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 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.
Highlights
Supplementary information The online version of this article contains supplementary material, which is available to authorized users.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]
In order to investigate the proteomic landscape of SI-NET, and especially to identify proteins connected to disease progression and metastatic spread, we performed in-depth proteomics profiling of 14 primary SI-NETs of which 7 presented with liver metastases, while 7 were without any detectable liver metastasis at the time of surgery (Supplementary Table S1)
A NEDD8 correlation analysis revealed that one of the genes with the highest NEDD8 correlation across the different cancer types was RBX1 (Fig. 1e, f). These results indicate a co-regulation of NEDD8 and RBX1 at the transcriptional level, underscoring the functional association between these proteins and suggesting that some cancer types rely more on processes that are dependent on cullin-RING ubiquitin ligases (CRLs) activity for protein degradation
Summary
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 develop from enterochromaffin cells, are positive for neuroendocrine markers, and the diagnosis is based on Cambridge, UK 6 Department of Clinical Genetics, Linköping University Hospital, Linköping, Sweden 7 Uppsala Clinical Research Center, Uppsala University, Uppsala, Sweden 8 Department of Breast, Endocrine Tumours and Sarcoma, Karolinska University Hospital Solna, Stockholm, Sweden histopathological examination [2]. The frequent expression of somatostatin receptors in SINETs has led to the development and successful application of somatostatin analog treatment. This treatment may improve symptoms and in some patients delay tumor progression, additional therapeutic strategies need to be identified and developed for patients with metastatic disease
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