Carcinogenesis vol.33 no.4 pp.799–803, 2012 doi:10.1093/carcin/bgs018 Advance Access publication January 19, 2012
Kinome expression profiling identifies IKBKE as a predictor of overall survival in clear cell renal cell carcinoma patients Michelle A.T.Hildebrandt, Weiqi Tan, Pheroze Tamboli1, Maosheng Huang, Yuanqing Ye, Jie Lin, Ju-Seog Lee2, Christopher G.Wood3 and Xifeng Wu Department of Epidemiology, 1Department of Pathology, 2Department of Systems Biology and 3Department of Urology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA To whom correspondence should be addressed. Department of Epidemiology, Unit 1340, The University of Texas MD Anderson Cancer Center, 1155 Pressler Boulevard, Houston, TX 77030, USA. Tel: þ1 713 792 2242; Fax: þ1 713 792 4657; Email:
[email protected]
There are 516 known kinases in the human genome. Because of their important role maintaining proper cellular function, they are often misregulated during tumorigenesis and associated with clinical outcomes in cancer patients, including clear cell renal cell carcinoma (ccRCC). However, less is known about the global expression status of these genes in renal cell carcinoma and their association with clinical outcomes. We performed a systematic analysis of gene expression for 503 kinases in 93 tumor samples and adjacent normal tissues. Expression patterns for 41 kinases were able to clearly differentiate tumor and normal samples. Expression of I-kappa-B kinase epsilon (IKBKE) was associated with a 5.3-fold increased risk of dying [95% confidence interval (CI): 1.93–14.59, P-value: 0.0012]. Individuals with high IKBKE expression were at a significantly increased risk of death (hazard ratio: 3.34, 95% CI: 1.07–10.40, P-value: 0.038) resulting in a significantly reduced overall survival time compared with those with low IKBKE tumor expression (P-value: 0.049). These results for IKBKE were validated in a replication population consisting of 237 ccRCC patients (P-value: 0.0021). Furthermore, IKBKE was observed to be higher expressed in tumors compared with adjacent normal tissues (P-value < 1027). IKBKE is a member of the nuclear factor-kappaB (NF-kB) signaling pathway and interestingly, gene expression patterns for other members of the NF-kB pathway were not associated with survival, suggesting that IKBKE gene expression may be an independent marker of variation in overall survival. Overall, these results support a novel role for IKBKE expression in modulating overall survival in ccRCC patients.
Introduction Kinase genes comprise an estimated 2% of the protein-coding component of the human genome (1). These proteins are involved in nearly all signal transduction processes within the cell, including essential cellular functions, such as apoptosis, metabolism, cell growth, cell cycle regulation, cell movement and development; pathways often misregulated in cancer. Kinases have been implicated not only in the progression from normal to tumor but also response to therapy, survival and other clinical outcomes—this includes clear cell renal cell carcinoma (ccRCC) (2,3). However, less is known about the global expression status of these proteins in ccRCC and association with clinical outcomes.
Abbreviations: AKT, v-akt murine thymoma viral oncogene homolog 1; ccRCC, clear cell renal cell carcinoma; CI, confidence interval; IKBKE, I-kappa-B kinase epsilon; mTOR, mammalian target of rapamycin; NF-jB, nuclear factor-kappaB; PI3K, phosphoinositide-3-kinase; RCC, renal cell carcinoma; VEGFR, vascular endothelial growth factor receptor.
