Takao Kamai, Department of Urology, Dokkyo University School of Medicine, 880 Kitakobayashi Mibu-machi, Shimotsuga-gun, Tochigi 321–0293, Japan.
Objective To clarify the role of Rho small GTP-binding protein (Rho) in the progression of testicular germ cell tumour (GCT), by examining the expression levels of mRNAs of Rho genes in testicular GCT.
Patients and methods The mRNA levels of the RhoA, RhoB and RhoC genes were analysed in the surgical specimens of testicular GCT tissues from 45 consecutive Japanese patients, and in the corresponding unaffected tissue originating from the same patient, using reverse transcription-polymerase chain reaction. The expression levels in tumour tissues were compared with those in unaffected tissues and the relationship between their expression levels in tumours and tumour stage evaluated. The expression levels of mRNAs of the Rho genes were also evaluated between tumours with seminoma only, and mixed tumours with seminoma and nonseminoma.
Results The mRNA levels of RhoA were greater in tumour tissues than in unaffected tissues of the resected testis (P < 0.01); the mRNAs of RhoB and RhoC were not detected in either tissue. The increase in RhoA mRNA levels was related to tumour stage (P < 0.05). The mRNA levels of RhoA in seminomatous and nonseminomatous areas where both were present were higher than those in tumours with seminoma only (P < 0.05).
Conclusions These results suggest that RhoA is involved in testicular germinal epithelial carcinogenesis and progression in testicular GCT, indicating that RhoA may be a useful prognostic marker for progression in testicular GCT.
The peak incidence of testicular germ cell tumour (GCT) is at 20–40 years old. Over 90% of patients with newly diagnosed testicular GCT are cured using effective combinations of surgery, radiotherapy and cisplatin-containing chemotherapy . However, the prognosis of patients with a high stage at presentation and with recurrent tumour is poorer than expected. The molecular mechanism of progression and metastasis in testicular GCT has yet to be elucidated. If there were more precise tumour markers for biological behaviour to predict progression and metastasis, new treatment strategies could be offered to patients.
Metastasis is the most common complication in human tumours that causes death; metastasis usually involves the transport of tumour cells through the fluid spaces of the body ; cell migration also plays a central role in the metastasis of malignant tumour. The Rho small GTP-binding protein (Rho) family, consisting of the Rho, Rac, and CDC42 subfamilies, regulates cell migration through the re-organization of the actin cytoskeleton. The Rho subfamily, consisting of RhoA, RhoB and RhoC, regulates the formation of stress fibres and focal adhesions in cells [3,4].
RhoA is over-expressed in breast, lung and colon carcinomas , RhoB in breast carcinoma  and RhoC in pancreas carcinoma . The over-expression of Rho correlated with a higher stage in these carcinomas [5,7] and Rho probably contributes to the metastatic phenotype . These observations suggest that Rho may be associated with carcinogenesis, progression and metastasis in human tumours, but there are no available data on its role in testicular GCT. To determine whether Rho is involved in the carcinogenesis of germinal epithelium, and progression and metastasis in testicular GCT, we compared the expression levels of mRNA of Rho genes in testicular GCT tissue with those of the corresponding unaffected tissue originating from the same patient, using RT-PCR. The relationship between the expression levels in tumours and tumour stage was also examined.
Patients and methods
Surgical specimens of testicular GCTs obtained between 1995 and 1999 from 45 consecutive Japanese patients (mean age 35.6 years, range 16–46) with newly diagnosed primary testicular GCT were examined; 27 patients had seminoma only and 18 had combined seminoma and nonseminoma. All patients routinely underwent imaging studies (CT and/or MRI) before surgery to obtain information for standard staging. The median (range) follow-up was 29 (3–60) months. In all patients three sites each of tumour and unaffected tissue of the resected testis were sampled. The resected tissues were embedded in OCT tissue compound (Miles, Elkhart, IN, USA) and stored at −80 °C, as described previously . The clinical stage was classified using the TNM staging system .
