Drug-resistant Prostate Cancer Research Articles

MP20-11 ENZALUTAMIDE RESISTANCE RESULTS IN IMMUNOSUPPRESSIVE ALTERATIONS IN PROSTATE TUMOR IMMUNE MICROENVIRONMENT

You have accessJournal of UrologyCME1 Apr 2023MP20-11 ENZALUTAMIDE RESISTANCE RESULTS IN IMMUNOSUPPRESSIVE ALTERATIONS IN PROSTATE TUMOR IMMUNE MICROENVIRONMENT Pengfei Xu, Joy C. Yang, Bo Chen, Christopher Nip, Jonathan E Van Dyke, Christopher P. Evans, William J. Murphy, and Chengfei Liu Pengfei XuPengfei Xu More articles by this author , Joy C. YangJoy C. Yang More articles by this author , Bo ChenBo Chen More articles by this author , Christopher NipChristopher Nip More articles by this author , Jonathan E Van DykeJonathan E Van Dyke More articles by this author , Christopher P. EvansChristopher P. Evans More articles by this author , William J. MurphyWilliam J. Murphy More articles by this author , and Chengfei LiuChengfei Liu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Emerging data suggested that enzalutamide-treated prostate cancer patients with increased programmed death-ligand 1 (PD-L1) expression may benefit from anti-PD-L1 treatment. Unfortunately, the Phase III IMbassador250 trial revealed that the combination of atezolizumab (PD-L1 inhibitor) and enzalutamide failed to extend overall survival in patients with castration-resistant prostate cancer (CRPC). The mechanisms underlying treatment failure remain unknown. In this study, we investigated the regulation of immunosuppressive signaling in enzalutamide resistant prostate cancer and characterized the immune infiltrating cells in murine prostate tumors. METHODS: The expression of interferon gamma-related genes was determined using qRT-PCR and/or western blotting. Androgen receptor (AR) and AR-V7 levels were downregulated using specific siRNA. Myc-CaP cells were chronically exposed to increasing concentrations of enzalutamide (5-50 μM) for >12 months, and the cells resistant to enzalutamide were referred to as Myc-CaP MDVR. The gene-regulating mechanisms in drug-resistant prostate cancer cells were determined by RNA sequencing analyses. Myc-CaP and Myc-CaP MDVR xenograft tumors were established in FVB mice, and tumor-infiltrating lymphocytes were isolated using Ficoll. The stained immune cells were determined by flow cytometry, and the data were analyzed using Flowjo. RESULTS: Immune-related signaling pathways (interferon alpha/gamma response, T cell activation, and cell chemotaxis pathways) were suppressed in C4-2B MDVR cells (p<0.001). However, PD-L1 expression was highly upregulated and negatively regulated by AR in C4-2B MDVR cells. Enzalutamide treatment decreased CD8+ T cell (14.8% vs. 10.3%, p<0.05) but increased monocytic myeloid-derived suppressor cell (M-MDSC) population (23.0% vs. 34.2%, p<0.05) and PD-L1 expression in murine Myc-CaP tumors. Consistently, chemotaxis and immune response-regulating signaling pathways were suppressed, and PD-L1 expression was increased in enzalutamide-resistant Myc-CaP MDVR cells. Notably, MDSC populations were significantly increased in Myc-CaP MDVR orthotopic tumors compared with those in Myc-CaP parental tumors (4.0% vs. 1.6%, p<0.01). Co-culturing bone marrow cells with Myc-CaP MDVR cells significantly promoted MDSC differentiation and polarized macrophages from the M1 to M2 phase. CONCLUSIONS: Immunosuppressive signaling is activated in enzalutamide resistant prostate cancer cells. The activated immune-related signatures involved in T cells, MDSC, and macrophages may reduce the efficacy of immune checkpoint inhibitors in enzalutamide-resistant prostate cancer. Source of Funding: This work was supported in part by grants NIH/NCI R37CA249108 (C, Liu) and R01CA251253 (C, Liu) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e279 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Pengfei Xu More articles by this author Joy C. Yang More articles by this author Bo Chen More articles by this author Christopher Nip More articles by this author Jonathan E Van Dyke More articles by this author Christopher P. Evans More articles by this author William J. Murphy More articles by this author Chengfei Liu More articles by this author Expand All Advertisement PDF downloadLoading ...

