Abstract
Background/Aim: Kidney and brain expressed protein (KIBRA), a member of the WW domain-containing protein family, has an important role in tumour growth and progression in various cancers. However, the pathological significance of KIBRA expression in clear cell renal cell carcinoma (ccRCC) tissues is not fully understood. The aim of this study was to clarify the pathological significance and prognostic roles of KIBRA expression in patients with ccRCC. Materials and Methods: KIBRA immunoreactivity, proliferation index (PI; with anti-Ki-67 antibody), apoptotic index (AI; using anti-cleaved caspase-3), and large tumour suppressor kinases (LATS-2) were evaluated in 157 ccRCC specimens by immunohistochemistry. Fifty normal kidney tissues were also evaluated as controls. The relationships between KIBRA expression and these cancer-related variables as well as clinicopathological features and survival were analysed. Results: Moderate to strong immunoreactivity of KIBRA was identified in all normal kidney tissues; however, ccRCC cells with strong KIBRA expression was rare. The immunoreactivity score (IRS) of KIBRA was negatively associated with grade, T stage, tumour diameter, and metastasis. Kaplan–Meier survival curves showed that high KIBRA expression was a favourite predictor for overall survival. KIBRA IRS was negatively associated with PI and positively associated with the IRS of LATS-2 by univariate analysis. In addition, multivariate analysis showed that KIBRA was significantly associated with PI. Conclusion: KIBRA demonstrated important roles as a tumour suppressor in ccRCC. In addition, its expression was significantly associated with survival in these patients. Several such KIBRA-related functions were speculated to be modulated by cancer cell proliferation and LATS-2.
Renal cell carcinoma (RCC) is one of the major urological cancer types, and metastasis and local invasion are frequently detected at diagnosis or following radical surgery (1). The pathological characteristics of RCC are regulated by various signalling pathways and molecules, and in recent years, several investigators suggested that the Hippo pathway plays critical roles in the malignant behaviours of RCC (2-4). The Hippo pathway consists of various intracellular molecules and is regarded as the regulator of organ size control and tissue growth during embryonic development (5, 6). However, in addition to RCC, the Hippo pathway is reported to be associated with carcinogenesis and malignant aggressiveness (5, 7-10), and it is thought that regulation of this pathway may be helpful in the development of new therapeutic agents for many types of malignancies including RCC (11-13). Therefore, understanding the regulatory mechanisms of the Hippo pathway is important in determining treatment and observation strategies for RCC patients.
Kidney and brain expressed protein (KIBRA), which is classified as a WW domain tandem-containing protein, is an important upstream regulator of the Hippo pathway (14-16), and associations between KIBRA and the Hippo pathway have been detected in a variety of cancers (15-17); however, there is no general agreement about the pathological roles of KIBRA in malignant cells. In short, several studies showed that KIBRA functions as a tumour suppressor but there is also evidence that KIBRA has oncogenic functions (15-19). Regarding RCC, there is little information on the clinicopathological roles of KIBRA in these patients because only one report has focused on the epigenetic mechanisms of KIBRA protein and mRNA expression regulation in clear cell RCC (ccRCC) and this was conducted in a relatively small study population (20).
In recent years, regulation of the core Hippo kinase cascade by KIBRA was reported to be one of the critical determinants of KIBRA-induced pathological activities (16). In addition, several investigators suggested that KIBRA modulated the functions of the Hippo pathway via interactions with large tumour suppressor kinase (LATS), which is a core component of the Hippo pathway (21-23). Furthermore, there is a report that KIBRA stabilizes LATS-2 through inhibition of ubiquitination and regulates LATS-2 expression (21). Importantly, LATS-2 is associated with carcinogenesis, malignant behaviours, and prognosis in various cancer types including RCC (3, 24, 25). Thus, KIBRA is speculated to modulate malignant aggressiveness via regulation of LATS-2 in human RCC tissues. However, there is no evidence about such a correlation in patients with ccRCC. In addition to LATS-2, KIBRA also plays important roles in the regulation of cell proliferation and apoptosis under various pathological conditions including malignancy (26-28). Cancer cell proliferation and apoptosis are closely associated with tumour growth, progression, and outcomes in patients with RCC (29-31), but there is no information on the pathological roles of KIBRA in the regulation of cancer cell proliferation and apoptosis in these patients.
