PACT is requisite for prostate cancer cell proliferation

PACT is requisite for prostate cancer cell proliferation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article PACT is requisite for prostate cancer cell proliferation Peter Leedman, Dianne Beveridge, Andrew Woo, Kirsty Richardson, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4121983/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract PACT (encoded by the PRKRA gene) is a double-stranded RNA binding protein that has two main functions in mammals: facilitation of antiviral defense mechanisms via the activation of protein kinase RNA (PKR) and retinoic acid-inducible gene 1 (RIG-1), and PACT is also a member of the cytoplasmic RNA-induced silencing complex. We previously described an alternate role for PACT as a modulator of nuclear receptor (NR)-regulated gene expression. Here, we investigated the role of PACT in prostate cancer (PCa) using a loss-of-function approach. Depletion of PACT in PCa cell lines resulted in a reduction in cell proliferation; however, they were viable. RNA-sequencing analysis of LNCaP PCa cells ± PACT revealed a depletion of biological processes involved in cell cycle, mitochondrial function, and NR-response pathways in the PACT knockout (KO) cells. In the PACT KO cells, downregulated genes included H2AFJ, PSMD5, AQP3, TMEM45B , SLC22A3, and KLK3 (prostate specific antigen, PSA), and siRNA mediated knockdown of these genes reduced cell growth and proliferation in LNCaP cells. Taken together, these data provide support for PRKRA as a proproliferative gene in PCa and targeting PRKRA , or the genes that are downregulated in PACT KO cells via siRNA therapies, could benefit PCa patient survival. Biological sciences/Cancer/Urological cancer/Prostate cancer Biological sciences/Molecular biology/Non-coding RNAs/siRNAs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Prostate cancer (PCa) is the fourth most commonly diagnosed cancer worldwide, and in 2020 accounted for 3.8% cancer related death in men( 1 ). The increasing awareness of PCa, coupled with improved detection methods, and the development of biomarkers for screening (e.g., Prostate-Specific Antigen (PSA), has attributed to an escalation in PCa diagnoses in recent years( 2 ). However, due to early diagnosis the majority of PCa patients have localized, low-risk disease, do not require treatment or surgery, and have a good prognosis( 3 ). At disease onset, PCa tumor growth is predominantly androgen-dependent, laying the foundation for the development of therapies targeting the androgen receptor (AR) and it’s signaling pathways, and these drugs include the anti-androgens Abiraterone acetate and Enzalutamide( 4 ). Inevitably, the PCa tumors become resistant to the drugs, and the disease transforms into castrate resistant PCa (CRPC)( 4 – 6 ). Current chemotherapies for the treatment of CRPC are predominantly taxane-based (e.g., docetaxel), however their efficacy is poor due to drug resistance, prompting clinical trials with platinum-based therapies (e.g., cisplatin)( 7 , 8 ). Immunotherapy (e.g., sipuleucel-T), and management of bone metastasis with bone-targeted therapy (e.g., radium-233), have also provided survival benefits for patients with CRPC( 8 – 11 ). Recent advances in systemic therapies for the effective treatment of CRPC include targeting the highly expressed prostate-specific membrane antigen (PSMA) with radioligand therapy i.e., 177 Lu-PMSA-617, polyadenosine diphosphate-ribosome polymerase inhibitors (PARPi) e.g., Talazoparib( 11 – 13 ), and ultrasound therapy, particularly when combined with anticancer drugs contained within or on ‘microbubbles’ or targeted nanobubbles to PSMA for targeted drug delivery and release( 14 – 17 ). Despite these advances in treatment options for CRPC, the disease remains incurable, emphasizing the unmet clinical need for new PCa therapeutics. PACT was initially discovered as a facilitator of mammalian anti-viral defense mechanisms by activating the double-stranded RNA-activated protein kinase (PKR)( 18 ). In addition, PACT activates retinoic acid-induced gene 1 (RIG-1) mediated signaling to regulate interferon induction following viral infection( 19 , 20 ). Subsequently, PACT, together with transactivation response RNA binding protein (TRBP) and Dicer, has been described as an integral member of the cytoplasmic RNA-induced silencing complex (RISC), which regulates the processing of microRNAs for the targeted silencing of gene expression through mRNA cleavage, or translational repression ( 21 , 22 ). We previously reported PACT to be a nuclear receptor (NR)-coactivator, which regulated AR activity and downstream gene expression in PCa, providing linkages between NR-coregulators and the microRNA processing machinery( 23 ). PACT overexpression promoted tumorigenesis in other cancers, including epithelial skin cancer, hepatocellular carcinoma, colorectal adenocarcinoma, and pancreatic cancer( 24 – 27 ), and disseminated chemoresistance in mucinous ovarian and basal-like breast cancers( 28 , 29 ). In addition, there are several other studies supporting PACT’s requisite role in the facilitation of cell growth, including in ear development and hearing, in germline stem cell fate, in postnatal anterior pituitary proliferation, and in skull and brain development( 30 – 34 ). In the present study, we investigated the role of PACT in PCa using a loss-of-function approach, to further understand its potential as a prospective PCa therapeutic. Depletion of PACT from human PCa cell lines resulted in a significant reduction in cell proliferation, cell cycle arrest at G0/G1, and an increase in apoptosis. RNA-sequencing analysis of differentially expressed genes between LNCaP parental and LNCaP PACT knockout cells, revealed a reduction in biological processes and hallmark gene sets pertaining to cell proliferation when PACT was depleted. Small interfering RNA (siRNA) mediated targeting of several downregulated genes; including H2AFJ, PSMD5, AQP3, TMEM45B , SLC22A3 , and KLK3 (PSA), in LNCaP parental cells recapitulated the functional effects of PACT knockdown. These data collectively support the notion that PACT is proproliferative in PCa, and the potential to therapeutically target PACT, or the genes that are downregulated in the absence of PACT, could be of great benefit to overall CRPC patient survival. RESULTS PACT sustains proliferation in prostate cancer cells To identify the functional effects of PACT expression on PCa cell growth we performed loss-of-function studies in LNCaP, C4-2B, 22Rv1, PC3, and DU145 human PCa cell lines. We initially used siRNA to mediate the transient knockdown of PACT in the cell lines. We firstly assessed three different PACT siRNAs (si-PACT) in LNCaP cells (Supplementary Fig. S1A) and selected #s16334 for use in subsequent experiments. We transiently transfected the PCa cells with si-PACT or a negative control siRNA (si-NC) and assessed cell proliferation using a Cell Titer end-point assay and the xCELLigence real time system. The cells with siRNA mediated knockdown of PACT exhibited a growth reduction as compared to the si-NC transfected cells using both methods of evaluation (Fig. 1A), indicating the requisite role for PACT in cell proliferation. As an alternate approach to reducing PACT via siRNA, we used CRISPR-Cas9 to knock PACT out of the androgen-dependent LNCaP cells. PCR and sequencing confirmed correct targeting of CRISPR-Cas9 to the PACT locus (Supplementary Fig. S2A), and verification of PACT protein depletion was by western blot (Fig. 1B (i)). There was a significant reduction in the proliferation of the PACT knockout (KO) cells compared to the parental cells as assessed by Cell Titer assay (Fig. 1B (ii)), which was in concordance with the PACT siRNA knockdown data (Fig. 1A). Furthermore, colony formation assays showed that LNCaP parental cells formed more colonies than their PACT KO counterparts (Fig. 1B (iii)), indicating a requirement for PACT in cell proliferation, albeit dispensable for cell survival. To further investigate if the reduced cell proliferation in PACT KO cells is directly due to the absence of PACT, we used lentiviral transduction of PACT cDNA to reconstitute PACT protein in the cells (confirmed by western blotting, Fig. 1C (i)). Re-expression of PACT in the PACT KO cells rescued the reduced cell proliferation phenotype back to the level equivalent to the LNCaP parental cells in Cell Titer and colony formation assays (Fig. 1C (ii-iii)). The loss-of-function studies using siRNA and CRISPR KO, together with the rescue experiment, suggest a direct role for PACT in sustaining the proliferation of PCa cells. PACT expression positively correlates with genes involved in metabolic processes Based on the loss-of-function studies using established PCa cell lines, PACT is required for sustaining the proliferation of PCa cells. To investigate if this association with proliferation exists in clinical samples, we interrogated the Prostate Adenocarcinoma samples within the TCGA PanCancer Atlas. Using the Database for Annotation, Visualization and Integrated Discovery (DAVID) we performed gene ontology (GO) pathway analyses on the top 200 genes which showed positive or negative correlation to PRKRA/ PACT expression (see gene lists in Supplementary Tables 1 and 2). The biological processes of topmost co-expressed genes included those involved with mitochondrial function and associated metabolic pathways e.g., ATP5PB (Fig. 2A). In contrast, the genes that are inversely correlated to PACT are enriched with biological processes involving epigenetic regulation and chromatin organization e.g., ASXL1 (Fig. 2B). Based on the requirement for PACT in PCa cell proliferation, and the co-expressed gene pathways in patients, we hypothesize that functionally PACT enhances cell growth by regulating metabolic processes. PACT regulates PCa cell cycle progression To explore the alterations in gene expression upon PACT depletion in PCa cells, we performed RNA-Sequencing (RNA-seq) analysis of LNCaP parental versus PACT CRISPR KO cells. Using ≥1.5 absolute fold change (FC) (0.58 log 2 FC) and a False Discovery Rate (FDR) of p<0.05, we identified 718 differentially expressed genes (DEGs) between the two groups; 343 DEGs were downregulated, and 375 DEGs were upregulated in the absence of PACT (Fig. 3A and Supplementary Tables 3 and 4). DAVID GO and Kyoto Encyclopedia of Genes and Genomes (KEGG)(35) pathway analyses of the downregulated DEGs identified mitotic nuclear division, cell division, cell cycle, and steroid hormone biosynthesis as the most enriched biological processes (Fig. 3B). The pathway analyses of the upregulated genes were mixed and less significant than the downregulated processes, without a clear trend (Fig. 3C). The enrichment of pathways relating to cell division in the downregulated DEGs in the PACT KO cells corroborated with the reduced proliferation phenotypes observed in the PACT loss-of-function studies. To confirm the finding, we performed cell cycle analyses in LNCaP PCa cells with either transient (siRNA) or stable (CRISPR) mediated PACT depletion. Using propidium iodide (PI) staining and flow cytometry we detected an increase in cell cycle arrest at G0/G1 and a block in transition to S phase using both methods of PACT knockdown (Fig. 3D). Due to the cell cycle arrest at G0/G1, we also investigated the effect of PACT reduction on apoptosis in LNCaP cells using annexin V/PI staining and flow cytometry and observed an increase in apoptotic cells with PACT depletion (Fig. 3E). Taken together, these data support the notion that the growth reduction in PACT KO cells is, in part, via alteration of cell cycle progression and apoptosis, further validating the proproliferative function of PACT in PCa growth. Targeted knockdown of genes downregulated in the absence of PACT affects PCa cell proliferation We validated five of the most downregulated and three of the most upregulated genes in the PACT KO cells from the RNA-seq. H2AFJ (H2A histone family member J) , PSMD5 (proteasome 26s non-ATPase subunit, 5) , AQP3 (Aquaporin 3), TMEM45B (transmembrane protein 45B), and SLC22A3 (Solute carrier family 22 member 3) were respectively downregulated 10.76, 7.18, 5.88, 4.63, and 3.9 log 2 fold change; and NOVA1 (neuro-oncological ventral antigen-1), PXDN (Peroxidasin), and RASSF2 (Ras association domain-containing protein 2),were respectively upregulated 2.77, 2.33, and 1.4 log 2 fold change, in the PACT KO cells (Supplementary Tables 3 and 4). RT-qPCR quantification of each of these genes in LNCaP parental versus PACT KO cells verified the RNA-seq data (Fig. 4A). We next used siRNA to transiently knockdown H2AFJ , PSMD5 , AQP3 , TMEM45B , and SLC22A3 gene expression in LNCaP PCa cells, to assess the functional effects of these molecules on PCa growth. We initially evaluated three different siRNAs for each gene (Supplementary Fig. S3A (i-v)) and selected optimal siRNAs for our experiments (#s31462 H2AFJ ; #SI03167108 PSMD5 ; #s1521 AQP3 ; #s42359 TMEM45B ; and #s13107 SLC22A3 ). We transiently transfected LNCaP cells with either gene-specific siRNA or si-NC and assessed cell proliferation using a Cell Titer assay at 2 to 6 days post-transfection. Gene-specific siRNA transfected cells exhibited a substantial growth reduction as compared to the si-NC transfected cells for each gene (Fig. 4B), which supports the notion that downregulating PACT, or the genes which are downregulated in the absence of PACT could also benefit PCa patient prognosis by reducing the tumor growth. PACT modulates key androgen receptor signaling molecules We next interrogated the entire RNA-seq expression data using Gene Set Enrichment Analysis (GSEA). The GO biological processes and Hallmark gene sets (FDR<0.25, p<0.01) both showed the depletion of gene sets relating to cell cycle and proliferation in the KO cells (e.g., regulation of cell cycle G1/S phase transition, and G2/M checkpoint) (Fig. 5A (i and iii) and 5B (i)). Moreover, biological processes relating to RNA transport and localization, chromosome organization and DNA repair were also depleted in the PACT KO cells (Fig. 5A (i-ii) and 5B (i)), congruent with the reported roles of PACT; acting via PKR and the RISC complex(18, 21). Interestingly, some of the depleted Hallmark gene sets in the PACT KO cells were NR-function related, including fatty acid metabolism, cholesterol homeostasis, estrogen response late, and bile acid metabolism (Fig. 5B (i-v)), and as we previously described PACT as a NR-coregulator regulating AR activity and downstream gene expression in PCa, we performed GSEA against Hallmark for Androgen