- Email: [email protected]
Kinesin Family Member 11 mRNA Expression Predicts Prostate Cancer Aggressiveness
Original Study
Kinesin Family Member 11 mRNA Expression Predicts Prostate Cancer Aggressiveness Xuan-Mei Piao,1 Young Joon Byun,1 Pildu Jeong,1 Yun-Sok Ha,2 Eun Sang Yoo,2 Seok Joong Yun,1 Wun-Jae Kim1 Abstract In the present study, we used 195 prostate tissue samples obtained from patients with prostate cancer (PCa) and benign prostatic hyperplasia to evaluate the value of KIF11 as a cancer biomarker for PCa using real-time polymerase chain reaction. Our results suggest that KIF11 has potential value as a prognostic marker for PCa. Background: KIF11 (kinesin family member 11), a molecular motor protein, is essential to mitosis and cell cycle progression. Inhibitors of KIF11 have been developed as chemotherapeutic agents for the treatment of various cancers. Regarding prostate cancer (PCa), clinical trials using KIF11 inhibitors for the treatment of castration-resistant PCa have been initiated. We hypothesized that a relationship might exist between KIF11 expression and PCa. To investigate the functional activities and clinical usefulness of KIF11 in PCa, we used quantitative real-time reverse transcriptase polymerase chain reaction to monitor the KIF11 expression patterns. Materials and Methods: Tissue samples from 134 patients with PCa were analyzed using gene expression profiling and compared with tissues from 61 patients with benign prostatic hyperplasia. KIF11 expression was evaluated by real-time reverse transcriptase polymerase chain reaction and compared with indicators of tumor aggressiveness, such as prostate-specific antigen (PSA) levels, Gleason score (GS), and tumor stage (TNM stage). Results: KIF11 mRNA expression in tissue was significantly greater in PCa patients with elevated serum PSA levels ( 10 ng/mL), GS 8, T stage T3, or metastatic disease than in those with PSA levels < 10 ng/mL, GS of 7, or T2 stage. Additionally, the expression was remarkably greater in patients with a GS of 9 than in patients with a GS of 3þ4. Conclusion: KIF11 expression might be indicative of PCa aggressiveness and could be useful as a prognostic marker for patients with PCa. Clinical Genitourinary Cancer, Vol. -, No. -, --- ª 2016 Elsevier Inc. All rights reserved. Keywords: Gleason score, KIF11, Metastasis, PCa, Prostate-specific antigen
Introduction Prostate cancer (PCa) is a remarkably heterogeneous disease that can be indolent or very aggressive, often metastasizing to bone and other organs and resulting in significant morbidity and mortality.1 The most widely used methods to distinguish between aggressive and indolent tumors are the measurement of prostate-specific antigen (PSA) and histopathologic grading using Gleason scoring and TNM stage.2 However, these methods have inherent limitations.3 X.-M.P. and Y.J.B. contributed equally. 1 Department of Urology, College of Medicine, Chungbuk National University, Cheongju, Republic of Korea 2 Department of Urology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea
Submitted: Jul 31, 2016; Revised: Oct 3, 2016; Accepted: Oct 10, 2016 Address for correspondence: Seok Joong Yun, MD, PhD, Department of Urology, Institute for Tumor Research, and College of Medicine, Chungbuk National University, 776 1sunhwan-ro, Seowon-gu, Cheonju 362-711, Korea E-mail contact: [email protected]
1558-7673/$ - see frontmatter ª 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clgc.2016.10.005
PSA lacks specificity, has high false-positive rates, and can lead to overdiagnosis and overtreatment of PCa. Although assessment based on Gleason score (GS) and TNM staging is a powerful prognostic indicator, it cannot accurately predict the aggressiveness of the disease, because tumors with similar histologic patterns can result in different clinical outcomes.2,4,5 Therefore, it is of great clinical importance to establish better prognostic markers. KIF11 (kinesin family member 11), also known as kinesin-5, is a molecular motor protein that is essential in mitosis.6 It is encoded by the KIF11 gene (also called BimC, Eg5, and N-2). It mediates centrosome separation and formation of the bipolar mitotic spindle, which is important for cell mitosis. Inactivation of KIF11 causes improper cell division and cell cycle arrest during mitosis, eventually leading to apoptotic cell death.7-9 KIF11 mutation has been described in various cancers, and clinical trials using KIF11 inhibitors as chemotherapeutic agents are underway. Antimitotic drugs, such as docetaxel (a member of the taxane family), are currently used in clinical PCa treatment. Thus, KIF11 inhibitors, such as ispinesib, which is also an antimitotic drug, are expected to
Clinical Genitourinary Cancer Month 2016
-1
KIF11 Expression in PCa effectively treat PCa. However, unlike docetaxel, which targets microtubules, ispinesib blocks the function of key enzymes involved in mitosis.10 Therefore, KIF11 inhibitors can target PCa that is androgen independent and largely docetaxel resistant.11 In addition, recent findings have demonstrated that patients with a high GS respond better to taxane-based therapy.12 Thus, although the role of KIF11 in PCa remains unclear, we postulated that KIF11 could be a prognostic indicator of tumor aggressiveness. The present study was designed to measure the expression levels of KIF11 in human PCa tissue and to examine KIF11 mRNA levels with respect to PCa clinicopathologic characteristics.
