Transgelin Contributes to a Poor Response of Metastatic Renal Cell Carcinoma to Sunitinib Treatment

Abstract

Renal cell carcinoma (RCC) represents about 2–3% of all cancers with over 400,000 new cases per year. Sunitinib, a vascular endothelial growth factor tyrosine kinase receptor inhibitor, has been used mainly for first-line treatment of metastatic clear-cell RCC with good or intermediate prognosis. However, about one-third of metastatic RCC patients do not respond to sunitinib, leading to disease progression. Here, we aim to find and characterize proteins associated with poor sunitinib response in a pilot proteomics study. Sixteen RCC tumors from patients responding (8) vs. non-responding (8) to sunitinib 3 months after treatment initiation were analyzed using data-independent acquisition mass spectrometry, together with their adjacent non-cancerous tissues. Proteomics analysis quantified 1996 protein groups (FDR = 0.01) and revealed 27 proteins deregulated between tumors non-responding vs. responding to sunitinib, representing a pattern of deregulated proteins potentially contributing to sunitinib resistance. Gene set enrichment analysis showed an up-regulation of epithelial-to-mesenchymal transition with transgelin as one of the most significantly abundant proteins. Transgelin expression was silenced by CRISPR/Cas9 and RNA interference, and the cells with reduced transgelin level exhibited significantly slower proliferation. Our data indicate that transgelin is an essential protein supporting RCC cell proliferation, which could contribute to intrinsic sunitinib resistance.

A large‐scale assay library for targeted protein quantification in renal cell carcinoma tissues

“…To provide a more comprehensive library of targeted proteomics assays, we fractionated peptides from a large set of RCC tissues and adjacent non-tumor samples, analyzed the fractions on QExactive HF-X mass spectrometer and constructed a new library of targeted proteomics assays for LC-MS/MS analysis of RCC tissue samples in DIA or SRM mode. Moreover, we demonstrate applicability of the library on two previously published [16,17] and two newly generated RCC datasets, showing increased number of quantified peptides and proteins, depending on the size of the library and LC-MS/MS instrumentation.…”

“…Only slightly increased number of protein ID is given by originally used instrumentation and highlights a correct FDR control in the dataset by Spectronaut software that did not lead to false positive identifications using a larger library. Another public dataset, dataset B [17], included samples from 16 RCC tumors and 16 adjacent non‐cancerous tissues. FASP‐digested samples were measured on Impact II qTOF mass spectrometer (Bruker Daltonics).…”

“…The number of quantified peptides and proteins groups increased more than twice from 11,536 to 25,687 peptides and from 1996 to 4492 protein groups (FDR = 1%) in dataset B using the new fractionated library compared to previous unfractionated sample pools (Data File S1B). Dataset C contained the same 16 RCC tumor samples from previous study (dataset B [17]), of which eight responded and eight did not respond to sunitinib treatment, that were remeasured on QExactive HF‐X LC‐MS/MS system (Thermo Fisher Scientific) (Supplementary Methods, Data File S2). Data analysis led to quantification of 32,794 peptides and 5181 protein groups (FDR = 1%), further increasing proteome coverage of RCC tissue at protein level for about 15% using a Orbitrap‐based MS instrumentation with higher resolution compared to dataset B (Data File S1C).…”

“…The number of quantified peptides and proteins groups increased more than twice from 11,536 to 25,687 peptides and from 1996 to 4492 protein groups (FDR = 1%) in dataset B using the new fractionated library compared to previous unfractionated sample pools (Data File S1B). Dataset C contained the same 16 RCC tumor samples from previous study (dataset B [17]), of which eight responded and eight did not respond to sunitinib treatment, that were remeasured on QExactive HF-X LC-MS/MS system (Thermo Fisher Scientific) (Supplementary Methods, Data File S2).…”

Catechol-O-methyl transferase suppresses cell invasion and interplays with MET signaling in estrogen dependent breast cancer

“…Samples were mixed in a thermomixer (600 rpm, 1 min, 37 °C) and cleaved overnight (37 °C, without shaking). On the next day, the peptides were eluted by centrifugation www.nature.com/scientificreports/ (14,000 g, 30 min, 20 °C) prior to desalting on MicroSpin C18 columns (The Nest Group, Inc., USA) as previously described 15 . The eluates were dried in SpeedVac (Thermo Fisher) and stored at − 20 °C.…”