RNA extraction and RT-PCR
The total RNA of tumour and unaffected tissues was extracted using the Catrimox-14™ RNA isolation kit (TaKaRa Biomedicals, Otsu, Japan) according to the manufacturer's protocol. The quantity and purity of the RNA prepared from each sample was determined by the ratio of the optical density at 260 nm to that at 280 nm. The mRNAs of each tissue were extracted using Oligotex-dT30 ‘Super’ in accordance with the recommendations of the manufacturer (TaKaRa Biomedicals).
Gene expressions were determined by RT-PCR as described previously [7,10], using a RNA PCR kit incorporating avian myeloblastosis virus-derived RT and Taq DNA polymerase according to the manufacturer's protocol (TaKaRa Biomedicals). Briefly, the RT reaction mixture contained 1 µg of total RNA, 5 mmol/L MgCl2, 1 × RNA PCR buffer, 1 mmol/L dNTP, 1 U/µL RNase inhibitor, 0.25 U/µL RT, 0.125 µmol/L oligo dT and 8.5 µL Rnase-free dH2O in a total volume of 20 µL. The mixture was incubated at 42°C for 30 min, heated to 99°C for 10 min and then cooled to 4°C. The PCR mixture contained 2 µL of cDNA, 2.5 mmol/L MgCl2, 1 × RNA PCR buffer, 0.2 µmol/L Rho primers, 0.2 µmol/L β2-microglobulin primers and 2.5 U/100 µL Taq DNA polymerase in a total volume of 80 µL for each reaction. The PCR primers for RhoA, RhoB, RhoC and β2-microglobulin gene amplification were chosen as described previously  (Table 1). The PCR conditions were: 32 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 1 min and extension at 72 °C for 1 min in a thermal cycler (TaKaRa Biomedicals). All RT-PCR experiments were routinely controlled by omitting the RT. The PCR products were examined by electrophoresis on a Spreadex EL 800 gel (Elchrom Scientific AG, Cham, Switzerland) at 120 V for 60 h at 55 °C. The gels were stained with Gel Star (TaKaRa Biomedicals) and photographed in an ultraviolet transilluminator system (Funakoshi Inc., Tokyo, Japan). The blot membrane was scanned with a PDI imaging scanner (Agfa-Gevaert Japan Ltd, Tokyo, Japan) and analysed with imaging software. The gene expression of Rho was derived as the relative yield of the PCR product from the target sequences to that from the β2-microglobulin gene, as described previously . The mean values were calculated for the three sections of tissues. The results from RT-PCR were analysed statistically using the Mann–Whitney U-test , with P < 0.05 considered to indicate significant differences.
Table 1. The specific primers for RT-PCR
U, upstream primer; D, downstream primer.
The mRNA of RhoA was detected in both tumour and unaffected tissues (Fig. 1a). The mRNA levels of RhoA were significantly higher in tumour tissues than in the unaffected portions (P = 0.0023, Fig. 2a). In contrast to RhoA, the mRNA levels of RhoB and RhoC were either below the limit of detection or only very poorly detectable in both tissues (Figs 1b,c). The expression of RhoA mRNA in the primary lesion was higher in patients with lymph node and/or distant metastasis (stage II/III) than in those without (stage I; P = 0.0359, Fig. 2b). The mRNA levels of RhoA in tumours containing both seminomatous and nonseminomatous areas were higher than those in tumours with seminoma only (P = 0.0158, Fig. 2c).
Testicular GCT originates from the germinal epithelium; thus to consider possible variations among individuals in the expression level of mRNAs of the Rho genes, tumour tissue in testicular GCT and the corresponding unaffected tissue obtained from the same patient were compared. The present study showed that mRNA levels of RhoA were significantly higher in tumour tissues than in unaffected portions and that the levels in tumour tissues correlated with higher tumour stage. The mRNA levels of RhoA were significantly higher in tumours in which there were seminomatous and nonseminomatous areas than in those with seminoma only. To our knowledge, this is the first report of the relationship between Rho and testicular GCT; the results suggest that RhoA may be involved in the progression of testicular GCT.
