LncFALEC recruits ART5/PARP1 and promotes castration-resistant prostate cancer through enhancing PARP1-meditated self PARylation

Abstract

Accumulating evidence indicates that long noncoding RNAs (lncRNAs) are abnormal expression in various malignant tumors. Our previous research demonstrated that focally amplified long non-coding RNA (lncRNA) on chromosome 1 (FALEC) is an oncogenic lncRNA in prostate cancer (PCa). However, the role of FALEC in castration-resistant prostate cancer (CRPC) is poorly understood. In this study, we showed FALEC was upregulated in post-castration tissues and CRPC cells, and increased FALEC expression was associated with poor survival in post-castration PCa patients. RNA FISH demonstrated FALEC was translocated into nucleus in CRPC cells. RNA pulldown and followed Mass Spectrometry (MS) assay demonstrated FALEC directly interacted with PARP1 and loss of function assay showed FALEC depletion sensitized CRPC cells to castration treatment and restored NAD⁺. Specific PARP1 inhibitor AG14361 and NAD⁺ endogenous competitor NADP⁺ sensitized FALEC-deleted CRPC cells to castration treatment. FALEC increasing PARP1 meditated self PARylation through recruiting ART5 and down regulation of ART5 decreased CRPC cell viability and restored NAD⁺ through inhibiting PARP1meditated self PARylation in vitro. Furthermore, ART5 was indispensable for FALEC directly interaction and regulation of PARP1, loss of ART5 impaired FALEC and PARP1 associated self PARylation. In vivo, FALEC depleted combined with PARP1 inhibitor decreased CRPC cell derived tumor growth and metastasis in a model of castration treatment NOD/SCID mice. Together, these results established that FALEC may be a novel diagnostic marker for PCa progression and provides a potential new therapeutic strategy to target the FALEC/ART5/PARP1 complex in CRPC patients.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13402-023-00783-z.

Introduction

Prostate cancer is a major cause of male cancer-related death worldwide [1]. Androgen deprivation therapy (ADT) has been a standard treatment strategy for patients with advanced or metastatic prostate cancer [2]. However, patients treated with ADT inevitably develop castration-resistant prostate cancer (CRPC) within 18 to 24 months [3, 4]. DNA damage response (DDR) pathways are complex networks that transfer DNA damage signals to activate DNA repair and stabilize the genome. Failure to repair DNA damage reduces cell viability and induces cell apoptosis [5]. Several studies have demonstrated that ADT combined with radiotherapy is more effective than either ADT or radiotherapy alone for locally advanced prostate cancer [6, 7]. Recent studies have shed light on androgen receptor (AR) and DNA damage response (DDR) and repair, including double-strand breaks (DSB) and homologous recombination (HR) [8]. One of the earliest events in the DDR is the recruitment of poly (ADP-ribose) polymerase 1 (PARP1; also known as ARTD1) to diverse DNA lesions. PARP1 is an important cofactor for AR transcriptional activity [9]. Previous research showed that AR promotes a DNA damage response through the upregulation of γH2AX and RAD51 expression. Blocking AR signaling with ADT activates PARP signaling [10]. The role of PARP1 in the DDR has been studied extensively. Induction of DNA damage results in the rapid recruitment of PARP1 to the sites of damage through DNA-binding [11, 12]. This binding stimulates the catalytic activity of PARP1, which results in the synthesis of negatively charged poly ADP-ribose chains on itself and histone and non-histone proteins [13, 14]. Clinical inhibited catalytic PARP1, which are defective in genes associated with HR pathways giving rise to synthetic lethality, which significantly reduces cell viability and increases apoptosis [15]. When DNA damage is moderate, PARP1 is recruited to DNA damage lesions and activates DDR using NAD⁺ and ATP through catalyzing caspase-3 [16, 17].

Emerging evidence has demonstrated that long non-coding RNA (lncRNA) is involved in human disease and promoting cancer progression through the modulation of gene expression, chromatin modification, transcriptional and post-transcriptional regulation, and drug-resistance [18–22]. Novel diagnostic methods based on liquid biopsy technique containing circulating cell-free long non-coding RNAs (cflncRNAs) measurement has great prognostic/predictive value on advanced and metastasis prostate cancer [23]. Recently, it was shown that lnc-LBCS could inhibit castration resistance in prostate cancer by directly interacting with hnRNPK to suppress AR translation[24]. lncRNA HOXD-AS1 Regulates Proliferation and Chemo-Resistance of Castration-Resistant Prostate Cancer via Recruiting WDR5[25]. LncRNA-p21 could reverse enzalutamide-induced prostate cancer neuroendocrine differentiation via the modulation of EZH2/STAT3 signaling, which may represent a new therapeutic strategy against this disease [26]. LncRNAs have been associated with chemotherapy resistance in multiple tumor types, including lung [27, 28] and breast [29] cancer. Our previous research revealed that FALEC is an oncogenic lncRNA that promotes prostate cancer cell proliferation, invasion, and metastasis in the presence of HIF-1α [30]. Because the activity of the HR pathway is decreased and PARP1 upregulated in patients receiving ADT, we investigated the link between FALEC and PARP1, which potentially represents a biomarker and therapeutic strategy for CRPC combined with PARP1 inhibitors.

In this study, we showed that FALEC was upregulated in post-castration prostate cancer and CRPC (LNCaP-bic and LNCaP-AI) cells. FALEC recruited ART5 and PARP1 to enhance PARP1 meditated DNA repair and increased CRPC cell viability. Suppression of FALEC/ART5/PARP1 complex in vivo significantly inhibited CRPC cell derived tumor growth and prolonged mice survival.

Materials and Methods

Patient and tissue samples

This study was approved by the ethics committee of Shanghai General Hospital (Shanghai, China). Prostate cancer (n = 24) and benign prostatic hyperplasia patients tissue samples (n = 24) were obtained from patients of Shanghai General Hospital. PCa patient tissues were collected at the time when patients were diagnosed with advanced prostate cancer and after they received androgen deprivation therapy. The strategy of multiple biopsies was shown in Fig. 1A. All samples were collected with informed consent. The patient characteristics are listed in Table S1.

Fig. 1.

