Introduction
Renal cell carcinomas (RCCs) are a heterogeneous group of different kidney tumors, with clear cell RCC (ccRCC) being the most prevalent histological subtype (1,2). The majority of sporadic ccRCC cases are characterized by loss or inactivation of von Hippel-Lindau (VHL) tumor-suppressor gene, resulting in accumulation of hypoxia inducible factors (HIFs) and overexpression of HIF-driven genes that are partially responsible for cell proliferation, angiogenesis and tumor growth (1,2). If diagnosed at early stages, clear cell tumors are usually curable by surgical treatment; whereas advanced ccRCC are characterized by worse prognosis compared to non-clear cell subtypes of RCC (3). The majority patients usually report no symptoms and approximately a third of patients with ccRCC are diagnosed at the metastatic stage of the disease. Despite surgical treatment of primary disease, approximately half of the patients with primarily localized tumors will eventually develop metastases or recurrent disease (1,3). The molecular background of ccRCC has been extensively studied resulting in better understanding of the ccRCC molecular background and development of novel therapeutic strategies (2,4). However, the benefits of adjuvant treatments for advanced or metastatic ccRCC still require improvement (1). In addition, there is a lack of reliable molecular prognostic and predictive markers that could be routinely used for improved patient monitoring and targeted as effective treatment strategies (1,5).
Nuclear factor-κB (NF-κB) is a protein complex that controls the expression of genes involved in immune response and cell survival and is often upregulated in human cancer (6,7) including RCC (8). The classical (canonical) NF-κB pathway comprises heterodimer of the transcription factors RelA/p50. In the resting state, heterodimers are sequestered in the cytoplasm by NF-κB inhibitors (Iκ-B). Canonical NF-κB signaling can be induced by pro-inflammatory mediators including lipopolysaccharides, cytokines or CD40 ligand (6,7). Inhibitor of nuclear factor κB kinase subunit B (IKBKB or IKKβ) is part of the Iκ-B kinase (IKK) complex that activates the transcription factor NF-κB (9). Upon activation, IKBKB phosphorylates Iκ-B leading to its ubiquitination and degradation, releasing RelA/p50 from inhibition. Following translocation to the nucleus, RelA/p50 binds to κB sites within promoters and regulates the transcription of target genes to increase the expression of pro-survival and pro-inflammatory factors (6,7,9). Generation of IKBKB−/− mice and further experimentation performed using IKBKB-deficient cells demonstrated that this protein is essential for activation of NF-κB and functions as a dominant kinase in the canonical NF-κB cascade (6,10,11).
Sustained activation, defective regulation and overexpression of proteins of the NF-κB pathway is observed in certain tumors and tumor-derived cell lines, and is associated with the malignant phenotype in the majority of cases (12). NF-κB transcription factors have been extensively researched because of their involvement in stromal communication with cancer cells and role in establishing the tumor microenvironment. Cancer cells, a variety of non-cancerous tumor-associated immune cells and fibroblasts exhibit altered NF-κB signaling, which is often associated with intratumoral immunosuppression and development of multidrug resistance (7,12). In addition to its major role in the activation of NF-κB pathway, IKBKB was demonstrated to phosphorylate NF-κB-unrelated factors, including tumor suppressor p53 or forkhead box O3 transcription factors, targeting them for degradation by the ubiquitin-proteasome pathway (13,14). It was also demonstrated that IKBKB is a target for prolyl hydroxylase-mediated hydroxylation and, therefore, hypoxia increases the expression and activity of IKBKB in cultured cancer cells (15); whereas the protein product of VHL tumor suppressor gene (pVHL) negatively regulates IKBKB (16).
In the present study, the expression of IKBKB in tumors and matched non-cancerous renal tissues of patients with ccRCC was investigated. The association of IKBKB protein expression with clinicopathological parameters and survival of ccRCC patients was assessed.
Discussion
In the present study, three independent techniques (RT-qPCR, western blot analysis and IHC) were used to demonstrate that the expression level of IKBKB is decreased in ccRCC. The majority of the analyzed tumor specimens exhibited reduced levels of IKBKB mRNA and protein. However, an upregulated IKBKB protein level was associated with higher nuclear grade of the tumor and significantly shorter survival, suggesting an oncogenic role of this kinase in ccRCC.
