small and very few sections could be found with benign epithelial cells in the same area and as there was no surrounding benign prostate tissue in metastatic samples, this analysis focused only on primary adenocarcinomas. 2.3. Tissue microarray A tissue microarray (TMA) (Pantomics MTU951) containing 95 cases of 40 types of cancer and matched benign tissue from 27 organs/sites (1.5 mm) was used for staining with VKOR. A second tissue array (Pantomics PRO01P) containing 5 benign prostate samples, derived from men with no history of prostate cancer, was also stained for VKOR. Staining and scoring was performed by the study pathologist as described earlier for RP samples. 2.4. SNP sequencing rs2359612 was prospectively genotyped from 57 patients in institutional review board studies 11058 and 11020 using published primers [11]. Expected allele frequencies were derived from the 1,000 genomes project [12] and weighted by the proportion of each race. 2.5. Statistical considerations For VKOR expression scoring on a scale of 0–3, and a standard deviation from the mean of o0.2 for all groups, there was a 95% power to detect a 0.152 difference between means using 45 RP specimens. Too few metastatic samples were available for analysis for appropriate power to detect this difference. For single nucleotide polymorphism (SNP) genotyping, as we had no a priori expectations of differences between patient groups, no power calculations were performed; however, the analysis was post hoc. 3. Results 3.1. VKOR expression in prostate and other tissues RP tissue and metastatic biopsies from 45 to 9 patients, respectively, were stained for expression of VKOR (Fig. 2). VKOR expression was observed in both basal and luminal epithelial cells. There was a significant difference in VKOR expression between benign and cancerous or precancerous PIN areas of patient samples. VKOR expression was nearly always strong in benign areas and was nearly always very weak or nonexistent in PIN or cancerous areas, as determined by pathologist scoring (Fig. 2B) or image quantitation software (Fig. 2C). VKOR expression often provided a stark demarcation between benign and cancerous sections of
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the RP specimen (Fig. 2A). There were no significant differences found by Gleason grade. Samples of 5 normal prostate from men with no history of prostate cancer were also stained for VKOR expression (Fig. 2A). Strong epithelial expression was observed in these tissues as well, indicating that VKOR is expressed in benign tissue of prostate from both healthy and diseased men. Although progression to adenocarcinoma paralleled decreased VKOR expression, expression in metastatic biopsies was variable, with 3 of 9 having strong staining, with no apparent correlation to the area of the body from which the biopsy was taken. It is possible that VKOR is re-expressed in some metastatic cancers. VKOR is strongly expressed in a panel of prostate cancer cell lines derived from metastatic lesions (Fig. 2D). As warfarin usage has not been associated with a reduced risk of other cancers, the expression of VKOR in benign and cancerous sections of other tissues was quantified on a TMA (Fig. 2E). The TMA contained multiple cancers from 27 tissues, including prostate, most with matched benign tissue. Only prostate cancer demonstrated a pattern of high expression in benign tissue and low to no expression in cancerous tissue. Thus, the pattern of VKOR expression, like the sensitivity to warfarin, is unique to prostate cancer. Interestingly, lung tissue shows the opposite pattern to prostate, where VKOR expression increases in malignant tissue. The relevance of this observation is unknown, but it represents an important area of future investigation.
3.2. VKOR SNP analysis There are several known SNPs in the VKORC1 gene, which have been associated with different sensitivities to warfarin. rs2359612, located þ2255 of VKORC1, has been found to be associated with VKOR activity and warfarin dosage, as well as with risk of vascular disease [11,13]. The “C” allele was found to be dominant regarding risk for vascular disease. This SNP was evaluated in germline DNA from 57 COH patients with prostate cancer (Fig. 3A). The population carrier rate of a C (C/C or C/T), taking into account the ethnicities of the patients, was 0.67. In our patients, the frequency was 0.88, which was significantly higher than expected (P ¼ 0.0003). Thus, the proportion of the carrier C allele in our patients is significantly higher than the population carrier rate, suggesting that this SNP in VKOR is associated with development of prostate cancer, further implicating VKOR in the mechanism of warfarin-mediated prostate cancer prevention. Interestingly, this SNP is highly divergent across ethnicities (Fig. 3B). The C allele is highest in African-American men, who have the highest risk of prostate cancer, and lowest in East-Asian men, who have the lowest risk of prostate cancer [14]. Two other SNPs in the VKOR gene, rs7294 and rs9934438, have similar ethnic distributions. Although there are many such SNPs that vary in this way, this information, combined with our other data, strongly implicates VKORC1 in the development of prostate cancer.
