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Transcriptome and Proteome Analyses of TNFAIP8 Knockdown Cancer Cells Reveal New Insights into Molecular Determinants of Cell Survival and Tumor Progression

  • Timothy F. Day
  • Rajshree R. Mewani
  • Joshua Starr
  • Xin Li
  • Debyani Chakravarty
  • Habtom Ressom
  • Xiaojun Zou
  • Ofer Eidelman
  • Harvey B. Pollard
  • Meera Srivastava
  • Usha N. KasidEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1513)

Abstract

Tumor necrosis factor-α-inducible protein 8 (TNFAIP8) is the first discovered oncogenic and an anti-apoptotic member of a conserved TNFAIP8 or TIPE family of proteins. TNFAIP8 mRNA is induced by NF-kB, and overexpression of TNFAIP8 has been correlated with poor prognosis in many cancers. Downregulation of TNFAIP8 expression has been associated with decreased pulmonary colonization of human tumor cells, and enhanced sensitivities of tumor xenografts to radiation and docetaxel. Here we have investigated the effects of depletion of TNFAIP8 on the mRNA, microRNA and protein expression profiles in prostate and breast cancers and melanoma. Depending on the tumor cell type, knockdown of TNFAIP8 was found to be associated with increased mRNA expression of several antiproliferative and apoptotic genes (e.g., IL-24, FAT3, LPHN2, EPHA3) and fatty acid oxidation gene ACADL, and decreased mRNA levels of oncogenes (e.g., NFAT5, MALAT1, MET, FOXA1, KRAS, S100P, OSTF1) and glutamate transporter gene SLC1A1. TNFAIP8 knockdown cells also exhibited decreased expression of multiple onco-proteins (e.g., PIK3CA, SRC, EGFR, IL5, ABL1, GAP43), and increased expression of the orphan nuclear receptor NR4A1 and alpha 1 adaptin subunit of the adaptor-related protein complex 2 AP2 critical to clathrin-mediated endocytosis. TNFAIP8-centric molecules were found to be predominately implicated in the hypoxia-inducible factor-1α (HIF-1α) signaling pathway, and cancer and development signaling networks. Thus TNFAIP8 seems to regulate the cell survival and cancer progression processes in a multifaceted manner. Future validation of the molecules identified in this study is likely to lead to new subset of molecules and functional determinants of cancer cell survival and progression.

Key words

TNFAIP8 shRNA and siRNA RNA and microRNA arrays Antibody arrays Cell survival and proliferation Invasion and metastasis Cancer systems biology 

Notes

Acknowledgements

Timothy F. Day, Rajshree R. Mewani, and Joshua Starr contributed equally to this work. This work was supported by grants from the National Institutes of Health (CA68322, CA74175) and Department of Defense (PC074171). TFD was supported, in part, by predoctoral fellowship award from the Department of Defense (W81XWH-10-1-0107). LM2-4175 cells were a gift from Dr. Joan Massagué. Portions of the work presented in this chapter were carried out by TFD and DC toward their Ph.D. dissertations. UNK is a coinventor on patent application, “Anti-apoptotic gene SCC-S2 and diagnostic and therapeutic uses thereof,” (US 12/858,360). Several cell lines were obtained from the Tissue Culture Shared Resource of the Georgetown Lombardi Comprehensive Cancer Center. The RNA array and microRNA array studies were performed using the Genomics and Epigenomics Shared Resource of the Georgetown Lombardi Comprehensive Cancer Center. All shared resources were supported by the NIH Grant P30-CA51008.

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Timothy F. Day
    • 1
  • Rajshree R. Mewani
    • 1
  • Joshua Starr
    • 2
  • Xin Li
    • 1
  • Debyani Chakravarty
    • 1
  • Habtom Ressom
    • 1
  • Xiaojun Zou
    • 1
  • Ofer Eidelman
    • 2
  • Harvey B. Pollard
    • 2
  • Meera Srivastava
    • 2
  • Usha N. Kasid
    • 1
    Email author
  1. 1.Georgetown-Lombardi Comprehensive Cancer CenterGeorgetown University Medical CenterWashingtonUSA
  2. 2.Department of Anatomy, Physiology and Genetics, Institute for Molecular Medicine, Center for Medical ProteomicsUniformed Services University School of MedicineBethesdaUSA

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