LncRNA ERVH48-1 Contributes to the Drug Resistance of Prostate Cancer and Proliferation through Sponging of miR-4784 to the Activation of the Wnt/β-Catenin Pathway

Long noncoding RNAs (LncRNAs) are very important in the way that docetaxel resistance (DR) happens in prostate cancer (PCa) patients. ImmuneScore and StromalScore were calculated using PCa-related expression data from TCGA and the ESTIMATE algorithm. We finally found the DEGs that were related to the immune system and the stroma of the patients by making profiles of the DEGs in ImmuneScore and StromalScore. The CancerSubtypes algorithm identified prognosis-related PCa subtypes, and the GSVA assessed their pathway activity. A UniCox regression analysis was used to identify a prognosis-related differential gene set. We then used intersection analysis to identify immunological and prognostic (IP)-related genes (IPGs). The coexpression of long noncoding RNAs (lncRNAs) and IPGs was used to identify IP-related lncRNAs (IPLs). Three methods (SVM-RFE, random forest, and LASSO) were used to find genes that overlap in the GEO database. A gene signature was then validated by building an ROC curve. CIBERSORT technology was used to look at the possibility of a link between the gene signature and immune cells. LncRNA-miRNA pairs and miRNA-mRNA pairs from the miRDB and TargetScan databases were used to construct the ERVH48-1-miR-4784-WNT2B ceRNA regulation network. The concentration of docetaxel elevated the expression of ERVH48-1. Overexpression of ERVH48-1 increased PCa-DR cell proliferation, invasion, and migration while inhibiting apoptosis. ERVH48-1 increased the tumorigenicity of PCa-DR cells in nude mice. ERVH48-1, acting as a ceRNA, targeted miR-4784 to increase WNT2B expression. ICG001 therapy increased Wnt/-catenin signaling activity in PCa-DR cells by inhibiting ERVH48-1. Finally, ERVH48-1 increased docetaxel resistance in a WNT2B-dependent manner via the miR-4784/Wnt/-catenin pathway.

Bayesian Machine Learning Enables Identification of Transcriptional Network Disruptions Associated with Drug-Resistant Prostate Cancer.

The computational method TraRe built on Bayesian machine learning models for investigating transcriptional network structures shows that disruption of ELK3, MXD1, and MYB signaling cascades impacts abiraterone resistance in prostate cancer.

Allosteric inhibition of HSP70 in collaboration with STUB1 augments enzalutamide efficacy in antiandrogen resistant prostate tumor and patient-derived models

Ubiquitin proteasome activity is suppressed in enzalutamide resistant prostate cancer cells, and the heat shock protein 70/STIP1 homology and U-box-containing protein 1 (HSP70/STUB1) machinery are involved in androgen receptor (AR) and AR variant protein stabilization. Targeting HSP70 could be a viable strategy to overcome resistance to androgen receptor signaling inhibitor (ARSI) in advanced prostate cancer. Here, we showed that a novel HSP70 allosteric inhibitor, JG98, significantly suppressed drug-resistant C4–2B MDVR and CWR22Rv1 cell growth, and enhanced enzalutamide treatment. JG98 also suppressed cell growth in conditional reprogramed cell cultures (CRCs) and organoids derived from advanced prostate cancer patient samples. Mechanistically, JG98 degraded AR/AR-V7 expression in resistant cells and promoted STUB1 nuclear translocation to bind AR-V7. Knockdown of the E3 ligase STUB1 significantly diminished the anticancer effects and partially restored AR-V7 inhibitory effects of JG98. JG231, a more potent analog developed from JG98, effectively suppressed the growth of the drug-resistant prostate cancer cells, CRCs, and organoids. Notably, the combination of JG231 and enzalutamide synergistically inhibited AR/AR-V7 expression and suppressed CWR22Rv1 xenograft tumor growth. Inhibition of HSP70 using novel small-molecule inhibitors coordinates with STUB1 to regulate AR/AR-V7 protein stabilization and ARSI resistance.

The Small Molecule Antagonist KCI807 Disrupts Association of the Amino-Terminal Domain of the Androgen Receptor with ELK1 by Modulating the Adjacent DNA Binding Domain.