Materials and Methods Study Populations and Samples Prostate tissue samples from 134 patients with newly diagnosed PCa and 61 control patients with benign prostatic hyperplasia (BPH) were examined. Patients who had undergone radical prostatectomy (RP) or palliative transurethral resection of the prostate (TURP) from January 2007 to June 2015 were included in the present study, and all tissue samples were histologically confirmed to be primary adenocarcinoma of the prostate at our institution. The control patients underwent TURP for relief of severe lower urinary tract symptoms or urinary retention despite proper medication. All tumors were macroscopically dissected, typically within 15 minutes of surgical resection. Each specimen was confirmed by analysis of adjacent tissue in fresh frozen sections from the surgical specimens. All samples were re-examined by a pathologist to confirm the presence of cancerous or noncancerous tissue. The GS and TNM 2002 stage were used as prognostic factors. The GS was assigned to specimens obtained from TURP or RP. The tumor stage was estimated from specimens obtained from RP or from the computed tomography, magnetic resonance imaging, or bone scan findings. The institutional review board of Chungbuk National University approved the collection and analysis of all samples, and each subject provided written informed consent.
RNA Extraction From Tissues and Synthesis of cDNA Total RNA was extracted from tissues using TRIzol reagent (Invitrogen, Carlsbad, CA), as described previously, and stored at 80 C.13 Then, cDNA was synthesized from 1 mg of total RNA using the First Strand cDNA Synthesis Kit (Clontech Laboratories, Takara Bio, Otsu, Japan), according to the manufacturer’s protocol.
Real-Time Polymerase Chain Reaction
2
-
Real-time polymerase chain reaction (PCR) was performed using a Rotor Gene 6000 instrument (Corbett Research, Mortlake, VIC, Australia) to amplify tissue mRNA. Microreaction tubes (Corbett Research) containing SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Hercules, CA) were used for the real-time PCR reactions. The primers used for amplifying KIF11 mRNA from tissues (150 base pairs) were as follows: sense, 50 -CAAGAGACTGAACAGAGATG-30 ; and antisense, 50 -TCTCATGAGCTGCCTTACGT-30 . The control glyceraldehyde-3-phosphate dehydrogenase primers were as follows: sense, 50 -CATGTTCGTCATGGGTGTGA-30 ; and antisense, 50 -ATGGCATGGACTGTGGTCAT-30 .
Clinical Genitourinary Cancer Month 2016
Statistical Analysis The Mann-Whitney U test was used to examine tissue mRNA expression in PCa. Receiver operating characteristics curves were used to identify the optimal cutoff point for each risk score that yielded the greatest combined sensitivity and specificity. Statistical analysis was performed using IBM SPSS Statistics, version 23.0 (IBM Corp., Armonk, NY). P < .05 was considered significant.
Results The clinicopathologic characteristics of 134 patients with PCa and 61 control patients with BPH are listed in Table 1. The mean age of the PCa patients was 68 years (range, 42-86 years) and that of the BPH controls was 69 years (range, 46-85 years). The serum PSA levels were remarkably greater in the patients with PCa than in the patients with BPH (average, 224.47 ng/mL vs. 3.77 ng/mL). Of the 134 PCa patients, 111 (82.8%) underwent RP by open or laparoscopic procedures and 23 (17.2%) underwent TURP. All BPH patients also underwent TURP. The number of subjects with GS of 3þ4, 4þ3, and 8 was 39 (29.1%), 37 (27.6%), and 58 (43.3%), respectively. In addition, the disease stage was T2 in 43 patients (32.1%), T3-T4N0M0 in 61 (45.5%), and metastatic (any T stage with N1 or M1) in 30 patients (22.4%). KIF11 expression, however, was not significantly different between the PCa and BPH groups (P ¼ .089). The expression levels of KIF11 with respect to aggressive clinicopathologic characteristics are shown in Figure 1. KIF11 expression
Table 1 Clinicopathologic Features of PCa Patients and BPH Controls Variable
PCa
BPH
P Value
Patients (n)
134
61
Age (years)
68.49 6.73
68.69 7.07
.767a
PSA (ng/mL)
224.47 845.98
3.77 4.58
.000a
23 (17.2)
61 (100)
Surgery TURP RP
111 (82.8)
GS
NA
7 (3þ4)
39 (29.1)
7 (4þ3)
37 (27.6)
8
12 (9.0)
9
43 (32.1)
10
3 (2.2)
Stage T2N0M0
NA 43 (32.1)
T3-T4N0M0
61 (45.5)
Any T, N1 or M1
30 (22.4)
KIF11 expression, median (range) (IQR 105 copies/mg)
296.24 (66.60-214.80)
226.19 (48.23-141.72)
.089a
Data presented as mean standard deviation or n (%). Abbreviations: BPH ¼ benign prostatic hyperplasia; GS ¼ Gleason score; IQR ¼ interquartile range; PCa ¼ prostate cancer; PSA ¼ prostate-specific antigen; RP ¼ radical prostatectomy; TURP ¼ transurethral resection of the prostate. a The Mann-Whitney U test was used to compare the expression levels and clinical variables.
