C4-2 Cells Research Articles

Cell type matters: competence for alkaloid metabolism differs in two seed-derived cell strains of Catharanthus roseus

Since the discovery of the anticancer drugs vinblastine and vincristine, Catharanthus roseus has been intensively studied for biosynthesis of several terpene indole alkaloids (TIAs). Due to their low abundance in plant tissues at a simultaneously high demand, modes of production alternative to conventional extraction are mandatory. Plant cell fermentation might become one of these alternatives, yet decades of research have shown limited success to certain product classes, leading to the question: how to preserve the intrinsic ability to produce TIAs (metabolic competence) in cell culture? We used the strategy to use the developmental potency of mature embryos to generate such strains. Two cell strains (C1and C4) from seed embryos of Catharanthus roseus were found to differ not only morphologically, but also in their metabolic competence. This differential competence became manifest not only under phytohormone elicitation, but also upon feeding with alkaloid pathway precursors. The more active strain C4 formed larger cell aggregates and was endowed with longer mitochondria. These cellular features were accompanied by higher alkaloid accumulation in response to methyl jasmonate (MeJA) elicitation. The levels of catharanthine could be increased significantly, while the concurrent vindoline branch of the pathway was blocked, such that no bisindole alkaloids were detectable. By feeding vindoline to MeJA-elicited C4 cells, vincristine became detectable; however, only to marginal amounts. In conclusion, these results show that cultured cells are not “de-differentiated”, but can differ in metabolic competence. In addition to elicitation and precursor feeding, the cellular properties of the “biomatter” are highly relevant for the success of plant cell fermentation.

Combined treatment with anti-PSMA CAR NK-92 cell and anti-PD-L1 monoclonal antibody enhances the antitumour efficacy against castration-resistant prostate cancer.

BackgroundThe chimeric antigen receptor NK‐92 (CAR NK‐92) cell targeting the prostate‐specific membrane antigen (PSMA) has shown antitumour effects in castration‐resistant prostate cancer (CRPC). However, the expression changes of programmed death ligand 1 (PD‐L1) and its mechanisms on CAR NK‐92 and CRPC cells and the effect of the anti‐PD‐L1 monoclonal antibody (mAb) on PD‐L1 expressed on CAR NK‐92 cells remain unknown.MethodsHuman dendritic cells and CD8+ T cells were acquired from blood samples of healthy donors and cocultured with C4‐2 cells. Changes in PD‐L1 expression were detected by flow cytometry. Differential gene expressions were investigated by RNA sequence analysis, while the regulation of PD‐L1 molecular signaling was explored using western blotting. In vitro cytotoxicity was evaluated using the Cell Counting Kit‐8 assay and the bioluminescent intensity (BLI) of green fluorescent protein‐labelled C4‐2 cells. CRPC growth in vivo was monitored using callipers and BLI in male NOD/SCID mice subcutaneously injected with C4‐2 cells and treated intravenously with anti‐PD‐L1/PD‐1 mAb, CAR NK‐92 or cocultured CD8+ T cells.ResultsSignificantly upregulated expression of PD‐L1k was observed in cocultured C4‐2 and CAR NK‐92 cells. In addition, upregulation of PD‐L1 expression was dependent on interferon‐γ in C4‐2 cells, while it was dependent on direct cell‐to‐cell interaction via the NK group 2 member D/ phosphatidylinositol 3‐kinase/AKT pathway in CAR NK‐92 cells. The anti‐PD‐L1 mAb directly acted on PD‐L1 expressed on CAR NK‐92 cells and augmented the cytotoxicity of CAR NK‐92 cells against C4‐2 and CRPC cells from one patient in vitro. Anti‐PD‐L1 mAb significantly enhanced the antitumour effect of CAR NK‐92 cells against CRPC cells in vivo when compared to treatment with CAR NK‐92 cells or combined with anti‐PD‐1 mAb in the absence or presence of cocultured CD8+ T cells.ConclusionCombined treatment with CAR NK‐92 and anti‐PD‐L1 mAb improved the antitumour efficacy against CRPC, which is of extraordinary translational value in the clinical treatment of CRPC.

Enzalutamide-resistant related lncRNA NONHSAT210528 promotes the proliferation and invasion of prostate cancer.

BackgroundAs a new-generation androgen-receptor antagonist, enzalutamide is a first-choice drug for advanced prostate cancer (PCa) patients. However, secondary resistance to enzalutamide poses a new challenge in the treatment of cancer. Long non-coding RNA (lncRNA) regulates cell function through many levels and mechanisms, and also plays an important role in the biological behaviors of tumors.MethodsLncRNA microarrays were used to detect enzalutamide-resistant related lncRNA in Enzalutamide-resistant C4-2 (C4-2 ENZ-R) cells and corresponding parent cells. Cell Counting Kit 8, flow cytometry, and transwell assays were used to test the effect of lncRNA NONHSAT210528 on the function of PCa cells. RNA pulls down and the luciferase report gene was used to detect the competitive endogenous RNA (ceRNA) mechanism. The culture supernatant of C4-2 and C4-2b cells was transferred to the lower chamber for transwell assay of human umbilical endothelial cells (HUVECs).ResultsThe lncRNA microarray analysis showed that there were significant differences in the expression of many lncRNAs between the C4-2 ENZ-R and C4-2 cells. The real-time polymerase chain reaction (PCR) detection showed that the expression of lncRNA NONHSAT210528 was significantly higher in the C4-2 ENZ-R cells than the C4-2 cells. The Transwell assays showed that lncRNA NONHSAT210528 overexpression increased the invasion of the C4-2 and C4-2b cells. The cell-wound scratch and the transwell assays showed that the culture supernatant of C4-2 and C4-2b cells with overexpressed lncRNA NONHSAT210528 promoted the migration and invasion of HUVECs. Furthermore, lncRNA NONHSAT210528 regulated the expression of YOD1 dependent on miR-21.ConclusionsEnzalutamide-resistant related lncRNA NONHSAT210528 appears to promote the proliferation and invasion of PCa cells by functioning as a ceRNA and regulating the miR-21-5p/YOD1 signal pathway.

