The Journal of Pathology

Volume 236, Issue 1

Original Paper

Orphan nuclear receptor TLX functions as a potent suppressor of oncogene‐induced senescence in prostate cancer via its transcriptional co‐regulation of the CDKN1A (p21^WAF1^/^CIP1) and SIRT1 genes

Dinglan Wu

School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, People's Republic of China

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Shan Yu

Corresponding Author

School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, People's Republic of China

Correspondence to: S Yu and FL Chan, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, Hong Kong, People's Republic of China. E‐mail:

yushan771@hotmail.com

;

franky-chan@cuhk.edu.hk

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Lin Jia

School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, People's Republic of China

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Chang Zou

School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, People's Republic of China

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Zhenyu Xu

School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, People's Republic of China

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Lijia Xiao

School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, People's Republic of China

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Kam‐Bo Wong

School of Life Sciences, Chinese University of Hong Kong, Hong Kong, People's Republic of China

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Chi‐Fai Ng

Department of Surgery, Chinese University of Hong Kong, Hong Kong, People's Republic of China

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Franky L Chan

Corresponding Author

School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, People's Republic of China

Correspondence to: S Yu and FL Chan, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, Hong Kong, People's Republic of China. E‐mail:

yushan771@hotmail.com

;

franky-chan@cuhk.edu.hk

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Dinglan Wu

School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, People's Republic of China

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Shan Yu

Corresponding Author

School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, People's Republic of China

Correspondence to: S Yu and FL Chan, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, Hong Kong, People's Republic of China. E‐mail:

yushan771@hotmail.com

;

franky-chan@cuhk.edu.hk

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Lin Jia

School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, People's Republic of China

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Chang Zou

School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, People's Republic of China

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Zhenyu Xu

School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, People's Republic of China

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Lijia Xiao

School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, People's Republic of China

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Kam‐Bo Wong

School of Life Sciences, Chinese University of Hong Kong, Hong Kong, People's Republic of China

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Chi‐Fai Ng

Department of Surgery, Chinese University of Hong Kong, Hong Kong, People's Republic of China

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Franky L Chan

Corresponding Author

School of Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, People's Republic of China

Correspondence to: S Yu and FL Chan, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, Hong Kong, People's Republic of China. E‐mail:

yushan771@hotmail.com

;

franky-chan@cuhk.edu.hk

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First published: 02 January 2015

https://doi.org/10.1002/path.4505

Cited by: 17

No conflicts of interest were declared.

Abstract

Oncogene‐induced senescence is an important tumour‐suppressing mechanism to prevent both premalignant transformation and cancer progression. Overcoming this process is a critical step in early cancer development. The druggable orphan nuclear receptor TLX (NR2E1) is characterized as an important regulator of neural stem cells and is also implicated in the development of some brain tumours. However, its exact functional roles in cancer growth regulation still remain unclear. Here we report that TLX can act as a promoter of tumourigenesis in prostate cancer by suppressing oncogene‐induced senescence. We determined that TLX exhibited an increased expression in high‐grade prostate cancer tissues and many prostate cancer cell lines. Functional studies revealed that TLX could perform an oncogenic function in prostate cancer cells, as its knockdown triggered cellular senescence and cell growth arrest in vitro and in vivo, whereas its over‐expression promoted the malignant growth of prostate cancer cells. Furthermore, enhancement of TLX activity, by either ectopic expression or ligand stimulation, could potently prevent doxorubicin‐induced senescence in prostate cancer cells and also allow prostatic epithelial cells to escape oncogene‐induced senescence induced either by activated oncogene H‐Ras^G12V or knockdown of tumour suppressor PTEN, via a mechanism of direct but differential transcriptional regulation of two senescence‐associated genes, repression of CDKN1A and transactivation of SIRT1. Together, our present study shows, for the first time, that TLX may play an important role in prostate carcinogenesis through its suppression of oncogene‐induced senescence, and also suggests that targeting the senescence‐regulatory TLX is of potential therapeutic significance in prostate cancer. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Citing Literature

