Oncogene (2019) 38:6881–6897 https://doi.org/10.1038/s41388-019-0938-8 ARTICLE Proteomics identifies neddylation as a potential therapy target in small intestinal neuroendocrine tumors 1 2 1,3 1,4 1,4 1 Omid Fotouhi ● Hanna Kjellin ● C. Christofer Juhlin ● Yanbo Pan ● Mattias Vesterlund ● Mehran Ghaderi ● 5 4 6 7 2,8 Abdelhamid Yousef ● Hillevi Andersson-Sand ● Pedram Kharaziha ● Stefano Caramuta ● Magnus Kjellman ● 2,8 1 1,4 Jan Zedenius ● Catharina Larsson ● Lukas M. Orre Received: 26 October 2018 / Revised: 30 April 2019 / Accepted: 15 May 2019 / Published online: 12 August 2019 © The Author(s) 2019. This article is published with open access Abstract Patients with small intestinal neuroendocrine tumors (SI-NETs) frequently develop spread disease; however, the underlying molecular mechanisms of disease progression are not known and effective preventive treatment strategies are lacking. Here, protein expression profiling was performed by HiRIEF-LC-MS in 14 primary SI-NETs from patients with and without liver metastases detected at the time of surgery and initial treatment. Among differentially expressed proteins, overexpression of the ubiquitin-like protein NEDD8 was identified in samples from patients with liver metastasis. Further, NEDD8 correlation 1234567890();,: 1234567890();,: analysis indicated co-expression with RBX1, a key component in cullin-RING ubiquitin ligases (CRLs). In vitro inhibition of neddylation with the therapeutic agent pevonedistat (MLN4924) resulted in a dramatic decrease of proliferation in SI- NET cell lines. Subsequent mass spectrometry-based proteomics analysis of pevonedistat effects and effects of the proteasome inhibitor bortezomib revealed stabilization of multiple targets of CRLs including p27, an established tumor suppressor in SI-NET. Silencing of NEDD8 and RBX1 using siRNA resulted in a stabilization of p27, suggesting that the cellular levels of NEDD8 and RBX1 affect CRL activity. Inhibition of CRL activity, by either NEDD8/RBX1 silencing or pevonedistat treatment of cells resulted in induction of apoptosis that could be partially rescued by siRNA-based silencing of p27. Differential expression of both p27 and NEDD8 was confirmed in a second cohort of SI-NET using immunohistochemistry. Collectively, these findings suggest a role for CRLs and the ubiquitin proteasome system in suppression of p27 in SI-NET, and inhibition of neddylation as a putative therapeutic strategy in SI-NET. Introduction Small intestinal neuroendocrine tumor (SI-NET) is a rare disease with an incidence of around 1 per 100,000, but still the most common small intestinal malignancy [1]. SI-NETs fi Supplementary information The online version of this article (https:// develop from enterochromaf n cells, are positive for neu- doi.org/10.1038/s41388-019-0938-8) contains supplementary roendocrine markers, and the diagnosis is based on material, which is available to authorized users. * Catharina Larsson 4 SciLifeLab, Stockholm, Sweden [email protected] 5 * Lukas M. Orre Department of Pharmacology, Cambridge University, [email protected] Cambridge, UK 6 Department of Clinical Genetics, Linköping University Hospital, 1 Department of Oncology-Pathology, Karolinska Institutet, Linköping, Sweden Stockholm, Sweden 7 Uppsala Clinical Research Center, Uppsala University, 2 Department of Molecular Medicine and Surgery, Karolinska Uppsala, Sweden Institutet, Stockholm, Sweden 8 Department of Breast, Endocrine Tumours and Sarcoma, 3 Department of Pathology and Cytology, Karolinska University Karolinska University Hospital Solna, Stockholm, Sweden Hospital Solna, Stockholm, Sweden 6882 O. Fotouhi et al. histopathological examination [2]. The patients typically Importantly, it has been shown that substrate recognition present with one or more small primary tumors, often and activity of CRLs (such as SCFSkp2) is strongly depen- together with mesenteric lymph node metastases, and later dent on neddylation of the Cullin subunit [13]. The ned- development of distant metastases frequently to the liver. dylation process is similar to ubiquitination and includes Although the tumors are commonly slow growing based on E1, E2, and E3 enzymes that catalyze conjugation of target a low Ki-67 proliferation index, the clinical picture suggests proteins with the ubiquitin-like protein NEDD8 (neural that tumor cell disconnection and spreading occur early in precursor cell expressed, developmentally downregulated 8) the tumor development [3]. [14]. Cullin neddylation results in a conformational change The primary treatment of SI-NET is surgical. In addition, of the CRL complex, eliminating the binding site for the the frequent expression of somatostatin receptors in SI- CRL inhibitor Cand1 and, at the same time, providing a NETs has led to the