The androgen receptor (AR) is a crucial coactivator of ELK1 for prostate cancer (PCa) growth, associating with ELK1 through two peptide segments (358-457 and 514-557) within the amino-terminal domain (NTD) of AR. The small-molecule antagonist 5-hydroxy-2-(3-hydroxyphenyl)chromen-4-one (KCI807) binds to AR, blocking ELK1 binding and inhibiting PCa growth. We investigated the mode of interaction of KCI807 with AR using systematic mutagenesis coupled with ELK1 coactivation assays, testing polypeptide binding and Raman spectroscopy. In full-length AR, deletion of neither ELK1 binding segment affected sensitivity of residual ELK1 coactivation to KCI807. Although the NTD is sufficient for association of AR with ELK1, interaction of the isolated NTD with ELK1 was insensitive to KCI807. In contrast, coactivation of ELK1 by the AR-V7 splice variant, comprising the NTD and the DNA binding domain (DBD), was sensitive to KCI807. Deletions and point mutations within DBD segment 558-595, adjacent to the NTD, interfered with coactivation of ELK1, and residual ELK1 coactivation by the mutants was insensitive to KCI807. In a glutathione S-transferase pull-down assay, KCI807 inhibited ELK1 binding to an AR polypeptide that included the two ELK1 binding segments and the DBD but did not affect ELK1 binding to a similar AR segment that lacked the sequence downstream of residue 566. Raman spectroscopy detected KCI807-induced conformational change in the DBD. The data point to a putative KCI807 binding pocket within the crystal structure of the DBD and indicate that either mutations or binding of KCI807 at this site will induce conformational changes that disrupt ELK1 binding to the NTD. SIGNIFICANCE STATEMENT: The small-molecule antagonist KCI807 disrupts association of the androgen receptor (AR) with ELK1, serving as a prototype for the development of small molecules for a novel type of therapeutic intervention in drug-resistant prostate cancer. This study provides basic information needed for rational KCI807-based drug design by identifying a putative binding pocket in the DNA binding domain of AR through which KCI807 modulates the amino-terminal domain to inhibit ELK1 binding.

Front Cover: Hybrid Androgen Receptor Inhibitors Outperform Enzalutamide and EPI‐001 in in vitro Models of Prostate Cancer Drug Resistance (ChemMedChem 2/2023)

The Front Cover shows the design of the dual inhibitors starting from the FDA-approved enzalutamide (pink) and EPI-001 (green). On the right, in grey, we have the androgen receptor protein. Herein we present the schematic design of the most potent analogue in killing in vitro models of prostate cancer drug resistance, which was designed by covalently linking the two drugs using a PEG triazole linker (in cyan). The paper describes the synthesis of the compounds and their full biological characterisation. More information can be found in the Research Article by Radu Costin Bizga Nicolescu, Zoe R. Maylin Mohammad Asim, David R. Spring et al.

The Neuropilin-1/PKC axis promotes neuroendocrine differentiation and drug resistance of prostate cancer

BackgroundNeuroendocrine prostate cancer (NEPC) is a multi-resistant variant of prostate cancer (PCa) that has become a major challenge in clinics. Understanding the neuroendocrine differentiation (NED) process at the molecular level is therefore critical to define therapeutic strategies that can prevent multi-drug resistance.MethodsUsing RNA expression profiling and immunohistochemistry, we have identified and characterised a gene expression signature associated with the emergence of NED in a large PCa cohort, including 169 hormone-naïve PCa (HNPC) and 48 castration-resistance PCa (CRPC) patients. In vitro and preclinical in vivo NED models were used to explore the cellular mechanism and to characterise the effects of castration on PCa progression.ResultsWe show for the first time that Neuropilin-1 (NRP1) is a key component of NED in PCa cells. NRP1 is upregulated in response to androgen deprivation therapies (ADT) and elicits cell survival through induction of the PKC pathway. Downmodulation of either NRP1 protein expression or PKC activation suppresses NED, prevents tumour evolution toward castration resistance and increases the efficacy of docetaxel-based chemotherapy in preclinical models in vivo.ConclusionsThis study reveals the NRP1/PKC axis as a promising therapeutic target for the prevention of neuroendocrine castration-resistant variants of PCa and indicates NRP1 as an early transitional biomarker.

A ferroptosis-inducing biomimetic nanocomposite for the treatment of drug-resistant prostate cancer.

