World J Urol (2012) 30:559–565 DOI 10.1007/s00345-011-0767-z
ORIGINAL ARTICLE
Tyrosine kinase expression profile in clear cell renal cell carcinoma Turang E. Behbahani • Claudia Thierse • Claudia Baumann Daniel Holl • Patrick J. Bastian • Alexander von Ruecker • Stefan C. Mu¨ller • Jo¨rg Ellinger • Stefan Hauser
•
Received: 7 July 2011 / Accepted: 20 September 2011 / Published online: 4 October 2011 Ó Springer-Verlag 2011
Abstract Purpose To profile different tyrosine kinase (TK) expression patterns in clear cell renal carcinoma (ccRCC). Methods We analysed mRNA expression levels of 89 receptor and non-receptor TK in corresponding cancer and normal renal tissue from 5 patients with ccRCC using the TaqMan Low-Density Array technology. In order to confirm aberrant TK expressions, a subsequent analysis of 25 ccRCC and corresponding normal renal tissues was performed, applying quantitative real-time PCR. To confirm mRNA expression levels on protein level, we studied ERBB4 and HCK using immunohistochemistry. Results A total of 12 TK were significantly upregulated in ccRCC (ABL2, FLT1, BTK, HCK, JAK3, CSF1R, MET, JAK1, MATK, PTPRC, FYN and CSK), coherently 7 TK
demonstrated a down-regulation (ERBB4, PDGFRA, NRTK3, SYK, ERBB2, FGFR3 and PTK7). These findings were validated by the utilization of RT-PCR for ABL2, FLT1 BTK, HCK, JAK3, CSF1R, MET, JAK1, MATK and vice versa for ERBB4 and PDGFRA. Immunohistochemistry revealed ERBB4 expression to be significantly lower in ccRCC in comparison to papillary RCC, chromophobe RCC, renal oncocytoma and normal renal tissue (P \ 0.001). HCK protein expression was reduced in ccRCC in contrast to papillary RCC (P \ 0.001) or oncocytoma (P = 0.023), but similar to chromphobe RCC (P = 0.470), sarcomatoid RCC (P = 0.754) and normal renal tissue (P = 0.083). Neither ERBB4 nor HCK were correlated (P [ 0.05) with clinical– pathological parameters. Conclusion TK constitute valuable targets for pharmaceutical anti-cancer therapy. ERBB4 and HCK depict significantly lower expression levels in renal cancer tissues.
Jo¨rg Ellinger and Stefan Hauser are contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s00345-011-0767-z) contains supplementary material, which is available to authorized users. T. E. Behbahani C. Thierse C. Baumann D. Holl S. C. Mu¨ller J. Ellinger (&) S. Hauser (&) Klinik und Poliklinik fu¨r Urologie und Kinderurologie, Universita¨tsklinikum Bonn, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany e-mail:
[email protected] S. Hauser e-mail:
[email protected] P. J. Bastian Urologische Klinik und Poliklinik, Universita¨tsklinikum Mu¨nchen-Großhadern, Munich, Germany A. von Ruecker Institut fu¨r Pathologie, Universita¨tsklinikum Bonn, Bonn, Germany
Keywords Tyrosine kinase Renal cell carcinoma ErbB4 HCK
Introduction Tyrosine kinases (TK) are promising targets in pharmaceutical therapy for renal malignancies, which demonstrate an increased incidence over recent decades [1]; TK are central-signalling molecules that effect proliferation, differentiation, motility, cell death and survival [2]. So far, 58 receptor and 32 intracellular non-receptor TK have been identified [3]. Several examples exist where TK are directly involved in cancer formation via overexpression, gene mutations or chromosome translocations, such as in the signalling systems of VEGF and PDGF; Aberrant VEGF expression is an important feature in the pathogenesis of
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clear cell renal cell carcinoma (ccRCC) and the development of specific VEGF-directed approaches (anti-VEGF antibody bevacizumab; small molecule VEGF/PDGFR receptor inhibitor: sunitinib, sorafenib and pazopanib; reviewed in [4, 5]) opened promising treatment possibilities in metastatic renal cell carcinoma (RCC). Broad clinical application is also established for other TK inhibitors (EGFR-inhibitor: gefitinib; BCR/ABL-inhibitor: imatinib; ERBB2-inhibitor: trastuzumab). Until now, gene expression analyses in the kidney have focussed on one or a few single genes or have used microarray technology for generating large expression profiles, thereby accepting a reduced accuracy when compared to quantitative real-time PCR. With regard to TK, few have been analysed extensively and knowledge about many remains incomplete. In this study, we used an intermediatescale screening method (TaqMan Low-Density Array) to identify aberrantly expressed TK in patients with ccRCC. Subsequently, mRNA expression levels were assessed in a larger cohort using quantitative real-time PCR and confirmed by immunohistochemistry on protein level.
