Tumor Biol. (2013) 34:1887–1894 DOI 10.1007/s13277-013-0732-0

RESEARCH ARTICLE

Polo-like kinase 1 is overexpressed in renal cancer and participates in the proliferation and invasion of renal cancer cells Guojun Zhang & Zhe Zhang & Zhuogang Liu

Received: 7 February 2013 / Accepted: 28 February 2013 / Published online: 14 March 2013 # International Society of Oncology and BioMarkers (ISOBM) 2013

Abstract Polo-like kinase 1 (Plk1) is an interesting molecule both as a biomarker and as a target for highly specific cancer therapy for several reasons. However, the functional significance of Plk1 in renal cell carcinoma (RCC) has not been reported. To explore whether Plk1 plays a general role in renal carcinoma, we examined the expression of Plk1 protein in renal urothelial carcinoma and cell lines, and analyzed the relationship between Plk1 protein expression and development, proliferation, and invasion of renal carcinoma. Immunohistochemisty was used to detect the expression of Plk1 in 100 renal carcinoma tissues. Moreover, the expression of Plk1 was analyzed by western blot and realtime polymerase chain reaction (PCR) in 80 renal carcinoma tissues and 20 normal renal tissues. CCK-8 assay, colony formation assay, and Transwell assay were used to examine proliferation and invasion ability of renal cancer cells with treatment of scytonemin (the specific inhibitor of Plk1). Statistical analysis was used to discuss the association between Plk1 expression and clinicopathologic parameters, and proliferation and invasion ability of renal cancer cells. Plk1 expressions were greater in cancerous tissues than in normal tissues (P<0.05). With an increase in tumor grade and stage, tumor metastasis, and recurrence, the level of Plk1 increased significantly in renal cancerous tissues. Moreover, there was a G. Zhang : Z. Liu (*) Department of Hematology, Shengjing Hospital of China Medical University, Shenyang City 110022, People’s Republic of China e-mail: [email protected] G. Zhang e-mail: [email protected] Z. Zhang Department of Urology, The First Hospital of China Medical University, 155 Nanjing North Street, Heping District, Shenyang City, Liaoning Province 110001, People’s Republic of China e-mail: [email protected]

significantly higher expression of Plk1 in higher degree of malignant renal adenocarcinoma cell ACHN than that in renal adenocarcinoma cell 769-P. With increasing concentration of scytonemin, we found that cell proliferation and invasion activity decreased significantly. Plk1 expression status was closely correlated with important histopathologic characteristics (grades, stages, metastasis, and recurrence) of renal carcinomas. Furthermore, Plk1 played an important function on renal cancer cells' proliferation and invasion. Keywords Polo-like kinase 1 . Renal carcinoma . Histopathological grade . Clinical stage . Scytonemin

Introduction Renal cell carcinoma (RCC) of the clear cell type is the most common malignant tumor of the kidney and has a poor prognosis. Approximately one-third of patients with RCC develop metastasis, and systemic treatment using chemotherapy and/or cytokines has proved to be rather ineffective [1]. Cytogenetic and molecular investigations of sporadic RCC revealed frequent chromosomal abnormality [2]. Although these studies led to identification of some putative oncogenes, which were thought to be involved in carcinogenesis of RCC, molecular mechanisms regulating aggressive properties of RCC are still poorly understood. Polo-like kinases (Plks) belong to the family of mitotic serine/threonine kinases, which are highly conserved among eukaryotes [3]. Many reports said that Plks played pivotal roles in regulation of cell cycle progression that strongly promotes progression of cells through mitosis [4–6]. Polo-like kinase 1 (Plk1) promotes mitotic cell division and also contributes to accelerated proliferation in mammalian cells. Plk1 is overexpressed in many cancers and serves as a significant prognostic factor in cancers such as small-cell lung cancer,