It is estimated that .58 000 new cases of renal cell carcinoma (RCC) were diagnosed in 2010 (4). Surgery is often curative for early-stage local disease with 5 year survival rate of 90.8%. However, the prognosis for late-stage disease is much dimmer due to lack of effective treatment regimens with the 5 year survival rate of only 11.1% for metastatic RCC (5). Furthermore, RCC is often refractory to radiation and current chemotherapeutic agents. Because of this, molecular targeted therapies, such as the kinase inhibitors sorafenib, sunitinib, everolimus and temsirolimus have been developed (6). These agents have shown potential in treating advanced RCC with increased progression-free survival rates, although most patients do ultimately develop resistance. With the success of these agents being dependent on the expression of specific kinases by the tumor, it underscores the need to better understand the molecular profiles of ccRCC tumors to improve treatment response. In addition because of the frequent development of resistance, there is a strong need for the identification of novel drug targets. The purpose of this study was to profile the global expression of kinases in ccRCC tumor and adjacent normal tissue samples and to integrate this information with data obtained from in-depth patient interviews and chart reviews to increase our understanding of kinase expression profiles related to overall survival. Identification of kinase gene expression profiles associated with ccRCC development and clinical outcome may help to identify biomarkers to better understand a patient’s prognosis as well as provide evidence for potential targets for novel drug development. Materials and methods Patient populations Tumor and adjacent normal tissue for the discovery population were obtained from 93 newly diagnosed histological confirmed ccRCC patients recruited from The University of Texas MD Anderson Cancer Center (Houston, TX). A replication set of 237 ccRCC tumors was also obtained from MD Anderson Cancer Center. Cases were identified through a daily review of computerized appointment schedules for the Department of Urology and Genitourinary Medical Oncology. There were no restrictions on age, gender, ethnicity or clinical stage at recruitment. The primary eligibility criterion for participation in the study was that the cases were Texas residents. Informed consent was obtained from all patients and the study was approved by MD Anderson Cancer Center’s Institutional Review Board. Epidemiologic and clinical data collection In-person interviews by MD Anderson staff interviewers were conducted for study participants using a risk factor questionnaire that collected information on demographics, tobacco use history, medical history and family history of cancer. For smoking history, a never-smoker was defined as an individual who had never smoked or had smoked ,100 cigarettes. Those subjects who had quit smoking .12 months prior to diagnosis were considered former smokers. Clinical information was abstracted from medical records and included clinical stage, age at diagnosis, history of hypertension and body mass index. RNA isolation Approximately 20 mg of flash frozen tissue was brought to 20°C in RNAlater-ICE Frozen Tissue Transition Solution (Ambion, Austin, TX) to minimize RNA degradation. Total RNA was isolated using the mirVana RNA Isolation Kit (Ambion) following standard protocol. Microarray hybridization and data processing For the discovery population samples, labeled complementary RNAs were generated from 300 ng of total RNA with the Illumina TotalPrep RNA Amplification Kit (Ambion, San Diego, CA). Each labeled sample was then hybridized to Illumina Human-6 v2 Expression BeadChips and read using the BeadStation 500 scanner. Arrays were quantile normalized and the data were log2 transformed. Gene expression data for the 516 known human kinases (1) (http:// kinase.com/human/kinome, updated December 2007) were extracted from the
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dataset with 503 human kinases having available expression data available (Supplementary Table S1 is available at Carcinogenesis Online). Genes were excluded from further analysis if ,20% of the 93 tumor samples had ,1.5-fold difference from the median. In total, gene expression data from 131 kinases were analyzed. The same process was used to generate the nuclear factorkappaB (NF-jB) pathway dataset (Supplementary Table S2 is available at Carcinogenesis Online). TaqMan gene expression Gene expression assays (Applied Biosystems, Foster City, CA) were performed in the replication population for I-kappa-B kinase epsilon (IKBKE) and the endogenous control glyceraldehyde-3-phosphate dehydrogenase. Real-time PCR reactions were run in duplicate for each sample using the ABI Prism 7900 sequence detection system (Applied Biosystems). Water-only negative controls were also included on each reaction plate. Glyceraldehyde-3-phosphate dehydrogenase expression was used to calculate the relative quantification of IKBKE based on 2DDCt. Statistical analysis Comparison of patient characteristics between the discovery and validation populations was analyzed using Student’s t-test, Mann–Whitney test or Fisher’s exact test, as appropriate. For overall survival risk analysis, expression for each gene was fitted to the Cox proportional hazard model with calculations of hazard ratios and corresponding 95% confidence intervals (95% CI). Multivariable analysis was adjusted for age at diagnosis, gender, smoking status, body mass index at diagnosis, family history of any cancer, ethnicity, history of hypertension and clinical stage. Kaplan–Meier survival functions and log-rank tests were used to assess overall survival durations with regard to risk groupings. All statistical analyses were performed using STATA (version 10.1; College Station, TX). Gene expression comparisons of tumor and adjacent normal tissues were performed using BRB-ArrayTools developed by Dr Richard Simon and BRB-ArrayTools Development Team (verson 4.10; http://linus.nci.nih.gov/BRB-ArrayTools. html). Hierarchical clustering was performed using GenePattern (7) software (release 3.3).