Materials and methods Patients The renal tissue specimens were obtained from patients with undergoing surgery for renal tumours at the Department of Urology, University Hospital Bonn, Germany. All patients had given written informed consent; the study was approved by the local ethic committee.
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TK and five reference genes (HPRT1, UBC, G6PDH, POLR2B and 18sRNA) were spotted on 4 9 96-well cards (see Supplementary Table 1). Additionally, cancer and noncancerous RNA samples from 5 patients (see Supplementary Table 2) were used for Low-Density Arrays (see Supplementary Methods). Quantitative real-time PCR TK representing an aberrant mRNA expression were identified on Low-Density Arrays and subsequently evaluated by conventional quantitative real-time PCR (see Supplementary Table 3 for clinical–pathological parameters and Supplementary Methods for experimental details). Tissue microarray and immunohistochemistry A tissue microarray with 193 formalin-fixed, paraffinembedded RCC samples [ccRCC, n = 142; papillary RCC (n = 31; papillary RCC type I, n = 18; papillary RCC type II, n = 13); chromophobe RCC (n = 10); sarcomatoid RCC (n = 10)], 10 oncocytoma and 30 normal kidney tissue samples were used to confirm gene expression on protein level. The tissue microarray was described earlier [6] (see Supplementary Table 4 for clinical–pathological parameters). Tumour and normal adjacent tissue from the same patients has been used for the construction of the tissue microarray. Results were evaluated, applying the Remmele scoring system as reported earlier [6] (see Supplementary Methods).
Results RNA isolation and cDNA synthesis Low-density arrays From each patient, malignant and normal fresh-frozen tissue was used for RNA analysis. A 5-lm-thick haematoxylin/eosin-stained cryostat section was used for histological evaluation. Cases were only allowed for further processing if tissue was viable; areas of necrosis were not used. The total RNA was isolated from *20 mg fresh-frozen tissue using the AllPrep DNA/RNA Kit according to the manufacturer’s recommendations (Qiagen, Hilden, Germany); DNAse I (Invitrogen, Paisley, Scotland) treatment was performed on column. RNA (1 lg) was reverse transcribed using the Superscript III Kit (Invitrogen) in a final volume of 20 ll. Low-density arrays PCR were performed using the TaqMan Low-Density Array technology (Applied Biosystems, Foster City, CA, USA). A total of 89 primer/probe sets from receptor and non-receptor
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In order to identify novel aberrantly expressed TK in ccRCC, we analysed mRNA expression levels of all 89 receptor and non-receptor TK in corresponding cancer and normal renal tissue from five patients with metastatic ccRCC using the TaqMan Low-Density Array technology. Thereby, we identified 19 TK significantly (P = 0.016–0.032) up- or downregulated. TK with upregulated gene expression in ccRCC were ABL2 (mean up-regulation in cancer samples: 10.52-fold), FLT1 (8.32-fold), BTK (6.04-fold), HCK (5.90-fold), JAK3 (5.75-fold), CSF1R (4.33-fold), MET (4.04-fold), JAK1 (3.89-fold), MATK (3.41-fold), PTPRC (3.20-fold), FYN (3.02-fold) and CSK (2.44-fold) (see Supplementary Figure 1). Down-regulation was observed for ERBB4 (45.45-fold), PDGFRA (14.92-fold), NRTK3 (9.01-fold), SYK (6.76-fold), ERBB2 (2.99-fold), FGFR3 (2.81-fold) and PTK7 (2.25-fold) in RCC tissue (see Supplementary Figure 2).
