CYTOR drives prostate cancer progression via facilitating AR-V7 generation and its oncogenic signalling

To the Editor: There are no data regarding expressed and functional characterisation of cytoskeleton-related non-coding RNA has been reported in prostate cancer (PCa). Here, we report a cytoskeleton regulator RNA (CYTOR)-regulated process that mediates castration-resistant PCa (CRPC)-specific androgen receptor splice variant 7 (AR-V7) generation, and further explore the vulnerability of CRPC growth through CYTOR-targeted locked nucleic acid (LNA). We retrieved public castration-sensitive PCa (CSPC) datasets (n = 65), neuroendocrine PCa (NEPC) datasets (n = 49) and CRPC datasets (including two studies, n = 171 and n = 118).1 Across the above RNA-seq data, CYTOR was found to be upregulated in CRPC with low expression in CSPC and NEPC (Figure 1A). RNA in situ hybridisation (RISH) assays2 of our centre samples confirmed the public domain data (Figure 1B and Figure S1A). Consistent with tissue detection, androgen-influenced CYTOR revealed significant increase in our two continuous established castration-resistant cell lines: LNCaP-AI,3 C4-2 Enz-R (Figure 1C and Figure S1B–G). Additionally, progression was more common in CSPC with higher CYTOR expression (Figure 1D). Expression analysis of CYTOR in flash-frozen surgical specimens was conducted in 11 CRPC patients (Figure 1E). Patients with high expression of CYTOR received worse PSA response to subsequential enzalutamide than those with CYTOR low expression (Figure 1F). Then, gene functional assays suggested knockdown of CYTOR suppressed the cancer cells growth (Figure 1G–J). The above results hint the association of CYTOR with CRPC development and inferior clinical outcomes. As it is, the primary therapeutic intervention for advanced PCa is androgen-deprivation therapy (ADT) with the goal of castration to suppress androgen receptor (AR) signalling. Although most patients respond to ADT, some inevitably develop resistance and progress to CRPC because of AR-V7 expression.4 Extensively investigated AR-V7 is a typically truncated AR without the ligand-binding domain but retaining transcriptional-regulated activity to mediate ligand-independent AR signalling.5 RNA-seq analysis revealed many AR-V7 downstream genes were differentially regulated as CYTOR knockdown (Figure 2A, Table S2). Most of them were enriched in metabolic pathways (Figure 2B). We validated the downregulation of AR-V7 canonic-activated genes (Figure 2C) after silencing CYTOR. Interestingly, knockdown of CYTOR resulted in specific decrease of AR-V7 without concurrent decrease of full-length AR (AR-FL) (Figure 2D), suggesting the critical role of CYTOR in AR-V7 mRNA splicing process. Multiplexed RISH assays of CRPC specimens revealed colocalisation and positive correlation of CYTOR and AR-V7 (pre-mRNA accumulated in nuclei) (Figure 2E). Their positive correlation was also confirmed by RT-PCR in four flash-frozen specimens (Figure 2F). Because key RNA-binding protein families involved in alternative splicing may include serine/arginine-rich proteins (SR proteins) and heterogeneous nuclear ribonucleoproteins (hnRNPs), we conducted differential expression analysis of SR proteins and hnRNPs between LNCaP-AI and LNCaP cells by our published RNA-arrays (GSE124291), and screened six upregulated splicing factors in LNCaP-AI cells (>1.5-fold) (Figure 2G). We further confirmed the upregulation of three genes (Figure 2H and Figure S2A). By Human Splicing Finder,6 the similar consensus splice site value for splice junctions of intron 3/cryptic exon 3 (CE3) (80.38) (as in AR-V7) and intron 3/exon 4 (80.1) (as in AR-FL) (Figure S2B) suggested the existence of a mechanism for CRPC-specific CE3 splice site utilisation. Given the established role of SR proteins in binding to pre-mRNA that prevents exon skipping, and the classical role of hnRNPs as splicing repressors, we postulated that nuclear-localised SRSF4 and SRSF7 (Figure 2I) may repress CE3 skipping, thus ensuring the correct 5′ to 3′ linear order of exons (exon1-3/CE3) in AR-V7 mRNA. Indeed, knockdown of SRSF4 or SRSF7 resulted in decreased expression of AR-FL and AR-V7, while withoutimpact