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Opposite response of hTERT expression by androgen dose: Androgen-mediated inhibition of hTERT is mediated by the ING1 and ING2 tumor suppressors in prostate cancer cells

ABSTRACT: The catalytic subunit of the human telomerase (hTERT) is activated during tumorigenesis in many cancers including prostate cancer (PCa). Androgens mediate their effect through the androgen receptor (AR), a key factor controlling PCa growth. hTERT expression is known to be regulated by androgens, however contrarily observed to be inhibited or activated. Here, we reveal that androgens repress or activate hTERT expression in a concentration-dependent manner. Low physiological androgen levels activate while supraphysiological androgen levels (SAL) repress hTERT expression. We confirmed SAL-mediated gene repression of hTERT in native human PCa samples derived from patients treated ex vivo as well as in cancer spheroids derived from androgen-dependent and castration resistant PCa (CRPC) cells. Interestingly, based on chromatin immunoprecipitation (ChIP) data both a positive androgen response element (pARE) at -4kb distal and a negative response element (nARE) at the proximal -0.1kb promoter region were identified. Focusing on the nARE it was narrowed down to -121 to -58 bp in the core promoter region. ChIP experiments confirmed an androgen-dependent recruitment of AR and the tumor suppressors inhibitor of growth 1 and 2 (ING1 and ING2), recently identified as AR co-repressors. Mechanistically, the knockdown of either ING1 or ING2 reduced androgen-mediated repression of hTERT indicating that ING1 and ING2 mediate AR-regulated transrepression on the hTERT expression. Thus, our data suggest a dual, biphasic function of AR to control hTERT expression by transactivation and transrepression. The inhibition of hTERT by androgens by a nARE located in the proximal promoter region is mediated at least in part by the AR co-repressors ING1 and ING2.

ORGANISM(S): Homo sapiens

PROVIDER: GSE155528 | GEO | 2022/03/04

REPOSITORIES: GEO

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Project description:The bipolar androgen therapy (BAT) to treat prostate cancer includes cycles of supraphysiological androgen levels (SAL) under continuous androgen deprivation therapy (ADT). We showed previously that SAL induces cellular senescence in androgen-sensitive PCa cells and ex vivo in PCa tumor samples from patients that underwent radical prostatectomy. Here, we show that SAL induces cellular senescence in both, castration sensitive (CSPC) LNCaP and castration resistant PCa (CRPC) C4-2 cells through the cell cycle inhibitor p15. Treatment with the Akt inhibitor (Akti) potently inhibited SAL-induced expression of p15 and cellular senescence in both cell lines. Proximity ligation assays (PLA) combined with high-resolution laser-scanning microscopy indicate that SAL promotes interaction of endogenous androgen receptor (AR) with endogenous Akt in cytoplasm as well as in nucleus after 72 hours hormone treatment. This suggests that the AR interacts with Akt also in a long-term manner. Transcriptome sequencing (RNA-seq) comparing the SAL-induced transcriptomes of LNCaP with C4-2 cells as well as of Akti treated cells revealed landscapes for cell senescence. Interestingly, one of the identified genes is the lncRNASAT1. SAL treatment of native patient tumor samples ex vivo results in upregulation of lncRNASAT1. The lncRNASAT1 is down-regulated in PCa tumor tissues compared to non-tumor tissues of same patients indicating a tumor suppressive function. Knockdown indicates that the lncRNASAT1 is crucial for SAL-induced cancer cell senescence and regulates LNCaP cell proliferation being an upstream factor for pAkt and for p15. Further, knockdown of lncRNASAT1 inhibits LNCaP cell proliferation by low androgen levels but enhances proliferation by SAL. This suggests that lncRNASAT1 serves as a tumor suppressor with SAL. Interestingly, immunoprecipitation of AR detected lncRNASAT1 as an AR interacting partner that regulates AR target gene expression. Thus, we identified a novel pathway of androgen signaling as the AR- lncRNASAT1- Akt- p15INK4b- axis to mediate SAL-induced cellular senescence.

