The effects of IFITM1 and IFITM3 gene deletion on IFNγ stimulated protein synthesis

• Published in: Cellular signalling Vol. 60; pp. 39 - 56
• Main Authors: Gómez-Herranz, Maria, Nekulova, Marta, Faktor, Jakub, Hernychova, Lenka, Kote, Sachin, Sinclair, Elizabeth H., Nenutil, Rudolf, Vojtesek, Borivoj, Ball, Kathryn L., Hupp, Ted R.
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01.08.2019; Published by Elsevier Inc
• Subjects: Antigens, Differentiation - physiology; CAS9 gene editing; Cell Line; Cervical cancer; Female; Histocompatibility Antigens Class I - metabolism; Humans; IFITM1; Interferon; Membrane Proteins - physiology; MHC Class I molecule; Protein Biosynthesis - physiology; RNA-Binding Proteins - physiology; SILAC mass spectrometry; Uterine Cervical Neoplasms - metabolism; PBS; SBP; RT; IFITM1; PLA; MS; SWATH-IP; MHC; ISG15; NMWCO; CAS9 gene editing; IFITM1/3; IFN; UPLC; IRF1; FASP; Interferon; SILAC mass spectrometry; FA; MHC Class I molecule; HLA; B2M; Cervical cancer
• ISSN: 0898-6568; 1873-3913
• DOI: 10.1016/j.cellsig.2019.03.024

Interferon-induced transmembrane proteins IFITM1 and IFITM3 (IFITM1/3) play a role in both RNA viral restriction and in human cancer progression. Using immunohistochemical staining of FFPE tissue, we identified subgroups of cervical cancer patients where IFITM1/3 protein expression is inversely related to metastasis. Guide RNA-CAS9 methods were used to develop an isogenic IFITM1/IFITM3 double null cervical cancer model in order to define dominant pathways triggered by presence or absence of IFITM1/3 signalling. A pulse SILAC methodology identified IRF1, HLA-B, and ISG15 as the most dominating IFNγ inducible proteins whose synthesis was attenuated in the IFITM1/IFITM3 double-null cells. Conversely, SWATH-IP mass spectrometry of ectopically expressed SBP-tagged IFITM1 identified ISG15 and HLA-B as dominant co-associated proteins. ISG15ylation was attenuated in IFNγ treated IFITM1/IFITM3 double-null cells. Proximity ligation assays indicated that HLA-B can interact with IFITM1/3 proteins in parental SiHa cells. Cell surface expression of HLA-B was attenuated in IFNγ treated IFITM1/IFITM3 double-null cells. SWATH-MS proteomic screens in cells treated with IFITM1-targeted siRNA cells resulted in the attenuation of an interferon regulated protein subpopulation including MHC Class I molecules as well as IFITM3, STAT1, B2M, and ISG15. These data have implications for the function of IFITM1/3 in mediating IFNγ stimulated protein synthesis including ISG15ylation and MHC Class I production in cancer cells. The data together suggest that pro-metastatic growth associated with IFITM1/3 negative cervical cancers relates to attenuated expression of MHC Class I molecules that would support tumor immune escape. [Display omitted] •IFITM1/3 expression in cervical cancers inversely correlates with metastases.•Isogenic IFITM1 and IFITM3 null cervical cancer cells were developed.•Pulse SILAC approaches were used to define IFITM1/3 dependent signalling pathways.•The major IFITM1/3-interferon-γ dependent effectors are HLA-B and ISG15.•IFITM1/3 loss would be predicted to reduce HLA expression and ISG15ylation in vivo.