Number of times cited: 17

Zhu Wang, Dinglan Wu, Chi-Fai Ng, Jeremy Yuen-Chun Teoh, Shan Yu, Yuliang Wang and Franky L Chan, Nuclear receptor profiling in prostatospheroids and castration-resistant prostate cancer, Endocrine-Related Cancer, 25, 1, (35), (2018).
Crossref
Hiroaki Hiramatsu, Kazuyoshi Kobayashi, Kyousuke Kobayashi, Takeshi Haraguchi, Yasushi Ino, Tomoki Todo and Hideo Iba, The role of the SWI/SNF chromatin remodeling complex in maintaining the stemness of glioma initiating cells, Scientific Reports, 7, 1, (2017).
Crossref
G Cai, D Wu, Z Wang, Z Xu, K-B Wong, C-F Ng, F L Chan and S Yu, Collapsin response mediator protein-1 (CRMP1) acts as an invasion and metastasis suppressor of prostate cancer via its suppression of epithelial–mesenchymal transition and remodeling of actin cytoskeleton organization, Oncogene, 36, 4, (546), (2017).
Crossref
Qingxuan Hu, Gang Wang, Jianping Peng, Guofeng Qian, Wei Jiang, Conghua Xie, Yu Xiao and Xinghuan Wang, Knockdown of SIRT1 Suppresses Bladder Cancer Cell Proliferation and Migration and Induces Cell Cycle Arrest and Antioxidant Response through FOXO3a-Mediated Pathways, BioMed Research International, 2017, (1), (2017).
Crossref
Vineet K. Dhiman, Michael J. Bolt and Kevin P. White, Nuclear receptors in cancer — uncovering new and evolving roles through genomic analysis, Nature Reviews Genetics, (2017).
Crossref
Corinne Abbadie, Olivier Pluquet and Albin Pourtier, Epithelial cell senescence: an adaptive response to pre-carcinogenic stresses?, Cellular and Molecular Life Sciences, 10.1007/s00018-017-2587-9, 74, 24, (4471-4509), (2017).
Crossref
Guoqiang Sun, Qi Cui and Yanhong Shi, Nuclear Receptor TLX in Development and Diseases, Nuclear Receptors in Development and Disease, 10.1016/bs.ctdb.2016.12.003, (257-273), (2017).
Crossref
Marissa E. Maheu and Kerry J. Ressler, Developmental pathway genes and neural plasticity underlying emotional learning and stress-related disorders, Learning & Memory, 24, 9, (492), (2017).
Crossref
Christopher McNair, Kexin Xu, Amy C. Mandigo, Matteo Benelli, Benjamin Leiby, Daniel Rodrigues, Johan Lindberg, Henrik Gronberg, Mateus Crespo, Bram De Laere, Luc Dirix, Tapio Visakorpi, Fugen Li, Felix Y. Feng, Johann de Bono, Francesca Demichelis, Mark A. Rubin, Myles Brown and Karen E. Knudsen, Differential impact of RB status on E2F1 reprogramming in human cancer, Journal of Clinical Investigation, 10.1172/JCI93566, (2017)., (2017).
Crossref
Ximena Corso-Díaz, Charles N. de Leeuw, Vivian Alonso, Diana Melchers, Bibiana K. Y. Wong, René Houtman and Elizabeth M. Simpson, Co-activator candidate interactions for orphan nuclear receptor NR2E1, BMC Genomics, 17, 1, (2016).
Crossref
Tao Wang and Jian-Qiong Xiong, The Orphan Nuclear Receptor TLX/NR2E1 in Neural Stem Cells and Diseases, Neuroscience Bulletin, 32, 1, (108), (2016).
Crossref
Xiaoli Shi, Haohua Deng, Zhe Dai, Yancheng Xu, Xiaokan Xiong, Pei Ma and Jing Cheng, Nr2e1 Deficiency Augments Palmitate-Induced Oxidative Stress in Beta Cells, Oxidative Medicine and Cellular Longevity, 2016, (1), (2016).
Crossref
Francesco Crea, Erik Venalainen, Xinpei Ci, Hongwei Cheng, Larissa Pikor, Abhijit Parolia, Hui Xue, Nur Ridzwan Nur Saidy, Dong Lin, Wan Lam, Colin Collins and Yuzhuo Wang, The role of epigenetics and long noncoding RNA MIAT in neuroendocrine prostate cancer, Epigenomics, 8, 5, (721), (2016).
Crossref
Dinglan Wu, Alyson Cheung, Yuliang Wang, Shan Yu and Franky L. Chan, The emerging roles of orphan nuclear receptors in prostate cancer, Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 10.1016/j.bbcan.2016.06.001, 1866, 1, (23-36), (2016).
Crossref
Damien A Leach, Sue M Powell and Charlotte L Bevan, WOMEN IN CANCER THEMATIC REVIEW: New roles for nuclear receptors in prostate cancer, Endocrine-Related Cancer, 10.1530/ERC-16-0319, 23, 11, (T85-T108), (2016).
Crossref
Xiaoli Shi, Xiaokan Xiong, Zhe Dai, Haohua Deng, Li Sun, Xuemei Hu, Feng Zhou and Yancheng Xu, Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells, Biochemical and Biophysical Research Communications, 468, 1-2, (387), (2015).
Crossref
Xingjie Ma, Marine Warnier, Clotilde Raynard, Mylène Ferrand, Olivier Kirsh, Pierre‐Antoine Defossez, Nadine Martin and David Bernard, The nuclear receptor RXRA controls cellular senescence by regulating calcium signaling, Aging Cell, e12831, (2018).
Wiley Online Library