response. We observed the upregulation of Androgen response genes in the parental cells relative to the PACT KO cells, indicating a depletion of the gene set in the absence of PACT (Fig. 5C). We further scrutinized the RNA-seq data to determine if known androgen-regulated genes were differentially expressed in the PACT KO cells, and notably the Kallikrein family members KLK3 (Kallikrein related peptidase 3, Prostate-specific antigen, PSA), KLK2 (Human kallikrein), and KLK4 (PSA-related serine protease) were all downregulated (2.49, 0.83 and 0.47 log 2 fold change, respectively) in the PACT KO cells (Supplementary Table 3, and GSE253245). We treated LNCaP parental and PACT KO cells ± dihydrotestosterone (DHT) and validated the differential expression, and hormone responsiveness of these androgen-regulated genes (Fig. 5D). The PACT KO cells also exhibited a lower hormone mediated expression of each gene (Fig. 5D). We subsequently used siRNA to transiently knockdown PSA in LNCaP PCa cells to assess the functional effects of PSA gene expression on PCa cell growth. We initially tested three different PSA targeting siRNAs (Supplementary Fig. S3C) and selected #SI03078299 for our studies. siRNA-mediated knockdown of PSA resulted in a reduction in cell proliferation at 3- and 6-days post-transfection via a Cell Titer assay, reduced colony formation in clonogenicity assays, and induced cell cycle arrest at G0/G1 (Fig. 5E). This data supports the role of PACT in modulating key AR-signaling molecules, the targeting of which could abrogate the proproliferative function of PACT. TRBP has a compensatory role for cell survival when PACT is depleted from LNCaP PCa cells Our findings suggest that PACT sustains PCa cell proliferation, although it is dispensable for cell survival.Given that PACT and TRBP are both integral members of the RISC complex and co-regulate PKR activation, we next used si-TRBP to investigate the role of TRBP in LNCaP cells ± PACT. We firstly evaluated three different siRNAs targeting TRBP (Supplementary Fig. S3E) and selected #s13790 to use in subsequent experiments. We transiently transfected LNCaP parental cells with either si-PACT, si-TRBP or si-NC; and PACT KO (CRISPR) cells with either si-TRBP or si-NC, and assessed TRBP expression, cell proliferation using a Cell Titer assay, colony formation, and cell cycle. When PACT was depleted from LNCaP cells, there was an increase in the expression of TRBP (Fig. 6A). Parental LNCaP cells exhibited a substantial growth reduction with si-PACT or si-TRBP as compared to the si-NC transfected cells, with the levels comparable to PACT KO cells (Fig. 6B (i)). Additionally, in the PACT KO cells, when TRBP was also depleted the cells exhibited extremely poor viability and very few colonies formed in clonogenic assays (Fig. 6B). Cell cycle analysis of parental LNCaP cells transfected with each gene-specific siRNA or si-NC, or PACT KO cells transfected with si-TRBP or si-NC showed that PACT knockdown induced cell cycle arrest at G0/G1, and TRBP knockdown had no effect on cell cycle in parental cells, and a marginal, yet significant additive effect in the PACT KO cells (Fig. 6C). Taken together, these data suggest that both PACT and TRBP are essential for LNCaP PCa cell growth, and TRBP expression compensates when PACT is either depleted or knocked out from these cells. MATERIALS AND METHODS Cell culture LNCaP, C4-2B, 22Rv1, PC3, and DU145 PCa cells were from the American Type Culture Collection and cultured at 37˚C in 5% CO2 with RPMI-1640 supplemented with 10% fetal bovine serum (FBS). Transfection of siRNA molecules Cells were seeded into 6-well or 10 cm dishes and transfected with 20 nM siRNA using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Silencer Select siRNAs to PRKRA (IDs: s16334, s16335 and s16336), H2AFJ (IDs: s31461, s31462 and s31463), AQP3 (IDs: s1521, s1522 and s1523), TMEM45B (IDs: s42358, s42359 and s42360), SLC22A3 (IDs: s13105, s13106 and s13107), TRBP2 (IDs: s13790, s13791 and s13792), and a negative control siRNA (Cat #4390843) were from Thermo Fisher Scientific. siRNA FlexiTube GeneSolution for PSMD5 (GS5711) and KLK3 (GS354) were from Qiagen. RNA extraction, Reverse Transcription and Quantitative Polymerase Chain Reaction (RT-qPCR) Total RNA was extracted from cells using Trizol reagent (Thermo Fisher Scientific) as per the manufacturer’s instuctions. Total RNA (800 ng) was used to generate cDNA using a QuantiTect Reverse Transcription Kit (Qiagen), and PCR performed using SYBR SensiMix (Bioline) and the following QuantiTect primer assays (Qiagen): Hs_PRKRA_1_SG, Hs_H2AFJ_1_SG, Hs_PSMD5_1_SG, Hs_AQP3_1_SG, Hs_TMEM45B_1_SG, Hs_SLC22A31_1_SG, Hs_NOVA1_1_SG, Hs_PXDN_1_SG, Hs_RASSF2_1_SG, Hs_KLK3_1_SG, Hs_KLK2_1_SG, Hs_KLK4_1_SG, Hs_TARBP2_1_SG, Hs_GAPDH_2_SG, and Hs_HPRT1_1_SG. The 2 −ΔΔCt method was used to determine normalised gene expression. Targeted knockout of PACT using CRISPR-Cas 9 LNCaP PCa cells were grown to ~ 80% confluency in 10 cm dishes and transfected with 5 ug PACT CRISPR all in one vector gRNA + Cas9WT plasmid (Sigma; CRISPR target ID: HS0000133877; target gene ID: 8575 ( PRKRA )) using Lipofectamine 2000. 72 h post-transfection green fluorescent protein (GFP) expressing cells were isolated by FACS (FACSCAlibur, BD Biosciences) and seeded at low density in 10 cm dishes. Cells were grown until single colonies formed and selected clones verified to contain the PACT knockdown via western blotting, PCR, and DNA sequencing (see Supplementary Fig. S2 for primers). Stable reconstitution of PACT expression in PACT knockout LNCaP cells LNCaP PACT KO cells stably expressing PACT cDNA (pcDNA-PACT; a gift from Dong-Yan Jin (Addgene plasmid #15667; http://n2t.net/addgene:15667 ; RRID:Addgene_15667)( 36 ) were generated by lentiviral transduction as previously described( 37 ). Briefly, cells were infected with lentiviruses carrying LeGO-iG2-Empty (a gift from Boris Fehse (Addgene plasmid #27341; http://n2t.net/addgene:27341 ; RRID:Addgene_27341)( 38 ) or LeGO-iG2-pcDNA-PACT plasmids, and transduced cells stably expressing GFP were isolated by FACS. Validation of the ectopic expression of PACT in the isolated cells was with western blotting. Hormone treatment 2 x 10 5 LNCaP parental or PACT KO cells were plated into 6-well plates and allowed to settle overnight. Cells were replenished in media supplemented with 10% charcoal stripped FBS for 24 h prior to treatment with 10 nM DHT (Cayman Chemicals) or DMSO (Sigma) vehicle control. Harvesting of RNA or protein was at 24 h post-treatment. Cell proliferation and colony forming assays For cell growth and proliferation assays PCa cells (± 24 h siRNA transfection, as described above) were plated at a density of 5000 cells/well in either; 96 well plates and growth assessed at end-point 1–7 days post-seeding using a CellTiter 96 AQ ueous One Solution Cell Proliferation Assay (Promega) and the Fluostar OPTIMA microplate reader (BMG Scientific), or into 16 well xCELLigence E-plates and measurement of cell proliferation in real time using the xCELLigence instrument (ELITechGroup). For colony forming assays, cells were plated at a low density (1x10 4 cells in a 10 cm dish) and colonies were allowed to develop for ~ 2 weeks prior to staining with crystal violet for visualization as previously described( 39 ). Protein extraction and western immunoblotting Whole cell protein lysates were prepared using mid-RIPA lysis buffer (50mM Tris (pH 7.4), 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) and western blotting performed. Briefly, lysates were resolved on NuPAGE 4–12% Bis-Tris gels (Thermo Fisher Scientific) and transferred to PVDF membranes (Roche). Membranes were blocked in 10% skim milk/Tris-buffered saline Tween 20 and probed with antibodies to PACT (Santa Cruz sc-18768), PSA (DAKO A0562), β-actin (AbCam ab6276), or Tubulin (AbCam ab4074). Protein detection was with horseradish peroxidase-linked anti-goat IgG (AbCam ab6885), anti-mouse IgG (Amersham NA931), or anti-rabbit IgG (Amersham NA934) with Luminata Classico Western HRP substrate (Millipore), and visualization was with either ECLHyperfilm (GE Healthcare) or the iBright Imaging System (Thermo Fisher Scientific). RNA-sequencing (RNA-seq) For RNA-seq analysis, 1x10 6 LNCaP parental and LNCaP PACT CRISPR KO cells were plated into 10 cm dishes (in triplicate) and when the cells were ~ 80% confluent total RNA was extracted using Trizol. Confirmation of the quantity and integrity of extracted RNA was with the 2100 Bioanalyzer (Agilent Technologies), and gene expression profiling was performed at the Australian Genome Research Facility (AGRF) using the Illumina NovaSeq platform and standard protocols. The differential gene expression analysis was performed using edgeR (v3.22.3) and default TMM normalization methods. Cell cycle analysis and Annexin V-APC/PI apoptosis assay LNCaP PCa cells were transfected as described above with 20 nM gene specific siRNA or si-NC for 72 h, followed by collection of both floating and adherent cells for assaying. For cell cycle analysis cells were fixed with cold 100% ethanol, and stained with Propidium Iodide (PI) staining solution (25µg/ml PI and 0.25 µg/ml RNase A in PBS). For assessment of apoptosis, the Annexin V-APC/PI Apoptosis Detection Kit I (BD Biosciences) was used according to manufacturer’s instructions, with no stain, single stain and Camptothecin (10 µM, 24 h (Cayman Chemicals)) treated cells used to set gating strategies. Samples for both assays were analysed using the BD Accuri C6 Flow Cytometer and FlowJo Software (version 7.6.5). For LNCaP parental versus PACT KO cells, cells were seeded into 10cm dishes and assayed as above when ~ 80% confluent. Clinical datasets and pathway analyses The publically available Prostate Adenocarcinoma datasets contained within The Cancer Genome Atlas (TCGA), PanCancer Atlas (Cell, 2018), were accessed via the cBioPortal for Cancer Genomics ( https://www.cbioportal.org/ ). Database for Annotation, Visualization and Integrated Discovery (DAVID; version 2021) was used to perform gene ontology (GO) biological processes and KEGG (Kyoto Encyclopedia of Genes and Genomes)( 35 ) pathway analyses. Gene Set Enrichment Analysis (GSEA), incorporating the MSigDB (Human Molecular Signatures Database), was used to perform GO biological processes and Hallmark gene set analyses of the RNA-Seq data, as previously described( 40 , 41 ). Statistical analysis Graphing and analysis of data was with GraphPad Prism 8 software. Use of the unpaired t -test (two-tailed) determined significant differences in RT-qPCR, cell proliferation, colony formation, apoptosis, and cell cycle analyses. DISCUSSION There is a growing body of evidence that supports a requisite role for PACT in facilitating cellular growth and proliferation in tissue development and in numerous cancers (e.g., breast, liver and colorectal)( 24 – 34 ), however little is known about PACT’s role in PCa or prostate development. Here we used a loss-of-function approach to characterize PACT’s mode of action in PCa and found that the depletion of PACT from PCa cell lines resulted in a reduction in cell proliferation, cell cycle arrest at G0/G1, and an increase in apoptosis. Additionally, there was a decrease in various biological processes and gene sets pertaining to cellular growth and proliferation, and to nuclear receptor (NR) function (e.g., androgen response) in the PACT depleted cells. Further, the expression of the androgen-regulated PSA gene was downregulated in the PACT knockout cells, supporting the role of PACT in modulating key androgen receptor (AR) molecules, the targeting of which could abrogate the proproliferative function of PACT in PCa. The importance of PACT in the endocrine system has been demonstrated in mice where PACT depletion impaired the postnatal development of the anterior pituitary lobe, leading to decreased hormone levels, and defects in the ovaries, mammary glands, and in fertility( 32 ). NRs, such as the AR, control the expression of genes and gene networks involved in cellular energy production at a transcriptional level( 42 ), and our TCGA PCa patient data supports this notion as the biological processes of the top-most genes which co-express with PACT are associated with energy production, such as mitochondrial and metabolic pathways. Congruent with the patient data, when PACT was knocked out of LNCaP cells, the depleted hallmark gene sets included fatty acid metabolism, reactive oxygen species pathway, and cholesterol homeostasis. Our data suggests that in addition to PACT’s widely recognized roles with PKR and the RISC complex, PACT may also have a prominent role in hormone response and facilitation of the NR-related cellular function in PCa cells, and is consistent with our previous finding that PACT is a NR co-activator in PCa cells( 23 ). PSA is commonly used as a biomarker for PCa detection and disease progression, its expression is induced by androgens, and it is regulated transcriptionally by the AR( 43 , 44 ). There is much warranted support for the clinical application of PSA and other kallikreins e.g., KLK2 and KLK4, as targeted therapies in PCa( 45 – 47 ). Niu and colleagues reported that tissue PSA, independent of its protease activity, is attributable to AR-mediated PCa tumour growth( 45 ), and our data using either siPACT or siPSA where we observed a decrease in cell proliferation, and cell cycle arrest at G0/G1, further corroborates the concept of PSA being a promising therapeutic target for the treatment of PCa. We validated five of the most downregulated genes in LNCaP PACT KO cells ( H2AFJ, PSMD5, AQP3, TMEM45B , and SLC22A3 ), and siRNA mediated targeting of these genes in LNCaP cells recapitulated the functional effects of PACT knockdown, and furthermore in the context of this study, these genes have reported roles as oncogenes in cancer. H2AFJ is involved in nucleosome DNA packaging into chromatin, and is implicated to be an oncogene in various cancers; it is overexpressed in luminal breast and prostate cancers( 48 ), and its elevated levels in Glioblastoma Multiforme, and colorectal cancer, is reported to be associated with therapeutic resistance( 49 , 50 ). PSMD5 is a component of the 26S proteosome involved in cellular protein degradation, and in the context of cancer, high levels of PSMD5 expression in patients with prostate adenocarcinoma had poorer overall survival( 51 ), and in multiple myeloma PSMD5 promoter hypermethylation resulted in resistance to proteosome inhibitors( 52 ), suggestive of an oncogenic role of PSMD5 in these scenarios. In contrast, PSMD5 was reported to be reduced in colorectal