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4
A
H&E
VKOR
4x
B
VKOR - benign
4x
C
20x
Quantitation software Relative VKOR staining intensity
Pathologist scoring
20x
1.5
1
*
0.5
0 Benign Primary
VKOR B-actin
DU-145
PC-3
22Rv1
LAPC-4
E LNCaP
D
Tissue
Normal
Cancer
Adrenal Gland None Bladder Strong Breast None Bone No data Brain None Esophagus Strong Stomach None Small Intestine Weak Colon Weak Rectum Weak Kidney None Liver None
None Strong None None None Strong None Weak Weak Weak None None
Tissue
Normal
Cancer
Lung None Lymphoma, Lymph Node No data Head & Neck, Oral Cavity No data Head & Neck, Salivary Gland None Ovary None Pancreas Weak Prostate Strong Skin Weak Testis None Thyroid None Uterus, Cervix Weak Uterus, Endometrium None
Strong None Strong None None Weak None Weak None None Weak None
Fig. 2. VKOR expression in prostate and other cancers. (A) Representative staining of a radical prostatectomy slide (left, with H&E comparison) and benign prostate slide (right) with the VKOR antibody. Areas of benign tissue have strong VKOR staining, which is strikingly lost in areas of cancerous tissue. (B and C) Quantification of VKOR staining. (B) A trained pathologist scored the intensity of staining (0–3) in benign, PIN, and primary adenocarcinomous areas of RP slides (n ¼ 45) and in prostate cancer metastases (n ¼ 9). VKOR staining was significantly higher in benign cells than in PIN or carcinoma areas (standard error is shown, * P o 0.05). (C) Image J was used to quantify VKOR staining in primary cancer and adjacent benign areas of RP slides. Staining is expressed relative to primary cancer areas (standard error is shown, * P o 0.001). (D) Expression of VKOR in prostate cancer cell lines. VKOR expression is strong in all cell lines, all of which were developed from metastatic lesions. (E) Summary of results of VKOR staining of a tissue microarray. A trained pathologist scored the intensity of staining as none, weak, or strong. Prostate cancer was the only cancer in which VKOR staining was strong in normal cells but absent in cancer cells. Color version of figure is available online.
4. Discussion Overwhelming evidence indicates that long-term warfarin usage is associated with a decreased risk of prostate
cancer diagnosis. Although these data are necessarily retrospective in nature, as a prospective study testing the effects of the anticoagulant warfarin is not feasible owing to safety concerns, the 4 largest experiments contain more than
B.Y. Tew et al. / Urologic Oncology: Seminars and Original Investigations ] (2016) 1–6 Allele frequencies of rs2359612 expected in general population and in patient cohort (n=57) SNP Genotype T/T T/C or C/C
A
Population
0.331
0.669
Patient Sample
0.123
0.877
p-value
0.0003
B Allele frequencies of rs2359612 and prostate cancer risk across ethnicities SNP Genotype
T/T
T/C or C/C
Prostate Cancer Incidence (per 100,000)
African American
0.033
0.967
236.0
Hispanic
0.199
0.801
125.9
White/European
0.166
0.834
146.9
East Asian
0.836
0.164
85.4
Fig. 3. SNPs in the VKOR gene are associated with prostate cancer risk. (A) VKOR SNP rs2359612 was prospectively sequenced in an institutional series of patients with prostate cancer (n ¼ 57). Expected population allele frequencies based on the ethnicities of patients in the cohort were derived from values in the 1,000 genomes dataset. The carrier “C” allele was found at a significantly higher frequency in patients with prostate cancer than would be expected in the population. (B) rs2359612 allele frequencies have a similar trend as prostate cancer incidence across ethnicities. Color version of figure is available online.