FALEC and PARP1 activity are upregulated in post-castration prostate cancer. (A) The strategy of tissues obtained from prostate cancer patient. (B) FALEC RNA FISH in prostate cancer pre- and post-castration treatment (n = 24). (C) Quantification of the data presented in (B). (D) Relative FALEC expression levels (qRT-PCR) in tumors from pre- and post-castration prostate cancer patients (n = 24). (E) Kaplan–Meier analysis of overall survival based on FALEC expression levels in post-castration prostate cancer patients (n = 24). (F) IHC staining of HR-associated proteins (BRCA1, BRCA2, p-ATM, and p-ATR) in pre- and post-castration prostate cancers. (G) PARP1 and PAR (left) IF in pre- and post-castration prostate cancers (quantitative data, right, scale bar = 50 µm). Data are shown as mean ± SD. *p < 0.05, ** p < 0.01 and *** p < 0.001

Cell culture and reagents

LNCaP cells were obtained from the Chinese Academy of Sciences Committee on Type Culture Collection Cell Bank (Shanghai, China). All cell lines were cultured in RPMI-1640 medium (R8758, Sigma-Aldrich; Merck, Darmstadt, Germany) supplemented with 10% fetal bovine serum and maintained in 5% CO₂ at 37℃. These cell lines were recently authenticated by the Chinese Academy of Sciences Committee using short tandem repeat (STR) profiling. LNCaP castration-resistant sublines were established as previously described [25]. Briefly, to obtain LNCaP-bic cells, we cultured LNCaP cells in phenol red free RPMI-1640 containing 10% FBS. Bicalutamide was supplemented at a starting concentration of 3 mM, with a weekly increment of 30% concentration, ultimately being maintained at 15 mM. To construct LNCaP-AI cells, we cultured LNCaP cells in phenol red free RPMI-1640 containing 10% charcoal stripped FBS (12676-029, Gibco). Both sublines were passaged 3 days at 1:3–1:4 ratio for 6 months, and the cultural medium was refreshed thrice per week. The LNCaP cells began to undergo obvious growth inhibition 5 days after androgen ablation and the inhibition continued for 4 months; then the proliferation accelerated gradually until reaching a comparable rate of normal LNCaP cells. All drugs used in this study were purchased from Target Mol (Boston, MA, USA). For in vitro assay the concentration of AG14361 was 0.5 µM. The doses of AG14361 used in the in vivo experiments were 30 mg/kg. The antibodies and culture reagents used in this study are listed below.

Bicalutamide (cat no. T6002), and the PARP1 inhibitor AG14361 (cat no. T6339) were obtained commercially from TargetMol (Boston, MA, USA). NADP⁺ (cat no. 1184-16-3) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Cell culture media and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). The antibodies against cleaved caspase-3 (cat no. ab2302), BRCA1 (cat no. ab238983), BRCA2 (cat no. ab123491), p-ATM (cat no. ab81292), p-ATR (cat no. 227,851), PARP1 (cat no. ab191217), and PARP2 (cat no. ab115620) were obtained from Abcam (Cambridge, MA). The anti-PAR antibody (cat no. 4335-MC-100) was from Trevigen (Gaithersburg, MD, USA). PARP10 (cat no. PA5-88774), pro-caspase-3 (cat no. MA1-91636), and GRP78 (cat no. MA5-27686) antibodies from Invitrogen (Carlsbad, CA, USA). The antibodies against CHOP (cat no. 2895), GAPDH (cat no. 5174), β-actin (cat no. 4970), and histone H3 (cat no. 4499) were purchased from Cell Signaling Technology (Danvers, MA, USA), and the anti-ART5 antibody (1:800, cat no. H00116969-B01) was from Novus (Centennial, USA). All antibodies were diluted according to the manufacturers’ recommendations and listed as Table S2.

RNA isolation and quantitative reverse transcription polymerase chain reaction (qRT-PCR)

Total RNA was extracted using Trizol (15,596,026, Invitrogen, Grand Island, NY). cDNA was reverse transcribed from 2 µg total RNA using SuperScript III reverse transcriptase (18,080,085, Invitrogen, USA). The mRNA expression was analyzed by qRT-PCR using PowerUp™ SYBR® Green Master Mix (A25742, Thermo Scientific, Waltham, MA, USA). Relative mRNA expression levels were quantified using the 2^−ΔΔCt method and using β-actin as the control. The specific primers used for the genes of interest are listed in Table S3.

Total protein extraction and western blotting

Cells were lysed in RIPA buffer (P0013C, Beyotime, Suzhou, China) containing 1% (v/v) protease inhibitor cocktail (11,873,580,001, Roche, Indianapolis, IN). The amount of protein in each sample was determined using the Pierce BCA Protein Assay (23,227, Thermo Scientific, Waltham, MA, USA). Total protein (20ug) was separated on 7.5 to 12.5% gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (IPVH00010, Millipore, Billerica, MA, USA). The membranes were blocked with skim milk and then incubated with primary antibodies and HRP-conjugated secondary antibodies. Protein bands were visualized by ECL (P10300, NCM, Suzhou, China) using the ECL Detection System (Thermo Scientific, Waltham, MA, USA).

Immunofluorescence (IF) and immunohistochemistry (IHC)

Formalin-fixed, paraffin-embedded tissue samples were cut into 4-µm-thick sections. Antigen retrieval was performed using a pressure cooker for 3 min in 0.01 M citrate buffer (pH 6.0). Cells for IF were washed with PBS and fixed in 4% paraformaldehyde for 20 min at room temperature followed by treatment with 0.05% Triton X-100 at 4℃ for 5 min. Sections and cells were blocked with 1% BSA for 1.5 h at room temperature and incubated with primary antibodies at 4℃ overnight. The samples were incubated with secondary antibodies conjugated with Alexa fluor or HRP for 1 h at room temperature. For IF, sections or cells were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) to detect nuclei and visualized by fluorescence microscopy. For IHC, the secondary antibody was diluted to 1:750 for recognizing primary antibodies. The staining for IHC was visualized using the VECTASTAIN ABC peroxidase system and peroxidase substrate DAB kit.

RNA fluorescence in situ hybridization (FISH)

Cells were washed three times in PBS and fixed with 4% formaldehyde for 20 min at room temperature and then incubated with 10% trypsin for 3 min and 0.05% Triton X-100 for 5 min at 4 °C. The cells were washed with 75% ethanol at 4 °C, 75% ethanol at -20 °C, 85% ethanol at -20 °C, and absolute ethanol at -20 °C for 5 min each. Tissue samples were deparaffinized, rehydrated, and then digested for 10 min at 37 °C with 10% trypsin prior to fixing with 4% paraformaldehyde.