Second-generation androgen receptor (AR) inhibitors such as enzalutamide are the first-line treatments for castration-resistant prostate cancer (CRPC). Resistance to enzalutamide will greatly increase the difficulty of prostate cancer treatment and reduce the survival time of patients. However, drug-resistant cancer cells seem to be more sensitive to ferroptosis. Therefore, we constructed a biomimetic tumor-targeting magnetic lipid nanoparticle (t-ML) to codeliver dihomo-γ-linolenic acid (DGLA) and 2,4-dienoyl-CoA reductase 1 (DECR1) siRNA (t-ML@DGLA/siDECR1). DGLA is a dietary polyunsaturated fatty acid (PUFA), while DECR1 is overexpressed in prostate cancer and can inhibit the generation of PUFAs. The combination of DGLA and siDECR1 can efficiently induce ferroptosis by peroxidation of PUFAs, which has been verified both in vitro and in vivo. With the assistance of an external magnet, t-ML showed good tumor targeting ability and biocompatibility, and t-ML@DGLA/siDECR1 exhibited significant ferroptosis induction and tumor suppression capabilities. Moreover, in a nude mouse model of prostate cancer fed on a high-fat diet (HFD), there was no distant organ metastasis when the tumor-bearing mice were treated with t-ML@DGLA/siDECR1 and an external magnet, with upregulated PUFAs and downregulated monounsaturated fatty acids (MUFAs). Hence, this study has broadened the way of treating drug-resistant prostate cancer based on ferroptosis induction.

Down-Regulation of lncRNA MBNL1-AS1 Promotes Tumor Stem Cell-like Characteristics and Prostate Cancer Progression through miR-221-3p/CDKN1B/C-myc Axis

The recurrence, progression, and drug resistance of prostate cancer (PC) is closely related to the cancer stem cells (CSCs). Therefore, it is necessary to find the key regulators of prostate cancer stem cells (PCSCs). Here, we analyzed the results of a single-class logistic regression machine learning algorithm (OCLR) to identify the PCSC-associated lncRNA MBNL1-AS1. The effects of MBNL1-AS1 on the stemness of CSCs was assessed using qPCR, western blot and sphere-forming assays. The role of MBNL1-AS1 in mediating the proliferation and invasion of the PC cell lines was examined using Transwell, wounding-healing, CCK-8, EdU and animal assays. Dual-luciferase and ChIRP assays were used to examine the molecular mechanism of MBNL1-AS1 in PCSCs. MBNL1-AS1 was shown to be negatively correlated with stemness index (mRNAsi), and even prognosis, tumor progression, recurrence, and drug resistance in PC patients. The knockdown of MBNL1-AS1 significantly affected the stemness of the PC cells, and subsequently their invasive and proliferative abilities. Molecular mechanism studies suggested that MBNL1-AS1 regulates CDKN1B through competitive binding to miR-221-3p, which led to the inhibition of the Wnt signaling pathway to affect PCSCs. In conclusion, our study identified MBNL1-AS1 as a key regulator of PCSCs and examined its mechanism of action in the malignant progression of PC.

Hybrid Androgen Receptor Inhibitors Outperform Enzalutamide and EPI-001 in in vitro Models of Prostate Cancer Drug Resistance.

Androgen receptor targeted therapies for prostate cancer have serious limitations in advanced stages of the disease. While resistance to the FDA-approved enzalutamide is extensively documented, novel therapies based on epichlorohydrin scaffolds (EPI) are currently in clinical trials, but display suboptimal pharmacokinetics. Herein, we report the synthesis and biological characterisation of a novel class of compounds designed through covalently linking enzalutamide and EPI-001 through various triazole based linkers. The compounds display an 18 to 53 fold improvement in the cell killing potency towards C4-2b prostate cancer (PCa) cells compared to the gold standards of therapy, enzalutamide and EPI-001. The most promising compounds were proven to exhibit their toxicity exclusively through androgen receptor (AR) mediated pathways. This work sets the basis for the first class of hybrid AR inhibitors which successfully combine two drug moieties - EPI-001 and enzalutamide - into the same molecule.

Androgen Metabolism and Response in Prostate Cancer Anti-Androgen Therapy Resistance.

All aspects of prostate cancer evolution are closely related to androgen levels and the status of the androgen receptor (AR). Almost all treatments target androgen metabolism pathways and AR, from castration-sensitive prostate cancer (CSPC) to castration-resistant prostate cancer (CRPC). Alterations in androgen metabolism and its response are one of the main reasons for prostate cancer drug resistance. In this review, we will introduce androgen metabolism, including how the androgen was synthesized, consumed, and responded to in healthy people and prostate cancer patients, and discuss how these alterations in androgen metabolism contribute to the resistance to anti-androgen therapy.

Long-Term Fenofibrate Treatment Stimulates the Phenotypic Microevolution of Prostate Cancer Cells In Vitro.

