DAB2IP regulates autophagy in prostate cancer in response to combined treatment of radiation and a DNA-PKcs inhibitor

Abstract

Radiation therapy (RT) is an effective strategy for the treatment of localized prostate cancer (PCa) as well as local invasion. However, some locally advanced cancers develop radiation resistance and recur after therapy; therefore, the development of radiation-sensitizing compounds is essential for treatment of these tumors. DOC-2/DAB2 interactive protein (DAB2IP), which is a novel member of the Ras-GTPase activating protein family and a regulator of phosphatidylinositol 3-kinase-Akt activity, is often downregulated in aggressive PCa. Our previous studies have shown that loss of DAB2IP results in radioresistance in PCa cells primarily because of accelerated DNA double-strand break (DSB) repair kinetics, robust G(2)/M checkpoint control, and evasion of apoptosis. A novel DNA-PKcs inhibitor NU7441 can significantly enhance the effect of radiation in DAB2IP-deficient PCa cells. This enhanced radiation sensitivity after NU7441 treatment is primarily due to delayed DNA DSB repair. More significantly, we found that DAB2IP-deficient PCa cells show dramatic induction of autophagy after treatment with radiation and NU7441. However, restoring DAB2IP expression in PCa cells resulted in decreased autophagy-associated proteins, such as LC3B and Beclin 1, as well as decreased phosphorylation of S6K and mammalian target of rapamycin (mTOR). Furthermore, the presence of DAB2IP in PCa cells can lead to more apoptosis in response to combined treatment of NU7441 and ionizing radiation. Taken together, NU7441 is a potent radiosensitizer in aggressive PCa cells and DAB2IP plays a critical role in enhancing PCa cell death after combined treatment with NU7441 and radiation.

Figure 1

NU7441 increases the radiosensitivity in both DAB2IP-negative and DAB2IP-positive PCa cell lines. (A) The SF analysis of C4-2 D2 and C4-2 neo cells after combined treatment with 1 µM NU7441 and IR as indicated. (B) SF analysis of DAB2IP-knockdown PC3 cells (PC3-KD) and PC3 Con cells after treatment with 1 µM NU7441 and IR as indicated. (C) The survival curves of PC3 KD cells after combination treatment with 0.5 to 5 µM NU7441 and irradiation at doses of 0 to 8 Gy. In all studies, results are expressed as the means ± SD from three independent experiments.

Figure 2

NU7441 blocks IR-induced DNA DSB repair in DAB2IP-positive and DAB2IP-negative PCa cells. PCa cells were treated with 2 µM NU7441 for 30 minutes followed by IR (2 Gy); samples were collected at the indicated time points after IR, immune-stained for 53BP1 (red foci) and phospho-γH2AX (green foci), and counted (average, 50 nuclei). (A) C4-2 D2 and C4-2 neo cells. (B) PC3-KD cells. Quantitative analysis of DNA repair kinetics between C4-2 cells (C) and PC3KD cells (D).

Figure 3

Cell cycle analysis in DAB2IP-negative and DAB2IP-positive PCa cells after treatment with NU7441 and IR. C4-2 D2 and C4-2 neo cells were treated with IR (2 Gy), NU7441 (2 µM), and IR + NU7441 as indicated. Samples were collected at 0, 2, 6, and 24 hours post-treatment. Propidium iodide (PI) staining was used to detect the distribution of cells after various treatments. (A and B) C4-2 neo cells and (C and D) C4-2D2 cells.

Figure 4

DAB2IP expression suppressed IR- or NU7441 + IR-induced autophagy in PCa cells. C4-2 neo and D2 cells were treated with NU744, IR, and IR + NU7441 for 72 hours and then stained with AO. (A) Fluorescent images of AVO-positive cells. (B) Flow cytometry analysis to assess autophagy and (C) immunofluorescence staining of LC3B antibody. Experiments in A to C were performed under identical conditions. (D) SF analysis of C4-2 neo cells after combined treatment of IR and an autophagy inhibitor Baf A1 as indicated.

Figure 5

DAB2IP inactivates mTOR-S6K pathway and suppresses the expression of autophagy-associated proteins. (A) The phosphorylation of mTOR and S6K and the expression of autophagy-associated Beclin 1 and LC3B were determined by Western blot analysis 24 hours after IR (5 Gy). (B) The phosphorylation of mTOR and S6K and the expression of autophagy-associated Beclin 1 and LC3B were determined by Western blot analysis at the indicated time points.

Figure 6

DAB2IP promotes apoptosis in response to IR + NU7441. (A) IR + NU7441-induced apoptosis was determined by DAPI staining. Cells were treated with +/- NU7441 and IR (10 Gy) for 12 hours and the cells were stained with DAPI; the representative fluorescence images are shown. White arrows indicated the mitotic cells and the red arrows represent the apoptotic cells. (B) Quantitative analysis of apoptotic cells. The data are presented as the means ± SD of three independent experiments. (C) Analysis of PARP cleavage. Cells were lysed 12 hours after exposure to IR or IR + NU7441 and subjected to Western blot analysis.

Figure 7

IHC staining of DAB2IP in high-risk PCa patients. (A) Representative patients with positive DAB2IP expression. (B) Representative patient with DAB2IP loss. (C) BRFS plotted by patients' DAB2IP status.