Previous studies using patients with RCC, cell lines and xenograft animal models of RCC identified IKBKB as a factor associated with the progression of the disease and poor patient outcomes. A dataset from TCGA containing 469 ccRCC cases revealed that copy number alterations of IKBKB were rare in this cancer (~1% of tumors), and no alterations in the IKBKB sequence were reported in this dataset (2,25). NF-κB pathway genes rarely contain mutations in cancer (12). Constitutive activation of NF-κB in certain cancers is usually mediated by alterations of upstream regulators and/or altered expression levels of NF-κB-associated genes (12). In the current study, IKBKB expression was also investigated using the TCGA data provided by the cBio Cancer Genomics Portal (2,25) setting a z-score threshold for 1.5-fold change. This evaluation revealed that the expression level of IKBKB transcript determined by RNA-Seq was downregulated in 7% and upregulated in 6% of ccRCC cases, while IKBKB protein levels measured by reverse-phase protein array remained unaltered in all 469 samples included in the study (2,25). Although these expression data are not in line with the results of the current study, this may be attributed to different sensitivities and specificities of methods used. However, the prognostic significance of IKBKB reported in this study is in accordance with analyses based on previously published study by Peri et al (26). Meta-analysis of ccRCC expression datasets identified a subset of genes including IKBKB and NF-κB mediators, matrix metalloproteinase 9, proteasome subunit β 9 and superoxide dismutase 2, whose elevated expression levels were associated with worse patient outcomes (26). Another study established a model linking pVHL loss to increased activation of NF-κB, potentially elucidating a previously reported observation that loss of may be associated with the chemoresistance of ccRCC to tumor necrosis factor therapy (27). Recently, it has been reported that IKBKB protein may indirectly have a role in stabilizing HIF1α and HIF2α in RCC cells via activation of NF-κB essential modulator (NEMO) protein, a regulatory subunit of the IKK complex (28). In addition, it was demonstrated that the degree of NEMO protein expression was downregulated in 62.8% tumor samples derived from 250 patients with ccRCC, and it was positively associated with the progression of ccRCC (28). This observation seems to be analogous to the finding that IKBKB immunoreactivity was decreased in the majority of analyzed ccRCC specimens, while increased IKBKB protein expression was associated with higher tumor grade and poorer patient outcomes. However, additional studies are required to investigate unknown links between the IKBKB and NEMO proteins and potential common mechanisms underlying their altered expression in ccRCC.
Beneficial effects of decreased IKBKB expression and/or IKBKB protein activity on the effectiveness of different anticancer approaches were reported previously (29). Treatment of metastatic RCC cell lines, ACHN and SN12K1, with an anti-oxidant agent, pyrrolidine dithiocarbamate, decreased expression level of IKBKB and other components of NF-κB signaling, as assessed by western blotting (30). The resulting inhibition of NF-κB pathways exerted anti-proliferative, pro-apoptotic and anti-angiogenic effects in the tested cell lines (30,31) and sensitized them to cisplatin treatment (32). Further experiments performed in a xenograft animal model of RCC demonstrated that the antioxidant treatment reduced cancer cell proliferation and attenuated tumor progression, with decreased expression of NF-κB proteins and upstream kinases, IKBKB and IKKA (33). Notably, accumulation of reactive oxygen species in the kidneys of superoxide-deficient mice resulted in decreased expression of IKBKB (34), suggesting that the influence of oxidative stress on expression of NF-κB-associated genes is tissue- or disease-specific. Another study performed in RCC cell lines demonstrated that IKBKB and RelA (p65) were required for the oncogenic properties of microRNA-21 (miR-21) in ACHN cells (35). In turn, miR-21 indirectly increased IKBKB phosphorylation, which upregulated NF-κB and mechanistic target of rapamycin complex 1 signaling, resulting in enhanced proliferation, migration and invasiveness of ACHN and 786-O cell lines (35,36). Transfection of RCC cell lines with a plasmid expressing IKBKB attenuated sunitinib-induced p53 promoter transcriptional activity (37). These reports suggest that increased IKBKB content in cancer cells can compromise the effectiveness of anticancer therapy, suggesting potential usefulness of IKBKB as both prognostic and predictive marker in ccRCC.
Altered IKBKB expression and/or IKBKB kinase activity were previously reported to be associated the with occurrence and progression of several types of human cancer, including breast cancer, pancreatic cancer, thyroidal C-cells carcinoma, acute myeloid leukemia and ovarian cancer (7,8,12,29,38). However, the available data concerning IKBKB expression are inconclusive. IKBKB transcript levels were significantly upregulated in human hepatocellular carcinoma compared with adjacent normal tissue (39). Cultured Hs578T breast cancer cells exhibited aberrant expression and activity of IKBKB compared with untransformed mammary epithelial cells (40). Reduced IKBKB expression were demonstrated in glioblastoma tissues at the mRNA and protein levels (41). However, downregulation of IKBKB was attributed to microglia/macrophages infiltrating advanced glioblastoma tumors, indicating the important role of this kinase for the local microenvironment and antitumor response (41).
In conclusion, to the best of our knowledge, the present study is the first comprehensive investigation analyzing IKBKB expression at the mRNA and protein levels in a cohort of patients with ccRCC. The results suggest that the three techniques applied to determine the IKBKB expression, RT-qPCR, western blotting and IHC, should be used as a complementary rather than alternative methods. However, additional methodological studies are required to compare these and other available assays before conclusions are made. The number of patients included in the study was enough to disclose associations of IKBKB protein expression level with clinicopathological parameters and survival of patients with ccRCC. Therefore, the findings of the present study demonstrate that the expression of IKBKB protein may be of clinical relevance in ccRCC. The elevated content of IKBKB in the ccRCC tumor tissue may be useful as a potential marker of prognosis, and suggests that the application of anti-NF-κB treatments may sensitize ccRCC cells to certain adjuvant therapies. Further studies using a larger number of patients are required to support this suggestion, and validate prognostic or predictive value of IKBKB in ccRCC.