The cells and tissue sections were incubated with hybridization solution (RiboBio, Guangzhou, China) at 50 °C for 2 h. Hybridization with the Cy3-labeled anti-FALEC oligodeoxynucleotide probe (RiboBio) was performed in hybridization solution for 16 h at 50 °C. After the hybridization, cells were washed in 2× SSC for 5 min at 50 °C and then 50% deionized formamide in 4× SSC at 50 °C for 25 min. After incubation with fluorescein-conjugated reagent and staining results were visualized using fluorescence microscopy.

RNA pull-down assay

The interaction between FALEC and proteins was detected by using The Pierce Magnetic RNA Protein pull-down Kit (Thermo Fisher Scientific) according to thermal manufacturer’s protocol. RNA was labeled with the included Thermo Scientific Pierce RNA 3´ Desthiobiotinylation Kit, and then the labeled RNA was added to the streptavidin magnetic beads and incubated for 30 min at room temperature with agitation for finishing the binding of RNA to the beads. The labeled RNA were mixed and co-incubated with lysate for 60 min at 4℃ with rotation. The RNA-protein complex was washed by Elution Buffer and put into a magnetic stand to remove the beads. The obtained protein samples were subjected to LC-MS/MS. The MS data were analyzed using MaxQuant software version 1.6.0.16. MS data were searched against the UniProtKB Rattus norvegicus database. All experiments were performed in triplicate, and data shown in the figures are representative of all of the experimental replicates.

RNA immunoprecipitation (RIP) assay

The RIP assay was performed using the Magna RIP™ RNA-Binding Protein Immunoprecipitation kit (17–700, Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. ART5 antibodies were purchased from Novus Biologicals USA. Normal mouse IgG (Cell Signaling Technology, Danvers, MA, USA) was used as a negative control. Briefly, after treatment, cells were fixed with 4% paraformaldehyde and lysed on ice with lysis buffer containing RNase inhibitor. The magnetic beads were incubated with the antibodies to form a magnetic bead-antibody complex, which was then incubated with the cell lysates. After isolation and purification, the purified RNA was subjected to qRT-PCR analysis.

Cell viability

Cell viability was measured using the CCK-8 (HY-K0301, Med Chem Express). Cells (2000 per well) were plated in 96-well plates and treated with the indicated agents until the control (vehicle)-treated cells reached 95% confluence. At each time point, CCK-8 reagent (20 µL per well) was added to each well for 2 h. The absorption values at 450 nm were measured using a multi-well plate reader (BioTek, Winooski, VT, USA).

Plasmid construction and lentiviral transfection

Plasmids carrying FALEC or ART5 shRNA or negative control shRNA were generated by OBiO (Shanghai, China). To knockdown FALEC or ART5, LNCaP-bic and LNCaP-AI cells (1 × 10⁵ per well) were seeded in 6-well plates. After they reached approximately 75% confluency, culture medium was changed into fresh culture medium containing either lentiviral particles (OBiO, Shanghai, China) containing FALEC-shRNA, negative control or ART5-shRNA and negative control using FuGENE6 (1,814,443, Roche, Indianapolis, IN) according to the manufacturer’s instructions and relative sequences are shown in Table S2. After 24 h transfection, culture medium was replaced with fresh medium containing puromycin of 1.5 µg/ml to select and create a stable line.

Co-immunoprecipitation (Co-IP) assay

Co-IP was performed using the Pierce Co-Immunoprecipitation Kit (26,149, Thermo Scientific) as described previously [31]. Briefly, cell extracts were incubated with PARP1 (1:500; Abcam, London, UK) or ART5 (1:500; Novus, Centennial, USA) antibodies at 4 °C overnight. PARP1 or ART5-binding proteins were precipitated with agarose beads and analyzed by western blotting. Normal IgG was used as a negative control.

Colony formation assay

Cells (1000 cells per well) were plated in 6-well plates and then treated with the indicated agents. After two weeks, cells were fixed with 4% paraformaldehyde for 10 min at room temperature, stained with crystal violet for 20 min, and washed under running water. After the plates were air-dried, they were scanned, and the stained areas were measured using ImageJ. The experiments were performed in triplicate.

Flow cytometry

Apoptosis was measured by flow cytometry using the FITC-Annexin V apoptosis detection kit (556,547, BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions. After treatment, cells were dissociated using 0.25% trypsin, washed with cold PBS, and resuspended in binding buffer. The cells were stained with Annexin V-FITC and PI solution in the dark and analyzed using a BD Accuri C6 flow cytometer (BD Biosciences, San Diego, CA, USA).

Animal experiments

All animal studies were performed with approval from the committee of Shanghai General Hospital according to institutional guidelines. Briefly, NOD/SCID mice (Shanghai General Hospital; 4 to 6 weeks) were castrated and subcutaneously implanted with LNCaP-bic or LNCaP-AI cells (1 × 10⁶) (n = 8 mice/group). When the average tumor size reached approximately 50 mm³, Lentivirus with either shART5 or shFALEC combined with AG14361 or the combination of all three agents were intratumorally injected into NOD/SCID mice. As a control, a group of mice was injected with PBS combined with scramble lentivirus. After seven weeks, all mice were euthanized, and necropsies were performed. Primary tumors were excised, embedded in paraffin, and fixed with formalin. Tumor sections were stained with cleaved caspase-3 by IHC.

In vivo lung metastasis model was established by injecting CRPC cells into castrated NOD/SCID mouse tail. Briefly, LNCaP-AI cells (1 × 10⁶) (n = 3 mice/group) were injected into castrated mouse tail and lentivirus with either shART5 or shFALEC combined with AG14361 or the combination of all three agents were intraperitoneal injected into NOD/SCID mice. As a control, a group of mice was injected with PBS combined with scramble lentivirus. Six weeks after initial injection, IVIS-200 bioluminescence and fluorescence imaging system (Caliper Life Sciences Inc, Hopkinton, MA, USA) was used to measure the luciferase value of each group. After 8 weeks, all mice were sacrificed and necropsies were performed. Various parameters including the number, weight, and location of individual tumor nodules were evaluated.

Statistical analysis

Statistical analysis and scientific graphing were performed using GraphPad Prism 8.0 to evaluate differences among experimental group. All experiments were performed in triplicate. All data are presented as means ± SD from three independent experiments, each of which were measured in triplicate. Differences between two groups were analyzed using the Student’s t-test. One-way analysis of variance (ANOVA) was performed for comparison between the different groups. Differences in survival were analyzed with Kaplan-Meier curves using the log-rank test. p < 0.05 was considered statistically significant. In all the figures, one to four asterisks respectively indicate p < 0.05, p < 0.01, p < 0.001 and p < 0.0001.