Xuan-Mei Piao et al Figure 1 Comparison of Aggressive Clinicopathologic Characteristics in Prostate Cancer. (A) KIF11 mRNA Expression Was Greater in Patients With Elevated Serum Prostate-Specific Antigen (PSA) Levels (‡ 10 ng/mL). (B) KIF11 mRNA Expression Was Greater in Patients With Gleason Score (GS) ‡ 8. (C) Expression of KIF11 Was Remarkably Greater in GS ‡ 9 Than in GS 3D4 and Showed an Increasing Trend With an Increasing GS. (D) Compared With Patients With T2 Stage Disease, the Expression of KIF11 Was Significantly Greater in Patients With Disease Stage ‡ T3 or Any T Plus N1 or M1
showed no significant differences between patients in the < 69 and 69 age groups (P ¼ .251). In contrast, KIF11 expression was significantly greater in the patients with elevated serum PSA levels when comparing patients with a serum PSA level of 10 ng/mL and those with a PSA level < 10 ng/mL (P ¼ .019; Figure 1A). Increased expression of KIF11 was also found in PCa patients with a GS of 8 compared with those with a GS of 7 (P ¼ .011) (Figure 1B). In addition, because GS 7 differs between GS 3þ4 and GS 4þ3 and the prognosis of those with GS 4þ3 is worse than that of those with GS 3þ4, we compared KIF11 expression in specimens with GS 3þ4, GS 4þ3, GS 8, and GS 9. KIF11 expression was remarkably greater in specimens with GS 9 than in those with GS 3þ4 (P ¼ .004; Figure 1C). In addition, KIF11 expression was upregulated in PCa patients with stage T3 or metastatic disease compared with PCa patients with localized stage T2N0M0 disease (Figure 1D). The trend of KIF11 expression with increasing GS is shown in Figure 2; increased KIF11 expression was seen when the GS was greater.
Discussion In the present study, we found that KIF11 mRNA expression in PCa tissue was significantly greater in patients with serum PSA levels of 10 ng/mL than in those with PSA levels < 10 ng/mL. In addition, KIF11 expression levels in PCa patients with GS 8 were greater than those in patients with GS 7. Moreover, KIF11 expression was upregulated in patients with more aggressive PCa. The most widely used pretreatment risk assessment tools for PCa are PSA, GS, and clinical TNM stage.14 These markers fare well in identifying patients at risk of aggressive disease but have limited ability to predict the presence of indolent disease.15,16 Moreover, for a substantial proportion (20%-60%) of men classified as having low-risk disease, these current pretreatment assessment tools underestimate the true tumor grade and, although less commonly, the true stage.17 The lack of predictive specificity of PSA for PCa has led to both unnecessary biopsies and overdiagnosis of indolent cancers.18 Hence, the establishment of reliable methods to identify patients with indolent and clinically significant cancers is an
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KIF11 Expression in PCa Figure 2 Comparison of KIF11 mRNA Expression With Different Gleason Scores (GSs). KIF11 Expression Showed an Increasing Trend With Increasing GS
important challenge in the diagnosis of PCa. Thus, we focused on finding a reliable marker to assess PCa progression. KIF11 is a mitotic kinesin that plays an important role in mitosis.6 Cancer and mitosis are closely related. Cancer cells undergo uncontrolled abnormal mitosis at a much more rapid rate than normal cells. Without mitosis, cancerous cells would be unable to form tumors and spread throughout the body.19,20 As cancer progresses, more and more abnormal mitosis can occur. Higher GSs and TNM stages are predictive of PCa aggressiveness. Tumors with a GS > 8 are more aggressive, and those with a GS < 7 have a better prognosis. In the present study, KIF11 expression was greater in PCa samples with a GS of 8 and stage of T3 or metastatic disease. Dysregulated mitosis might be the reason KIF11 expression was greater in more aggressive PCa. KIF11 expression can considered to be replace the role of GS, such as is shown in our results (Figure 2), because KIF11 tended to have greater expression with increasing GS. Accordingly, the GS could be inferred from the KIF11 expression. The GS for PCa is an established prognostic indicator worldwide in research and daily practice.21 However, like all histologic grading methods, GS has an intrinsic degree of subjectivity.22 Thus, grading errors result according to pathologist experience and observer variability. Unlike GS, determination of KIF11 expression is objective. Thus, KIF11 has potential as a prognostic marker in PCa. In addition, KIF11 could be used in planning patient treatment and predicting the response to a specific therapy. KIF11 is highly expressed in proliferating compared with nonproliferating cells and in tumor tissue relative to normal tissue.23 In in vitro experiments, cells treated with the prototype KSP (kinesin
4
-
Clinical Genitourinary Cancer Month 2016
spindle protein, KIF11) inhibitor, monastrol, displayed abnormal, monopolar spindles with chromosomes attached by microtubules to a single pole, resulting in deranged cell division, mitotic cell cycle arrest, and apoptosis.24,25 Accordingly, several studies were performed to explore the potential use of KIF11 inhibitors as chemotherapeutic drugs for the treatment of PCa.6-8,10,11,24-30 However, the results showed that the KIF11 inhibitors were not as effective as hoped. Although additional studies focusing on KIF11 inhibitors as an anticancer therapy are ongoing, little research has been performed on the role of KIF11 in PCa. To the best of our knowledge, only 1 study has been reported documenting the expression of KIF11 in PCa.26 In that study, immunohistochemical methods were used to measure the expression of KIF11 in PCa tissues. Eg5 (KIF11)-positive nuclei were observed more frequently in stage T4 tumors (P ¼ .04), GS 8-10 tumors (P ¼ .08), and metastasized tumors (P < .01). Thus, the investigators concluded that KIF11 expression might be an independent parameter for tumor aggressiveness. In the present study, we performed gene expression analysis, and our results indicated that KIF11 might be a gene related to PCa aggressiveness. However, the question remains as to why KIF11 expression was not upregulated in PCa samples compared with BPH samples, although KIF11 is related to mitosis. Disrupted passage through mitosis often leads to chromosome mis-segregation and the production of aneuploid progeny. Aneuploidy has long been recognized as a frequent characteristic of cancer cells and a possible cause of tumorigenesis.19 However, the link between the mitotic checkpoint and the apoptotic machinery remains unclear. Therefore, additional studies to delineate the role of KIF11 in cancer are needed.
