REV1 Inhibition Enhances Radioresistance and Autophagy

Abstract

Cancer therapy resistance is a persistent clinical challenge. Recently, inhibition of the mutagenic translesion synthesis (TLS) protein REV1 was shown to enhance tumor cell response to chemotherapy by triggering senescence hallmarks. These observations suggest REV1's important role in determining cancer cell response to chemotherapy. Whether REV1 inhibition would similarly sensitize cancer cells to radiation treatment is unknown. This study reports a lack of radiosensitization in response to REV1 inhibition by small molecule inhibitors in ionizing radiation-exposed cancer cells. Instead, REV1 inhibition unexpectedly triggers autophagy, which is a known biomarker of radioresistance. We report a possible role of the REV1 TLS protein in determining cancer treatment outcomes depending upon the type of DNA damage inflicted. Furthermore, we discover that REV1 inhibition directly triggers autophagy, an uncharacterized REV1 phenotype, with a significant bearing on cancer treatment regimens.

Keywords: REV1; autophagy; etoposide; ionizing radiations; radioresistance; translesion synthesis.

Figure 1

REV1 inhibition does not sensitize cancer cells to ionizing radiation. (A) Relative cell survival of HCT116 (colorectal), HT1080 (fibrosarcoma), mouse embryonic fibroblasts (MEF), and REV1 KO (knockout; MEFs) cells in response to increasing amounts of ionizing radiation at doses of 1 and 4 Gy and REV1 inhibitor drugs at a 1 μM concentration: 4 (7922759), JH-1 (JH-RE-06), and JH-2 (JH-RE06.NaOH). (B) Relative cell survival of HT1080 cells with 0, 10, and 100 Gy. (C) Relative cell survival in REV1 KO cells after treatment with increasing doses of IR at 0, 1, and 4 Gy (left graph) and with 10 mM cisplatin and 4 Gy of radiation (right graph). (D) Western blot showing γH2AX in HCT116 cells treated with 1, 4, and 10 Gy of radiation at 24 h. Graph shows the relative quantification of the Western blots. p-Values are * p < 0.05, ** p < 0.01, and *** p < 0.001. Error bars represent standard deviations. p-Values were calculated by 2-way ANOVA. N = 6 for all values.

Figure 2

REV1 inhibition does not sensitize cancer cells to ionizing radiation. (A) Graphs show relative luminescence with increasing incubation times (24, 48, and 72 h) with drug 4 (7922759) and increasing doses of IR (0, 1, and 4 Gy) in the HT1080 and HCT116 cell lines. Also shown are relative luminescence intensities in MEF REV1 KO cells after exposure to 1 and 4 Gy ionizing radiation and incubation for 24, 48, and 72 h. (B) Relative luminescence in HT1080 treated with 0, 5, 15, and 30 μM of JH-RE-06 with 1 Gy of IR exposure. (C) Relative luminescence in REM and REM RR cells in response to treatment with JH-RE-06, JH-RE-06.NaOH, and drug 5 (4053831). p-Values are * p < 0.05, ** p < 0.01, and **** p < 0.0001. Error bars represent standard deviations. p-Values were calculated by 2-way ANOVA. N = 6 for all values.

Figure 3

REV1 inhibition triggers autophagy. (A) Immunofluorescence images show autophagy flux (green) in MEF WT cells treated with chloroquine (positive control), drug 4 (7922759), and JH-RE-06, with REV1KO cells as validation controls. The graph shows relative quantification of the cells expressing the green fluorescence signal. p-Values are **** p < 0.0001. Error bars represent standard deviations. p-Values were calculated by t-test. N ≥ 50. Images are at 40×. (B) Representative image of a Western blot showing expression of p62 and LC3a in MEF REV1 KO cells compared to the WT MEF. Graph shows relative quantification.

Figure 4

Autophagy inhibition has a narrow range for sensitizing cancer cells treated with ionizing radiation and REV1 inhibitors. (A) Graphs show relative luminescence in HT1080 cells treated with 0 or 4 Gy ionizing radiation in the presence of increasing doses of autophagy inhibitor BFA and 1 uM of JH-RE-06.NaOH. (B) Representative Western blot images show expression patterns of p62 and LC3a/b in HT1080 cells treated with JH-RE-06.NaOH at 1 mM, BFA at 50 mM, and ionizing radiation at 0 and 4 Gy, respectively. Graphs show relative quantification of p62 and LC3a/b expression in HT1080 cells from the Western blots above. p-Values are * p < 0.05, ** p < 0.01, and *** p < 0.001. Error bars represent standard deviations. p values were calculated by 2-way ANOVA. N = 6 for all values.