727 Resistance to enzalutamide and abiraterone drives tumor phenotypic plasticity and resistance to immune-mediated cytotoxicity

BackgroundBackground: Treatment of patients with castration-resistant prostate cancer (CRPC) includes the use of next-generation hormonal therapies such as abiraterone or enzalutamide. Although these agents extend survival, a significant proportion of patients exhibit primary or acquired resistance to treatment. In recent years, immune checkpoint blockade has led to remarkable responses in patients with several tumor types, however, CRPC has remained resistant to immunotherapy. Previous studies have demonstrated that different tumor variants could emerge along the progression of prostate cancer, including tumors undergoing phenotypic plasticity in the context of an epithelial-mesenchymal transition. Our laboratory and others have shown that phenotypic plasticity is a driver of resistance to immunotherapy. Based on this knowledge, we investigated whether changes in tumor phenotype could affect the response of CRPC to immune-based therapies, and ways this can be mitigated.MethodsThe androgen sensitive LNCAP prostate cancer cell line was used to derive LNCAP cells resistant to enzalutamide (LNCAP-EnzaR) or abiraterone (LNCAP-AbiR). Resistant cell lines and parental LNCAP cells were comparatively evaluated for features of EMT and neuroendocrine phenotype via RT-PCR, ELISA, western blot, immunofluorescence, and RNAseq. Changes in the susceptibility to NK-cell mediated cytotoxicity were evaluated with NK cells isolated from peripheral blood from healthy donors. LNCAP-EnzaR cells were also grown in vivo in NSG MHC-deficient mice, and tumors were characterized for phenotypic markers and potential therapeutic targets.ResultsAcquisition of resistance to both enzalutamide and abiraterone was associated with a significant increase in mesenchymal tumor features, including high levels of vimentin and fibronectin, and the loss of epithelial features and cell-to-cell attachments. LNCAP-Enza-R and LNCAP-AbiR cells showed a significant reduction (up to 90%) in susceptibility to NK-cell mediated cytotoxicity and antibody-dependent cell cytotoxicity (ADCC), compared with parental cells. These results prompted us to investigate approaches to improve immune-mediated lysis, including inhibition of estrogen receptor 1 (ESR1), which was identified as highly upregulated in LNCAP-EnzaR cells via RNAseq analysis. In a xenograft model of LNCAP-EnzaR cells, we corroborated the maintenance of tumor phenotypic plasticity and the expression of actionable targets.ConclusionsOur data indicates that acquisition of resistance to androgen receptor inhibition is associated with marked reduction of susceptibility to immune attack, and the acquisition of tumor phenotypic plasticity. Future studies will investigate approaches that revert tumor plasticity, including blockade of ESR1, TGF-beta or IL-8, for potential improvement of tumor susceptibility to immune attack in CRPC.Ethics ApprovalPBMCs were obtained from healthy donors at the NIH Clinical Center Blood Bank (NCT00001846). All animal studies were approved and conducted in accordance with an IACUC-approved animal protocol (LTIB-57) with the approval the NIH/NCI Institutional Animal Care and Use Committee.

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Elevated Expression Levels of PC3-Secreted Microprotein (PSMP) in Prostate Cancer Associated With Increased Xenograft Growth and Modification of Immune-Related Microenvironment

Frontiers in Oncology ◽

10.3389/fonc.2019.00724 ◽

2019 ◽

Vol 9 ◽

Author(s):

Xiaolei Pei ◽

Danfeng Zheng ◽

Shaoping She ◽

Zhiwei Fang ◽

Shiying Zhang ◽

...

Keyword(s):