Results Discovery and replication populations The patient characteristics for the discovery and replication populations are shown in Table I. The populations are well matched on all Table I. Host Characteristics Discovery N (%) Total 93 Age, mean (SD) 59.10 (10.26) Gender Female 39 (41.94) Male 54 (58.06) Ethnicity Caucasian 66 (71.74) Non-Caucasian 26 (28.26) Smoking Status Never 49 (52.69) Ever 28 (30.11) Former/Current 16 (17.20) BMI, mean (SD) 30.70 (6.52) History of hypertension No 41 (44.09) Yes 52 (55.91) Family history of cancer No 40 (43.01) Yes 53 (56.99) Stage I 41 (44.09) II 11 (11.83) III 26 (27.96) IV 15 (16.13) Dead No 71 (77.17) Yes 21 (22.83) BMI, body mass index; SD, standard deviation.
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Replication N (%) 237 59.68 (10.56)
P-value
0.65
70 (29.54) 167 (70.46)
0.031
189 (79.75) 48 (20.25)
0.12
109 (46.38) 93 (39.57) 33 (14.04) 30.36 (6.61)
0.27 0.68
108 (45.57) 129 (54.43)
0.81
75 (31.65) 162 (68.35)
0.051
94 17 80 45
(39.83) (7.20) (33.90) (19.07)
0.39
185 (78.06) 52 (21.94)
0.86
variables (P-value , 0.05) with the exception of gender. There was an excess of male patients in the replication population (P-value: 0.031). The median overall survival time for both groups was .67 months and was not statistically significant (P-value: 0.95). Kinase gene expression differences between tumor and adjacent normal tissues Utilizing expression data from adjacent normal tissues obtained at the same time as resection of the ccRCC tumor, we sought to identify a kinase-based gene expression signature for differences between ccRCC and normal. Fourty-one kinases were differentially expressed between tumor and adjacent normal at a false discovery rate of 1 107 (Figure 1a). Two major branches were evident differentiating tumor and normal samples, with the exception of 15 adjacent normal samples that had similar expression patterns as the tumors. Analysis of demographics (age at diagnosis, gender and ethnicity), epidemiology (smoking status, family history of cancer, family history of kidney cancer and body mass index) and clinical characteristics (stage, grade, performance status and outcome) of these 15 individuals showed no statistical differences from the rest of the patient population, suggesting that the clustering of these normal tissue samples is due to a field effect from the tumor. A majority (N 5 55, 76.4%) of the significantly differentially expressed kinases were upregulated in the tumor tissues compared with the adjacent normal, including two vascular endothelial growth factor receptors (VEGFR), FLT1 encoding for VEGFR1 and KDR for VEGFR2. Kinase gene expression associated with overall survival From the analysis of the 131 kinase genes, only one gene, inhibitor of kappa light polypeptide gene enhancer in B-cells kinase epsilon or IKBKE, was found to be significantly associated with overall survival at P-value ,0.01. Increased expression of IKBKE resulted in a 5.30fold increased risk of death (95% CI: 1.93–14.59, P-value: 0.0012) in multivariable analysis. This significant association was also observed in unadjusted Cox regression modeling, suggesting that expression of IKBKE is a strong prognostic factor independent of other factors influencing overall survival (hazard ratio: 5.63, 95% CI: 2.30– 13.81, P-value , 0.001). We then used the expression profile of IKBKE to split our patient population into two groups based on the median IKBKE expression level. Patients with high IKBKE expression had a 3-fold increased risk of death compared with those with low IKBKE expression (Table II, 95% CI: 1.07–10.40, P-value: 0.038). Furthermore, differences in IKBKE expression levels translated into a significant difference in overall survival times (Figure 2a; log-rank P-value: 0.049). The association of IKBKE expression and overall survival was replicated in analysis of additional ccRCC tumors (P-value: 0.0021). Patients with the highest IKBKE expression were at a 2.74fold increased risk of dying (95% CI: 1.05–7.15, P-value: 0.039) compared with those with the lowest IKBKE expression (Table III). This resulted in a significant reduction in median survival time by 40.4 months from over 126.1 months to only 85.7 months for those with the highest expression (Figure 2b; P-value: 0.006). Because of the significant effect of IKBKE expression on overall survival, we went back to our tumor and adjacent normal samples and observed that IKBKE was significantly higher expressed in tumors compared with adjacent normal (P-value , 1 107; Figure 1b). Tumors had a 1.3-fold increase in IKBKE expression. Furthermore, a trend toward increased expression by tumor grade was evident (data not shown). NF-jB signaling associated with overall survival IKBKE (also known as IKK-i) is an IKK-related kinase involved in regulation of NF-jB signaling. We next determined if global NF-jB signaling was also associated with a poor prognosis. Toward this, we selected an additional 41 genes within the NF-jB signaling pathway
IKBKE as a predictor of overall survival in ccRCC
Fig. 1. Kinase gene expression in 93 paired ccRCC tumors and adjacent normal tissues. (A) Global expression patterns for the top 41 differentially expressed kinase genes. (B) IKBKE expression differences between tumor and adjacent normal tissues.