Fenofibrate is a widely used anti-hyperlipidemic agonist of peroxisome proliferator-activated receptor alpha (PPARα). As a metabolic blocker, fenofibrate interferes with cancer promotion/progression via its misbalancing effects on cellular metabolism. However, the consequences of its long-term application for patients with diagnosed drug-resistant cancers are unknown. We addressed this point by tracing the phenotypic microevolution of naïve and drug-resistant prostate cancer PC3_DCX20 cells that underwent a long-term exposition to 10 μM and 50 μM fenofibrate. Their resistance to fenofibrate, metabolic profile and invasive phenotype were estimated in the control conditions and under fenofibrate-induced stress. Apparently, drug efflux systems are not effective against the cytostatic FF action. However, wtPC3 and PC3_DCX20 cells that survived the long-term 50 μM fenofibrate treatment gave rise to lineages that displayed an increased proliferation rate, lower motility in the control conditions and enhanced fenofibrate resistance. Attenuated fenofibrate bioavailability modified the pattern of PC3 microevolution, as illustrated by phenotypic differences between wtPC3/PC3_DCX20 lineages propagated in the presence of 50 μM and 10 μM fenofibrate. Collectively, our observations indicate that fenofibrate acts as a selective factor that affects prostate cancer microevolution. We also pinpoint potential consequences of long-term exposition of prostate cancer patients to metabolic blockers.

Polygodial, a Sesquiterpene Dialdehyde, Activates Apoptotic Signaling in Castration-Resistant Prostate Cancer Cell Lines by Inducing Oxidative Stress.

Prostate cancer (PCa) is the second leading cause of cancer death among men in the United States. Surgery, radiation therapy, chemotherapy, and androgen deprivation therapy are currently the standard treatment options for PCa. These have poor outcomes and result in the development of castration-resistant prostate cancer (CRPC), which is the foremost underlying cause of mortality associated with PCa. Taxanes, diterpene compounds approved to treat hormonal refractory PCa, show poor outcomes in CRPC. Polygodial (PG) is a natural sesquiterpene isolated from water pepper (Persicaria hydropiper), Dorrigo pepper (Tasmannia stipitata), and mountain pepper (Tasmannia lanceolata). Previous reports show that PG has an anticancer effect. Our results show that PG robustly inhibits the cell viability, colony formation, and migration of taxane-resistant CRPC cell lines and induces cell cycle arrest at the G0 phase. A toxicity investigation shows that PG is not toxic to primary human hepatocytes, 3T3-J2 fibroblast co-cultures, and non-cancerous BPH-1 cells, implicating that PG is innocuous to healthy cells. In addition, PG induces oxidative stress and activates apoptosis in drug-resistant PCa cell lines. Our mechanistic evaluation by a proteome profiler-human apoptotic array in PC3-TXR cells shows that PG induces upregulation of cytochrome c and caspase-3 and downregulation of antiapoptotic markers. Western blot analysis reveals that PG activates apoptotic and DNA damage markers in PCa cells. Our results suggest that PG exhibits its anticancer effect by promoting reactive oxygen species generation and induction of apoptosis in CRPC cells.

Cytotoxic N-Methylpretrichodermamide B Reveals Anticancer Activity and Inhibits P-Glycoprotein in Drug-Resistant Prostate Cancer Cells.

N-methylpretrichodermamide B (NB) is a biologically active epidithiodiketopiperazine isolated from several strains of the algae-derived fungus Penicillium sp. Recently, we reported the first data on its activity in human cancer cells lines in vitro. Here, we investigated the activity, selectivity, and mechanism of action of NB in human prostate cancer cell lines, including drug-resistant subtypes. NB did not reveal cross-resistance to docetaxel in the PC3-DR cell line model and was highly active in hormone-independent 22Rv1 cells. NB-induced cell death was stipulated by externalization of phosphatidylserine and activation of caspase-3. Moreover, inhibition of caspase activity by z-VAD(OMe)-fmk did not affect NB cytotoxicity, suggesting a caspase-independent cell death induced by NB. The compound has a moderate p-glycoprotein (p-gp) substrate-like affinity and can simultaneously inhibit p-gp at nanomolar concentrations. Therefore, NB resensitized p-gp-overexpressing PC3-DR cells to docetaxel. A kinome profiling of the NB-treated cells revealed, among other things, an induction of mitogen-activated protein kinases JNK1/2 and p38. Further functional analysis confirmed an activation of both kinases and indicated a prosurvival role of this biological event in the cellular response to the treatment. Overall, NB holds promising anticancer potential and further structure–activity relationship studies and structural optimization are needed in order to improve its biological properties.