English

Cancer is one of the major public health problems of the 21st century. Its incidence is increasing in both developed and low-income countries. This trend is justified by the change in people's lifestyles. Cancer of the prostate is one of major cancer types affecting male population. The present study focused on the use of methanolic extract of Striga hermonthica (Delile) Benth. to evaluate total phenolics and flavonoids contents, antioxidant activities, anti-proliferative and anti-migratory effects, using ATCC prostate cancer cell lines (22RV1, DU145, C4-2 and RWPE-1). Total phenolics and flavonoids content were estimated using spectrophotometric methods. Antioxidant potential was evaluated using three methods while cytotoxicity assay, anti-migratory assay and western blot analysis were used for the determination of extracts anti-proliferative activity on prostate cancer cells. Therefore, to assess apoptosis induction in prostate cancer cell lines, the effect of S. hermonthica (Delile) Benth. methanolic extract was investigated on procaspase-3, procaspase-8, and cyclin-B2 expression. A range of half-dilution concentrations beginning from 500 µg/mL have been prepared to determine the IC50 in the various tests. Antioxidant compounds were shown to be promising biomolecules, with particular interest in cancer management. Among plant derivative compounds, phenolics and derived molecules have endorsed antioxidant capacities and potential anti-proliferative activities. The best inhibition was obtained on C4-2 cells with an IC50 = 65.76 µg/mL and a selectivity index of 2.35. The results obtained from this study could partially justify the traditional indications of this plant in the treatment of prostate cancer. Key words: Plant derived products, anti-tumoral, prostate cancer, antioxidants.

Allogeneic Expanded Human Peripheral NK Cells Control Prostate Cancer Growth in a Preclinical Mouse Model of Castration-Resistant Prostate Cancer.

Adoptive allogeneic natural killer (NK) cell therapy has shown promise in treating castration-resistant prostate cancer (CRPC), which is the terminal stage of prostate cancer (PCa) and incurable. Thus, we employed an efficient manufacturing method for the large-scale ex vivo expansion of high-quality NK cells from peripheral blood of healthy donors. In the present study, we evaluated the in vitro cytotoxicity of NK cells against human PCa cell lines and in vivo antitumor activity in a preclinical mouse model of CRPC. CCK-8 results demonstrated that the NK cells exerted potent cytotoxicity against all PCa cell lines in vitro. The NK cells were activated when cocultured with PCa C4-2 cells, evidenced by upregulation of the degranulation marker CD107a and secretion of cytokines (TNF-α and IFN-γ). In a xenograft mouse model of CRPC, the caliper, CT, and ultrasonography examination results showed that the size of tumors treated with NK cells was significantly smaller than that in the control group. Moreover, ultrasonography examination also indicated that the NK cell treatment evidently reduced the blood supply of the tumors and HE staining results demonstrated that the NK treatment increased the proportion of necrosis in the tumor specimen compared to PBS treatment. Meanwhile, the NK cell treatment did not cause significant serum IL-6 elevation. Therefore, our study suggested that the expanded NK cells exhibited significant cytotoxicity against PCa cell lines in vitro and excellent therapeutic efficacy against CRPC in a xenograft mouse model, which was of great value for the clinical treatment of CRPC.

Different Complement Activation Pathways Underly Cognitive Impairment and Type 2 Diabetes Mellitus Combined With Cognitive Impairment.

BackgroundThe immune response and the complement system are associated with cognitive impairment and diabetes mellitus, respectively. Activation of the complement system in these diseases occurs mainly through either the classical pathway or the alternative pathway. However, the specific complement proteins involved in the development of the type 2 diabetes mellitus (T2DM) and cognitive impairment are still unclear. Here, we investigated complement proteins in serum from patients with T2DM, cognitive impairment, or both T2DM and cognitive impairment.ObjectiveTo investigate the levels of serum immune complement proteins in patients with T2DM, cognitive impairment, or T2DM combined with cognitive impairment and the associations between these complement proteins and risk factors for T2DM or cognitive impairment.MethodsClinical markers were collected from blood samples of 264 participants. Luminex multiplex assays were used to detect serum complement proteins. All statistical analyses were performed using Prism or R studio.ResultsThere was a difference in serum levels of the complement proteins C1q, C3, C3b, and FH between the three different groups. Hyperglycemia was significantly correlated with elevated C3b or reduced C3, C1q, and FH. In addition, hyperlipidemia was positively correlated with elevated levels of C3, C4, C1q, and FH proteins. There was an association between C1q, C3, C4, and FH and β-pancreas cell function, whereas only FH was associated with insulin resistance. Higher serum C1q was significantly associated with an increased risk of cognitive impairment.ConclusionSerum levels of complement proteins were closely associated with hyperglycemia and hyperlipidemia. We found that classical complement pathway activation mainly occurred in the cognitive impairment only group, whereas the alternative pathway may reflect T2DM and T2DM with cognitive impairment.

