Identification of BAG3 target proteins in anaplastic thyroid cancer cells by proteomic analysis

BAG3 protein is an apoptosis inhibitor and is highly expressed in Anaplastic Thyroid Cancer. We investigated the entire set of proteins modulated by BAG3 silencing in the human anaplastic thyroid 8505C cancer cells by using the Stable-Isotope Labeling by Amino acids in Cell culture strategy combined with mass spectrometry analysis. By this approach we identified 37 up-regulated and 54 down-regulated proteins in BAG3-silenced cells. Many of these proteins are reportedly involved in tumor progression, invasiveness and resistance to therapies. We focused our attention on an oncogenic protein, CAV1, and a tumor suppressor protein, SERPINB2, that had not previously been reported to be modulated by BAG3. Their expression levels in BAG3-silenced cells were confirmed by qRT-PCR and western blot analyses, disclosing two novel targets of BAG3 pro-tumor activity. We also examined the dataset of proteins obtained by the quantitative proteomics analysis using two tools, Downstream Effect Analysis and Upstream Regulator Analysis of the Ingenuity Pathways Analysis software. Our analyses confirm the association of the proteome profile observed in BAG3-silenced cells with an increase in cell survival and a decrease in cell proliferation and invasion, and highlight the possible involvement of four tumor suppressor miRNAs and TP53/63 proteins in BAG3 activity.

[1]  Craig E. Higgins,et al.  Redox-Induced Src Kinase and Caveolin-1 Signaling in TGF-β1-Initiated SMAD2/3 Activation and PAI-1 Expression , 2011, PloS one.

[2]  M. Todaro,et al.  The antiapoptotic protein BAG3 is expressed in thyroid carcinomas and modulates apoptosis mediated by tumor necrosis factor-related apoptosis-inducing ligand. , 2007, The Journal of clinical endocrinology and metabolism.

[3]  C. Behl.  Breaking BAG: The Co-Chaperone BAG3 in Health and Disease. , 2016, Trends in pharmacological sciences.

[4]  M. Shah,et al.  Role of BRAF in Thyroid Oncogenesis , 2011, Clinical Cancer Research.

[5]  Yonglin Lu,et al.  HMGA2 regulates lung cancer proliferation and metastasis , 2017, Thoracic cancer.

[6]  A. Khar,et al.  Differential gene expression during apoptosis induced by a serum factor: Role of mitochondrial F0-F1 ATP synthase complex , 2005, Apoptosis.

[7]  S. Chi,et al.  Far upstream element-binding protein-1, a novel caspase substrate, acts as a cross-talker between apoptosis and the c-myc oncogene , 2009, Oncogene.

[8]  R. Franco,et al.  BAG3 protein is overexpressed in human glioblastoma and is a potential target for therapy. , 2011, The American journal of pathology.

[9]  G. T. Bowden,et al.  Differential Gene Expression during , 2016 .

[10]  Li-Jun Bi,et al.  Bcl2-associated Athanogene 3 Interactome Analysis Reveals a New Role in Modulating Proteasome Activity* , 2013, Molecular & Cellular Proteomics.

[11]  X. Meng,et al.  Induction of BAG2 protein during proteasome inhibitor‐induced apoptosis in thyroid carcinoma cells , 2008, British journal of pharmacology.

[12]  F. Sotgia,et al.  Caveolae and signalling in cancer , 2015, Nature Reviews Cancer.

[13]  Anwar Hossain,et al.  Mir-17-5p Regulates Breast Cancer Cell Proliferation by Inhibiting Translation of AIB1 mRNA , 2006, Molecular and Cellular Biology.

[14]  Akhilesh Pandey,et al.  Quantitative proteomics using stable isotope labeling with amino acids in cell culture , 2008, Nature Protocols.

[15]  Hua-Qin Wang,et al.  BAG3 regulates ECM accumulation in renal proximal tubular cells induced by TGF-β1. , 2015, American journal of translational research.

[16]  M. Mann,et al.  A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC) , 2006, Nature Protocols.

[17]  Hu Li,et al.  Hsp70-Bag3 interactions regulate cancer-related signaling networks. , 2014, Cancer research.