1888

colon cancer, and ovarian cancer [7–9]. In addition, high expression levels of Plk1 in melanoma and breast cancer correlate well with metastatic potential of these tumors [10, 11]. Plk1 overexpression may contribute to deregulation of cell proliferation during oncogenesis by overcoming mitotic checkpoints. Thus, among mammalian Plks, Plk1 exhibits the most striking promotion of neoplastic transformation. An oncogenic role for Plk1 has been hypothesized because its constitutive expression in NIH3T3 fibroblasts causes oncogenic foci formation and is tumorigenic in nude mice [12]. Furthermore, Plk1 is overexpressed in a variety of human tumors, including some urinary tract tumors [13, 14]. Intriguingly, Plk1 expression directly correlates with tumor genetic instability and patient prognosis in several tumor types, indicating its involvement in carcinogenesis and its potential as a therapeutic target. Use of different Plk1 inhibitors has increased our knowledge of mitotic regulation and allowed us to assess their ability to suppress tumor growth in vivo. Scytonemin, a small molecule inhibitor of Plk1, exhibits antiproliferative and antiinflammatory properties in some tumor cells. For example, scytonemin can significantly constrain osteosarcoma cell growth and induce apoptosis. In the present study, we observed that there was a high level of Plk1 expressions in renal cancer tissues and renal cancer cell lines. Overexpression of Plk1 in tumors closely correlates with a higher malignant degree in patients. Furthermore, a small molecule inhibitor for PLK1 significantly inhibits renal cancer cell growth and invasion. These data suggest that the role of Plk1 can be important in renal cancer.

Tumor Biol. (2013) 34:1887–1894 Table 1 Clinicopathologic characteristics of renal cell carcinoma patients Clinicopathologic characteristics

n (%)

Age (years) Gender

Median age (range) Male Female Left Right Transperitoneal radical nephrectomy Lumbar radical nephrectomy Lumbar nephrectomy T1 T2 T3 T4 I

52 (33–68) 58 (58) 42 (42) 55 (55) 45 (45) 81 (81) 15 (15) 4 (4) 48 (48) 15 (15) 31 (31) 6 (6) 27 (27)

II III IV No Yes No Yes

35 (35) 22 (22) 16 (16) 71 (71) 29 (29) 78 (78) 22 (22)

Tumor side Surgery

pT stage

Fuhrman grade

Metastasis Recurrence

Materials and methods

(included in the 100 cases) of tumor and 20 nontumorous tissues that were made sure to be free of malignant cells were quickly frozen in a deep freezer until protein extraction. The study was conducted according to an institutional review board-approved protocol, and written informed consent was obtained from all patients for surgery and research purposes.

Patients' information

Immunohistochemistry and evaluation

From 2005 to 2012, at the Department of Urology, the First Hospital of China Medical University in China, a total of 100 consecutive patients (58 men and 42 women; age range 33– 68 years) were with diagnosed primary RCC. Clinical information was retrieved from medical records. All patients routinely underwent computed tomography before surgery for preoperative staging. All enrolled patients underwent curative surgical resection without having chemotherapy or radiation therapy. Fifty-five cases had left RCC and 45 cases had right RCC. Eighty-one cases were treated with transperitoneal radical nephrectomy, 15 cases were treated with lumbar radical nephrectomy, and four cases were treated with lumbar nephrectomy. Two consulting pathologists retrospectively and independently reviewed hematoxylin and eosin-stained tissue slides according to the World Health Association classification. The Fuhrman scale was used to assess nuclear grade. Tumor stage was assigned according to the 2002 TNM classification. Clinicopathological characteristics are shown in Table 1. Eighty cases

Formalin-fixed, paraffin-embedded tissue blocks were cut into 4-μm sections and subjected to immunohistochemistry with mouse monoclonal antibody for human Plk1 (1:200 dilutions; Santa Cruz Biotechnology, Dallas, TX, USA). Immunohistochemistry was carried out using the streptavidin–peroxidaseconjugated method. Negative controls were prepared by substituting phosphate-buffered saline (PBS) for primary antibody. Immunohistochemical staining was performed according to previously published methods with minor modification [13]. All the immunoreactions were separately evaluated by two senior pathologists. Immunoreactivity for Plk1 was considered positive when brown particles appeared in cytoplasm. Intensity of Plk1 immunostaining (1 = weak, 2 = moderate, and 3 = intense) and percentage of positive tumor cells (0 % = negative, 1–50 % = 1, 51–75 % = 2, ≥76 % = 3) were assessed in at least five high power fields (×400 magnification). Scores of each tumorous sample were multiplied to give a final score of 0, 1, 2, 3, 4, 6, or 9, and the tumors were