tumourigenesis and silenced with disease progression( 53 ). AQP3, TMEM45B, and SLC22A3 are all proteins involved in transmembrane transport of various drugs and molecules, and their aberrant overexpression has been implicated in tumourigenesis in numerous cancers (e.g., colorectal, lung, and pancreatic( 54 – 56 ); gastric, and lung( 57 , 58 ); and lung, colorectal, and prostate( 59 – 61 ), respectively). Significantly, in prostate cancer, AQP3 has been reported to promote cell motility and invasion, and its inhibition increases the sensitivity of cancer cells to cryotherapy( 62 , 63 ). Furthermore, both AQP3 and TMEM45B have the potential as predictive biomarkers for PCa progression and metastasis( 64 , 65 ). Additionally, AQP3 and SLC22A3 were identified as androgen-responsive genes via RNA expression profiling of a normal prostate epithelial cell line ( 66 ). More detailed and extensive studies are necessitated to determine the benefits of targeting these genes as potential therapies for the treatment of CRPC. RASSF2, NOVA1, and PXDN were upregulated in the LNCaP PACT KO cells. In a variety of tumours, including prostate and lung, RASSF2 is inactivated by promoter methylation and has the properties of being a tumour suppressor( 67 , 68 ), which supports our observation of increased RASSF2 in the less proliferative PACT KO cells. In contrast, NOVA1 and PXDN are potential oncogenes in numerous cancers (e.g., head and neck carcinoma, and lung cancer; and ovarian, and prostate cancers, respectively) with their overexpression being associated with poor prognoses and tumour progression( 69 – 72 ). The LNCaP cells with either PACT depletion or knockout showed a reduction in proliferation but were viable. PACT and TRBP within the RISC complex regulate the abundance and biogenesis of microRNAs; however, each of them is dispensable in the absence of the other, and their precise role in microRNA processing is not entirely understood( 73 , 74 ). PACT and TRBP interact with PKR and RIG-1 to regulate stress response, viral defense, apoptosis and gene expression, albeit in opposing manners( 18 , 20 , 75 , 76 ). Cellular stress leads to the PACT phosphorylation and initiation of PKR-dependent apoptosis( 32 ), while the overexpression and hyperphosphorylation of TRBP inhibits oxidative stress-induced apoptosis and promotes cell survival via PKR inhibition( 77 ). We hypothesize that the compensatory overexpression of TRBP and the subsequent inhibition of PKR have kept the PACT-depleted LNCaP cells viable, and indeed the depletion of TRBP in PACT KO cells significantly reduced the cell viability. More studies are needed to comprehensively decipher the changes in the molecular landscape in the absence of PACT in PCa cells. There is an urgent need for new treatments for advanced CRPC. RNA-based therapeutics is a rapidly advancing field, with the recent FDA approval of several siRNA based drugs e.g., Inclisiran ( 78 ). In that context, our data provides a foundation for further work to develop siPACT or siPSA as RNA-based therapeutics for the treatment of CRPC, as their siRNA mediated targeting results in a decrease in PCa cell growth and proliferation. In addition, site specific delivery of these siRNAs to the prostate could be achieved by harnessing of the PSMA using nanoparticle or microbubble guided ultrasound technologies, allowing for more effective treatments for patients with PCa. Declarations ACKNOWLEDGEMENTS The authors would like to thank Dr. Tasnuva Kabir for helpful discussions regarding this article. AUTHOR CONTRIBUTIONS DJB, AJW and PJL designed the experiments and drafted the manuscript. DJB, AJW, KLR, RAMB, LMS, and MS performed the experiments. AJW, KLR and DJB performed the bioinformatics/pathways and clinical data set analyses. ADR contributed valuable clinical input. FUNDING The National Health and Medical Research Council of Australia supported this work. COMPETING INTERESTS The authors declare no competing interests. DATA AVAILABILITY The RNA-Seq data is available in the Gene Expression Omnibus under Accession Number GSE253245. References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209-49. Litwin MS, Tan H-J. The Diagnosis and Treatment of Prostate Cancer: A Review. JAMA. 2017;317(24):2532-42. Kinsella N, Helleman J, Bruinsma S, Carlsson S, Cahill D, Brown C, et al. Active surveillance for prostate cancer: a systematic review of contemporary worldwide practices. Transl Androl Urol. 2018;7(1):83-97. Imamura Y, Sadar MD. Androgen receptor targeted therapies in castration-resistant prostate cancer: Bench to clinic. Int J Urol. 2016;23(8):654-65. Grossmann M, Cheung AS, Zajac JD. Androgens and prostate cancer; pathogenesis and deprivation therapy. Best Practice & Research Clinical Endocrinology & Metabolism. 2013;27(4):603-16. Kingsley LA, Fournier PG, Chirgwin JM, Guise TA. Molecular biology of bone metastasis. Mol Cancer Ther. 2007;6(10):2609-17. Ruiz de Porras V, Font A, Aytes A. Chemotherapy in metastatic castration-resistant prostate cancer: Current scenario and future perspectives. Cancer Lett. 2021;523:162-9. Cai M, Song XL, Li XA, Chen M, Guo J, Yang DH, et al. Current therapy and drug resistance in metastatic castration-resistant prostate cancer. Drug Resist Updat. 2023;68:100962. Kantoff PW, Higano CS, Shore ND, Berger ER, Small EJ, Penson DF, et al. Sipuleucel-T immunotherapy for castration-resistant prostate cancer. N Engl J Med. 2010;363(5):411-22. Parker C, Nilsson S, Heinrich D, Helle SI, O'Sullivan JM, Fosså SD, et al. Alpha emitter radium-223 and survival in metastatic prostate cancer. N Engl J Med. 2013;369(3):213-23. Hatano K, Nonomura N. Systemic Therapies for Metastatic Castration-Resistant Prostate Cancer: An Updated Review. World J Mens Health. 2023;41. Sartor O, de Bono J, Chi KN, Fizazi K, Herrmann K, Rahbar K, et al. Lutetium-177–PSMA-617 for Metastatic Castration-Resistant Prostate Cancer. New England Journal of Medicine. 2021;385(12):1091-103. Tisseverasinghe S, Bahoric B, Anidjar M, Probst S, Niazi T. Advances in PARP Inhibitors for Prostate Cancer. Cancers (Basel). 2023;15(6). Lee HJ, Yoon YI, Bae YJ. Theragnostic ultrasound using microbubbles in the treatment of prostate cancer. Ultrasonography. 2016;35(4):309-17. Wang Y, De Leon AC, Perera R, Abenojar E, Gopalakrishnan R, Basilion JP, et al. Molecular imaging of orthotopic prostate cancer with nanobubble ultrasound contrast agents targeted to PSMA. Scientific Reports. 2021;11(1):4726. He M, Cao Y, Chi C, Zhao J, Chong E, Chin KXC, et al. Unleashing novel horizons in advanced prostate cancer treatment: investigating the potential of prostate specific membrane antigen-targeted nanomedicine-based combination therapy. Front Immunol. 2023;14:1265751. Lopez W, Nguyen N, Cao J, Eddow C, Shung KK, Lee NS, et al. Ultrasound Therapy, Chemotherapy and Their Combination for Prostate Cancer. Technol Cancer Res Treat. 2021;20:15330338211011965. Patel RC, Sen GC. PACT, a protein activator of the interferon-induced protein kinase, PKR. EMBO J. 1998;17(15):4379-90. Vaughn LS, Chukwurah E, Patel RC. Opposite actions of two dsRNA-binding proteins PACT and TRBP on RIG-I mediated signaling. Biochemical Journal. 2021;478(3):493-510. Kok KH, Lui PY, Ng MH, Siu KL, Au SW, Jin DY. The double-stranded RNA-binding protein PACT functions as a cellular activator of RIG-I to facilitate innate antiviral response. Cell Host Microbe. 2011;9(4):299-309. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116(2):281-97. Ha M, Kim VN. Regulation of microRNA biogenesis. Nature Reviews Molecular Cell Biology. 2014;15(8):509-24. Redfern AD, Colley SM, Beveridge DJ, Ikeda N, Epis MR, Li X, et al. RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators. Proc Natl Acad Sci U S A. 2013;110(16):6536-41. Sand M, Skrygan M, Georgas D, Arenz C, Gambichler T, Sand D, et al. Expression levels of the microRNA maturing microprocessor complex component DGCR8 and the RNA-induced silencing complex (RISC) components argonaute-1, argonaute-2, PACT, TARBP1, and TARBP2 in epithelial skin cancer. Mol Carcinog. 2012;51(11):916-22. Wang F, Zhu L, Xue Q, Tang C, Tang W, Zhang N, et al. Novel lncRNA AL033381.2 Promotes Hepatocellular Carcinoma Progression by Upregulating PRKRA Expression. Oxidative medicine and cellular longevity. 2022;2022:1125932-. Chiosea S, Acquafondata M, Luo J, Kuan S, Seethala R. DICER1 and PRKRA in Colon Adenocarcinoma. Biomark Insights. 2008;3:253-8. Qiu J, Feng M, Yang G, Su D, Zhao F, Liu Y, et al. PRKRA promotes pancreatic cancer progression by upregulating MMP1 transcription via the NF-κB pathway. Heliyon. 2023;9(6):e17194. Hisamatsu T, McGuire M, Wu SY, Rupaimoole R, Pradeep S, Bayraktar E, et al. PRKRA/PACT Expression Promotes Chemoresistance of Mucinous Ovarian Cancer. Mol Cancer Ther. 2019;18(1):162-72. Wei L, Wang W, Yao J, Cui Z, Xu Z, Ding H, et al. PACT promotes the metastasis of basal-like breast cancer through Rac1 SUMOylation and activation. Oncogene. 2022;41(37):4282-94. Rowe TM, Rizzi M, Hirose K, Peters GA, Sen GC. A role of the double-stranded RNA-binding protein PACT in mouse ear development and hearing. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(15):5823-8. Fujitani K, Otomo A, Nagayama Y, Tachibana T, Kato R, Kawashima Y, et al. PACT/PRKRA and p53 regulate transcriptional activity of DMRT1. Genet Mol Biol. 2020;43(2):e20190017. Peters GA, Seachrist DD, Keri RA, Sen GC. The double-stranded RNA-binding protein, PACT, is required for postnatal anterior pituitary proliferation. Proceedings of the National Academy of Sciences. 2009;106(26):10696. Dickerman BK, White CL, Chevalier C, Nalesso V, Charles C, Fouchécourt S, et al. Missense mutation in the second RNA binding domain reveals a role for Prkra (PACT/RAX) during skull development. PloS one. 2011;6(12):e28537-e. Yong Y, Meng Y, Ding H, Fan Z, Tang Y, Zhou C, et al. PACT/RAX Regulates the Migration of Cerebellar Granule Neurons in the Developing Cerebellum. Scientific Reports. 2015;5(1):7961. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28(1):27-30. Kok KH, Ng MH, Ching YP, Jin DY. Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA. J Biol Chem. 2007;282(24):17649-57. Woo AJ, Patry CA, Ghamari A, Pregernig G, Yuan D, Zheng K, et al. Zfp281 (ZBP-99) plays a functionally redundant role with Zfp148 (ZBP-89) during erythroid development. Blood Adv. 2019;3(16):2499-511. Weber K, Bartsch U, Stocking C, Fehse B. A multicolor panel of novel lentiviral "gene ontology" (LeGO) vectors for functional gene analysis. Mol Ther. 2008;16(4):698-706. Beveridge DJ, Richardson KL, Epis MR, Brown RAM, Stuart LM, Woo AJ, et al. The tumor suppressor miR-642a-5p targets Wilms Tumor 1 gene and cell-cycle progression in prostate cancer. Sci Rep. 2021;11(1):18003. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102(43):15545-50. Liberzon A, Birger C, Thorvaldsdóttir H, Ghandi M, Mesirov JP, Tamayo P. The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst. 2015;1(6):417-25. Scholtes C, Giguère V. Transcriptional control of energy metabolism by nuclear receptors. Nature Reviews Molecular Cell Biology. 2022;23(11):750-70. Kim J, Coetzee GA. Prostate specific antigen gene regulation by androgen receptor. J Cell Biochem. 2004;93(2):233-41. Hong SK. Kallikreins as Biomarkers for Prostate Cancer. BioMed Research International. 2014;2014:526341. Niu Y, Yeh S, Miyamoto H, Li G, Altuwaijri S, Yuan J, et al. Tissue prostate-specific antigen facilitates refractory prostate tumor progression via enhancing ARA70-regulated androgen receptor transactivation. Cancer Res. 2008;68(17):7110-9. Fuhrman-Luck RA, Loessner D, Clements JA. Kallikrein-Related Peptidases in Prostate Cancer: From Molecular Function to Clinical Application. EJIFCC. 2014;25(3):269-81. Bonk S, Kluth M, Jansen K, Hube-Magg C, Makrypidi-Fraune G, Höflmayer D, et al. Reduced KLK2 expression is a strong and independent predictor of poor prognosis in ERG-negative prostate cancer. Prostate. 2020;80(13):1097-107. Redon CE, Schmal Z, Tewary G, Mangelinck A, Courbeyrette R, Thuret JY, et al. Histone Variant H2A.J Is Enriched in Luminal Epithelial Gland Cells. Genes (Basel). 2021;12(11). Lee HH, Lin CH, Lin HY, Kuei CH, Zheng JQ, Wang YH, et al. Histone 2A Family Member J Drives Mesenchymal Transition and Temozolomide Resistance in Glioblastoma Multiforme. Cancers (Basel). 2019;12(1). Wang X, Ghareeb WM, Lu X, Huang Y, Huang S, Chi P. Coexpression network analysis linked H2AFJ to chemoradiation resistance in colorectal cancer. J Cell Biochem. 2019;120(6):10351-62. Yi X, Li J, Zheng X, Xu H, Liao D, Zhang T, et al. Construction of PANoptosis signature: Novel target discovery for prostate cancer immunotherapy. Molecular Therapy - Nucleic Acids. 2023;33:376-90. Haertle L, Barrio S, Munawar U, Han S, Zhou X, Simicek M, et al. Single-Nucleotide Variants and Epimutations Induce Proteasome Inhibitor Resistance in Multiple Myeloma. Clin Cancer Res. 2023;29(1):279-88. Levin A, Minis A, Lalazar G, Rodriguez J, Steller H. PSMD5 Inactivation Promotes 26S Proteasome Assembly during Colorectal Tumor Progression. Cancer Res. 2018;78(13):3458-68. Li A, Lu D, Zhang Y, Li J, Fang Y, Li F, et al. Critical role of aquaporin-3 in epidermal growth factor-induced migration of colorectal carcinoma cells and its clinical significance. Oncol Rep. 2013;29(2):535-40. Xia H, Ma YF, Yu CH, Li YJ, Tang J, Li JB, et al. Aquaporin 3 knockdown suppresses tumour growth and angiogenesis in experimental non-small cell lung cancer. Exp Physiol. 2014;99(7):974-84. Silva PM, da Silva IV, Sarmento MJ, Silva Í C, Carvalho FA, Soveral G, et al. Aquaporin-3 and Aquaporin-5 Facilitate Migration and Cell-Cell Adhesion in Pancreatic Cancer by Modulating Cell Biomechanical Properties. Cells. 2022;11(8). Shen K, Yu W, Yu Y, Liu X, Cui X. Knockdown of TMEM45B inhibits cell proliferation and invasion in gastric cancer. Biomed Pharmacother. 2018;104:576-81. Hu R, Hu F, Xie X, Wang L, Li G, Qiao T, et al. TMEM45B, up-regulated in human lung cancer, enhances tumorigenicity of lung cancer cells. Tumour Biol. 2016;37(9):12181-91. Nguyen T-A, Le M-K, Nguyen P-T, Tran NQV, Kondo T, Nakao A. SLC22A3 that encodes organic cation transporter-3 is associated with prognosis and immunogenicity of human lung squamous cell carcinoma. Translational Lung Cancer Research. 2023;12(10):1972-86. Ren A, Sun S, Li S, Chen T, Shu Y, Du M, et al. Genetic variants in SLC22A3 contribute to the susceptibility to colorectal cancer. Int J Cancer. 