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Quantitative real-time PCR In order to confirm aberrant TK expression, mRNA from 25 cancer and corresponding normal tissues from nephrectomy specimens was analysed using quantitative real-time PCR. Genes with more than 3.3-fold up-/downregulation in Low-Density Arrays were chosen for confirmatory experiments. We confirmed up-regulation of ABL2 (mean up-regulation: 3.42; P \ 0.001), FLT1 (4.50; P \ 0.001), BTK (6.02; P \ 0.001), HCK (7.21; P \ 0.001), JAK3 (10.42; P \ 0.001), CSF1R (11.02; P \ 0.001), MET (4.47; P \ 0.001), JAK1 (2.86; P \ 0.001), MATK (5.11; P \ 0.001) and MET (4.47; P \ 0.001). ERBB4 (212.80; P \ 0.001) and PDGFRA (4.96; P \ 0.001) were significantly downregulated. Surprisingly, we noticed that SYK—identified as downregulated in the low-density arrays—was expressed at higher levels in RCC compared to normal renal tissue (5.55; P \ 0.001). NTRK3 (1.19; P = 0.473) was not differentially expressed (Fig. 1). Immunohistochemistry In order to confirm aberrant mRNA expression levels exemplary on the protein level, we selected ABL2, HCK, JAK3, SYK and ERBB4 for immunohistochemical analysis. Antibodies graded for immunohistochemistry with FFPE tissue were not commercially available for BTK, CSF1R, JAK1, MATK and MET. FLT1 and PDGFRA have been extensively studied before, and overexpression in RCC is established for these proteins. Unfortunately, antibodies against JAK3, SYK, ABL2 and SYK did not produce specific and reproducibly staining results with FFPE tissues. ERBB4 expression was significantly lower in ccRCC (mean: 5.6; 95% CI = 5.21–6.00) than in papillary RCC (mean: 7.31; 95% confidence interval = 6.62–8.01; P \ 0.001), chromophobe RCC (mean: 5.0; 95% confidence interval = 3.45–6.55; P \ 0.001), renal oncocytoma (mean: 7.5; 95% confidence interval = 5.81–9.19; P \ 0.001), and normal renal tissue (mean: 6.4; 95% confidence interval = 5.52–7.28; P \ 0.001); sarcomatoid RCC levels were similar (mean: 5.6; 95% confidence interval = 4.71–6.54; P = 0.051). Surprisingly, HCK protein expression was reduced in ccRCC compared to papillary RCC (P \ 0.001) or oncocytoma (P = 0.023), but similar to chromophobe RCC (P = 0.470), sarcomatoid RCC (P = 0.754) and normal renal tissue (P = 0.083). See Fig. 2. Neither ERBB4 nor HCK were correlated (all P [ 0.05) with clinical–pathological parameters (pT-stage, Fuhrman grading, lymph node involvement and surgical margins) in the whole RCC cohort. If these analyses were restricted to patients with ccRCC, we observed a significant correlation between HCK protein levels and grading (P = 0.022; see Supplementary Figure 3). Neither ERBB4
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(P = 0.797, hazard ratio 1.04, 95% confidence interval 0.77–1.39) nor HCK (P = 0.164, hazard ratio 1.16, 95% confidence interval 0.942–1.427) were correlated with cancer-specific survival, respectively, progression-free survival (ERBB4: P = 0.255, hazard ratio 1.01, 95% confidence interval 0.936–1.28; HCK: (P = 0.320, hazard ratio 1.06, 95% confidence interval 0.94-1.20) in patients with ccRCC. Similar results were observed for the whole RCC cohort for progression-free survival (ERBB4: P = 0.804, hazard ratio 1.02, 95% confidence interval 0.902–1.14; HCK: (P = 0.874, hazard ratio 1.01, 95% confidence interval 0.89–1.15) and cancer-specific survival (ERBB4: P = 0.943, hazard ratio 0.99, 95% confidence interval 0.81–1.22; HCK: (P = 0.154, hazard ratio 1.14, 95% confidence interval 0.95–1.37).