Supporting Information

Filename	Description
path4505-sup-0001-AppendixS1.docWord document, 36 KB	AppendixS1. Supplementary materials and methods
path4505-sup-0002-figS1.tifTIFF image, 2.5 MB	FigureS1. Up‐regulation of TLX in prostate cancer cell lines. (A) Real‐time PCR analysis of TLX in PrEC cells and a panel of prostatic cell lines. Most prostate cancer cell lines expressed TLX transcripts at significantly higher levels, with the highest in NCI‐H660, as compared to primary cultured PrEC cells and immortalized prostatic epithelial cell lines; results were obtained from three independent experiments; ^p <* 0.05; ^p < 0.01. (B, C) Validation of specificities of TLX antibodies. (B) Representative immunoblots of FLAG‐tagged wild‐type TLX or its truncated mutant ΔZF1, ectopically expressed in transfected HEK293 cells and bacterially expressed recombinant His‐tagged TLX with a rabbit polyclonal antibody (pAb) and a commercial mAb. Both TLX antibodies recognized specifically the FLAG–TLX, FLAG–TLXΔZF1 and His–TLX fusion proteins. (C) Immunocytochemistry of TLX in parent DU145 and DU145–shTLX cells. The nuclear TLX immunosignals were significantly reduced in DU145–shTLX cells, as compared to those in parental DU145 cells.
path4505-sup-0003-figS2.tifTIFF image, 1.1 MB	FigureS2. Flow‐cytometric analysis of (A) LNCaP–shTLX and (B) DU145–shTLX infectants: (top) representative DNA histograms; (bottom) graphs showing cell cycle distribution as percentages of cells at each phase. Both LNCAP–shTLX and DU145–shTLX infectants had significantly higher percentages of cells at G₀–G₁ phase than their respective Scramble–shTLX infectants. No significant apoptosis was detected in shTLX infectants, as shown by the absence of a sub‐G₁ population.
path4505-sup-0004-figS3.tifTIFF image, 6.4 MB	FigureS3. TLX over‐expression enhances the malignant phenotype of prostate cancer. (A) Relative TLX mRNA levels in representative LNCaP–TLX and PC‐3–TLX infectants, assayed by real‐time PCR analysis. (B) In vitro proliferation by MTT assay or cell counting; LNCaP–TLX and PC‐3–TLX infectants proliferated at faster rates than the empty vector pBABE infectants. (C) Soft agar assay; TLX infectants showed higher colony formation efficiency in soft agar than pBABE infectants. (D) Transwell invasion assay: (top) representative micrographs show the crystal violet‐stained migrated LNCaP–TLX infectants; (bottom) graph shows the invasion capacity of LNCaP–TLX and LNCaP–pBABE infectants. LNCaP–TLX infectants showed significantly higher invasion capacity than LNCaP–pBABE infectants. (E) Wound‐healing assay; the leading edges of wounded areas are defined by dotted lines. All PC‐3–TLX infectants showed faster closure of wounded monolayers at 48 h post‐scrape as compared to vector control. (F) In vitro proliferation upon hypoxia exposure (1% O₂, 4 days) by MTT assay. LNCaP–TLX infectants proliferated at faster rates than LNCaP–pBABE infectants under hypoxic conditions.