2.5 million subjects combined, and each reaches a very similar conclusion: warfarin usage of more than a half of a year reduces the risk of a prostate cancer diagnosis 25%. Although men on warfarin tend to be less healthy, and therefore less likely to undergo aggressive screening for prostate cancer, the association between warfarin use and decreased risk of prostate cancer is not likely attributable to ascertainment bias. Several of the studies control for age of screening and most importantly, the Schulman et al. [5], study compared men with 6 months warfarin usage with 6 weeks of warfarin usage, thus mitigating any potential ascertainment bias. Recall bias is also not an issue with most of these studies, as prerecorded prescription histories were prospectively collected through patient registries. Having established the association between warfarin use and the decreased risk of prostate cancer diagnosis, it is now essential to elucidate the mechanism by which warfarin acts. It is possible that warfarin could prevent the development of new lesions or the growth of existing lesions or both. Although this cannot be accurately determined from existing literature or our study, here we demonstrate that the molecular target of warfarin, VKOR, is expressed in benign prostate cells but not in most cancerous cells in a series of 54 patient samples. The expression of VKOR in benign prostate tissue suggests that it has the potential to mediate the chemoprotective effects of warfarin. In contrast, the near absence of VKOR expression in PIN and primary cancers regardless of grade suggests that warfarin is unlikely to be useful to control the growth of cancers once they develop, at least using a VKOR-mediated mechanism. Interestingly, Tagalakis et al. [15] demonstrated that contrary to warfarin’s association with the reduced risk of prostate cancer diagnosis, men treated with warfarin after diagnosis with
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prostate cancer have worse prostate cancer–specific and overall survival. A larger, more recent study reached similar conclusions [16]. Our VKOR expression data fit with a model, wherein VKOR mediates the chemoprotective effects of warfarin but does not influence the effect of warfarin on the growth of established cancers. We also demonstrated that the C allele of rs2359612 SNP in the VKOR gene is found at a higher rate in our prospective series of patient with prostate cancer than would be expected in the general population. Unfortunately, rs2359612 was not included in the collaborative ovarian, prostate, and breast gene-environment study (GOGS) dataset [17], which has been used to detect associations between polymorphisms and prostate cancer risk. A detailed analysis of rs2359612 in this dataset and others like, it is warranted. Although no direct connection can be assumed, the ethnic distribution of rs2359612 is intriguing as it is parallel when compared with prostate cancer risk across ethnicities. It is challenging to envision warfarin as a suitable strategy for preventing prostate cancer as, in the aged population that develops prostate cancer, the risks of bleeding associated with warfarin would likely outweigh any preventative benefit. However, understanding the mechanistic connection between warfarin usage and prostate cancer risk may allow us to separate the effects on cancer from the effects on coagulation. Based on the data presented herein, our laboratory is actively investigating potential mechanisms of VKOR action in prostate cells and identifying targets downstream of VKOR inactivation that may represent more reasonable drug targets. As noted previously, warfarin regulates the gamma-carboxylation of target proteins through its effects on VKOR. For instance, GAS6, a ligand for the AXL tyrosine kinase, is a gammacarboxylated protein that is potently inhibited by warfarin [18]. It has been shown that activation of AXL may promote growth of prostate cancer in preclinical models [19,20]. Although cabozantinib, a potent inhibitor of AXL [21], failed in phase III trials in prostate cancer, there was substantial enthusiasm surrounding phase II data suggesting potent anticancer activity with disappearance of bony lesions on nuclear medicine scans [22]. Thus, it is possible that GAS6/AXL signaling contributes to the effect of VKOR inhibition on prostate cancer development. Several limitations of this work should be noted. First, validation in larger series would be necessary to more convincingly demonstrate the presence and absence of VKOR in benign and malignant prostate tissue, respectively. A relatively limited number of metastatic samples were explored, and we are currently developing a much larger repository of paired primary and metastatic samples through a prospective study. A second limitation is the inherent challenge of characterizing VKOR expression by immunohistochemistry, which is subject to interpreter bias and potential intrapatient heterogeneity. This is mitigated by the use of a second quantitation method, which reached the same conclusion that VKOR expression is significantly
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higher in benign prostate glands vs. cancerous areas. Although the difference is not as great as the pathologist scoring system, this is likely owing to the difficulties of removing the white space of the lumen in the image analysis, which leads to underestimations of staining intensity. Because VKOR is expressed in so few lesions, it was not possible to derive any associations between VKOR staining and National Comprehensive Cancer Network or other risk categories or any specific clinical outcome. Despite these limitations, our immunohistochemistry data provide a compelling suggestion that the association between warfarin use and reduced prostate cancer incidence may be tied to expression of VKOR. Supporting this is the strong association observed between a functional VKOR SNP and prostate cancer incidence. If validated in larger multi-institutional series, drugs that target proteins downstream of VKOR and that lack the deleterious anticoagulant effects of warfarin could be explored as a possible means of chemoprevention. Acknowledgments S.L.N. is the Morris and Horowitz Families Endowed Professor and B.Y.T. was supported by the Dr. Norman and Melinda Payson Graduate School Fellowship. References [1] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin 2016;66:7. [2] Tagalakis V, Tamim H, Blostein M, Collet JP, Hanley JA, Kahn SR. Use of warfarin and risk of urogenital cancer: a population-based, nested case-control study. Lancet Oncol 2007;8:395. [3] Pengo V, Noventa F, Denas G, Pengo MF, Gallo U, Grion AM, et al. Long-term use of vitamin K antagonists and incidence of cancer: a population-based study. Blood 2011;117:1707. [4] Pottegard A, Friis S, Hallas J. Cancer risk in long-term users of vitamin K antagonists: a population-based case-control study. Int J Cancer 2013;132:2606. [5] Schulman S, Rhedin AS, Lindmarker P, Carlsson A, Larfars G, Nicol P, et al. A comparison of six weeks with six months of oral anticoagulant therapy after a first episode of venous thromboembolism. Duration of Anticoagulation Trial Study Group. N Engl J Med 1995;332:1661. [6] Blumentals WA, Foulis PR, Schwartz SW, Mason TJ. Does warfarin therapy influence the risk of bladder cancer? Thromb Haemost 2004;91:801. [7] Oldenburg J, Watzka M, Rost S, Muller CR. VKORC1: molecular target of coumarins. J Thromb Haemost 2007;5(Suppl. 1):S1.