Discussion A better understanding of molecular pathways regarding RCC has led to the integration and increasing application of molecularly targeted therapies in patients with advanced diseases. TK expression profiles allow valuable insight into regulative cancer activities, such as proliferation, migration and diversification of malignant cells. Regarding the variety of TK expression alterations associated with ccRCC, our study demonstrated a significant down-regulation of the epidermal growth factor receptor (EGFR) TK ERBB4 applying RT-PCR and immunohistochemical analysis, which confirmed a down-regulation for ERBB4. Our results indicate that not all isoforms of the EGF receptor (ERBB14) are overexpressed and correspond to results of Thomasson et al. [7], who outline a significant down-regulation of ERBB4 (P = 0.04) and ERBB2 (P = 0.02) among 31 patients with RCC via PCR and immunostaining. Despite investigations indicating unfavourable outcomes for ERBB4 expression in colon cancer or medulloblastoma [8] associated with decreased overall survival, our findings suggest ERBB4 to act as a tumour suppressor in RCC. Interestingly, ERBB4 overexpression is shown to increase cyclooxigenase-2 (COX-2) and cell survival in colon epithelial cells [9]. A prognostic evaluation of COX-2 in RCC associates COX-2 protein expression with slow development of metastases and favourable prognosis [10], which suggests ERBB4 to potentially function as a tumour suppressor. The consistent down-regulation of ERBB4 involves difficulties in obtaining correlations between its expression and cancer characteristics as well as clinical parameters. Since a downregulated, or even absent, receptor does not represent a sufficient pharmaceutical target, the molecular pathways that lead to its down-regulation should be taken into closer consideration. Liu et al. [11] depict dysregulated microRNA to be inversely correlated with
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Fig. 1 Hierarchical clustering (normal tissues are indicating with ‘N’, RCC tissues with ‘T’) and boxplot analysis of relative mRNA expression in renal cell carcinoma (RCC) and normal renal (NORM) tissue. n.d. not determined (Ct [ 40). The expression profile of 13 tyrosine kinases was assessed using quantitative real-time PCR in 25
clear cell renal cell carcinoma (RCC) samples and normal adjacent renal tissue (NORM); the boxplots represent x-fold expression changes. Tyrosine kinase expression is significantly different in malignant and normal renal tissue
expression of tumour suppressor genes in RCC. The authors outline the miR-224, a microRNA which is associated with the fragile site on chromosome Chr-Xq28, to be upregulated in RCC and demonstrate it to target ERBB4 that will causally be downregulated. These findings underline the existing controversy concerning the introduction of EGFR
inhibitors in advanced renal cancers and are supported by findings of Zhou et al. [12] who state that the miR-200 family plays an important role in the silencing of ERBB4, implicating advantages in tumour invasion and migration. In order to implement EGFR inhibitors into therapeutical regimens, Dawson et al. [13] conducted a phase II trial
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Fig. 2 Distribution of HCK and ERBB4 levels in the different histological subtypes of renal cell carcinoma and oncocytoma as determined using immunohistochemistry. The protein expression was assessed semi-quantitatively using the Remmele Score
to examine the outcome of gefitinib in advanced stage and recurrent RCC, which revealed unsatisfying results and is concluded to be without significant conventional activity in RCC. Further studies are constrained not to focus on the ERBB4 receptor itself, rather to investigate mediators that play a key role in its silencing. These conclusions are limited because our study design did not solely include metastatic RCC specimen which aggravates comparisons with other studies. Platelet-derived growth factor receptors (PDGFR) represent TK, which lead to tumour growth, supported by the recruitment of pericytes to capillaries [14] and constitute principal receptor targets to antiangiogenic TK inhibitors such as sunitinib, sorafenib or pazopanib (for PDGFRA). Although the efficacy of PDGFR-inhibitors in metastatic RCC has been demonstrated in recent studies, [15] our examinations revealed a down-regulation of PDGFRA in advanced, mainly non-metastatic, renal carcinomas. These results seem to be consistent with results of Tawfik et al. [16], who could demonstrate an association of overall survival with PDGFRA values as a single significant variable on multivariate analysis. Nevertheless, sunitinib demonstrated solid results in a recent phase III trial and revealed significantly increased response rates in prior untreated patients with RCC (47% vs. 12% with IFN-alpha) as well as an increased median progression-free survival (HR 0.54)