The main aim of this study was to clarify the pathological significance and prognostic roles of KIBRA expression in ccRCC patients by immunohistochemical analyses of a relatively large study population. In addition, we also investigated the relationships between KIBRA expression and LATS-2 expression, cancer cell proliferation, and apoptosis in ccRCC tissues.
Materials and Methods
Patients. Formalin-fixed and paraffin-embedded sections were generated from the surgical specimens of 157 ccRCC patients (male=117, 74.5%) at Nagasaki University Hospital (Nagasaki, Japan). Mean±SD age at diagnosis was 60.9±12.4 years. Consecutive specimens were examined in the present study but some specimens were excluded because of low cancer cell numbers (<500) resulting from their use in previous investigations (32). We also excluded patients who received neo-adjuvant therapy, including immunotherapy and molecular targeted therapy. All patients were evaluated by chest X-ray, ultrasonography, and computed tomography (CT), and bone scanning. As a control, KIBRA immunoreactivity was also evaluated in 50 normal kidney tissues. The study protocol met the ethical standards of the Human Ethics Review Committee of Nagasaki University Hospital (No. 12052899: Nagasaki). Written informed consent was obtained from all patients prior to enrolment.
Immunohistochemistry. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded sections. Five-micrometre-thick sections were deparaffinized and rehydrated, and antigen retrieval was performed at 96°C for 40 min in 0.01 M sodium citrate buffer (pH 6.0). All sections were then immersed in 3% hydrogen peroxide for 30 min and incubated overnight at 4°C with an anti-KIBRA antibody (Abcam, Cambridge, UK) and an anti-LATS-2 antibody (Abcam, Boston, MA, USA). Next, all sections were incubated at room temperature with peroxidase, according to the labelled polymer method, using EnVision+ Peroxidase (Dako Corp., Glostrup, Denmark) for 60 min. The peroxidase reaction was visualized using a liquid 3,3-diaminobenzidine tetrahydrochloride (DAB) substrate kit (Thermo Fisher Scientific, Rockford, IL, USA). Proliferation index (PI) and apoptotic index (PI) were evaluated according to previous reports (30, 33).
Evaluation. KIBRA and LATS-2 expression was assessed semi-quantitatively according to the percentage of positively stained cancer cells in 200 randomly selected high-power fields. Their expression levels were quantified using the immunoreactivity score (IRS), where IRS=staining intensity × percentage of positive cells. Staining intensity was defined as 0 (no staining), 1 (weak), 2 (moderate), and 3 (strong), and the percentage of positively stained cancer cells was scored on a scale of 0 to 3 according to the percentage of cells (0%, 1-50%, 51%-100% for scores 0, 1, and 2 respectively). Finally, scores 4-6 were judged as high expression of KIBRA and LATS-2. PI and AI represented the percentage of Ki-67-positive and cleaved caspase-3-positive cells, respectively.
Statistical analysis. Data are expressed as the mean±SD. Student’s t-test was used to compare the continuous variables and Scheffé’s method was used for multiple comparisons of the data. Chi-squared test was used for categorical comparisons of the data. Pearson’s correlation coefficient (r) was used to evaluate the relationship between each variable. Differences in survival were assessed using Kaplan–Meier curves and the log-rank test. To clarify the pathological roles of KIBRA expression, univariate and multivariate logistic regression analyses were performed, and results were described as odds ratios (ORs) with 95% confidence intervals (CIs) and p-values. All statistical analyses were two-sided and performed using StatView for Windows version 5.0 (Abacus Concepts, Inc., Berkeley, CA, USA). p<0.05 was considered to indicate statistical significance.
