Contributed equally
Targeting protein for Xenopus kinesin-like protein 2 (TPX2) activates Aurora kinase A during mitosis and targets its activity to the mitotic spindle, serving an important role in mitosis. It has been associated with different types of cancer and is considered to promote tumor growth. The aim of the present study was to explore the role of TPX2 in diagnosing prostate cancer (PCa). It was identified that TPX2 expression in PCa tissues was increased compared with benign prostate tissues. Microarray analysis demonstrated that TPX2 was positively associated with the Gleason score, tumor-node-metastasis (TNM) stage, clinicopathological stage, metastasis, overall survival and biochemical relapse-free survival.
Prostate cancer (PCa) is the most commonly diagnosed cancer in men. In 2016, there were an estimated 1,685,210 incident cases of PCa in the USA (
Targeting protein for Xenopus kinesin-like protein 2 (TPX2) belongs to the Microtubule-associated protein family. All members of this family contain a conserved TPX2 motif that interacts with microtubules (
Previous evidence indicated that increased TPX2 expression was observed in different cancer types: It has been demonstrated that high TPX2 expression was associated with tumor progression and poor survival rate of gastric cancer (
These procedures were approved by the Research Ethics Committee of The Third Affiliated Hospital of Guangzhou Medical University (Guangzhou, China). A tissue microarray (TMA) containing 73 primary PCa tissues and 7 adjacent noncancerous prostate tissues was purchased from Alenabio (cat no. PR803c; Xi'an, China). None of the patients from which the samples were taken had undergone chemotherapy or radiotherapy prior to surgery. The Taylor dataset (Gene Expression Omnibus accession no., GSE21032), an online PCa dataset that included 150 primary PCa tissues and 29 adjacent noncancerous prostate tissues, was downloaded from
Proteins of LNCap, PC3 and DU145 cells were extracted by RIPA lysis buffer (cat no. P0013B; Beyotime Institute of Biotechnology, Shanghai, China) for 30 min at 4°C, using the protease inhibitor phenylmethylsulfonyl fluoride (100 mM; cat no. ST506; Beyotime Institute of Biotechnology) and the supernatant liquid was centrifuged at 13780 × g. Protein quantification was performed using BCA (cat no. 23227; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and 15 µg protein/lane was separated using 10% SDS-PAGE and transferred to nitrocellulose membranes. Subsequent to blocking with 10% skim milk (Kang Long Group, Inc., Inwood, NY, USA) for 1 h at room temperature, the membrane was incubated with the TPX2 polyclonal rabbit antibody (dilution, 1:100; cat no. HPA005487; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and β-actin antibody (dilution, 1:200; cat no. BM0627; Boster Biological Technology, Pleasanton, CA, USA) at 4°C overnight. Subsequent to washing three times with Tris-buffered saline with 0.25% Tween-20, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit IgG; dilution, 1:400; cat no. BA1060; Boster Biological Technology) for a further 2 h at room temperature. Finally, the immunoreactive protein bands were detected by a Chemiluminescence imaging analysis system (5200; Tanon Science and Technology Co., Ltd., Shanghai, China). The relative density of protein expression was quantified using Image J software 1.46 (National Institutes of Health, Bethesda, MD, USA).
Immunohistochemistry staining of the tissue microarray slice was conducted with the DAKO EnVision system (Dako Diagnostics, Zug, Switzerland) on Dako automated immunostaining instruments, according to the protocol of our previous studies (
The three human PCa DU145, PC3 (CVCL_0035) and LNCaP cell lines used in the present study were purchased from the American Type Culture Collection (Manassas, VA, USA) and were maintained in the high-glucose Dulbecco's modified Eagle's medium (DMEM; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). The cells were cultured at 37°C in a 5% CO2 humidified incubator. The TPX2 coding sequence (Genebank: NM_012112.4) was synthesized and cloned into lentivirus-vector by Shanghai Generay Biotech Co., Ltd. (Shanghai, China), and the plasmid was verified by sequencing. The lentivirus of TPX2-overexpresion and blank vector were produced by Guangzhou HYY Medical Science Co., Ltd. (Guangzhou, China). Subsequently, the LNCaP, DU145 and PC3 cell lines were transfected by the lentivirus of TPX2-overexpresion and blank vector, as the negative control, and finally the stable cell line was produced through puromycin treatment.