MiRNAs in prostate cancer: Intercellular and extracellular communications.

Prostate cancer is the most prevalent male cancer in Western Europe and North America. Although new drugs were recently approved, clinical challenges such as accurately predicting and screening drug-resistant prostate cancer remain. microRNAs are short noncoding RNA molecules that participate in gene regulation at the post-transcriptional level by targeting messenger RNAs. There is accumulating evidence that intracellular microRNAs play important roles as promoters or inhibitors of prostate cancer progression. Additionally, recent studies showed that microRNAs are encapsulated in extracellular vesicles and shuttled into the extracellular space. Transfer of extracellular microRNAs contributes to intercellular communication between prostate cancer cells and components of the tumor microenvironment, which can promote prostate cancer progression. Furthermore, due to their encapsulation in extracellular vesicles, extracellular microRNAs can be stably present in body fluids which contain high levels of RNase. Thus, circulating microRNAs have great potential as noninvasive diagnostic and prognostic biomarkers for prostate cancer. Here, we summarize the roles of intracellular and extracellular microRNAs in prostate cancer progression and discuss the potential of microRNA-based therapeutics as a novel treatment strategy for prostate cancer.

New diterpenes from the marine sponge Spongionella sp. overcome drug resistance in prostate cancer by inhibition of P-glycoprotein

Spongian diterpenes are a group of marine natural compounds possessing various biological activities. However, their anticancer activity is still poorly studied and understood. We isolated six spongian diterpenes from the marine sponge Spongionella sp., including one new spongionellol A and five previously known molecules. The structures were elucidated using a detailed analysis MS and NMR spectra as well as by comparison with previously reported data. Two of them, namely, spongionellol A and 15,16-dideoxy-15α,17β-dihydroxy-15,17-oxidospongian-16-carboxylate-15,17-diacetate exhibited high activity and selectivity in human prostate cancer cells, including cells resistant to hormonal therapy and docetaxel. The mechanism of action has been identified as caspase-dependent apoptosis. Remarkably, both compounds were able to suppress expression of androgen receptor (AR) and AR-splice variant 7, as well as AR-dependent signaling. The isolated diterpenes effectively inhibited drug efflux mediated by multidrug-resistance protein 1 (MDR1; p-glycoprotein). Of note, a synergistic effect of the compounds with docetaxel, a substrate of p-glycoprotein, suggests resensitization of p-glycoprotein overexpressing cells to standard chemotherapy. In conclusion, the isolated spongian diterpenes possess high activity and selectivity towards prostate cancer cells combined with the ability to inhibit one of the main drug-resistance mechanism. This makes them promising candidates for combinational anticancer therapy.

Abstract 3810: Muscle invasion produced drug-resistant and bone metastatic prostate cancer

Abstract Overtreatment of prostate cancer is a significant source of patient morbidity and cost. The human prostate is bounded by a smooth muscle capsule, and aggressive tumors invade through the muscle layer, called extracapsular extension (ECE), to escape organ confinement. The presence of ECE defines pT3a pathologic stage and is associated with increased risk of biochemical recurrence, metastases, and cancer-specific mortality. Although muscle invasion is required for ECE and metastatic spread, both the invasive tumor network phenotypes and muscle responses are understudied. The goal was to probe the molecular events of ECE to understand this critical step in metastasis. Muscle invasion of prostate cancer cell lines was tested in vivo by injecting cells into the peritoneal cavity of male NSG mice. The cells colonize the inferior surface of the muscular respiratory diaphragm and invade through to the superior surface. To explore transcriptional regulators, we performed whole genome RNAseq on cells from three compartments: (1) “Inferior” non-invading cells on the underside of the diaphragm; (2) “Muscle-resident” cells that have invaded and now reside within the diaphragm muscle; and (3) “Superior” cells that have completely traversed the diaphragm. Tumors cells reaching the superior side of the diaphragm were established ex vivo as polyclonal cell lines termed the “KM” series. RNAseq reveals 1,482 differentially expressed sequences (DES) between Inferior and Muscle-resident cells, 253 DES between Muscle-resident and Superior cells, and 896 DES between Inferior and Superior cells (padj &amp;lt;0.05, |fold change| ≥ 1.3). Further, we identified 84 DES that overlap between the Superior vs. Muscle-resident and Inferior vs. Muscle-resident groups, highlighting candidate gene changes unique to cancer cells in a muscle microenvironment. Importantly, 4,076 differentially expressed, mouse-specific RNA sequences (padj &amp;lt; 0.05, |fold change| ≥ 2.0) highlight pathways within muscle that respond to the presence of invasive tumors. Strikingly, the KM series have aggressive features including newly acquired bone metastasis and resistance to a taxane. The gene expression patterns imply a dominant effect of the muscle microenvironment in evoking a new and transient tumor transcriptional response as revealed by the DESs. Independent of this response, the KM sub-populations successfully navigating the muscle were more aggressive in laboratory assays, including bone metastatic potential and chemotherapeutic resistance. These studies support the hypothesis that successful invasion into and through a contractile muscle layer results in aggressive cancer cells. Using the in vivo assay provides the unique ability to parse out unexplored patterns of changes that occur in early, muscle-invasive disease to improve prostate cancer patient selection for definitive treatment versus active surveillance. Citation Format: Kendra D. Marr, Beatrice S. Knudsen, Jaime M. Gard, Malia Bird, Raymond B. Nagle, Anne E. Cress. Muscle invasion produced drug-resistant and bone metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3810.

