Human Prostate Cancer Cell Lines Research Articles

Cytotoxic activities of metallic nanoparticles biosynthesized using plant extracts and pure compounds from Cephalaria species

Green nanotechnology has been focused on new concepts and fundamental research to produce nanoparticles in high rates and low risks by the use of different sources such as microorganisms and plants. Due to the several constituents in secondary metabolites, plants play an important role in nanomaterial synthesis known as ecofriendly methods which acquired important potential in different studies. The current work aimed to synthesize different silver and zinc-based nanoparticles using Cephalaria tchihatchewii via phytobiological methods involving metallic solutions, organic extracts, and isolated compounds. Sixteen types of nanoparticles were determined in between 50 and 297 nm as diameter and characterized using ultraviolet-visible spectrophotometer, dynamic light scattering, scanning electron microscopy and fourier transform infrared spectroscopy. The cytotoxic activities of nanoparticles were evaluated against human lung cancer (A549), breast cancer (MDA-MB-231), prostate cancer (PC3), and healthy lung (CCD-34Lu) cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide MTT assay utilizing doxorubicin as a positive control. Against PC3 cells, the cytotoxic activity of silver nitrate and loganin-AgNPs show significant inhibition with IC50 values of 2.39 ± 0.16 and 7.84 ± 1.21 respectively compared with doxorubicin (12.99 ± 1.57 µg/mL). A moderate inhibition activity was observed for Zinc sulfate, elmalienoside-B-AgNPs, methyl glycoside-AgNPs, and luteolin-7-O-glycoside-AgNPs with IC50 values of 22. 27± 0.18, 11.59 ± 0.82, 12.43 ± 0.37 and 13.26 ± 1.63 µg/mL, respectively. The inhibition activity against human breast cancer cells MDA-MB-231 was significantly reported with silver nitrate (2.25 ± 0.62 µg/mL), and elmalienoside-B-AgNPs (11.48 ± 0.46 µg/mL) compared with doxorubicin (11.61± 0.68 µg/mL). The cytotoxicity activities, make Cephalaria tchihatchewii plant a suitable candidate in different biopharmaceutical usages especially in anticancer activities. This study will provide a clear perspective on nanoparticles containing green synthesized pure compounds and the reaction mechanism of their activities.

Role of KDM2B epigenetic factor in regulating calcium signaling in prostate cancer cells

KDM2B, a histone lysine demethylase, is expressed in a plethora of cancers. Earlier studies from our group, have showcased that overexpression of KDM2B in the human prostate cancer cell line DU-145 is associated with cell adhesion, actin reorganization, and improved cancer cell migration. In addition, we have previously examined changes of cytosolic Ca2+, regulated by the pore-forming proteins ORAI and the Ca2+ sensing stromal interaction molecules (STIM), via store-operated Ca2+ entry (SOCE) in wild-type DU-145. This study sought to evaluate the impact of KDM2B overexpression on the expression of key molecules (SGK1, Nhe1, Orai1, Stim1) and SOCE. Furthermore, this is the first study to evaluate KDM2B expression in circulating tumor cells (CTCs) from patients with prostate cancer. mRNA levels for SGK1, Nhe1, Orai1, and Stim1 were quantified by RT-PCR. Calcium signals were measured in KDM2B-overexpressing DU-145 cells, loaded with Fura-2. Blood samples from 22 prostate cancer cases were scrutinized for KDM2B expression using immunofluorescence staining and the VyCAP system. KDM2B overexpression in DU-145 cells increased Orai1, Stim1, and Nhe1 mRNA levels and significantly decreased Ca2+ release. KDM2B expression was examined in 22 prostate cancer patients. CTCs were identified in 45 % of these patients. 80 % of the cytokeratin (CK)-positive patients and 63 % of the total examined CTCs exhibited the (CK + KDM2B + CD45−) phenotype. To conclude, this study is the first to report increased expression of KDM2B in CTCs from patients with prostate cancer, bridging in vitro and preclinical assessments on the potentially crucial role of KDM2B on migration, invasiveness, and ultimately metastasis in prostate cancer.

Maramycin, a Cytotoxic Isoquinolinequinone Terpenoid Produced through Heterologous Expression of a Bifunctional Indole Prenyltransferase/Tryptophan Indole-Lyase in S. albidoflavus.

