Ras homolog gene family, member A (RhoA) has been reported as essential to the invasion process and aggressiveness of numerous cancers. However, there are only sparse data on the expression and activity of RhoA in clinically localised prostate cancer. In numerous cancers, tumour cells at the invasive front demonstrate more aggressive behaviour in comparison with the cells in the central regions. In the present study, the expression and activity of RhoA was evaluated in 34 paraffin-embedded and 20 frozen prostate tissue specimens obtained from 45 patients treated with radical prostatectomy for clinically localised cancer. The expression patterns of RhoA were assessed by immunohistochemical staining and western blotting. Additional comparisons were performed between the tumour centre, tumour front and distant peritumoural tissue. RhoA activity was assessed by G-LISA. Associations between RhoA expression and the clinical features and outcome of the patients were also analysed. The present study found an increasing gradient of expression from the centre to the periphery of index tumour foci. RhoA expression was significantly increased at the tumour front compared to the tumour centre, which was determined using immunohistochemistry (P=0.001). Increased RhoA expression was associated with poor tumour differentiation in the tumour front (P=0.044) and tumour centre (P=0.039). Subsequent to a median follow-up period of 52 months, the rate of prostate-specific antigen (PSA) relapse was increased in patients with higher RhoA expression at the tumour front when compared with patients with lower RhoA expression (62.5 vs. 35.0%), although the difference was not significant (P=0.09). There was no association between RhoA expression and the PSA level or pathological stage in the present study. In conclusion, RhoA expression was increased at the tumour front and was associated with poor tumour differentiation in the tumour front and tumour centre, indicating the potential role of RhoA in prostate cancer. RhoA expression may also act as a prognostic factor in prostate cancer. The present data provide a foundation for novel therapeutic approaches by targeting RhoA in prostate cancer.
Prostate cancer is the most prevalent malignancy and the second leading cause of cancer-associated mortality in men in Western countries (
The molecular mechanisms that differentiate cancers with an invasive phenotype from those that remain latent within the gland continue to be largely unknown. Numerous factors have been implicated in the process of prostate cancer invasion and metastasis, but the initial molecular transforming events that occur in prostatic epithelial cells and the local microenvironment remain unknown (
Ras homolog gene family, member A (RhoA) guanosine triphosphatase (GTPase) is a well-established regulator of the dynamic and spatiotemporal rearrangement of the cell cytoskeleton during cell migration (
Although a previous study has indicated that overexpression of RhoA is present in prostate cancer tissues (
The aim of the present study was to evaluate and compare the expression and activity of RhoA in specimens obtained from radical prostatectomy (RP) performed on patients treated for localised or locally advanced prostate cancer, and to assess the value of RhoA as a prognostic marker of aggressiveness in prostate lesions. Three zones were compared, consisting of the tumour centre, the tumour front and peritumoural tissue.
All human tissues were obtained from the Department of Surgical Pathology, Cochin Hospital (Paris, France), in accordance with the ethical policies and procedures of the Institutional Review Board of Cochin Hospital. All patients provided informed consent prior to participation in the present study. Details that may disclose the identity of the enrolled subjects have been omitted. Tumour tissues were graded according to the modified Gleason grading system (
In total, 34 patients diagnosed with clinically localised prostate cancer that had been treated with RP were enrolled in the present study. PSA relapse was experienced by 16 patients during the median follow-up duration of 52 months. Clinical data records included the patient age, PSA level, pathological stage, Gleason score and follow-up times.
Fresh-cut sections (5-µm) were selected from formalin-fixed paraffin-embedded tissue blocks that contained tissue with the highest density of tumour cells and the highest Gleason scores, termed the index tumour. The sections were deparaffinised with xylene (Sigma-Aldrich, St. Louis, MO, USA) twice for 5 min, rehydrated through an alcohol gradient, blocked with 0.3% H2O2/methanol (Sigma-Aldrich) at room temperature for 30 min, and heated for 40 min at 96°C in 10 mM sodium-citrate solution (pH 6.0; Sigma-Aldrich) for antigen retrieval. The slides were then incubated with goat anti-human polyclonal anti-RhoA antibody (dilution, 1:50; catalogue number, SC32039, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C, and were then incubated with biotinylated rabbit anti-goat secondary antibody (dilution, 1:200; catalogue number, BA-5000; Vector Laboratories, Burlingame, CA, USA) at room temperature for 60 min. This was followed by incubation with Vectastain Elite ABC Reagent (catalogue number, PK-7100; Vector Laboratories) for 30 min. Peroxidase activity was examined using 3,3′-diaminobenzidine (catalogue number, D4418; Sigma-Aldrich). The sections were counterstained using hemalum and mounted using Permount (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
Two pathologists independently scored the tissue staining for RhoA. The central region, which was fully composed of cancerous glands, was defined as the tumour centre. The edges of the tumour foci were termed the tumour front. Peritumoural benign prostate glands that did not exhibit intraepithelial prostatic neoplasia and were located on the same slide acted as the control, and were termed the peritumoural prostatic tissues. A semi-quantitative ordinal and categorical method (
A total of 20 tissue samples were obtained from 11 patients that had undergone RP for the treatment of clinically localised or locally advanced prostate cancer. Immediately subsequent to surgical removal, small sections of tissue were dissected, snap-frozen and stored in liquid nitrogen. Histological analysis of the frozen sections was then performed. The percentage of tumour cells in each sample was evaluated. In total, 12 samples contained 50–90% cancerous cells and were labelled as tumour tissues, and 8 samples containing no cancerous tissue were termed peritumoural prostatic tissue. These samples were then subjected to western blot analysis for RhoA expression and G-LISA analysis for RhoA activity. The proteins were extracted on ice with cold RIPA lysis buffer (catalogue number, 9806; Cell Signaling Technology, Inc., Danvers, MA, USA) containing a protease and phosphatase inhibitor (catalogue number, 88669; Thermo Fisher Scientific, Inc.).
