Targeting PHB1 to inhibit castration-resistant prostate cancer progression in vitro and in vivo

Targeting PHB1 to inhibit castration-resistant prostate cancer progression in vitro and in vivo | 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 Research Article Targeting PHB1 to inhibit castration-resistant prostate cancer progression in vitro and in vivo Junmei Liu, Ranran Zhang, Tong Su, Qianqian Zhou, Lin Gao, Zongyue He, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2325130/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 May, 2023 Read the published version in Journal of Experimental & Clinical Cancer Research → Version 1 posted 4 You are reading this latest preprint version Abstract Background Castration-resistant prostate cancer (CRPC) is currently the main challenge for prostate cancer (PCa) treatment, and there is an urgent need to find novel therapeutic targets and drugs. Prohibitin (PHB1) is a multifunctional chaperone/scaffold protein that is upregulated in various cancers and plays a pro-cancer role. FL3 is a synthetic flavagline drug that inhibits cancer cell proliferation by targeting PHB1. However, the biological functions of PHB1 in CRPC and the effect of FL3 on CRPC cells remain to be explored. Methods Several public datasets were used to analyze the association between the expression level of PHB1 and PCa progression as well as PCa patient outcomes. The expression of PHB1 in human PCa specimens and PCa cell lines was examined by immunohistochemistry (IHC), qRT-PCR, and western blotting. Then both the biological roles of PHB1 in castration resistance and underlying mechanisms were investigated by gain/loss-of-function analyses. Next, in vitro and in vivo a series of experiments were conducted to investigate the anti-cancer effects of FL3 on CRPC cells as well as the underlying mechanisms. Results PHB1 expression was significantly upregulated in CRPC and was associated with poor prognosis. PHB1 promoted castration resistance of PCa cells under androgen deprivation conditions. PHB1 is an androgen receptor (AR) suppressive gene and androgen deprivation promotes the PHB1 expression and its nucleus-cytoplasm translocation. FL3, alone or combined with the antiandrogen drug Enzalutamide (ENZ), suppressed CRPC cells especially ENZ-sensitive AR + CRPC cells both in vitro and in vivo . By targeting the PHB1 protein, FL3 promoted its trafficking from plasma membrane and mitochondria to nucleus, which in turn inhibited AR signaling as well as MAPK signaling, but promoted apoptosis. Conclusion Our data indicated that PHB1 is abnormally upregulated in CRPC and involved in castration resistance and provided a novel rational therapeutic approach for CRPC. CRPC prohibitin Enzalutamide FL3 androgen receptor prostate flavagline Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Prostate cancer (PCa) is a malignant tumor that poses a severe threat to males [ 1 ] . Androgen deprivation therapy (ADT) is the primary therapy for locally advanced or metastatic PCa, but most patients will gradually develop into castration-resistant prostate cancer (CRPC) after initial treatment [ 2 ] . The occurrence and development of CRPC is a complex process involved and driven by multiple molecular pathways [ 3 ] . Although reactivation of the AR signal pathway is a critical driver of CRPC, and AR-targeted drugs such as ENZ have also significantly improved the clinical outcomes of CRPC patients, disease resistance still makes the cure of CRPC a serious challenge [ 3 – 6 ] . In recent years, drugs targeting other CRPC-driver genes, such as AKT inhibitor ipatasertib, PARP inhibitors olaparib and rucaparib, have been developed and applied clinically one after another [ 7 ] , but which are far from enough to completely cure CRPC. Therefore, searching for novel targets and targeted drugs, is still an urgent problem to be solved. Prohibitin (PHB1/PHB) is a ubiquitously expressed protein conserved throughout evolution [ 8 ] . PHB1 gene is located on chromosome 17q21 and encodes a 32 kDa protein composed of 272 amino acids [ 9 , 10 ] . PHB1 gene was initially cloned based on the ability of its own 3’UTR to induce growth arrest in mammalian cells, and numerous subsequent studies have found that PHB1 is a multifunctional protein that is involved in cell growth, proliferation, apoptosis, and metabolism [ 11 – 13 ] . In recent years, a large number of studies have shown that PHB1 expression is upregulated in a variety of clinical tumor specimens including lung cancer, diffuse large B-cell lymphomas (DLBCL), cervical cancer, bladder cancer, glioblastoma, endometrial cancer, and gastric cancer [ 14 – 20 ] . Due to its low mutation rate and essential function, PHB1 is a suitable target for drug administration [ 21 , 22 ] . Flavaglines are a class of natural products isolated from the medicinal plant Aglaia , which can exert a unique anti-cancer activity by binding to PHB1/PHB2 [ 18 ] . FL3, a synthetic derivative of flavagline with higher tumor cell specificity and lower normal cell cytotoxicity compared to natural flavaglines, has presented potent tumor suppressive effect by targeting PHB1 in a variety of cancers such as lung cancer, neuroblastoma, and DLBCL [ 19 , 20 , 23 ] . However, the role of PHB1 in PCa is still controversial. Bevan et al. found that PHB1 is downregulated in LNCaP cells by dihydrotestosterone (DHT) stimulation after androgen starvation for 24h [ 24 , 25 ] , while overexpression of PHB1 inhibited cell growth in LNCaP cells [ 25 , 26 ] . Contrarily, Zhu et al. reported that mitochondria-localized PHB1 suppresses TGF-β induced apoptosis in CRPC cell line PC3 [ 27 ] . In addition, Ummanni and Cho, respectively, reported that PHB1 was upregulated in PCa specimens and positively correlated with the degree of malignancy [ 28 , 29 ] . Therefore, we speculate that PHB1 may be involved in the castration resistance. In this study, we explored the role of PHB1 in CRPC progression and assessed the effect of FL3 in CRPC therapy. Methods Reagents Fetal bovine serum (FBS), charcoal-stripped fetal bovine serum (CSS, depleted androgen and any other steroid), Bovine pituitary extract (BPE), and cell culture mediums were purchased from Gibco (NY, USA). ENZ was purchased from MedChemExpress (NJ, USA). FL3 was synthesized in Dr. Laurent Désaubry’s lab according to a described procedure [ 30 ] . DHT was purchased from Meilunbio (Dalian, China). Epidermal growth factor (EGF) was obtained from Peprotech (NJ, USA). Glutamine PenStrep was obtained from Invitrogen. (CA, UAS). Serial dilutions of all drugs were made using DMSO. Bioinformatics Analysis The mRNA expression data of PHB1 were extracted from GEO ( http://www.ncbi.nlm.nih.gov/geo ) and cBioPortal ( http://www.cbioportal.org/ ) database. The data used for Kaplan-Meier survival analysis were from TCGA ( https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga/using-tcga ) and cBioPortal ( http://www.cbioportal.org/ ) database. Immunohistochemistry(Ihc) The cancer-adjacent normal tissues and PCa tissues were collected from undergoing radical prostatectomy for PCa at Qilu Hospital, Shandong University (Jinan, China) and made into tissue microarray (TMA). TMA was stained with H&E to be reviewed by two pathologists according to the WHO histologic classification of PCa, and the IHC staining was performed with Universal two-step detection kit (PV9000, Zhongshan Golden Bridge Biotechnology Co, Beijing, China). For PHB1 expression in tissues, the staining intensity was scored from 1 to 3: 1, weak; 2, moderate; 3, strong. The information of antibody used is summarized in Supplementary Table S2 Cell Lines And Cell Culture Human prostate epithelial cell line RWPE-1 was purchased from National Collection of Authenticated Cell Cultures (Shanghai, China). PCa cell lines LNCaP, C4-2B, 22Rv1 and PC-3 were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). RWPE-1 cells were cultured in Keratinocyte serum-free medium supplemented with EGF, BPE, and 1% glutamine PenStrep. LNCaP, C4-2B, and 22Rv1 cells were cultured in RPMI-1640 with 10% FBS or charcoal-stripped fetal bovine serum (CSS). PC-3 cell was cultured in F12-K with 10% FBS or CSS. To establish derived LNCaP-AI (androgen-independent) cell line, LNCaP cells were maintained in RPMI-1640 supplemented with 10% CSS for at least 3 months [ 31 ] . All cells were cultured at 37°C in an atmosphere of 5% CO 2 and used up to 15 passages maximum. After the last experiment, all cells were confirmed mycoplasma free using GMyc-PCR Mycoplasma Test Kit (40601ES10, YeSen Biotech, Shanghai, China). Qrt-pcr Total RNA was isolated using TRIzol reagent (Invitrogen, CA, UAS), and 1 µg of total RNA was used as template for the first strand cDNA synthesis using the ReverTra Ace qPCR RT Kit (TOYOBO, Osaka, Japan). qPCR reactions were carried out with a FastStart Universal SYBR Green Master Mix (Roche, Mannheim, Germany). Data were normalized by the expression level of β-actin in each sample. The primer sequences used are listed in Supplementary Table S1. Western Blotting Whole-cell lysates were prepared by lysing the cells in ice-cold RIPA buffer (P0013C, Beyotime, Shanghai, China) with 1% protease inhibitor cocktail (NCM Biotech, Suzhou, China). 20 µg total protein was separated by SDS-PAGE and transferred to the PVDF membrane, which were then incubated with corresponding antibodies. The information of antibodies is summarized in Supplementary Table S2. Plasmids, Sirna, And Cell Transfection Expression vector pENTER-PHB1 and its control vector was designed and constructed by Vigene Bioscience (Shandong, China). siNC and siPHB1 were synthesized by Jikai company (Shanghai, China). The target sequences are listed in Supplementary Table S3. Lipofectamine 3000 (Invitrogen, CA, USA) was used for transient transfection following the manufacturer’s instruction. The transfection efficiency was confirmed using qRT-PCR and Western blotting. MTS assay Cell viability and cell proliferation were determined by MTS assay with MTS Cell Proliferation Assay Kit (BestBio, Shanghai, China). LNCaP cells (3.0×10 3 cells/well), C4-2B cells (2.0×10 3 cells/well), 22Rv1 (1.5×10 3 cells/well), or PC3 cells (1.5×10 3 cells/well) were plated in 96-well plate. After cells adhering to the wall, 10 µl MTS was added to each well at 0, 24, 48, and 72 h, respectively, and incubated at 37°C for 2h. Then the absorbance was measured at 490 nm. Cell viability was expressed as the percentage of absorbance of cells treated with FL3 or Enzalutamide compared with cells treated with DMSO. Experiments were performed in triplicate and repeated three times. Transwell Assay Transwell invasion/migration assays were performed using Transwell Chambers with or without coated Matrigel (Corning, NY, USA). RPMI-1640 or F12-K medium with 10% FBS was added to the lower chamber as a chemoattractant for cells. LNCaP (10.0×10 4 cells/well), C4-2B (10.0×10 4 cells/well), 22Rv1 (7.0×10 4 cells/well), or PC3 cells (7.0×10 4 cells/well) were plated in the upper chamber with RPMI-1640 or F12-K medium. After 24h of incubation, the migrated and invasive cells were fixed with 4% paraformaldehyde (Biosharp, Beijing, China) and stained with crystal violet solution (Solarbio, Beijing China). 5 fields of view were randomly selected for counting transmembrane cells under the light microscope using a 40 × magnification. Plate Colony Formation Assay The clonogenic ability of PCa cells was measured by plate colony formation assay. LNCaP (300 cells/well), C4-2B (300 cells/well), 22Rv1 (200 cells/well) or PC3 cells (200 cells/well) were plated in 6-well plate. 14d after initial seeding, cells were fixed with 4% paraformaldehyde and stained with crystal violet solution. Colonies containing more than 50 cells were counted and plotted. Experiments were repeated independent three times. Subcellular Fractionation Nucleus and cytosol proteins were separated using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, Nantong, China) following the manufacturer’s instruction. plasma membrane extracts were prepared using Minute™ Plasma Membrane Protein Isolation and Cell Fractionation Kit (Invent Biotechnologies, Eden Prairie, USA) according to manufacturer’s manual. Immunofluorescence (If) IF was performed as previously described [ 32 ] . Pre-treated LNCaP (4 × 10 4 cells/well) and C4-2B (2 × 10 4 cells/well) cells were seeded on glass coverslips in 24-well plates, respectively. Mitochondrial labelling was performed using MitoTracker Deep Red FM (40743ES, Yeasen Biotech, Shanghai, China) following the manufacturer’s instruction. Nucleus was stained with DAPI (H-1200, VECTASHIELD, CA, USA). Antibodies used is summarized in Supplementary Table S2. Cells were observed under Laser Confocal Microscope LSM 980 (Zeiss, Oberkochen, Germany). Images taken were appropriately processed with the ZEN software program. Experiments were repeated three independent times. Co-immunoprecipitation (Co-ip) Co-IP assays were performed according to the instruction of BeyoMag™ Protein A/G Magnetic Beads for IP (Beyotime, Shanghai, China). 