Xuan-Mei Piao et al Conclusion KIF11 mRNA expression in prostate tissue is a significant and independent predictor of aggressiveness that could be useful for making treatment decisions on a case-by-case basis.
Clinical Practice Points PCa is the most common malignancy affecting Western men,
and the incidence and mortality is increasing in Korea. Nevertheless, accurate, noninvasive, and convenient diagnostic
and prognostic markers for PCa are still lacking. KIF11 mRNA expression was upregulated in PCa tissues with
aggressive characteristics. KIF11 could be used as a prognostic marker for PCa, and further
research of KIF11 could help researchers determine the mechanisms of PCa aggressiveness.
Acknowledgments The biospecimens used in the present study were provided by the Chungbuk National University Hospital, a member of the National Biobank of Korea, which is supported by the Ministry of Health, Welfare, and Family Affairs. All samples derived from the National Biobank of Korea were obtained with informed consent under institutional review board-approved protocols. The authors thank Ms Eun-Ju Shim from the National Biobank of Korea at Chungbuk National University Hospital for sample preparation and her excellent technical assistance.
Disclosure This research was supported by the International Science and Business Belt Program through the Ministry of Science, ICT and Future Planning (2015K000284), Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (No. NRF2014R1A2A1A09006983), Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (No. NRF2015R1A2A2A03004100) and by the Technological Innovation R&D Program (S2316843) funded by the Small and Medium Business Administration (SMBA, Korea).
References 1. Saini S. PSA and beyond: alternative prostate cancer biomarkers. Cell Oncol 2016; 39:97-106. 2. Otero JR, Gomez BG, Juanatey FC, Touijer KA. Prostate cancer biomarkers: an update. Urol Oncol Semin Orig Invest 2014; 32:252-60. 3. Tewari AK. Prostate Cancer: A Comprehensive Perspective. London: Springer Science & Business Media; 2013.
4. Shen MM, Abate-Shen C. Molecular genetics of prostate cancer: new prospects for old challenges. Genes Dev 2010; 24:1967-2000. 5. Schoenborn JR, Nelson P, Fang M. Genomic profiling defines subtypes of prostate cancer with the potential for therapeutic stratification. Clin Cancer Res 2013; 19: 4058-66. 6. Wojcik EJ, Buckley RS, Richard J, Liu L, Huckaba TM, Kim S. Kinesin-5: crossbridging mechanism to targeted clinical therapy. Gene 2013; 531:133-49. 7. Davis DA, Sarkar SH, Hussain M, Li Y, Sarkar FH. Increased therapeutic potential of an experimental anti-mitotic inhibitor SB715992 by genistein in PC-3 human prostate cancer cell line. BMC Cancer 2006; 6:1. 8. Grimes SR. A good molecular target for prostate cancer chemotherapy. Asian J Androl 2011; 13:207. 9. Venere M, Horbinski C, Crish JF, et al. The mitotic kinesin KIF11 is a driver of invasion, proliferation, and self-renewal in glioblastoma. Sci Transl Med 2015; 7:304ra143. 10. Blagden S, Molife L, Seebaran A, et al. A phase I trial of ispinesib, a kinesin spindle protein inhibitor, with docetaxel in patients with advanced solid tumours. Br J Cancer 2008; 98:894-9. 11. Beer TM, Goldman B, Synold TW, et al. Southwest Oncology Group phase II study of ispinesib in androgen-independent prostate cancer previously treated with taxanes. Clin Genitourin Cancer 2008; 6:103-9. 12. van Soest RJ, de Morrée ES, Shen L, Tannock IF, Eisenberger MA, de Wit R. Initial biopsy Gleason score as a predictive marker for survival benefit in patients with castration-resistant prostate cancer treated with docetaxel: data from the TAX327 study. Eur Urol 2014; 66:330-6. 13. Yun SJ, Yan C, Jeong P, et al. Comparison of mRNA, protein, and urinary nucleic acid levels of S100A8 and S100A9 between prostate cancer and BPH. Ann Surg Oncol 2015; 22:2439-45. 14. Cooperberg MR, Pasta DJ, Elkin EP, et al. The University of California, San Francisco cancer of the prostate risk assessment score: a straightforward and reliable preoperative predictor of disease recurrence after radical prostatectomy. J Urol 2005; 173:1938-42. 15. Dong F, Kattan MW, Steyerberg EW, et al. Validation of pretreatment nomograms for predicting indolent prostate cancer: efficacy in contemporary urological practice. J Urol 2008; 180:150-4. 16. Ploussard G, Epstein JI, Montironi R, et al. The contemporary concept of significant versus insignificant prostate cancer. Eur Urol 2011; 60:291-303. 17. Klein EA, Cooperberg MR, Magi-Galluzzi C, et al. A 17-gene assay to predict prostate cancer aggressiveness in the context of Gleason grade heterogeneity, tumor multifocality, and biopsy undersampling. Eur Urol 2014; 66:550-60. 18. Cary KC, Cooperberg MR. Biomarkers in prostate cancer surveillance and screening: past, present, and future. Ther Adv Urol 2013; 5:318-29. 