Discussion
Table II. IKBKE gene expression and overall survival a
Expression levels
Alive N (%)
Dead N (%)
b
Low High
39 (84.8) 32 (69.6)
7 (15.2) 14 (30.4)
1 (reference) 3.34
HR
95% CI
P-value
1.07–10.40
0.038
a
Expression levels: split into low and high groups based on median IKBKE expression for all subjects. b HR: adjusted hazard ratio by age at diagnosis, gender, smoking status, body mass index at diagnosis, family history of any cancer, ethnicity, history of hypertension and clinical stage.
(Supplementary Table S2 is available at Carcinogenesis Online), focusing on genes upstream of the core NF-jB/IKK/IjB complex. Gene expression data from our Illumina BeadChips were used to determine associations with overall survival and recurrence. Interestingly, only IKBKE expression was associated with overall survival among these genes at P ,0.01.
Because of their central role in cell signaling, kinases have become attractive drug targets for pharmaceutical companies (8). However, the success of these therapies for improving clinical outcomes in ccRCC patients is dependent on the expression profiles of target kinases within the tumor. In this study, we comprehensively profiled the gene expression of all known kinases in 93 ccRCC tumors and replicated our findings in an additional 237 tumors. High IKBKE expression was consistently found to result in an increased risk of dying and a decreased median survival time. This is the first study to implicate IKBKE as playing a role in ccRCC. IKBKE encodes for an inducible IKK-related kinase. This kinase was first identified as a member of the NF-jB complex following activation by lipopolysaccharides and phorbol esters (9,10). Further studies demonstrated that IKBKE stimulates NF-jB signaling through activation of cRel and p65/RelA (11,12). IKBKE has also been shown to be involved in regulation of interferon regulatory factor transcription factors during the innate immune response (13,14).
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Fig. 2. IKBKE expression in ccRCC tumors and overall survival in (A) discovery population and (B) replication population. Numbers in parentheses are number of events by the total number of patients in each group. MST, median survival time in months. Table III. IKBKE gene expression and overall survival in replication population a
Expression levels
Alive N (%)
Dead N (%)
b
Low Medium High P for trend
68 (86.08) 66 (83.54) 51 (64.56)
11 (13.92) 13 (16.46) 28 (35.44)
1(reference) 1.73 2.74
HR
95% CI
P-value
0.51–5.84 1.05–7.15
0.38 0.039 0.031
a
Expression levels: split into low, medium and high groups based on tertiles of IKBKE expression for all subjects. b HR: adjusted hazard ratio by age at diagnosis, gender, smoking status, body mass index at diagnosis, family history of any cancer, ethnicity, history of hypertension and clinical stage.