Tumor Biol. (2013) 34:1887–1894

finally determined as negative: score 0; lower expression: score ≤4; or higher expression: score ≥6. Real-time PCR analysis Total RNA was isolated from cultured cells and frozen tissues with ice-cold TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's specifications. Concentration of RNA was determined by Thermo Scientific NanoDrop ND-100 (Wilmington, DE, USA). Then, reverse transcription of 2 μl of total RNA was performed using Perfect Real-Time SYBR® PrimeScript® RT-PCR Kit (Takara, Kyoto, Japan). Real-time PCR analysis of the cDNA was quantified using Thermal Cycler Dice™ Real-Time System TP800

Fig. 1 Expressions of Plk1 in renal cancer tissues and normal renal tissues. a Band intensities of western blot indicated significant upregulation in renal cancer tissues (T1, T2, and T3) in comparison with that in the normal renal tissues (N1, N2, and N3). β-Actin was used as a loading control to assure equal amounts of protein in all lanes. b The ratio between the optical densities of Plk1 and β-actin of the same tissue though western blot was calculated and expressed graphically. Significant differences of Plk1 protein expression between tumor (T) and normal tissues (N) were analyzed statistically and Plk1

1889

(Takara, Kyoto, Japan). The RT reaction system was kept in 50 °C for 2 min and heated to 95 °C for 10 min. Then, the PCR reaction was done by 45 cycles, denaturing the mixture at 95 ° C for 15 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s. β-Actin was chosen as the internal control. Primer sequences used for Plk1 and β-actin were as follows: Plk1 sense primer: 5′-CAAGAAGAATGATACAGTA-3′, Plk1 antisense primer: 5′-GGATATAGCCAGAAGTAA-3′, β-actin sense primer: 5′-CTCCATCCTGGCCTCGCTGT-3′, and βactin antisense primer: 5′-GCTGTCACCTTCACCGTTC-3′. Negative controls consisted of distilled H2O. The expression levels of Plk1 were normalized with β-actin using a 2−ΔΔCT method. A melting curve analysis was performed for the PCR products to evaluate primer specificity.

protein expression was obviously lower in renal cancer tissues (P< 0.05). c The ratio between the CT value of Plk1 and β-actin of the same tissue by real-time PCR was calculated and expressed graphically. Significant differences of Plk1 mRNA level between tumor (T) and normal tissues (N) were analyzed statistically and Plk1 mRNA level was obviously lower in renal cancer tissues (P<0.05). The data were representative of three individual experiments. d Plk1 immunostaining in the normal renal tissue and the renal cancer. (×400)

1890

Tumor Biol. (2013) 34:1887–1894

Western blot Frozen tissues (including renal tumor tissues and normal renal tissues) or cells were washed twice with ice-cold PBS, homogenized on ice in 10 volumes (w/v) of lysis buffer containing 20 mM Tris–HCl, 1 mM ethylene diamine tetraacetie acid (EDTA), 50 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 1 % Triton X-100, 1 mM phenylmethanesulfonyl fluoride (PMSF), and phosphatase inhibitor using a homogenizer (Heidoph, DLA×900, Germany). The homogenate was centrifuged at 10,000 rpm for 30 min at 4 °C. The supernatant was collected and stored at −70 °C. Protein content was determined by the bicinchonininc acid (BCA) assay (BCA protein assay kit23227, Pierce Biotechnology, USA). From each sample preparation, 80 μg of total protein was separated by 8 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and then transferred to polyvinylidene fluoride (PVDF) blot membranes. Total protein extracts were analyzed by immunoblot with indicated antibodies following SDS-PAGE analysis. Immunoblots were performed using mouse monoclonal antibodies for Plk1 (Abcam, Cambridge, MA, USA) and mouse monoclonal antibody for β-actin (Abcam, Hong Kong, a housekeeping protein used as a loading control to assure equal amounts of protein in all lanes). After blocking nonspecific binding with 5 % bovine serum albumin (BSA) in thermomorphic biphasic amine solvent (TBS) (pH 7.5) containing 0.05 % Tween 20 (TBS+Tween (TBST)), primary antibodies were incubated on the membranes for Plk1 (1:2000) and β-actin (1:2000) overnight at 4 °C in TBST. Following three times washes in TBST, the membranes were incubated for 2 h at 37 °C with goat polyclonal secondary antibody to mouse IgG (1:5,000, Abcam, Hong Kong) labeled with horseradish peroxidase. The proteins were detected using an ECL Table 2 Relationship between Plk1 expression and clinicopathological features of renal cell carcinoma