The Androgen Hormone-Induced Increase in Androgen Receptor Protein Expression Is Caused by the Autoinduction of the Androgen Receptor Translational Activity

The androgen receptor (AR) plays a central role in prostate, muscle, bone and adipose tissue. Moreover, dysregulated AR activity is a driving force in prostate cancer (PCa) initiation and progression. Consequently, antagonizing AR signalling cascades via antiandrogenic therapy is a crucial treatment option in PCa management. Besides, very high androgen levels also inhibit PCa cells’ growth, so this effect could also be applied in PCa therapy. However, on the molecular and cellular level, these mechanisms have hardly been investigated so far. Therefore, the present study describes the effects of varying androgen concentrations on the viability of PCa cells as well as localization, transactivation, and protein stability of the AR. For this purpose, cell viability was determined via WST1 assay. Alterations in AR transactivity were detected by qPCR analysis of AR target genes. A fluorescent AR fusion protein was used to analyse AR localization microscopically. Changes in AR protein expression were detected by Western blot. Our results showed that high androgen concentrations reduce the cell viability in LNCaP and C4-2 cell lines. In addition, androgens have been reported to increase AR transactivity, AR localization, and AR protein expression levels. However, high androgen levels did not reduce these parameters. Furthermore, this study revealed an androgen-induced increase in AR protein synthesis. In conclusion, inhibitory effects on cell viability by high androgen levels are due to AR downstream signalling or non-genomic AR activity. Moreover, hormonal activation of the AR leads to a self-induced stabilization of the receptor, resulting in increased AR activity. Therefore, in clinical use, a therapeutic reduction in androgen levels represents a clinical target and would lead to a decrease in AR activity and, thus, AR-driven PCa progression.

Citrus Flavone Tangeretin Inhibits CRPC Cell Proliferation by Regulating Cx26, AKT, and AR Signaling.

Prostate cancer (PCa) progression depends on the action of androgen receptors (AR). Therefore, preventing ligand-mediated activation of AR is the first-line treatment strategy for metastatic PCa. Androgen deprivation therapy (ADT) can inhibit ligand binding to AR and alleviate PCa progression initially. However, due to the adaptation of PCa and recovery of AR signaling, castration-resistant prostate cancer (CRPC) eventually develops. Exploring novel dietary compounds that can target AR signaling appears to be a viable alternative therapeutic option for CRPC. In the present study, compounds from the citrus fruits were focused upon, which contain various flavonoid ingredients. Key components contained within orange peel, which is frequently used in traditional Chinese medicine, and downstream targets were first analyzed using network pharmacology approach. Notably, it was found that tangeretin, an active ingredient from orange peel, can significantly inhibit CRPC cell (C4-2 and Du145 cells) proliferation and migration whilst also synergistically increasing the sensitivity of CRPC cells to anti-tumor drugs sorafenib or cisplatin. Tangeretin also significantly reduced AR and AKT expressions in C4-2 cells and signal transducer and activator of transcription 3 expression in the androgen-insensitive cell line Du145. In addition, tangeretin increased the expression of both connexin26 (Cx26) and gap junction function, which may mediate the bystander effects of cisplatin or sorafenib. Taken together, the present study revealed a novel molecular mechanism by which tangeretin may inhibit the proliferation of CRPC cells, by affecting the Cx26/AKT/AR pathway, to synergistically increase the sensitivity of CRPC cells to sorafenib and cisplatin.

In This Issue

See article, p. 16.B-precursor acute lymphoblastic leukemia (B-ALL) relapse often involves the central nervous system (CNS). In this article, Vanner, Dobson, et al. characterize B-ALL subclones with differential CNS involvement in patient-derived xenografts. By comparing transcriptional profiles, the authors identify complement component C3 and stem cell, metabolic, and mRNA translation pathways enriched in subclones with preferential CNS residence regardless of mutational composition. These findings are validated by proteomics and by genetic or pharmacologic targeting of the candidate pathways. The protein translation inhibitor omacetaxine mepesuccinate strongly reduces CNS infiltration.See article, p. 32.In this study, Petti et al. evaluated mechanisms that contribute to the relapse of patients with acute myeloid leukemia (AML) after therapy. Bone marrow samples from presentation and relapse from six patients with normal karyotype AML were subjected to enhanced whole-genome sequencing to identify all somatic mutations and their evolution. Single-cell RNA sequencing defined transcriptional and cellular changes at relapse. Each case had a unique set of genetic and transcriptional adaptations associated with relapse, but most acquired a conserved relapse-enriched leukemia cell (RELC) signature that had features of stemness, quiescence, and adhesion.See article, p. 50.Diffuse large B-cell lymphomas (DLBCL) are dependent on anaplerotic glutaminolysis driven by SIRT3. Li et al. show that the metabolic collapse induced by SIRT3 depletion leads to marked repression of ATF4 target genes that are highly expressed in DLBCL. ATF4 is required for DLBCL cell proliferation and is translationally activated when sensing depletion of nonessential amino acids (NEAA). Reciprocally, loss of SIRT3 induces accumulation of NEAAs due to induction of autophagy, which in turn suppresses ATF4 translation, further aggravates nutrient starvation, and ultimately leads to cell death, which is further enhanced by exposure to GCN2 inhibitors.See article, p. 66.Although 80% to 90% of patients treated with CD19 CAR T-cell therapy for ALL achieve remission, half will relapse. Biomarkers predicting relapse have so far been unknown. In this study, Pulsipher et al. hypothesize that the presence of minimal residual disease detected by next-generation sequencing (NGS-MRD) after tisagenlecleucel therapy would predict relapse of both CD19+ and CD19− blasts. They demonstrate that any level of NGS-MRD positivity was highly associated with relapse risk. A combination of early loss of B-cell aplasia with NGS-MRD >0 predicted relapse with sufficient time to intervene with various approaches aimed at preventing relapse.