Abstract 301: Repurposing engineered thymoquinone nanoparticles to inhibit drug-resistant prostate cancer

Abstract Despite advanced treatment strategies and therapeutics for prostate cancer (PCa), the metastatic castration-resistant prostate cancer (mCRPC) mechanism remains obscure. Although docetaxel (DTX) and other anti-mitotic chemotherapeutic drug are considered the first line drugs for PCa patients; the resulting side effects and drug resistance increase the demand for an alternative such as natural compounds for effective treatment. However, the natural compounds that achieved remarkable height to overcome drug resistance, their bioavailability and targeted delivery during treatment has become a new challenge. As prostate-specific membrane antigen (PSMA) receptors are expressed on the surface of most PCa cells, we anticipated that these receptors may provide the potential target for effective therapy. Considering these, we employed the natural compound, thymoquinone (TQ)-encapsulated planetary ball-milled nanoparticles (PBM-NPs) formulated with starch (FDA approved, natural polysaccharides) inner core and biocompatible and biodegradable co-polymers poly (ε-caprolactone)/poly (ethylene glycol), functionalized with a PSMA aptamer (A10), which can selectively recognize and bind to PSMA membrane. The functionalities of this strategy were analyzed by targeting A10-TQ-PBM-NP in DTX-resistant cells (C4-2B-R and LNCaP-R), which overexpresses PSMA protein and multidrug resistance (MDR) gene ABCB1, and validated through flow cytometry, immunofluorescence, qRT-PCR, and western blot techniques. The results showed that the aptamer-based nano delivery of a TQ can modulate the cellular and molecular pathways responsible for DTX resistance in PCa cells through the EGFR and ZEB1 dependent mechanism. The A10-TQ-PBM-NPs counteract ATP-binding cassette (ABC)-transporter-mediated chemoresistance in PCa cells. In both cell lines, these NPs rapidly internalized and induced significant cell death at five times lower concentrations than non-conjugated PBM-TQ-NP. Further, A10-TQ-PBM-NPs have high selectivity to the PSMA receptor and downregulated the EGFR dependent pathway, Akt-1, ERK1/2, NF-kB, STAT3, and epithelial-mesenchymal transition (EMT) markers in both C4-2B-R and LNCaP-R cells, thus constraining cell survival, proliferation, and migration. The A10-TQ-PBM-NP bioconjugates also reveal remarkable efficacy and reduced toxicity as measured by body weight loss in the LNCaP-R cells xenograft mice. Altogether, our findings demonstrate that targeting PCa cells with PBM-NPs may reverse the MDR and could be used as potential therapeutic options for patients. Citation Format: Santosh Kumar Singh, Manoj Kumar Mishra, Rajesh Singh. Repurposing engineered thymoquinone nanoparticles to inhibit drug-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 301.

LncRNA-DANCR Promotes Taxol Resistance of Prostate Cancer Cells through Modulating the miR-33b-5p-LDHA Axis.