[18]  K. Itahana,et al.  Mitochondrial p32 is a critical mediator of ARF-induced apoptosis. , 2008, Cancer cell.

[19]  J. Koo,et al.  Expression of Caveolin-1, Caveolin-2 and Caveolin-3 in Thyroid Cancer and Stroma , 2012, Pathobiology.

[20]  M. Yutsudo,et al.  Multi-functional gene ASY/Nogo/RTN-X/RTN4: Apoptosis, tumor suppression, and inhibition of neuronal regeneration , 2004, Apoptosis.

[21]  Xiaoli Liu,et al.  Targeting TGF-β1 inhibits invasion of anaplastic thyroid carcinoma cell through SMAD2-dependent S100A4-MMP-2/9 signalling. , 2016, American journal of translational research.

[22]  X. Xu,et al.  MicroRNA-133a functions as a tumor suppressor in gastric cancer. , 2014, Journal of biological regulators and homeostatic agents.

[23]  G. Botti,et al.  BAG3 down-modulation reduces anaplastic thyroid tumor growth by enhancing proteasome-mediated degradation of BRAF protein. , 2012, The Journal of clinical endocrinology and metabolism.

[24]  M. Lai,et al.  Effect of silencing of high mobility group A2 gene on gastric cancer MKN-45 cells. , 2013, World journal of gastroenterology.

[25]  V. de Laurenzi,et al.  BAG3: a multifaceted protein that regulates major cell pathways , 2011, Cell Death and Disease.

[26]  Linfu Xie,et al.  MicroRNA-124-3p inhibits cell migration and invasion in bladder cancer cells by targeting ROCK1 , 2013, Journal of Translational Medicine.

[27]  B. Baradaran,et al.  HMGI-C suppressing induces P53/caspase9 axis to regulate apoptosis in breast adenocarcinoma cells , 2016, Cell cycle.

[28]  A. Ribechini,et al.  Anaplastic thyroid carcinoma: from clinicopathology to genetics and advanced therapies , 2017, Nature Reviews Endocrinology.

[29]  Cao Huang,et al.  Pathogenic mutation of UBQLN2 impairs its interaction with UBXD8 and disrupts endoplasmic reticulum‐associated protein degradation , 2014, Journal of neurochemistry.

[30]  G. Chiappetta,et al.  IKKγ protein is a target of BAG3 regulatory activity in human tumor growth , 2010, Proceedings of the National Academy of Sciences.

[31]  David R. Croucher,et al.  Revisiting the biological roles of PAI2 (SERPINB2) in cancer , 2008, Nature Reviews Cancer.

[32]  Y. Nikiforov,et al.  American Thyroid Association guidelines for management of patients with anaplastic thyroid cancer. , 2012, Thyroid : official journal of the American Thyroid Association.

[33]  S. Grimm,et al.  Adenine Nucleotide Translocase-1, a Component of the Permeability Transition Pore, Can Dominantly Induce Apoptosis , 1999, The Journal of cell biology.

[34]  G. Melino,et al.  How the TP53 Family Proteins TP63 and TP73 Contribute to Tumorigenesis: Regulators and Effectors , 2014, Human mutation.

[35]  M. Walter,et al.  Knockdown of HSPA9 induces TP53-dependent apoptosis in human hematopoietic progenitor cells , 2017, PloS one.

[36]  K. Thiel,et al.  Knockdown of MTDH Sensitizes Endometrial Cancer Cells to Cell Death Induction by Death Receptor Ligand TRAIL and HDAC Inhibitor LBH589 Co-Treatment , 2011, PloS one.

[37]  L. Pfeffer,et al.  miR-203 Functions as a Tumor Suppressor by Inhibiting Epithelial to Mesenchymal Transition in Ovarian Cancer. , 2015, Journal of cancer science & therapy.

[38]  E. White,et al.  Btf, a Novel Death-Promoting Transcriptional Repressor That Interacts with Bcl-2-Related Proteins , 1999, Molecular and Cellular Biology.

[39]  Mei Xu,et al.  Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors , 2015, Molecular Cancer Therapeutics.

[40]  Soo-Jin Jeong,et al.  Down-regulation of phosphoglucomutase 3 mediates sulforaphane-induced cell death in LNCaP prostate cancer cells , 2010, Proteome Science.