Acquisition of epithelial-mesenchymal transition and cancer stem cell phenotypes is associated with activation of the PI3K/Akt/mTOR pathway in prostate cancer radioresistance

Abstract

Radioresistance is a major challenge in prostate cancer (CaP) radiotherapy (RT). In this study, we investigated the role and association of epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) and the PI3K/Akt/mTOR signaling pathway in CaP radioresistance. We developed three novel CaP radioresistant (RR) cell lines (PC-3RR, DU145RR and LNCaPRR) by radiation treatment and confirmed their radioresistance using a clonogenic survival assay. Compared with untreated CaP-control cells, the CaP-RR cells had increased colony formation, invasion ability and spheroid formation capability (P<0.05). In addition, enhanced EMT/CSC phenotypes and activation of the checkpoint proteins (Chk1 and Chk2) and the PI3K/Akt/mTOR signaling pathway proteins were also found in CaP-RR cells using immunofluorescence, western blotting and quantitative real-time PCR (qRT-PCR). Furthermore, combination of a dual PI3K/mTOR inhibitor (BEZ235) with RT effectively increased radiosensitivity and induced more apoptosis in CaP-RR cells, concomitantly correlated with the reduced expression of EMT/CSC markers and the PI3K/Akt/mTOR signaling pathway proteins compared with RT alone. Our findings indicate that CaP radioresistance is associated with EMT and enhanced CSC phenotypes via activation of the PI3K/Akt/mTOR signaling pathway, and that the combination of BEZ235 with RT is a promising modality to overcome radioresistance in the treatment of CaP. This combination approach warrants future in vivo animal study and clinical trials.

Figure 1

Different radiosensitivity to RT in CaP-RR and CaP-control cells. CaP-RR and CaP-control cells were seeded in 10 cm dishes and treated with 2–10 Gy radiation. The colonies that formed after 10–12 d incubation were counted to calculate the survival fractions. (a) Survival fractions in CaP-RR cells (PC-3RR, DU145RR and LNCaPRR) were significantly increased compared with those in CaP-control cells (P<0.01). (b) Typical images are shown for colony growth in CaP-RR and CaP-control cells after exposure to different radiation doses. Images were taken using a Sony camera (Tokyo, Japan). All data used in RR cell lines were based on cells between 5 and 6 weeks post radiation treatment. All results were from three independent experiments (Mean±S.D., n=3)

Figure 2

Matrigel invasion and spheroid formation in CaP-RR and CaP-control cells. (a) The cells were incubated 12 h for the invasion assay. The invasive potential in CaP-RR cells was significantly increased in PC-3RR, DU145RR and LNCaPRR cells compared with CaP-control cells (★, P<0.01). (b) Representative images for CaP cell invasion and migration in CaP-RR and CaP-control cells are shown. Magnification: × 200 in all images. (c) The spheroid formation ability in CaP-RR cells was significantly enhanced compared with CaP-control cells (★, P<0.01) after 5 days culture. (d) Representative images for spheroid formation in CaP-RR cells and CaP-control cells are shown. The upper images are from CaP-control cells (cell numbers <50/spheroid), whereas the lower images were from CaP-RR cells (cell numbers >50/spheroid). All data used in RR cell lines were based on cells between 5 and 6 weeks post radiation treatment. All results were from three independent experiments (Mean±S.D., n=3)

Figure 3

EMT phenotypic expression in CaP-RR and CaP-control cells. Reduced expression of membranous E-cadherin and increased membranous or cytoplasmic expression of N-cadherin, Vimentin, OCT3/4, SOX2 and αSMA were found in CaP-RR cells. (a) Representative immunofluorescence images of E-cadherin, N-cadherin, Vimentin, OCT3/4, SOX2 and αSMA (green) are shown for CaP-RR and CaP-control cells. Nuclei are stained with PI (red). Magnification: all images × 400. (b) Western blotting results were consistent with immunofluorescence staining results. β-Tubulin was used as a loading control. (c) Phenotypic changes of EMT markers in CaP-RR cells using immunofluorescence and western blotting were further confirmed by qRT-PCR (♦, P<0.01). All data used in RR cell lines were based on cells between 5 and 6 weeks post radiation treatment