The mRNAs of RhoB and RhoC were not detected in either tissue, indicating a possible role only for RhoA in the carcinogenesis of testicular germinal epithelium. However, RhoA is over-expressed in breast, lung and colon carcinomas , and RhoB in breast carcinoma . In pancreatic carcinoma, although the mRNA of RhoA, RhoB and RhoC genes were expressed, only the mRNA levels of the RhoC gene were significantly higher in tumour than in unaffected tissues . Although it is likely that RhoA, RhoB and RhoC share common functions in regulating stress-fibre formation , they differ in subcellular location  and regulation of expression [13,14]. These observations suggest that there are tumour-specific and/or organ-specific changes in the expression of RhoA, RhoB and RhoC.
Rho is involved in the regulation of cytoskeletal organization [3,4]; RhoA regulates the microfilament network  and cadherin-dependent cell-cell contact . Over-expression of RhoA promotes invasive ability in vitro and in vivo. Furthermore, Rho contributes to the metastatic phenotype ; the higher the tumour stage, the higher the expression levels of Rho in breast, lung, colon and pancreatic carcinomas [5,7]. In the present study, the expression of RhoA mRNA in the primary lesion was significantly higher in stage II/III than in stage I disease. Taken together, these results suggest that Rho plays an important role in tumour progression and metastasis, indicating that it may be a useful prognostic marker.
Testicular GCTs are classified as seminoma or nonseminoma, reflecting their origin in primordial germ cells and their remarkable ability to differentiate in vivo. Seminoma has the best prognosis; nonseminomatous tumours are more clinically aggressive than seminomatous tumours . In the current study, mixed tumours with seminoma and nonseminoma correlated with higher stage (P = 0.0013, data not shown). The mRNA levels of RhoA were significantly higher in mixed tumours than in seminoma only, suggesting that increased expression levels of RhoA mRNA were associated with the biological aggressiveness of nonseminomatous tumours. As neoplasms are biologically heterogeneous and contain subpopulations of cells with different angiogenic, invasive and metastatic properties, their response to therapeutic agents is likewise heterogeneous . Therefore, it is important to understand the biological differences between seminoma and nonseminoma in testicular GCT. Further investigations, including the mRNA levels of Rho genes in each cell type, should be undertaken to elucidate the metastatic characteristics of each cell type.
Therapy for GCT is based largely on the stage, histological differentiation and serum values of AFP, βhCG and LDH . Cisplatin-based chemotherapy is a widespread and promising chemotherapeutic regimen for testicular GCT, because it is effective . However, despite the extensive evaluation of many different treatments, some metastatic or recurrent tumours in which previous high-dose chemotherapy with stem-cell rescue has failed remain highly resistant to systemic therapy [19–21]. Furthermore, high-dose chemotherapy may be a risk factor for secondary leukaemia [22,23]; new treatment strategies are needed.
A specific Rho-associated serine-threonine protein kinase (ROCK) inhibitor (Y-27632) blocked both the Rho-mediated activation of actomyosin and the invasive activity of cultured rat MM1 hepatoma cells; continuous delivery of this inhibitor reduced the dissemination of MM1 cells implanted into the peritoneal cavity of syngeneic rats . This suggests that the ROCK inhibitor may be a potential therapeutic target for preventing cancer invasion and metastasis.
Mutations of Rho have not been identified in human tumours [7,25]. Although mutation was not investigated in the present study, mutational activation of Rho genes in testicular GCT will be determined in forthcoming studies. In the present study, all 29 patients with stage I and 15 with stage II disease are well with no evidence of disease after systemic therapy; only one patient with stage III disease is alive, with metastatic disease. The follow-up in the present study was too short to draw definitive conclusions about the possible relationship between mRNA levels of Rho genes and prognosis; this relationship will be assessed in forthcoming studies with more patients. It is likely that RhoA is a useful prognostic marker in patients with testicular GCT.
This research was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan to T.K.
T. Kamai, MD, Urologist.
K. Arai, MD, Urologist.
T. Tsujii, MD, PhD, Urologist.
M. Honda, MD, PhD, Urologist.
K. Yoshida, MD, PhD, Urologist, Chairman of Department.