Results
KIBRA expression. Representative examples of KIBRA expression in normal kidney and ccRCC tissue are shown in Figure 1A and B, respectively. Moderate to strong expression of KIBRA was observed in all normal kidney tissues (Figure 1A). In contrast, some regions in ccRCC tissues showed moderate to strong KIBRA expression (Figure 1B), but most ccRCC cells had no to weak staining (Figure 1A). In short, KIBRA expression in ccRCC tissues was clearly lower compared with that in normal kidney. Finally, the mean±SD KIBRA IRS in ccRCC was 3.74±1.57.
Representative images of KIBRA expression in human renal cell carcinoma (RCC) tissues. KIBRA was mainly detected in the cytoplasm. (A) Most cancer cells had no to weak expression, although normal tubules had moderate to strong expression (T, tumour cells; N, normal kidney tissue). (B) Moderate to strong expression of KIBRA in cancer cells.
Correlation with clinicopathological features. KIBRA expression was not significantly associated with age at diagnosis and sex (p=0.700 and p=0.518, respectively). IRSs of KIBRA for each pathological feature are shown in Figure 2. The IRS of KIBRA in grade 3/4 disease (2.20±1.44) was significantly lower (p<0.001) than that in grade 1 (4.34±1.27) or grade 2 (1.95±1.40; Figure 2A). A similar negative relationship was shown for T stage and metastasis (Figure 2B and C). Furthermore, the IRS of KIBRA was closely and negatively correlated with maximum tumour diameter (r=0.57, p<0.001).
Immunoreactivity scores are shown according to nuclear grade, T stage, and metastasis (in A-C, respectively).
Survival analyses. Regarding the prognostic role of KIBRA expression, Kaplan–Meier survival curves showed that high KIBRA expression was a better predictive factor for overall survival (log rank p<0.001; Figure 3). When a similar analysis was performed using the univariate Cox regression hazard model, the IRS of KIBRA had significantly lower risk compared with high KIBRA expression [hazard ratio (HR)=0.12, 95%CI=0.05-0.29, p<0.001]. In addition, multivariate analysis of KIBRA IRS, grade, T stage, and metastasis demonstrated that KIBRA was an independent predictor for overall survival (HR=0.60, 95%CI=0.37-0.97, p=0.036).
Kaplan–Meier survival curves showed that patients with high KIBRA expression had better overall survival compared with those with low KIBRA expression (log rank p<0.001).
Correlation of KIBRA with cancer cell proliferation, apoptosis, and LATS-2 expression. The relationships between KIBRA IRS and PI, AI, and LATS-2 IRS are shown in Table I. The IRS of KIBRA was negative correlated to PI (r=−0.43, p<0.001). In contrast, its IRS tended to be positively correlated to AI; however, it did not reach significance (r=0.151, p=0.077). However, the IRS of KIBRA was positively associated with the IRS of LATS-2 (r=0.32, p<0.001). Moreover, we found that the IRS of LATS-2 was significantly associated with PI (r=−0.031, p<0.001). Based on these results, we analysed the anti-proliferative effect of KIBRA and LATS-2 on cancer cell proliferation by univariate and multivariate analyses (Table II). We found that KIBRA expression was independently associated with PI by multivariate analysis (OR=0.25, 95%CI=0.13-0.50, p<0.001).
KIBRA expression and cell proliferation, apoptosis, and LATS-2.
Regression analyses of KIBRA and LATS-2 for proliferation index.
Discussion
Our results first showed that KIBRA immunoreactivity in ccRCC tissues was remarkably lower than that in normal kidney tissues. KIBRA has contrary pathological characteristics that are either oncogenic or tumour suppressive and are speculated to be dependent on cancer type. KIBRA promoter methylation was positively associated with ccRCC, and its mRNA expression in carcinomatous tissues was lower than that in non-tumour kidney tissues (20). Although the numbers of patients in this previous study were relatively small (n=8 in the methylation study, n=32 in the mRNA study), their results suggested that KIBRA is a tumour suppressor in ccRCC. In this study, we confirmed this finding in a larger study population. In addition, our results demonstrated that KIBRA expression was negatively associated with malignant aggressiveness and worse prognosis in patients with ccRCC. Unfortunately, there was no report regarding the relationships between KIBRA immunoreactivity and pathological features, tumour size, and prognosis in these patients. However, several investigations supported our results that KIBRA functions as a tumour suppressor and a better predictor in other cancers. For example, low KIBRA expression was associated with higher histological grade, advanced stage, metastasis, poor event-free survival, and high risk of death in breast cancer patients (34-36). In addition, KIBRA was reported to reduce the invasive ability of lung cancer and hepatocellular carcinoma cells (37, 38). Thus, while it is known that KIBRA can promote tumour development and is positively associated with poor prognosis in a variety of cancers (39, 40), we speculate that KIBRA is a tumour suppressor in ccRCC.