detection system (Pierce, Rockford, IL, USA), as directed by the manufacturer. Specific bands for Plk1 and β-actin were identified by prestained protein molecular weight marker (MBI Fermentas, USA). The EC3 Imaging System (UVP Inc., PA, USA) was used to catch up the specific bands, and the optical density of each band was measured using Image J software. The ratio between the optical density of interest proteins and βactin of the same sample was calculated as relative content and expressed graphically. Cell lines and cultures The following two renal adenocarcinoma cell lines with different invasiveness abilities were selected: invasively growing ACHN cells and poorly invasive cells 769-P. They were obtained from the American Type Culture Collection (Shanghai, China). The cells were maintained in the culture medium recommended by the American Type Culture Collection and were harvested by using treatment with 0.25 % trypsin (Invitrogen, USA) when they were in the logarithmic phase of growth for use in the following experiments. Cell proliferation assays ACHN cells (1×104 cells per well) in logarithmic growth phase were cultured per well with a total of 100 μl of medium (96-well plates) in a triplicate pattern and treated with different concentrations of scytonemin for 12, 24, 48, and 72 h. Each well received 20 μl CCK-8 solution and incubated for a further 4 h. Finally, the optical density value (A) of each well was measured at a measurement wavelength of 450 nm using a plate reader (Model 680, BioRad, UK). Cell growth inhibition ratio was calculated as (1

Clinicopathologic characteristics

Group

Age (years)

<52 ≥52 Male Female Left Right Transperitoneal radical nephrectomy Lumbar (radical) nephrectomy T1 T2–T4 I–II III–IV No Yes No Yes

Gender Tumor side Surgery

pT stage Fuhrman grade Metastasis Recurrence

No. of cases

Cases with high Plk1 expression (%)

53 47 58 42 55 45 81

37 (69.8) 40 (85.1) 43 (74.1) 34 (81.0) 46 (83.6) 31 (68.9) 60 (74.1)

19 48 52 62 38

17 (89.5) 30 (62.5) 47 (90.4) 39 (62.9) 33 (86.8)

71 29 78 22

45 (63.4) 27 (93.1) 51 (65.4) 21 (95.5)

X2

P

2.483

0.115

0.312

0.576

2.264

0.132

1.283

0.257

9.441

0.002

5.562

0.018

7.609

0.006

6.277

0.012

Tumor Biol. (2013) 34:1887–1894

−A490 of experimental well/A490 of blank control well)× 100 %. Each assay was repeated at least three times. Colony formation assay ACHN cells were incubated either in the absence or presence of different concentrations of scytonemin for 24, 48, and 72 h before reseeding in soft agar at low density. After trypsinization and counting, the cells were resuspended in medium containing 0.3 % agarose and 0.5 ml containing 500 cells was added to each well of 48-well plates coated with polyHEMA. The cells were incubated at 37 °C in a humidified incubator with 5 % CO2 in air for 14 days and colonies were counted in an inverted phase contrast microscope (Olympus, Tokyo, Japan). Transwell cell invasion assay