[8] Wang Y, Zhen Y, Shi Y, Chen J, Zhang C, Wang X, et al. Vitamin k epoxide reductase: a protein involved in angiogenesis. Mol Cancer Res 2005;3:317. [9] Berkner KL. Expression of recombinant vitamin K-dependent proteins in mammalian cells: factors IX and VII. Methods Enzymol 1993;222:450. [10] Pal SK, He M, Tong T, Wu H, Liu X, Lau C, et al. RNA-seq reveals aurora kinase-driven mTOR pathway activation in patients with sarcomatoid metastatic renal cell carcinoma. Mol Cancer Res 2015;13:130. [11] Wang Y, Zhang W, Zhang Y, Yang Y, Sun L, Hu S, et al. VKORC1 haplotypes are associated with arterial vascular diseases (stroke, coronary heart disease, and aortic dissection). Circulation 2006; 113:1615. [12] Genomes Project C, Abecasis GR, Auton A, Brooks LD, DePristo MA, Durbin RM, et al. An integrated map of genetic variation from 1,092 human genomes. Nature 2012;491:56. [13] Wadelius M, Chen LY, Downes K, Ghori J, Hunt S, Eriksson N, et al. Common VKORC1 and GGCX polymorphisms associated with warfarin dose. Pharmacogenomics J 2005;5:262. [14] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin 2015;65:5. [15] Tagalakis V, Tamim H, Blostein M, Hanley JA, Kahn SR. Risk of prostate cancer death in long-term users of warfarin: a populationbased case-control study. Cancer Causes Control 2013;24:1079. [16] O’Rorke MA, Murray LJ, Hughes CM, Cantwell MM, Cardwell CR. The effect of warfarin therapy on breast, colorectal, lung, and prostate cancer survival: a population-based cohort study using the Clinical Practice Research Datalink. Cancer Causes Control 2015;26:355. [17] Eeles RA, Olama AA, Benlloch S, Saunders EJ, Leongamornlert DA, Tymrakiewicz M, et al. Identification of 23 new prostate cancer susceptibility loci using the iCOGS custom genotyping array. Nat Genet 2013;45:385. [18] Nakano T, Kawamoto K, Kishino J, Nomura K, Higashino K, Arita H. Requirement of gamma-carboxyglutamic acid residues for the biological activity of Gas6: contribution of endogenous Gas6 to the proliferation of vascular smooth muscle cells. Biochem J 1997;323(Pt 2):387. [19] Sainaghi PP, Castello L, Bergamasco L, Galletti M, Bellosta P, Avanzi GC. Gas6 induces proliferation in prostate carcinoma cell lines expressing the Axl receptor. J Cell Physiol 2005;204:36. [20] Shiozawa Y, Pedersen EA, Patel LR, Ziegler AM, Havens AM, Jung Y, et al. GAS6/AXL axis regulates prostate cancer invasion, proliferation, and survival in the bone marrow niche. Neoplasia 2010;12:116. [21] Zhou L, Liu XD, Sun M, Zhang X, German P, Bai S, et al. Targeting MET and AXL overcomes resistance to sunitinib therapy in renal cell carcinoma. Oncogene 2016;35:2687. [22] Smith MR, Sweeney CJ, Corn PG, Rathkopf DE, Smith DC, Hussain M, et al. Cabozantinib in chemotherapy-pretreated metastatic castration-resistant prostate cancer: results of a phase II nonrandomized expansion study. J Clin Oncol 2014;32:3391.