A Cell Counting Kit-8 (CCK-8) assay (cat. no., C0038; Beyotime Institute of Biotechnology, Shanghai, China) was used to evaluate the proliferation of PCa cells. A total of ~2×103 LnCap and PC3 cells/well were seeded into 96-well plates and cultured for 24, 48, and 72 h at 37°C. Cells were then incubated with 10 µl CCK-8 for 2 h at 37°C. The absorbance at a wavelength of 450 nm was measured with a spectrophotometer (Multiskan™ MK3, Thermo Fisher Scientific, Inc.). Data are presented as mean ± standard deviation (SD) of three independent experiments.
Apoptosis was analyzed using an Annexin V- allophycocyanin (APC)/7-aminoactinomyocin (7-AAD) Apoptosis Detection kit (BD Pharmingen; BD Biosciences, Franklin Lakes, NJ, USA). The cultured cells were collected and suspended in 1× Binding Buffer (from the apoptosis detection kit) followed by incubation with Annexin V-APC/7AAD for 15 min at room temperature (Annexin V-APC/7AAD kit, cat no. 4224750). Apoptotic analyses were conducted on a BD FACSCalibur flow cytometer (BD Biosciences), and a minimum of 1×106 cell counts were used for each experimental sample. Docetaxel (10 nmol; Shanghai Yu Yan Biotech Co, Ltd, Shanghai, China) was added to the PC3 cells for 48 h at 37°C and the experiments were repeated. Only PC3 cells were used due to patients with androgen-dependent prostate cancer primarily receiving emasculation therapy, with patients with non-androgen-dependent prostate cancer being treated with chemotherapy. LNCap is an androgen-dependent cell and PC3 is a non-androgen-dependent cell (
The scratch wound healing assay was performed to evaluate the migratory ability of the PCa cells. The transfections with the TPX2 or negative control vectors were performed when the cells (LnCap and PC3) reached 80–90% confluence using Lipofectamine 2000 reagent (cat no. 11668019; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. After 24 h transfection, a scratch was made with a 10 µl pipette tip. The cells were then returned to the incubator until the indicated times (24, 48 and 72 h) at 37°C. Representative images were captured, and the distance that the cells that had migrated from the wound edge were counted at each time point use ImageJ Pro-Plus 6.0 (National Institutes of Health, Bethesda, MD, USA). Data are presented as mean ± SD of three independent experiments.
The CytoSelect Cell Migration and Invasion kit (Cell Biolabs, Inc., San Diego, CA, USA) was used according to the manufacturer's protocol. After 24 h transfection, PCa cells were re-suspended in a serum-free RPMI-1640 medium (cat no. sh30809.01B; HyClone; GE Healthcare Life Sciences) to a density of 25×104/ml. The suspended cells (200 µl) were seeded in the upper chambers, and the lower chamber contained 10% fetal bovine serum as a chemoattractant. Following incubation at 37°C for 48 h, the cells that had migrated through the membrane were stained by 0.1% crystal violet (cat no. BS234b; Biosharp, Hefei, China) at room temperature for 15 min and quantified by counting 9 independent symmetrical visual fields under a light microscope (Nikon Corporation). Data are presented as mean ± SD of three independent experiments.
The software of SPSS 13.0 for Windows (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. Continuous variables are presented as mean ± SD. The Kaplan-Meier method was used for the survival analysis, and a log-rank test was used to analyze the difference between survival times. The Student's t-test was used for data analysis. P<0.05 was considered to indicate a statistically significant difference.