Folic Acid-Modified miR-491-5p-Loaded ZIF-8 Nanoparticles Inhibit Castration-Resistant Prostate Cancer by Regulating the Expression of EPHX1.

Epoxide hydrolase 1 (EPHX1) has been reported to be related to the development of several tumors. However, the regulation of castration-resistant prostate cancer (CRPC) development by EPHX1 has not been reported. We used proteomic technology and found that the EPHX1 protein was highly expressed in CRPC tissues and the CRPC cell line C4-2. We performed screening and found that EPHX1 is a direct target of miR-491-5p. High miR-491-5p expression significantly reduced the EPHX1 level in C4-2 cells and inhibited C4-2 cell proliferation and migration. Zeolite imidazolate framework-8 (ZIF-8) has good thermal stability, a simple synthesis method, tumor site stability, and specific acid responsiveness. We synthesized ZIF-8 nanodrug vectors to deliver miR-491-5p into C4-2 cells. After loading miR-491-5p into ZIF-8, we modified the ZIF-8 surface with folic acid (FA) as the target group (FA@ZIF-8). Our synthesized nanodrug carrier showed less cytotoxicity to C4-2 cells even at 200 μg/ml. Modified FA could increase the efficiency of nanomaterial entry into C4-2 cells. FA@miR-491-5p@ZIF-8 could stably release miR-491-5p for a long period in both phosphate-buffered saline (pH 7.4) and acetate buffer (pH 4.8), and miR-491-5p was released faster at the beginning of the experiment in acetate buffer (pH 4.8). FA@miR-491-5p@ZIF-8 significantly reduced C4-2 cell proliferation and migration, and FA@miR-491-5p@ZIF-8 had a better effect than miR-491-5p alone. In vivo, FA@miR-491-5p@ZIF-8 significantly inhibited CRPC growth in nude mice. Overall, we verified that miR-491-4p regulated CRPC development by targeting EPHX1. The drug nanocarrier FA@miR-491-5p@ZIF-8 not only significantly reduced C4-2 CRPC cell proliferation and migration but also significantly inhibited CRPC growth. Our research provides a theoretical basis for treatment and treatment strategies for CRPC.

Long Non-coding RNA AFAP1-AS1 Facilitates Prostate Cancer Progression by Regulating miR-15b/IGF1R Axis.

Prostate cancer (PCa) is a commonly diagnosed malignant cancer and is the second- highest cause of cancer death in men worldwide. Enzalutamide is the second-generation inhibitor of androgen receptor signaling and is the fundamental drug for the treatment of advanced PCa. However, the disease will eventually progress to metastatic castration-resistant prostate cancer (CRPC) and aggressive neuroendocrine prostate cancer (NEPC) because of androgen-deprivation therapy (ADT) resistance. The aim of the study was to investigate the role of long non-coding RNA (lncRNA) AFAP1-AS1 in ADT resistance. Quantitative real-time PCR analysis (qPCR) was used to assess the expression of AFAP1-AS1 in PCa cell lines and tissues. Cell proliferation and invasion were assessed after AFAP1-AS1 knockdown using Cell Counting Kit (CCK)-8 and Transwell assay, respectively. A dual-luciferase reporter gene assay was carried out to validate the regulatory relationship among AFAP1-AS1, microRNA (miR)-15b, and insulin-like growth factor1 receptor (IGF1R). AFAP1-AS1 level was markedly increased in castration-resistant C4-2 cells and NE-like cells (PC3, DU145, and NCI-H660), compared with androgen-sensitive LNCaP cells. Enzalutamide treatment increased the expression of AFAP1-AS1 in vitro and in vivo. Functionally, AFAP1-AS1 knockdown repressed tumor cell proliferation and invasion. Mechanistically, AFAP1-AS1 functioned as an oncogene in PCa through binding to miR-15b and destroying its tumor suppressor function. Finally, we identified that AFAP1-AS1 up-regulated IGF1R expression by competitively binding to miR-15b to de-repress IGF1R. AFAP1-AS1 facilitates PCa progression by regulating miR-15b/IGF1R axis, indicating that AFAP1-AS1 may serve as a diagnostic biomarker and therapeutic target for PCa.

