Laboratory-Kidney cancer
Expression of Ring Box-1 protein and its relationship with Fuhrman grade
and other clinical-pathological parameters in renal cell cancer
Emre Altintas, M.D.
a,*
, Mehmet Kaynar, M.D.
b, Z. Esin Celik, M.D.
c, Murat Celik, M.D.
d,
Ozcan Kilic, M.D.
b, Murat Akand, M.D.
b, Serdar Goktas, M.D.
baAkcakale State Hospital, Department of Urology, Sanl{urfa, Turkey bSelcuk University, School of Medicine, Department of Urology, Konya, Turkey cSelcuk University, School of Medicine, Department of Pathology, Konya, Turkey
d
Kars Harakani State Hospital, Department of Pathology, Kars, Turkey
Received 22 June 2019; received in revised form 17 September 2019; accepted 22 September 2019
Abstract
Objective: To determine the relationship between RING-box protein 1 (RBX-1) expression and renal cell carcinoma (RCC) with prog-nostic factors.
Methods: A total of 88 patients who underwent radical/partial nephrectomy between January 2009 and January 2016 have been included in our study. The age, gender, tumor location, tumor size, and tumor-node-metastasis stage of each patient was evaluated. From the best sec-tions in hematoxylin-eosin stained pathology preparasec-tions, tumor histological subtype, Fuhrman nuclear grade, lymphovascular invasion, renal artery/vein invasion, capsule invasion, perirenal fatty tissue invasion, and tumor grade were evaluated. Participants were divided into 2 groups according to Fuhrman grade. Fuhrman grades 1 to 2 comprised Group 1, and Fuhrman grades 3 to 4 comprised Group 2. An immu-noreactivity scoring system was used to evaluate RBX-1 expression.
Results: Upon examining all histological subtypes together, it was observed that RBX-1 expression was statistically higher in Group 2 compared to Group 1 (P< 0.008). Upon examining clear RCC cases, it was observed again that Group 2 had a higher RBX-1 expression than Group 1 (P< 0.009). RBX-1 expression was not associated with clinical-pathological parameters including tumor grade, lymphovascu-lar invasion, capsule invasion, or perirenal invasion.
Conclusion: RBX-1 expression is closely associated with a highly important prognostic factor in RCC—Fuhrman grade—and it shows promise as a prognostic marker. Further studies are required to reveal the importance of RBX-1 in RCC prognosis and treatment.Ó 2019 Elsevier Inc. All rights reserved.
Keywords: Renal cell cancer; Ring box protein-1; Fuhrman grading system; Immunohistochemistry
1. Introduction
Around the world, renal cell carcinoma (RCC) is the sixth most commonly diagnosed cancer among men and tenth among women[1]. RCC is most commonly observed between the ages of 60 to 70, and the male/female ratio is 3:2[2]. According to the latest data reported by the World Health Organization, there are 140,000 deaths every year
due to RCC[3]. The gold standard in the treatment of RCC is partial or radical nephrectomy. Because the condition does not benefit from classical chemotherapy and radiother-apy and has high mortality levels, diagnosis, treatment, and prognosis are extremely important[4]. While tumor grade and Fuhrman grade are the most important prognostic fac-tors in RCC, anatomical, histological, clinical, and molecu-lar factors are also important for determining RCC prognosis [5,6]. Recently, studies have been performed on various molecular markers such as carbonic anhydrase (CAIX), hypoxia-inducible factor, p53, p21, and osteopon-tin [7−9]. However, none of the molecular markers for
Funding: None declared.
*Corresponding Author. Tel.: +90-505-932-3338; fax: 0-414-318-9422 E-mail address:dr.e.altintas@gmail.com(E. Altintas).
https://doi.org/10.1016/j.urolonc.2019.09.019 1078-1439/Ó 2019 Elsevier Inc. All rights reserved.
RCC have been found to be useful in prognostic models or clinical practices.
The ubiquitin-proteasome system, which contains cellu-lar proteins such as tumor protein p53, cyclin-dependent kinase inhibitor 1b (CDKN1B/p27/Kip1), and cyclins, plays an important role in the protection and regulation of cellular hemostasis. The Skp1/Cullin/RBX-1/F-box protein (SCF) complex forms the largest group of the ubiquitin ligase fam-ily and plays a role in the regulation of functions such as cell growth, signal conduction, DNA replication, and gene transcription[10]. In recent studies, it has been shown that RING-box protein 1 (RBX-1) is effective not only in the regulation of physiological cellular functions, but also in the development of cancer [11,12]. The RBX-1 protein was first detected in yeast, and it was discovered to be a basic component of von Hippel-Lindau (VHL) tumor suppressor complex, as well as both human and yeast SCF complexes [11−15]. Overexpression of RBX-1 has been observed in lung, liver, colon, stomach, and ovarian cancers [13,16,17]. In recent studies, RBX-1 overexpression was observed in bladder and prostate cancers[18−20].