path4505-sup-0005-figS4.tifTIFF image, 2.8 MB	FigureS4. Ectopic TLX expression inhibits cellular senescence and confers resistance to Dox treatment in prostate cancer cells. (A) Evaluation of SA‐β‐Gal activity in LNCaP–TLX infectants: (top) micrographs show SA‐β‐Gal staining in representative LNCaP–TLX and LNCaP–pBABE infectants; (bottom) graph shows SA‐β‐Gal‐positive cells (%) in LNCaP infectants; very few or no SA‐β‐Gal‐positive cells were detected in LNCaP–TLX as compared to LNCaP–pBABE infectants. (B) Evaluation of SA‐β‐Gal staining in LNCaP–TLX infectants expressing intact or truncated TLX mutants: (top) schematic diagram shows the wild‐type intact TLX [385 amino acids (aa)] and truncated mutants TLXΔZF1 (364 aa) and TLXDLBD–AF2 (340 aa); (bottom) graph shows the scores of SA‐β‐Gal‐positive cells in LNCaP–TLX infectants. TLX‐mutant infectants exhibited similar SA‐β‐Gal staining scores as the LNCaP–pBABE infectants, as compared to infectants generated by intact TLX, which showed significant reduction of SA‐β‐gal‐positive cells. (C) Evaluation of growth responses of LNCaP–TLX infectants towards Dox treatment (0.1–20 nm, 24 h) by MTT assay. LNCaP–TLX infectants were more resistant to Dox at different doses than LNCaP–pBABE infectants. (D) Immunoblot analysis of cleaved caspase‐3 (apoptosis marker) and p53 expression status in Dox‐treated LNCaP–TLX infectants. Upon low‐dose Dox treatment (0.1–5 nm), cleaved capase‐3 (19 and 17 kDa) was not detected in either LNCaP–TLX or LNCaP–pBABE infectants. However, upon Dox treatment at high dose (20 nm), cleaved caspase‐3 and significant accumulated levels of p53 were detected in LNCaP–pBABE but not in LNCaP–TLX infectants; ^p <* 0.05; ^p < 0.01.
path4505-sup-0006-figS5.tifTIFF image, 5.1 MB	FigureS5. Suppression of Dox‐induced senescence by TLX ligand ccrp2 in prostate cancer cells. Cytochemical staining of SA‐β‐Gal activity was performed in parental (A) LNCaP and (B) PC‐3 cells with Dox treatment (1 nm), alone or combined treatment with ccrp2, for 24 h. In combined Dox–ccrp2 treatment, cells were pretreated with 10 µm ccrp2 for 8 h, followed by Dox treatment. (top) Micrographs show the SA‐β‐Gal staining in representative cells; (bottom) graph shows the statistics of SA‐β‐Gal‐positive cells (%). Both LNCaP and PC‐3 cells pretreated with ccrp2 displayed a significantly less SA‐β‐Gal‐positive population upon Dox treatment.
path4505-sup-0007-figS6.tifTIFF image, 3.1 MB	FigureS6. Ectopic TLX expression enhances in vitro invasion capacity in PrEC cells with Ras^G12V transduction or PTEN knockdown. (A, B) Transwell invasion assay: (left) representative micrographs show the crystal violet‐stained invaded PrEC cells; (right) quantitative analysis shows that co‐infection with TLX could significantly enhance the invasion capacity of Ras^G12V‐ and shPTEN‐transduced PrEC cells, as compared to cells infected with empty vector or singly with Ras^G12V or shPTEN; ^p <* 0.05 versus vector control.