Results

FALECand PARP1 activity are upregulated in post-castration prostate cancer

Our previous results demonstrated that lncRNA FALEC was upregulated in prostate cancer and positively correlated with patient survival and clinical features [30]. To further explore the role of FALEC expression in CRPC, we obtained matched pairs of prostate cancer tissues pre- and post-castration. FISH and qRT-PCR was used to detect the variation of FALEC level in these biopsy samples. The strategies for tissue obtained was shown in Fig. 1A. The results showed that FALEC was upregulated in patients who received Bicalutamide (Bic) drug castration treatment (Fig. 1B, C, D). We also evaluated FALEC expression with post-castration treatment for 12 months. The Kaplan-Meier curve showed that high FALEC expression was associated with poor patient survival after receiving ADT (Fig. 1E). Furthermore, high FALEC expression was negatively correlated with the time progression to CRPC (Table S1).

Previous studies reported that AR regulated DNA repair and PARP1 activity [32, 33]. Studies have shown that ADT can induce HR deficiency and PARP1 activation in prostate cancer [33]. In the current study, we detected HR-associated protein expression in tumor tissues, which had significantly higher FALEC levels after Bic treatment. Whereas, BRCA1, BRCA2, p-ATM, and p-ATR were down-regulated in Bic treated prostate cancers (Fig. 1F). Analysis of HR protein levels in PCa tissues collected from all 24 patients pre- and post-ADT showed that such phenomena were not limited to single patient (Figure S1A). In consideration of PARP1 is the major enzyme responsible for over 90% of the PARylation including itself observed in vivo [34], we investigated the link between FALEC expression and PARylation of PARP1 in post-castration prostate cancer tissues. Immunofluorescence result showed PARP1 meditated PARylation was activated after Bic administration to patients (Fig. 1G). Together, these data indicated that FALEC and PARP1 activity is upregulated in patients that received androgen-deprivation therapy, and patients with high FALEC expression was positively associated with poor survival, and those disease rapid progression patients displayed high FALEC expression. Thus, FALEC may serve as a prognostic and therapeutic target for prostate cancer progression.

FALEC promotes castration-resistant through PARP1-meditated self PARylation

To clarify the mechanism of the upregulation of FALEC in CRPC, we generated LNCaP cell castration-resistant sublines. To mimic the in vivo castration condition, we artificially depleted androgen in culture medium for the in vitro assay. Castration resistance effect was confirmed by cell viability assays (Figure S1B). Cleaved caspase-3 levels were not increased in LNCaP-bic and LNCaP-AI following Bic treatment compared to the normal LNCaP cells (Figure S1C). Bic treatment did not increase cell apoptosis in the LNCaP-bic and LNCaP-AI cells (Figure S1D). We also evaluated the FALEC expression levels in the LNCaP-bic and LNCaP-AI cells by qRT-PCR and observed that FALEC expression was upregulated in both CRPC cells (Fig. 2A). RNA FISH result showed that the nuclear expression of FALEC was increased in LNCaP-bic and LNCaP-AI compared to the normal LNCaP cells (Fig. 2B). PARP1 was previously reported to be one of the most important enzymes which consumed NAD⁺ to catalyze PARylation and functions in the supplementary pathway activated during the DDR under HR-deficiency in CRPC [11, 12]. Thus, we measured the level of PARP1-meditated PARylation and HR-associated proteins in FALEC depleted CRPC cells in vitro. We found that PARP1 meditated self PARylation was significantly increased in both LNCaP-bic and LNCaP-AI cells, while BRCA1, BRCA2, p-ATR, and p-ATM were downregulated (Fig. 2C, D). The total ATM and ATR protein levels in LNCaP-bic and LNCaP-AI cells also decreased (Fig. 2E). To further clarify the link of FALEC and PARP1 in CRPC, we conducted RNA pull-down followed by LC-MS/MS assay, and the list of candidate proteins were shown in Table S4. Among these proteins, PARP1 is responsible for protein ribosylation and has the top priority (Table S4). Moreover, the RNA pull-down and RIP assay further confirmed that FALEC could directly interact with PARP1 in CRPC cells (Fig. 2F, G). However, inhibited PARP1 with AG14361 did not alter FALEC expression in CRPC cells (Fig. 2H). To investigate the regulatory role of FALEC to PARP1, we performed qRT-PCR and Western blot assay. We did not observe changes in PARP1 mRNA or protein expression, which suggesting the regulation was not at transcriptional or translational level (Fig. 2I). However, knocking down FALEC significant reducing PARP1 meditated self PARylation in CRPC cells (Fig. 3D). Together, these data suggested that FALEC appears to positively regulate PARP1-meditated self PARylation by directly interacting with PARP1 in CRPC cells.

Fig. 2.

FALEC positively regulates PARP1-meditated PAR synthesis in CRPC cells. (A) FALEC expression increased dramatically in LNCaP-bic and LNCaP-AI cells (qRT-PCR). (B) FALEC RNA FISH in CRPC cells. Result showed nucleus expression of FALEC was increased in LNCaP-bic and LNCaP-AI cells. (C) IP result showed PARP1-meditated self PARylation was increased in LNCaP-bic and LNCaP-AI cells. (D) Western blot analysis showed BRCA1, BRCA2, p-ATR, and p-ATM was increased in CRPC cells. (E) Western blots of the protein levels of total ATM, ATR in LNCaP, LNCaP-bic and LNCaP-AI cells. (F) RNA-pull down assay showed FALEC was directly interacted with PARP1 in LNCaP-bic and LNCaP-AI cells instead of LNCaP cells. (G) RIP assay. FALEC was detected in anti-PARP1 group, but not IgG. (H) Expression of FALEC was not influenced by inhibition of PARP1 by using AG14361. (I) Expression of PARP1 was not altered by FALEC knockdown in RNA and protein level. Data are shown as mean ± SD. *p < 0.05, ** p < 0.01 and *** p < 0.001

Fig. 3.