10 µg antibody in a 1:50 dilution was incubated with 20 µl Protein A/G Magnetic Beads for 1h at temperature. The cell lysate or nuclear extract, leaving 50 µl as input, was incubated with antibody-coated immunomagnetic beads at 4℃ overnight. The immuno-complex was collected and was analyzed by Western blotting. The information of antibodies is summarized in Supplementary Table S2 Tumor Xenografts 5-week-old male BALB/c nude mice were purchased from Weitonglihua Biotechnology (Beijing, China). A total of 6.0 × 10 6 C4-2B cells were mixed with Matrigel (1:1) and injected subcutaneously into the mice (n = 6/group). After the mice were surgically castrated, they were randomized into 4 groups (n = 6/group) and treated with indicated drugs and concentrations. ENZ and FL3 were administered by oral gavage and subcutaneously every two days, respectively. Body weight and tumor volume was measured and recorded twice a week. Tumor tissues were harvested and weighed after 15 times of treatment. Tumor volume was calculated with the following formula: tumor volume = length × width 2 × 0.5. All animal experiments followed a protocol approved by the Shandong University Animal Care Committee (Document No. ECSBMSSDU2019-2-019). Flow Cytometry Dead Cell Apoptosis Kit (Invitrogen, CA, USA) was used according to the manufacturer's instruction. C42B cells treated with FL3 or DMSO were harvested and dealed with Annexin V-FITC and propidium iodide, followed by apoptosis analysis with CytoFLEX S (1720610S-2, BECKMAN COULTER, CA, USA). Experiments were repeated three independent times. Pattern Drawing The pattern diagram was drawn using the Figdraw software (ResearchHome, Zhejiang, China). Statistical analysis For in vitro experiment, experiments were carried out at least in triplicate to confirm reproducibility and presented as mean ± SD. In the xenograft studies, tumor sizes were served as the primary response measure when the mice were sacrificed. Statistical analysis was carried out with GraphPad Prism 8.0 using a t-test and two-way ANOVA. Significance was determined at *P < 0.05, **P < 0.01 and ***P < 0.001. Result PHB1 expression is significantly upregulated in CRPC and is associated with poor prognosis. To explore its role in castration resistance, we first investigated the association between the expression level of PHB1 and PCa progression. By analyzing public datasets, we found that PHB1 mRNA expression was upregulated in localized PCa tissues compared with benign prostate tissues (GSE35988) [ 33 ] (Fig. S1 A). Higher mRNA levels of PHB1 were associated with higher Gleason score (P = 0.0054) (Fig. S1 B). More importantly, analysis of three independent datasets (GSE35988, GES74367 and Fred Hutchinson Cancer Research Center-2016) [ 33 – 35 ] showed significantly increased PHB1 expression in CRPC tissues compared to primary PCa tissues (Fig. 1 A). Using IHC, we examined PHB1 protein expression in 105 PCa and 5 cancer-adjacent normal tissues. The results showed that PHB1 expression was significantly higher in PCa tissues than that in adjacent normal tissues and was positively correlated with the Gleason score (Fig. 1 B). Similarly, the expression of PHB1 was higher in LNCaP cells with ADPC phenotype than that in normal prostate epithelial RWPE-1 cells, while in CRPC cell lines (C4-2B, 22Rv1, DU145, PC3), PHB1 expression was higher than that in LNCaP cells (Fig. 1 C). Finally, we also performed Kaplan-Meier survival analysis using the TCGA database ( https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga/using-tcga ) and cBioPortal database ( http://www.cbioportal.org/ ) to explore the clinicopathological significance of PHB1 expression in PCa patients. As shown in Fig. 1 D, compared with the low PHB1 expression group, the high expression group had shorter progression-free survival (TCGA), overall survival (MCTP-2012) [ 33 ] , and disease-free survival (MSCKK-2010) [ 36 ] . The above results suggested that increased PHB1 expression positively correlate with the degree of malignancy and may participate in the castration resistance of PCa. Phb1 Is Involved In The Development Of Crpc To explore whether PHB1 is involved in the castration resistance process of PCa, we performed a series of gain/loss-of-function experiments in ADPC (LNCaP), AR + CRPC (C4-2B, 22Rv1) and AR − CRPC (PC-3) cell lines. Cells were grown in the medium with 10% CSS for 3d to mimic the ADT environment. We then overexpressed PHB1 in LNCaP cells whereas applied PHB1 siRNA to knockdown PHB1 expression in C4-2B, 22Rv1, and PC3 cells, respectively. As shown in Fig. 2 , under CSS condition, PHB1 overexpression enhanced the proliferation, invasion, migration, and clonogenicity of LNCaP cells, whereas PHB1 knockdown inhibited the cell growth, invasion, migration and clonogenic growth of C4-2B, 22Rv1, and PC-3 cells. Together, these data indicate that PHB1 is involved in the development of CRPC. Androgen Depletion Upregulates The Phb1 Expression And Promotes Its Nucleus-cytoplasm Translocation To elucidate the mechanism by which PHB1 promotes castration resistance in PCa, we first determined whether the AR signaling pathway regulates PHB1 expression. Androgen-starved LNCaP cells were stimulated with DHT for indicated times (0\6\12\24\48 h) or concentrations (0\0.01\0.1\1\10 nM), respectively. Western blot and qRT-PCR results showed that DHT treatment inhibited the expression of PHB1 in a time/concentration-dependent manner (Fig. 3 A), indicating that PHB1 is an androgen suppressive gene, which is consistent with Bevan's results [ 24 ] . Furthermore, the GSE59986 [ 37 ] dataset analysis revealed that PHB1 mRNA expression in LNCaP cells was upregulated under chronic androgen depletion. Meanwhile, comparison of PHB1 protein expression level between LNCaP and LNCaP-AI (established by our lab) cells presented similar trend (Fig. 3 B). Given that the subcellular localization of PHB1 determines its functions [ 18 ] , we thereby investigated whether ADT affects the subcellular distribution of PHB1. After culturing LNCaP cells under CSS condition for 3d, subcellular fractionation results showed that CSS culture resulted in decreased nuclear PHB1 protein abundance but increased cytoplasmic and membrane-associate PHB1 compared to FBS culture (Fig. 3 C). Further, colocalizaion analysis using IF assay revealed that in nucleus, PHB1 signal was significantly attenuated, and the PHB1:AR colocalization signal almost disappeared (Fig. 3 D), which was confirmed by Co-IP experiments (Fig. 3 E). In addition, the phosphorylation levels of c-Raf ser338 and MEK were increased after 3d of CSS culture, and this effect was partially attenuated by knockdown of PHB1 (Fig. 3 F). Finally, CSS culture for 3d also significantly enhanced PHB1 fluorescence signal in mitochondria compared with FBS culture (Fig. 3 G). These data suggest that ADT simultaneously upregulates PHB1 expression and modulates its subcellular distribution, which in turn affects the status of AR signaling as well as mitochondrial apoptosis pathway and c-Raf/MAPK pathway. FL3 has a stronger inhibition effect on ENZ-sensitive CRPC, particularly combined with ENZ. To characterize the potential role of FL3 in CRPC therapy, we firstly determined the half-maximal inhibitory concentration (IC50) values of FL3 on different PCa cell lines. The results showed that C4-2B cells was mostly sensitive to FL3 (IC50 17.50 nM), followed by LNCaP (IC50 35.99 nM), 22Rv1 (IC50 43.05 nM) and PC3 (IC50 57.04 nM) (Fig. 4 A). Based on the measured IC50 values, transwell assay results further showed that 20/40/80 nM FL3 inhibited migration and invasion of all 4 cell lines in a concentration-dependent manner (Fig. S2 A-D). Given that FL3 was more cytotoxic to ENZ-sensitive AR + CRPC cell line C4-2B [ 38 ] than to ADPC cell line LNCaP, ENZ-resistant AR + CRPC cell line 22Rv1 [ 6 , 39 ] and AR − cell line PC-3, we then compared the effect of FL3, ENZ treatment alone or in combination on the proliferation of AR + cell lines LNCaP, C4-2B and 22Rv1 using MTS. As shown in Fig. 4 B, although the low-dose combination of FL3 and ENZ does not effectively inhibit cell proliferation, but the combination of 40 nM FL3 with 20 µM ENZ significantly inhibited the growth of LNCaP, C4-2B, and 22Rv1 cells in 70%, 81%, and 54%inhibition rate (P < 0.001), respectively, which were higher than that by treatment with either 20 µM ENZ (55%, 52%, 39% suppression, P < 0.01) or 40 nM FL3 (52%, 77%, 35% suppression, P < 0.01) alone. These results indicate that FL3 significantly enhanced the sensitivity of C4-2B cells to ENZ. Next, we examined the impacts of FL3 on tumor growth of C4-2B xenograft in castrated nude mice. Mice were grouped randomly and treated with DMSO (control)/ENZ/FL3/FL3 plus ENZ, respectively. ENZ or FL3 treatment alone reduced both weight (ENZ vs. control: 983.3 ± 213.7 vs. 2566.7 ± 578.5 mg; FL3 vs. control: 1183.3 ± 231.7 vs. 2566.7 ± 578.5 mg) and volume (ENZ vs. control: 872.2 ± 284.1 vs. 1809 ± 218.8 mm 3 ; FL3 vs. control: 944.2 ± 205 mm 3 vs. 1809 ± 218.8 mm 3 ) of the tumor, and the combined treatment with ENZ and FL3 caused stronger inhibition (Weight, 233.3 ± 103.3 mg; Volume, 453.7 ± 73.1 mm 3 ) of tumor growth than that with FL3 or ENZ alone (Fig. 4 C). Moreover, there were no significant changes in body weights of all group’s mice (Fig. S3 A) as well as vital organ (liver and lung) histology (Fig. S3 B). In addition, transwell assays on C4-2B cells showed that the number of migrated cells in the combined administration group was further attenuated compared with the single drug administration groups (Fig. 4 D). Fl3 Inhibits Crpc By Affecting The Subcellular Distribution Of Phb1 To explore the molecular mechanism underlying FL3-mediated tumor suppression in CRPC, we first analyzed the expression and subcellular distribution of PHB1 in C4-2B cells upon exposure to FL3. The subcellular fractionation results showed that after treatment with 20nM FL3 for 48 h, the transfer of PHB1 from the cytoplasm and plasma membrane to the nucleus increased compared to DMSO control without changing total PHB1 protein level (Fig. 5 A). Colocalization analysis using IF assays revealed that FL3 promoted the nuclear translocation of PHB1 protein and consequently enhanced the co-localization of PHB1:AR significantly (Fig. 5 B), which was further confirmed by Co-IP experiments (Fig. 5 C). Moreover, the two essential AR target genes, PSA and TMPRSS2, were downregulated at the mRNA level by FL3 treatment (Fig. 5 D), suggesting that FL3 inhibited AR transcriptional activity by promoting the PHB1:AR interaction. In addition, FL3 significantly inhibited the EGF-induced phosphorylation of P-c-Raf Ser338 and P-MEK in C4-2B cells (Fig. 5 E). And FL3 also decreased the colocalization signals of PHB1 with mitochondria in the cytoplasm (Fig. 5 F), along with increased apoptosis rate in C4-2B cells (Fig. 5 G and Fig. S4). These data indicate that FL3 may exert its anticancer effect via reversing the subcellular distribution alteration of PHB1 in CRPC. Discussion It has been widely reported that PHB1 is upregulated in a variety of tumors and has a tumor-promoting effect [ 40 – 42 ] , but there are few reports on the role of PHB1 in CRPC. In the current study, we showed that PHB1 was highly expressed in CRPC compared with that of localized PCa and was positively correlated with poor prognosis in clinical setting. Further gain/loss-of-function analysis showed that PHB1 can promote the proliferation, invasion and metastasis of all 4 tested PCa cell lines under CSS condition. These results indicated that PHB1 is involved in castration resistance and is one of the potential driving factors for CRPC. PHB1 is a multifunctional molecular chaperone/scaffold protein that primarily localizes to mitochondria, plasma membrane and nucleus [ 10 , 14 – 20 , 43 ] . Mitochondrial PHB1 involves in stabilization of mitochondria and suppress apoptosis [ 44 – 46 ] . Nuclear PHB1 as a cofactor mediate transcriptional regulation through interacting with several transcription factors [ 47 , 48 ] . Cell membrane-localized PHB1 is required for K-Ras-induced c-Raf activation [ 49 ] . Numerous studies have shown that the specific role of PHB1 depends on its abundance and subcellular distribution characteristic of specific tumor types. PHB1 can shuttle between subcellular compartments under the induction of regulatory factors, and in turn mediate the regulation of several signal pathways [ 25 , 26 , 50 , 51 ] Previously, Bevan et al . reported that PHB1 is an androgen-repressive gene in the LNCaP cells (also validated by us in this study), and ectopic overexpression of PHB1 inhibited androgen-mediated cell growth in LNCaP cells. By contrast, our result showed that PHB1 overexpression under CSS condition promoted the cell growth of LNCaP cells. To this reason, we observe the effect of ADT on PHB1 expression and its subcellular distribution. As expected, PHB1 expression was significantly upregulated under chronic CSS culture. Next, we found that ADT promoted translocation of PHB1 from the nucleus to mitochondria and plasma membrane, leading to mitochondrial PHB1 increase, nuclear PHB1:AR colocalization decrease, and activation of c-Raf/MAPK signaling. As a hormone (androgen) sensitive disease, reactivation of AR signaling remains dominant in the molecular mechanism of castration resistance, and alterations in cofactors of the AR pathway are one of the reactivation factors as well as potential therapeutic targets of CRPC [ 52 – 58 ] . Previous reports have indicated that nuclear PHB1 mainly as corepressor interact with other transcription factors or corepressors such as Rb, E2F, N-CoR and HDAC1 [ 52 , 53 , 59 , 60 ] . Bevan