2019;145(1):154-63. Grisanzio C, Werner L, Takeda D, Awoyemi BC, Pomerantz MM, Yamada H, et al. Genetic and functional analyses implicate the NUDT11, HNF1B, and SLC22A3 genes in prostate cancer pathogenesis. Proc Natl Acad Sci U S A. 2012;109(28):11252-7. de Almeida A, Parthimos D, Dew H, Smart O, Wiltshire M, Errington RJ. Aquaglyceroporin-3's Expression and Cellular Localization Is Differentially Modulated by Hypoxia in Prostate Cancer Cell Lines. Cells. 2021;10(4). Ismail M, Bokaee S, Davies J, Harrington KJ, Pandha H. Inhibition of the aquaporin 3 water channel increases the sensitivity of prostate cancer cells to cryotherapy. British Journal of Cancer. 2009;100(12):1889-95. Kushwaha PP, Verma S, Gupta S. Aquaporins as Prognostic Biomarker in Prostate Cancer. Cancers. 2023;15(2):331. Luo F, Yang K, Wang YZ, Lin D. TMEM45B is a novel predictive biomarker for prostate cancer progression and metastasis. Neoplasma. 2018;65(5):815-21. Bolton EC, So AY, Chaivorapol C, Haqq CM, Li H, Yamamoto KR. Cell- and gene-specific regulation of primary target genes by the androgen receptor. Genes Dev. 2007;21(16):2005-17. Yi Y, Nandana S, Case T, Nelson C, Radmilovic T, Matusik RJ, et al. Candidate metastasis suppressor genes uncovered by array comparative genomic hybridization in a mouse allograft model of prostate cancer. Mol Cytogenet. 2009;2:18. Clark J, Freeman J, Donninger H. Loss of RASSF2 Enhances Tumorigencity of Lung Cancer Cells and Confers Resistance to Chemotherapy. Mol Biol Int. 2012;2012:705948. Kim EK, Cho YA, Seo MK, Ryu H, Cho BC, Koh YW, et al. NOVA1 induction by inflammation and NOVA1 suppression by epigenetic regulation in head and neck squamous cell carcinoma. Sci Rep. 2019;9(1):11231. Qu L, Tian Y, Wang F, Li Z. NOVA1 promotes NSCLC proliferation and invasion by activating Wnt/β-catenin signaling. BMC Cancer. 2022;22(1):1091. Zheng YZ, Liang L. High expression of PXDN is associated with poor prognosis and promotes proliferation, invasion as well as migration in ovarian cancer. Ann Diagn Pathol. 2018;34:161-5. Dougan J, Hawsawi O, Burton LJ, Edwards G, Jones K, Zou J, et al. Proteomics-Metabolomics Combined Approach Identifies Peroxidasin as a Protector against Metabolic and Oxidative Stress in Prostate Cancer. Int J Mol Sci. 2019;20(12). Kim Y, Yeo J, Lee Jung H, Cho J, Seo D, Kim J-S, et al. Deletion of Human tarbp2 Reveals Cellular MicroRNA Targets and Cell-Cycle Function of TRBP. Cell Reports. 2014;9(3):1061-74. Pullagura SRN, Buaas B, Gray N, Krening LC, Srivastava A, Braun RE. Functional Redundancy of DICER Cofactors TARBP2 and PRKRA During Murine Embryogenesis Does Not Involve miRNA Biogenesis. Genetics. 2018;208(4):1513-22. Heyam A, Lagos D, Plevin M. Dissecting the roles of TRBP and PACT in double-stranded RNA recognition and processing of noncoding RNAs. Wiley Interdiscip Rev RNA. 2015;6(3):271-89. Park H, Davies MV, Langland JO, Chang HW, Nam YS, Tartaglia J, et al. TAR RNA-binding protein is an inhibitor of the interferon-induced protein kinase PKR. Proc Natl Acad Sci U S A. 1994;91(11):4713-7. Chukwurah E, Patel RC. Stress-induced TRBP phosphorylation enhances its interaction with PKR to regulate cellular survival. Scientific Reports. 2018;8(1):1020. Traber GM, Yu AM. RNAi-Based Therapeutics and Novel RNA Bioengineering Technologies. J Pharmacol Exp Ther. 2023;384(1):133-54. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files SupplementaryDataFiguresXLegendsXTables.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4121983","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":292640753,"identity":"b5ba59cd-3ca2-4b37-91a3-bc4a11d4f16d","order_by":0,"name":"Peter Leedman","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-1831-4804","institution":"Harry Perkins Institute of Medical Research","correspondingAuthor":true,"prefix":"","firstName":"Peter","middleName":"","lastName":"Leedman","suffix":""},{"id":292640754,"identity":"7d066a07-8951-4b03-bbfd-60def3cb3e16","order_by":1,"name":"Dianne Beveridge","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Dianne","middleName":"","lastName":"Beveridge","suffix":""},{"id":292640755,"identity":"0483094b-1a9c-4d7f-98f8-2f1898206e84","order_by":2,"name":"Andrew Woo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"","lastName":"Woo","suffix":""},{"id":292640756,"identity":"072e6335-fb78-42d1-8b76-602148b52c8d","order_by":3,"name":"Kirsty Richardson","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kirsty","middleName":"","lastName":"Richardson","suffix":""},{"id":292640757,"identity":"571515cf-02fe-4acf-a4f1-95f4bc23a3b1","order_by":4,"name":"Rikki Brown","email":"","orcid":"https://orcid.org/0000-0001-5970-7236","institution":"Harry Perkins Institute of Medical Research","correspondingAuthor":false,"prefix":"","firstName":"Rikki","middleName":"","lastName":"Brown","suffix":""},{"id":292640758,"identity":"dc9b01df-09ad-45fe-9c83-2fb2950d917f","order_by":5,"name":"Lisa Stuart","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lisa","middleName":"","lastName":"Stuart","suffix":""},{"id":292640759,"identity":"4f01cb5b-d8ce-42d1-8f25-71de47a47af3","order_by":6,"name":"Manjot Singh","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Manjot","middleName":"","lastName":"Singh","suffix":""},{"id":292640760,"identity":"0b5e8826-6246-48ce-b916-df89e6d08cf2","order_by":7,"name":"Andrew Redfern","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"","lastName":"Redfern","suffix":""}],"badges":[],"createdAt":"2024-03-18 09:50:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4121983/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4121983/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55329718,"identity":"bcb6f397-e0b7-4c37-86f4-9013b0aa2e8e","added_by":"auto","created_at":"2024-04-25 19:03:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":209279,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePACT sustains proliferation in prostate cancer cells. (A) \u003c/strong\u003eLNCaP, C4-2B, 22Rv1, PC3, and DU145 PCa cells were treated with negative control siRNA (si-NC) or PACT siRNA (si-PACT) for one day and then harvested for RNA extraction or for seeding into appropriate plates for 72 h for \u003cstrong\u003e(i) \u003c/strong\u003ecell titre proliferation assay; and \u003cstrong\u003e(ii)\u003c/strong\u003e xCELLigence assay. RT-qPCR for PACT gene expression is shown in Supplementary Fig. S1B (i-v). \u003cstrong\u003e(B) \u003c/strong\u003eComparison of LNCaP parental and CRISPR PACT knockout (PACT KO) cells \u003cstrong\u003e(i)\u003c/strong\u003e western blot for PACT expression, tubulin loading control; \u003cstrong\u003e(ii)\u003c/strong\u003e cell titre proliferation assay; and \u003cstrong\u003e(iii)\u003c/strong\u003e colony forming assays.\u003cstrong\u003e (C)\u003c/strong\u003e Comparison of LNCaP parental and PACT KO cells stably overexpressing empty vector (EV) or PACT cDNA (PACT OE) \u003cstrong\u003e(i)\u003c/strong\u003e western blot for PACT expression, b-actin loading control; (ii) cell titre proliferation assay; and \u003cstrong\u003e(iii)\u003c/strong\u003e colony forming assays.\u003cstrong\u003e \u003c/strong\u003eError bars = SD and are representative of three independent experiments. *p\u0026lt;0.05, **p\u0026lt;0.005 relative to si-NC or parental cells. \u003csup\u003e##\u003c/sup\u003ep\u0026lt;0.005 PACT KO PACT OE relative to PACT KO EV. Original western blots are shown in Supplementary Fig. S2 (B-C).\u003c/p\u003e","description":"","filename":"Binder51.png","url":"https://assets-eu.researchsquare.com/files/rs-4121983/v1/6734bc6bb00a5b3c21fd56b5.png"},{"id":55329715,"identity":"c4c4db6d-369c-496f-955c-b713cf60ea3f","added_by":"auto","created_at":"2024-04-25 19:03:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":82788,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePACT expression positively correlates with genes involved in metabolic processes. (A) (i)\u003c/strong\u003eGene ontology (GO) Biological Processes analyses of the top 200 genes whose expression was positively correlated with PACT expression (P\u0026lt;0.05, and Spearman’s correlation values highest to lowest), and \u003cstrong\u003e(ii)\u003c/strong\u003e ATP5BP gene expression positively correlated to PACT mRNA expression. \u003cstrong\u003e(B) (i)\u003c/strong\u003e GO Biological Processes analyses of the top 200 genes whose expression was negatively correlated with PACT expression (P\u0026lt;0.05, and Spearman’s correlation values highest to lowest), and \u003cstrong\u003e(ii)\u003c/strong\u003e Expression of ASLX1 negatively correlated to PACT expression. The mRNA expression was batch normalized from Illumina HISeq_RNA Seq V2, and the Spearman’s correlation, p-values, and q-values are indicated in Supplementary Tables 1 and 2.\u003c/p\u003e","description":"","filename":"Binder52.png","url":"https://assets-eu.researchsquare.com/files/rs-4121983/v1/3c4b55c2418d7bc63f9dccbf.png"},{"id":55329719,"identity":"ad229ef1-5560-411e-b8f8-af42246e603f","added_by":"auto","created_at":"2024-04-25 19:03:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":73317,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePACT regulates PCa cell cycle progression. (A) \u003c/strong\u003eVolcano plot of the differentially expressed genes between the LNCaP parental versus LNCaP PACT CRISPR knockout (PACT KO) cell lines, using ≥1.5 absolute fold change (FC) (0.58 log\u003csub\u003e2\u003c/sub\u003e FC) and a False Discovery Rate (FDR) of p\u0026lt;0.05, with blue dots representing genes downregulated, and red dots representing genes upregulated with PACT KO.\u003cstrong\u003e (B-C) \u003c/strong\u003eDAVID gene ontology (GO) enrichment analyses of the pathways downregulated \u003cstrong\u003e(B)\u003c/strong\u003e and upregulated \u003cstrong\u003e(C)\u003c/strong\u003e with PACT KO; \u003cstrong\u003e(i)\u003c/strong\u003e GO Biological processes, and \u003cstrong\u003e(ii) \u003c/strong\u003eKEGG pathways.\u003cstrong\u003e (D-E)\u003c/strong\u003e Flow cytometry cell cycle analysis \u003cstrong\u003e(D) \u003c/strong\u003eand apoptosis analysis \u003cstrong\u003e(E)\u003c/strong\u003e of LNCaP cells ± PACT; \u003cstrong\u003e(i) \u003c/strong\u003eLNCaP cells transfected with si-NC or si-PACT (20 nM) for 72 h, and \u003cstrong\u003e(ii)\u003c/strong\u003e LNCaP parental and LNCaP PACT KO cells assayed 72 h post-plating. n = 3; *p\u0026lt;0.05, **p\u0026lt;0.005 relative to si-NC or parental cells. See Supplementary Fig. S1C (i-ii) for RT-qPCR validation of PACT knockdown.\u003c/p\u003e","description":"","filename":"Binder53.png","url":"https://assets-eu.researchsquare.com/files/rs-4121983/v1/ad6acc44f2a38a009f1af8ed.png"},{"id":55329716,"identity":"12ca7f3a-259e-4f8d-a0dd-fab6e17ae112","added_by":"auto","created_at":"2024-04-25 19:03:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":35496,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeted knockdown of genes downregulated in the absence of PACT affects PCa cell proliferation. (A) \u003c/strong\u003eRT-qPCR validation of differentially expressed genes from the RNA-seq in LNCaP parental and LNCaP PACT KO cells. Expression of mRNAs is normalised to HPRT housekeeping gene expression, calculated using the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method, and relative to parental LNCaP.\u003cstrong\u003e \u003c/strong\u003eError bars = SE; n = 3; **p\u0026lt;0.005 relative to parental cells. \u003cstrong\u003e(B)\u003c/strong\u003e Transient transfection of LNCaP cells was with 20 nM gene specific siRNAs or si-NC and the effects of gene knockdown measured via cell proliferation assay at 2- to 6-days post-transfection. See Supplementary Fig. S3B for validation of siRNA mediated gene knockdown. Error bars = SD; n = 3; *p\u0026lt;0.05, **p\u0026lt;0.005 relative to si-NC.\u003c/p\u003e","description":"","filename":"Binder54.png","url":"https://assets-eu.researchsquare.com/files/rs-4121983/v1/0e40c37a25cec083861b6106.png"},{"id":55329721,"identity":"6cae9d1d-0707-41d5-b2e3-41b45f9e7901","added_by":"auto","created_at":"2024-04-25 19:03:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":271926,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePACT modulates key androgen receptor signaling molecules.\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003eGene Set Enrichment Analysis (GSEA) and representative enrichment score plots of the RNA-Seq data;\u003cstrong\u003e (A)\u003c/strong\u003e Biological processes, \u003cstrong\u003e(B)\u003c/strong\u003e Hallmark gene sets, and \u003cstrong\u003e(C)\u003c/strong\u003e Androgen response Hallmark. The y-axis and the green line show the enrichment score for each gene, illustrated as a vertical line plotted in rank order of the most gene abundance (red, left) to the least gene abundance (blue, right) within the indicated samples (as log\u003csub\u003e2\u003c/sub\u003eFC/comparison); the black vertical lines correspond to member genes from the set. NES normalized enrichment score, FDR false discovery rate. Pathways ranked by the p-value result of a Fisher’s exact test. \u003cstrong\u003e(D)\u003c/strong\u003e LNCaP parental and PACT KO cells were treated for 24 h ± DHT \u003cstrong\u003e(i)\u003c/strong\u003e PSA mRNA and protein expression (b-actin loading control (for original, uncropped western see Supplementary Fig. S2D)), \u003cstrong\u003e(ii)\u003c/strong\u003e KLK2 mRNA expression, and \u003cstrong\u003e(iii)\u003c/strong\u003e KLK4 mRNA expression. \u003cstrong\u003e(E)\u003c/strong\u003e LNCaP cells were transiently transfected with 20 nM PSA siRNA (si-PSA) or negative control siRNA (si-NC) and assayed for the effects of gene knockdown via \u003cstrong\u003e(i)\u003c/strong\u003e cell proliferation at 3 d and 6 d post-transfection; \u003cstrong\u003e(ii)\u003c/strong\u003e colony formation at ~ 2 weeks post-transfection; and \u003cstrong\u003e(iii)\u003c/strong\u003e flow cytometry cell cycle analysis 72 h post-transfection. See Supplementary Fig. S3D (i-iii) for validation of siRNA mediated PSA knockdown. Expression of mRNAs via RT-qPCR is normalised to GAPDH housekeeping gene expression, calculated using the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method, and relative to parental LNCaP (no treatment); Error bars = SE; n = 3; **p\u0026lt;0.005 relative to parental cells (no treatment); \u003csup\u003e#\u003c/sup\u003ep\u0026lt;0.05 and \u003csup\u003e##\u003c/sup\u003ep\u0026lt;0.005 LNCaP +DHT relative to PACT KO +DHT; \u003csup\u003e$$\u003c/sup\u003ep\u0026lt;0.005 PACT KO relative to PACT KO +DHT. For cell proliferation and colony forming assays; Error bars = SD; n = 3; *p\u0026lt;0.05, **p\u0026lt;0.005 relative to si-NC.