Abstract
BackgroundA key feature of clear cell renal cell carcinoma (ccRCC) is the inactivation of the von Hippel-Lindau tumour suppressor protein (pVHL) that leads to the activation of hypoxia-inducible factor (HIF) pathway also in well-oxygenated conditions. Important regulator of HIF-α, prolyl hydroxylase PHD3, is expressed in high amounts in ccRCC. Although several functions and downstream targets for PHD3 in cancer have been suggested, the role of elevated PHD3 expression in ccRCC is not clear.MethodsTo gain insight into the functions of high PHD3 expression in ccRCC, we used PHD3 knockdown by siRNA in 786-O cells under normoxic and hypoxic conditions and performed discovery mass spectrometry (LC-MS/MS) of the purified peptide samples. The LC-MS/MS results were analysed by label-free quantification of proteome data using a peptide-level expression-change averaging procedure and subsequent gene ontology enrichment analysis.ResultsOur data reveals an intriguingly widespread effect of PHD3 knockdown with 91 significantly regulated proteins. Under hypoxia, the response to PHD3 silencing was wider than under normoxia illustrated by both the number of regulated proteins and by the range of protein expression levels. The main cellular functions regulated by PHD3 expression were glucose metabolism, protein translation and messenger RNA (mRNA) processing. PHD3 silencing led to downregulation of most glycolytic enzymes from glucose transport to lactate production supported by the reduction in extracellular acidification and lactate production and increase in cellular oxygen consumption rate. Moreover, upregulation of mRNA processing-related proteins and alteration in a number of ribosomal proteins was seen as a response to PHD3 silencing. Further studies on upstream effectors of the translational machinery revealed a possible role for PHD3 in regulation of mTOR pathway signalling.ConclusionsOur findings suggest crucial involvement of PHD3 in the maintenance of key cellular functions including glycolysis and protein synthesis in ccRCC.
Highlights
A key feature of clear cell renal cell carcinoma is the inactivation of the von Hippel-Lindau tumour suppressor protein that leads to the activation of hypoxia-inducible factor (HIF) pathway in well-oxygenated conditions
Experimental setup and the effect of PHD3 silencing on 786-O global proteome We undertook a task of characterizing the protein expression of clear cell renal cell carcinoma (ccRCC) cell line 786-O using proteome-based techniques with PHD3 depletion. 786-O cell line has a high basal PHD3 expression both in normoxia and hypoxia
Protein and messenger RNA (mRNA) expression efficiently. siPHD3#1 was selected to be used for further experiments as it is the most widely used and the best verified sequence for its PHD3 specificity in the literature
Summary
A key feature of clear cell renal cell carcinoma (ccRCC) is the inactivation of the von Hippel-Lindau tumour suppressor protein (pVHL) that leads to the activation of hypoxia-inducible factor (HIF) pathway in well-oxygenated conditions. Clear cell renal cell carcinoma (ccRCC), the most common subtype of human kidney carcinomas, is commonly associated with the inactivating mutations of VHL leading to a loss of function of the von Hippel‐Lindau tumour suppressor protein (pVHL). This leads to constant activation of hypoxia signalling pathway paving the way for malignant progression (reviewed in [1,2,3]). As compared to other cancer types, in ccRCC, the differential activation of target genes leads to contrasting effects on cancer progression, as high HIF-2α expression enhances tumorigenic activity, whereas high HIF-1α has tumour suppressive effect [11,12,13]
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