et al . firstly found that PHB1 is also a corepressor of AR, and it represses ligand-dependent AR activity by forming part of transcriptional repressive complex with AR through indirect interaction [ 25 , 26 ] . Thus, ADT-caused loss of nuclear PHB1 should be one of the reasons explained reactivation of AR signaling in CRPC. Furthermore, PHB1 translocated to the plasma membrane and mitochondria could further facilitate promoting castration resistance by enhancing MAPK signaling pathway activation and stabilizing mitochondria. Therefore, PHB1 could be a novel potential therapeutic target for CRPC. As a small-molecule drug targeting PHB1, FL3 has been shown to suppress the growth of several tumors including urothelial carcinoma, colorectal cancer, and DLBCL [ 20 , 61 , 62 ] . However, the anticancer effect and underlying mechanism of FL3 in PCa remains unclear. In this study, we first demonstrated that nanomolar FL3 significantly suppressed the cell viability, invasion and migration of 4 tested PCa cell lines, especially C4-2B cells exhibited the greatest sensitivity to FL3. Because FL3 could promote cytoplasmic-nucleus translocation of PHB1 in DLBCL cells [ 20 , 62 ] , and PHB1 is required for AR-transcriptional repression mediated by androgen antagonists bicalutamide, for that PHB1 is recruited to androgen response promoters such as PSA gene by bicalutamide-bound AR, where they form a AR-corepressor complex with other corepressors and release coactivator P300 [ 63 ] . Thus, we infer that FL3 should be suitable for combination with new generation of antiandrogenic drug ENZ [ 5 , 64 ] on AR + ENZ-sensitive CRPC cell line C4-2B. As expected, 40 nM FL3 combined with 20 µM ENZ administration on C4-2B still present the most inhibition effect than that treatment with FL3 or ENZ alone both in vitro and in vivo . Next, a series of mechanism analysis experiments confirmed that FL3 led to cytoplasmic-nucleus translocation of PHB1 without downregulating PHB1 in C4-2B cells, along with AR signaling repressing due to reflowed nuclear PHB1 synergistically enhance the inhibitory effect of ENZ on AR signaling. Meanwhile, reversal of PHB1 subcellular distribution also inhibited c-Raf/MAPK signaling and promoted apoptosis. These results suggest that FL3 is suitable for PCa treatment, especially as an adjuvant chemotherapy drug in combination with ENZ to treat ENZ-sensitive AR + CRPC. In conclusion, in this study, we firstly showed that ADT triggered the role switch of PHB1 from anti-tumor in ADPC to pro-tumor in CRPC by upregulating PHB1 and more importantly, inducing its nucleus-cytoplasmic translocation, while FL3 inhibit PCa by reversing the ADT-induced subcellular distribution alteration of PHB1, particularly reflow nuclear PHB1 synergistically enhances the anti-cancer effect of ENZ by form transcriptional repressive complex with AR-ENZ complex in ENZ-sensitive CRPC (Fig. 6 ), indicating that targeting PHB1 is a promising strategy as adjuvant therapy for ENZ-sensitive CRPC, reducing ENZ dosage and adverse reactions. Abbreviations CRPC Castration-resistant prostate cancer PCa Prostate cancer PHB1 Prohibitin AR Androgen receptor ADT Androgen deprivation therapy ENZ Enzalutamide DHT Dihydrotestosterone CSS Charcoal-stripped fetal bovine serum FBS Fetal bovine serum TMPRSS2 Transmembrane protease serine 2 Declarations Acknowledgements Not applicable. Authors’ contributions Study concept and design: JL, WC and BH. Data acquisition: JL, RZ, TS, LG, ZH, XW and FS. Analysis and interpretation of the data: JL, RZ, QZ, YX, WC and XW. Paper preparation: JL, RZ and WC. Critical review: WC, BH, and LD. The authors read and approved the final manuscript. Funding This work was supported by the National Natural Science Foundation of China [81972413], Natural Science Foundation of Shandong Province [ZR2018MH025, ZR2018MH030], and Primary Research & Development Plan of Shandong Province [2019GSF108076]. Ethics approval The use of clinical samples was approved by the ethics committee of Shandong University and informed consent was obtained from all patients (reference number ECSBMSSDU2019-1-007). All animal experimental protocols were performed following the Ethical Animal Care and Use Committee of Shandong University (reference number ECSBMSSDU2019-2-019). Consent for publication All authors consent to the publication of this article. Competing interests The authors declare no competing interests. References Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021. Scher HI, Sawyers CL. Biology of progressive, castration-resistant prostate cancer: directed therapies targeting the androgen-receptor signaling axis. J Clin Oncol. 2005;23(32):8253–61. Bai B, Chen Q, Jing R, et al. Molecular Basis of Prostate Cancer and Natural Products as Potential Chemotherapeutic and Chemopreventive Agents. Front Pharmacol. 2021;12:738235. Kobayashi T, Inoue T, Kamba T, Ogawa O. Experimental evidence of persistent androgen-receptor-dependency in castration-resistant prostate cancer. Int J Mol Sci. 2013;14(8):15615–35. Scher HI, Fizazi K, Saad F, et al. Increased survival with enzalutamide in prostate cancer after chemotherapy. N Engl J Med. 2012;367(13):1187–97. Antonarakis ES, Lu C, Wang H, et al. AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer. N Engl J Med. 2014;371(11):1028–38. Cornford P, Bellmunt J, Bolla M, et al. EAU-ESTRO-SIOG Guidelines on Prostate Cancer. Part II: Treatment of Relapsing, Metastatic, and Castration-Resistant Prostate Cancer. Eur Urol. 2017;71(4):630–42. Mishra S, Murphy LC, Murphy LJ. The Prohibitins: emerging roles in diverse functions. J Cell Mol Med. 2006;10(2):353–63. Mishra S, Ande SR, Nyomba BL. The role of prohibitin in cell signaling. FEBS J. 2010;277(19):3937–46. Theiss AL, Sitaraman SV. The role and therapeutic potential of prohibitin in disease. Biochim Biophys Acta. 2011;1813(6):1137–43. Peng YT, Chen P, Ouyang RY, Song L. Multifaceted role of prohibitin in cell survival and apoptosis. Apoptosis. 2015;20(9):1135–49. Ande SR, Xu Y, Mishra S. Prohibitin: a potential therapeutic target in tyrosine kinase signaling. Signal Transduct Target Ther. 2017;2:17059. Signorile A, Sgaramella G, Bellomo F, De Rasmo D. Prohibitins. A Critical Role in Mitochondrial Functions and Implication in Diseases. Cells. 2019. 8(1). Chiu CF, Ho MY, Peng JM, et al. Raf activation by Ras and promotion of cellular metastasis require phosphorylation of prohibitin in the raft domain of the plasma membrane. Oncogene. 2013;32(6):777–87. Jiang L, Dong P, Zhang Z, et al. Akt phosphorylates Prohibitin 1 to mediate its mitochondrial localization and promote proliferation of bladder cancer cells. Cell Death Dis. 2015;6(2):e1660. Huang H, Zhang S, Li Y, et al. Suppression of mitochondrial ROS by prohibitin drives glioblastoma progression and therapeutic resistance. Nat Commun. 2021;12(1):3720. Yang B, Chen R, Liang X, et al. Estrogen Enhances Endometrial Cancer Cells Proliferation by Upregulation of Prohibitin. J Cancer. 2019;10(7):1616–21. Wang D, Tabti R, Elderwish S, et al. Prohibitin ligands: a growing armamentarium to tackle cancers, osteoporosis, inflammatory, cardiac and neurological diseases. Cell Mol Life Sci. 2020;77(18):3525–46. Yurugi H, Marini F, Weber C, et al. Targeting prohibitins with chemical ligands inhibits KRAS-mediated lung tumours. Oncogene. 2017;36(33):4778–89. Bentayeb H, Aitamer M, Petit B, et al. Prohibitin (PHB) expression is associated with aggressiveness in DLBCL and flavagline-mediated inhibition of cytoplasmic PHB functions induces anti-tumor effects. J Exp Clin Cancer Res. 2019;38(1):450. Sato T, Sakamoto T, Takita K, Saito H, Okui K, Nakamura Y. The human prohibitin (PHB) gene family and its somatic mutations in human tumors. Genomics. 1993;17(3):762–4. White KA, Lange EM, Ray AM, Wojno KJ, Cooney KA. Prohibitin mutations are uncommon in prostate cancer families linked to chromosome 17q. Prostate Cancer Prostatic Dis. 2006;9(3):298–302. MacArthur IC, Bei Y, Garcia HD, et al. Prohibitin promotes de-differentiation and is a potential therapeutic target in neuroblastoma. JCI Insight. 2019. 5(10). Gamble SC, Odontiadis M, Waxman J, et al. Androgens target prohibitin to regulate proliferation of prostate cancer cells. Oncogene. 2004;23(17):2996–3004. Gamble SC, Chotai D, Odontiadis M, et al. Prohibitin, a protein downregulated by androgens, represses androgen receptor activity. Oncogene. 2007;26(12):1757–68. Dart DA, Spencer-Dene B, Gamble SC, Waxman J, Bevan CL. Manipulating prohibitin levels provides evidence for an in vivo role in androgen regulation of prostate tumours. Endocr Relat Cancer. 2009;16(4):1157–69. Zhu B, Zhai J, Zhu H, Kyprianou N. Prohibitin regulates TGF-beta induced apoptosis as a downstream effector of Smad-dependent and -independent signaling. Prostate. 2010;70(1):17–26. Ummanni R, Junker H, Zimmermann U, et al. Prohibitin identified by proteomic analysis of prostate biopsies distinguishes hyperplasia and cancer. Cancer Lett. 2008;266(2):171–85. Cho SY, Kang S, Kim DS, et al. HSP27, ALDH6A1 and Prohibitin Act as a Trio-biomarker to Predict Survival in Late Metastatic Prostate Cancer. Anticancer Res. 2018;38(11):6551–60. Thuaud F, Bernard Y, Türkeri G, et al. Synthetic analogue of rocaglaol displays a potent and selective cytotoxicity in cancer cells: involvement of apoptosis inducing factor and caspase-12. J Med Chem. 2009;52(16):5176–87. McNaughton M, Pitman M, Pitson SM, Pyne NJ, Pyne S. Proteasomal degradation of sphingosine kinase 1 and inhibition of dihydroceramide desaturase by the sphingosine kinase inhibitors, SKi or ABC294640, induces growth arrest in androgen-independent LNCaP-AI prostate cancer cells. Oncotarget. 2016;7(13):16663–75. Sun F, Wang X, Hu J, et al. RUVBL1 promotes enzalutamide resistance of prostate tumors through the PLXNA1-CRAF-MAPK pathway. Oncogene. 2022;41(23):3239–50. Grasso CS, Wu YM, Robinson DR, et al. The mutational landscape of lethal castration-resistant prostate cancer. Nature. 2012;487(7406):239–43. Kumar A, Coleman I, Morrissey C, et al. Substantial interindividual and limited intraindividual genomic diversity among tumors from men with metastatic prostate cancer. Nat Med. 2016;22(4):369–78. Roudier MP, Winters BR, Coleman I, et al. Characterizing the molecular features of ERG-positive tumors in primary and castration resistant prostate cancer. Prostate. 2016;76(9):810–22. Taylor BS, Schultz N, Hieronymus H, et al. Integrative genomic profiling of human prostate cancer. Cancer Cell. 2010;18(1):11–22. Akamatsu S, Wyatt AW, Lin D, et al. The Placental Gene PEG10 Promotes Progression of Neuroendocrine Prostate Cancer. Cell Rep. 2015;12(6):922–36. Verma S, Shankar E, Chan ER, Gupta S. Metabolic Reprogramming and Predominance of Solute Carrier Genes during Acquired Enzalutamide Resistance in Prostate Cancer. Cells. 2020. 9(12). Dyshlovoy SA, Otte K, Tabakmakher KM, et al. Synthesis and anticancer activity of the derivatives of marine compound rhizochalin in castration resistant prostate cancer. Oncotarget. 2018;9(24):16962–73. Wu TF, Wu H, Wang YW, et al. Prohibitin in the pathogenesis of transitional cell bladder cancer. Anticancer Res. 2007;27(2):895–900. Tsai HW, Chow NH, Lin CP, Chan SH, Chou CY, Ho CL. The significance of prohibitin and c-Met/hepatocyte growth factor receptor in the progression of cervical adenocarcinoma. Hum Pathol. 2006;37(2):198–204. Ryu JW, Kim HJ, Lee YS, et al. The proteomics approach to find biomarkers in gastric cancer. J Korean Med Sci. 2003;18(4):505–9. Sripathi SR, He W, Atkinson CL, et al. Mitochondrial-nuclear communication by prohibitin shuttling under oxidative stress. Biochemistry. 2011;50(39):8342–51. Artal-Sanz M, Tavernarakis N. Prohibitin and mitochondrial biology. Trends Endocrinol Metab. 2009;20(8):394–401. Osman C, Merkwirth C, Langer T. Prohibitins and the functional compartmentalization of mitochondrial membranes. J Cell Sci. 2009;122(Pt 21):3823–30. Rizwani W, Alexandrow M, Chellappan S. Prohibitin physically interacts with MCM proteins and inhibits mammalian DNA replication. Cell Cycle. 2009;8(10):1621–9. Wang S, Nath N, Adlam M, Chellappan S. Prohibitin, a potential tumor suppressor, interacts with RB and regulates E2F function. Oncogene. 1999;18(23):3501–10. Yang J, Li B, He QY. Significance of prohibitin domain family in tumorigenesis and its implication in cancer diagnosis and treatment. Cell Death Dis. 2018;9(6):580. Rajalingam K, Wunder C, Brinkmann V, et al. Prohibitin is required for Ras-induced Raf-MEK-ERK activation and epithelial cell migration. Nat Cell Biol. 2005;7(8):837–43. Rastogi S, Joshi B, Fusaro G, Chellappan S. Camptothecin induces nuclear export of prohibitin preferentially in transformed cells through a CRM-1-dependent mechanism. J Biol Chem. 2006;281(5):2951–9. Luan Z, He Y, Alattar M, Chen Z, He F. Targeting the prohibitin scaffold-CRAF kinase interaction in RAS-ERK-driven pancreatic ductal adenocarcinoma. Mol Cancer. 2014;13:38. Rastogi S, Joshi B, Dasgupta P, Morris M, Wright K, Chellappan S. Prohibitin facilitates cellular senescence by recruiting specific corepressors to inhibit E2F target genes. Mol Cell Biol. 2006;26(11):4161–71. Fusaro G, Wang S, Chellappan S. Differential regulation of Rb family proteins and prohibitin during camptothecin-induced apoptosis. Oncogene. 2002;21(29):4539–48. Cai Z, Chen W, Zhang J, Li H. Androgen receptor: what we know and what we expect in castration-resistant prostate cancer. Int Urol Nephrol. 