Prostate Cancer ◽

Neutralizing Antibodies ◽

Epithelial Mesenchymal Transition ◽

Macrophage Polarization ◽

Xenograft Model ◽

Metastatic Progression ◽

Male Mortality ◽

Mesenchymal Transition ◽

Monocyte Migration

Prostate cancer (PCa), especially metastatic PCa, is one of the main cancer types accounting for male mortality worldwide. Over decades, researchers have tried to search for effective curative methods for PCa, but many attempts have failed. The therapeutic failure of PCa is usually due to off-target or side effects; thus, finding a key molecule that could prevent PCa metastatic progression has become the most important goal for curing aggressive PCa. In this study, we collected hundreds of PCa tissues and serum and urine samples from patients to verify the upregulated expression of PC3-secreted microprotein (PSMP) in PCa tumor tissues with high Gleason scores. According to biopsy results, PSMP expression was found related to extraprostatic extension (EPE), contributing to PCa metastasis. Mechanistically, recombinant PSMP protein could promote the proliferation both in vitro and in vivo, and rhPSMP could promote epithelial–mesenchymal transition (EMT) of PC3 in vitro. Additionally, PSMP could also influence cytokine production in the xenograft model and monocyte migration and macrophage polarization in vitro. Our most important finding was that neutralizing antibodies against PSMP could suppress xenograft PC3 growth and promote the survival of PC3 metastatic mice model, providing an effective option to cure human PCa.

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Histological examination of the cellular reactions around schistosomula of Schistosoma mansoni in the lungs of sublethally irradiated and unirradiated, immune and control rats

Parasitology ◽

10.1017/s0031182000059680 ◽

1989 ◽

Vol 98 (1) ◽

pp. 57-65 ◽

Cited By ~ 14

Author(s):

Dario A. A. Vignali ◽

S. N. Klaus ◽

Q. D. Bickle ◽

M. G. Taylor

Keyword(s):

Schistosoma Mansoni ◽

Mononuclear Cells ◽

Significant Proportion ◽

Immune Serum ◽

Immune Recognition ◽

Cellular Infiltration ◽

Cellular Composition ◽

Immune Mediated ◽

Immune Attack ◽

And Control

SummaryHistopathological data on the cellular reactions (foci) around Schistosoma mansoni schistosomula in the lungs of both irradiated (750 rad) and unirradiated, passively immunized and normal rats were consistent with the idea that a significant proportion of immune-mediated attrition in passively immunized rats occurs in the lungs. In unirradiated rats, immune serum elicited an enhanced (i.e. larger) and accelerated (i.e. more rapidly developing) inflammatory cellular infiltration around lung-stage parasites when administered 5 days post-infection, when the parasites were already in the lungs. This demonstrated the antigenicity of lung-stage schistosomula and their potential as targets for immune attack. In irradiated rats, innate immunity was decreased as judged by an increase in the number of worms recovered by portal perfusion, and was accompanied by an overall decreased percentage of trapped parasites compared with unirradiated controls, suggesting that trapping in the lungs is involved in innate, as well as acquired immunity. In contrast to the results in unirradiated rats, passive transfer of immune serum into irradiated recipients did not result in larger lung foci than in the NRS-recipients. However, there was evidence of an accelerated response resulting in an essentially similar ratio of trapped parasites (VRS- compared with NRS-recipients) in irradiated rats, as compared with unirradiated rats, reflecting the similar levels of resistance manifested in both groups of rats. This also lent credence to the notion that it was the speed of immune recognition of the migrating schistosomula and the establishment of trapping foci that were of greater importance rather than the size of the enveloping granulomata. Investigations into the cellular composition of the foci surrounding trapped parasites in unirradiated rats revealed a predominance of mononuclear cells, with equal proportions of lymphocytes and macrophages. Eosinophils represented less than 3% of the cellular composition of the foci and were typically distant from the parasites themselves, arguing against their role in specific immunity in this model. Irradiation of recipient rats resulted in a corresponding increase in the percentage of macrophages in lung foci.

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microRNA-802 inhibits epithelial-mesenchymal transition through targeting flotillin-2 in human prostate cancer

Bioscience Reports ◽

10.1042/bsr20160521 ◽

2017 ◽

Vol 37 (2) ◽

Cited By ~ 14

Author(s):

Dawei Wang ◽

Guoliang Lu ◽

Yuan Shao ◽

Da Xu

Keyword(s):