\u003c/p\u003e","description":"","filename":"Binder55.png","url":"https://assets-eu.researchsquare.com/files/rs-4121983/v1/edb5b010a5848b3fecc1270e.png"},{"id":55329720,"identity":"5ab5ab4b-f3d4-4ca7-a8c1-44f13a85fc81","added_by":"auto","created_at":"2024-04-25 19:03:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":45074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTRBP has a compensatory role for cell survival when PACT is depleted from LNCaP PCa cells. \u003c/strong\u003eLNCaP parental or PACT KO cells were transiently transfected with either si-NC, si-PACT or si-TRBP at 20 nM, and subsequently assayed for \u003cstrong\u003e(A)\u003c/strong\u003e TRBP mRNA expression at 72 h post-transfection; \u003cstrong\u003e(B) (i)\u003c/strong\u003e cell viability with cell titre assay at 72 h post-transfection; \u003cstrong\u003e(ii)\u003c/strong\u003e colony formation at ~2 weeks post-transfection, and \u003cstrong\u003e(C)\u003c/strong\u003e flow cytometry cell cycle analysis at 72 h post-transfection. See Supplementary Fig. S3F (i-iii) for validation of siRNA mediated PACT or TRBP knockdown. Expression of TRBP mRNA is normalised to GAPDH housekeeping gene expression, calculated using the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method, and relative to si-NC parental LNCaP. Error bars = SE (A) or SD (B-C); n = 3; *p\u0026lt;0.05, **p\u0026lt;0.005 relative to si-NC parental cells;\u003csup\u003e #\u003c/sup\u003ep\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003ep\u0026lt;0.005 si-NC PACT KO relative to si-TRBP PACT KO.\u003c/p\u003e","description":"","filename":"Binder56.png","url":"https://assets-eu.researchsquare.com/files/rs-4121983/v1/6fe7450493e1ffc58b70d9ff.png"},{"id":56670078,"identity":"a4a6859c-3bd9-4733-83f1-0d66bf1447bd","added_by":"auto","created_at":"2024-05-17 14:23:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1464733,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4121983/v1/c2a74a0c-7310-4053-a7ad-7c84ff2d4cbd.pdf"},{"id":55329717,"identity":"9c9d31ea-e4cf-4cd8-a1db-6491bde18157","added_by":"auto","created_at":"2024-04-25 19:03:16","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5970232,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryDataFiguresXLegendsXTables.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4121983/v1/c601769fb6bbd458b60a6c2b.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"PACT is requisite for prostate cancer cell proliferation","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eProstate cancer (PCa) is the fourth most commonly diagnosed cancer worldwide, and in 2020 accounted for 3.8% cancer related death in men(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The increasing awareness of PCa, coupled with improved detection methods, and the development of biomarkers for screening (e.g., Prostate-Specific Antigen (PSA), has attributed to an escalation in PCa diagnoses in recent years(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). However, due to early diagnosis the majority of PCa patients have localized, low-risk disease, do not require treatment or surgery, and have a good prognosis(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). At disease onset, PCa tumor growth is predominantly androgen-dependent, laying the foundation for the development of therapies targeting the androgen receptor (AR) and it\u0026rsquo;s signaling pathways, and these drugs include the anti-androgens Abiraterone acetate and Enzalutamide(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Inevitably, the PCa tumors become resistant to the drugs, and the disease transforms into castrate resistant PCa (CRPC)(\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Current chemotherapies for the treatment of CRPC are predominantly taxane-based (e.g., docetaxel), however their efficacy is poor due to drug resistance, prompting clinical trials with platinum-based therapies (e.g., cisplatin)(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Immunotherapy (e.g., sipuleucel-T), and management of bone metastasis with bone-targeted therapy (e.g., radium-233), have also provided survival benefits for patients with CRPC(\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Recent advances in systemic therapies for the effective treatment of CRPC include targeting the highly expressed prostate-specific membrane antigen (PSMA) with radioligand therapy i.e., \u003csup\u003e177\u003c/sup\u003eLu-PMSA-617, polyadenosine diphosphate-ribosome polymerase inhibitors (PARPi) e.g., Talazoparib(\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), and ultrasound therapy, particularly when combined with anticancer drugs contained within or on \u0026lsquo;microbubbles\u0026rsquo; or targeted nanobubbles to PSMA for targeted drug delivery and release(\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Despite these advances in treatment options for CRPC, the disease remains incurable, emphasizing the unmet clinical need for new PCa therapeutics.\u003c/p\u003e \u003cp\u003ePACT was initially discovered as a facilitator of mammalian anti-viral defense mechanisms by activating the double-stranded RNA-activated protein kinase (PKR)(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). In addition, PACT activates retinoic acid-induced gene 1 (RIG-1) mediated signaling to regulate interferon induction following viral infection(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Subsequently, PACT, together with transactivation response RNA binding protein (TRBP) and Dicer, has been described as an integral member of the cytoplasmic RNA-induced silencing complex (RISC), which regulates the processing of microRNAs for the targeted silencing of gene expression through mRNA cleavage, or translational repression (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe previously reported PACT to be a nuclear receptor (NR)-coactivator, which regulated AR activity and downstream gene expression in PCa, providing linkages between NR-coregulators and the microRNA processing machinery(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). PACT overexpression promoted tumorigenesis in other cancers, including epithelial skin cancer, hepatocellular carcinoma, colorectal adenocarcinoma, and pancreatic cancer(\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), and disseminated chemoresistance in mucinous ovarian and basal-like breast cancers(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). In addition, there are several other studies supporting PACT\u0026rsquo;s requisite role in the facilitation of cell growth, including in ear development and hearing, in germline stem cell fate, in postnatal anterior pituitary proliferation, and in skull and brain development(\u003cspan additionalcitationids=\"CR31 CR32 CR33\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, we investigated the role of PACT in PCa using a loss-of-function approach, to further understand its potential as a prospective PCa therapeutic. Depletion of PACT from human PCa cell lines resulted in a significant reduction in cell proliferation, cell cycle arrest at G0/G1, and an increase in apoptosis. RNA-sequencing analysis of differentially expressed genes between LNCaP parental and LNCaP PACT knockout cells, revealed a reduction in biological processes and hallmark gene sets pertaining to cell proliferation when PACT was depleted. Small interfering RNA (siRNA) mediated targeting of several downregulated genes; including \u003cem\u003eH2AFJ, PSMD5, AQP3, TMEM45B\u003c/em\u003e, \u003cem\u003eSLC22A3\u003c/em\u003e, and \u003cem\u003eKLK3\u003c/em\u003e (PSA), in LNCaP parental cells recapitulated the functional effects of PACT knockdown. These data collectively support the notion that PACT is proproliferative in PCa, and the potential to therapeutically target PACT, or the genes that are downregulated in the absence of PACT, could be of great benefit to overall CRPC patient survival.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003ePACT sustains proliferation in prostate cancer cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the functional effects of PACT expression on PCa cell growth we performed loss-of-function studies in LNCaP, C4-2B, 22Rv1, PC3, and DU145 human PCa cell lines. We initially used siRNA to mediate the transient knockdown of PACT in the cell lines. We firstly assessed three different PACT siRNAs (si-PACT) in LNCaP cells (Supplementary Fig. S1A) and selected #s16334 for use in subsequent experiments. We transiently transfected the PCa cells with si-PACT or a negative control siRNA (si-NC) and assessed cell proliferation using a Cell Titer end-point assay and the xCELLigence real time system. The cells with siRNA mediated knockdown of PACT exhibited a growth reduction as compared to the si-NC transfected cells using both methods of evaluation (Fig. 1A), indicating the requisite role for PACT in cell proliferation. \u003c/p\u003e\n\u003cp\u003eAs an alternate approach to reducing PACT via siRNA, we used CRISPR-Cas9 to knock PACT out of the androgen-dependent LNCaP cells. PCR and sequencing confirmed correct targeting of CRISPR-Cas9 to the PACT locus (Supplementary Fig. S2A), and verification of PACT protein depletion was by western blot (Fig. 1B (i)). There was a significant reduction in the proliferation of the PACT knockout (KO) cells compared to the parental cells as assessed by Cell Titer assay (Fig. 1B (ii)), which was in concordance with the PACT siRNA knockdown data (Fig. 1A). Furthermore, colony formation assays showed that LNCaP parental cells formed more colonies than their PACT KO counterparts (Fig. 1B (iii)), indicating a requirement for PACT in cell proliferation, albeit dispensable for cell survival. \u003c/p\u003e\n\u003cp\u003eTo further investigate if the reduced cell proliferation in PACT KO cells is directly due to the absence of PACT, we used lentiviral transduction of PACT cDNA to reconstitute PACT protein in the cells (confirmed by western blotting, Fig. 1C (i)). Re-expression of PACT in the PACT KO cells rescued the reduced cell proliferation phenotype back to the level equivalent to the LNCaP parental cells in Cell Titer and colony formation assays (Fig. 1C (ii-iii)). The loss-of-function studies using siRNA and CRISPR KO, together with the rescue experiment, suggest a direct role for PACT in sustaining the proliferation of PCa cells. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePACT expression positively correlates with genes involved in metabolic processes \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the loss-of-function studies using established PCa cell lines, PACT is required for sustaining the proliferation of PCa cells. To investigate if this association with proliferation exists in clinical samples, we interrogated the Prostate Adenocarcinoma samples within the TCGA PanCancer Atlas. Using the Database for Annotation, Visualization and Integrated Discovery (DAVID) we performed gene ontology (GO) pathway analyses on the top 200 genes which showed positive or negative correlation to \u003cem\u003ePRKRA/\u003c/em\u003ePACT expression (see gene lists in Supplementary Tables 1 and 2). The biological processes of topmost co-expressed genes included those involved with mitochondrial function and associated metabolic pathways e.g., ATP5PB (Fig. 2A). In contrast, the genes that are inversely correlated to PACT are enriched with biological processes involving epigenetic regulation and chromatin organization e.g., ASXL1 (Fig. 2B). Based on the requirement for PACT in PCa cell proliferation, and the co-expressed gene pathways in patients, we hypothesize that functionally PACT enhances cell growth by regulating metabolic processes. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePACT regulates PCa cell cycle progression \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the alterations in gene expression upon PACT depletion in PCa cells, we performed RNA-Sequencing (RNA-seq) analysis of LNCaP parental versus PACT CRISPR KO cells. Using \u0026ge;1.5 absolute fold change (FC) (0.58 log\u003csub\u003e2\u003c/sub\u003e FC) and a False Discovery Rate (FDR) of p\u0026lt;0.05, we identified 718 differentially expressed genes (DEGs) between the two groups; 343 DEGs were downregulated, and 375 DEGs were upregulated in the absence of PACT (Fig. 3A and Supplementary Tables 3 and 4). DAVID GO and Kyoto Encyclopedia of Genes and Genomes (KEGG)(35) pathway analyses of the downregulated DEGs identified mitotic nuclear division, cell division, cell cycle, and steroid hormone biosynthesis as the most enriched biological processes (Fig. 3B). The pathway analyses of the upregulated genes were mixed and less significant than the downregulated processes, without a clear trend (Fig. 3C). \u003c/p\u003e\n\u003cp\u003eThe enrichment of pathways relating to cell division in the downregulated DEGs in the PACT KO cells corroborated with the reduced proliferation phenotypes observed in the PACT loss-of-function studies. To confirm the finding, we performed cell cycle analyses in LNCaP PCa cells with either transient (siRNA) or stable (CRISPR) mediated PACT depletion. Using propidium iodide (PI) staining and flow cytometry we detected an increase in cell cycle arrest at G0/G1 and a block in transition to S phase using both methods of PACT knockdown (Fig. 3D). Due to the cell cycle arrest at G0/G1, we also investigated the effect of PACT reduction on apoptosis in LNCaP cells using annexin V/PI staining and flow cytometry and observed an increase in apoptotic cells with PACT depletion (Fig. 3E). Taken together, these data support the notion that the growth reduction in PACT KO cells is, in part, via alteration of cell cycle progression and apoptosis, further validating the proproliferative function of PACT in PCa growth.