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. 2014 Jan 28;111(4):E484-91.
doi: 10.1073/pnas.1323253111. Epub 2014 Jan 13.

RIG-I-like receptor LGP2 protects tumor cells from ionizing radiation

Ryan C Widau  1 , Akash D Parekh, Mark C Ranck, Daniel W Golden, Kiran A Kumar, Ravi F Sood, Sean P Pitroda, Zhengkai Liao, Xiaona Huang, Thomas E Darga, David Xu, Lei Huang, Jorge Andrade, Bernard Roizman, Ralph R Weichselbaum, Nikolai N Khodarev
Affiliations

RIG-I-like receptor LGP2 protects tumor cells from ionizing radiation

Ryan C Widau et al. Proc Natl Acad Sci U S A. .

Abstract

An siRNA screen targeting 89 IFN stimulated genes in 14 different cancer cell lines pointed to the RIG-I (retinoic acid inducible gene I)-like receptor Laboratory of Genetics and Physiology 2 (LGP2) as playing a key role in conferring tumor cell survival following cytotoxic stress induced by ionizing radiation (IR). Studies on the role of LGP2 revealed the following: (i) Depletion of LGP2 in three cancer cell lines resulted in a significant increase in cell death following IR, (ii) ectopic expression of LGP2 in cells increased resistance to IR, and (iii) IR enhanced LGP2 expression in three cell lines tested. Studies designed to define the mechanism by which LGP2 acts point to its role in regulation of IFNβ. Specifically (i) suppression of LGP2 leads to enhanced IFNβ, (ii) cytotoxic effects following IR correlated with expression of IFNβ inasmuch as inhibition of IFNβ by neutralizing antibody conferred resistance to cell death, and (iii) mouse embryonic fibroblasts from IFN receptor 1 knockout mice are radioresistant compared with wild-type mouse embryonic fibroblasts. The role of LGP2 in cancer may be inferred from cumulative data showing elevated levels of LGP2 in cancer cells are associated with more adverse clinical outcomes. Our results indicate that cytotoxic stress exemplified by IR induces IFNβ and enhances the expression of LGP2. Enhanced expression of LGP2 suppresses the IFN stimulated genes associated with cytotoxic stress by turning off the expression of IFNβ.

Keywords: DHX58; cytoplasmic sensor; innate immunity; interferon beta.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Identification of LGP2 as prosurvival ISG. In each cell line tested, 89 screened genes were ranked according to the ability of corresponding siRNAs to suppress cell viability as measured by CellTiter-Glo luminescent assay (Promega). FDR-corrected significance values for each gene across all tested cell lines were estimated by rank aggregation approach (Methods). Data are presented as negative log-transformed FDRs for each gene on the basal level (closed triangles, right y axis) and 48 h after irradiation at 3 Gy (open diamonds, left y axis).
Fig. 2.
Fig. 2.
Knockdown of LGP2 enhances radiation-induced killing. Cell death was quantified by flow cytometric analysis using Annexin V and propidium iodide staining. Tumor cells were treated with IR (5 Gy) 24 h posttransfection with the indicated siRNA. (A) Graphical representation of flow cytometric data in WiDr cells that were collected 48 h post-IR treatment. (B) Quantification of flow cytometric experiments in D54, WiDr, and Scc61 cells collected 48 h post-IR treatment. The data are represented as fold change relative to siNT at 0 Gy. (C and D) Clonogenic survival curves in D54 (C) and Scc61 (D) cells transiently transfected with siNT or siLGP2 and irradiated at 0, 3, 5, or 7 Gy. Data are represented in a semilog scale. Western blots are representative of siRNA-mediated knockdown of LGP2. In all experiments, data are presented as mean values of at least three independent measurements; error bars are SDs, and significance was assessed using two-tailed t test (*P < 0.05).
Fig. 3.
Fig. 3.
Overexpression of LGP2 inhibits radiation-induced killing. D54 cells were stably transfected by full-size p3xFLAG–CMV10–LGP2 (LGP2) or control p3xFLAG–CMB10 (Flag). Selected clones were propagated, plated in six-well plates, and irradiated at 0, 5, and 7 Gy. (A) Crystal violet staining of survived colonies 12 d after irradiation of cells, transfected with Flag (Upper) or LGP2 (Lower). (B) Quantification of survival fraction of mock-transfected and LGP-transfected cells (Methods). Representative Western blot of stable Flag and LGP2 clone is inserted into B.
Fig. 4.
Fig. 4.
LGP2 is radioinducible. D54, WiDr, and Scc61 cells were irradiated at 6 Gy; 72 h post-IR, cell lysates were analyzed by Western blotting.
Fig. 5.
Fig. 5.
IR induces cytotoxic IFNβ response. (A) Radiation-induced expression of IFNβ mRNA. IFNβ expression in D54, WiDr, SCC61, and HEK293 cells treated with or without 6 Gy IR was measured by qRT-PCR and normalized to GAPDH expression. Data are expressed as fold change relative to nonirradiated cells. (B) Radiation-induced activation of IFNβ promoter. HEK293 cells were transiently cotransfected with pGL3–Ifnβ and pRL–SV40. Firefly luciferase was normalized to Renilla luciferase and is expressed relative to nonirradiated cells at each collection time. (C) Type I IFN receptor (IFNAR1) is needed for cytotoxicity induced by IR. WT and IFNAR1−/− MEFs were treated with the indicated doses of IR and collected 96 h post-IR. Viability was determined by methylene blue staining and extraction, followed by spectrophotometric quantification. Viability is shown relative to nonirradiated control cells. Data are represented as means with SDs for assays performed at least in triplicate.
Fig. 6.
Fig. 6.
LGP2 inhibits IR-induced cytotoxic IFNβ. (A) LGP2 suppresses IR-induced activation of IFNβ promoter. HEK293 cells were stably transduced with shRNA directed to LGP2 or shNT. Cells were transfected with pGL3–Ifnβ and pRL–SV40, irradiated (indicated dose), and collected 72 h after IR. Firefly luciferase activity was normalized to Renilla luciferase activity and is expressed relative to nonirradiated cells. (B) Neutralizing antibodies to IFNβ prevent cytotoxic effects of LGP2 depletion. D54 cells were depleted of LGP2 with siRNA (Fig. 2C) and irradiated at 0, 3, or 6 Gy in the presence or absence of neutralizing antibody to IFNβ (1 μg/mL). Cell viability was assessed 96 h post-IR using methylene blue assay. Data are normalized to nontargeting siRNA at 0 Gy and represented as means with error bars showing SDs for assays performed at least in triplicate. Significance was measured using two-tailed t test (*P < 0.05).
Fig. 7.
Fig. 7.
Expression of LGP2 is associated with poor overall survival in patients with GBM. (A) Expression of ISGs and LGP2 in the Phillips database (37) (n = 77). Yellow represents up-regulated and blue down-regulated genes. Rows correspond to patients, and columns correspond to individual genes in IRDS signature (10, 13). (B) Kaplan–Meier survival of LGP2-high (LGP2+) and LGP2-low (LGP2–) patients from the Phillips et al. database. (C) Expression of ISGs and LGP2 in the TCGA database (n = 382) and (D) survival of LGP2+ and LGP2– patients in CGA database. P values represent Cox proportional hazards test.
Fig. 8.
Fig. 8.
Activation of IFNβ by IR is suppressed by LGP2. Acute response to IR leads to activation of IFNβ and induction of ISGs with cytotoxic functions (A). Chronic exposure to cytotoxic stress leads to constitutive expression of some ISGs with prosurvival functions and LGP2-dependent suppression of the autocrine IFNβ loop (B).
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