2018;50(10):1753–64. Sharifi N. Mechanisms of androgen receptor activation in castration-resistant prostate cancer. Endocrinology. 2013;154(11):4010–7. Culig Z. Androgen Receptor Coactivators in Regulation of Growth and Differentiation in Prostate Cancer. J Cell Physiol. 2016;231(2):270–4. Biron E, Bédard F. Recent progress in the development of protein-protein interaction inhibitors targeting androgen receptor-coactivator binding in prostate cancer. J Steroid Biochem Mol Biol. 2016;161:36–44. Shiota M, Yokomizo A, Fujimoto N, Naito S. Androgen receptor cofactors in prostate cancer: potential therapeutic targets of castration-resistant prostate cancer. Curr Cancer Drug Targets. 2011;11(7):870–81. Wang S, Fusaro G, Padmanabhan J, Chellappan SP. Prohibitin co-localizes with Rb in the nucleus and recruits N-CoR and HDAC1 for transcriptional repression. Oncogene. 2002;21(55):8388–96. Wang S, Nath N, Fusaro G, Chellappan S. Rb and prohibitin target distinct regions of E2F1 for repression and respond to different upstream signals. Mol Cell Biol. 1999;19(11):7447–60. Jackson DN, Alula KM, Delgado-Deida Y, et al. The Synthetic Small Molecule FL3 Combats Intestinal Tumorigenesis via Axin1-Mediated Inhibition of Wnt/β-Catenin Signaling. Cancer Res. 2020;80(17):3519–29. Yuan G, Chen X, Liu Z, et al. Flavagline analog FL3 induces cell cycle arrest in urothelial carcinoma cell of the bladder by inhibiting the Akt/PHB interaction to activate the GADD45α pathway. J Exp Clin Cancer Res. 2018;37(1):21. Dai Y, Ngo D, Jacob J, Forman LW, Faller DV. Prohibitin and the SWI/SNF ATPase subunit BRG1 are required for effective androgen antagonist-mediated transcriptional repression of androgen receptor-regulated genes. Carcinogenesis. 2008;29(9):1725–33. Lanz C, Bennamoun M, Macek P, Cathelineau X, Sanchez-Salas R. The importance of antiandrogen in prostate cancer treatment. Ann Transl Med. 2019;7(Suppl 8):362. 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11:55:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2325130/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2325130/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13046-023-02695-0","type":"published","date":"2023-05-20T20:51:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":30024219,"identity":"b0f656a4-e6bf-43b9-9186-349063cdee6d","added_by":"auto","created_at":"2022-12-07 17:20:16","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5487281,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of PHB1 is elevated in CRPC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Dataset analysis of PHB1 expression level in CRPC tissues compared with primary PCa tissues. Left plot: GSE 35988; Middle plot: GSE 74367; Right plot: Kumar A et al. 2016. \u003cstrong\u003eB. \u003c/strong\u003eRepresentative IHC images of PHB1 expression in PCa patient samples (above) and the quantification of IHC scores (below). \u003cstrong\u003eC\u003c/strong\u003e. qRT-PCR and Western blot analysis of PHB1 expression levels in human normal prostate cell RWPE-1 and 6 human PCa cell lines. \u003cstrong\u003eD.\u003c/strong\u003e Kaplan-Meier survival analysis in three referenced datasets. Left: the progression free survival analysis of the TCGA database; Middle: the overall survival analysis derived from Grasso CS et al (2012);\u003cstrong\u003e \u003c/strong\u003eRight: the disease free survival analysis derived from Taylor BS et al (2010). Data are represented as mean ± SD of three independent experiments. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2325130/v1/be0e27bfca713407135f5cf2.jpg"},{"id":30023029,"identity":"a3b3598e-40da-4579-8e89-7b2b413e9889","added_by":"auto","created_at":"2022-12-07 17:04:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5763929,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePHB1 promotes the development of CRPC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder CSS condition, MTS assay, transwell assay and plate colony formation assay were performed on LNCaP with ectopic PHB1 overexpression as well as C4-2B, 22Rv1 and PC-3 with PHB1 knockdown to assess \u003cstrong\u003eA.\u003c/strong\u003e The effect of PHB1 on cell proliferation of the 4 tested PCa cell lines. \u003cstrong\u003eB.\u003c/strong\u003e The effect of PHB1 on invasion/migration activity of the 4 tested PCa cell lines. \u0026nbsp;Left: representative images of cell invasion and migration; Right: quantitative results of invasion and migration assays of triplicate experiments. \u003cstrong\u003eC.\u003c/strong\u003e The effect of PHB1 on clonogenic ability of the 4 tested PCa cell lines. Data are represented as mean ± SD of three independent experiments. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2325130/v1/457d18037c5542eb8983d085.jpg"},{"id":30023030,"identity":"5cd62581-6780-4dd7-ac95-acf1f22f64a0","added_by":"auto","created_at":"2022-12-07 17:04:16","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4705329,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe androgen deprivation upregulated PHB1 expression and altered the subcellular distribution of PHB1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLNCaP cells were cultured under CSS condition for 3d and then were used for the following series of experiments except B. \u003cstrong\u003eA.\u003c/strong\u003e qRT-PCR and Western blot analysis for the effect of DHT on the expression level of PHB1 at indicated times (above) and in indicated concentrations (below). \u003cstrong\u003eB.\u003c/strong\u003e The effect of long-term androgen deprivation on expression of PHB1. Above: dataset analysis of mRNA expression in LNCaP cells following chronic androgen deprivation (GSE 59986); Below: Western blot analysis for comparison of the protein expression levels of PHB1 between LNCaP and LNCaP-AI cells. \u003cstrong\u003eC. \u003c/strong\u003eSubcellular fractionation and\u003cstrong\u003e \u003c/strong\u003eWestern blot analysis of PHB1 distribution in nuclear (N), cytosol (C), and membrane. \u003cstrong\u003eD.\u003c/strong\u003e Colocalization \u0026nbsp;analysis between PHB1 and AR by IF. PHB1 was detected using anti-PHB1. AR was detected using anti-AR. DAPI was used for the visualization of nuclear staining by laser confocal microscopy. PHB1: green; AR: red; DAPI: blue. Scale bars, 10 µm. \u003cstrong\u003eE. \u003c/strong\u003eCo-IP assay of \u0026nbsp;PHB1:AR interaction in whole cell lysate (left) and nuclear extract (right). \u003cstrong\u003eF.\u003c/strong\u003e Western blot detection of P-c-Raf\u003csup\u003eSer338\u003c/sup\u003e and P-MEK level without/with PHB1 knockdown. \u003cstrong\u003eG.\u003c/strong\u003e Colocalization analysis between PHB1 and mitochondria by IF. PHB1 was detected using anti-PHB1. Mito-Tracker and DAPI were used for the visualization of mitochondrial and nuclear staining by laser confocal microscopy. PHB1: green; Mito-Tracker: red; DAPI: blue. Scale bars, 10 µm. Data are represented as mean ± SD of three independent experiments.*P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2325130/v1/06e0f924e11dcb5787a501bf.jpg"},{"id":30023702,"identity":"3353afd8-2624-4079-b8a9-ed97dee9cb2b","added_by":"auto","created_at":"2022-12-07 17:12:16","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3774914,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombine ENZ with FL3 can improve the therapeutic effect of ENZ on CRPC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eIC50 values of FL3 in LNCaP, C2-2B, 22Rv1 and PC-3 cells determined by MTS assay, respectively.\u003cstrong\u003e B.\u003c/strong\u003e MTS assay showing the cell viability of LNCaP, C4-2B, and 22Rv1 cells treated with ENZ, FL3, and FL3 plus ENZ in indicated concentrations for 48h. \u003cstrong\u003eC. \u003c/strong\u003eC4-2B tumor xenograft showing the effect of ENZ, FL3, and FL3 plus ENZ in the indicated concentrations for 30d (n=6). Above: photographs of C4-2B tumors collected from sacrificed tumor-bearing mice in each group; Middle: analysis of tumor weight of each group ; Below: analysis of tumor volume of each group. \u003cstrong\u003eD. \u003c/strong\u003eTranswell assays showing the effect of FL3, ENZ, and FL3 plus ENZ on C4-2B cell migration. Data are represented as mean ± SD of three independent experiments. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2325130/v1/18b8fcd726148ee568b6d68b.jpg"},{"id":30023032,"identity":"0381fb49-f688-4b59-be41-389410b2963d","added_by":"auto","created_at":"2022-12-07 17:04:16","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4368850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFL3 treatment reversed the subcellular distribution changes of PHB1 induced by ADT.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC4-2B cells were treated with DMSO or FL3 20nM/40nM for 48h. \u003cstrong\u003eA.\u003c/strong\u003e Subcellular fractionation and Western blot analysis showing the effect of FL3 on PHB1 expression and subcellular distribution. Left: total expression; Middle: nuclear (N) and cytosol (C) expression; Right: plasma membranes expression. \u003cstrong\u003eB.\u003c/strong\u003e Colocalization analysis between PHB1 and AR by IF. \u0026nbsp;PHB1 was detected using anti-PHB1. AR was detected using anti-AR. DAPI was used for the visualization of nuclear staining by laser scanning microscopy. PHB1, green; AR, red; DAPI, blue. Scale bars, 10 µm. \u003cstrong\u003eC. \u003c/strong\u003eCo-IP assay for PHB1:AR interaction using the whole cell lysate (left) and nuclear extract (right). \u003cstrong\u003eD.\u003c/strong\u003e qRT-PCR detection of the expression of PHB1, PSA, and TMPRSS2. \u003cstrong\u003eF.\u003c/strong\u003e Colocalization analysis between PHB1 and mitochondria by IF. PHB1 was detected using anti-PHB1. Mito-Tracker and DAPI were used for the visualization of mitochondria and nuclear staining by laser confocal microscopy. PHB1: green; Mito-Tracker: red; DAPI: blue. Scale bars, 10 µm. \u003cstrong\u003eG. \u003c/strong\u003eApoptosis analysis of C42B cells after staining with Annexin-V and PI using flow cytometer. The bar diagram represents the statistical result. Data are represented as mean ± SD of three independent experiments. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2325130/v1/794bf82940e964c6721e0882.jpg"},{"id":30023028,"identity":"98798e72-71b7-435a-8bff-a31124c045cf","added_by":"auto","created_at":"2022-12-07 17:04:16","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1050349,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram showing the mechanism by which PHB1 promotes castration resistance and FL3 inhibits CRPC.\u003c/strong\u003eADT induces the translocation of PHB1 from nucleus to mitochondria and plasma membrane in ADPC, thereby facilitates reactivation of AR signaling, stabilization of mitochondria and activation of MAPK signaling. While FL3 promotes the translocation of PHB1 from plasma membrane and mitochondria to nucleus in CRPC, thereby inhibits the AR signaling through PHB1:AR interacting as well as MAPK signaling, but promotes apoptosis.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2325130/v1/763352fca56482a643ab2a84.jpg"},{"id":44728920,"identity":"7e2a57bc-3dc8-4a37-be8d-4475be8d0047","added_by":"auto","created_at":"2023-10-16 21:09:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1535343,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2325130/v1/aac6c699-f5ae-4d55-9ee7-1c8d2ee22fc6.pdf"},{"id":30023034,"identity":"1f822025-e04e-4c36-a263-50e3ca84d87a","added_by":"auto","created_at":"2022-12-07 17:04:16","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":1025550,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiguresandLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-2325130/v1/3b80336007267ce198593446.docx"},{"id":30023701,"identity":"5b2c76c8-cc54-4545-a1a8-af47589d4e2a","added_by":"auto","created_at":"2022-12-07 17:12:16","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":21491,"visible":true,"origin":"","legend":"","description":"","filename":"TableS13.PrimersAntibodiessiRNAsInformations.docx","url":"https://assets-eu.researchsquare.com/files/rs-2325130/v1/e33559cbcec43ec53a654d6e.docx"}],"financialInterests":"","formattedTitle":"Targeting PHB1 to inhibit castration-resistant prostate cancer progression in vitro and in vivo","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProstate cancer (PCa) is a malignant tumor that poses a severe threat to males\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Androgen deprivation therapy (ADT) is the primary therapy for locally advanced or metastatic PCa, but most patients will gradually develop into castration-resistant prostate cancer (CRPC) after initial treatment\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The occurrence and development of CRPC is a complex process involved and driven by multiple molecular pathways\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Although reactivation of the AR signal pathway is a critical driver of CRPC, and AR-targeted drugs such as ENZ have also significantly improved the clinical outcomes of CRPC patients, disease resistance still makes the cure of CRPC a serious challenge\u003csup\u003e[\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. In recent years, drugs targeting other CRPC-driver genes, such as AKT inhibitor ipatasertib, PARP inhibitors olaparib and rucaparib, have been developed and applied clinically one after another\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e, but which are far from enough to completely cure CRPC. Therefore, searching for novel targets and targeted drugs, is still an urgent problem to be solved.