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTargeted knockdown of genes downregulated in the absence of PACT affects PCa cell proliferation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe validated five of the most downregulated and three of the most upregulated genes in the PACT KO cells from the RNA-seq. \u003cem\u003eH2AFJ \u003c/em\u003e(H2A histone family member J)\u003cem\u003e, PSMD5 \u003c/em\u003e(proteasome 26s non-ATPase subunit, 5)\u003cem\u003e, AQP3 \u003c/em\u003e(Aquaporin 3),\u003cem\u003eTMEM45B \u003c/em\u003e(transmembrane protein 45B), and \u003cem\u003eSLC22A3 \u003c/em\u003e(Solute carrier family 22 member 3) were respectively downregulated 10.76, 7.18, 5.88, 4.63, and 3.9 log\u003csub\u003e2\u003c/sub\u003e fold change; and \u003cem\u003eNOVA1 \u003c/em\u003e(neuro-oncological ventral antigen-1), \u003cem\u003ePXDN\u003c/em\u003e (Peroxidasin), and\u003cem\u003e RASSF2 \u003c/em\u003e(Ras association domain-containing protein 2),were respectively upregulated 2.77, 2.33, and 1.4 log\u003csub\u003e2\u003c/sub\u003e fold change, in the PACT KO cells (Supplementary Tables 3 and 4). RT-qPCR quantification of each of these genes in LNCaP parental versus PACT KO cells verified the RNA-seq data (Fig. 4A). \u003c/p\u003e\n\u003cp\u003eWe next used siRNA to transiently knockdown \u003cem\u003eH2AFJ\u003c/em\u003e, \u003cem\u003ePSMD5\u003c/em\u003e, \u003cem\u003eAQP3\u003c/em\u003e, \u003cem\u003eTMEM45B\u003c/em\u003e, and \u003cem\u003eSLC22A3\u003c/em\u003e gene expression in LNCaP PCa cells, to assess the functional effects of these molecules on PCa growth. We initially evaluated three different siRNAs for each gene (Supplementary Fig. S3A (i-v)) and selected optimal siRNAs for our experiments (#s31462 \u003cem\u003eH2AFJ\u003c/em\u003e; #SI03167108 \u003cem\u003ePSMD5\u003c/em\u003e; #s1521 \u003cem\u003eAQP3\u003c/em\u003e; #s42359 \u003cem\u003eTMEM45B\u003c/em\u003e; and #s13107 \u003cem\u003eSLC22A3\u003c/em\u003e). We transiently transfected LNCaP cells with either gene-specific siRNA or si-NC and assessed cell proliferation using a Cell Titer assay at 2 to 6 days post-transfection. Gene-specific siRNA transfected cells exhibited a substantial growth reduction as compared to the si-NC transfected cells for each gene (Fig. 4B), which supports the notion that downregulating PACT, or the genes which are downregulated in the absence of PACT could also benefit PCa patient prognosis by reducing the tumor growth.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePACT modulates key androgen receptor signaling molecules\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next interrogated the entire RNA-seq expression data using Gene Set Enrichment Analysis (GSEA). The GO biological processes and Hallmark gene sets (FDR\u0026lt;0.25, p\u0026lt;0.01) both showed the depletion of gene sets relating to cell cycle and proliferation in the KO cells (e.g., regulation of cell cycle G1/S phase transition, and G2/M checkpoint) (Fig. 5A (i and iii) and 5B (i)). Moreover, biological processes relating to RNA transport and localization, chromosome organization and DNA repair were also depleted in the PACT KO cells (Fig. 5A (i-ii) and 5B (i)), congruent with the reported roles of PACT; acting via PKR and the RISC complex(18, 21). \u003c/p\u003e\n\u003cp\u003eInterestingly, some of the depleted Hallmark gene sets in the PACT KO cells were NR-function related, including fatty acid metabolism, cholesterol homeostasis, estrogen response late, and bile acid metabolism (Fig. 5B (i-v)), and as we previously described PACT as a NR-coregulator regulating AR activity and downstream gene expression in PCa, we performed GSEA against Hallmark for Androgen response. We observed the upregulation of Androgen response genes in the parental cells relative to the PACT KO cells, indicating a depletion of the gene set in the absence of PACT (Fig. 5C).\u003c/p\u003e\n\u003cp\u003eWe further scrutinized the RNA-seq data to determine if known androgen-regulated genes were differentially expressed in the PACT KO cells, and notably the Kallikrein family members \u003cem\u003eKLK3\u003c/em\u003e (Kallikrein related peptidase 3, Prostate-specific antigen, PSA), \u003cem\u003eKLK2\u003c/em\u003e (Human kallikrein), and \u003cem\u003eKLK4\u003c/em\u003e (PSA-related serine protease) were all downregulated (2.49, 0.83 and 0.47 log\u003csub\u003e2\u003c/sub\u003e fold change, respectively) in the PACT KO cells (Supplementary Table 3, and GSE253245). We treated LNCaP parental and PACT KO cells \u0026plusmn; dihydrotestosterone (DHT) and validated the differential expression, and hormone responsiveness of these androgen-regulated genes (Fig. 5D). The PACT KO cells also exhibited a lower hormone mediated expression of each gene (Fig. 5D). \u003c/p\u003e\n\u003cp\u003eWe subsequently used siRNA to transiently knockdown PSA in LNCaP PCa cells to assess the functional effects of PSA gene expression on PCa cell growth. We initially tested three different PSA targeting siRNAs (Supplementary Fig. S3C) and selected #SI03078299 for our studies. siRNA-mediated knockdown of PSA resulted in a reduction in cell proliferation at 3- and 6-days post-transfection via a Cell Titer assay, reduced colony formation in clonogenicity assays, and induced cell cycle arrest at G0/G1 (Fig. 5E). \u003c/p\u003e\n\u003cp\u003eThis data supports the role of PACT in modulating key AR-signaling molecules, the targeting of which could abrogate the proproliferative function of PACT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTRBP has a compensatory role for cell survival when PACT is depleted from LNCaP PCa cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur findings suggest that PACT sustains PCa cell proliferation, although it is dispensable for cell survival.Given that PACT and TRBP are both integral members of the RISC complex and co-regulate PKR activation, we next used si-TRBP to investigate the role of TRBP in LNCaP cells \u0026plusmn; PACT. We firstly evaluated three different siRNAs targeting TRBP (Supplementary Fig. S3E) and selected #s13790 to use in subsequent experiments. We transiently transfected LNCaP parental cells with either si-PACT, si-TRBP or si-NC; and PACT KO (CRISPR) cells with either si-TRBP or si-NC, and assessed TRBP expression, cell proliferation using a Cell Titer assay, colony formation, and cell cycle. When PACT was depleted from LNCaP cells, there was an increase in the expression of TRBP (Fig. 6A). Parental LNCaP cells exhibited a substantial growth reduction with si-PACT or si-TRBP as compared to the si-NC transfected cells, with the levels comparable to PACT KO cells (Fig. 6B (i)). Additionally, in the PACT KO cells, when TRBP was also depleted the cells exhibited extremely poor viability and very few colonies formed in clonogenic assays (Fig. 6B). Cell cycle analysis of parental LNCaP cells transfected with each gene-specific siRNA or si-NC, or PACT KO cells transfected with si-TRBP or si-NC showed that PACT knockdown induced cell cycle arrest at G0/G1, and TRBP knockdown had no effect on cell cycle in parental cells, and a marginal, yet significant additive effect in the PACT KO cells (Fig. 6C). \u003c/p\u003e\n\u003cp\u003eTaken together, these data suggest that both PACT and TRBP are essential for LNCaP PCa cell growth, and TRBP expression compensates when PACT is either depleted or knocked out from these cells. \u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eLNCaP, C4-2B, 22Rv1, PC3, and DU145 PCa cells were from the American Type Culture Collection and cultured at 37˚C in 5% CO2 with RPMI-1640 supplemented with 10% fetal bovine serum (FBS).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eTransfection of siRNA molecules\u003c/h2\u003e \u003cp\u003eCells were seeded into 6-well or 10 cm dishes and transfected with 20 nM siRNA using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions. Silencer Select siRNAs to \u003cem\u003ePRKRA\u003c/em\u003e (IDs: s16334, s16335 and s16336), \u003cem\u003eH2AFJ\u003c/em\u003e (IDs: s31461, s31462 and s31463), \u003cem\u003eAQP3\u003c/em\u003e (IDs: s1521, s1522 and s1523), \u003cem\u003eTMEM45B\u003c/em\u003e (IDs: s42358, s42359 and s42360), \u003cem\u003eSLC22A3\u003c/em\u003e (IDs: s13105, s13106 and s13107), \u003cem\u003eTRBP2\u003c/em\u003e (IDs: s13790, s13791 and s13792), and a negative control siRNA (Cat #4390843) were from Thermo Fisher Scientific. siRNA FlexiTube GeneSolution for \u003cem\u003ePSMD5\u003c/em\u003e (GS5711) and \u003cem\u003eKLK3\u003c/em\u003e (GS354) were from Qiagen.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction, Reverse Transcription and Quantitative Polymerase Chain Reaction (RT-qPCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cells using Trizol reagent (Thermo Fisher Scientific) as per the manufacturer\u0026rsquo;s instuctions. Total RNA (800 ng) was used to generate cDNA using a QuantiTect Reverse Transcription Kit (Qiagen), and PCR performed using SYBR SensiMix (Bioline) and the following QuantiTect primer assays (Qiagen): Hs_PRKRA_1_SG, Hs_H2AFJ_1_SG, Hs_PSMD5_1_SG, Hs_AQP3_1_SG, Hs_TMEM45B_1_SG, Hs_SLC22A31_1_SG, Hs_NOVA1_1_SG, Hs_PXDN_1_SG, Hs_RASSF2_1_SG, Hs_KLK3_1_SG, Hs_KLK2_1_SG, Hs_KLK4_1_SG, Hs_TARBP2_1_SG, Hs_GAPDH_2_SG, and Hs_HPRT1_1_SG. The 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method was used to determine normalised gene expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTargeted knockout of PACT using CRISPR-Cas 9\u003c/h2\u003e \u003cp\u003eLNCaP PCa cells were grown to ~\u0026thinsp;80% confluency in 10 cm dishes and transfected with 5 ug PACT CRISPR all in one vector gRNA\u0026thinsp;+\u0026thinsp;Cas9WT plasmid (Sigma; CRISPR target ID: HS0000133877; target gene ID: 8575 (\u003cem\u003ePRKRA\u003c/em\u003e)) using Lipofectamine 2000. 72 h post-transfection green fluorescent protein (GFP) expressing cells were isolated by FACS (FACSCAlibur, BD Biosciences) and seeded at low density in 10 cm dishes. Cells were grown until single colonies formed and selected clones verified to contain the PACT knockdown via western blotting, PCR, and DNA sequencing (see Supplementary Fig. S2 for primers).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStable reconstitution of PACT expression in PACT knockout LNCaP cells\u003c/h2\u003e \u003cp\u003eLNCaP PACT KO cells stably expressing PACT cDNA (pcDNA-PACT; a gift from Dong-Yan Jin (Addgene plasmid #15667; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://n2t.net/addgene:15667\u003c/span\u003e\u003cspan address=\"http://n2t.net/addgene:15667\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; RRID:Addgene_15667)(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) were generated by lentiviral transduction as previously described(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Briefly, cells were infected with lentiviruses carrying LeGO-iG2-Empty (a gift from Boris Fehse (Addgene plasmid #27341; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://n2t.net/addgene:27341\u003c/span\u003e\u003cspan address=\"http://n2t.net/addgene:27341\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; RRID:Addgene_27341)(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) or LeGO-iG2-pcDNA-PACT plasmids, and transduced cells stably expressing GFP were isolated by FACS. Validation of the ectopic expression of PACT in the isolated cells was with western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHormone treatment\u003c/h2\u003e \u003cp\u003e2 x 10\u003csup\u003e5\u003c/sup\u003e LNCaP parental or PACT KO cells were plated into 6-well plates and allowed to settle overnight. Cells were replenished in media supplemented with 10% charcoal stripped FBS for 24 h prior to treatment with 10 nM DHT (Cayman Chemicals) or DMSO (Sigma) vehicle control. Harvesting of RNA or protein was at 24 h post-treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation and colony forming assays\u003c/h2\u003e \u003cp\u003eFor cell growth and proliferation assays PCa cells (\u0026plusmn;\u0026thinsp;24 h siRNA transfection, as described above) were plated at a density of 5000 cells/well in either; 96 well plates and growth assessed at end-point 1\u0026ndash;7 days post-seeding using a CellTiter 96 AQ\u003csub\u003eueous\u003c/sub\u003e One Solution Cell Proliferation Assay (Promega) and the Fluostar OPTIMA microplate reader (BMG Scientific), or into 16 well xCELLigence E-plates and measurement of cell proliferation in real time using the xCELLigence instrument (ELITechGroup). For colony forming assays, cells were plated at a low density (1x10\u003csup\u003e4\u003c/sup\u003e cells in a 10 cm dish) and colonies were allowed to develop for ~\u0026thinsp;2 weeks prior to staining with crystal violet for visualization as previously described(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eProtein extraction and western immunoblotting\u003c/h2\u003e \u003cp\u003eWhole cell protein lysates were prepared using mid-RIPA lysis buffer (50mM Tris (pH 7.4), 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) and western blotting performed. Briefly, lysates were resolved on NuPAGE 4\u0026ndash;12% Bis-Tris gels (Thermo Fisher Scientific) and transferred to PVDF membranes (Roche). Membranes were blocked in 10% skim milk/Tris-buffered saline Tween 20 and probed with antibodies to PACT (Santa Cruz sc-18768), PSA (DAKO A0562), β-actin (AbCam ab6276), or Tubulin (AbCam ab4074). Protein detection was with horseradish peroxidase-linked anti-goat IgG (AbCam ab6885), anti-mouse IgG (Amersham NA931), or anti-rabbit IgG (Amersham NA934) with Luminata Classico Western HRP substrate (Millipore), and visualization was with either ECLHyperfilm (GE Healthcare) or the iBright Imaging System (Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRNA-sequencing (RNA-seq)\u003c/h2\u003e \u003cp\u003eFor RNA-seq analysis, 1x10\u003csup\u003e6\u003c/sup\u003e LNCaP parental and LNCaP PACT CRISPR KO cells were plated into 10 cm dishes (in triplicate) and when the cells were ~\u0026thinsp;80% confluent total RNA was extracted using Trizol. Confirmation of the quantity and integrity of extracted RNA was with the 2100 Bioanalyzer (Agilent Technologies), and gene expression profiling was performed at the Australian Genome Research Facility (AGRF) using the Illumina NovaSeq platform and standard protocols. The differential gene expression analysis was performed using edgeR (v3.22.3) and default TMM normalization methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle analysis and Annexin V-APC/PI apoptosis assay\u003c/h2\u003e \u003cp\u003eLNCaP PCa cells were transfected as described above with 20 nM gene specific siRNA or si-NC for 72 h, followed by collection of both floating and adherent cells for assaying. For cell cycle analysis cells were fixed with cold 100% ethanol, and stained with Propidium Iodide (PI) staining solution (25\u0026micro;g/ml PI and 0.25 \u0026micro;g/ml RNase A in PBS). For assessment of apoptosis, the Annexin V-APC/PI Apoptosis Detection Kit I (BD Biosciences) was used according to manufacturer\u0026rsquo;s instructions, with no stain, single stain and Camptothecin (10 \u0026micro;M, 24 h (Cayman Chemicals)) treated cells used to set gating strategies. Samples for both assays were analysed using the BD Accuri C6 Flow Cytometer and FlowJo Software (version 7.6.5). For LNCaP parental versus PACT KO cells, cells were seeded into 10cm dishes and assayed as above when ~\u0026thinsp;80% confluent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eClinical datasets and pathway analyses\u003c/h2\u003e \u003cp\u003eThe publically available Prostate Adenocarcinoma datasets contained within The Cancer Genome Atlas (TCGA), PanCancer Atlas (Cell, 2018), were accessed via the cBioPortal for Cancer Genomics (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cbioportal.org/\u003c/span\u003e\u003cspan address=\"https://www.cbioportal.