path4505-sup-0008-figS7.tifTIFF image, 1.3 MB	FigureS7. (A) ChIP–PCR of CDKN1A (p21) promoter. A proximal p21 promoter fragment (222 bp) was strongly amplified in immunoprecipitated DNA extracted from HEK293 cells transfected with TLX. Weak signals were also detected in cells transfected with empty vector pcDNA3.1 or truncated mutants TLXΔZF1 and TLXΔLBD–AF2, attributed to endogenous TLX expression in HEK293 cells. No signal was detected in the negative control without antibody. (B) Luciferase reporter assay of p21 gene promoter transactivation in HEK293 cells upon ccrp2 treatment (0, 1, 10 µm) for 24 h. Ectopic expression of TLX by transient transfection of pcDNA3.1–TLX was used as a positive control. p21–Luc reporter could be significantly transrepressed by the TLX ligand ccrp2 in a dose‐dependent manner. (C) ChIP–PCR of the SIRT1 promoter. An intense SIRT1 promoter fragment (162 bp) could be amplified in immunoprecipitated DNA extracted from HEK293 cells transfected with TLX. Weak signals were also amplified in cells transfected with truncated mutants TLXΔZF1 and TLXΔLBD–AF2. (D) Luciferase reporter assay of SIRT1 gene promoter transactivation in HEK293 cells upon ccrp2 treatment; pcDNA3.1–TLX was used as a positive control; SIRT1–Luc reporter could be significantly transactivated by ccrp2 in a dose‐dependent manner; ^p <* 0.05, ^p < 0.01, versus 0 µm ccrp2 treatment.
path4505-sup-0009-figS8.tifTIFF image, 3.4 MB	FigureS8. TLX‐mediated repression of p21 in prostate cancer cells is p53‐independent. (A) Immunoblot analysis of p53 and p21 expression in LNCaP–TLX infectants treated with Dox (24 h) for induction of senescence. Upon Dox treatment (0.1–2.5 nm), increase of p21 levels was induced in a dose‐dependent manner in LNCaP–pBABE infectants, but undetected or barely detected in LNCaP–TLX infectants. A slight increase of p53 levels was detected in both LNCaP–TLX and LNCaP–pBABE infections upon Dox treatment. (B) Immunoblot analysis of p53 and p21 expression in LNCaP–TLX infectants upon treatment with a p53 stabilizer, Nutlin‐3. Upon Nutlin‐3 treatment (1–2.5 nm), accumulation of p53 levels was detected in both LNCaP–TLX and LNCaP–pBABE infectants. However, increase of p21 levels was only detected in LNCaP–pBABE infectants but not LNCaP–TLX infectants, in which p21 remained barely detected or not detected.

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Volume236, Issue1

May 2015

Pages 103-115

The Journal of Pathology

Orphan nuclear receptor TLX functions as a potent suppressor of oncogene‐induced senescence in prostate cancer via its transcriptional co‐regulation of the CDKN1A (p21^WAF1^/^CIP1) and SIRT1 genes

Abstract

Number of times cited: 17

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The Journal of Pathology

Orphan nuclear receptor TLX functions as a potent suppressor of oncogene‐induced senescence in prostate cancer via its transcriptional co‐regulation of the CDKN1A (p21WAF1/CIP1) and SIRT1 genes

Abstract

Number of times cited: 17

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Orphan nuclear receptor TLX functions as a potent suppressor of oncogene‐induced senescence in prostate cancer via its transcriptional co‐regulation of the CDKN1A (p21^WAF1^/^CIP1) and SIRT1 genes