The androgen receptor\u2014lncRNASAT1-AKT-p15 axis mediates androgen-induced cellular senescence in prostate cancer cells

The bipolar androgen therapy (BAT) to treat prostate cancer (PCa) includes cycles of supraphysiological androgen levels (SAL) under androgen-deprivation therapy (ADT). We showed previously that SAL induces cellular senescence in androgen-sensitive PCa cells and in ex vivo-treated patient PCa tumor samples. Here, we analyzed the underlying molecular pathway and reveal that SAL induces cellular senescence in both, castration-sensitive (CSPC) LNCaP and castration-resistant PCa (CRPC) C4-2 cells through the cell cycle inhibitor p15INK4b and increased phosphorylation of AKT. Treatment with the AKT inhibitor (AKTi) potently inhibited SAL-induced expression of p15INK4b and cellular senescence in both cell lines. Proximity-ligation assays (PLA) combined with high-resolution laser-scanning microscopy indicate that SAL promotes interaction of endogenous androgen receptor (AR) with AKT in the cytoplasm as well as in the nucleus detectable after three days. Transcriptome sequencing (RNA-seq) comparing the SAL-induced transcriptomes of LNCaP with C4-2 cells as well as with AKTi-treated cell transcriptomes revealed landscapes for cell senescence. Interestingly, one of the identified genes is the lncRNASAT1. SAL treatment of native patient tumor samples ex vivo upregulates lncRNASAT1. In PCa tumor tissues, lncRNASAT1 is downregulated compared with nontumor tissues of the same patients. Knockdown indicates that the lncRNASAT1 is crucial for SAL-induced cancer-cell senescence as an upstream factor for pAKT and for p15INK4b. Further, knockdown of lncRNASAT1 enhances cell proliferation by SAL, suggesting that lncRNASAT1 serves as a tumor suppressor at SAL. Interestingly, immunoprecipitation of AR detected lncRNASAT1 as an AR-interacting partner that regulates AR target-gene expression. Similarly, RNA-ChIP experiments revealed the interaction of AR with lncRNASAT1 on chromatin. Thus, we identified a novel AR-lncRNASAT1-AKT-p15INK4b signaling axis to mediate SAL-induced cellular senescence.

PD05-02 TARGETING TR4 NUCLEAR RECEPTOR WITH ANTAGONIST BEXAROTENE INCREASES DOCETAXEL SENSITIVITY TO BETTER SUPPRESS THE METASTATIC CASTRATION-RESISTANT PROSTATE CANCER PROGRESSION

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) I (PD05)1 Sep 2021PD05-02 TARGETING TR4 NUCLEAR RECEPTOR WITH ANTAGONIST BEXAROTENE INCREASES DOCETAXEL SENSITIVITY TO BETTER SUPPRESS THE METASTATIC CASTRATION-RESISTANT PROSTATE CANCER PROGRESSION Linyi Hu, Yin Sun, Jie Luo, Xiang He, Meihua Ye, Gonghui Li, Yong Zhang, Jian Bai, Dahong Zhang, and Chawnshang Chang Linyi HuLinyi Hu More articles by this author , Yin SunYin Sun More articles by this author , Jie LuoJie Luo More articles by this author , Xiang HeXiang He More articles by this author , Meihua YeMeihua Ye More articles by this author , Gonghui LiGonghui Li More articles by this author , Yong ZhangYong Zhang More articles by this author , Jian BaiJian Bai More articles by this author , Dahong ZhangDahong Zhang More articles by this author , and Chawnshang ChangChawnshang Chang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001969.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Resistance to docetaxel (DTX) in metastatic castration-resistant prostate cancer (mCRPC) is a significant clinical problem as half of patients fail to respond to this therapy. The TR4 nuclear receptor has been reported to play an important role in prostate cancer (PCa) progression, however, its linkage to the DTX resistance remains unclear. Here we investigated the role of TR4 in DTX sensitivity in mCRPC cells and patients. Mechanism dissections revealed that TR4 might alter the DTX chemo-sensitivity via modulating the TR4/lincRNA-p21/HIF-1α/VEGF-A signaling. METHODS: We firstly investigated the TR4 expression in PCa cell lines (PC-3, Du-145, C4-2, and 22RV-1), non-tumor prostate cell lines (WPMY-1 and BPH-1), and human PCa clinical tissues from the public database Oncomine. Furthermore, we retrospectively investigated the TR4 expression through IHC staining in clinical samples from mCRPC patients treated with DTX chemotherapy. To confirm whether TR4 could influence chemo-sensitivity, we altered TR4 level in PCa cells. We also investigated the TR4 expression in DTX-resistant C4-2 and 22RV-1 cells, and its impact on tumor sphere formation and DTX-sensitivity. We established a reporter assay in PC-3 cells with the promoter of TR4 target gene (Oxytocin) and applied PITCh system with CRISPR-CAS9 technique to establish stable cell lines with luciferase inserted at the genomic location in the Oxytocin gene in 293T cells. These reporter systems were used to test whether Bexarotene (Bex, a derivative of retinoid) could influence DTX chemo-sensitivity through TR4 transactivation. Furthermore, based on bioinformatics prediction as well as chromatin-immunoprecipitation (ChIP) analysis, whether TR4 could regulate lincRNA-p21 expression via direct binding to the potential TR4-response-elements (TR4REs) upstream of lincRNA-p21 was tested. Newly identified TR4/lincRNA-p21/HIF-1alpha/VEGF-A signaling was also examined through gene correlation analysis based on mCRPC dataset from GEO, as well as HIF-1alpha inhibitor. The clinical significance of these findings was tested with staining of HIF-1α, VEGF-A, TR4 and P63 in mCRPC tissues between biopsy samples at first diagnosis and surgically resected samples after DTX treatment. RESULTS: We found TR4 was upregulated after DTX chemotherapy in the mCRPC cells and patients, and TR4 expression is correlated with DTX sensitivity with a higher level conferring chemo-resistance. Targeting TR4 with an antagonist Bexarotene suppressed the TR4 transactivation with increased DTX chemo-sensitivity. Mechanism dissection studies revealed that TR4 might alter the DTX chemo-sensitivity via modulating the TR4/lincRNA-p21/HIF-1α/VEGF-A signaling. CONCLUSIONS: We identify a novel role of TR4 that may function via altering the TR4/lincRNA-p21/HIF-1α/VEGF-A signaling to modulate the DTX-resistance in PCa, and targeting this newly identified signaling with Bex, a FDA-approved drug, may lead to increase DTX chemo-sensitivity to better suppress the mCRPC progression. Source of Funding: NIH grants (CA155477 and CA156700), George Whipple Professorship Endowment and Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW104-TDU-B-212-113002) © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e55-e56 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Linyi Hu More articles by this author Yin Sun More articles by this author Jie Luo More articles by this author Xiang He More articles by this author Meihua Ye More articles by this author Gonghui Li More articles by this author Yong Zhang More articles by this author Jian Bai More articles by this author Dahong Zhang More articles by this author Chawnshang Chang More articles by this author Expand All Advertisement Loading ...