Prostate cancer (PCa) is one of the most common malignancies in men with high death rate worldwide. Paclitaxel (Taxol) is a widely used anticancer agent. Despite recent improvements in clinical application and research, development of drug resistance limits the efficacy of the Taxol-based chemotherapy. Previous studies revealed that the long noncoding RNA DANCR positively regulated progression of prostate cancer. However, the precise roles of DANCR in the Taxol sensitivity of PCa and the underlying molecular mechanisms remain largely unknown. Here, we report that the expressions of DANCR were significantly upregulated and miR-33b-5p were downregulated in prostate tumor specimens and cells as well as the Taxol-resistant prostate cancer cell line (PC3-TXR). Silencing DANCR or overexpressing miR-33b-5p effectively enhanced the Taxol sensitivity of PCa cells. Bioinformatics analysis, RNA pull-down assay, and luciferase assay consistently illustrated that DANCR was associated with miR-33b-5p, leading to downregulation of miR-33b-5p in PCa. Interestingly, glucose metabolism of PC3-TXR cells was remarkedly elevated. The glucose uptake, extracellular acidification rate (ECAR), and glycolysis speed-limiting enzyme expressions were significantly promoted in PC3-TXR cells. We further identified the glucose metabolism enzyme; LDHA was a direct target of miR-33b-5p in PCa cells. LDHA restoration attenuated miR-33b-5p-mediated PTX sensitization. Finally, the rescue of miR-33b-5p in DANCR-overexpressing PC3-TXR cells successfully overrode the DANCR-promoted Taxol resistance. In summary, this study uncovered biological roles and molecular mechanisms of the DANCR-promoted chemoresistance, contributing to the development of noncoding RNA-based therapeutic strategies against drug-resistant prostate cancer.

Lipin Expression Correlates to Differences in Phospholipid Profiles in Drug‐Resistant Prostate Cancer Cells

PURPOSEThe association between clinical outcomes of prostate cancer progression and lipid remodeling is not well understood. Although it is known that increases in select circulating lipids correlate to decreased patient survival, the mechanisms mediating alterations in these lipids are not fully explained. We addressed this gap‐in‐knowledge using in vitro models of non‐cancerous, hormone‐sensitive, castration‐resistant prostate cancer (CRPC), and docetaxel resistant (DR) CRPC cell lines combined with quantitative HPLC‐ESI‐Orbitrap‐MS lipidomic analysis.HYPOTHESISWe hypothesized that the expression of lipin (phosphatidic acid phosphatase), an enzyme mediating the conversion of phosphatidic acid to diacylglycerol, correlates to changes in lipidomic profiles in prostate cancer cells. We further hypothesized that the lipidomic profile of metastatic CRPC cell lines (PC‐3 and DU‐145) will shift as they are undergoing multiple treatments of docetaxel.METHODSLipids were isolated using the Bligh‐Dyer extraction from non‐cancerous, hormone‐sensitive, CRPC, and DR‐CRPC cell lines. Lipids were initially analyzed using an untargeted shotgun approach (ESI‐MS), followed by a targeted‐based quantitative HPLC‐ESI‐Orbitrap‐MS approach. Post data acquisition were based on multiple methods including isotope, carbon number (to internal standards) and ionization efficiency‐based corrections. Online resources and software's utilized included MetaboAnalyst, LIPIDMATCH, LIPID MAPS, XCMS, and MZmine.Lipin‐1 expression in cells was determined using immunoblot analysis. Docetaxel‐sensitive traits were obtained after five treatments of 1 nM docetaxel between passages of the CRPC cell line PC‐3. Results were demonstrated by a 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) assay.RESULTSOur data showed significant changes in the abundance of lipid species between the various cell lines and a correlation between select lipid species and the progression of prostate cancer. Prominent among these were the levels of phosphatidic acid (PA) and diacylglycerol (DAG). Most notably, PA levels in cells tended to inversely correlate with DAG levels. These levels correlated to the decreased expression on lipin, specifically lipin‐1.CONCLUSIONSDistinct phospholipid classes were observed to have increased levels in prostate cancer cells, as compared to non‐cancer cells. The protein expression of lipin‐1 in most prostate cell lines correlated to the abundance of PA, excluding the DR cell lines. This suggests that altered lipin expression correlates to decreased survival. Additionally, dosing the parent and treated PC‐3 cells with a range of docetaxel indicated that the 5x1nM treated cells had a larger cytotoxic effect to docetaxel, as compared to the non‐treated cells.REFERENCESIngram, L.M., et al., Identification of lipidomic profiles associated with drug‐resistant prostate cancer cells. Lipids Health Dis, 2021. 20(1): p. 15.