Cell cycle pathology commonly observed in various can-cer types plays an important role in RCC. Due to chemo-therapy and radiochemo-therapy inefficiency in RCC, molecular markers are increasingly important for prognosis evaluation after treatment. This relationship between RBX-1 expres-sion and some cancer types has suggested that it may play a role in RCC. In the literature search, no studies on RBX-1 and RCC were detected. For this reason, the purpose of this study is to evaluate RBX-1 expression and its relationship with RCC prognostic factors.
2. Methods
After obtaining ethics approval (Sel¸cuk University, ethics committee no:2015/299), the pathology data of 112 cases of patients who underwent surgery for RCC in Sel¸cuk University Faculty of Medicine Urology Clinic between the dates 01/01/2009 and 01/01/2016 were examined. A total of 88 cases were included in the study; 24 cases were excluded because they were diagnosed by another patholo-gist in an external center, and pathology preparations were not available or complete. Age, gender, tumor location, tumor size, and tumor-node-metastasis stage were evalu-ated retrospectively from medical records of participants who underwent radical or partial nephrectomy for RCC. Hematoxylin-eosin stained preparations in the pathology archive were evaluated with regard to histological subtype of tumor, Fuhrman nuclear grade, presence of lymphovas-cular (LV) invasion, presence of renal artery/vein invasion, capsule and perirenal fatty tissue invasion, sarcomatoid degeneration, and pathological tumor grade. Participants with Fuhrman grades 1 to 2 were placed in Group 1, and Fuhrman grade 3 to 4 participants were placed in Group 2. In each case, the block of the best preparation representing the tumor and involving nontumor renal tissue was selected.
Clinical and pathological grading of the cases was done based on the American Joint Committee on Cancer 2017 tumor-node-metastasis RCC grading system.
2.1. Immunohistochemical staining procedure
In pathology laboratory, sections 4 microns thick were placed on Poly-L-Lysin-coated, positively charged slides with the help of a microtome. They were then deparaffi-nized at 60˚C for 60 minutes in the oven, stained with anti-RBX-1 antibody in a semi-automatic Ventana BenchMark XT (Roche, U.S.A) immunohistochemistry staining device. 2.2. Immunohistochemical evaluation and scoring
In light microscopy examination of immunohistochemis-try stained preparations, cells were examined at 100X mag-nification in 10 different microscope areas, and scoring was performed according to staining density of positively stained cells. The dominant staining location (cytoplasmic/ nuclear) was determined. Staining density was scored as 0 ( ), 1 (+), 2 (++), or 3 (+++), according to staining inten-sity. An immunoreactivity scoring system was used for determining RBX-1 expression levels [18,19]. The percent-age of RBX-1 positive cells were denoted as: 0 (negative), 1 (1%−33%), 2 (34%−66%), and 3 (67%−100%). Accord-ing to this system, stainAccord-ing intensity was determined by multiplying the staining density values by the percentage of RBX-1 positive cells [18,19].
2.3. Statistical analysis
Statistical analysis was performed using the IBM SPSS Statistics for Windows, Version 22.0 (Armonk, NY: IBM Corp.). The correlation between RBX-1 expression and clini-cal pathologiclini-cal results was evaluated statisticlini-cally. Kruskal Wallis, chi-square and Mann-Whitney U tests were used in determining the relationship between Fuhrman grade of RBX-1 expression, tumor grade, and other pathological prog-nostic parameters. A value of P< 0.05 was assumed to be sta-tistically significant (Fig. 1).
3. Results
Data on the demographic characteristics, tumor location, and tumor size for each of the participants included in the study is presented in Table 1. RBX-1 staining was not observed in 3 of the 88 participants (2 with clear RCC and 1 with papillary RCC). Fuhrman grading was not performed by pathology in 11 of the 88 cases (2 with clear RCC, 2 with papillary RCC, and 7 with chromophobe RCC).
Groups 1 and 2 were evaluated with regard to RBX-1 protein staining level according to staining intensities of +, ++, and +++ (upon the integration of nuclear and cyto-plasmic staining). It was observed that Group 2 was more intensely stained with RBX-1, and this was statistically
significant (P < 0.008) (Table 2). Participants with clear RCC were separated again according to Fuhrman grade as Group 1 and Group 2, and the relationship between these subgroups and RBX-1 protein staining was examined. It was observed that Group 2 was significantly more intensely stained with RBX-1 protein than Group 1 (P < 0.009) (Table 2).
When RBX-1 staining level was grouped as +, ++, and +++, no significant relationship was observed between clin-ical-pathological parameters such as tumor grade, tumor size, tumor subtype, LV invasion, capsule invasion, perire-nal invasion, surgical margin border positivity, and RBX-1 (Table 3).
