Original article
Receptor activator of NF-κB (RANK)-mediated induction of metastatic spread and association with poor prognosis in renal cell carcinoma

https://doi.org/10.1016/j.urolonc.2018.07.013Get rights and content

Highlights

  • Receptor activator of NFκB Ligand (RANK) and its receptor RANK expression in primary clear cell renal cell carcinoma (ccRCC) tissue is associated advanced clinicopathological features of ccRCC patients.

  • RANKL expression in primary ccRCC tissue is associated with short cancer specific survival (CSS) while RANK expression is associated with both short recurrence free survival (RFS) and CSS.

  • RANK expression is an independent prognostic factor for RFS and CSS in multivariable analyses.

  • Treating RANK positive ccRCC cells with RANKL results in increased proliferation, soft agar growth, and colony formation which can be reversed with an NFκB inhibitor and with a combination of osteoprotegerin (OPG) and RANKL in vitro.

  • RANKL inhibition combined with standard of care treatment may be a promising approach to improve ccRCC patients’ survival.

Abstract

Background

Inhibition of the receptor activator of NF-κB ligand (RANKL) has become a standard of care supportive treatment to prevent skeletal related events in cancer patients. Moreover, RANKL inhibition has been implicated with better survival outcome in lung cancer, while RANKL expression induces tumor progression and metastatic spread in vivo in breast cancer. Whether RANK/RANKL may have an impact on the pathogenesis of clear cell renal cell carcinoma (ccRCC) is currently unknown.

Patients and Methods

A retrospective tissue micro array (TMA)-study was carried out determining the expression of RANK/RANKL in primary tumors of 306 ccRCC patients. Additionally, 24 ccRCC cell lines were employed for in vitro analyses of the RANK/RANKL axis including cell proliferation, migration and anchorage independent growth.

Results

RANK (+) vs. RANK (−) tumors had both worse cancer specific survival (CSS) (6.3 vs. 1.3 years; p < 0.001) and recurrence free survival (RFS) (9.9 vs. 5.8 years; p < 0.001). RANK (+) (HR 2.21; p < 0.001) was an independent prognostic factor for CSS and RFS (HR 4.98; p < 0.001). RANKL treatment resulted in increased proliferation, soft agar growth, and colony formation of RANK (+) RCC cell lines, which could be reversed by treatment with an NF-κB inhibitor and with a combination of osteoprotegrin and RANKL in vitro.

Conclusions

RANK is expressed in ccRCC tissue, correlates with clinicopathological features, survival outcome, and when stimulated with RANKL can induce ccRCC progression in vitro. Consequently, RANKL inhibition combined with standard of care treatment may be a promising approach to improve ccRCC patient's survival.

Introduction

According to histological criteria, renal cell carcinoma (RCC) can be classified into 3 major subtypes: clear cell renal cell carcinoma (ccRCC), papillary RCC, and chromophobe RCC [1]. The most frequent subtype is ccRCC, accounting for approximately 75% of all RCC cases [1]. Metastatic renal cell carcinoma (mRCC) is a chemo- and irradiation-resistant tumor with a historical median overall survival (OS) of RCC patients of approximately 10 months [2]. Despite the recent success of new treatment strategies with targeted therapies and specific immunotherapies in the treatment of mRCC, many tumors are resistant to current standard of care agents and only a few patients can be durably cured with standard of care therapies [3], [4], [5]. For example, the Checkmate 214 study investigated the treatment with Ipilimumab/Nivolumab vs. Sunitinib, has demonstrated that 20% of patients in the intermediate and high-risk groups according to the International mRCC Database Consortium criteria demonstrate no response to this checkpoint inhibitor therapy [6]. Furthermore, 17% and 19% demonstrated primary resistance to Sunitinib and Pazopanib, respectively, in the Comparz trial that investigated the noninferiority of Pazopanib vs. Sunitinib [7]. Ipilimumab/Nivolumab is the new standard of care treatment for intermediate and high-risk mRCC patients while Sunitinib and Pazopanib still the standard treatments in mRCC patients with a favorable risk profile according to International metastatic renal cell carcinoma Database Consortium criteria [8]. Thus, there is still a substantial number of patients with primary treatment resistance to standard of care therapies and thus, there is an urgent need for the identification and development of novel alternative therapies that improve the outcome of RCC patients.

The receptor activator of NF-κB ligand (RANKL) and its receptor receptor activator of NF-Κb (RANK) have been characterized as a central regulator of osteoclastogenesis and lymphogenesis [9]. The third important member in the RANKL/RANK axis is osteoprotegrin (OPG), a natural decoy receptor for RANKL, which blocks the RANK/RANKL interaction and RANKL-induced signaling pathways [10]. Next to its physiological role in the development of the lymphatic system and bone turnover, RANK/RANKL may play a key role in oncogenic processes [11], [12]. In breast cancer, RANKL can initiate tumor formation and metastatic spread via its receptor RANK [10], [13], [14], [15] In addition, inhibition of RANKL has been shown to prolong survival of lung cancer patients [16].