Depletion of FALEC and PARP1 partial reverses CRPC progression. (A) Viability of LNCaP-bic and LNCaP-AI cells following treatment with shFALEC alone or in combination with 0.5 µM AG14361 showed FALEC-deleted synthetically reduced CRPC cell viability with PARP1 inhibition. (B) Flow cytometry (left) showed that shFALEC alone slightly increased apoptosis, whereas shFALEC combined with AG14361 (0.5 µM) significantly increased apoptotic in LNCaP-bic and LNCaP-AI cells (quantitative data, right) (C) Expression of AR was not influenced by FALEC knockdown by using shRNA. (D) shFALEC combined with AG14361 (0.5 µM) increased cleaved caspase-3, CHOP, and GRP78 protein levels in LNCaP-bic and LNCaP-AI cells. The levels of these proteins were not affected by AG14361 alone (left). The quantitative data for the western blots are presented on the right. (E) Western blot result showed that shFALEC augmented AG14361 (0.5 µM) efficacy to suppress PARP1-meditated self PARylation in LNCaP-bic and LNCaP-AI cells. Data are shown as mean ± SD. *p < 0.05, ** p < 0.01 and *** p < 0.001

Depletion of FALEC and PARP1 partial reverses CRPC progression

To further investigate the role of FALEC and PARP1 enzyme activity in CRPC, we pharmacologically inhibited PARP1 in LNCaP-bic and LNCaP-AI cells with AG14361. This treatment increased cell apoptosis in CRPC cells (Figure S2A). FALEC knockdown using shRNA (Figure S2B), in combination with AG14361, further reduced cell viability and increased apoptosis in the LNCaP-bic and LNCaP-AI cells (Fig. 3A, B). Noteworthy, FALEC depletion had no influence on AR signaling (Fig. 3C).Previous studies showed that castration induces ER stress, which promotes prostate cancer cell apoptosis through the increased cleavage of caspase-3 [17, 35]. Therefore, we measured the expression of ER stress-associated protein following FALEC-deleted and PARP1 inhibition in CRPC cells. The results showed that shFALEC combined with AG14361 significantly increased GRP78 and CHOP expression and cleaved caspase-3 protein levels in LNCaP-bic and LNCaP-AI cells compared to either treatment alone (Fig. 3D). The combination of shFALEC and AG14361 also significantly reduced PARP1-meditated self PARylation (Fig. 3E). Together, these data suggest the FALEC posttranscriptional enhanced PARP1 enzyme activity and impaired ER stress to promote CRPC.

FALEC recruits ART5 and decreases NAD⁺in CRPC cells

ADP-ribosyl transferases (ARTs) are enzymes that use NAD⁺ as a substrate [36]. ART5 is a member of the mono-ADP-ribosyl transferase family that transfers ADP-ribose from NAD⁺ to proteins [37]. The MS data and subsequently deep analysis, including Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Protein-Protein Interaction Networks (PPI), elicited that the most enriched parameter in MS results is Ribosome-related pathway and having binding function (Figure S3A-C). The PPI network showed the most interactive proteins (Figure S3D). FALEC might interacted with ART5 and closely correlated with NAD⁺ function in CRPC cells. To confirm whether FALEC directly interacted ART5, we conducted biotin-linked FALEC RNA pull-down and RIP assay. We observed that FALEC could physically interact with ART5 (Fig. 4A), which was confirmed by RNA immunoprecipitation (Fig. 4B). ART5 is an NAD⁺ transfer enzyme. The protein level of ART5 increased in LNCaP-bic and LNCaP-AI cells (Fig. 4C). We measured the NAD⁺ levels in the blood and urine of post-castration prostate cancer patients (patients 1, 4, 7–9). NAD⁺ was decreased in both the blood and urine of post-castration prostate cancer patients compared to the benign prostatic hyperplasia patients (Figure S2C). It should be noted that patients 1, 4, and 9, who expressed higher levels of FALEC, had much lower NAD⁺ levels than patients 7 and 8, who expressed relatively low FALEC levels (Fig. 1D). In addition, the NAD⁺ levels in CRPC cells were lower than those of the hormone-sensitive cells (Fig. 4D). Either FALEC or ART5 knockdown restored the NAD⁺ levels in CRPC cells (Fig. 4E, S4F).

Fig. 4.

FALEC directly interacting with ART5 and decreases NAD⁺. (A) RNA-pull down assay showed FALEC was directly interacted with ART5 in LNCaP-bic and LNCaP-AI cells. (B) FALEC was detected in the immuneprecipitate of ART5 but not IgG. (C) Western blots of ART5 protein level in LNCaP, LNCaP-bic and LNCaP-AI cells. (D) NAD⁺ concentrations were reduced in LNCaP-bic and LNCaP-AI cells compared to hormone-sensitive LNCaP. (E) FALEC knockdown restored NAD⁺ concentrations. (F) Apoptosis (left) was significantly increased in shFALEC-treated LNCaP-bic and LNCaP-AI cells in the presence of 1 mM NADP⁺ (quantitative data are presented on the right). (G) shFALEC combined with additional NADP⁺ (0.5mM) increased cleaved caspase-3 levels in LNCaP-bic and LNCaP-AI cells (left). The data were quantified by GraphPad Prism 8.0 (right). Data are shown as mean ± SD. Data are shown as mean ± SD. *p < 0.05, ** p < 0.01 and *** p < 0.001

NAD⁺ is a well-known ADP-ribosylation donor for PARylation. Thus, most PARP inhibitors compete with NAD⁺ in the catalytic cage of enzymes [38, 39]. To understand the function of NAD⁺ in the upregulation of PARylation that meditates castration resistance, we used an endogenous NAD⁺ antagonist (NADP⁺) that acts as a negative regulator to suppress ADP-ribosylation [40]. Apoptosis was significantly increased in the castration-resistant LNCaP-bic and LNCaP-AI cells when NADP⁺ treatment was combined with shFALEC compared to NADP⁺ alone (Fig. 4F, G). These results suggested that FALEC increased the consumption of NAD⁺ by recruiting ART5 and loss of FALEC restored NAD⁺ levels and hormone sensitivity.

Downregulation of ART5 reduces castration-resistant cell viability

To understand ART5 function in castration-resistant cells, we conducted loss of function assay by knocking down ART5 (Figure S4A). Immunofluorescence assay showed that ART5 knockdown in LNCaP-bic and LNCaP-AI cells increased cleaved caspase-3 levels in the cytoplasm and suppressed PAR levels in the nucleus (Figure S4B). Downregulation of ART5 increased apoptosis and cleaved-caspase-3 in CRPC cells (Figure S4C, D). Knocking down ART5 also decreased PARP1-meditated PARylation in these castration-resistant cell lines (Figure S4E). Moreover, the downregulation of ART5 increased the hormone sensitivity of LNCaP-bic and LNCaP-AI cells, this effect was abolished by NADP⁺ (Figure S2D). It is noteworthy that shART5 restored NAD⁺ levels in LNCaP-bic and LNCaP-AI cells (Figure S4F). However, knocking down ART5 did not influence FALEC expression (Figure S2E). These data suggested that the downregulation of ART5 could abrogate castration resistance by modulating NAD⁺ consumption in a FALEC-independent manner.