To assess TPX2 protein expression in PCa, a prostate tissue chip containing 7 benign prostate tissues and 73 prostate cancer tissues was evaluated by immunohistochemistry (
As the tissue chip data did not contain additional clinicopathological parameters, including metastasis, PSA level or biochemical recurrence time, the Taylor dataset, an online PCa dataset, was used for the analysis of survival and prognosis. The results of the analysis of the Taylor dataset revealed that PCa at later pathological stages (III–IV) demonstrated increased TPX2 expression compared with PCa in earlier pathological stages (I–II). In addition, statistical analyses of the Taylor dataset also indicated that PCa with a Gleason Score ≥8 exhibited a higher expression of TPX2 compared with those with a Gleason Score <8 (P=0.004) at the mRNA level (
The Kaplan-Meier method was then used to analyze the association of TPX2 expression levels with the biochemical recurrence-free time and the overall survival time of patients with PCa in the Taylor dataset. The median TPX2 expression (~6.03) in all PCa patients was used as the cutoff to divide all PCa tissues into high (n=74) and low (n=74) TPX2 expression groups. As demonstrated in
To assess the expression of TPX2 in human PCa cells, the expression of TPX2 in three established PCa cell lines: LNCaP; DU145; and PC3, was first assessed. Notably, TPX2 was highly expressed in the DU145 cell line, and weakly expressed in the LNCaP and PC3 cell lines (
The data indicated that the LNCaP-TPX2 cells exhibited fewer apoptotic cells compared with the vector-transfected LNCaP cells (
To identify whether TPX2 promoted cell mobility, A Transwell assay was used to assess the migration activity of LNCaP and PC3 cells with or without forced expression of TPX2. The results indicated that the migratory ability of the LNCaP-Tpx2 cells was increased compared with the LNCaP-NC cells (
Although early detection of PCa through serum testing of PSA, and improved procedures of surgical intervention and radiation therapy have significantly decreased the number of fatalities, the prognosis of late-stage PCa remains poor (
Aberrant activity of TPX2 has been confirmed in several types of cancer (
Chemotherapy has served a pivotal role in the treatment of metastatic castration-resistant prostate cancer (mCRPC) since 2004, when docetaxel-based chemotherapy first exhibited modest improvement in survival time compared with mitoxantrone-based therapy as a first-line chemotherapy (
In conclusion, the present study suggested that the overexpression of TPX2 improved the proliferative, invasive and migratory abilities and inhibited apoptosis of PCa cell lines. Overexpression of TPX2 was more frequently observed in high TNM and clinicopathological staging. Statistical analyses of the online Taylor dataset revealed that high TPX2 levels were associated with high Gleason scores, metastasis and PSA rising again following treatment, indicating that high-level TPX2 in PCa tissues indicated poor prognosis. Therefore, the preliminary conclusion of the present study is that the overexpression of TPX2 serves as a potential biomarker for PCa diagnosis and prognosis.
Previous studies have demonstrated that the proliferation and invasion of prostate cancer cells may be promoted by inhibiting the expression of TPX2 (
Not applicable.
The present study was supported by grants from the Guangzhou Health Science and Technology General Guidance project [Health Science and technology (2017) 6], Guangzhou Medical University Fund (grant no., 2015C26), Natural Science Foundation of Guangdong Province (grant no., 2014A030310088), the Zhejiang Open Foundation of the Most Important (grant no., YFKJ003), the Fundamental Research Funds for the Central Universities (grant nos., 2017PY023 and 2017MS127).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
WDZ supervised the entire study, participated in study design and coordination. JZ, RYH, FNJ, DXC, CW, ZDH and YXL performed most of the experiments and statistical analyses, and drafted the manuscript. All authors read and approved the final manuscript.
These procedures were approved by the Research Ethics Committee of The Third Affiliated Hospital of Guangzhou Medical University (Guangzhou, China).
Not applicable.