\u003c/p\u003e \u003cp\u003eProhibitin (PHB1/PHB) is a ubiquitously expressed protein conserved throughout evolution\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. PHB1 gene is located on chromosome 17q21 and encodes a 32 kDa protein composed of 272 amino acids\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. PHB1 gene was initially cloned based on the ability of its own 3\u0026rsquo;UTR to induce growth arrest in mammalian cells, and numerous subsequent studies have found that PHB1 is a multifunctional protein that is involved in cell growth, proliferation, apoptosis, and metabolism\u003csup\u003e[\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. In recent years, a large number of studies have shown that PHB1 expression is upregulated in a variety of clinical tumor specimens including lung cancer, diffuse large B-cell lymphomas (DLBCL), cervical cancer, bladder cancer, glioblastoma, endometrial cancer, and gastric cancer \u003csup\u003e[\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Due to its low mutation rate and essential function, PHB1 is a suitable target for drug administration\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Flavaglines are a class of natural products isolated from the medicinal plant \u003cem\u003eAglaia\u003c/em\u003e, which can exert a unique anti-cancer activity by binding to PHB1/PHB2\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. FL3, a synthetic derivative of flavagline with higher tumor cell specificity and lower normal cell cytotoxicity compared to natural flavaglines, has presented potent tumor suppressive effect by targeting PHB1 in a variety of cancers such as lung cancer, neuroblastoma, and DLBCL\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHowever, the role of PHB1 in PCa is still controversial. Bevan \u003cem\u003eet al.\u003c/em\u003e found that PHB1 is downregulated in LNCaP cells by dihydrotestosterone (DHT) stimulation after androgen starvation for 24h\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, while overexpression of PHB1 inhibited cell growth in LNCaP cells \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Contrarily, Zhu \u003cem\u003eet al.\u003c/em\u003e reported that mitochondria-localized PHB1 suppresses TGF-β induced apoptosis in CRPC cell line PC3\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. In addition, Ummanni and Cho, respectively, reported that PHB1 was upregulated in PCa specimens and positively correlated with the degree of malignancy\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Therefore, we speculate that PHB1 may be involved in the castration resistance. In this study, we explored the role of PHB1 in CRPC progression and assessed the effect of FL3 in CRPC therapy.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents\u003c/h2\u003e \u003cp\u003eFetal bovine serum (FBS), charcoal-stripped fetal bovine serum (CSS, depleted androgen and any other steroid), Bovine pituitary extract (BPE), and cell culture mediums were purchased from Gibco (NY, USA). ENZ was purchased from MedChemExpress (NJ, USA). FL3 was synthesized in Dr. Laurent D\u0026eacute;saubry\u0026rsquo;s lab according to a described procedure\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. DHT was purchased from Meilunbio (Dalian, China). Epidermal growth factor (EGF) was obtained from Peprotech (NJ, USA). Glutamine PenStrep was obtained from Invitrogen. (CA, UAS). Serial dilutions of all drugs were made using DMSO.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBioinformatics Analysis\u003c/h3\u003e\n\u003cp\u003eThe mRNA expression data of PHB1 were extracted from GEO (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/geo\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/geo\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and cBioPortal (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cbioportal.org/\u003c/span\u003e\u003cspan address=\"http://www.cbioportal.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database. The data used for Kaplan-Meier survival analysis were from TCGA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga/using-tcga\u003c/span\u003e\u003cspan address=\"https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga/using-tcga\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and cBioPortal (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cbioportal.org/\u003c/span\u003e\u003cspan address=\"http://www.cbioportal.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry(Ihc)\u003c/h3\u003e\n\u003cp\u003eThe cancer-adjacent normal tissues and PCa tissues were collected from undergoing radical prostatectomy for PCa at Qilu Hospital, Shandong University (Jinan, China) and made into tissue microarray (TMA). TMA was stained with H\u0026amp;E to be reviewed by two pathologists according to the WHO histologic classification of PCa, and the IHC staining was performed with Universal two-step detection kit (PV9000, Zhongshan Golden Bridge Biotechnology Co, Beijing, China). For PHB1 expression in tissues, the staining intensity was scored from 1 to 3: 1, weak; 2, moderate; 3, strong. The information of antibody used is summarized in Supplementary Table S2\u003c/p\u003e\n\u003ch3\u003eCell Lines And Cell Culture\u003c/h3\u003e\n\u003cp\u003eHuman prostate epithelial cell line RWPE-1 was purchased from National Collection of Authenticated Cell Cultures (Shanghai, China). PCa cell lines LNCaP, C4-2B, 22Rv1 and PC-3 were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). RWPE-1 cells were cultured in Keratinocyte serum-free medium supplemented with EGF, BPE, and 1% glutamine PenStrep. LNCaP, C4-2B, and 22Rv1 cells were cultured in RPMI-1640 with 10% FBS or charcoal-stripped fetal bovine serum (CSS). PC-3 cell was cultured in F12-K with 10% FBS or CSS. To establish derived LNCaP-AI (androgen-independent) cell line, LNCaP cells were maintained in RPMI-1640 supplemented with 10% CSS for at least 3 months\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. All cells were cultured at 37\u0026deg;C in an atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e and used up to 15 passages maximum. After the last experiment, all cells were confirmed mycoplasma free using GMyc-PCR Mycoplasma Test Kit (40601ES10, YeSen Biotech, Shanghai, China).\u003c/p\u003e\n\u003ch3\u003eQrt-pcr\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated using TRIzol reagent (Invitrogen, CA, UAS), and 1 \u0026micro;g of total RNA was used as template for the first strand cDNA synthesis using the ReverTra Ace qPCR RT Kit (TOYOBO, Osaka, Japan). qPCR reactions were carried out with a FastStart Universal SYBR Green Master Mix (Roche, Mannheim, Germany). Data were normalized by the expression level of β-actin in each sample. The primer sequences used are listed in Supplementary Table S1.\u003c/p\u003e\n\u003ch3\u003eWestern Blotting\u003c/h3\u003e\n\u003cp\u003eWhole-cell lysates were prepared by lysing the cells in ice-cold RIPA buffer (P0013C, Beyotime, Shanghai, China) with 1% protease inhibitor cocktail (NCM Biotech, Suzhou, China). 20 \u0026micro;g total protein was separated by SDS-PAGE and transferred to the PVDF membrane, which were then incubated with corresponding antibodies. The information of antibodies is summarized in Supplementary Table S2.\u003c/p\u003e\n\u003ch3\u003ePlasmids, Sirna, And Cell Transfection\u003c/h3\u003e\n\u003cp\u003eExpression vector pENTER-PHB1 and its control vector was designed and constructed by Vigene Bioscience (Shandong, China). siNC and siPHB1 were synthesized by Jikai company (Shanghai, China). The target sequences are listed in Supplementary Table S3. Lipofectamine 3000 (Invitrogen, CA, USA) was used for transient transfection following the manufacturer\u0026rsquo;s instruction. The transfection efficiency was confirmed using qRT-PCR and Western blotting.\u003c/p\u003e\n\u003ch3\u003eMTS assay\u003c/h3\u003e\n\u003cp\u003eCell viability and cell proliferation were determined by MTS assay with MTS Cell Proliferation Assay Kit (BestBio, Shanghai, China). LNCaP cells (3.0\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well), C4-2B cells (2.0\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well), 22Rv1 (1.5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well), or PC3 cells (1.5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well) were plated in 96-well plate. After cells adhering to the wall, 10 \u0026micro;l MTS was added to each well at 0, 24, 48, and 72 h, respectively, and incubated at 37\u0026deg;C for 2h. Then the absorbance was measured at 490 nm. Cell viability was expressed as the percentage of absorbance of cells treated with FL3 or Enzalutamide compared with cells treated with DMSO. Experiments were performed in triplicate and repeated three times.\u003c/p\u003e\n\u003ch3\u003eTranswell Assay\u003c/h3\u003e\n\u003cp\u003eTranswell invasion/migration assays were performed using Transwell Chambers with or without coated Matrigel (Corning, NY, USA). RPMI-1640 or F12-K medium with 10% FBS was added to the lower chamber as a chemoattractant for cells. LNCaP (10.0\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well), C4-2B (10.0\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well), 22Rv1 (7.0\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well), or PC3 cells (7.0\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well) were plated in the upper chamber with RPMI-1640 or F12-K medium. After 24h of incubation, the migrated and invasive cells were fixed with 4% paraformaldehyde (Biosharp, Beijing, China) and stained with crystal violet solution (Solarbio, Beijing China). 5 fields of view were randomly selected for counting transmembrane cells under the light microscope using a 40 \u0026times; magnification.\u003c/p\u003e\n\u003ch3\u003ePlate Colony Formation Assay\u003c/h3\u003e\n\u003cp\u003eThe clonogenic ability of PCa cells was measured by plate colony formation assay. LNCaP (300 cells/well), C4-2B (300 cells/well), 22Rv1 (200 cells/well) or PC3 cells (200 cells/well) were plated in 6-well plate. 14d after initial seeding, cells were fixed with 4% paraformaldehyde and stained with crystal violet solution. Colonies containing more than 50 cells were counted and plotted. Experiments were repeated independent three times.\u003c/p\u003e\n\u003ch3\u003eSubcellular Fractionation\u003c/h3\u003e\n\u003cp\u003eNucleus and cytosol proteins were separated using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, Nantong, China) following the manufacturer\u0026rsquo;s instruction. plasma membrane extracts were prepared using Minute\u0026trade; Plasma Membrane Protein Isolation and Cell Fractionation Kit (Invent Biotechnologies, Eden Prairie, USA) according to manufacturer\u0026rsquo;s manual.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence (If)\u003c/h3\u003e\n\u003cp\u003eIF was performed as previously described\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Pre-treated LNCaP (4 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) and C4-2B (2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) cells were seeded on glass coverslips in 24-well plates, respectively. Mitochondrial labelling was performed using MitoTracker Deep Red FM (40743ES, Yeasen Biotech, Shanghai, China) following the manufacturer\u0026rsquo;s instruction. Nucleus was stained with DAPI (H-1200, VECTASHIELD, CA, USA). Antibodies used is summarized in Supplementary Table S2. Cells were observed under Laser Confocal Microscope LSM 980 (Zeiss, Oberkochen, Germany). Images taken were appropriately processed with the ZEN software program. Experiments were repeated three independent times.\u003c/p\u003e\n\u003ch3\u003eCo-immunoprecipitation (Co-ip)\u003c/h3\u003e\n\u003cp\u003e Co-IP assays were performed according to the instruction of BeyoMag\u0026trade; Protein A/G Magnetic Beads for IP (Beyotime, Shanghai, China). 10 \u0026micro;g antibody in a 1:50 dilution was incubated with 20 \u0026micro;l Protein A/G Magnetic Beads for 1h at temperature. The cell lysate or nuclear extract, leaving 50 \u0026micro;l as input, was incubated with antibody-coated immunomagnetic beads at 4℃ overnight. The immuno-complex was collected and was analyzed by Western blotting. The information of antibodies is summarized in Supplementary Table S2\u003c/p\u003e\n\u003ch3\u003eTumor Xenografts\u003c/h3\u003e\n\u003cp\u003e5-week-old male BALB/c nude mice were purchased from Weitonglihua Biotechnology (Beijing, China). A total of 6.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e C4-2B cells were mixed with Matrigel (1:1) and injected subcutaneously into the mice (n\u0026thinsp;=\u0026thinsp;6/group). After the mice were surgically castrated, they were randomized into 4 groups (n\u0026thinsp;=\u0026thinsp;6/group) and treated with indicated drugs and concentrations. ENZ and FL3 were administered by oral gavage and subcutaneously every two days, respectively. Body weight and tumor volume was measured and recorded twice a week. Tumor tissues were harvested and weighed after 15 times of treatment. Tumor volume was calculated with the following formula: tumor volume\u0026thinsp;=\u0026thinsp;length \u0026times; width\u003csup\u003e2\u003c/sup\u003e \u0026times; 0.5. All animal experiments followed a protocol approved by the Shandong University Animal Care Committee (Document No. ECSBMSSDU2019-2-019).