IKBKE knockout mice have less inflammatory cytokine production, which was shown to be protective against the effects of obesity and other chronic inflammatory conditions (15). As for a role in cancer, IKBKE has been found to be upregulated in breast, endometrial and ovarian cancers as well as gliomas (16–21). Our analysis of paired adjacent normal and ccRCC tissues also demonstrate a similar significant upregulation of IKBKE in tumor tissue. Furthermore, the studies in ovarian cancer and glioma demonstrated that IKBKE overexpression was associated with increased progression, poor prognosis and resistance to therapy, similar to our current observations. These previous findings lend support that IKBKE functions as an oncogene and plays a significant role in cancer development and clinical outcomes. We also took advantage of the adjacent normal tissues to identify kinase-specific expression patterns for tumor and normal tissues. A clear separation was evident, with the exception of 15 adjacent normal tissues that clustered within the tumors. There were no differences among these 15 individuals compared with the other 78 individuals in
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demographic, epidemiology and clinical variables, suggesting the presence of a field effect with the initiation of changes in pathologically normal tissue that mirror tumor tissues. IKBKE was one of these top kinases with a 1.3-fold increase in tumor gene expression, and other upregulated kinases include two VEGFRs, the primary targets for both sorafenib and sunitinib. The most upregulated kinase in our study was PNCK, encoding for a calcium/calmodulin-dependent serine/threonine kinase. This kinase was expressed 18 times higher in tumor tissue compared with normal. Interestingly, this kinase was identified in a previous expression analysis, ccRCC as being highly upregulated (22) and has recently been shown to induce degradation of epidermal growth factor receptor through the heat shock protein-90 chaperone system (23). Epidermal growth factor receptor is known to be altered in ccRCC and was also two times higher expressed in tumor tissues compared with normal in this study. Future studies in an independent set of tumor and adjacent normal tissues, ideally on an independent analytical platform, such as reverse transcription–PCR, will be needed to confirm the kinase signature. The phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene homolog/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway is often activated in ccRCC tumors and has become an attractive target of drug development for ccRCC. However, resistance is a frequent occurrence to mTOR inhibitors and progression-free survival is improved by only a few months (24). Combinatorial approaches including with other kinase inhibitors, such as sorafenib and sunitinib, are being taken in attempt to improve outcomes. Previous functional studies have shown that IKBKE plays a role in modulating PI3K/AKT/mTOR signaling through the direct phosphorylation and activation of AKT (25). Although requiring validation and replication, our results preliminarily suggest that the addition of an IKBKE inhibitor with PI3K/AKT/mTOR inhibitors may enhance the effectiveness of these agents. This approach would shut down signaling through the PI3K/AKT/mTOR signaling pathway at two sites—AKT and mTOR—potentially overcoming resistance and improving response. Indeed, a recent preclinical study demonstrated that an AKT inhibitor was able to abrogate the phosphorylation and activation of AKT in RCC cell lines, following treatment with an mTOR inhibitor (26). Several studies have attempted to elucidate gene expression signatures for clinical outcomes, such as overall survival and metastasis for RCC (27–32). Unfortunately, there is little overlap in the findings generated from these studies due to a wide variation in microarray platforms (complementary DNA spotted arrays with varying number of clones included and various oligonucleotide arrays), sample size, study design and analytical approaches utilized (33). This lack of constancy underscores the need for a focused approach that includes an independent validation utilizing a different technical platform. Additionally as with all microarray-based experiments, multiple comparisons are a concern. The strength of this study is that we had a large independent sample set available for replication utilizing an independent analytical platform. This allowed us to replicate our findings obtained from expression microarrays using TaqMan expression assays to minimize the possibility of a false positive finding. We were also able to integrate epidemiology and clinical variables into the data analysis to take into account other factors that influence outcomes and would potentially confound the results. Through a comprehensive approach that integrated gene expression with epidemiological and clinical variables, this study identified IKBKE as a mediator of ccRCC overall survival and brings forth this kinase as a potential target for drug development. Overall, these results help to better understand the molecular mechanisms underlying ccRCC clinical outcomes and suggest that further study of IKBKE in ccRCC is warranted. Supplementary material Supplementary Tables S1 and S2 can be found at http://carcin. oxfordjournals.org/.
IKBKE as a predictor of overall survival in ccRCC
Funding Supported in part by National Institute of Health grant (R01 CA098897 to X.W.) and MD Anderson’s Center for Translational and Public Health Genomics. Conflict of Interest Statement: None declared.
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