development and successful application more open structure favoring polyubiquitination [15]. of somatostatin analog treatment. Although this treatment Here we performed in-depth proteomics profiling of 14 may improve symptoms and also in some patients delay primary SI-NET tumors with focus on proteome-level tumor progression, additional therapeutic strategies need to differences between tumors from patients with and without be identified and developed for patients with metastatic liver metastasis. This clinical proteomics analysis impli- disease. cated differences in the ubiquitination machinery, with SI-NETs are characterized by a few recurrent genetic higher expression of NEDD8 and RBX1 in tumors from aberrations such as copy number loss of chromosome 18, patients with liver metastasis. To further investigate the which is seen in >70% of the cases [4]. The high frequency importance of these findings, inhibition of neddylation was of this event spawned a search for putative tumor supressors performed in SI-NET cell lines resulting in reduced pro- located on chromosome 18 in SI-NET, resulting in the liferation and induction of apoptosis. Proteomics profiling identification of DCC (deleted in colorectal cancer) as the so of the effects of neddylation inhibition in two SI-NET cell far best candidate [5]. Even though SI-NETs are in general lines resulted in identification of multiple upregulated mutationally quiet, the application of genome-wide proteins known to be substrates of CRLs including p27. sequencing identified CDKN1B as a recurrently mutated Our findings thus suggest deregulation of CRLs as well as gene in SI-NET with a frequency of 8% [6]. In addition, a an alternative route for CDKN1B/p27 inactivation in SI- broad molecular subtyping of SI-NET was recently reported NET with possible connections to metastatic spread and where genomic, epigenomic, and transcriptomic profiles therapy. were used to suggest three molecular subtypes of SI-NET with differences in progression-free survival [7]. However, the molecular background of SI-NET development, specific Results oncogenic drivers, and mechanisms of metastatic spread still remain largely unknown. In addition, no study so far Proteomics profiling of primary SI-NETs indicate has integrated proteome-level information in the analysis of NEDD8 and RBX1 overexpression in patients with SI-NET from primary tumors. liver metastases CDKN1B encodes p27, a bona-fide tumor suppressor that controls cell cycle entry through inhibition of cyclin/cyclin In order to investigate the proteomic landscape of SI-NET, dependent kinase (CDK) activity. It has been thoroughly and especially to identify proteins connected to disease demonstrated that the CDK inhibitor p27 is regulated pri- progression and metastatic spread, we performed in-depth marily by ubiquitination and subsequent proteasomal proteomics profiling of 14 primary SI-NETs of which 7 degradation [8], and that the ubiquitin E3-ligase responsible presented with liver metastases, while 7 were without any for the ubiquitination is the SCFSkp2 Cullin Ring Ligase detectable liver metastasis at the time of surgery (Supple- (CRL) complex [9]. The SCFSkp2 complex was later shown mentary Table S1). High-resolution isolectric focusing to include Skp1, Cullin-1, Rbx1, and Skp2 as the core liquid chromatography–tandem mass spectrometry (HiR- complex components [10]. Increased degradation of p27 in IEF-LC-MS)-based proteomics combined with relative cancer has also been firmly established beginning with a quantification of proteins between samples (iTRAQ-label- seminal study where increased p27-specific proteolytic ing) resulted in the identification and quantification of 6775 activity was coupled to low abundance of p27, which in turn proteins across all 14 SI-NET samples (Supplementary was demonstrated as a powerful negative prognostic factor Table S2). Unsupervised analysis using principal compo- in colorectal cancer [11]. An overwhelming abundance of nent analysis of the 14 samples revealed no major batch reports since then connects overexpression of Skp2 (the F- effects between the 2 separate quantitative proteomics box protein that recognize p27) with loss of p27 protein and experiments (2 iTRAQ 8-plex kits), but indicated 2 poten- poor prognosis in a long list of different cancer types [12]. tial outliers in the analysis (sample Screen-8 and -12; Proteomics identifies neddylation as a potential therapy target in small intestinal neuroendocrine tumors 6883 Supplementary Fig. S1a, b). Unsupervised Spearman’s correlation across the different cancer types was RBX1 (Fig. hierarchical clustering, however, did not indicate the two 1e, f). These results indicate a co-regulation of NEDD8 and samples as outliers. Instead, this clustering resulted