RCC is frequently associated with the development of bone metastases, which further correlates with a poor prognosis of RCC patients treated with either immunotherapy or targeted therapies [17], [18]. Thus, it seems plausible that the RANKL/RANK axis may be involved in the development of (bone) metastases and progression in this disease. Two studies have associated RANKL and a RANK/OPG ratio with advanced tumor stages, shorter progression free survival, OS, and time to metastatic spread in ccRCC [18], [19]. However, to the best of our knowledge a comprehensive analysis of a large patient cohort investigating RANK/RANKL protein expression combined with functional analyses has not yet been published in RCC.

Therefore, the current study aims to provide both a correlation of RANKL/RANK expression with clinicopathological features and survival outcome as well as broad mechanistic in vitro studies regarding the effects of RANKL, OPG, and a NF-κB pathway inhibitor on proliferation, migration, and angiogenesis of RANK (+) vs. RANK (−) RCC cell lines.

Section snippets

Tissue micro array (TMA) analysis

The study cohort was comprised of 306 ccRCC patients who were surgically treated at the University of California Los Angeles (UCLA) between 1989 and 2000. Clinical and pathologic data were retrospectively gathered from the UCLA kidney cancer database and electronic charts. The study was approved from the UCLA institutional review board (UCLA IRB #02-355). Clinicopathological data included age, gender, Eastern Cooperative Oncology Group performance status [20], TNM stages (seventh edition of

RANK and RANKL expression on the TMA in correlation to clinicopathological features of ccRCC patients

RANKL expression was found in 18/306 (6%), and RANK expression in 53/306 (17%) lesions of ccRCC patients (Supplementary Figure 1A). As shown in Table 1, ccRCC patients with RANKL (+) had more advanced Fuhrman grades (P = 0.003), while the comparison of all other clinicopathological features did not demonstrate any differences between RANKL (+) and RANKL (−) ccRCC. In contrast, patients with RANK (+) ccRCC lesions more often had distant metastases (P = 0.009), more advanced ECOG PS (P = 0.020),

Discussion

Previous studies have demonstrated that RANK expressing breast, lung, and prostate cancer cells as well as melanoma cells, exhibit an increased migration and invasion [10]. Additionally, this finding was paralleled by increased expression of proteins that are involved in invasion and migration, such as VEGF, the matrix metallo proteinase MMP9 and several members of the CXCL family [9], [10], [27]. These results were confirmed by our study demonstrating a higher proliferation and migration

Acknowledgments

The UCLA tissue micro array (UCLA TMA) was initially constructed by David Seligson, MD and Jonathan Said, MD. All tumors on the TMA had been pathologically re-evaluated before inclusion.

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      As a corollary, the K5:RANKL transcriptomic dataset is also closely related to the human kidney renal clear cell carcinoma signature (Fig. 5A (KIRC)). This finding is interesting as RANKL/RANK signaling has recently been linked to poor prognosis in patients diagnosed with renal clear cell carcinoma [61] and supports the K5:RANKL transcriptomic signature as a powerful informational resource with which to molecularly understand RANKL-dependent cancers outside the head and neck cancer group. In addition to RNA expression, we validated at the protein level the upregulation of three of these genes (PTHRP; POSTN; and SPP1) in the K5:RANKL salivary gland tumor.

    Trial registration: This is a non-interventional retrospective study. The study has been approved by the local UCLA IRB (protocol #02-355).

    Funding: The current study was supported with material and financial support by AMGEN™; Dr. Brandstetter and Dr. Dougall were employees of AMGEN when the study was performed. In addition, the study was supported by the Deutsche Krebshilfe project (BS). All other authors declare no conflict of interest in regard to the current study.

    Authors contributions:

    André Steven: Conduction of in vitro experiments, design of experiments, data analysis, wrote and approved the manuscript.

    Sandra Leisz: Conduction of in vitro experiments, design of experiments, data analysis, wrote and approved the manuscript.

    Sebastian Fussek: Data acquisition, data analyses, approval of the manuscript.

    Behdokht Nowroozizadeh: Data analysis, evaluated the tissue micro array, approval of the manuscript.

    Jiaoti Huang: Data analysis, evaluated the tissue micro array, approval of the manuscript.

    Daniel Brandstetter: Material support; optimized and conducted the staining of the TMA, approval of the manuscript.

    Bill C. Dougall: Material support, design of the experiments, staining of the array, approval of the manuscript.

    Martin Burchardt: Data analysis, material support, approval of the manuscript.

    Arie S. Belldegrun: Data analysis, material support, approval of the manuscript.

    Barbara Seliger: Designed the in vitro experiments; data analysis and interpretation, wrote and approved the manuscript.

    Allan Pantuck: Designed the TMA study; data analysis and interpretation, wrote the manuscript and approved the manuscript.

    Nils Kroeger: Designed the TMA study; wrote the manuscript, data analysis and interpretation, wrote and approved the manuscript.

    1

    Nils Kroeger, Barbara Seliger, and Allan Pantuck are co-contributing senior authors to this work.

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