4. Discussion
In this study, we evaluated RBX-1 expression in RCC and its relationship with other prognostic factors. We have observed that RBX-1 expression is higher in cancer cells with high Fuhrman grades. This relationship between Fuhr-man grade and RBX-1 expression is observed both upon evaluating clear RCC cases within themselves, and upon evaluating all RCC cases together. Our data also revealed RBX-1 protein was expressed both in the nucleus and cyto-plasm. This was in accordance with other studies performed with RBX-1 protein [18]. While it was particularly expressed in the cytoplasm in papillary RCC, it was
Fig. 1. According to immunohistochemical RBX-1 expression (a) clear RCC (+) RBX-1 expression (£200), (b) chromophobe RCC (++) RBX-1 expression (£200),(c) clear RCC (+++) RBX-1 expression (£200)(d) papillary RCC (+++) RBX-1 expression (£200). RBX = RING-box protein; RCC = renal cell carcinoma.
Table 1
Demographics of patients according to tumor subtypes
Clear RCC Papillary RCC Chromophobe RCC
Age 32−79 (59.8) 39−70 (55.6) 49−68 (58.1)
Gender M:36, F:30 M:11, F:2 M:3, F:6
Tumor location Right:26, left:40 Right:4, left:9 Right:7, left:2 Tumor size (mm) 17−100 mm(47,2) 25−80 mm(49,2) 27−105 mm(63,2)
expressed both in the nucleus and cytoplasm in clear RCC and chromophobe RCC.
VHL tumor suppressor gene on chromosome 3p25.5 undergoes mutation in most human kidney cancers [21−23]. VHL protein is a component of a complex con-taining Elongin B, Elongin C, and Cullin 2 transcription associated proteins [24−26]. In 1 study, it was observed that an endogenous VHL complex contained an evolution-arily conserved protein associated with Cullin, which was RBX-1 protein[27]. The yeast homolog of RBX-1 is a potent activator of the SCF complex containing Cdc53,
which plays a role in the regulation of cyclin-dependent kinase inhibitor Sic1 and the G1-S cell cycle [27]. In a study by Kamura et al., the determination that RBX-1 is an important component of both VHL and SCF complexes provided information about the development of possible RCC after VHL mutations. RBX-1 protein, which is over-expressed in various cancer tissues including lung, colon, ovary, stomach, and recently bladder and prostate cancer, is an important part of SCF (SKp1-Cullin-F-box proteins) and has many physiological tasks including cell growth, DNA replication, signal conduction, and gene transcription [28,29]. It was determined under in vivo conditions that RBX-1 targeted disruption of p27 at the early stage of embryological development and ensured cell proliferation [30].
One method of determining the potential role of a certain overexpressed gene in human cancer cells reveals whether this gene is required for silencing cancer cell growth and improving survival. This strategy was considered in a study performed on RBX-1 overexpressed in human cancers and revealed that RBX-1 siRNA silencing in multiple cancer cell lines suppressed cancer cell growth, and this occurred as the result of initiation of apoptosis and aging by RBX-1 silencing [6,31−33].
In a study by Jia et al., RBX-1 silencing induced G2/M arrest, apoptosis, and aging, and tumor cell growth was sig-nificantly inhibited in the result [13]. In another study, autophagy and p21 induced cell senescence were induced with the result of RBX-1 silencing, and the growth of liver cells was suppressed[16]. Considered together, these stud-ies suggest that RBX-1 may be a therapeutic target with regard to cancer treatment. Suppression of RBX-1 expression promises hope for treatment of RCC cancer that has been unresponsive to classical chemotherapy and radiotherapy.