Prostate Cancer ◽

Cell Proliferation ◽

Cell Lines ◽

Cell Apoptosis ◽

Epithelial Mesenchymal Transition ◽

Xenograft Model ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Mesenchymal Transition

miRNAs are a class of non-coding RNAs that exert critical roles in various biological processes. The aim of the present study was to identify the functional roles of miR-802 in regulating epithelial–mesenchymal transition (EMT) in prostate cancer (PCa). miR-802 expression was detected in 73 pairs of PCa samples and PCa cell lines (PC3 and DU145 cells) by qRT-PCR. Cell proliferation was detected using MTT assay, and cell apoptosis was evaluated using flow cytometry. Transwell assay was conducted to investigate cell migration and invasion. Expression analysis of a set of EMT markers was performed to explore whether miR-802 is involved in EMT program. Xenograft model was established to investigate the function of miR-802 in carcinogenesis in vivo. The direct regulation of Flotillin-2 (Flot2) by miR-802 was identified using luciferase reporter assay. miR-802 was remarkably down-regulated in PCa tissues and cell lines. Gain-of-function trails showed that miR-802 serves as an ‘oncosuppressor’ in PCa through inhibiting cell proliferation and promoting cell apoptosis in vitro. Overexpression of miR-802 significantly suppressed in vivo PCa tumor growth. Luciferase reporter analysis identified Flot2 as a direct target of miR-802 in PCa cells. Overexpressed miR-802 significantly suppressed EMT, migration and invasion in PCa cells by regulating Flot2. We identified miR-802 as a novel tumor suppressor in PCa progression and elucidated a novel mechanism of the miR-802/Flot2 axis in the regulation of EMT, which may be a potential therapeutic target.

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Androgen receptor as a regulator of ZEB2 expression and its implications in epithelial-to-mesenchymal transition in prostate cancer

Endocrine Related Cancer ◽

10.1530/erc-13-0514 ◽

2014 ◽

Vol 21 (3) ◽

pp. 473-486 ◽

Cited By ~ 18

Author(s):

Sheeba Jacob ◽

S Nayak ◽

Gwendolyn Fernandes ◽

R S Barai ◽

S Menon ◽

...

Keyword(s):

Prostate Cancer ◽

Androgen Receptor ◽

Cancer Metastasis ◽

Epithelial To Mesenchymal Transition ◽

Negative Regulator ◽

Lncap Cells ◽

Mesenchymal Transition ◽

Positive Regulator ◽

And Migration ◽

Zeb2 Expression

Zinc finger E-box-binding protein 2 (ZEB2) is known to help mediate the epithelial-to-mesenchymal transition, and thereby it facilitates cancer metastasis. This study was initiated to explore whether ZEB2 expression differs in prostate cancer (PCa,n=7) and benign prostatic hyperplasia (BPH,n=7) tissues. In PCa tissues, the levels of both immunoreactive ZEB2 and androgen receptor (AR) were found to be significantly higher (P<0.05) when compared with BPH tissues. Co-regulation of AR and ZEB2 prompted us to investigate the role of androgenic stimuli in ZEB2 expression. ZEB2 expression was found to be significantly (P<0.05) upregulated after androgen stimulation and downregulated following AR silencing in LNCaP cells, an androgen-dependent PCa cell line. This finding suggested AR as a positive regulator of ZEB2 expression in androgen-dependent cells. Paradoxically, androgen-independent (AI) cell lines PC3 and DU145, known to possess low AR levels, showed significantly (P<0.05) higher expression of ZEB2 compared with LNCaP cells. Furthermore, forced expression of AR in PC3 (PC3-AR) and DU145 (DU-AR) cells led to reductions in ZEB2 expression, invasiveness, and migration. These cells also exhibited an increase in the levels of E-cadherin (a transcriptional target of ZEB2). Co-transfection ofARandZEB2cDNA constructs prevented the decline in invasiveness and migration to a significant extent. Additionally, ZEB2 downregulation was associated with an increase in miR200a/miR200b levels in PC3-AR cells and with a decrease in miR200a/miR200b levels in AR-silenced LNCaP cells. Thus, AR acts as a positive regulator of ZEB2 expression in androgen-dependent cells and as a negative regulator in AI PCa cells.

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ISL1 Romotes Enzalutamide Resistance in Castration-resistant Prostate Cancer (CRPC) through Epithelial to Mesenchymal Transition (EMT)

10.21203/rs.3.rs-249256/v1 ◽

2021 ◽

Author(s):

Jae Duck Choi ◽

Tae Jin Kim ◽

Byong Chang Jeong ◽

Hwang Gyun Jeon ◽

Seong Soo Jeon ◽

...