Lysates were centrifuged at 12,000 × g for 20 min at 4°C, and the supernatant was collected. Total protein concentrations were determined using a bicinchoninic acid assay (BCA) protein-assay kit (catalogue number, 23250; Thermo Fisher Scientific, Inc.). Equal quantities of protein were subjected to 12% SDS-PAGE and then electrically transferred onto nitrocellulose membranes (Hybond-C; Amersham Biosciences, Uppsala, Sweden). The membranes were blocked for 1 h with 5% (w/v) non-fat milk in phosphate-buffered saline with 0.1% (v/v) Tween-20 (PBST; Sigma-Aldrich), and incubated with polyclonal goat anti-human RhoA (dilution, 1:250; catalogue number, SC32039; Santa Cruz Biotechnology, Inc.) and monoclonal mouse anti-human β-actin (dilution, 1:500; catalogue number, SC47778; Santa Cruz Biotechnology, Inc.) primary antibodies overnight at 4°C. Finally, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, consisting of the polyclonal rabbit anti-goat immunoglobulin G (IgG)-HRP antibody (dilution, 1:5,000; catalogue number, SC2768; Santa Cruz Biotechnology, Inc.) and polyclonal goat anti-mouse IgG-HRP antibody (dilution, 1:5,000; catalogue number, SC2005; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature.
Subsequent to washing three times with PBST, the proteins were visualised using an enhanced chemiluminescence Prime Western Blotting Detection kit (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Images of the protein bands were captured using a digital imaging system (Image Quant LAS; GE Healthcare Bio-Sciences), and densitometric measurements of band intensity in the western blots were performed using ImageJ software (National Institutes of Health, Bethesda, MA, USA). The results are representative of three independent experiments.
RhoA activity was assayed using a G-LISA RhoA Activation Assay Biochem kit (cat. no. BK124; Cytoskeleton, Inc., Denver, CO, USA), according to the manufacturer's instructions. Briefly, the prostate tissue samples were homogenised in ice-cold lysis buffer with a protease-inhibitor cocktail, and then centrifuged at 450 × g at 4°C for 1 min. The supernatants were harvested and protein concentrations were measured using the Precision Red Advanced Protein Assay Reagent (catalogue number, ADV02; Cytoskeleton, Inc.) and were finally equalised with ice-cold lysis buffer to 1.0 mg/ml. Equalised prostate-tissue protein extractions were transferred to a Rho-GTP-binding protein pre-coated plate (Cytoskeleton, Inc.). The plate was placed on an orbital microplate shaker (SSM1; Bibby Scientific Limited Group, Staffordshire, UK) at 0.72 × g for 30 min at 4°C, and then incubated with monoclonal mouse anti-human anti-RhoA primary antibody (cat. no. GL01A; 1:250; Cytoskeleton, Inc.), followed by a polyclonal goat anti-mouse horseradish-conjugated secondary antibody (cat. no. GL02; 1:62.5; Cytoskeleton, Inc.), on an orbital microplate shaker (SSM1; Bibby Scientific Limited Group) at 0.72 × g at room temperature, for 45 min each. The plate was then incubated with the HRP detection reagent at 37°C for 15 min. Subsequent to the addition of HRP stop buffer, absorbance was read at 490 nm using an ELx808 Absorbance Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA).
Comparisons between data were performed using the χ2 test with a Yates continuity correction for independent samples, and the McNemar test was conducted for paired samples. Continuous data from two independent groups were compared using the Mann-Whitney U test. All tests performed were two tailed. P<0.05 was considered to indicate a statistically significant difference.
The clinicopathological features of the study population are summarised in
RhoA expression was significantly decreased in prostate cancer tissue specimens compared with the peritumoural tissue specimens (P<0.05;
To further evaluate the patterns of RhoA expression in prostate tissue specimens, immunohistochemistry was performed in 34 paraffin-embedded tissue specimens excised by RP. Analysis of the intensity of staining enabled the detection of various patterns of expression throughout the tissues. In the distant peritumoural tissue, immunohistological analysis revealed moderate or strong staining for RhoA expression in all tissues (
In the prostate cancer tissue specimens, RhoA expression was significantly increased in high-grade tumours that demonstrated a Gleason score >3+4, regardless of the location of the tumour cells, such as the tumour centre or front (
To the best of our knowledge, the present study is the first to evaluate RhoA expression not only in the centre of tumours, but also at the tumour front and in peritumoural tissues. The current study identified significantly increased RhoA expression at the tumour front compared with the tumour centre.