1891

in renal cancer samples in comparison with the normal renal samples. The western blot of six samples is shown in Fig. 1a, and the optical density of the tumorous (T) and normal (N) tissues was measured and expressed graphically (Fig. 1b). The data by real-time PCR were examined in Fig. 1c. Moreover, immunohistochemistry was used to determine the expression of Plk1 in tumorous and normal tissues (Fig. 1d). More Plk1 expression in renal cancer suggested that Plk1 might be involved in renal carcinogenesis. Relationship of Plk1 expression with clinicopathologic characters Comparative analysis about relationship of Plk1 expression with clinicopathologic characters by immunohistochemistry are shown in Table 2. There was an obvious difference of

Cell invasion assay was performed using a 24-well Transwell chamber (Costar, MA, USA). At 48 h following the treatment of different concentrations of scytonemin, ACHN cells (1×104) were detached and seeded in the upper chamber of an 8 μm pore size that was coated with Matrigel and inserted in the 24-well plate. Then, the cells were cultured in serum-free medium for another 12 h. Cells were allowed to invade forward to Dulbecco’s modification of Eagle’s medium (DMEM) containing 10 % fatal bovine serun (FBS) in the bottom chamber. The noninvasive cells on the upper membrane surface were removed with a cotton tip, and the invasive cells attached to the lower membrane surface were fixed with 4 % paraformaldehyde and stained with hematoxylin. The number of invasive cells was counted in five randomly selected high power fields under microscope. Data presented are representative of three individual wells. Statistical analysis SPSS 13.0 was used to perform data analysis. The t test was used to analyze the data from real-time PCR and western blot in the tissues and cells. The χ2 test was used to evaluate the association between Plk1 expression and clinicopathologic variables. Spearman's correlation test was used to analyze the rank data and Fisher's exact test to compare the different rates. P values <0.05 were considered statistically significant.

Results Expression of Plk1 in renal cancer tissues and normal renal tissues Real-time PCR and western blot analysis were used to evaluate Plk1 expression in renal carcinoma tissues and normal renal tissues. The increased Plk1 expression could be detected

Fig. 2 Expressions of Plk1 in 769-P cell line and ACHN cell line. a Band intensities indicated higher expression level of Plk1 in ACHN cells in comparison with that in 769-P cells. β-Actin was used as a loading control to assure equal amounts of protein in all lanes. b The ratio between the optical densities of Plk1 and β-actin of the same group though western blot was calculated and expressed graphically. c The ratio between the CT value of Plk1 and β-actin of the same group by real-time PCR was calculated and expressed graphically. The data were representative of three individual experiments

1892

Plk1 expression in renal carcinoma tissues with different histopathology grades, clinical stages, tumor metastases, and recurrences. The expression of Plk1 in the renal cancer tissues increased with increasing tumor stage and grade. These data suggested that the expression of Plk1 was closely related to differentiation, malignancy, metastasis, and recurrence of RCC. In other words, high expression of Plk1 was the Fig. 3 Proliferation and invasion ability of ACHN were inhibited by scytonemin. a CCK-8 assay was used to examine ACHN cell proliferation. The cell proliferation was inhibited on dose-dependent correlation with the increasing concentration of scytonemin. b Colony formation assay was used to examine ACHN cell proliferation. The cell proliferation was inhibited on dose-dependent correlation with the increasing concentration of scytonemin. *P<0.05, relative to former concentration scytonemin group. #P<0.05, relative to former time point. c Transwell invasion assay was used to examine ACHN cell invasion. The cell invasion was inhibited on dose-dependent correlation with the increasing concentration of scytonemin. The data are representative of three individual experiments

Tumor Biol. (2013) 34:1887–1894

important feature of poor differentiated and aggressive renal carcinoma, which were useful indicators for prognosis of RCC. Plk1 expression in renal cancer cell lines The above studies have reported that Plk1 expression was correlated with progression of renal cancer tissues. Next,

Tumor Biol. (2013) 34:1887–1894

western blot was used to determine the expression of Plk1 in human renal cancer cell lines ACHN and 769-P. The results showed that high invasive renal adenocarcinoma ACHN cells have intense Plk1 expression compared with renal adenocarcinoma 769-P cells (Fig. 2a, b, and c). It further confirmed that tumor malignancy grade was positive in correlation with Plk1 expression. Scytonemin, Plk1-specific inhibitor, could inhibit proliferation and invasion ability of ACHN For further determination of the Plk1's role in renal cancer, we used the specific inhibitor of Plk1, syctonemin, which treated renal adenocarcinoma ACHN cell. With increasing concentration of scytonemin, we found that cell proliferation and invasion activity decreased significantly (Fig. 3a–c). It demonstrated that Plk1 played an important function on renal cancer cells' proliferation and invasion.