The authors declare that they have no competing interests.
targeting protein for Xenopus kinesin-like protein 2
prostate cancer
prostate specific antigen
clinical tumor-node-metastasis
Immunohistochemical staining for TPX2 protein expression in PCa and adjacent benign tissues. (A) The whole scanned image of the tissue array (B) Statistical analyses indicated an increased immunoreactivity score in cancer tissues compared with benign tissues. (C) Enlarged image of BPH tissue indicated that TPX2 was only weakly expressed in benign tissues. Red box demonstrates the blue box at a magnification. (D) Enlarged image of PCa tissue demonstrated that TPX2 was highly abundant in the nucleus and cytoplasm of tumor cells. Red box demonstrates the blue box at a magnification. TPX2, Targeting protein for Xenopus kinesin-like protein 2; PCa, prostate cancer; BPH, benign prostatic hypertrophy. *P<0.05.
High TPX2 expression is associated with poor prognosis in patients with PCa. (A) Kaplan-Meier analysis of the BCR time of patients with PCa with high TPX2 expression levels was shorter compared with those with low TPX2 expression levels (P=0.007). (B) The overall survival time of patients with PCa was not associated with TPX2 expression levels (P=0.120). TPX2, Targeting protein for Xenopus kinesin-like protein 2; PCa, prostate cancer; BCR, biochemical recurrence-free time.
TPX2 expression and function in prostate tumor cell lines. (A) Western blotting detected TPX2 protein expression in PCa cell lines. TPX2 was highly expressed in the DU145 cell line, while expressed at low levels in the PC3 and LNCaP cell lines. The CCK8 assay indicated that the overexpression of TPX2 in (B) LNCaP and (C) PC3 cells promoted the proliferation rate of PCa cells compared with the control group. The flow cytometry assay conducted in (D) LNCaP-NC and (E) LNCaP-TPX2 cells suggested that TPX2 inhibited cell cycle transit from G0/G1 to S/M phase in LNCaP-TPX2 cells. (F) The average proportion of cells in S+G2 phase was 40.607±2.172 in the LNCaP-TPX2 group, while it was 47.09±1.804 in the LNCaP-NC group (P=0.016). (G) PC3-NC and (H) PC3-TPX2 cells were also examined using flow cytometry. (I) No significant difference in PC3 cells was observed (P>0.05). TPX2, Targeting protein for Xenopus kinesin-like protein 2; PCa, prostate cancer; NC, negative control; NS, not significant. *P<0.05.
Overexpression of TPX2 inhibits apoptosis in LNCaP and PC3 cells. Cells overexpressing TPX2 were stained using Annexin V, and it was demonstrated that TPX2 decreased the rate of apoptosis in (A) LNCaP-NC and (B) LNCaP-TPX2 cells. (C) The apoptotic rate in the LNCaP-TPX2 group was 2.83±0.42, while it was 4.25±0.13 in the LNCaP-NC group (P=0.005). No difference in the apoptotic rate was observed between (D) PC3-NC and (E) PC3-TPX2 cells treated with docetaxel. (F) The apoptosis rates in the PC3-TPX2 and PC3-NC groups were similar (36.92±1.49 vs. 38.49±2.36; P=0.383). TPX2, Targeting protein for Xenopus kinesin-like protein 2; PCa, prostate cancer; NC, negative control; NS, not significant. *P<0.05.
Overexpression of TPX2 promotes the migration of LNCaP and PC3 cells. A Transwell assay was used to detect migration in (A) LNCaP-NC and (B) LNCaP-TPX2 cells. (C) The migration cell number in LNCaP-TPX2 group was 366.7±24.44, while it was 117.3±48.39 in LNCaP-NC group (P=0.022). A Transwell assay was also performed in (D) PC3-NC and (E) PC3-TPX2 cells. (F) The migration cell number in PC3-TPX2 group was increased compared with the PC3-NC group (260.0±8.0 vs. 150.7±18.04; P=0.041). Magnification of all images, ×400. TPX2, Targeting protein for Xenopus kinesin-like protein 2; PCa, prostate cancer; NC, negative control. *P<0.05.