ART5 is required for FALEC/PARP1-meditated castration resistance

The role of PARP1 in the DDR has been studied extensively. PARP1-meditated PARylation is required to recruit proteins to finish DNA damage repair [11, 12]. To further investigate whether ART5 was involved in the FALEC/PARP1 regulatory complex in castration resistance, we used an RNA pull-down assay to study the relationship between FALEC and PARP1. Thus far, our data suggested that both FALEC and ART5 positively regulated PAR synthesis and induced apoptosis in castration-resistant prostate cancer cells. The RNA pull-down assay showed that FALEC directly interacted with PARP1 in the presence of ART5 (Fig. 5A). Meanwhile, IP analysis showed that FALEC directly interacted with ART5 and PARP1 (Fig. 5B). Thus, we speculated that ART5 is essential for the effect of FALEC on PARP1 activity. Knockdown of both FALEC and ART5 reduced PAR upregulation to a greater extent than FALEC knockdown alone (Fig. 5C). The double knockdown of FALEC and ART5 also partially abolished the clonality and cleaved-caspase-3 expression of castration-resistant cells (Fig. 5D-G and S6A). These data indicated that ART5 was essential for FALEC positively regulates PARP1 activity.

Fig. 5.

ART5 is essential for FALEC/PARP1-induced castration-resistance. (A) RNA pull-down assay showed ART5 was essential for directly interaction of FALEC and PARP1. (B) IP analysis showed the interaction between ART5 and PARP1 in the presence or absence of LncFALEC. (C) Western blot analysis for PARP1-meditated PAR levels showed that either shFALEC or AG14361 (0.5 µM) alone could inhibit PAR synthesis; however, knockdown of both ART5 and FALEC abrogated this inhibitory effect. (D) LNCaP-bic and LNCaP-AI colony formation. The number of colonies significantly increased following treatment with shFALEC or AG14361 (0.5 µM) alone; however, the number of colonies decreased with the double knockdown of FALEC and ART5 compared to shFALEC alone. (E) The numbers of colonies were quantified using Image J. (F) Apoptosis in LNCaP-bic and LNCaP-AI cells. Apoptosis increased after FALEC knockdown or PARP1 inhibition (0.5 µM AG14361); however, apoptosis levels were restored after knockdown of both FALEC and ART5. (G) Apoptosis was quantified by GraphPad Prism 8.0. Data are shown as mean ± SD. *p < 0.05, ** p < 0.01 and *** p < 0.001

Because our previous data demonstrated that FALEC physically interacted with ART5, we investigated whether ART5 was recruited to the sites of DNA damage to enhance PARP1-meditated self PARylation in CRPC cells. Co-IP experiments showed that ART5 directly interacted with PARP1, but not PARP2 or PARP10 in LNCaP-bic cells (Figure S5A, B). In addition, ART5 was essential for PARP1-meditated self PARylation in LNCaP-bic cells (Figure S5C). We observed upregulated ART5 in nucleus by Western blot assay (Figure S5D). We also observed up-regulated PAR and ART5 expression in the nucleus of both LNCaP-bic and LNCaP-AI cells compared to the hormone-sensitive LNCaP cells (Figure S5E). We further evaluated the ART5 and PAR levels in pre- and post-castration prostate cancer tissues. This analysis showed that both ART5 and PAR were increased in the post-castration prostate cancer (Figure S5F-a, b). The quantification data of all 24 samples were shown in supplementary figure S6B. Suppression of ART5 and PARP1 in CRPC cells by shRNA and AG14361, respectively, significantly increased apoptosis (Figure S5G). Moreover, suppression of the ART5/PARP1 complex activity restored NAD⁺ levels (Figure S6C). Together, these data suggest that ART5 is recruited to the complex and essential for PARP1-meditated self PARylation in the castration-resistance prostate cancer. Downregulation of ART5 impaired the function of the FALEC/ART5/PARP1 complex in CRPC.

Depleting FALEC/ART5/PARP1 increases castration efficacy and reduces metastasis CRPC in vivo

To further investigate the role of the FALEC/ART5/PARP1 complex in castration resistance in vivo, we generated LNCaP-bic and LNCaP-AI tumor xenografts in castrated NOD/SCID mice. The NOD/SCID mice in control group, which was treated with castration alone, showed progressively increasing tumor volumes. As expected, ART5 knockdown slightly suppressed xenograft growth and decreased tumor volume. However, shART5 combined with AG14361 potently inhibited castration-resistant cells derived tumor growth compared to the vehicle control. Furthermore, complete suppression of the FALEC/ART5/PARP1 complex by knockdown of both FALEC and ART5 with shRNA and treatment with AG14361 significantly inhibited tumor proliferation and decreased the tumor volume compared to each treatment alone (Fig. 6A, B, C). The knockdown efficiency of these models was shown in Figure S6D. Although ART5 was significantly downregulated in the experimental groups compared to the control group, we did not observe a difference in PARP1 expression between the control and shART5 groups (Figure S6E). Of note, suppression of the FALEC/ART5/PARP1 complex increased the survival time of the tumor-bearing mice compared to each treatment alone (Figure S6F). To further confirm this finding, we measured cleaved caspase-3 levels in the xenografts. Inhibition of the FALEC/ART5/PARP1 complex significantly increased cleaved caspase-3 levels (Fig. 6D, E).

Fig. 6.

Suppression of FALEC/ART5/PARP1 complex activity augments efficacy in vivo. (A) Tumor growth curves for LNCaP-bic and LNCaP-AI derived xenografts (n = 8). Triple inhibition of the FALEC/ART5/PARP1 complex was accomplished by intratumoral injection using lentivirus with shART5, shFALEC combined with AG14361 (30 mg/kg), thus suppressing tumor growth in castrated NOD/SCID mice. (B) Weights of LNCaP-bic and LNCaP-AI derived tumors (n = 8). Suppression of FALEC/ART5/PARP1 complex activity reduced tumor volume and weight. (C) Representative LNCaP-bic and LNCaP-AI derived xenografts (n = 8). (D) IHC staining for cleaved caspase-3 levels in the xenografts. Inhibition of the FALEC/ART5/PARP1 complex increased cleaved caspase-3 levels in the tumors. (E) Quantification of the data presented in (D). Data are shown as mean ± SD. *p < 0.05, ** p < 0.01 and *** p < 0.001

Metastasis is the worst ending of CRPC, lung is the frequent victim organ for metastatic CRPC [41, 42]. To further investigation target FALEC/ART5/PARP1 complex to CRPC lung metastasis, we established in vivo CRPC cell metastasis model via tail injection into castrated NOD/SCID mouse. With our expect, complete depletes FALEC/ART5/PARP1 complex by knockdown both FALEC and ART5 with shRNA and targeted PARP1 with AG14361 significantly reduced the CRPC lung metastasis compared to each treatment alone (Fig. 7A, B, C, D). The schematic illustration of how increased FALEC/ART5/PARP1 activity could meditate castration resistance is shown in Fig. 7E. These data demonstrated that suppression of FALEC/ART5/PARP1 activity in vivo could sensitize CRPC cell to castration treatment and reduced lung metastasis.