\u003c/p\u003e\n\u003ch3\u003eFlow Cytometry\u003c/h3\u003e\n\u003cp\u003eDead Cell Apoptosis Kit (Invitrogen, CA, USA) was used according to the manufacturer's instruction. C42B cells treated with FL3 or DMSO were harvested and dealed with Annexin V-FITC and propidium iodide, followed by apoptosis analysis with CytoFLEX S (1720610S-2, BECKMAN COULTER, CA, USA). Experiments were repeated three independent times.\u003c/p\u003e\n\u003ch3\u003ePattern Drawing\u003c/h3\u003e\n\u003cp\u003eThe pattern diagram was drawn using the Figdraw software (ResearchHome, Zhejiang, China).\u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eFor \u003cem\u003ein vitro\u003c/em\u003e experiment, experiments were carried out at least in triplicate to confirm reproducibility and presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. In the xenograft studies, tumor sizes were served as the primary response measure when the mice were sacrificed. Statistical analysis was carried out with GraphPad Prism 8.0 using a t-test and two-way ANOVA. Significance was determined at *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Result","content":"\u003cp\u003e\u003cstrong\u003ePHB1 expression is significantly upregulated in CRPC and is associated with poor prognosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore its role in castration resistance, we first investigated the association between the expression level of PHB1 and PCa progression. By analyzing public datasets, we found that PHB1 mRNA expression was upregulated in localized PCa tissues compared with benign prostate tissues (GSE35988)\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e (Fig. S1 A). Higher mRNA levels of PHB1 were associated with higher Gleason score (P\u0026thinsp;=\u0026thinsp;0.0054) (Fig. S1 B). More importantly, analysis of three independent datasets (GSE35988, GES74367 and Fred Hutchinson Cancer Research Center-2016)\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e showed significantly increased PHB1 expression in CRPC tissues compared to primary PCa tissues (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Using IHC, we examined PHB1 protein expression in 105 PCa and 5 cancer-adjacent normal tissues. The results showed that PHB1 expression was significantly higher in PCa tissues than that in adjacent normal tissues and was positively correlated with the Gleason score (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Similarly, the expression of PHB1 was higher in LNCaP cells with ADPC phenotype than that in normal prostate epithelial RWPE-1 cells, while in CRPC cell lines (C4-2B, 22Rv1, DU145, PC3), PHB1 expression was higher than that in LNCaP cells (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). Finally, we also performed Kaplan-Meier survival analysis using the TCGA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga/using-tcga\u003c/span\u003e\u003c/span\u003e) and cBioPortal database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cbioportal.org/\u003c/span\u003e\u003c/span\u003e) to explore the clinicopathological significance of PHB1 expression in PCa patients. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD, compared with the low PHB1 expression group, the high expression group had shorter progression-free survival (TCGA), overall survival (MCTP-2012)\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e, and disease-free survival (MSCKK-2010)\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. The above results suggested that increased PHB1 expression positively correlate with the degree of malignancy and may participate in the castration resistance of PCa.\u003c/p\u003e\n\u003ch3\u003ePhb1 Is Involved In The Development Of Crpc\u003c/h3\u003e\n\u003cp\u003eTo explore whether PHB1 is involved in the castration resistance process of PCa, we performed a series of gain/loss-of-function experiments in ADPC (LNCaP), AR\u003csup\u003e+\u003c/sup\u003e CRPC (C4-2B, 22Rv1) and AR\u003csup\u003e\u0026minus;\u003c/sup\u003e CRPC (PC-3) cell lines. Cells were grown in the medium with 10% CSS for 3d to mimic the ADT environment. We then overexpressed PHB1 in LNCaP cells whereas applied PHB1 siRNA to knockdown PHB1 expression in C4-2B, 22Rv1, and PC3 cells, respectively. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, under CSS condition, PHB1 overexpression enhanced the proliferation, invasion, migration, and clonogenicity of LNCaP cells, whereas PHB1 knockdown inhibited the cell growth, invasion, migration and clonogenic growth of C4-2B, 22Rv1, and PC-3 cells. Together, these data indicate that PHB1 is involved in the development of CRPC.\u003c/p\u003e\n\u003ch3\u003eAndrogen Depletion Upregulates The Phb1 Expression And Promotes Its Nucleus-cytoplasm Translocation\u003c/h3\u003e\n\u003cp\u003eTo elucidate the mechanism by which PHB1 promotes castration resistance in PCa, we first determined whether the AR signaling pathway regulates PHB1 expression. Androgen-starved LNCaP cells were stimulated with DHT for indicated times (0\\6\\12\\24\\48 h) or concentrations (0\\0.01\\0.1\\1\\10 nM), respectively. Western blot and qRT-PCR results showed that DHT treatment inhibited the expression of PHB1 in a time/concentration-dependent manner (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA), indicating that PHB1 is an androgen suppressive gene, which is consistent with Bevan\u0026apos;s results\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Furthermore, the GSE59986\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e dataset analysis revealed that PHB1 mRNA expression in LNCaP cells was upregulated under chronic androgen depletion. Meanwhile, comparison of PHB1 protein expression level between LNCaP and LNCaP-AI (established by our lab) cells presented similar trend (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003eGiven that the subcellular localization of PHB1 determines its functions\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, we thereby investigated whether ADT affects the subcellular distribution of PHB1. After culturing LNCaP cells under CSS condition for 3d, subcellular fractionation results showed that CSS culture resulted in decreased nuclear PHB1 protein abundance but increased cytoplasmic and membrane-associate PHB1 compared to FBS culture (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). Further, colocalizaion analysis using IF assay revealed that in nucleus, PHB1 signal was significantly attenuated, and the PHB1:AR colocalization signal almost disappeared (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD), which was confirmed by Co-IP experiments (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE). In addition, the phosphorylation levels of c-Raf \u003csup\u003eser338\u003c/sup\u003e and MEK were increased after 3d of CSS culture, and this effect was partially attenuated by knockdown of PHB1 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF). Finally, CSS culture for 3d also significantly enhanced PHB1 fluorescence signal in mitochondria compared with FBS culture (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG). These data suggest that ADT simultaneously upregulates PHB1 expression and modulates its subcellular distribution, which in turn affects the status of AR signaling as well as mitochondrial apoptosis pathway and c-Raf/MAPK pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFL3 has a stronger inhibition effect on ENZ-sensitive CRPC, particularly combined with ENZ.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo characterize the potential role of FL3 in CRPC therapy, we firstly determined the half-maximal inhibitory concentration (IC50) values of FL3 on different PCa cell lines. The results showed that C4-2B cells was mostly sensitive to FL3 (IC50 17.50 nM), followed by LNCaP (IC50 35.99 nM), 22Rv1 (IC50 43.05 nM) and PC3 (IC50 57.04 nM) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Based on the measured IC50 values, transwell assay results further showed that 20/40/80 nM FL3 inhibited migration and invasion of all 4 cell lines in a concentration-dependent manner (Fig. S2 A-D). Given that FL3 was more cytotoxic to ENZ-sensitive AR\u003csup\u003e+\u003c/sup\u003e CRPC cell line C4-2B\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e than to ADPC cell line LNCaP, ENZ-resistant AR\u003csup\u003e+\u003c/sup\u003e CRPC cell line 22Rv1\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e and AR\u003csup\u003e\u0026minus;\u003c/sup\u003e cell line PC-3, we then compared the effect of FL3, ENZ treatment alone or in combination on the proliferation of AR\u003csup\u003e+\u003c/sup\u003e cell lines LNCaP, C4-2B and 22Rv1 using MTS. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, although the low-dose combination of FL3 and ENZ does not effectively inhibit cell proliferation, but the combination of 40 nM FL3 with 20 \u0026micro;M ENZ significantly inhibited the growth of LNCaP, C4-2B, and 22Rv1 cells in 70%, 81%, and 54%inhibition rate (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), respectively, which were higher than that by treatment with either 20 \u0026micro;M ENZ (55%, 52%, 39% suppression, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) or 40 nM FL3 (52%, 77%, 35% suppression, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) alone. These results indicate that FL3 significantly enhanced the sensitivity of C4-2B cells to ENZ.\u003c/p\u003e\n\u003cp\u003eNext, we examined the impacts of FL3 on tumor growth of C4-2B xenograft in castrated nude mice. Mice were grouped randomly and treated with DMSO (control)/ENZ/FL3/FL3 plus ENZ, respectively. ENZ or FL3 treatment alone reduced both weight (ENZ vs. control: 983.3\u0026thinsp;\u0026plusmn;\u0026thinsp;213.7 vs. 2566.7\u0026thinsp;\u0026plusmn;\u0026thinsp;578.5 mg; FL3 vs. control: 1183.3\u0026thinsp;\u0026plusmn;\u0026thinsp;231.7 vs. 2566.7\u0026thinsp;\u0026plusmn;\u0026thinsp;578.5 mg) and volume (ENZ vs. control: 872.2\u0026thinsp;\u0026plusmn;\u0026thinsp;284.1 vs. 1809\u0026thinsp;\u0026plusmn;\u0026thinsp;218.8 mm\u003csup\u003e3\u003c/sup\u003e; FL3 vs. control: 944.2\u0026thinsp;\u0026plusmn;\u0026thinsp;205 mm\u003csup\u003e3\u003c/sup\u003e vs. 1809\u0026thinsp;\u0026plusmn;\u0026thinsp;218.8 mm\u003csup\u003e3\u003c/sup\u003e) of the tumor, and the combined treatment with ENZ and FL3 caused stronger inhibition (Weight, 233.3\u0026thinsp;\u0026plusmn;\u0026thinsp;103.3 mg; Volume, 453.7\u0026thinsp;\u0026plusmn;\u0026thinsp;73.1 mm\u003csup\u003e3\u003c/sup\u003e) of tumor growth than that with FL3 or ENZ alone (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). Moreover, there were no significant changes in body weights of all group\u0026rsquo;s mice (Fig. S3 A) as well as vital organ (liver and lung) histology (Fig. S3 B). In addition, transwell assays on C4-2B cells showed that the number of migrated cells in the combined administration group was further attenuated compared with the single drug administration groups (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\n\u003ch3\u003eFl3 Inhibits Crpc By Affecting The Subcellular Distribution Of Phb1\u003c/h3\u003e\n\u003cp\u003eTo explore the molecular mechanism underlying FL3-mediated tumor suppression in CRPC, we first analyzed the expression and subcellular distribution of PHB1 in C4-2B cells upon exposure to FL3. The subcellular fractionation results showed that after treatment with 20nM FL3 for 48 h, the transfer of PHB1 from the cytoplasm and plasma membrane to the nucleus increased compared to DMSO control without changing total PHB1 protein level (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Colocalization analysis using IF assays revealed that FL3 promoted the nuclear translocation of PHB1 protein and consequently enhanced the co-localization of PHB1:AR significantly (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB), which was further confirmed by Co-IP experiments (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). Moreover, the two essential AR target genes, PSA and TMPRSS2, were downregulated at the mRNA level by FL3 treatment (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD), suggesting that FL3 inhibited AR transcriptional activity by promoting the PHB1:AR interaction. In addition, FL3 significantly inhibited the EGF-induced phosphorylation of P-c-Raf\u003csup\u003eSer338\u003c/sup\u003e and P-MEK in C4-2B cells (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). And FL3 also decreased the colocalization signals of PHB1 with mitochondria in the cytoplasm (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF), along with increased apoptosis rate in C4-2B cells (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG and Fig. S4). These data indicate that FL3 may exert its anticancer effect via reversing the subcellular distribution alteration of PHB1 in CRPC.