on CYTOR (Figure 2J). The catRAPID strength algorithm computed output suggested the high specificity of CYTOR–SRSF4 interaction and CYTOR–SRSF7 interaction, respectively (Figure 2K,L).7 RNA immunoprecipitation results revealed both SRSF4 and SRSF7 proteins interacted with CYTOR, AR-V7 pre-mRNA and AR-V7 mRNA (Figure 2K,L), indicating nuclear binding of SRSF4 and SRSF7 to AR-V7 pre-mRNA and CYTOR was responsible for AR-V7 generation, even though there was weak interaction of SRSF4 and SRSF7 (Figure 2M). According to the functional interaction of CYTOR and SRSF4/7 proteins, we hypothesised that CYTOR may recognise AR-V7 pre-mRNA to induce this splicing process. Toward this end, maximum entropy modeling was used to collect motifs in the intron3/CE3 flanking sequence and identified the 3′ motif (3′ site of intron 3) and the 5′ motif (first 20 bp of CE3) (Figure 3A–C).6 The complementary sequence of the 5′ motif in the sequence of CYTOR (5′-UUCCAACCGC-3′) suggested that CYTOR may recognise the 5′ motif of CE3 (5′-GGGUUGGCAA-3′) to initiate the splicing process (Figure 3C). Next, we designed an 18 bp antisense oligonucleotides (ASO) to the 5′ motif of CE3 (ASOCE3) to prevent the recognition. The ASOCE3 suppressed, in a concentration- and time-dependent manner, the expression of AR-V7 mRNA (Figure 3D). We then designed an 18 bp ASOCYTOR to the complementary sequence of CE3 5′ motif in CYTOR. ASOCYTOR inhibited expression of AR-V7 mRNA without interfering CYTOR expression (Figure 3E and Figure S2C). Also as shown in C4-2 Enz-R cells, the ASOCE3 and ASOCYTOR prevented the generation of AR-V7 mRNA (Figure 3F,G). To validate this splicing model, truncated mutant assays confirmed the pivotal role of 5′-UUCCAACCGC-3′ in CYTOR on AR-V7 expression (Figure S2D,E). Together, CYTOR/SRSF4/SRSF7 complex interacts with AR-V7 pre-mRNA to regulate its splicing by recognising a specific signal element in CE3 (Figure 3H). Then LNAs GapmeRCYTOR were designed to silence CYTOR (Figure 4A). In C4-2 Enz-R cells, AR-V7 expression was largely suppressed in parallel with the silenced pattern of CYTOR in a concentration- and time-dependent manner (Figure 4B–E). GapmeRCYTOR could attenuate the resistance of enzalutamide significantly in vitro (Figure 4F). We then established in vivo mouse models and found that enzalutamide significantly suppressed C4-2 tumours, shCYTOR- and GapmeRCYTOR-treated C4-2 Enz-R tumours (Figure 4G,H). The expressions of CYTOR and AR-V7 were validated in each group (Figure S2F,G). As such, our data suggested that on-target effect of CYTOR knockdown with GapmeRCYTOR can be used as an option in castration resistance to provide therapeutic efficacy. In conclusion, we propose the importance of a novel complex composed of CYTOR/SRSF4/SRSF7 that mediates AR-V7 generation, and a critical role in suppressing PCa progression by targeting CYTOR/AR-V7 axis with shRNA or preclinical LNA GapmerCYTOR. Some of the biospecimens used in the present study were provided by the Chungbuk National University Hospital, a member of the National Biobank of Korea, which is supported by the Ministry of Health, Welfare, and Family Affairs. All samples derived from the National Biobank of Korea were obtained with informed consent under institutional review board-approved protocols. The authors wish to thank Ms. Eun-Ju Shim from the National Biobank of Korea at Chungbuk National University Hospital for the sample preparations and her excellent technical assistance. The authors declare they have no conflicts of interest. National Natural Science Foundation of China, Grant Numbers: 91959114, 81872106, 82072851, 81872100, 81972654, 82273262; National Natural Science Foundation of China, International (Regional) Cooperation and Exchange Program, Grant Number: 82061160493; Natural Science Foundation of Tianjin, Grant Numbers: 18PTLCSY00030, 21JCQNJC01700; Tianjin Key Medical Discipline (Specialty) Construction Project, Grant Numbers: TJYXZDXK-023A, TJYXZDXK-065B; The Second Hospital of Tianjin Medical University, Grant Number: 2020ydey01; Scientific Research Project of Tianjin Education Commission, Grant Number: 2021KJ225 Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.