For the first time, we found that KIBRA expression was negatively associated with cancer cell proliferation and positively associated with LATS-2 expression in human ccRCC tissues. Unfortunately, there was no general agreement regarding the relationship between KIBRA expression and cancer cell proliferation in malignant tissues. However, several investigators suggested that KIBRA has crucial roles in the suppression of several cancers (23, 38, 41), and our results support their opinion. Nonetheless, various studies demonstrated that KIBRA is one of the major upstream regulators of the Hippo pathway (16, 21), and LATS-2 was reported to be such a KIBRA-related molecule in a variety of cancers (23, 38). From these observations, we hypothesized that KIBRA might affect malignant behaviours via regulation of LATS-2 expression, as supported by our results.
Our results also showed that LATS-2 expression had an anti-proliferative effect in these tissues. Furthermore, multivariate analyses demonstrated that KIBRA expression was significantly associated with cancer cell proliferation by a mechanism independent of LATS-2. Although previous reports showed that KIBRA regulated cell proliferation in various cancers, regulation of Hippo pathway-related molecules, such as YAP and LATS, was suggested as a representative molecular mechanism of such KIBRA-induced anti-proliferative activity (42-44). However, we should note that KIBRA can modulate cancer cell proliferation through Hippo pathway-independent mechanisms (26, 27, 43). Unfortunately, our study design cannot provide information regarding the detailed molecular mechanism of the anti-proliferative effects of KIBRA on ccRCC; however, we considered that KIBRA may modulate ccRCC via LATS-2-independent systems in these patients.
This study has several limitations. First, experiments in RCC cell lines and animal models were not performed. We understand that these experiments are important to determine the detailed pathological and molecular mechanisms of KIBRA-related malignant aggressiveness in ccRCC. However, there were no data on the pathological significance of KIBRA immunoreactivity in patients with ccRCC. Therefore, we believe that this study has significant value for determining the direction of further basic research. Another limitation is that the relationships between KIBRA and other core components of the Hippo pathway, such as YAP/TAZ and LATS-1, were not analysed in this study. In recent years, we reported that LATS-2 plays important roles as a tumour suppressor in prostate cancer in vivo and in vitro (25). Therefore, we paid special attention to LATS-2 in this study. We also emphasized that further in vivo and in vitro studies are essential to answer these questions and elucidate the pathological significance and prognostic roles of LATS-2 in ccRCC.
In conclusion, KIBRA immunoreactivity was negatively associated with grade, T stage, tumour diameter, and metastasis in ccRCC. In addition, it is a better predictor of survival in these patients. KIBRA immunoreactivity is significantly correlated with PI and LATS-2 expression, and LATS-2 expression was also correlated with PI by univariate analysis. However, multivariate analysis showed that KIBRA was independently associated with PI, but not with LATS-2 expression. Finally, in patients with ccRCC, KIBRA was speculated to act as a tumour suppressor via regulation of cancer cell proliferation and LATS-2 expression by a complex mechanism.
Acknowledgements
The Authors thank H. Nikki March, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.
Footnotes
Authors’ Contributions
Conception or design of the work: K.O., K.M., and Y. Nagashima; acquisition of data: T.M., Y.N., and K.M.; analysis or interpretation of data: K.M. and Y.M.; and drafting or revising work: Y. Nagashima and K.O.
Conflicts of Interest
The Authors declare that they have no conflicts of interest in relation to this study.
- Received November 17, 2022.
- Revision received November 30, 2022.
- Accepted December 1, 2022.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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