19. Weaver BA, Cleveland DW. Decoding the links between mitosis, cancer, and chemotherapy: the mitotic checkpoint, adaptation, and cell death. Cancer Cell 2005; 8:7-12. 20. Rath O, Kozielski F. Kinesins and cancer. Nat Rev Cancer 2012; 12:527-39. 21. Humphrey PA. Gleason grading and prognostic factors in carcinoma of the prostate. Mod Pathol 2004; 17:292-306. 22. Humphrey PA. Prostate Pathology: Grading of Prostatic Carcinoma. Chicago: American Society for Clinical Pathology; 2003. 23. Hegde P, Cogswell J, Carrick K, et al. Differential gene expression analysis of kinesin spindle protein in human solid tumors. Proc Am Soc Clin Oncol 2003; 22:535. 24. Mayer TU, Kapoor TM, Haggarty SJ, King RW, Schreiber SL, Mitchison TJ. Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotypebased screen. Science 1999; 286:971-4. 25. Cochran JC, Gatial JE, Kapoor TM, Gilbert SP. Monastrol inhibition of the mitotic kinesin Eg5. J Biol Chem 2005; 280:12658-67. 26. Wissing MD, De Morrée ES, Dezentjé VO, et al. Nuclear Eg5 (kinesin spindle protein) expression predicts docetaxel response and prostate cancer aggressiveness. Oncotarget 2014; 5:7357. 27. Hayashi N, Koller E, Fazli L, Gleave ME. Effects of Eg5 knockdown on human prostate cancer xenograft growth and chemosensitivity. Prostate 2008; 68:1283-95. 28. Wiltshire C, Singh BL, Stockley J, et al. Docetaxel-resistant prostate cancer cells remain sensitive to S-trityl-L-cysteineemediated Eg5 inhibition. Mol Cancer Ther 2010; 9:1730-9. 29. Xing N-D, Ding S-T, Saito R, et al. A potent chemotherapeutic strategy in prostate cancer: S-(methoxytrityl)-L-cysteine, a novel Eg5 inhibitor. Asian J Androl 2011; 13:236-41. 30. Lad L, Luo L, Carson JD, et al. Mechanism of inhibition of human KSP by ispinesib. Biochemistry 2008; 47:3576-85.
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Kinesin Family Member 11 mRNA Expression Predicts Prostate Cancer Aggressiveness Xuan-Mei Piao,1 Young Joon Byun,1 Pildu Jeong,1 Yun-Sok Ha,2 Eun Sang Yoo,2 Seok Joong Yun,1 Wun-Jae Kim1 Abstract In the present study, we used 195 prostate tissue samples obtained from patients with prostate cancer (PCa) and benign prostatic hyperplasia to evaluate the value of KIF11 as a cancer biomarker for PCa using real-time polymerase chain reaction. Our results suggest that KIF11 has potential value as a prognostic marker for PCa. Background: KIF11 (kinesin family member 11), a molecular motor protein, is essential to mitosis and cell cycle progression. Inhibitors of KIF11 have been developed as chemotherapeutic agents for the treatment of various cancers. Regarding prostate cancer (PCa), clinical trials using KIF11 inhibitors for the treatment of castration-resistant PCa have been initiated. We hypothesized that a relationship might exist between KIF11 expression and PCa. To investigate the functional activities and clinical usefulness of KIF11 in PCa, we used quantitative real-time reverse transcriptase polymerase chain reaction to monitor the KIF11 expression patterns. Materials and Methods: Tissue samples from 134 patients with PCa were analyzed using gene expression profiling and compared with tissues from 61 patients with benign prostatic hyperplasia. KIF11 expression was evaluated by real-time reverse transcriptase polymerase chain reaction and compared with indicators of tumor aggressiveness, such as prostate-specific antigen (PSA) levels, Gleason score (GS), and tumor stage (TNM stage). Results: KIF11 mRNA expression in tissue was significantly greater in PCa patients with elevated serum PSA levels ( 10 ng/mL), GS 8, T stage T3, or metastatic disease than in those with PSA levels < 10 ng/mL, GS of 7, or T2 stage. Additionally, the expression was remarkably greater in patients with a GS of 9 than in patients with a GS of 3þ4. Conclusion: KIF11 expression might be indicative of PCa aggressiveness and could be useful as a prognostic marker for patients with PCa. Clinical Genitourinary Cancer, Vol. -, No. -, --- ª 2016 Elsevier Inc. All rights reserved. Keywords: Gleason score, KIF11, Metastasis, PCa, Prostate-specific antigen
Introduction Prostate cancer (PCa) is a remarkably heterogeneous disease that can be indolent or very aggressive, often metastasizing to bone and other organs and resulting in significant morbidity and mortality.1 The most widely used methods to distinguish between aggressive and indolent tumors are the measurement of prostate-specific antigen (PSA) and histopathologic grading using Gleason scoring and TNM stage.2 However, these methods have inherent limitations.3 X.-M.P. and Y.J.B. contributed equally. 1 Department of Urology, College of Medicine, Chungbuk National University, Cheongju, Republic of Korea 2 Department of Urology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea
Submitted: Jul 31, 2016; Revised: Oct 3, 2016; Accepted: Oct 10, 2016 Address for correspondence: Seok Joong Yun, MD, PhD, Department of Urology, Institute for Tumor Research, and College of Medicine, Chungbuk National University, 776 1sunhwan-ro, Seowon-gu, Cheonju 362-711, Korea E-mail contact: [email protected]
1558-7673/$ - see frontmatter ª 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clgc.2016.10.005
PSA lacks specificity, has high false-positive rates, and can lead to overdiagnosis and overtreatment of PCa. Although assessment based on Gleason score (GS) and TNM staging is a powerful prognostic indicator, it cannot accurately predict the aggressiveness of the disease, because tumors with similar histologic patterns can result in different clinical outcomes.2,4,5 Therefore, it is of great clinical importance to establish better prognostic markers. KIF11 (kinesin family member 11), also known as kinesin-5, is a molecular motor protein that is essential in mitosis.6 It is encoded by the KIF11 gene (also called BimC, Eg5, and N-2). It mediates centrosome separation and formation of the bipolar mitotic spindle, which is important for cell mitosis. Inactivation of KIF11 causes improper cell division and cell cycle arrest during mitosis, eventually leading to apoptotic cell death.7-9 KIF11 mutation has been described in various cancers, and clinical trials using KIF11 inhibitors as chemotherapeutic agents are underway. Antimitotic drugs, such as docetaxel (a member of the taxane family), are currently used in clinical PCa treatment. Thus, KIF11 inhibitors, such as ispinesib, which is also an antimitotic drug, are expected to
Clinical Genitourinary Cancer Month 2016
-1
KIF11 Expression in PCa effectively treat PCa. However, unlike docetaxel, which targets microtubules, ispinesib blocks the function of key enzymes involved in mitosis.10 Therefore, KIF11 inhibitors can target PCa that is androgen independent and largely docetaxel resistant.11 In addition, recent findings have demonstrated that patients with a high GS respond better to taxane-based therapy.12 Thus, although the role of KIF11 in PCa remains unclear, we postulated that KIF11 could be a prognostic indicator of tumor aggressiveness. The present study was designed to measure the expression levels of KIF11 in human PCa tissue and to examine KIF11 mRNA levels with respect to PCa clinicopathologic characteristics.
Materials and Methods Study Populations and Samples Prostate tissue samples from 134 patients with newly diagnosed PCa and 61 control patients with benign prostatic hyperplasia (BPH) were examined. Patients who had undergone radical prostatectomy (RP) or palliative transurethral resection of the prostate (TURP) from January 2007 to June 2015 were included in the present study, and all tissue samples were histologically confirmed to be primary adenocarcinoma of the prostate at our institution. The control patients underwent TURP for relief of severe lower urinary tract symptoms or urinary retention despite proper medication. All tumors were macroscopically dissected, typically within 15 minutes of surgical resection. Each specimen was confirmed by analysis of adjacent tissue in fresh frozen sections from the surgical specimens. All samples were re-examined by a pathologist to confirm the presence of cancerous or noncancerous tissue. The GS and TNM 2002 stage were used as prognostic factors. The GS was assigned to specimens obtained from TURP or RP. The tumor stage was estimated from specimens obtained from RP or from the computed tomography, magnetic resonance imaging, or bone scan findings. The institutional review board of Chungbuk National University approved the collection and analysis of all samples, and each subject provided written informed consent.
RNA Extraction From Tissues and Synthesis of cDNA Total RNA was extracted from tissues using TRIzol reagent (Invitrogen, Carlsbad, CA), as described previously, and stored at 80 C.13 Then, cDNA was synthesized from 1 mg of total RNA using the First Strand cDNA Synthesis Kit (Clontech Laboratories, Takara Bio, Otsu, Japan), according to the manufacturer’s protocol.
Real-Time Polymerase Chain Reaction
2
-
Real-time polymerase chain reaction (PCR) was performed using a Rotor Gene 6000 instrument (Corbett Research, Mortlake, VIC, Australia) to amplify tissue mRNA. Microreaction tubes (Corbett Research) containing SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Hercules, CA) were used for the real-time PCR reactions. The primers used for amplifying KIF11 mRNA from tissues (150 base pairs) were as follows: sense, 50 -CAAGAGACTGAACAGAGATG-30 ; and antisense, 50 -TCTCATGAGCTGCCTTACGT-30 . The control glyceraldehyde-3-phosphate dehydrogenase primers were as follows: sense, 50 -CATGTTCGTCATGGGTGTGA-30 ; and antisense, 50 -ATGGCATGGACTGTGGTCAT-30 .