RhoA has previously been reported to be involved in prostate cancer invasion. Hodge
The microenvironment of a tumour is highly complex and varies between locations (
The present study indicates that prostate cancer cells at the tumour front demonstrate a more aggressive phenotype, and express specific and different features compared with cells at the centre of the tumour. Chemokine (C-X-C motif) receptor 4 (CXCR4), which is implicated in tumour invasion through the extracellular matrix, is specifically expressed at the tumour front of prostate tumours, whereas the expression of CXCR4 at the centre of tumours is low (
As RhoA is implicated in cancer-cell invasion, it was hypothesized that cancer cells expressing RhoA at the tumour front may demonstrate increased mobility and aggressiveness (
Although a previous study by Schmidt
RhoA is also involved in secretory granule trafficking and exocytosis (
The present study has several limitations, the most important being the small sample size, which may have biased the statistical analysis. A larger cohort is required to reliably test the association between RhoA expression and other adverse clinical and pathological features. Another limitation of the current study is the absence of whole gland analysis. Only the expression in the index tumour was focused on, and contiguous or distant tumours may not have exhibited the same profile of RhoA expression. Additional analyses should be conducted on more samples to assess the downstream effectors for the whole prostatic gland.
In conclusion, the present study has identified increased RhoA expression in prostate tumour fronts and has determined associations between increased RhoA expression and cancer relapse, increased RhoA expression and increased Gleason score. This indicates an association between RhoA expression and aggressiveness of the prostate cancer. The present findings may provide a foundation for novel therapeutic approaches that may inhibit the clinical aggressiveness of prostate cancer.
The authors would like to thank Dr T. Hua-Huy (Service de Physiologie, Cochin Hospital, Paris, France) for aiding with the present study and comments on the manuscript. This study was supported by the Program of International Science & Technology Cooperation (grant no. 2012DFG31440), awarded by the Ministry of Science and Technology, People's Republic of China. The authors thank NewMed Publishing Services for providing
RhoA expression and activity in prostate cancer tissue specimens. (A) Representative western blots revealing RhoA expression in three prostate tumour tissue samples (T1-T3) and three peritumoral tissues (P1-P3). (B) Quantitative analysis of RhoA expression. The expression of RhoA in prostate cancer tissue specimens is significantly decreased compared with the peritumoral prostatic tissue samples (*P<0.05). (C) RhoA activity was increased in the tumour tissue samples compared with peritumoral prostatic tissues. However, this was not statistically significant (P=0.35). RhoA, Ras homolog gene family, member A.
RhoA expression in prostate cancer, as assessed by immunohistochemistry. (A) An increasing gradient in the level of RhoA expression was identified between the tumour centre and the periphery of the index tumour foci. The expression of RhoA in (B) the prostate cancer tumour centre is significantly decreased compared with the expression at (D) the tumor front. However, (C) the adjacent or (E and F) distal peritumoral prostate gland demonstrated the highest expression of RhoA. (G) Individual intensities of each sample and median intensities in each group. (A and E) Magnification, ×50. (B-D and F) Magnification, ×400. **P<0.01. RhoA, Ras homolog gene family, member A.
Association between the GS, PSA relapse and RhoA expression. (A) In the tumour centre and front, patients with poor tumor differentiation demonstrated a significantly increased expression of RhoA, as indicated by a GS of 4+3 or higher and a percentage of RhoA staining score ≥2 (tumour centre, 15 vs. 50%, P=0.044; tumour front, 46 vs. 88%, P=0.039). *P<0.05, high-grade differentiation vs. low-grade differentiation. (B) Patients that developed relapse possessed increased RhoA expression in tumour fronts compared to those that did not relapse. However, this difference was not statistically significant (62.5 vs. 35.0%; P=0.089). RhoA, Ras homolog gene family, member A; PSA, prostate-specific antigen; GS, Gleason score.
Demographics of patients with prostate cancer and the characteristics of tissue specimens assessed by IHC or WB and G-LISA.
Cohort, n (%) | ||
---|---|---|
Characteristics | IHC | WB and G-LISA |
Total | 34 (100.0) | 11 (100) |
Median age, years (range) | 62 (50–73) | 61 (49–71) |
Median PSA level, ng/ml (range) | 8.66 (2.5–30) | 9.2 (4.4–30) |
Tumour stage | ||
T2 | 17 (50.0) | 6 (55) |
T3 | 17 (50.0) | 5 (45) |
Gleason score | ||
3+3 | 15 (44.0) | 5 (45) |
3+4 | 11 (32.0) | 3 (27) |
4+3 | 6 (18.0) | 2 (18) |
4+4 | 2 (6.0) | 1 (9) |
Median follow-up period, months (range) | 52 (7–112) | N/A |
PSA relapse | 16 (47.1) | N/A |
PSA relapse is defined as an increase in total serum PSA of >0.2 ng/ml over two consecutive measurements. IHC, immunohistochemistry; WB, western blotting; PSA, prostate-specific antigen; N/A, not assessed.