A Systematic Comparison of Antiandrogens Identifies Androgen Receptor Protein Stability as an Indicator for Treatment Response.

Antiandrogen therapy is a primary treatment for patients with metastasized prostate cancer. Whilst the biologic mechanisms of antiandrogens have been extensively studied, the operating protocols used for the characterization of these drugs were not identical, limiting their comparison. Here, the antiandrogens Bicalutamide, Enzalutamide, Apalutamide, and Darolutamide were systematically compared using identical experimental setups. Androgen-dependent LNCaP and LAPC4 cells as well as androgen-independent C4-2 cells were treated with distinct concentrations of antiandrogens. Androgen receptor (AR)-mediated gene transactivation was determined using qPCR. Cell viability was measured by WST1 assay. Protein stability and AR localization were determined using western blot. Response to the tested antiandrogens across cellular backgrounds differed primarily in AR-mediated gene transactivation and cell viability. Antiandrogen treatment in LNCaP and LAPC4 cells resulted in AR protein level reduction, whereas in C4-2 cells marginal decreased AR protein was observed after treatment. In addition, AR downregulation was already detectable after 4 h, whereas reduced AR-mediated gene transactivation was not observed before 6 h. None of the tested antiandrogens displayed an advantage on the tested parameters within one cell line as opposed to the cellular background, which seems to be the primary influence on antiandrogen efficacy. Moreover, the results revealed a prominent role in AR protein stability. It is one of the first events triggered by antiandrogens and correlated with antiandrogen efficiency. Therefore, AR stability may surrogate antiandrogen response and may be a possible target to reverse antiandrogen resistance.

KAT2A-mediated AR translocation into nucleus promotes abiraterone-resistance in castration-resistant prostate cancer

Abiraterone, a novel androgen synthesis inhibitor, has been approved for castration-resistant prostate cancer (CRPC) treatment. However, most patients eventually acquire resistance to this agent, and the underlying mechanisms related to this resistance remain largely unelucidated. Lysine acetyltransferase 2 A (KAT2A) has been reported to enhance transcriptional activity for certain histone or non-histone proteins through the acetylation and post-translational modification of the androgen receptor (AR). Therefore, we hypothesised that KAT2A might play a critical role in the resistance of prostate tumours to hormonal treatment. In this study, we found that KAT2A expression was increased in abiraterone-resistant prostate cancer C4-2 cells (C4-2-AbiR). Consistently, elevated expression of KAT2A was observed in patients with prostate cancer exhibiting high-grade disease or biochemical recurrence following radical prostatectomy, as well as in those with poor clinical survival outcomes. Moreover, KAT2A knockdown partially re-sensitised C4-2-AbiR cells to abiraterone, whereas KAT2A overexpression promoted abiraterone resistance in parental C4-2 cells. Consistent with this finding, KAT2A knockdown rescued abiraterone sensitivity and inhibited the proliferation of C4-2-AbiR cells in a mouse model. Mechanistically, KAT2A directly acetylated the hinge region of the AR, and induced AR translocation from the cytoplasm to the nucleus, resulting in increased transcriptional activity of the AR-targeted gene prostate specific antigen (PSA) leading to resistance to the inhibitory effect of abiraterone on proliferation. Taken together, our findings demonstrate a substantial role for KAT2A in the regulation of post-translational modifications in AR affecting CRPC development, suggesting that targeting KAT2A might be a potential strategy for CRPC treatment.

Radionuclide Imaging of the Gut-Brain Axis in Parkinson Disease.

