Dataset Information

The Prostate Cancer mTOR Chromatin-Bound Interactome

ABSTRACT: Growing studies support a direct role for nuclear mTOR in gene regulation and chromatin structure. Still, the scarcity of known chromatin-bound mTOR partners limits our understanding of how nuclear mTOR controls transcription. Herein, we comprehensively mapped the mTOR chromatin-bound interactome in four cellular models of prostate cancer (PCa) identifying a conserved 67-protein interaction network enriched for epigenetic and transcription factors as well as SUMOylation machinery in both androgen-dependent and -independent cells. Notably, SUMO2/3 and nuclear pore protein NUP210 are among the strongest interactors while the androgen receptor (AR) is the dominant androgen-inducible mTOR partner. Further investigation showed that NUP210 facilitates mTOR nuclear trafficking, that mTOR, AR and NuRD act as a functional transcriptional complex, and that androgens dictate mTOR-SUMO2/3 promoter-enhancer specificity. This work identifies a vast network of mTOR-associated nuclear complexes advocating novel molecular strategies to modulate mTOR-dependent gene regulation with evident implications for PCa and other diseases.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Prostate Epithelium Cell Line, Prostate Cancer Cell Line, Cell Line Cell

DISEASE(S): Prostate Adenocarcinoma,Prostate Carcinoma

SUBMITTER: Vincent Giguere

LAB HEAD: Vincent Giguere

PROVIDER: PXD026101 | Pride | 2022-04-01

REPOSITORIES: Pride

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Publications

The mTOR chromatin-bound interactome in prostate cancer.

Dufour Catherine R CR Scholtes Charlotte C Yan Ming M Chen Yonghong Y Han Lingwei L Li Ting T Xia Hui H Deng Qiyun Q Vernier Mathieu M Giguère Vincent V

Cell reports 20220301 12

A growing number of studies support a direct role for nuclear mTOR in gene regulation and chromatin structure. Still, the scarcity of known chromatin-bound mTOR partners limits our understanding of how nuclear mTOR controls transcription. Herein, comprehensive mapping of the mTOR chromatin-bound interactome in both androgen-dependent and -independent cellular models of prostate cancer (PCa) identifies a conserved 67-protein interaction network enriched for chromatin modifiers, transcription fact ...[more]

PMID: 35320709

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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).

Project description:Reactivation of androgen receptor (AR) may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 in the N-terminal transactivation domain. In this study, the role of this phosphorylation site was investigated by characterizing the phosphorylation site mutant in the context of full length and truncated AR lacking the ligand-binding domain. The Y267F mutant showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. Expression of AR responsive genes in cells expressing truncated AR wt and AR-Y267F mutant under androgen deprived conditions was assessed by microarray gene expression analysis. The AR pathway score was increased in cells expressing truncated AR wt and decreased in cells expressing truncated AR-Y267F. These results support the concept that phosphorylation of Tyr-267 is required for AR nuclear translocation and recruitment and DNA binding and transcription of AR responsive genes. Gene expression profiling was performed using RNA samples from LNCaP cells expressing truncated AR-wt and truncated AR-Y267F growing in androgen deprived conditions. The RNA sample from vector control cells were used as reference sample. Two biological replicates were used per each cell line.

Dataset Information

The Prostate Cancer mTOR Chromatin-Bound Interactome

Publications

The mTOR chromatin-bound interactome in prostate cancer.

Similar Datasets

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