Project description:The androgen receptor (AR) plays a key role in progression to incurable androgen-ablation resistant prostate cancer (PCA). We have identified three novel AR splice variants lacking the ligand binding domain (designated as AR3, AR4 and AR5) in hormone insensitive PCA cells. AR3, one of the major splice variants expressed in human prostate tissues, is constitutively active and its transcriptional activity is not regulated by androgens or antiandrogens. Immunohistochemistry analysis on tissue microarrays containing 429 human prostate tissue samples shows that AR3 is significantly upregulated during PCA progression and AR3 expression level is correlated with the risk of tumor recurrence after radical prostatectomy. Overexpression of AR3 confers ablation-independent growth of PCA cells while specific knock-down of AR3 expression (without altering AR level) in hormone resistant PCA cells attenuates their growth under androgen-depleted conditions in both cell culture and xenograft models, suggesting an indispensable role of AR3 in ablation-independent growth of PCA cells. Furthermore, AR3 may play a distinct yet essential role in ablation-independent growth through regulating a unique set of genes including AKT1, which are not regulated by the prototype AR. Our data suggest that aberrant expression of AR splice variants may be a novel mechanism underlying ablation-independence during PCA progression and AR3 may serve as a prognostic marker to predict patient outcome in response to hormonal therapy. Given that these novel AR splice variants are not inhibited by currently available anti-androgen drugs, development of new drugs targeting these AR isoforms may potentially be effective for treatment of ablation-resistant PCA. Total RNA was extracted from CWR-R1 and 22Rv1 cells treated with shAR3-1, shARa and the scrambled shRNA control, respectively. Each of CWR-R1 and 22Rv1 cells treated with shAR3-1 was compared with the scrambled shRNA control. The same experiments were performed for the cells treated with shARa.

Project description:Androgens drive the onset and progression of prostate cancer (PCa) by modulating androgen receptor (AR) transcriptional activity. Although several microarray-based studies have identified androgen-regulated genes, here we identify in-parallel global androgen-dependent changes in both gene and alternative mRNA isoform expression by exon-level analyses of the LNCaP transcriptome. While genome-wide gene expression changes correlated well with previously-published studies, we additionally uncovered a subset of 226 novel androgen-regulated genes. Gene expression pathway analysis of this subset revealed gene clusters associated with, and including the tyrosine kinase LYN, as well as components of the mTOR (mammalian target of rapamycin) pathway, which is commonly dysregulated in cancer. We also identified 1279 putative androgen-regulated alternative events, of which 325 (~25%) mapped to known alternative splicing events or alternative first/last exons. We selected 30 androgen-dependent alternative events for RT-PCR validation, including mRNAs derived from genes encoding tumour suppressors and cell cycle regulators. Of seven positively-validating events (~23%), five events involved transcripts derived from known AR gene targets. In particular, we found a novel androgen-dependent mRNA isoform derived from an alternative internal promoter within the TSC2 tumour suppressor gene, which is predicted to encode a protein lacking an interaction domain required for mTOR inhibition. We confirmed that expression of the alternative TSC2 mRNA isoform was directly regulated by androgens. Furthermore, by chromatin immunoprecipitation, we observed recruitment of AR to the alternative promoter region at early timepoints following androgen stimulation, which correlated with expression of alternative transcripts. Together, our data suggest that alternative mRNA isoform expression might mediate the cellular response to androgens, and may have roles in clinical PCa. Total 8 samples were analysed. 4 control (LNCaP cells grown in RPMI-1640 media with charcoal-stripped 10% FBS devoid of steroids) and 4 treatment (LNCaP cells grown in media devoid of steroid, and subsequently treated with 10nM R1881 synthetic androgen analogue for 24 hours).

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Opposite response of hTERT expression by androgen dose: Androgen-mediated inhibition of hTERT is mediated by the ING1 and ING2 tumor suppressors in prostate cancer cells

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