Clinical Genitourinary Cancer Month 2016
Statistical Analysis The Mann-Whitney U test was used to examine tissue mRNA expression in PCa. Receiver operating characteristics curves were used to identify the optimal cutoff point for each risk score that yielded the greatest combined sensitivity and specificity. Statistical analysis was performed using IBM SPSS Statistics, version 23.0 (IBM Corp., Armonk, NY). P < .05 was considered significant.
Results The clinicopathologic characteristics of 134 patients with PCa and 61 control patients with BPH are listed in Table 1. The mean age of the PCa patients was 68 years (range, 42-86 years) and that of the BPH controls was 69 years (range, 46-85 years). The serum PSA levels were remarkably greater in the patients with PCa than in the patients with BPH (average, 224.47 ng/mL vs. 3.77 ng/mL). Of the 134 PCa patients, 111 (82.8%) underwent RP by open or laparoscopic procedures and 23 (17.2%) underwent TURP. All BPH patients also underwent TURP. The number of subjects with GS of 3þ4, 4þ3, and 8 was 39 (29.1%), 37 (27.6%), and 58 (43.3%), respectively. In addition, the disease stage was T2 in 43 patients (32.1%), T3-T4N0M0 in 61 (45.5%), and metastatic (any T stage with N1 or M1) in 30 patients (22.4%). KIF11 expression, however, was not significantly different between the PCa and BPH groups (P ¼ .089). The expression levels of KIF11 with respect to aggressive clinicopathologic characteristics are shown in Figure 1. KIF11 expression
Table 1 Clinicopathologic Features of PCa Patients and BPH Controls Variable
PCa
BPH
P Value
Patients (n)
134
61
Age (years)
68.49 6.73
68.69 7.07
.767a
PSA (ng/mL)
224.47 845.98
3.77 4.58
.000a
23 (17.2)
61 (100)
Surgery TURP RP
111 (82.8)
GS
NA
7 (3þ4)
39 (29.1)
7 (4þ3)
37 (27.6)
8
12 (9.0)
9
43 (32.1)
10
3 (2.2)
Stage T2N0M0
NA 43 (32.1)
T3-T4N0M0
61 (45.5)
Any T, N1 or M1
30 (22.4)
KIF11 expression, median (range) (IQR 105 copies/mg)
296.24 (66.60-214.80)
226.19 (48.23-141.72)
.089a
Data presented as mean standard deviation or n (%). Abbreviations: BPH ¼ benign prostatic hyperplasia; GS ¼ Gleason score; IQR ¼ interquartile range; PCa ¼ prostate cancer; PSA ¼ prostate-specific antigen; RP ¼ radical prostatectomy; TURP ¼ transurethral resection of the prostate. a The Mann-Whitney U test was used to compare the expression levels and clinical variables.
Xuan-Mei Piao et al Figure 1 Comparison of Aggressive Clinicopathologic Characteristics in Prostate Cancer. (A) KIF11 mRNA Expression Was Greater in Patients With Elevated Serum Prostate-Specific Antigen (PSA) Levels (‡ 10 ng/mL). (B) KIF11 mRNA Expression Was Greater in Patients With Gleason Score (GS) ‡ 8. (C) Expression of KIF11 Was Remarkably Greater in GS ‡ 9 Than in GS 3D4 and Showed an Increasing Trend With an Increasing GS. (D) Compared With Patients With T2 Stage Disease, the Expression of KIF11 Was Significantly Greater in Patients With Disease Stage ‡ T3 or Any T Plus N1 or M1
showed no significant differences between patients in the < 69 and 69 age groups (P ¼ .251). In contrast, KIF11 expression was significantly greater in the patients with elevated serum PSA levels when comparing patients with a serum PSA level of 10 ng/mL and those with a PSA level < 10 ng/mL (P ¼ .019; Figure 1A). Increased expression of KIF11 was also found in PCa patients with a GS of 8 compared with those with a GS of 7 (P ¼ .011) (Figure 1B). In addition, because GS 7 differs between GS 3þ4 and GS 4þ3 and the prognosis of those with GS 4þ3 is worse than that of those with GS 3þ4, we compared KIF11 expression in specimens with GS 3þ4, GS 4þ3, GS 8, and GS 9. KIF11 expression was remarkably greater in specimens with GS 9 than in those with GS 3þ4 (P ¼ .004; Figure 1C). In addition, KIF11 expression was upregulated in PCa patients with stage T3 or metastatic disease compared with PCa patients with localized stage T2N0M0 disease (Figure 1D). The trend of KIF11 expression with increasing GS is shown in Figure 2; increased KIF11 expression was seen when the GS was greater.