[17]. Similar results, compared to placebo, have been reported by the TARGET trial for sorafenib [18], which included 903 patients with advanced RCC who had failed a prior standard therapy. The discrepancy between a silenced PDGFRA and increased progression-free survival in patients being treated with sorafenib (as a PDGFR inhibitor) can be engendered by its ability to inhibit the constitutive activation of the B-raf pathway, which has been described in more than 50% of RCCs, according to Turner et al. [19]. Beside various potential tumour suppressors, our study illustrates different TK over expressions that might function as oncogenes in RCC development and progression and allegorize optional targets for TK inhibitors. In correspondence to our results, Wu et al. [20] outline a baseline up-regulation of janus kinases (JAK) (JAK3 and JAK1) in von Hippel-Lindau disease (VHL) patients with RCC. The authors state that JAK kinases promote the invasiveness of RCC by suppression of cytokine-mediated pathways. Furthermore, an inhibition of the activation of the JAK-STAT3 pathway in renal cancer cells could be shown to decrease the proliferation and mobility capacities of these cells [21]. So far, there is only little evidence for a clinical efficiency by implementing JAK kinase inhibitors into treatment regimens for patients with RCC. Anyhow, Vaddi et al. [22] indicate that selective JAK kinase inhibition in epithelial cancer cell lines, such as RCC, significantly
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improves the outcome of molecularly targeted agents, which is consistent to our results. In order to assess disease stabilization, rather than tumour shrinkage, blood levels of the vascular endothelial growth factor receptor-1 (FLT-1) above the median are allied with unstable disease and poor survival under antiangiogenic therapy with razoxane [23]. Our low-density arrays and RT-PCR confirm the upregulated gene expression of FLT1 in patients with RCC and may underline the function of FLT1 as a proangiogenic factor on vessels, macrophages and tumour stroma. Especially patients with high pretreatment FLT1 levels may benefit from an anti-VEGF therapy, as VEGF upregulates FLT1 [24]. Additionally, a FLT1-expression on macrophages offers new treatment options via inhibiting their angiogenic migration into tumours. Beside JAK and FLT-1, screening of receptor genes in patients with RCC revealed the haematopoietic cell kinase (HCK) to be associated with angiogenesis and can be shown to be upregulated in our specimen, while divergent results were gained concerning gene expression and analyses on protein level which might be consistent with the implementation of different specimens or external influences, such as microRNA [25]. Additional analyses revealed an overexpression of colonystimulating factor receptor-1 in RCC (CSF1R), which is shown to suppress apoptosis in bladder cancer cells and to promote survival in these cells [26]. Similar results have not been published for RCC. Reflecting the variety of analysed and published TK expression profiles, there is an increasing demand for further prospective studies. Beside established anti-VEGF therapy regimens with sunitinib, sorafenib or pazopanib, new agents such as axitinib, tivozanib, dovitinib and cediranib should be taken into close consideration, especially in sorafenib-resistant patients or to reduce related toxicities [27]. Particularly anti-VEGF resistant patients with RCC should consider a therapy by inhibiting the mammalian target of rapamycin pathway with temsirolimus or everolimus, which both were evaluated in phase III trials to be clinically effective for RCC that showed progression on anti-VEGF therapy [28]. Interestingly, SYK mRNA expression was downregulated in the low-density arrays, but upregulated in the conventional qPCR experiments. Possible explanations are the use of different samples in these experiments, but also alternative splice variants may be expressed at different levels, and differences of the PCR amplicons may lead to his observation. So far, SYK has not been investigated in RCC before, and future investigations are warranted to clarify our finding. Although we did not check RNA integrity and did not validate low-density arrays via conventional qPCR with the same patients, it underlines future directions to apply molecularly targeted therapies in progressed RCC and expresses the need of further investigations in terms of new
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combination regimens, the establishment of new biomarkers to predict the responsiveness and to reduce toxic side effects of these regimens. Furthermore, the deficient correlation of TK with clinical and histological characteristics of RCC accentuates the requirement of valuable biomarkers in view of accuracy and reliability [29]. Beside established laboratory parameters for risk stratification and prognosis, yet there is an insufficient implementation of molecular markers such as TK, CAIX and B7-H1/4, which might promise precious insight into the guidance of patients towards safe therapy options [30]. Acknowledgments assistance. Conflict of interest of interest.
We
thank
Doris
Schmidt
for
technical
The authors declare that they have no conflict
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