Keyword(s):

Prostate Cancer ◽

Cell Growth ◽

Epithelial Mesenchymal Transition ◽

Mrna Level ◽

Xenograft Model ◽

Resistant Cell Line ◽

Castration Resistant Prostate Cancer ◽

Mesenchymal Transition ◽

Knock Down ◽

Castration Resistant

Abstract Abnormal expression of insulin gene enhancer-binding protein 1 (ISL1) has been demonstrated to be closely associated with cancer development and progression in several cancers. However, little is known about ISL1 expression in metastatic castration-resistant prostate cancer (CRPC). ISL1 has also been recognized as a positive modulator of epithelial-mesenchymal transition (EMT). In this study, we focused on ISL1 which showed maximum upregulation at the mRNA level in the enzalutamide-resistant cell line. Accordingly, we found that ISL1 was overexpressed in enzalutamide-resistant C4-2B cells and its expression was significantly related to EMT. Our findings reveal the important role of ISL1 in androgen receptor (AR)-dependent prostate cancer cell growth; ISL1 knockdown reduced the AR activity and cell growth. ISL1 knockdown using small-interfering RNA inhibited AR, PSA, and EMT-related protein expression in C4-2B ENZR cells. In addition, knock-down ISL1 reduced the levels of AKT and p65 phosphorylation in C4-2B ENZR cells and these suggest that knock-down ISL1 suppresses EMT in part by targeting the AKT/ NF-κB pathway. Further, ISL1 downregulation could effectively inhibit tumor growth in a human CRPC xenograft model. Together, the present study shows that downregulation of ISL1 expression is necessary for overcoming enzalutamide resistance and improving the survival of CRPC patients.

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Zebrafish Microenvironment Elevates EMT and CSC-Like Phenotype of Engrafted Prostate Cancer Cells

Cells ◽

10.3390/cells9040797 ◽

2020 ◽

Vol 9 (4) ◽

pp. 797

Author(s):

Lanpeng Chen ◽

Maciej Boleslaw Olszewski ◽

Marianna Kruithof-de Julio ◽

B. Ewa Snaar-Jagalska

Keyword(s):

Prostate Cancer ◽

Single Cell ◽

Epithelial Mesenchymal Transition ◽

Early Stage ◽

Xenograft Model ◽

Light Sheet ◽

Hematopoietic Tissue ◽

Cell Dynamics ◽

Mesenchymal Transition ◽

Cell Subpopulations

To visually and genetically trace single-cell dynamics of human prostate cancer (PCa) cells at the early stage of metastasis, a zebrafish (ZF) xenograft model was employed. The phenotypes of intravenously transplanted fluorescent cells were monitored by high-resolution, single-cell intravital confocal and light-sheet imaging. Engrafted osteotropic, androgen independent PCa cells were extravasated from caudle vein, invaded the neighboring tissue, proliferated and formed experimental metastases around caudal hematopoietic tissue (CHT) in four days. Gene expression comparison between cells in culture and in CHT revealed that engrafted PCa cells responded to the ZF microenvironment by elevating expression of epithelial–mesenchymal transition (EMT) and stemness markers. Next, metastatic potentials of ALDHhi cancer stem-like cells (CSCs) and ALDHlow non-CSCs were analyzed in ZF. Engraftment of CSCs induced faster metastatic onset, however after six days both cell subpopulations equally responded to the ZF microenvironment, resulting in the same increase of stemness genes expression including Nanog, Oct-4 and Cripto. Knockdown of Cripto significantly reduced the vimentin/E-cadherin ratio in engrafted cells, indicating that Cripto is required for transduction of the microenvironment signals from the ZF niche to increase mesenchymal potential of cells. Targeting of either Cripto or EMT transcriptional factors Snail 1 and Zeb1 significantly suppressed metastatic growth. These data indicated that zebrafish microenvironment governed the CSC/EMT plasticity of human PCa cells promoting metastasis initiation.

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ISL1 promotes enzalutamide resistance in castration-resistant prostate cancer (CRPC) through epithelial to mesenchymal transition (EMT)

Scientific Reports ◽

10.1038/s41598-021-01003-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jae Duck Choi ◽

Tae Jin Kim ◽

Byong Chang Jeong ◽

Hwang Gyun Jeon ◽

Seong Soo Jeon ◽

...