Discussion Many studies demonstrate that there are overexpressions of Plk1 in several human cancers and Plk1 overexpression correlates with tumor progression and patient survival [15–17]. It has been reported that the level of Plk1 transcripts in tumor samples could be linked to prognosis of patients with nonsmall-cell lung cancer [17]. Our data also show that high expression level of Plk1 is strongly correlated with clinicopathologic parameters in renal carcinoma patients. The correlation between Plk1 expression with clinical stage and histological grade of a tumor has potential to aid clinicians in their search for improving treatment decisions for different cancer patients. Plk1 was first cloned and reported by Golsteyn in 1994 [18]. Subsequent studies have shown that Plk1 alone was the important protein activating the cdc2–cyclin B complex, which is also known as mitosis-promoting factor (MPF) [19]. In transition of G2 to M phase, Plk1 activates cdc25, which is a promoter of cdc2–cyclin B complex and also plays a key role in bipolar spindle formation, centrosome maturation, and separation of the chromosome [20, 21]. Expression of Plk1 is clearly increased in G2/M phase transition, and the expression of Plk1 on both the mRNA and protein levels is positively correlated to cell proliferation activity [22]. Similar to other reports of Plk1 involvement in proliferation of cell types such as breast cancer, lung cancer, prostate cancer, and esophageal cancer, we have also shown that scytonemin, a small molecule inhibitor of Plk1, significantly reduced cell proliferation and cell invasion [13, 22, 23]. Plk1 is, therefore, a promising gene candidate for targeted therapy in treating not only renal cancer but other solid

1893

tumors as well. The requirement of Plk1 in tumorigenesis of renal cancer and other cancers implies it has a critical role in the final pathway for cancer cell proliferation and invasion. We have demonstrated that Plk1 was overexpressed in renal cancer tissues and is correlated with important histopathologic characteristics (grades and stages). By using the specific inhibitor, we showed that Plk1 inhibitor inhibited cell proliferation and invasion of renal cancer cells. Therefore, Plk1 is likely to play an important role in oncogenesis and progression of renal cancer. Further research is needed to prove the value of Plk1 as a molecular target, such as the relationship between Plk1 expression and 5-year survival rate of patients, and interaction between Plk1 and other molecules.

