Abstract
Fractionated ionizing radiation combined with surgery or hormone therapy represents the first-choice treatment for medium to high-risk localized prostate carcinoma. One of the main reasons for the failure of radiotherapy in prostate cancer is radioresistance and further dissemination of surviving cells. In this study, exposure of four metastasis-derived human prostate cancer cell lines (DU145, PC-3, LNCaP and 22RV1) to clinically relevant daily fractions of ionizing radiation (35 doses of 2 Gy) resulted in generation of two radiation-surviving populations: adherent senescent-like cells expressing common senescence-associated markers and non-adherent anoikis-resistant stem cell-like cells with active Notch signaling and expression of stem cell markers CD133, Oct-4, Sox2 and Nanog. While a subset of the radiation-surviving adherent cells resumed proliferation shortly after completion of the irradiation regimen, the non-adherent cells started to proliferate only on their reattachment several weeks after the radiation-induced loss of adhesion. Like the parental non-irradiated cells, radiation-surviving re-adherent DU145 cells were tumorigenic in immunocompromised mice. The radiation-induced loss of adhesion was dependent on expression of Snail, as siRNA/shRNA-mediated knockdown of Snail prevented cell detachment. On the other hand, survival of the non-adherent cells required active Erk signaling, as chemical inhibition of Erk1/2 by a MEK-selective inhibitor or Erk1/2 knockdown resulted in anoikis-mediated death in the non-adherent cell fraction. Notably, whereas combined inhibition of Erk and PI3K–Akt signaling triggered cell death in the non-adherent cell fraction and blocked proliferation of the adherent population of the prostate cancer cells, such combined treatment had only marginal if any impact on growth of control normal human diploid cells. These results contribute to better understanding of radiation-induced stress response and heterogeneity of human metastatic prostate cancer cells, document treatment-induced plasticity and phenotypically distinct cell subsets, and suggest the way to exploit their differential sensitivity to radiosensitizing drugs in overcoming radioresistance.
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Abbreviations
- Akt (PKB):
-
protein kinase B
- 53BP1:
-
p53 binding protein 1
- AKTi:
-
Akt inhibitor
- APC:
-
allophycocyanin
- Bcl-XL:
-
BCL-2-like 1, long isoform
- Bim (EL):
-
Bcl-2 interacting mediator of cell death, extra large form
- BrdU:
-
5-bromo-2'-deoxyuridine
- CaP:
-
prostate carcinoma
- DAPI:
-
4',6-diamidino-2-phenylindole
- DLL1:
-
delta-like 1
- DLL4:
-
delta-like 4
- DNA:
-
deoxyribonucleic acid
- Dox:
-
doxycycline
- Doxo:
-
doxorubicine
- ECM:
-
extracellular matrix
- EdU:
-
5-ethynyl-2’-deoxyuridine
- EGF:
-
epidermal growth factor
- EMT:
-
epithelial-to-mesenchymal transition
- ERK1 (MAPK3):
-
mitogen-activated protein kinase 3
- ERK2 (MAPK1):
-
mitogen-activated protein kinase 1
- ERKi:
-
Erk inhibitor
- FACS:
-
fluorescence-activated cell sorting
- FAK:
-
focal adhesion kinase
- FGF:
-
fibroblast growth factors
- fIR:
-
fractionated irradiation
- fRT:
-
fractionated radiotherapy
- GAPDH:
-
glyceraldehyde 3-phosphate dehydrogenase
- Gy:
-
gray
- H2AX:
-
H2A histone family, member X
- Hes1:
-
hairy and enhancer-of-split 1
- Hey1:
-
hairy/enhancer-of-split related with YRPW motif 1
- HTS:
-
high throughput sampler
- IL:
-
interleukin
- IR:
-
irradiation
- ITGA2:
-
integrin, alpha 2
- LAMC2:
-
laminin, gamma 2
- LAMA3:
-
laminin, alpha 3
- MACS:
-
magnetic-activated cell sorting
- MET:
-
mesenchymal-epithelial transition
- MMP-7:
-
matrix metallopeptidase 7
- NFkappaB:
-
nuclear factor kappa B
- Oct-4:
-
octamer-binding transcription factor-4
- PI3K:
-
phosphoinositide-3-kinase
- PMA:
-
phorbol 12-myristate 13-acetate
- PML:
-
promyelocytic leukemia protein
- PML NBs:
-
promyelocytic leukemia nuclear bodies
- qRT–PCR:
-
quantitative real time polymerase chain reaction
- RNA:
-
ribonucleic acid
- RPL37a:
-
ribosomal protein L37a
- S.D.:
-
standard deviation
- SA-β-gal:
-
senescence-associated beta-galactosidase
- siNT:
-
Non-targeting siRNA
- STAT:
-
signal transducers and activators of transcription
- CTCs:
-
circulating tumor cells
- Twist1:
-
Twist-related protein 1
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Acknowledgements
This study was supported by Grant Agency of the Czech Republic (Project 13-17658S), Institutional Grant (Project RVO 68378050), DiaNa21 (Smartbrain s.r.o.), the Danish Research Council (DFF-1331-00262B), the Lundbeck Foundation (R93-A8990), the Danish National Research Foundation, the European Commission (Project DDResponse 259893) and the Grant Agency of the Ministry of Health of the Czech Republic (Project NT14461). LK, SH and TI were supported in part by the Faculty of Science, Charles University, Prague. We acknowledge P. Hamerlik for helpful discussions, D. Hladovcova for help with the mouse model, M. Vancurova for technical assistance with cell culture, O. Horvath and Z. Cimburek for their help with flow cytometry and cell sorting, R. Liska and S. Pavelka for help with cell irradiation, L. Andera for providing the anti-Bcl-XL and RPL37a forward and reverse primers for qRT–PCR, J. Švadlenka for providing the pCDH-EFI-Neo-empty and pCDH-EFI-Neo-Bcl-XL lentiviral vectors, T. Vomastek for the anti-Erk2 antibody and P. Draber for the anti-gamma-tubulin antibody.
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Kyjacova, L., Hubackova, S., Krejcikova, K. et al. Radiotherapy-induced plasticity of prostate cancer mobilizes stem-like non-adherent, Erk signaling-dependent cells. Cell Death Differ 22, 898–911 (2015). https://doi.org/10.1038/cdd.2014.97
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DOI: https://doi.org/10.1038/cdd.2014.97
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