Keyword(s):

Prostate Cancer ◽

Cell Growth ◽

Epithelial Mesenchymal Transition ◽

Mrna Level ◽

Xenograft Model ◽

Resistant Cell Line ◽

Castration Resistant Prostate Cancer ◽

Mesenchymal Transition ◽

Knock Down ◽

Castration Resistant

AbstractAbnormal expression of insulin gene enhancer-binding protein 1 (ISL1) has been demonstrated to be closely associated with cancer development and progression in several cancers. However, little is known about ISL1 expression in metastatic castration-resistant prostate cancer (CRPC). ISL1 has also been recognized as a positive modulator of epithelial–mesenchymal transition (EMT). In this study, we focused on ISL1 which showed maximum upregulation at the mRNA level in the enzalutamide-resistant cell line. Accordingly, we found that ISL1 was overexpressed in enzalutamide-resistant C4-2B cells and its expression was significantly related to EMT. Our findings reveal the important role of ISL1 in androgen receptor (AR)-dependent prostate cancer cell growth; ISL1 knockdown reduced the AR activity and cell growth. ISL1 knockdown using small-interfering RNA inhibited AR, PSA, and EMT-related protein expression in C4-2B ENZR cells. In addition, knock-down ISL1 reduced the levels of AKT and p65 phosphorylation in C4-2B ENZR cells and these suggest that knock-down ISL1 suppresses EMT in part by targeting the AKT/NF-κB pathway. Further, ISL1 downregulation could effectively inhibit tumor growth in a human CRPC xenograft model. Together, the present study shows that downregulation of ISL1 expression is necessary for overcoming enzalutamide resistance and improving the survival of CRPC patients.

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Transcriptomic Analysis of LNCaP Tumor Xenograft to Elucidate the Components and Mechanisms Contributed by Tumor Environment as Targets for Dietary Prostate Cancer Prevention Studies

Nutrients ◽

10.3390/nu13031000 ◽

2021 ◽

Vol 13 (3) ◽

pp. 1000

Author(s):

Lu Yu ◽

Robert W. Li ◽

Haiqiu Huang ◽

Quynhchi Pham ◽

Liangli Yu ◽

...

Keyword(s):

Prostate Cancer ◽

Interleukin 1 ◽

Xenograft Model ◽

Human Prostate ◽

Tumor Xenograft ◽

Xenograft Tumor ◽

Lncap Cells ◽

Experimental Treatments

LNCaP athymic xenograft model has been widely used to allow researchers to examine the effects and mechanisms of experimental treatments such as diet and diet-derived cancer preventive and therapeutic compounds on prostate cancer. However, the biological characteristics of human LNCaP cells before/after implanting in athymic mouse and its relevance to clinical human prostate outcomes remain unclear and may dictate interpretation of biological efficacies/mechanisms of diet/diet-derived experimental treatments. In this study, transcriptome profiles and pathways of human prostate LNCaP cells before (in vitro) and after (in vivo) implanting into xenograft mouse were compared using RNA-sequencing technology (RNA-seq) followed by bioinformatic analysis. A shift from androgen-responsive to androgen nonresponsive status was observed when comparing LNCaP xenograft tumor to culture cells. Androgen receptor and aryl-hydrocarbon pathway were found to be inhibited and interleukin-1 (IL-1) mediated pathways contributed to these changes. Coupled with in vitro experiments modeling for androgen exposure, cell-matrix interaction, inflammation, and hypoxia, we identified specific mechanisms that may contribute to the observed changes in genes and pathways. Our results provide critical baseline transcriptomic information for a tumor xenograft model and the tumor environments that might be associated with regulating the progression of the xenograft tumor, which may influence interpretation of diet/diet-derived experimental treatments.

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RANK- and c-Met-mediated signal network promotes prostate cancer metastatic colonization

Endocrine Related Cancer ◽

10.1530/erc-13-0548 ◽

2014 ◽

Vol 21 (2) ◽

pp. 311-326 ◽

Cited By ~ 48

Author(s):

Gina Chia-Yi Chu ◽

Haiyen E Zhau ◽

Ruoxiang Wang ◽

André Rogatko ◽

Xu Feng ◽

...