Discussion In the present study, we found that KIF11 mRNA expression in PCa tissue was significantly greater in patients with serum PSA levels of 10 ng/mL than in those with PSA levels < 10 ng/mL. In addition, KIF11 expression levels in PCa patients with GS 8 were greater than those in patients with GS 7. Moreover, KIF11 expression was upregulated in patients with more aggressive PCa. The most widely used pretreatment risk assessment tools for PCa are PSA, GS, and clinical TNM stage.14 These markers fare well in identifying patients at risk of aggressive disease but have limited ability to predict the presence of indolent disease.15,16 Moreover, for a substantial proportion (20%-60%) of men classified as having low-risk disease, these current pretreatment assessment tools underestimate the true tumor grade and, although less commonly, the true stage.17 The lack of predictive specificity of PSA for PCa has led to both unnecessary biopsies and overdiagnosis of indolent cancers.18 Hence, the establishment of reliable methods to identify patients with indolent and clinically significant cancers is an
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KIF11 Expression in PCa Figure 2 Comparison of KIF11 mRNA Expression With Different Gleason Scores (GSs). KIF11 Expression Showed an Increasing Trend With Increasing GS
important challenge in the diagnosis of PCa. Thus, we focused on finding a reliable marker to assess PCa progression. KIF11 is a mitotic kinesin that plays an important role in mitosis.6 Cancer and mitosis are closely related. Cancer cells undergo uncontrolled abnormal mitosis at a much more rapid rate than normal cells. Without mitosis, cancerous cells would be unable to form tumors and spread throughout the body.19,20 As cancer progresses, more and more abnormal mitosis can occur. Higher GSs and TNM stages are predictive of PCa aggressiveness. Tumors with a GS > 8 are more aggressive, and those with a GS < 7 have a better prognosis. In the present study, KIF11 expression was greater in PCa samples with a GS of 8 and stage of T3 or metastatic disease. Dysregulated mitosis might be the reason KIF11 expression was greater in more aggressive PCa. KIF11 expression can considered to be replace the role of GS, such as is shown in our results (Figure 2), because KIF11 tended to have greater expression with increasing GS. Accordingly, the GS could be inferred from the KIF11 expression. The GS for PCa is an established prognostic indicator worldwide in research and daily practice.21 However, like all histologic grading methods, GS has an intrinsic degree of subjectivity.22 Thus, grading errors result according to pathologist experience and observer variability. Unlike GS, determination of KIF11 expression is objective. Thus, KIF11 has potential as a prognostic marker in PCa. In addition, KIF11 could be used in planning patient treatment and predicting the response to a specific therapy. KIF11 is highly expressed in proliferating compared with nonproliferating cells and in tumor tissue relative to normal tissue.23 In in vitro experiments, cells treated with the prototype KSP (kinesin
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spindle protein, KIF11) inhibitor, monastrol, displayed abnormal, monopolar spindles with chromosomes attached by microtubules to a single pole, resulting in deranged cell division, mitotic cell cycle arrest, and apoptosis.24,25 Accordingly, several studies were performed to explore the potential use of KIF11 inhibitors as chemotherapeutic drugs for the treatment of PCa.6-8,10,11,24-30 However, the results showed that the KIF11 inhibitors were not as effective as hoped. Although additional studies focusing on KIF11 inhibitors as an anticancer therapy are ongoing, little research has been performed on the role of KIF11 in PCa. To the best of our knowledge, only 1 study has been reported documenting the expression of KIF11 in PCa.26 In that study, immunohistochemical methods were used to measure the expression of KIF11 in PCa tissues. Eg5 (KIF11)-positive nuclei were observed more frequently in stage T4 tumors (P ¼ .04), GS 8-10 tumors (P ¼ .08), and metastasized tumors (P < .01). Thus, the investigators concluded that KIF11 expression might be an independent parameter for tumor aggressiveness. In the present study, we performed gene expression analysis, and our results indicated that KIF11 might be a gene related to PCa aggressiveness. However, the question remains as to why KIF11 expression was not upregulated in PCa samples compared with BPH samples, although KIF11 is related to mitosis. Disrupted passage through mitosis often leads to chromosome mis-segregation and the production of aneuploid progeny. Aneuploidy has long been recognized as a frequent characteristic of cancer cells and a possible cause of tumorigenesis.19 However, the link between the mitotic checkpoint and the apoptotic machinery remains unclear. Therefore, additional studies to delineate the role of KIF11 in cancer are needed.
Xuan-Mei Piao et al Conclusion KIF11 mRNA expression in prostate tissue is a significant and independent predictor of aggressiveness that could be useful for making treatment decisions on a case-by-case basis.
Clinical Practice Points PCa is the most common malignancy affecting Western men,
and the incidence and mortality is increasing in Korea. Nevertheless, accurate, noninvasive, and convenient diagnostic
and prognostic markers for PCa are still lacking. KIF11 mRNA expression was upregulated in PCa tissues with
aggressive characteristics. KIF11 could be used as a prognostic marker for PCa, and further
research of KIF11 could help researchers determine the mechanisms of PCa aggressiveness.
Acknowledgments The biospecimens used in the present study were provided by the Chungbuk National University Hospital, a member of the National Biobank of Korea, which is supported by the Ministry of Health, Welfare, and Family Affairs. All samples derived from the National Biobank of Korea were obtained with informed consent under institutional review board-approved protocols. The authors thank Ms Eun-Ju Shim from the National Biobank of Korea at Chungbuk National University Hospital for sample preparation and her excellent technical assistance.
Disclosure This research was supported by the International Science and Business Belt Program through the Ministry of Science, ICT and Future Planning (2015K000284), Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (No. NRF2014R1A2A1A09006983), Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (No. NRF2015R1A2A2A03004100) and by the Technological Innovation R&D Program (S2316843) funded by the Small and Medium Business Administration (SMBA, Korea).
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