Fig. 7.

Depleting FALEC/ART5/PARP1 complex reduces lung metastasis of CRPC. (A) Lung metastasis model (n = 3) was established by tail injection of 1 × 10⁶ LNCaP-AI-luc cells into nude mice, pharm treatment was used (including PBS, shART5, shART5 combined with AG14361 or triple combination) once tail injection was finished. IVIS-200 bioluminescence system was used to monitor lung metastasis once a week, node mice was sacrificed after 6 weeks. (B) Metastatic lung was displayed as indicated (brightness, n = 3). (C-D) Quantification of lung metastasis nudes and lung weight was displayed as indicated (n = 3). (E) Schematic diagram for the synergistic suppression of PARP1-meditated PAR synthesis by FALEC and PARP1 inhibition to induce castration-resistance of prostate cancer. Data are shown as mean ± SD. *p < 0.05, ** p < 0.01 and *** p < 0.001

Discussion

Emerging evidence indicates that lncRNAs function as regulatory molecules in carcinogenesis and drug resistance [43, 44]. Recently, studies have demonstrated that lncRNAs frequently act as scaffolds in protein complexes to regulate gene expression [45, 46]. Several lncRNAs are upregulated during CRPC progression. Prostate Cancer Gene Expression Marker 1 is induced by ADT to promote prostate cancer proliferation [47]. CTBP1-AS promotes cell cycle progression and proliferation through the inhibition of p53 and CTBP1 expression[48], HULLK is a novel AR-regulated lncRNA that overlaps with the LCK gene and promotes prostate cancer cell growth [49]. Our previous research demonstrated that FALEC is upregulated in prostate cancer and promotes cell proliferation and invasion in vitro and in vivo [30]. ADT is currently the most effective treatment for advanced prostate cancer; however, almost all men treated with ADT will eventually develop CRPC [4]. In this study, we investigated variations of FALEC expression in CRPC. We recruited patients who were diagnosed with prostate cancer and received ADT. RNA FISH and qRT-PCR analysis revealed that ADT increased FALEC expression in the tumors from these patients, and high FALEC expression was positively correlated with poor survival. Prostate cancer-associated lncRNAs have been identified in both hormone-sensitive and hormone-refractory [25]. Thus, lncRNAs may act as diagnostic or therapeutic targets to prostate cancer progression. Besides, LncRNAs regulate the cellular signaling pathway in the metastasis prostate cancer and CRPC [44, 50].

Increased DDR and HR deficiency, which lead to increased PARP1 enzymatic activity and enhanced complementary DNA repair pathway activation, have been observed in prostate cancer following ADT [33]. We verified that ADT induced HR deficiency in prostate cancer by detecting HR-associated protein, BRCA1, BRCA2, p-ATM, and p-ATR. We also evaluated PARP1 and PAR levels in pre- and post-castration prostate cancer patient samples. We observed PARP1 associated PARylation was upregulated in post-castration prostate cancer. This finding might be due to the HR deficiency caused PARP1 activation. These results suggested that FALEC expression and PARP1 enzymatic activity were both upregulated in post-castration prostate cancer and might be positively associated with disease progression.

To explain the upregulation of FALEC expression and PARP1 activity in castration-resistant prostate cancer, we established hormone sensitive LNCaP cell castration-resistant sublines LNCaP-bic and LNCaP-AI. We observed nuclear expression of FALEC was upregulated in castration-resistant LNCaP-bic and LNCaP-AI cells. These data were in line with the data obtained from post-resistant prostate cancer tissues. We also observed increased PARP1-meditated self PARylation and HR-associated BRCA1 and BRCA2 levels in CRPC cells, which is consistent with what we observed in post-castration prostate cancer tissues. Recently, it was revealed that lncRNAs could mediate promote castration-resistance of prostate cancer by sponging miR-200b to upregulate E2F3 and ZEB1 [27]. According to previous researches, PARP1 meditated PARylation occurred in nucleus [10]. We speculated that FALEC could induce prostate cancer cell resistant to castration therapy through enhancing PARP1 activity. To further clarify the mechanism of FALEC-facilitated, PARP1-meditated self PARylation of castration-resistance in prostate cancer, we applied biotin-linked FALEC RNA pull-down and MS assay to directly explore FALEC interacted proteins. We observed the function of proteins interacted with FALEC mainly focus on DNA damage responses, protein complex binding and nuclear ribosylation. Among these proteins, PARP1 is responsible for protein ribosylation and has the top priority. Besides, we also noticed that knocking down FALEC significantly reduced PARP1 meditated self PARylation instead of PARP1 itself. Thus, we speculated that the upregulation of FALEC could induce prostate cancer cell resistant to castration therapy through enhancing PARP1 activity. Surprisingly, the knockdown of FALEC cooperated with PARP1 inhibition increased the sensitivity of castration-resistant cells. Besides, we observed FALEC increased PARP1 enzymatic activity was AR-independent. Furthermore, FALEC-deplete LNCaP-bic and LNCaP-AI cells showed low PARylation and high ER stress-associated GRP78/CHOP/cleaved caspase-3 expression. To date, there have been no other reports on the relationship between the upregulation of FALEC and PARP1 enzymatic activity in castration-resistance prostate cancer. Our findings identified increased FALEC and PARP1-meditated self PARylation as a mechanism of castration-resistance prostate cancer.

PARP1-meditated self PARylation frequently cooperates with functional proteins to finish DNA damage repair [11, 12]. To further clarify the mechanism of FALEC-facilitated, PARP1-meditated self PARylation of castration-resistance in prostate cancer, we re-analyzed our MS data. We observed among these FALEC interplayed proteins, the ART5 is an NAD⁺ transfer enzyme that utilizes NAD⁺ as a substrate showed highest priority. Further, we observed FALEC decreased NAD⁺ through recruiting ART5. The PARP1 enzyme plays a central role in producing PAR in response to DNA damage [33, 40]. Thus, we hypothesized that FALEC might recruit ART5 to enhance PARP1-meditated self PARylation. In the current study, FALEC was proved to directly interact with ART5 and PARP1 in castration-resistant prostate cancer cells. These data indicated that the FALEC meditated directly interaction of PARP1 and ART5 might be functional complex in CRPC.