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIt has been widely reported that PHB1 is upregulated in a variety of tumors and has a tumor-promoting effect \u003csup\u003e[\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e, but there are few reports on the role of PHB1 in CRPC. In the current study, we showed that PHB1 was highly expressed in CRPC compared with that of localized PCa and was positively correlated with poor prognosis in clinical setting. Further gain/loss-of-function analysis showed that PHB1 can promote the proliferation, invasion and metastasis of all 4 tested PCa cell lines under CSS condition. These results indicated that PHB1 is involved in castration resistance and is one of the potential driving factors for CRPC.\u003c/p\u003e \u003cp\u003ePHB1 is a multifunctional molecular chaperone/scaffold protein that primarily localizes to mitochondria, plasma membrane and nucleus\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Mitochondrial PHB1 involves in stabilization of mitochondria and suppress apoptosis\u003csup\u003e[\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Nuclear PHB1 as a cofactor mediate transcriptional regulation through interacting with several transcription factors\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. Cell membrane-localized PHB1 is required for K-Ras-induced c-Raf activation\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. Numerous studies have shown that the specific role of PHB1 depends on its abundance and subcellular distribution characteristic of specific tumor types. PHB1 can shuttle between subcellular compartments under the induction of regulatory factors, and in turn mediate the regulation of several signal pathways\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e \u003cp\u003ePreviously, Bevan \u003cem\u003eet al\u003c/em\u003e. reported that PHB1 is an androgen-repressive gene in the LNCaP cells (also validated by us in this study), and ectopic overexpression of PHB1 inhibited androgen-mediated cell growth in LNCaP cells. By contrast, our result showed that PHB1 overexpression under CSS condition promoted the cell growth of LNCaP cells. To this reason, we observe the effect of ADT on PHB1 expression and its subcellular distribution. As expected, PHB1 expression was significantly upregulated under chronic CSS culture. Next, we found that ADT promoted translocation of PHB1 from the nucleus to mitochondria and plasma membrane, leading to mitochondrial PHB1 increase, nuclear PHB1:AR colocalization decrease, and activation of c-Raf/MAPK signaling. As a hormone (androgen) sensitive disease, reactivation of AR signaling remains dominant in the molecular mechanism of castration resistance, and alterations in cofactors of the AR pathway are one of the reactivation factors as well as potential therapeutic targets of CRPC\u003csup\u003e[\u003cspan additionalcitationids=\"CR53 CR54 CR55 CR56 CR57\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e. Previous reports have indicated that nuclear PHB1 mainly as corepressor interact with other transcription factors or corepressors such as Rb, E2F, N-CoR and HDAC1\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/sup\u003e. Bevan \u003cem\u003eet al\u003c/em\u003e. firstly found that PHB1 is also a corepressor of AR, and it represses ligand-dependent AR activity by forming part of transcriptional repressive complex with AR through indirect interaction\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Thus, ADT-caused loss of nuclear PHB1 should be one of the reasons explained reactivation of AR signaling in CRPC. Furthermore, PHB1 translocated to the plasma membrane and mitochondria could further facilitate promoting castration resistance by enhancing MAPK signaling pathway activation and stabilizing mitochondria. Therefore, PHB1 could be a novel potential therapeutic target for CRPC.\u003c/p\u003e \u003cp\u003eAs a small-molecule drug targeting PHB1, FL3 has been shown to suppress the growth of several tumors including urothelial carcinoma, colorectal cancer, and DLBCL\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/sup\u003e. However, the anticancer effect and underlying mechanism of FL3 in PCa remains unclear. In this study, we first demonstrated that nanomolar FL3 significantly suppressed the cell viability, invasion and migration of 4 tested PCa cell lines, especially C4-2B cells exhibited the greatest sensitivity to FL3. Because FL3 could promote cytoplasmic-nucleus translocation of PHB1 in DLBCL cells\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/sup\u003e, and PHB1 is required for AR-transcriptional repression mediated by androgen antagonists bicalutamide, for that PHB1 is recruited to androgen response promoters such as PSA gene by bicalutamide-bound AR, where they form a AR-corepressor complex with other corepressors and release coactivator P300\u003csup\u003e[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]\u003c/sup\u003e. Thus, we infer that FL3 should be suitable for combination with new generation of antiandrogenic drug ENZ\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]\u003c/sup\u003e on AR\u003csup\u003e+\u003c/sup\u003e ENZ-sensitive CRPC cell line C4-2B. As expected, 40 nM FL3 combined with 20 \u0026micro;M ENZ administration on C4-2B still present the most inhibition effect than that treatment with FL3 or ENZ alone both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Next, a series of mechanism analysis experiments confirmed that FL3 led to cytoplasmic-nucleus translocation of PHB1 without downregulating PHB1 in C4-2B cells, along with AR signaling repressing due to reflowed nuclear PHB1 synergistically enhance the inhibitory effect of ENZ on AR signaling. Meanwhile, reversal of PHB1 subcellular distribution also inhibited c-Raf/MAPK signaling and promoted apoptosis. These results suggest that FL3 is suitable for PCa treatment, especially as an adjuvant chemotherapy drug in combination with ENZ to treat ENZ-sensitive AR\u003csup\u003e+\u003c/sup\u003e CRPC.\u003c/p\u003e \u003cp\u003eIn conclusion, in this study, we firstly showed that ADT triggered the role switch of PHB1 from anti-tumor in ADPC to pro-tumor in CRPC by upregulating PHB1 and more importantly, inducing its nucleus-cytoplasmic translocation, while FL3 inhibit PCa by reversing the ADT-induced subcellular distribution alteration of PHB1, particularly reflow nuclear PHB1 synergistically enhances the anti-cancer effect of ENZ by form transcriptional repressive complex with AR-ENZ complex in ENZ-sensitive CRPC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), indicating that targeting PHB1 is a promising strategy as adjuvant therapy for ENZ-sensitive CRPC, reducing ENZ dosage and adverse reactions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCRPC Castration-resistant prostate cancer\u003c/p\u003e\u003cp\u003ePCa Prostate cancer\u003c/p\u003e\u003cp\u003ePHB1 Prohibitin\u003c/p\u003e\u003cp\u003eAR Androgen receptor\u003c/p\u003e\u003cp\u003eADT Androgen deprivation therapy\u003c/p\u003e\u003cp\u003eENZ Enzalutamide\u003c/p\u003e\u003cp\u003eDHT Dihydrotestosterone\u003c/p\u003e\u003cp\u003eCSS Charcoal-stripped fetal bovine serum\u003c/p\u003e\u003cp\u003eFBS Fetal bovine serum\u003c/p\u003e\u003cp\u003eTMPRSS2 Transmembrane protease serine 2\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy concept and design: JL, WC and BH. Data acquisition: JL, RZ, TS, LG, ZH, XW and FS. Analysis and interpretation of the data: JL, RZ, QZ, YX, WC and XW. Paper preparation: JL, RZ and WC. Critical review: WC, BH, and LD. The authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China [81972413], Natural Science Foundation of Shandong Province [ZR2018MH025, ZR2018MH030], and Primary Research \u0026amp; Development Plan of Shandong Province [2019GSF108076].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe use of clinical samples was approved by the ethics committee of Shandong University and informed consent was obtained from all patients\u0026nbsp;(reference number ECSBMSSDU2019-1-007). All animal experimental protocols were performed following the Ethical Animal Care and Use Committee of Shandong University (reference number ECSBMSSDU2019-2-019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors consent to the publication of this article.\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"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScher HI, Sawyers CL. Biology of progressive, castration-resistant prostate cancer: directed therapies targeting the androgen-receptor signaling axis. J Clin Oncol. 2005;23(32):8253\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBai B, Chen Q, Jing R, et al. Molecular Basis of Prostate Cancer and Natural Products as Potential Chemotherapeutic and Chemopreventive Agents. Front Pharmacol. 2021;12:738235.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKobayashi T, Inoue T, Kamba T, Ogawa O. Experimental evidence of persistent androgen-receptor-dependency in castration-resistant prostate cancer. Int J Mol Sci. 2013;14(8):15615\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScher HI, Fizazi K, Saad F, et al. Increased survival with enzalutamide in prostate cancer after chemotherapy. N Engl J Med. 2012;367(13):1187\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAntonarakis ES, Lu C, Wang H, et al. AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer. N Engl J Med. 2014;371(11):1028\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCornford P, Bellmunt J, Bolla M, et al. EAU-ESTRO-SIOG Guidelines on Prostate Cancer. Part II: Treatment of Relapsing, Metastatic, and Castration-Resistant Prostate Cancer. Eur Urol. 2017;71(4):630\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMishra S, Murphy LC, Murphy LJ. The Prohibitins: emerging roles in diverse functions. J Cell Mol Med. 2006;10(2):353\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMishra S, Ande SR, Nyomba BL. The role of prohibitin in cell signaling. FEBS J. 2010;277(19):3937\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTheiss AL, Sitaraman SV. The role and therapeutic potential of prohibitin in disease. Biochim Biophys Acta. 2011;1813(6):1137\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng YT, Chen P, Ouyang RY, Song L. Multifaceted role of prohibitin in cell survival and apoptosis. Apoptosis. 2015;20(9):1135\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnde SR, Xu Y, Mishra S. Prohibitin: a potential therapeutic target in tyrosine kinase signaling. Signal Transduct Target Ther. 2017;2:17059.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSignorile A, Sgaramella G, Bellomo F, De Rasmo D. Prohibitins. A Critical Role in Mitochondrial Functions and Implication in Diseases. Cells. 2019. 8(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChiu CF, Ho MY, Peng JM, et al. Raf activation by Ras and promotion of cellular metastasis require phosphorylation of prohibitin in the raft domain of the plasma membrane. Oncogene. 2013;32(6):777\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang L, Dong P, Zhang Z, et al. Akt phosphorylates Prohibitin 1 to mediate its mitochondrial localization and promote proliferation of bladder cancer cells. Cell Death Dis. 2015;6(2):e1660.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang H, Zhang S, Li Y, et al. Suppression of mitochondrial ROS by prohibitin drives glioblastoma progression and therapeutic resistance. Nat Commun. 2021;12(1):3720.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang B, Chen R, Liang X, et al. Estrogen Enhances Endometrial Cancer Cells Proliferation by Upregulation of Prohibitin. J Cancer. 2019;10(7):1616\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang D, Tabti R, Elderwish S, et al. Prohibitin ligands: a growing armamentarium to tackle cancers, osteoporosis, inflammatory, cardiac and neurological diseases. Cell Mol Life Sci. 2020;77(18):3525\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYurugi H, Marini F, Weber C, et al. Targeting prohibitins with chemical ligands inhibits KRAS-mediated lung tumours. Oncogene. 2017;36(33):4778\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBentayeb H, Aitamer M, Petit B, et al. Prohibitin (PHB) expression is associated with aggressiveness in DLBCL and flavagline-mediated inhibition of cytoplasmic PHB functions induces anti-tumor effects. J Exp Clin Cancer Res. 2019;38(1):450.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSato T, Sakamoto T, Takita K, Saito H, Okui K, Nakamura Y. The human prohibitin (PHB) gene family and its somatic mutations in human tumors. Genomics. 1993;17(3):762\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWhite KA, Lange EM, Ray AM, Wojno KJ, Cooney KA. Prohibitin mutations are uncommon in prostate cancer families linked to chromosome 17q. Prostate Cancer Prostatic Dis. 2006;9(3):298\u0026ndash;302.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMacArthur IC, Bei Y, Garcia HD, et al. Prohibitin promotes de-differentiation and is a potential therapeutic target in neuroblastoma. JCI Insight. 2019. 5(10).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGamble SC, Odontiadis M, Waxman J, et al. Androgens target prohibitin to regulate proliferation of prostate cancer cells. Oncogene. 2004;23(17):2996\u0026ndash;3004.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGamble SC, Chotai D, Odontiadis M, et al. Prohibitin, a protein downregulated by androgens, represses androgen receptor activity. Oncogene. 2007;26(12):1757\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDart DA, Spencer-Dene B, Gamble SC, Waxman J, Bevan CL. Manipulating prohibitin levels provides evidence for an in vivo role in androgen regulation of prostate tumours. Endocr Relat Cancer. 