Overexpression of TPX2 enhanced wound healing of LNCaP and PC3 cells. Wound healing assay analysis was performed at 0, 24 and 48 h with or without TPX2 overexpression. The migratory ability was increased in (A) LNCaP-TPX2 and (B) PC3-TPX2 cells at 24 and 48 h. TPX2, Targeting protein for Xenopus kinesin-like protein 2; NC, negative control. **P<0.01.
Clinical features of patients.
Dataset | ||
---|---|---|
Clinical features | TMA |
Taylor dataset |
Benign tissue, cases | 7 | 29 |
Prostate cancer, cases | 73 | 150 |
Age, years | ||
Mean | 68.29±10.20 | 58.33±7.10 |
<66 | 23 | 124 |
≥66 | 44 | 26 |
Serum PSA | ||
<4 | – | 21 |
4–10 | – | 81 |
≥10 | – | 42 |
Gleason score | ||
≤6 | – | 41 |
7 | – | 76 |
≥8 | – | 22 |
Pathological stage | ||
I–II | 43 | 86 |
III–IV | 27 | 55 |
TNM stage | ||
≤T2A | 45 | 135 |
≥T2B | 27 | 10 |
Immunohistochemical analysis. TMA, tissue microarray; PSA, prostate serum antigen; TNM, tumor-node-metastasis.
Association of TPX2 expression with clinicopathological characteristics of prostate cancer.
Dataset | ||||||
---|---|---|---|---|---|---|
TMA dataset |
Taylor dataset | |||||
Clinical features | Case | Mean ± SD | P-value | Case | Mean ± SD | P-value |
Tissue | ||||||
Benign | 7 | 2.43±1.72 | 0.019 | 29 | 5.79±0.16 | <0.001 |
Cancer | 73 | 5.12±2.91 | – | 150 | 6.20±0.61 | – |
Age | ||||||
<66 | 24 | 5.17±2.88 | 0.930 | 12 | 56.18±0.57 | 0.421 |
≥66 | 49 | 5.10±2.95 | – | 25 | 6.29±0.75 | – |
Serum PSA | ||||||
<10 | – | – | – | 105 | 6.08±0.47 | 0.009 |
≥10 | – | – | – | 42 | 6.37±0.63 | – |
Gleason score | ||||||
≤7 | – | – | – | 117 | 6.05±0.44 | 0.004 |
≥8 | – | – | – | 22 | 6.59±0.77 | – |
Pathological stage | ||||||
I–II | 43 | 4.26±2.37 | 0.002 | 86 | 6.03±0.41 | 0.007 |
III–IV | 27 | 6.63±3.25 | – | 55 | 6.30±0.66 | – |
TNM stage | ||||||
≤T2A | 45 | 4.16±2.25 | 0.001 | 135 | 6.14±0.55 | 0.028 |
≥T2B | 27 | 6.78±3.22 | – | 10 | 6.55±0.76 | – |
Metastasis | ||||||
No | – | – | – | 122 | 6.02±0.37 | <0.001 |
Yes | – | – | – | 28 | 6.96±0.81 | – |
PSA rise following treatment | ||||||
Negative | – | – | – | 104 | 6.01±0.37 | 0.001 |
Positive | – | – | – | 36 | 6.46±0.73 | – |
Overall survival | ||||||
Alive | – | – | – | 131 | 6.46±0.73 | 0.002 |
Succumbed | – | – | – | 19 | 6.90±0.96 | – |
Data correspond with immunoreactive score from immunohistochemical analysis; TMA, tissue microarray; PSA, prostate serum antigen; TNM, Tumor-Node-Metastasis; SD, standard deviation; TPX2, Targeting protein for Xenopus kinesin-like protein 2.