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Database for Annotation, Visualization and Integrated Discovery (DAVID; version 2021) was used to perform gene ontology (GO) biological processes and KEGG (Kyoto Encyclopedia of Genes and Genomes)(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) pathway analyses. Gene Set Enrichment Analysis (GSEA), incorporating the MSigDB (Human Molecular Signatures Database), was used to perform GO biological processes and Hallmark gene set analyses of the RNA-Seq data, as previously described(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphing and analysis of data was with GraphPad Prism 8 software. Use of the unpaired \u003cem\u003et\u003c/em\u003e-test (two-tailed) determined significant differences in RT-qPCR, cell proliferation, colony formation, apoptosis, and cell cycle analyses.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThere is a growing body of evidence that supports a requisite role for PACT in facilitating cellular growth and proliferation in tissue development and in numerous cancers (e.g., breast, liver and colorectal)(\u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28 CR29 CR30 CR31 CR32 CR33\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), however little is known about PACT\u0026rsquo;s role in PCa or prostate development. Here we used a loss-of-function approach to characterize PACT\u0026rsquo;s mode of action in PCa and found that the depletion of PACT from PCa cell lines resulted in a reduction in cell proliferation, cell cycle arrest at G0/G1, and an increase in apoptosis. Additionally, there was a decrease in various biological processes and gene sets pertaining to cellular growth and proliferation, and to nuclear receptor (NR) function (e.g., androgen response) in the PACT depleted cells. Further, the expression of the androgen-regulated PSA gene was downregulated in the PACT knockout cells, supporting the role of PACT in modulating key androgen receptor (AR) molecules, the targeting of which could abrogate the proproliferative function of PACT in PCa.\u003c/p\u003e \u003cp\u003eThe importance of PACT in the endocrine system has been demonstrated in mice where PACT depletion impaired the postnatal development of the anterior pituitary lobe, leading to decreased hormone levels, and defects in the ovaries, mammary glands, and in fertility(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). NRs, such as the AR, control the expression of genes and gene networks involved in cellular energy production at a transcriptional level(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), and our TCGA PCa patient data supports this notion as the biological processes of the top-most genes which co-express with PACT are associated with energy production, such as mitochondrial and metabolic pathways. Congruent with the patient data, when PACT was knocked out of LNCaP cells, the depleted hallmark gene sets included fatty acid metabolism, reactive oxygen species pathway, and cholesterol homeostasis. Our data suggests that in addition to PACT\u0026rsquo;s widely recognized roles with PKR and the RISC complex, PACT may also have a prominent role in hormone response and facilitation of the NR-related cellular function in PCa cells, and is consistent with our previous finding that PACT is a NR co-activator in PCa cells(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePSA is commonly used as a biomarker for PCa detection and disease progression, its expression is induced by androgens, and it is regulated transcriptionally by the AR(\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). There is much warranted support for the clinical application of PSA and other kallikreins e.g., KLK2 and KLK4, as targeted therapies in PCa(\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Niu and colleagues reported that tissue PSA, independent of its protease activity, is attributable to AR-mediated PCa tumour growth(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), and our data using either siPACT or siPSA where we observed a decrease in cell proliferation, and cell cycle arrest at G0/G1, further corroborates the concept of PSA being a promising therapeutic target for the treatment of PCa.\u003c/p\u003e \u003cp\u003eWe validated five of the most downregulated genes in LNCaP PACT KO cells (\u003cem\u003eH2AFJ, PSMD5, AQP3, TMEM45B\u003c/em\u003e, and \u003cem\u003eSLC22A3\u003c/em\u003e), and siRNA mediated targeting of these genes in LNCaP cells recapitulated the functional effects of PACT knockdown, and furthermore in the context of this study, these genes have reported roles as oncogenes in cancer. H2AFJ is involved in nucleosome DNA packaging into chromatin, and is implicated to be an oncogene in various cancers; it is overexpressed in luminal breast and prostate cancers(\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e), and its elevated levels in Glioblastoma Multiforme, and colorectal cancer, is reported to be associated with therapeutic resistance(\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). PSMD5 is a component of the 26S proteosome involved in cellular protein degradation, and in the context of cancer, high levels of PSMD5 expression in patients with prostate adenocarcinoma had poorer overall survival(\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e), and in multiple myeloma PSMD5 promoter hypermethylation resulted in resistance to proteosome inhibitors(\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e), suggestive of an oncogenic role of PSMD5 in these scenarios. In contrast, PSMD5 was reported to be reduced in colorectal tumourigenesis and silenced with disease progression(\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). AQP3, TMEM45B, and SLC22A3 are all proteins involved in transmembrane transport of various drugs and molecules, and their aberrant overexpression has been implicated in tumourigenesis in numerous cancers (e.g., colorectal, lung, and pancreatic(\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e); gastric, and lung(\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e); and lung, colorectal, and prostate(\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e), respectively). Significantly, in prostate cancer, AQP3 has been reported to promote cell motility and invasion, and its inhibition increases the sensitivity of cancer cells to cryotherapy(\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). Furthermore, both AQP3 and TMEM45B have the potential as predictive biomarkers for PCa progression and metastasis(\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). Additionally, AQP3 and SLC22A3 were identified as androgen-responsive genes via RNA expression profiling of a normal prostate epithelial cell line (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). More detailed and extensive studies are necessitated to determine the benefits of targeting these genes as potential therapies for the treatment of CRPC.\u003c/p\u003e \u003cp\u003eRASSF2, NOVA1, and PXDN were upregulated in the LNCaP PACT KO cells. In a variety of tumours, including prostate and lung, RASSF2 is inactivated by promoter methylation and has the properties of being a tumour suppressor(\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e), which supports our observation of increased RASSF2 in the less proliferative PACT KO cells. In contrast, NOVA1 and PXDN are potential oncogenes in numerous cancers (e.g., head and neck carcinoma, and lung cancer; and ovarian, and prostate cancers, respectively) with their overexpression being associated with poor prognoses and tumour progression(\u003cspan additionalcitationids=\"CR70 CR71\" citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe LNCaP cells with either PACT depletion or knockout showed a reduction in proliferation but were viable. PACT and TRBP within the RISC complex regulate the abundance and biogenesis of microRNAs; however, each of them is dispensable in the absence of the other, and their precise role in microRNA processing is not entirely understood(\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e). PACT and TRBP interact with PKR and RIG-1 to regulate stress response, viral defense, apoptosis and gene expression, albeit in opposing manners(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e). Cellular stress leads to the PACT phosphorylation and initiation of PKR-dependent apoptosis(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), while the overexpression and hyperphosphorylation of TRBP inhibits oxidative stress-induced apoptosis and promotes cell survival via PKR inhibition(\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e). We hypothesize that the compensatory overexpression of TRBP and the subsequent inhibition of PKR have kept the PACT-depleted LNCaP cells viable, and indeed the depletion of TRBP in PACT KO cells significantly reduced the cell viability. More studies are needed to comprehensively decipher the changes in the molecular landscape in the absence of PACT in PCa cells.\u003c/p\u003e \u003cp\u003eThere is an urgent need for new treatments for advanced CRPC. RNA-based therapeutics is a rapidly advancing field, with the recent FDA approval of several siRNA based drugs e.g., \u003cem\u003eInclisiran\u003c/em\u003e(\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e). In that context, our data provides a foundation for further work to develop siPACT or siPSA as RNA-based therapeutics for the treatment of CRPC, as their siRNA mediated targeting results in a decrease in PCa cell growth and proliferation. In addition, site specific delivery of these siRNAs to the prostate could be achieved by harnessing of the PSMA using nanoparticle or microbubble guided ultrasound technologies, allowing for more effective treatments for patients with PCa.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Dr. Tasnuva Kabir for helpful discussions regarding this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDJB, AJW and PJL designed the experiments and drafted the manuscript. DJB, AJW, KLR, RAMB, LMS, and MS performed the experiments. AJW, KLR and DJB performed the bioinformatics/pathways and clinical data set analyses. ADR contributed valuable clinical input.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe National Health and Medical Research Council of Australia supported this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA-Seq data is available in the Gene Expression Omnibus under Accession Number GSE253245.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021;71(3):209-49.\u003c/li\u003e\n\u003cli\u003eLitwin MS, Tan H-J. The Diagnosis and Treatment of Prostate Cancer: A Review. JAMA. 2017;317(24):2532-42.\u003c/li\u003e\n\u003cli\u003eKinsella N, Helleman J, Bruinsma S, Carlsson S, Cahill D, Brown C, et al. Active surveillance for prostate cancer: a systematic review of contemporary worldwide practices. Transl Androl Urol. 2018;7(1):83-97.\u003c/li\u003e\n\u003cli\u003eImamura Y, Sadar MD. Androgen receptor targeted therapies in castration-resistant prostate cancer: Bench to clinic. Int J Urol. 2016;23(8):654-65.\u003c/li\u003e\n\u003cli\u003eGrossmann M, Cheung AS, Zajac JD. Androgens and prostate cancer; pathogenesis and deprivation therapy. Best Practice \u0026amp; Research Clinical Endocrinology \u0026amp; Metabolism. 2013;27(4):603-16.\u003c/li\u003e\n\u003cli\u003eKingsley LA, Fournier PG, Chirgwin JM, Guise TA. Molecular biology of bone metastasis. Mol Cancer Ther. 2007;6(10):2609-17.\u003c/li\u003e\n\u003cli\u003eRuiz de Porras V, Font A, Aytes A. Chemotherapy in metastatic castration-resistant prostate cancer: Current scenario and future perspectives. Cancer Lett. 2021;523:162-9.\u003c/li\u003e\n\u003cli\u003eCai M, Song XL, Li XA, Chen M, Guo J, Yang DH, et al. Current therapy and drug resistance in metastatic castration-resistant prostate cancer. Drug Resist Updat. 2023;68:100962.\u003c/li\u003e\n\u003cli\u003eKantoff PW, Higano CS, Shore ND, Berger ER, Small EJ, Penson DF, et al. Sipuleucel-T immunotherapy for castration-resistant prostate cancer. N Engl J Med. 2010;363(5):411-22.\u003c/li\u003e\n\u003cli\u003eParker C, Nilsson S, Heinrich D, Helle SI, O\u0026apos;Sullivan JM, Foss\u0026aring; SD, et al. Alpha emitter radium-223 and survival in metastatic prostate cancer. N Engl J Med. 2013;369(3):213-23.\u003c/li\u003e\n\u003cli\u003eHatano K, Nonomura N. Systemic Therapies for Metastatic Castration-Resistant Prostate Cancer: An Updated Review. World J Mens Health. 2023;41.\u003c/li\u003e\n\u003cli\u003eSartor O, de Bono J, Chi KN, Fizazi K, Herrmann K, Rahbar K, et al. Lutetium-177\u0026ndash;PSMA-617 for Metastatic Castration-Resistant Prostate Cancer. New England Journal of Medicine. 2021;385(12):1091-103.\u003c/li\u003e\n\u003cli\u003eTisseverasinghe S, Bahoric B, Anidjar M, Probst S, Niazi T. Advances in PARP Inhibitors for Prostate Cancer. Cancers (Basel). 2023;15(6).\u003c/li\u003e\n\u003cli\u003eLee HJ, Yoon YI, Bae YJ. Theragnostic ultrasound using microbubbles in the treatment of prostate cancer. Ultrasonography. 2016;35(4):309-17.\u003c/li\u003e\n\u003cli\u003eWang Y, De Leon AC, Perera R, Abenojar E, Gopalakrishnan R, Basilion JP, et al. Molecular imaging of orthotopic prostate cancer with nanobubble ultrasound contrast agents targeted to PSMA. Scientific Reports. 2021;11(1):4726.\u003c/li\u003e\n\u003cli\u003eHe M, Cao Y, Chi C, Zhao J, Chong E, Chin KXC, et al. Unleashing novel horizons in advanced prostate cancer treatment: investigating the potential of prostate specific membrane antigen-targeted nanomedicine-based combination therapy. Front Immunol. 2023;14:1265751.\u003c/li\u003e\n\u003cli\u003eLopez W, Nguyen N, Cao J, Eddow C, Shung KK, Lee NS, et al. Ultrasound Therapy, Chemotherapy and Their Combination for Prostate Cancer. Technol Cancer Res Treat. 2021;20:15330338211011965.\u003c/li\u003e\n\u003cli\u003ePatel RC, Sen GC. PACT, a protein activator of the interferon-induced protein kinase, PKR. EMBO J. 1998;17(15):4379-90.\u003c/li\u003e\n\u003cli\u003eVaughn LS, Chukwurah E, Patel RC. Opposite actions of two dsRNA-binding proteins PACT and TRBP on RIG-I mediated signaling. Biochemical Journal. 2021;478(3):493-510.\u003c/li\u003e\n\u003cli\u003eKok KH, Lui PY, Ng MH, Siu KL, Au SW, Jin DY. The double-stranded RNA-binding protein PACT functions as a cellular activator of RIG-I to facilitate innate antiviral response. Cell Host Microbe. 2011;9(4):299-309.\u003c/li\u003e\n\u003cli\u003eBartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116(2):281-97.