In response to DNA damage, 90% of cellular NAD⁺ was consumed rapidly by PARP1 to catalyze extensive ADP-ribosylation at the sites of DNA lesions [51]. Previous study has demonstrated that NADP⁺ was a potent competitor to NAD⁺ to suppress PARylation of PARP1 [40]. Thus, we found FALEC depleted combined with NADP⁺ significantly increased the cell apoptotic rate and enhanced the cleaved caspase-3 activation in CRPC cells. This implied us PARP1 meditated PARylation which relied on NAD⁺ promote prostate cancer cells resistant to castration therapy and loss of FALEC impaired such effect. To clarify protein function of ART5, we applied loss of function assay in castration-resistant prostate cancer cells. ART5 was act as transfer ADP-ribose from NAD⁺ to proteins which shared the similar function with PARP1 [37]. We observed that ART5 knockdown significantly increased CRPC cell apoptosis and cleaved-caspase-3 expression. We also observed that ART5 knockdown restored the NAD⁺ levels in CRPC cells, which indicated that ART5 might have similar function to or cooperate with PARP1. However, knocking down ART5 did not alter the expression of FALEC which implied ART5 reversed castration-resistant was FALEC independent. Indeed, previous studies demonstrated that ART5 could transfer ADP-ribose from NAD⁺ to proteins [37]. Thus, we further explored the role of ART5 in FALEC/PARP1 complex. In this study, we found that ART5 formed a complex with PARP1, but not PARP2 or PARP10.The RNA pull-down assay demonstrated that FALEC formed a complex with PARP1 in the presence of ART5. Knocking down ART5 impaired the suppressive effect of FALEC-deplete and AG14361 on the PARylation of PARP1. Thus, ART5 was required for FALEC/PARP1 complex activity and ART5 deficiency decreased clone formation and apoptosis in the context of double inhibition FALEC and PARP1. Furthermore, ART5/PAR co-localized in the nucleus, where the PARylation process occurs for DNA damage repair. CRPC cell derived xenografts showed more sensitivity to castration treatment following the suppression of the FALEC/ART5/PARP1 complex. Suppression of this complex also prolonged mouse survival and was accompanied by significantly decreased tumor volume and slower tumor growth. Interestingly, knockdown of ART5 or PARP1 did not alter the expression of FALEC in vivo. Moreover, depleting FALEC/ART5/PARP1 complex could significantly reduce lung metastasis of CRPC. Meanwhile, HPF1 is also reported to be important for PARP1 activity, the function of HPF1 and its role in the progression of PARP1-meditated PARylation in CRPC will be further investigated in our research.

In this study, FALEC appears to act as a scaffold to assist the recruitment of PARP1 and ART5 to sites of DNA damage to enhance PARP1-meditated self PARylation in CRPC. Recently, studies showed that RNA-protein binding was not simply relayed on RNA binding domain, there also exist short repetitive amino acid motifs and DNA domains which function as RNA binding structure [52, 53]. In current research, we demonstrated lncRNA FALEC could directly interact with PARP1 and ART5 in CRPC context. However, in this study, we mainly focused on the scaffold function of FALEC to recruit ART5/PARP1, thus facilitating CRPC progression. We failed to determine the specific region of ART5 and PARP1 interaction and whether PARP1 influenced the function and expression of ART5. We were also unable to determine the mechanism by which the FALEC/ART5 combined and the complex translocation into the nucleus following DNA damage and induction of the DDR, which need be further studied. Meanwhile, Geisler et al. reported that the altered expression of LncRNA was attributed to various epigenetic factors, as well as transcriptional or post-transcriptional level [54]. The expression regulatory mechanism of lncFALEC was complicated, thereby we need more experimental methods to explore its regulating model. In future, motif analysis of lncFALEC was needed to demonstrate whether there are key molecules which influence its expression pattern. The cause of the upregulation of FALEC and its regulatory mechanism in CRPC need to be further addressed. Epigenetic modifications, such as m6A methylation of RNA, may also be involved. Additional research with more patients is needed to confirm the current findings. This study revealed a novel FALEC/ART5/PARP1 complex and the role of this complex in CRPC. In the future, we will explore the role of this complex in energy metabolism and the tricarboxylic acid cycle because the function of this complex is related to NAD⁺ biogenesis and consumption. The data generated in the current study have expanded our understanding of lncRNAs in prostate cancer progression and identified FALEC/ART5/PARP1 as potential diagnostic and therapeutic targets for CRPC.

Electronic supplementary material

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Abbreviations

CRPC

Castration-resistant prostate cancer

ADT

Androgen deprivation therapy

FISH

Fluorescence in situ hybridization

CCK8

Cell counting kit-8

MS

Mass spectrometry

PARP

Poly ADP-ribose polymerase

ART5

ADP-Ribosyltransferase 5

FALEC

Focally amplified lncRNA on chromosome 1

NAD

Nicotinamide adenine dinucleotide

HR

Homologous recombination

DDR

DNA damage repair

AR

Androgen receptor

BCA

Bicinchoninic acid

RIP

RNA binding protein immunoprecipitation

Author contribution

Study design: Feng Sun, Fei Shi, Zheng Zhu, Shujie Xia, Bangmin Han; Data collection: Fei Shi, Lei Wu, Zheng Zhou, Di Cui; Data analysis: Lei Wu, Menghao Sun, Zheng Zhu, Zheng Deng; Manuscript preparation: Fei Shi, Menghao Sun, Di Cui. All authors have read and approved the final manuscript.

Funding

This study was supported in part by grants from the Youth Fund Project of the National Natural Science Foundation (#81602252), the “Chen Guang” project supported by the Shanghai Municipal Education Commission and Shanghai Education Development Foundation (#16CG10) and Postgraduate Research & Practice Innovation Program of Jiangsu Province (#KYCX22_1836).

Data Availability

Please contact author for data requests.

Declarations

Ethics approval and consent to participate

All experiments and procedures in the research involving human participants are in accordance with the ethical standards of the Research Ethics Committee of the Shanghai General Hospital. Informed consents have been acquired. Animal research has been approved and carried out strictly following the institutional ethical guidelines of the Committee on the Use of Live Animals of Shanghai General Hospital.

Disclosure Statement

We have no competing interests.