Figure 4

CSC phenotypic expression in CaP-RR and CaP-control cells. The enhanced CSC phenotypes were seen in CaP-RR (PC-3RR, DU145RR and LNCaPRR) cells. (a) Representative immunofluorescence images of membranous or cytoplasmic expression of CD44, CD44v6, CD326, ALDH1, Nanog and Snail are shown in CaP-RR and CaP-control cells. Nuclei were stained with PI (red). Magnification × 400 in all images. (b) Western blotting results were consistent with the immunofluorescence staining results. GAPDH was used as a loading control. (c) The phenotypic changes of CSC in CaP-RR cells were further confirmed by qRT-PCR (♦, P<0.01). All data used in RR cell lines were based on cells between 5 and 6 weeks post radiation treatment. All results were from three independent experiments (Mean±S.D., n=3)

Figure 5

CaP-RR cells activate checkpoint proteins and the PI3K/Akt/mTOR pathway, and the effect of RT and BEZ235 on the expression of PI3K/Akt/mTOR pathway proteins. Two markers (Chk1 and Chk2) involved in radiation checkpoint and eight signal transduction molecules (mTOR, p-mTOR, Akt, p-Akt, 4EBP1, p-4EBP1, S6K and p-S6K) were assessed to investigate the roles of checkpoint proteins or PI3K/Akt/mTOR signaling proteins in CaP radioresistance. (a) The levels of p-Chk1 and p-Chk2, p-Akt, p-mTOR, p-S6K and p-4EBP1 were increased in CaP-RR cells compared with CaP-control cells, whereas no change was found in total of Chk1, Chk2, Akt, mTOR, S6K and 4EBP1 proteins in both CaP-RR and control cells. Representative results are shown. (b) After 12 h treatment by 6 Gy RT alone or combination treatment with RT and BEZ235 (BEZ235 was used for 12 h prior RT and then 6 Gy was applied. Cell lysate was extracted after RT applied for 12 h). Reduced levels of p-Akt, p-mTOR, p-S6K and p-4EBP1 were detected in CaP-RR cells in combination treatment compared with single 6 Gy treatment. β-Tubulin was used as a loading control. All data used in RR cell lines were based on cells between 5 and 6 weeks post radiation treatment. All data were from three independent experiments (Mean±S.D., n=3). p-mTOR: phosphorylated-mTOR; p-Akt: phosphorylated-Akt; p-4EBP1: phosphorylated-4EBP1; p-S6K: phosphorylated-S6K

Figure 6

Effect of BEZ235 and RT on the expression of EMT/CSCs and radiosensitivity in CaP-RR cells. Combination treatment with BEZ235 and RT reversed EMT expression and reduced the levels of CSC marker expression in CaP-RR cells compared with the RT alone. (a) CaP-RR cells were treated with single RT (6 Gy) for 12 h or combination (BEZ235 was used for 12 h prior RT and then 6 Gy was applied. Cell lysate was extracted 12 h after RT. Typical results for EMT/CSC phenotypic changes after different treatments are shown. β-Tubulin was used as a loading control. (b) Colony formation was significantly reduced in combination treatment with BEZ235 and RT compared with single BEZ236, 6 Gy RT alone or vehicle control in CaP-RR cells (★, P<0.01). (c) Typical images of colony growth for the different treatments are shown. (d) Combination treatment induced more apoptotic cells in CaP-RR cells compared with single BEZ235 or RT or control. All data used in RR cell lines were based on cells between 5 and 6 weeks post radiation treatment. All data were from three independent experiments (n=3)

Figure 7

Diagram showing the model proposed for the association of EMT, CSCs and the PI3K/Akt/mTOR signaling pathway in CaP radioresistance and the effect of possible action of BEZ235 on radiosensitivity. (a) RT can induce CaP-RR cells with EMT and enhanced CSC phenotypes, and activation of the PI3K/Akt/mTOR signaling pathway, resulting in CaP metastasis and recurrence after RT. (b) A dual PI3K/mTOR inhibitor BEZ235 combined with RT can inactivate the PI3K/Akt/mTOR signaling pathway, reducing EMT and CSC phenotypes and leading to increased radiosensitivity. All data used in RR cell lines were based on cells between 5 and 6 weeks post radiation treatment