Integration of quantitative phosphoproteomics and transcriptomics revealed phosphorylation-mediated molecular events as useful tools for a potential patient stratification and personalized treatment of human nonfunctional pituitary adenomas

Abstract

Background

Invasiveness is a very challenging clinical problem in nonfunctional pituitary adenomas (NFPAs), and currently, there are no effective invasiveness-related molecular biomarkers. The post-neurosurgery treatment is much different as for invasive and noninvasive NFPAs. The aim of this study was to integrate phosphoproteomics and transcriptomics data to reveal phosphorylation-mediated molecular events for invasive characteristics of NFPAs to achieve a potential tool for patient stratification, and prognostic/predictive assessment to discriminate invasive from noninvasive NFPAs for personalized attitude.

Methods

The 6-plex tandem mass tag (TMT) labeling reagents coupled with TiO2 enrichment of phosphopeptides and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to identify and quantify each phosphoprotein and phosphosite in NFPAs and controls. Differentially expressed genes (DEGs) between invasive NFPA and control tissues were obtained from the Gene Expression Omnibus (GEO) database. The overlapping analysis was performed between phosphoprotiens and invasive DEGs. Gene Ontology (GO) enrichment, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and protein–protein interaction (PPI) analyses were used to analyze these overlapped molecules.

Results

In total, 1035 phosphoproteins with 2982 phosphorylation sites were identified in NFPAs vs. controls, and 2751 DEGs were identified in invasive NFPAs vs. controls. Overlapping analysis of these phosphoproteins and DEGs exposed 130 overlapped molecules (phosphoproteins; invasive DEGs). GO enrichment and KEGG pathway analyses of 130 overlapped molecules revealed multiple biological processes and signaling pathway network alterations, including cell–cell adhesion, platelet activation, GTPase signaling pathway, protein kinase signaling, calcium signaling pathway, estrogen signaling pathway, glucagon signaling pathway, cGMP–PKG signaling pathway, GnRH signaling pathway, inflammatory mediator regulation of TRP channels, vascular smooth muscle contraction, and Fc gamma R-mediated phagocytosis, which were obviously associated with tumor invasive characteristics. For 130 overlapped molecules, PPI network-based molecular complex detection (MCODE) identified 10 hub molecules, namely SLC2A4, TSC2, AKT1, SCG3, ALB, APOL1, ACACA, SPARCL1, CHGB, and IGFBP5. These hub molecules are involved in multiple signaling pathways and represent potential predictive/prognostic markers in NFPA patients as well as they represent potential therapeutic targets.

Conclusions

This study provided the first large-scale phosphoprotein profiling and phosphorylation-related signaling pathway network alterations in human NFPA tissues. Further, overlapping analysis of phosphoproteins and invasive DEGs revealed the phosphorylation-mediated signaling pathway network changes in invasive NFPAs. These findings are the precious resource for in-depth insight into the molecular mechanisms of NFPAs, as well as for the discovery of effective phosphoprotein biomarkers and therapeutic targets for invasive NFPAs.

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Abbreviations

ACACA:

acetyl-CoA carboxylase 1

ACN:

acetonitrile

AGC:

automatic gain control

AKT:

protein kinase B

AKT1:

RAC-alpha serine/threonine-protein kinase

ALB:

serum albumin

AMPK:

AMP-activated protein kinase

APOL1:

apolipoprotein L1

ATF2:

activating transcription factor 2

BP:

biological process

CC:

cellular component

cGMP:

cyclic nucleotide cGMP

CHGB:

secretogranin-1

DEG:

differentially expressed gene

DEP:

differentially expressed protein

DTT:

dithiothreitol

r-ERG channel:

rat ether-à-go-go-related (ERG) channel

ERK:

extracellular signal–regulated kinase

ESCRT:

endosomal sorting complex required for transport

ESI-TRAP:

electrospray ionization-ion trap

FC:

fold change

FDR:

false discovery rate

FGF-2:

fibroblast growth factor-2

FPA:

functional pituitary adenoma

GEO:

Gene Expression Omnibus

GH:

growth hormone

GH3:

pituitary growth hormone 3

GnRH:

gonadotropin-releasing hormone

GO:

Gene Ontology

HCD:

high-energy collision dissociation

HIF-1a:

hypoxia-inducible factor-1a

HPLC:

high-performance liquid chromatography

IGF-1:

insulin growth factor-1

IGFBP5:

insulin-like growth factor-binding protein 5

KEGG:

Kyoto Encyclopedia of Genes and Genomes

LC:

liquid chromatography

MAPK:

mitogen-activated protein kinase

MCODE:

molecular complex detection

MF:

molecular function

MMPs:

matrix metalloproteinases

MS/MS:

tandem mass spectrometry

mTOR:

mammalian target of rapamycin

NCBI:

National Center for Biotechnology Information

NFκB:

nuclear factor kappa-B

NFPA:

nonfunctional pituitary adenoma

NMR:

nuclear magnetic resonance

PACAP:

pituitary adenylyl cyclase activating polypeptide

PI3K:

phosphatidylinositol 3 kinase

PKG:

protein kinase G

PPI:

protein–protein interaction

PTM:

post-translational modification

PTTG:

pituitary tumor-transforming gene

RSK:

ribosomal S6 kinase

SCG3:

secretogranin-3

Ser or S:

serine

SLC2A4:

solute carrier family 2 facilitated glucose transporter member 4

S/N:

signal-to-noise

SNAP:

soluble N-ethylmaleimide-sensitive fusion attachment protein

SNARE:

soluble N-ethylmaleimide-sensitive factor attachment protein receptor

SPARCL1:

SPARC-like protein 1

TFA:

trifluoroacetic acid

Thr or T:

threonine

TMT:

tandem mass tag

TSC2:

Tuberin

TRH:

thyrotropin-releasing hormone

Tyr or Y:

tyrosine

VEGF:

vascular endothelial growth factor

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Acknowledgments

The authors also acknowledge Professor Xuejun Li from Xiangya Hospital and Professor Dominic M. Desiderio from University of Tennessee Health Science Center to assist in obtaining human tissues.

Funding

This work was supported by the Shandong First Medical University Talent Introduction Funds (to X.Z.), the Hunan Provincial Hundred Talent Plan (to X.Z.), the SCIBP supported project (No. SCIBP2019090006), and China “863” Plan Project (Grant No. 2014AA020610-1 to XZ).

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Authors

Contributions

D.L. analyzed data, prepared figures and tables, and wrote the manuscript draft. J.J., N.L., M.L., and S.W. participated in partial data analysis and bioinformatics. X.Z. conceived the concept, designed and instructed the experiments, analyzed the data, obtained the phosphoproteomic data, supervised the results, coordinated, wrote and critically revised the manuscript, and was responsible for its financial supports and the corresponding works. All authors approved the final manuscript.

Corresponding author

Correspondence to Xianquan Zhan.

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Competing interests

The authors declare that they have no conflict of interest.

Ethical approval

Four pituitary adenoma tissue samples, obtained from the Department of Neurosurgery of Xiangya Hospital, Central South University, were approved by the Xiangya Hospital Medical Ethics Committee of Central South University. Post-mortem control pituitary tissue samples, obtained from the Memphis Regional Medical Center (n = 5), were approved by the University of Tennessee Health Science Center Internal Review Board.

Additional information

Abbreviations for all particular genes and proteins can be found in the Supplemental Table 1 and the UniProtKB database at the following link: https://www.expasy.org/.

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Liu, D., Li, J., Li, N. et al. Integration of quantitative phosphoproteomics and transcriptomics revealed phosphorylation-mediated molecular events as useful tools for a potential patient stratification and personalized treatment of human nonfunctional pituitary adenomas. EPMA Journal 11, 419–467 (2020). https://doi.org/10.1007/s13167-020-00215-0

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Keywords

  • Nonfunctional pituitary adenomas
  • Invasiveness
  • Tandem mass tag (TMT) labeling
  • TiO2 enrichment
  • Quantitative phosphoproteomics
  • Transcriptomics
  • Differentially expressed genes
  • Overlapped molecule (phosphoprotein
  • invasive DEG)
  • Signaling pathway
  • Patient stratification
  • Prognostic/predictive assessment
  • Personalized treatment