1504 Introduction: Prostate-specific membrane antigen (PSMA) is an important biological target in molecular radiotherapy, for which there exists a variety of PSMA-targeting agents. In this work, we present a preclinical evaluation of PSMA-TO1, a ligand developed aiming at a prolonged circulating half-life through increased lipophilicity. The aim of this study was to directly compare PSMA-TO1 as an imaging and therapeutic agent with PSMA-11 and PSMA-617, the two best known PSMA ligands. First, we compared imaging properties, biodistribution and tumor uptake of 68Ga-/ 177Lu-labeled PSMA-TO1 versus PSMA-617 and PSMA-11 in a mouse model of prostate cancer. Second, we assessed treatment efficacy of 225Ac-labeled PSMA-TO1 and PSMA-617 against a mouse model of systemic disease. Methods: NSG mice bearing subcutaneous C4-2 tumors underwent PET/CT imaging with each of the following compounds: 68Ga-PSMA-11, 68Ga-PSMA-617, and 68Ga-PSMA-TO1 (n=3 mice) and tumor uptake was analyzed. We then performed a biodistribution study in mice with subcutaneous C4-2 tumors with either 30 MBq 177Lu-PSMA-617 or 177Lu-PSMA-TO1 (n=25 mice per ligand). Following treatment, mice were sacrificed at 5 time points up to 7 days after administration for tumor and organ collection and activity quantification. Finally, mice were inoculated via intracardiac injection of C4-2 cells to establish widespread microscopic visceral and bone lesions, as previously described (1). These mice were treated 5 weeks after inoculation with either 40 kBq 225Ac-PSMA-617 or 225Ac-PSMA-TO1 (n=10 mice per ligand, and n=5 for untreated controls) and efficacy was assessed by overall survival. Results: PET images of 68Ga-labeled PSMA-TO1, PSMA-11, and PSMA-617 all showed high accumulation in the tumor with the greatest uptake observed using PSMA-617, followed by PSMA-TO1 then PSMA-11. Serial ex-vivo counts of 177Lu-PSMA-617 and 177Lu-PSMA-TO1-treated tumors revealed faster tumor clearance for PSMA-617. While the tumor retention of PSMA-TO1 is superior to PSMA-617, kidney uptake is also increased in this mouse model. In the 225Ac survival study, both PSMA-617 and PSMA-TO1 significantly prolonged median survival from 7.7 weeks in untreated mice to 14.5 weeks (p<0.0001) and 17.8 weeks (p<0.0001), respectively. The survival difference observed in favor of the mice treated with 225Ac-PSMA-TO1 compared to those treated with 225Ac-PSMA-617 was significant (p=0.0002). There was no observed difference in toxicity between treatment groups. Conclusions: In a mouse model of prostate cancer, PSMA-TO1 exhibits increased tumor retention compared to PSMA-617, albeit at the expense of higher kidney uptake. The significant survival benefit incurred by mice treated with 225Ac-PSMA-TO1 versus PSMA-617 may support further investigations into the potential utility of this compound.

Abstract 2509: RB1-loss in castration-resistant prostate cancer reprograms androgen receptor signaling and confers vulnerability to LSD1 inhibition

Abstract Background: Retinoblastoma protein (Rb) functions to repress the transcription activity of E2Fs by forming an Rb-E2F repressor complex and the hyperphosphorylation of Rb during G1/S cell cycle transition results in its disassociation with E2Fs, thus inducing E2F-dependent transcription of DNA replication and cell cycle. Genomic RB1-loss is frequently occurred in castration-resistant prostate cancer (CRPC) (~10-15%) and is significantly associated with poor clinical outcomes. RB1-loss is also more frequently found in CRPC with neuroendocrine features (CRPC-NE) (~45-50%). However, it remains unclear on the global activities of Rb-E2F repressor complex functions in modulating transcriptional networks in CRPC. In this study, we performed a comprehensive analysis to functionally characterize the activities of Rb-E2F in multiple CRPC models and aimed to identify vulnerabilities in CRPC with RB1-loss. Methods: C4-2 cells (PTEN-loss, p53 positive) stably infected with doxycycline-inducible lentiviral shRNA against RB1 were generated. VCaP cells (PTEN positive, p53 R248W mut) that transfected with siRNAs against RB1 (siRB) were also examined. The chromatin landscape of Rb-E2F complex was determined by Rb ChIP-seq. Rb-E2F transcriptome was obtained from RNA-seq analysis in the C4-2 stable line treated with/out doxycycline and VCaP cells with siRB. An integrated analysis combines the ChIP-seq and RNA-seq data was also performed to characterize the direct targets of Rb-E2F. Furthermore, we have also assessed the effect of RB1 loss on AR transcriptional program using AR ChIP-seq and RNA-seq in cells treated with/out DHT. Results: Using an integrated cistromic and transcriptomic analysis, we have characterized Rb-E2F activities in multiple CRPC models by identifying the Rb-E2F directly regulated genes. Our results revealed that Rb-E2F has distinct binding sites and targets in TP53-mutated CRPC, and that the expression of Rb-E2F directly repressed genes is increased in the neuroendocrine (NE) subtype of CRPC. Furthermore, we have found that loss of Rb expression redistributes the chromatin binding of androgen receptor (AR), leading to the transcriptional activation of a subset of genes that are increased in CRPC-NE. Remarkably, we have found that the chromatin binding of E2F1 is regulated by an epigenetic factor LSD1/KDM1A (a lysine-specific demethylase that demethylates histone and non-histone proteins) and loss of Rb sensitizes the E2F signaling to the regulation by LSD1. Importantly, LSD1 inhibitor treatment was more effective in CRPC xenograft tumors with RB silencing. Conclusion: Taken together, these results characterized the direct transcriptomic changes in RB1-loss CRPC, indicated a reprogramming of AR signaling by loss of RB1, and suggested LSD1 as a potential therapeutic target in RB1-loss CRPC or CRPC-NE by modulating chromatin binding of E2Fs. Citation Format: Wanting Han, Mingyu Liu, Dong Han, Muqing Li, Anthia A. Toure, Susan C. Patalano-Salsman, Jill A. Macoska, Shuai Gao, Changmeng Cai. RB1-loss in castration-resistant prostate cancer reprograms androgen receptor signaling and confers vulnerability to LSD1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2509.