Keyword(s):

Prostate Cancer ◽

Epithelial To Mesenchymal Transition ◽

Neuroendocrine Differentiation ◽

Skeletal Metastasis ◽

Site Directed Mutagenesis ◽

Lncap Cells ◽

Mesenchymal Transition ◽

Premetastatic Niche ◽

Metastatic Colonization ◽

Metastatic Cells

Prostate cancer (PCa) metastasis to bone is lethal and there is no adequate animal model for studying the mechanisms underlying the metastatic process. Here, we report that receptor activator of NF-κB ligand (RANKL) expressed by PCa cells consistently induced colonization or metastasis to bone in animal models. RANK-mediated signaling established a premetastatic niche through a feed-forward loop, involving the induction of RANKL and c-Met, but repression of androgen receptor (AR) expression and AR signaling pathways. Site-directed mutagenesis and transcription factor (TF) deletion/interference assays identified common TF complexes, c-Myc/Max, and AP4 as critical regulatory nodes. RANKL–RANK signaling activated a number of master regulator TFs that control the epithelial-to-mesenchymal transition (Twist1, Slug, Zeb1, and Zeb2), stem cell properties (Sox2, Myc, Oct3/4, and Nanog), neuroendocrine differentiation (Sox9, HIF1α, and FoxA2), and osteomimicry (c-Myc/Max, Sox2, Sox9, HIF1α, and Runx2). Abrogating RANK or its downstream c-Myc/Max or c-Met signaling network minimized or abolished skeletal metastasis in mice. RANKL-expressing LNCaP cells recruited and induced neighboring non metastatic LNCaP cells to express RANKL, c-Met/activated c-Met, while downregulating AR expression. These initially non-metastatic cells, once retrieved from the tumors, acquired the potential to colonize and grow in bone. These findings identify a novel mechanism of tumor growth in bone that involves tumor cell reprogramming via RANK–RANKL signaling, as well as a form of signal amplification that mediates recruitment and stable transformation of non-metastatic bystander dormant cells.

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Galeterone and The Next Generation Galeterone Analogs, VNPP414 and VNPP433-3β Exert Potent Therapeutic Effects in Castration-/Drug-Resistant Prostate Cancer Preclinical Models In Vitro and In Vivo

Cancers ◽

10.3390/cancers11111637 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1637 ◽

Cited By ~ 2

Author(s):

Andrew K. Kwegyir-Afful ◽

Senthilmurugan Ramalingam ◽

Vidya P. Ramamurthy ◽

Puranik Purushottamachar ◽

Francis N. Murigi ◽

...

Keyword(s):

Prostate Cancer ◽

Xenograft Model ◽

Cancer Drug ◽

Therapeutic Effects ◽

Stem Cell Markers ◽

Next Generation ◽

In Vivo Models ◽

Mesenchymal Transition

These studies compared the efficacies of our clinical agent galeterone (Gal) and the FDA-approved prostate cancer drug, enzalutamide (ENZ) with two lead next generation galeterone analogs (NGGAs), VNPP414 and VNPP433-3β, using prostate cancer (PC) in vitro and in vivo models. Antitumor activities of orally administered agents were also assessed in CWR22Rv1 tumor-bearing mice. We demonstrated that Gal and NGGAs degraded AR/AR-V7 and Mnk1/2; blocked cell cycle progression and proliferation of human PC cells; induced apoptosis; inhibited cell migration, invasion, and putative stem cell markers; and reversed the expression of epithelial-to-mesenchymal transition (EMT). In addition, Gal/NGGAs (alone or in combination) also inhibited the growth of ENZ-, docetaxel-, and mitoxantrone-resistant human PC cell lines. The NGGAs exhibited improved pharmacokinetic profiles over Gal in mice. Importantly, in vivo testing showed that VNPP433-3β (at 7.53-fold lower equimolar dose than Gal) markedly suppressed (84% vs. Gal, 47%; p < 0.01) the growth of castration-resistant PC (CRPC) CWR22Rv1 xenograft tumors, with no apparent host toxicity. ENZ was ineffective in this CRPC xenograft model. In summary, our findings show that targeting AR/AR-V7 and Mnk1/2 for degradation represents an effective therapeutic strategy for PC/CRPC treatment and supports further development of VNPP433-3β towards clinical investigation.

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