2009;16(4):1157\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu B, Zhai J, Zhu H, Kyprianou N. Prohibitin regulates TGF-beta induced apoptosis as a downstream effector of Smad-dependent and -independent signaling. Prostate. 2010;70(1):17\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUmmanni R, Junker H, Zimmermann U, et al. Prohibitin identified by proteomic analysis of prostate biopsies distinguishes hyperplasia and cancer. Cancer Lett. 2008;266(2):171\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCho SY, Kang S, Kim DS, et al. HSP27, ALDH6A1 and Prohibitin Act as a Trio-biomarker to Predict Survival in Late Metastatic Prostate Cancer. Anticancer Res. 2018;38(11):6551\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThuaud F, Bernard Y, T\u0026uuml;rkeri G, et al. Synthetic analogue of rocaglaol displays a potent and selective cytotoxicity in cancer cells: involvement of apoptosis inducing factor and caspase-12. J Med Chem. 2009;52(16):5176\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcNaughton M, Pitman M, Pitson SM, Pyne NJ, Pyne S. Proteasomal degradation of sphingosine kinase 1 and inhibition of dihydroceramide desaturase by the sphingosine kinase inhibitors, SKi or ABC294640, induces growth arrest in androgen-independent LNCaP-AI prostate cancer cells. Oncotarget. 2016;7(13):16663\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun F, Wang X, Hu J, et al. RUVBL1 promotes enzalutamide resistance of prostate tumors through the PLXNA1-CRAF-MAPK pathway. Oncogene. 2022;41(23):3239\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrasso CS, Wu YM, Robinson DR, et al. The mutational landscape of lethal castration-resistant prostate cancer. Nature. 2012;487(7406):239\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar A, Coleman I, Morrissey C, et al. Substantial interindividual and limited intraindividual genomic diversity among tumors from men with metastatic prostate cancer. Nat Med. 2016;22(4):369\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoudier MP, Winters BR, Coleman I, et al. Characterizing the molecular features of ERG-positive tumors in primary and castration resistant prostate cancer. Prostate. 2016;76(9):810\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaylor BS, Schultz N, Hieronymus H, et al. Integrative genomic profiling of human prostate cancer. Cancer Cell. 2010;18(1):11\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkamatsu S, Wyatt AW, Lin D, et al. The Placental Gene PEG10 Promotes Progression of Neuroendocrine Prostate Cancer. Cell Rep. 2015;12(6):922\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerma S, Shankar E, Chan ER, Gupta S. Metabolic Reprogramming and Predominance of Solute Carrier Genes during Acquired Enzalutamide Resistance in Prostate Cancer. Cells. 2020. 9(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDyshlovoy SA, Otte K, Tabakmakher KM, et al. Synthesis and anticancer activity of the derivatives of marine compound rhizochalin in castration resistant prostate cancer. Oncotarget. 2018;9(24):16962\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu TF, Wu H, Wang YW, et al. Prohibitin in the pathogenesis of transitional cell bladder cancer. Anticancer Res. 2007;27(2):895\u0026ndash;900.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsai HW, Chow NH, Lin CP, Chan SH, Chou CY, Ho CL. The significance of prohibitin and c-Met/hepatocyte growth factor receptor in the progression of cervical adenocarcinoma. Hum Pathol. 2006;37(2):198\u0026ndash;204.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRyu JW, Kim HJ, Lee YS, et al. The proteomics approach to find biomarkers in gastric cancer. J Korean Med Sci. 2003;18(4):505\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSripathi SR, He W, Atkinson CL, et al. Mitochondrial-nuclear communication by prohibitin shuttling under oxidative stress. Biochemistry. 2011;50(39):8342\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArtal-Sanz M, Tavernarakis N. Prohibitin and mitochondrial biology. Trends Endocrinol Metab. 2009;20(8):394\u0026ndash;401.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOsman C, Merkwirth C, Langer T. Prohibitins and the functional compartmentalization of mitochondrial membranes. J Cell Sci. 2009;122(Pt 21):3823\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRizwani W, Alexandrow M, Chellappan S. Prohibitin physically interacts with MCM proteins and inhibits mammalian DNA replication. Cell Cycle. 2009;8(10):1621\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S, Nath N, Adlam M, Chellappan S. Prohibitin, a potential tumor suppressor, interacts with RB and regulates E2F function. Oncogene. 1999;18(23):3501\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang J, Li B, He QY. Significance of prohibitin domain family in tumorigenesis and its implication in cancer diagnosis and treatment. Cell Death Dis. 2018;9(6):580.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRajalingam K, Wunder C, Brinkmann V, et al. Prohibitin is required for Ras-induced Raf-MEK-ERK activation and epithelial cell migration. Nat Cell Biol. 2005;7(8):837\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRastogi S, Joshi B, Fusaro G, Chellappan S. Camptothecin induces nuclear export of prohibitin preferentially in transformed cells through a CRM-1-dependent mechanism. J Biol Chem. 2006;281(5):2951\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuan Z, He Y, Alattar M, Chen Z, He F. Targeting the prohibitin scaffold-CRAF kinase interaction in RAS-ERK-driven pancreatic ductal adenocarcinoma. Mol Cancer. 2014;13:38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRastogi S, Joshi B, Dasgupta P, Morris M, Wright K, Chellappan S. Prohibitin facilitates cellular senescence by recruiting specific corepressors to inhibit E2F target genes. Mol Cell Biol. 2006;26(11):4161\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFusaro G, Wang S, Chellappan S. Differential regulation of Rb family proteins and prohibitin during camptothecin-induced apoptosis. Oncogene. 2002;21(29):4539\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai Z, Chen W, Zhang J, Li H. Androgen receptor: what we know and what we expect in castration-resistant prostate cancer. Int Urol Nephrol. 2018;50(10):1753\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharifi N. Mechanisms of androgen receptor activation in castration-resistant prostate cancer. Endocrinology. 2013;154(11):4010\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCulig Z. Androgen Receptor Coactivators in Regulation of Growth and Differentiation in Prostate Cancer. J Cell Physiol. 2016;231(2):270\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBiron E, B\u0026eacute;dard F. Recent progress in the development of protein-protein interaction inhibitors targeting androgen receptor-coactivator binding in prostate cancer. J Steroid Biochem Mol Biol. 2016;161:36\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShiota M, Yokomizo A, Fujimoto N, Naito S. Androgen receptor cofactors in prostate cancer: potential therapeutic targets of castration-resistant prostate cancer. Curr Cancer Drug Targets. 2011;11(7):870\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S, Fusaro G, Padmanabhan J, Chellappan SP. Prohibitin co-localizes with Rb in the nucleus and recruits N-CoR and HDAC1 for transcriptional repression. Oncogene. 2002;21(55):8388\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S, Nath N, Fusaro G, Chellappan S. Rb and prohibitin target distinct regions of E2F1 for repression and respond to different upstream signals. Mol Cell Biol. 1999;19(11):7447\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJackson DN, Alula KM, Delgado-Deida Y, et al. The Synthetic Small Molecule FL3 Combats Intestinal Tumorigenesis via Axin1-Mediated Inhibition of Wnt/β-Catenin Signaling. Cancer Res. 2020;80(17):3519\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan G, Chen X, Liu Z, et al. Flavagline analog FL3 induces cell cycle arrest in urothelial carcinoma cell of the bladder by inhibiting the Akt/PHB interaction to activate the GADD45α pathway. J Exp Clin Cancer Res. 2018;37(1):21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai Y, Ngo D, Jacob J, Forman LW, Faller DV. Prohibitin and the SWI/SNF ATPase subunit BRG1 are required for effective androgen antagonist-mediated transcriptional repression of androgen receptor-regulated genes. Carcinogenesis. 2008;29(9):1725\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLanz C, Bennamoun M, Macek P, Cathelineau X, Sanchez-Salas R. The importance of antiandrogen in prostate cancer treatment. Ann Transl Med. 2019;7(Suppl 8):362.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-experimental-and-clinical-cancer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jecc","sideBox":"Learn more about [Journal of Experimental \u0026 Clinical Cancer Research](http://jeccr.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jecc/default.aspx","title":"Journal of Experimental \u0026 Clinical Cancer Research","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"CRPC, prohibitin, Enzalutamide, FL3, androgen receptor, prostate, flavagline","lastPublishedDoi":"10.21203/rs.3.rs-2325130/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2325130/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCastration-resistant prostate cancer (CRPC) is currently the main challenge for prostate cancer (PCa) treatment, and there is an urgent need to find novel therapeutic targets and drugs. Prohibitin (PHB1) is a multifunctional chaperone/scaffold protein that is upregulated in various cancers and plays a pro-cancer role. FL3 is a synthetic flavagline drug that inhibits cancer cell proliferation by targeting PHB1. However, the biological functions of PHB1 in CRPC and the effect of FL3 on CRPC cells remain to be explored.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSeveral public datasets were used to analyze the association between the expression level of PHB1 and PCa progression as well as PCa patient outcomes. The expression of PHB1 in human PCa specimens and PCa cell lines was examined by immunohistochemistry (IHC), qRT-PCR, and western blotting. Then both the biological roles of PHB1 in castration resistance and underlying mechanisms were investigated by gain/loss-of-function analyses. Next, \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e a series of experiments were conducted to investigate the anti-cancer effects of FL3 on CRPC cells as well as the underlying mechanisms.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ePHB1 expression was significantly upregulated in CRPC and was associated with poor prognosis. PHB1 promoted castration resistance of PCa cells under androgen deprivation conditions. PHB1 is an androgen receptor (AR) suppressive gene and androgen deprivation promotes the PHB1 expression and its nucleus-cytoplasm translocation. FL3, alone or combined with the antiandrogen drug Enzalutamide (ENZ), suppressed CRPC cells especially ENZ-sensitive AR\u003csup\u003e+\u003c/sup\u003e CRPC cells both in \u003cem\u003evitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. By targeting the PHB1 protein, FL3 promoted its trafficking from plasma membrane and mitochondria to nucleus, which in turn inhibited AR signaling as well as MAPK signaling, but promoted apoptosis.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur data indicated that PHB1 is abnormally upregulated in CRPC and involved in castration resistance and provided a novel rational therapeutic approach for CRPC.\u003c/p\u003e","manuscriptTitle":"Targeting PHB1 to inhibit castration-resistant prostate cancer progression in vitro and in vivo","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-12-07 17:04:10","doi":"10.21203/rs.3.rs-2325130/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2022-12-18T16:30:40+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-12-05T08:58:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-12-05T05:26:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Experimental \u0026 Clinical Cancer Research","date":"2022-11-30T20:24:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-experimental-and-clinical-cancer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jecc","sideBox":"Learn more about [Journal of Experimental \u0026 Clinical Cancer Research](http://jeccr.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jecc/default.aspx","title":"Journal of Experimental \u0026 Clinical Cancer Research","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3fdaef07-d494-49eb-b45a-48b4e4d62a71","owner":[],"postedDate":"December 7th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T20:58:31+00:00","versionOfRecord":{"articleIdentity":"rs-2325130","link":"https://doi.org/10.1186/s13046-023-02695-0","journal":{"identity":"journal-of-experimental-and-clinical-cancer-research","isVorOnly":false,"title":"Journal of Experimental \u0026 Clinical Cancer Research"},"publishedOn":"2023-05-20 20:51:01","publishedOnDateReadable":"May 20th, 2023"},"versionCreatedAt":"2022-12-07 17:04:10","video":"","vorDoi":"10.1186/s13046-023-02695-0","vorDoiUrl":"https://doi.org/10.1186/s13046-023-02695-0","workflowStages":[]},"version":"v1","identity":"rs-2325130","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2325130","identity":"rs-2325130","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}