Enzalutamide-Induced Upregulation of PCAT6 Promotes Prostate Cancer Neuroendocrine Differentiation by Regulating miR-326/HNRNPA2B1 Axis.

Our previous studies have demonstrated that Enzalutamide-induced upregulation of long non-coding RNA p21 (lncRNA-p21) facilitates prostate cancer (PCa) neuroendocrine differentiation (NED). Given the important role of lncRNAs in PCa pathogenesis, and given that lots of lncRNAs are dys-regulated in neuroendocrine PCa (NEPC) patients, we next explored the biological function and underlying mechanism of lncRNA-PCAT6 (PCAT6) in mediating Enzalutamide-induced NED. The level of PCAT6 in Enzalutamide-treated PCa cells and NEPC samples were assessed using quantitative RT-PCR (qPCR). The effect of PCAT6 on PCa cell proliferation, invasion, and NED was evaluated through CCK-8, transwell, qPCR, western blot analysis, Xenograft mouse model, and in vivo lung metastasis model. We found that PCAT6 was highly expressed in NE-like cells (PC3, DU145, and NCI-H660) compared with androgen-sensitive LNCaP cells. PCAT6 was also highly expressed in NEPC tissues. Enzalutamide treatment resulted in a significant increase of PCAT6 level in a dose- and time-dependent fashion. Functionally, PCAT6 overexpression promoted NED of C4-2 cells, as evidenced by an increased expression of NE markers (NSE, ChgA, and SYP), whereas PCAT6 knockdown in NCI-H661 cells repressed NED. Furthermore, PCAT6 overexpression promoted PCa cell proliferation and invasion in vitro and in vivo. Mechanistically, PCAT6 functioned as competing endogenous (ce) RNA via absorbing miR-326, thus resulting in a de-suppression of Hnrnpa2b1 target gene. The current results demonstrate that PCAT6 acted as a tumor activator in PCa progression by sponging miR-326 and increasing Hnrnpa2b1 expression and that the PCAT6/miR-326/Hnrnpa2b1 signaling might be a new therapeutic target for PCa.

Long noncoding RNA GAS5 interacts and suppresses androgen receptor activity in prostate cancer cells.

The androgen receptor (AR) plays an important role in the progression of prostate cancer and is the most important therapeutic target. However, androgen deprivation therapy will finally lead patients to progress to castration-resistant prostate cancer (CRPC). Here, we confirmed that GAS5, a long noncoding RNA, could interact and suppress AR transactivation in CRPC C4-2 cells. Knockdown GAS5 by short hairpin RNA would enhance the transcription of AR via promote AR recruitment to the promoter of its downstream target genes. Functionally, GAS5 overexpression inhibits cell proliferation partially through inhibiting AR transactivation in C4-2 cells. Moreover, knocking down GAS5 protects C4-2 cells from the docetaxel-induced cell apoptosis. In return, the suppressed AR was found to downregulate the GAS5 expression, which forms a feedback loop resulted in AR high transcription activity in CRPC. Collectively, our findings revealed the important role of GAS5 in AR axis activity regulation and CRPC progression. Targeting GAS5 to intervene the feedback loop might be a new potential therapeutic approach for the patients at CRPC stage.

Prostate cancer cells survive anti-androgen and mitochondrial metabolic inhibitors by modulating glycolysis and mitochondrial metabolic activities.

Most cancer cells are more glycolytic even under aerobic conditions compared with their normal counterparts. Recent evidence of tumor cell metabolism, however, shows that some tumors also increase mitochondrial oxidative phosphorylation (ox-phos) at some disease states during progression and/or development of drug resistance. Our data show that anti-androgen enzalutamide (ENZA) resistant prostate cancer (PCa) cells use more mitochondrial metabolism leading to higher ox-phos as compared to the ENZA-sensitive cells and can become vulnerable to mitochondrial metabolism targeted therapies. Seahorse assay, mass spectrometry and high resolution fluorescence confocal microscopy coupled with image analysis has been used to compare mitochondrial metabolism in ENZA-treated and -untreated anti-androgen-sensitive LNCaP and -resistant C4-2, CWR22ν1, and PCa2b cells. Ex vivo fluorescence microscopy and image analysis has been standardized to monitor mitochondrial electron transport (ETS) activity that likely increases ox-phos in circulating tumor cells (CTCs) isolated fom patients undergoing AR-targeted therapies. Our data show that PCa cells that are resistant to anti-androgen ENZA switch from glycolysis to ox-phos leading to an increased ETS activity. ENZA pretreated cells are more vulnerable to ETS component complex I inhibitor IACS-010759 (IACS) and mitochondrial glutaminase inhibitor CB-839 that reduces glutamate supply to tricarboxylic acid cycle. CTCs isolated from 6 of 20 patient blood samples showed relatively higher ETS activity than the rest of the patients. All six patients have developed ENZA resistance within less than 6 months of the sample collection. The enhanced growth inhibitory effects of mitochondrial metabolic inhibitors IACS and CB-839 in ENZA pretreated PCa cells provides a rationale for designing a drug combination trial. Patients can be selected for such trials by monitoring the mitochondrial ETS activities in their CTCs to maximize success.