References 1. Kyttaris VC. Kinase inhibitors: a new class of antirheumatic drugs. Drug Des Devel Ther. 2012;6:245–50. 2. Kuroda N, Shiotsu T, Hes O, Michal M, Shuin T, Lee GH. Acquired cystic disease-associated renal cell carcinoma with gain of chromosomes 3, 7, and 16, gain of chromosome X, and loss of chromosome Y. Med Mol Morphol. 2010;43(4):231–4. 3. Luo J, Liu X. Polo-like kinase 1, on the rise from cell cycle regulation to prostate cancer development. Protein Cell. 2012;3(3):182–97. 4. Sakai D, Dixon J, Dixon MJ, Trainor PA. Mammalian neurogenesis requires Treacle-Plk1 for precise control of spindle orientation, mitotic progression, and maintenance of neural progenitor cells. PLoS Genet. 2012;8(3):e1002566. 5. Kachaner D, Filipe J, Laplantine E, et al. Plk1-dependent phosphorylation of optineurin provides a negative feedback mechanism for mitotic progression. Mol Cell. 2012;45(4):553–66. 6. Vazquez-Martin A, Oliveras-Ferraros C, Cufí S, Menendez JA. Pololike kinase 1 regulates activation of AMP-activated protein kinase (AMPK) at the mitotic apparatus. Cell Cycle. 2011;10(8):1295–302. 7. Yoon HE, Kim SA, Choi HS, Ahn MY, Yoon JH, Ahn SG. Inhibition of Plk1 and Pin1 by 5′-nitro-indirubinoxime suppresses human lung cancer cells. Cancer Lett. 2012;316(1):97–104. 8. Francescangeli F, Patrizii M, Signore M, et al. Proliferation state and polo-like kinase1 dependence of tumorigenic colon cancer cells. Stem Cells. 2012;30(9):1819–30. 9. Peng DX, Luo M, Qiu LW, He YL, Wang XF. Prognostic implications of microRNA-100 and its functional roles in human epithelial ovarian cancer. Oncol Rep. 2012;27(4):1238–44. 10. de Oliveira JC, Brassesco MS, Pezuk JA, et al. In vitro PLK1 inhibition by BI 2536 decreases proliferation and induces cell-cycle arrest in melanoma cells. J Drugs Dermatol. 2012;11(5):587–92. 11. Maire V, Némati F, Richardson M, et al. Polo-like kinase 1: a potential therapeutic option in combination with conventional chemotherapy for the management of patients with triple-negative breast cancer. Cancer Res. 2013;73(2):813–23. 12. Zhang Y, Du XL, Wang CJ, et al. Reciprocal activation between PLK1 and Stat3 contributes to survival and proliferation of esophageal cancer cells. Gastroenterology. 2012;142(3):521–30.e3. 13. Zhang Z, Zhang G, Kong C. High expression of polo-like kinase 1 is associated with the metastasis and recurrence in urothelial carcinoma of bladder. Urol Oncol. 2011 Dec 20, [Epub ahead of print]. 14. Deeraksa A, Pan J, Sha Y, et al. Plk1 is upregulated in androgeninsensitive prostate cancer cells and its inhibition leads to necroptosis. Oncogene. 2012 Aug 13, [Epub ahead of print].

1894 15. Lee C, Fotovati A, Triscott J, et al. Polo-like kinase 1 inhibition kills glioblastoma multiforme brain tumor cells in part through loss of SOX2 and delays tumor progression in mice. Stem Cells. 2012;30(6):1064–75. 16. Pellegrino R, Calvisi DF, et al. Oncogenic and tumor suppressive roles of polo-like kinases in human hepatocellular carcinoma. Hepatology. 2010;51(3):857–68. 17. Medema RH, Lin CC, Yang JC. Polo-like kinase 1 inhibitors and their potential role in anticancer therapy, with a focus on NSCLC. Clin Cancer Res. 2011;17(20):6459–66. 18. Golsteyn RM, Schultz SJ, Bartek J, Ziemiecki A, Ried T, Nigg EA. Cell cycle analysis and chromosomal localization of human Plk1, a putative homologue of the mitotic kinases Drosophila polo and Saccharomyces cerevisiae Cdc5. J Cell Sci. 1994;107(Pt 6):1509–17. 19. Kettenbach AN, Schweppe DK, Faherty BK, Pechenick D, Pletnev AA, Gerber SA. Quantitative phosphoproteomics

Tumor Biol. (2013) 34:1887–1894

20.

21.

22.

23.

identifies substrates and functional modules of Aurora and Polo-like kinase activities in mitotic cells. Sci Signal. 2011;4(179):rs5. Lobjois V, Froment C, Braud E, et al. Study of the dockingdependent PLK1 phosphorylation of the CDC25B phosphatase. Biochem Biophys Res Commun. 2011;410(1):87–90. Smits VA, Klompmaker R, Arnaud L, Rijksen G, Nigg EA, Medema RH. Polo-like kinase-1 is a target of the DNA damage checkpoint. Nat Cell Biol. 2000;2(9):672–6. Zhang Z, Su WH, Feng C, et al. Polo-like kinase 1 may regulate G2/M transition of mouse fertilized eggs by means of inhibiting the phosphorylation of Tyr 15 of Cdc2. Mol Reprod Dev. 2007;74(10):1247–54. McInnes C, Mezna M, Fischer PM. Progress in the discovery of polo-like kinase inhibitors. Curr Top Med Chem. 2005;5(2):181– 97.