\u003c/li\u003e\n\u003cli\u003eHa M, Kim VN. Regulation of microRNA biogenesis. Nature Reviews Molecular Cell Biology. 2014;15(8):509-24.\u003c/li\u003e\n\u003cli\u003eRedfern AD, Colley SM, Beveridge DJ, Ikeda N, Epis MR, Li X, et al. RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators. Proc Natl Acad Sci U S A. 2013;110(16):6536-41.\u003c/li\u003e\n\u003cli\u003eSand M, Skrygan M, Georgas D, Arenz C, Gambichler T, Sand D, et al. Expression levels of the microRNA maturing microprocessor complex component DGCR8 and the RNA-induced silencing complex (RISC) components argonaute-1, argonaute-2, PACT, TARBP1, and TARBP2 in epithelial skin cancer. Mol Carcinog. 2012;51(11):916-22.\u003c/li\u003e\n\u003cli\u003eWang F, Zhu L, Xue Q, Tang C, Tang W, Zhang N, et al. Novel lncRNA AL033381.2 Promotes Hepatocellular Carcinoma Progression by Upregulating PRKRA Expression. Oxidative medicine and cellular longevity. 2022;2022:1125932-.\u003c/li\u003e\n\u003cli\u003eChiosea S, Acquafondata M, Luo J, Kuan S, Seethala R. DICER1 and PRKRA in Colon Adenocarcinoma. Biomark Insights. 2008;3:253-8.\u003c/li\u003e\n\u003cli\u003eQiu J, Feng M, Yang G, Su D, Zhao F, Liu Y, et al. PRKRA promotes pancreatic cancer progression by upregulating MMP1 transcription via the NF-\u0026kappa;B pathway. Heliyon. 2023;9(6):e17194.\u003c/li\u003e\n\u003cli\u003eHisamatsu T, McGuire M, Wu SY, Rupaimoole R, Pradeep S, Bayraktar E, et al. PRKRA/PACT Expression Promotes Chemoresistance of Mucinous Ovarian Cancer. Mol Cancer Ther. 2019;18(1):162-72.\u003c/li\u003e\n\u003cli\u003eWei L, Wang W, Yao J, Cui Z, Xu Z, Ding H, et al. PACT promotes the metastasis of basal-like breast cancer through Rac1 SUMOylation and activation. Oncogene. 2022;41(37):4282-94.\u003c/li\u003e\n\u003cli\u003eRowe TM, Rizzi M, Hirose K, Peters GA, Sen GC. A role of the double-stranded RNA-binding protein PACT in mouse ear development and hearing. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(15):5823-8.\u003c/li\u003e\n\u003cli\u003eFujitani K, Otomo A, Nagayama Y, Tachibana T, Kato R, Kawashima Y, et al. PACT/PRKRA and p53 regulate transcriptional activity of DMRT1. Genet Mol Biol. 2020;43(2):e20190017.\u003c/li\u003e\n\u003cli\u003ePeters GA, Seachrist DD, Keri RA, Sen GC. The double-stranded RNA-binding protein, PACT, is required for postnatal anterior pituitary proliferation. Proceedings of the National Academy of Sciences. 2009;106(26):10696.\u003c/li\u003e\n\u003cli\u003eDickerman BK, White CL, Chevalier C, Nalesso V, Charles C, Fouch\u0026eacute;court S, et al. Missense mutation in the second RNA binding domain reveals a role for Prkra (PACT/RAX) during skull development. PloS one. 2011;6(12):e28537-e.\u003c/li\u003e\n\u003cli\u003eYong Y, Meng Y, Ding H, Fan Z, Tang Y, Zhou C, et al. PACT/RAX Regulates the Migration of Cerebellar Granule Neurons in the Developing Cerebellum. Scientific Reports. 2015;5(1):7961.\u003c/li\u003e\n\u003cli\u003eKanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28(1):27-30.\u003c/li\u003e\n\u003cli\u003eKok KH, Ng MH, Ching YP, Jin DY. Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA. J Biol Chem. 2007;282(24):17649-57.\u003c/li\u003e\n\u003cli\u003eWoo AJ, Patry CA, Ghamari A, Pregernig G, Yuan D, Zheng K, et al. Zfp281 (ZBP-99) plays a functionally redundant role with Zfp148 (ZBP-89) during erythroid development. Blood Adv. 2019;3(16):2499-511.\u003c/li\u003e\n\u003cli\u003eWeber K, Bartsch U, Stocking C, Fehse B. A multicolor panel of novel lentiviral \u0026quot;gene ontology\u0026quot; (LeGO) vectors for functional gene analysis. Mol Ther. 2008;16(4):698-706.\u003c/li\u003e\n\u003cli\u003eBeveridge DJ, Richardson KL, Epis MR, Brown RAM, Stuart LM, Woo AJ, et al. The tumor suppressor miR-642a-5p targets Wilms Tumor 1 gene and cell-cycle progression in prostate cancer. Sci Rep. 2021;11(1):18003.\u003c/li\u003e\n\u003cli\u003eSubramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102(43):15545-50.\u003c/li\u003e\n\u003cli\u003eLiberzon A, Birger C, Thorvaldsd\u0026oacute;ttir H, Ghandi M, Mesirov JP, Tamayo P. The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst. 2015;1(6):417-25.\u003c/li\u003e\n\u003cli\u003eScholtes C, Gigu\u0026egrave;re V. Transcriptional control of energy metabolism by nuclear receptors. Nature Reviews Molecular Cell Biology. 2022;23(11):750-70.\u003c/li\u003e\n\u003cli\u003eKim J, Coetzee GA. Prostate specific antigen gene regulation by androgen receptor. J Cell Biochem. 2004;93(2):233-41.\u003c/li\u003e\n\u003cli\u003eHong SK. Kallikreins as Biomarkers for Prostate Cancer. BioMed Research International. 2014;2014:526341.\u003c/li\u003e\n\u003cli\u003eNiu Y, Yeh S, Miyamoto H, Li G, Altuwaijri S, Yuan J, et al. Tissue prostate-specific antigen facilitates refractory prostate tumor progression via enhancing ARA70-regulated androgen receptor transactivation. Cancer Res. 2008;68(17):7110-9.\u003c/li\u003e\n\u003cli\u003eFuhrman-Luck RA, Loessner D, Clements JA. Kallikrein-Related Peptidases in Prostate Cancer: From Molecular Function to Clinical Application. EJIFCC. 2014;25(3):269-81.\u003c/li\u003e\n\u003cli\u003eBonk S, Kluth M, Jansen K, Hube-Magg C, Makrypidi-Fraune G, H\u0026ouml;flmayer D, et al. Reduced KLK2 expression is a strong and independent predictor of poor prognosis in ERG-negative prostate cancer. Prostate. 2020;80(13):1097-107.\u003c/li\u003e\n\u003cli\u003eRedon CE, Schmal Z, Tewary G, Mangelinck A, Courbeyrette R, Thuret JY, et al. Histone Variant H2A.J Is Enriched in Luminal Epithelial Gland Cells. Genes (Basel). 2021;12(11).\u003c/li\u003e\n\u003cli\u003eLee HH, Lin CH, Lin HY, Kuei CH, Zheng JQ, Wang YH, et al. Histone 2A Family Member J Drives Mesenchymal Transition and Temozolomide Resistance in Glioblastoma Multiforme. Cancers (Basel). 2019;12(1).\u003c/li\u003e\n\u003cli\u003eWang X, Ghareeb WM, Lu X, Huang Y, Huang S, Chi P. Coexpression network analysis linked H2AFJ to chemoradiation resistance in colorectal cancer. J Cell Biochem. 2019;120(6):10351-62.\u003c/li\u003e\n\u003cli\u003eYi X, Li J, Zheng X, Xu H, Liao D, Zhang T, et al. Construction of PANoptosis signature: Novel target discovery for prostate cancer immunotherapy. Molecular Therapy - Nucleic Acids. 2023;33:376-90.\u003c/li\u003e\n\u003cli\u003eHaertle L, Barrio S, Munawar U, Han S, Zhou X, Simicek M, et al. Single-Nucleotide Variants and Epimutations Induce Proteasome Inhibitor Resistance in Multiple Myeloma. Clin Cancer Res. 2023;29(1):279-88.\u003c/li\u003e\n\u003cli\u003eLevin A, Minis A, Lalazar G, Rodriguez J, Steller H. PSMD5 Inactivation Promotes 26S Proteasome Assembly during Colorectal Tumor Progression. Cancer Res. 2018;78(13):3458-68.\u003c/li\u003e\n\u003cli\u003eLi A, Lu D, Zhang Y, Li J, Fang Y, Li F, et al. Critical role of aquaporin-3 in epidermal growth factor-induced migration of colorectal carcinoma cells and its clinical significance. Oncol Rep. 2013;29(2):535-40.\u003c/li\u003e\n\u003cli\u003eXia H, Ma YF, Yu CH, Li YJ, Tang J, Li JB, et al. Aquaporin 3 knockdown suppresses tumour growth and angiogenesis in experimental non-small cell lung cancer. Exp Physiol. 2014;99(7):974-84.\u003c/li\u003e\n\u003cli\u003eSilva PM, da Silva IV, Sarmento MJ, Silva \u0026Iacute; C, Carvalho FA, Soveral G, et al. Aquaporin-3 and Aquaporin-5 Facilitate Migration and Cell-Cell Adhesion in Pancreatic Cancer by Modulating Cell Biomechanical Properties. Cells. 2022;11(8).\u003c/li\u003e\n\u003cli\u003eShen K, Yu W, Yu Y, Liu X, Cui X. Knockdown of TMEM45B inhibits cell proliferation and invasion in gastric cancer. Biomed Pharmacother. 2018;104:576-81.\u003c/li\u003e\n\u003cli\u003eHu R, Hu F, Xie X, Wang L, Li G, Qiao T, et al. TMEM45B, up-regulated in human lung cancer, enhances tumorigenicity of lung cancer cells. Tumour Biol. 2016;37(9):12181-91.\u003c/li\u003e\n\u003cli\u003eNguyen T-A, Le M-K, Nguyen P-T, Tran NQV, Kondo T, Nakao A. SLC22A3 that encodes organic cation transporter-3 is associated with prognosis and immunogenicity of human lung squamous cell carcinoma. Translational Lung Cancer Research. 2023;12(10):1972-86.\u003c/li\u003e\n\u003cli\u003eRen A, Sun S, Li S, Chen T, Shu Y, Du M, et al. Genetic variants in SLC22A3 contribute to the susceptibility to colorectal cancer. Int J Cancer. 2019;145(1):154-63.\u003c/li\u003e\n\u003cli\u003eGrisanzio C, Werner L, Takeda D, Awoyemi BC, Pomerantz MM, Yamada H, et al. Genetic and functional analyses implicate the NUDT11, HNF1B, and SLC22A3 genes in prostate cancer pathogenesis. Proc Natl Acad Sci U S A. 2012;109(28):11252-7.\u003c/li\u003e\n\u003cli\u003ede Almeida A, Parthimos D, Dew H, Smart O, Wiltshire M, Errington RJ. Aquaglyceroporin-3\u0026apos;s Expression and Cellular Localization Is Differentially Modulated by Hypoxia in Prostate Cancer Cell Lines. Cells. 2021;10(4).\u003c/li\u003e\n\u003cli\u003eIsmail M, Bokaee S, Davies J, Harrington KJ, Pandha H. Inhibition of the aquaporin 3 water channel increases the sensitivity of prostate cancer cells to cryotherapy. British Journal of Cancer. 2009;100(12):1889-95.\u003c/li\u003e\n\u003cli\u003eKushwaha PP, Verma S, Gupta S. Aquaporins as Prognostic Biomarker in Prostate Cancer. Cancers. 2023;15(2):331.\u003c/li\u003e\n\u003cli\u003eLuo F, Yang K, Wang YZ, Lin D. TMEM45B is a novel predictive biomarker for prostate cancer progression and metastasis. Neoplasma. 2018;65(5):815-21.\u003c/li\u003e\n\u003cli\u003eBolton EC, So AY, Chaivorapol C, Haqq CM, Li H, Yamamoto KR. Cell- and gene-specific regulation of primary target genes by the androgen receptor. Genes Dev. 2007;21(16):2005-17.\u003c/li\u003e\n\u003cli\u003eYi Y, Nandana S, Case T, Nelson C, Radmilovic T, Matusik RJ, et al. Candidate metastasis suppressor genes uncovered by array comparative genomic hybridization in a mouse allograft model of prostate cancer. Mol Cytogenet. 2009;2:18.\u003c/li\u003e\n\u003cli\u003eClark J, Freeman J, Donninger H. Loss of RASSF2 Enhances Tumorigencity of Lung Cancer Cells and Confers Resistance to Chemotherapy. Mol Biol Int. 2012;2012:705948.\u003c/li\u003e\n\u003cli\u003eKim EK, Cho YA, Seo MK, Ryu H, Cho BC, Koh YW, et al. NOVA1 induction by inflammation and NOVA1 suppression by epigenetic regulation in head and neck squamous cell carcinoma. Sci Rep. 2019;9(1):11231.\u003c/li\u003e\n\u003cli\u003eQu L, Tian Y, Wang F, Li Z. NOVA1 promotes NSCLC proliferation and invasion by activating Wnt/\u0026beta;-catenin signaling. BMC Cancer. 2022;22(1):1091.\u003c/li\u003e\n\u003cli\u003eZheng YZ, Liang L. High expression of PXDN is associated with poor prognosis and promotes proliferation, invasion as well as migration in ovarian cancer. Ann Diagn Pathol. 2018;34:161-5.\u003c/li\u003e\n\u003cli\u003eDougan J, Hawsawi O, Burton LJ, Edwards G, Jones K, Zou J, et al. Proteomics-Metabolomics Combined Approach Identifies Peroxidasin as a Protector against Metabolic and Oxidative Stress in Prostate Cancer. Int J Mol Sci. 2019;20(12).\u003c/li\u003e\n\u003cli\u003eKim Y, Yeo J, Lee Jung H, Cho J, Seo D, Kim J-S, et al. Deletion of Human tarbp2 Reveals Cellular MicroRNA Targets and Cell-Cycle Function of TRBP. Cell Reports. 2014;9(3):1061-74.\u003c/li\u003e\n\u003cli\u003ePullagura SRN, Buaas B, Gray N, Krening LC, Srivastava A, Braun RE. Functional Redundancy of DICER Cofactors TARBP2 and PRKRA During Murine Embryogenesis Does Not Involve miRNA Biogenesis. Genetics. 2018;208(4):1513-22.\u003c/li\u003e\n\u003cli\u003eHeyam A, Lagos D, Plevin M. Dissecting the roles of TRBP and PACT in double-stranded RNA recognition and processing of noncoding RNAs. Wiley Interdiscip Rev RNA. 2015;6(3):271-89.\u003c/li\u003e\n\u003cli\u003ePark H, Davies MV, Langland JO, Chang HW, Nam YS, Tartaglia J, et al. TAR RNA-binding protein is an inhibitor of the interferon-induced protein kinase PKR. Proc Natl Acad Sci U S A. 1994;91(11):4713-7.\u003c/li\u003e\n\u003cli\u003eChukwurah E, Patel RC. Stress-induced TRBP phosphorylation enhances its interaction with PKR to regulate cellular survival. Scientific Reports. 2018;8(1):1020.\u003c/li\u003e\n\u003cli\u003eTraber GM, Yu AM. RNAi-Based Therapeutics and Novel RNA Bioengineering Technologies. J Pharmacol Exp Ther. 2023;384(1):133-54.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4121983/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4121983/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePACT (encoded by the \u003cem\u003ePRKRA\u003c/em\u003e gene) is a double-stranded RNA binding protein that has two main functions in mammals: facilitation of antiviral defense mechanisms via the activation of protein kinase RNA (PKR) and retinoic acid-inducible gene 1 (RIG-1), and PACT is also a member of the cytoplasmic RNA-induced silencing complex. We previously described an alternate role for PACT as a modulator of nuclear receptor (NR)-regulated gene expression. Here, we investigated the role of PACT in prostate cancer (PCa) using a loss-of-function approach. Depletion of PACT in PCa cell lines resulted in a reduction in cell proliferation; however, they were viable. RNA-sequencing analysis of LNCaP PCa cells ± PACT revealed a depletion of biological processes involved in cell cycle, mitochondrial function, and NR-response pathways in the PACT knockout (KO) cells. In the PACT KO cells, downregulated genes included \u003cem\u003eH2AFJ, PSMD5, AQP3, TMEM45B\u003c/em\u003e, \u003cem\u003eSLC22A3,\u003c/em\u003e and\u003cem\u003e KLK3\u003c/em\u003e (prostate specific antigen, PSA), and siRNA mediated knockdown of these genes reduced cell growth and proliferation in LNCaP cells. Taken together, these data provide support for \u003cem\u003ePRKRA\u003c/em\u003e as a proproliferative gene in PCa and targeting \u003cem\u003ePRKRA\u003c/em\u003e, or the genes that are downregulated in PACT KO cells via siRNA therapies, could benefit PCa patient survival.\u003c/p\u003e","manuscriptTitle":"PACT is requisite for prostate cancer cell proliferation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-25 19:03:10","doi":"10.21203/rs.3.rs-4121983/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a0263a73-8e22-4880-9e95-386980efc7f4","owner":[],"postedDate":"April 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":30832095,"name":"Biological sciences/Cancer/Urological cancer/Prostate cancer"},{"id":30832096,"name":"Biological sciences/Molecular biology/Non-coding RNAs/siRNAs"}],"tags":[],"updatedAt":"2024-05-17T14:15:12+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-25 19:03:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4121983","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4121983","identity":"rs-4121983","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}