Discussion
In efforts to develop more effective therapies, it is critical to identify prognostic factors with high predictive significance and improve the understanding of molecular mechanisms underlying CRPC development ( Ge et al., 2020 ; Zhang et al., 2022 ). Previous knowledge mainly focused on the screening of biomarkers that expressed differentially between tumors and normal samples ( He et al., 2021 ; Sharp et al., 2019 ). Nevertheless, bulk transcriptome profiling may ignore significant targets during the process. Thus, in the current study, we analyzed the scRNA-seq to identify top AR-related drivers and particularly screen the prognostic ARGs based on large PRAD cohorts. Then, we constructed the ARGs model integrating 10 genes to validate its predictive efficiency and robustness in internal and external cohorts. The ARGs score is an independent factor for PRAD progression and high levels of ARGs scores correlated with advanced stages, Gleason scores and tumor mutational burden. The consensus clustering analysis further classified the PRAD samples into two groups via ARGs gene set and found the highly enriched crosstalk in cluster 2 versus cluster 1. In addition, MTT assays were conducted to further narrow down the targets and we observed that ORC1 is a potential hit within these genes. Low-throughput validations revealed that targeting ORC1 could significantly suppress cell growth of PRAD cells. ORC1 expressed highly in PRAD samples relative to paired normal tissues and correlated with elevated Gleason scores. Kaplan-Meier analysis further suggested that ORC1 is a prognostic factor and patients with high ORC1 levels suffered from worse PFS outcomes. Mechanistically, AR could directly bind at the promoter of ORC1 that contributes to its enhanced transcription. Increased ORC1 levels could notably mediate the Enza-R process, whereas targeting ORC1 could render PRAD more sensitive to enzalutamide treatment.

As is well documented, aberrant activation of AR signaling could drive the progression and ADT resistance of PRAD, which is the main reason that contributes to limited efficacy of treatment ( Wen, Niu & Huang, 2020 ; VanDeusen et al., 2020 ). However, there is currently no systematical screening of AR-related drivers in PRAD. Meanwhile, previous studies have already established the corresponding risk models based on autophagy-related signature, long non-coding RNAs, as well as DEGs ( Hu et al., 2020 , 2021 ). Compared with these common models, the predictive efficiency and accuracy of ARGs are superior to most of these already established signature. In our model, we also analyzed the prognostic significance of well-known PRAD risk factors, including PSA levels and Gleason scores. Compared with other clinical factors, we found that ARGs signature and Gleason scores were all independent elements for PRAD PFS and prognosis, but not the T or N stages. Meanwhile, DCA indicated that incorporation of ARGs and Gleason scores could notably elevate the net benefit for PRAD patients. How to integrate the ARGs and Gleason scores together to enhance predictive significance remains an intriguing problem to elucidate. In addition, we also utilized the cluster analysis to stratify PRAD samples into two groups in which the patients had distinct outcomes. Cell cycle is the top hit that enriched in cluster 2 versus cluster 1, indicating the underlying relationships between ARGs gene set with cell cycle process. Intriguingly, within these 10 identified genes, ORC1, ORC1, CENPF and DYNLL1 were already indicated to participate in cell cycle. For instance, one study found that DEFA1B could interact with ORC1 which is indispensable to initiate DNA replication during the cell cycle and enhanced DEFA1B proteins could inhibit the ORC1 level in the cell nuclei. Li et al. (2021) also indicated that overexpression of CENPF could accelerate cell proliferation and cell cycle, which correlated with progression and poor prognosis of lung adenocarcinoma. Collectively, we speculated that activated cell cycle process linked the tight associations between ARGs and PRAD progression.

The initial step of DNA replication contains the ORC in eukaryotes, consisting of ORC1/2/3/4/5/6 ( Lee et al., 2021 ). As the largest subunit, ORC1 was originally recognized to be a component of DNA replication initiation complexes ( Kuo et al., 2012 ). Besides, ORC1 could also function as a transcriptional regulator of a list of target genes, including human aldolase B and c-Myc genes ( Eladl et al., 2021 ). Defective mutations of several ORC subunits in multicellular organisms would lead to pleiotropic phenotypes, like cell cycle arrest, zygotic lethality, as well as chromosomal abnormalities ( Chou et al., 2021 ). Previous studies have indicated that ORC1 levels were regulated during the cell division cycle, and ORC is a dynamic complex ( Li et al., 2020 ). ORC1 is ubiquitinated for destruction in the S phase entry, followed by the dissociation of ORC from chromosomes. While, ORC1 re-localizes to condensing chromatin during early mitosis and then exhibits various nuclear localization patterns during G1 phase, as evidenced by the time lapse and live cell images of human cells expressing fluorescently tagged ORC1 ( Higa et al., 2021 ). Xiong et al. (2020) indicated that ORC1 played important roles in the migration, invasion, apoptosis, and proliferation of glioma cells. Mechanistically, OCR1 inhibition could suppress the activation of the Extracellular Signal-Regulated Kinase/c-Jun N-terminal Kinase (ERK/JNK) signaling pathway, implicating that ORC1 could be a novel prognostic marker of glioma via the activation of the ERK/JNK signaling pathway ( Xiong et al., 2020 ). Besides, TRF2 is regarded to play an essential role in ORC and MCM loading at telomeres, and TRF2 directly binds to ORC complex via the ORC1 protein. The TRF2-ORC1 interaction thus promote the replication origins that are assembled at telomeres. The initiation events could have an important role in telomere maintenance because the persistent arrest of replication forks within a telomere would cause under replication for the absence of a converging fork. However, little research has elucidated the specific mechanisms that cause high levels of ORC1 in tumors. In this study, we utilized the ChIP-qPCR assay to find that AR could cooperate with H3K27ac, one active indicator of transcription, to promote the transcriptional activity of ORC1. ORC1 is the direct downstream target of AR in PRAD cells, which has never been reported. In line with the above knowledge, we found that ORC1 overexpression could significantly enhance cell stemness potentiality, as indicated by the sphere formation assays. Given that sustained activation of cell cycle and stemness features could contribute to Enza-R, we further demonstrated that ORC1 could mediate Enza-R based on the in vitro and in vivo assays.

However, this study still has several shortcomings that need to be further improved. First of all, we should have recruited more PRAD samples and cohorts to validate the predictive efficiency and robustness of ARGs model. Secondly, apart from ORC1, we observed that targeting other genes in ARGs signature could also reduce cell growth. Experimental assays were further warranted to validate the functional roles of other targets, such as ORC or SHANK3. Lastly, although we have elucidated the relationships between AR-activated ORC1 levels with Enza-R, the underlying mechanisms were still uncertain. Whether ORC1 regulates other unknown regulators to drive Enza-R process would provide more useful vulnerabilities for treatment. Previous studies also indicated that stabilizing androgen receptor in mitosis could inhibits prostate cancer progression, indicating that we should choose suitable conditions to interfere ARG signaling ( Vander Griend, Litvinov & Isaacs, 2007 ).

In summary, our study integrated the scRNA-seq and TCGA-PRAD data to identify a prognostic ARGs model for PRAD patients. The established ARGs score is a vital factor in PRAD that correlated with Gleason scores, tumor stages and progression-free survival outcomes. Experimental studies further screened that AR-activated ORC1 is an essential hit and could promote cell growth and stemness features. Targeting ORC1 renders PRAD cells more sensitive to enzalutamide therapy, which provides a valuable vulnerability that deserves further investigations.