In a study performed using tissue microarray genetic applications in the examination of the relationship between RCC development and Cullin 1, which is a complex com-prised of Skp1/RBX-1/F-box proteins and plays a role in cell cycle signal conduction and transcription, 34 normal kidney tissues and 307 RCC cases were examined. It was
Table 2
The relation between disease groups with regard to RBX-1 staining severity and Fuhrman grade
RBX-1 staining severity Group-1 (Fuhrman grade 1−2) Group-2 (Fuhrman grade 3−4) P value All subtypes + 21 4 *P < 0.008 ++ 23 17 +++ 4 8 Total (n = 77) 48(62.3%) 29 (37.7%) Clear RCC + 17 2 *P < 0.009 ++ 20 14 +++ 4 7 Total (n = 64) 41(64%) 23 (36%) Table 3
The relation between RBX-1 expression and clinical-pathological parameters Parameter RBX-1 expression Number + ++ +++ P value Grade I 58 16 33 9 II 16 9 5 2 P< 0.325 III 7 2 4 1 IV 4 0 3 1 Tumor size <40 mm 28 11 13 4 40−69 mm 41 8 26 7 P< 0.192 >69 mm 16 8 6 2 Tumor subtype Clear RCC 64 19 34 11 Papillary RCC 12 4 6 2 P< 0.713 Chromophobe RCC 9 4 5 0 LV invasion Yes 4 3 1 0 P< 0.155 No 81 24 44 13 Capsule invasion Yes 10 4 6 0 P< 0.353 No 75 23 39 13 Perirenal invasion Yes 6 3 2 1 P< 0.562 No 79 24 43 12
Surgical margin positivity
Yes 6 2 4 0 P< 0.543
No 79 25 41 13
determined that Cullin 1 expression was significantly higher in RCC tissues compared to normal kidney tissue. Cullin 1 expression showed correlation with RCC histology level (P = 0.007), tumor size (P = 0.013), and pT status [34]. It was also determined that inhibition of Cullin 1 prevents cyclin D1 and cyclin E2 expressions and causes increased expression of p21 and p27, which can keep the cell cycle at the G1 phase [34]. As a result, it is hypothesized that Cullin 1 inhibition may inhibit RCC cell migration and invasion.
The correlation of RBX-1 expression with Fuhrman grade suggests that RBX-1 may be a molecular parameter for RCC in future prospective and multi-center studies, and it may be a target for RCC treatment. Since Cullin 1 expres-sion showed a correlation with increases in RCC and RCC histological type, tumor size and pT status in a study done by Ping et al.[34], it was estimated that RBX-1 may be a marker in RCC development, treatment, and prognosis due to the fact that Cullin 1 and RBX-1 belong to the same complex.
CRL2 is a complex of cullin-2, REX1, adapter protein Elongin B / C and substrate receptor protein VHL. The best known substrate of CRL2 is hypoxia-inducible factor1a [35,36]. In the VHL, Cullin and Elongin B / C complex, Elongin C binds VHL to Cul2 and combines with elongin C, NEDD8 and Rbx1[8]. It was found that Cul-2 interacts with VHL but does not interact with SOCS-box proteins, suggesting that Cul-2-Rbx-1 complex of VHL is not the only substrate subunit. CRL3 complex is comprised of cullin-3, RBX1 and BTB (Bric-a-brac, Tramtrack, Broad-complex domain) proteins as substrate receptors. In a study examining the association of cul-3 and papillary RCC, a mutation in cul-3 showed that NRF-2 activity was particu-larly effective in the formation of sporadic pRCC[37].
When the studies investigating the relationship between Cullin1,2, and 3 and RCC are examined, RBX-1 protein was associated with cullins and they coexist in different complex structures. Since cullin 1 and 2 are associated with cRCC, cullin 3 is associated with pRCC, and the rbx-1 pro-tein is associated with cullins, we can suggests that rbx1 may be central in RCC development.
There are some inherit limitations of the current study. First,it was performed with a small number of participants from a single clinical center in a retrospective design. Sec-ond, a significant relationship could not be determined between RBX-1 expression and prognostic parameters including LV invasion, surgical margin positivity, perirenal invasion, and capsule invasion due to very small number of cases. Although, higher RBX-1 expression levels were observed in patients with stage 3 to 4 compared with patients with stage 1 to 2, no statistical significant was found. Finally, the lacking data of the association RBX-1 with Cullin 2 in the subgroup analysis is another limitation of the current paper. However, in TGCA dataset analysis we could not find a direct relationship between rbx-1 expression and RCC.
5. Conclusion
In RCC, RBX-1 protein expression is closely associated with Fuhrman grade in particular, and the intensity of RBX-1 expression increases with increasing Fuhrman grade. In order for RBX-1 to particularly constitute a molecular marker and a parameter for estimating prognosis, our study should be supported with future studies performed with a larger number of cases in varied populations. Financial disclosures
All authors declare that they have no conflict of interest or financial ties to disclose.
Author contributions
Study concept and design: Altıntas, Kaynar, E. Celik, M. Celik.
Acquisition of data: Altıntas, Kaynar, E. Celik, M. Celik Analysis and interpretation of data: Altıntas, Kaynar, E. Celik,
Drafting of the manuscript: Altıntas, Kaynar, Akand, E. Celik, Kilic
Critical revision of the manuscript for important intellec-tual content: Altıntas, Kaynar, Akand, Kilic, Goktas,
Statistical analysis: Altıntas, Kaynar
Obtaining funding: This research was supported by Sel¸cuk University Scientific Research Projects Coordinator. (project number: 16102007)
Administrative, technical, or material support: Altıntas, Kaynar, E.Celik, Goktas, Akand, Kilic
Supervision: Kaynar Acknowledgments
None declared. Reference
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