Isoquinolinequinones represent an important family of natural alkaloids with profound biological activities. Heterologous expression of a rare bifunctional indole prenyltransferase/tryptophan indole-lyase enzyme from Streptomyces mirabilis P8-A2 in S. albidoflavus J1074 led to the activation of a putative isoquinolinequinone biosynthetic gene cluster and production of a novel isoquinolinequinone alkaloid, named maramycin (1). The structure of maramycin was determined by analysis of spectroscopic (1D/2D NMR) and MS spectrometric data. The prevalence of this bifunctional biosynthetic enzyme was explored and found to be a recent evolutionary event with only a few representatives in nature. Maramycin exhibited moderate cytotoxicity against human prostate cancer cell lines, LNCaP and C4-2B. The discovery of maramycin (1) enriched the chemical diversity of natural isoquinolinequinones and also provided new insights into crosstalk between the host biosynthetic genes and the heterologous biosynthetic genes in generating new chemical scaffolds.

Biogenic silver nanoparticle synthesis using orange peel extract and its multifaceted biomedical application.

The aim of this study was to employ an agro-industrial byproduct, specifically Citrus sinensis peels, as a reservoir of polyphenols. The natural chemicals present in C. sinensis peels serve as reducing agents in an environmentally benign method for synthesizing silver nanoparticles (AgNPs). This methodology not only provides a more environmentally friendly method for synthesizing nanoparticles but also enhances the value of agricultural waste, emphasizing the sustainable utilization of resources. In our study, AgNPs were successfully synthesized using peel aqueous exact of C. sinensis and then their various biological activity has been investigated. The synthesized AgNPs were characterized by UV-vis spectroscopy, dynamic light scattering (DLS), scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), and transmission electron microscopy (TEM) analysis. Furthermore, their effectiveness in inhibiting growth and biofilm formation of Escherichia coli, Staphylococcus aureus, and Candida albicans has been investigated. The minimum inhibitory concentrations (MIC) for E. coli and S. aureus were both 32μg/mL, and for C. albicans, it was 128µg/mL. At 250µg/mL of AgNPs, 94% and 92% biofilm inhibition were observed against E. coli and S. aureus, respectively. Furthermore, AgNPs demonstrated significant toxic effects against human prostate cancer cell line DU145 as investigated by anti-apoptotic, 4',6-diamidino-2-phenylindole (DAPI), reactive oxygen species (ROS), and acridine orange/ethidium bromide (AO/EtBr) assays. We also conducted uptake analysis on these pathogens and cancer cell lines to preliminarily investigate the mechanisms underlying their toxic effects. These findings confirm that AgNPs can serve as a cost-effective, non-toxic, and environmentally friendly resource for green synthesis of medicinal AgNPs. Moreover, this approach offers an alternative recycling strategy that contributes to the sustainable use of biological by-products.

Effects of crotamine in human prostate cancer cell line

Our study presents the anticancer potential of crotamine from Crotalus durissus terrificus in human prostate cancer cell line DU-145. Crotamine isolation was conducted through RP-FPLC, its molecular mass analyzed by MALDI-TOF was 4881.4 kDa, and N-terminal sequencing confirmed crotamine identity. Crotamine demonstrated no toxicity and did not inhibit migration in HUVEC cells. Although no cell death occurred in DU-145 cells, crotamine inhibited their migration. Thus, crotamine presented potential to be a prototype of anticancer drug.

An efficient synthesis and anticancer evaluation of 1,2,3-triazolo-indole-2-carboxylate derivatives

A new series of 1,2,3-Triazole bearing Indole derivatives (6a–j, 8a & 9a) was developed and examined for their preliminary anticancer effects against five human cancer cell lines such as breast cancer cell line (MCF-7), human lung cancer cell line (A549), human colon cancer cell line (Colo-205), human prostate cancer cell line (DU-145) and human breast cancer cell line (MDA MB-231). The well-known chemotherapeutic agent as etoposide used as a positive control. Among synthesized compounds, six compounds (6a, 6b, 6c, 6d, 6f and 8a) were possessed more potent anticancer activities than standard drug.

Isolation, Identification and Chemical Modification of Bufadienolides from Bufo melanostictus Schneider and Their Cytotoxic Activities against Prostate Cancer Cells.

The traditional Chinese medicine toad venom (Venenum bufonis) has been extensively used to treat various diseases, including cancers, in China and other Southeast Asian countries. The major constituents of toad venom, e.g., bufadienolides and alkaloids, exhibit broad-spectrum pharmacological effects in cancers. Herein, two new bufadienolides (1 and 2), along with eleven known compounds (3-13) were successfully isolated from Bufo melanostictus Schneider. Their structures were elucidated by extensive spectroscopic data and X-ray diffraction analysis. Furthermore, four lactam derivatives were synthesized through the transformation of bufadienolides lactones. The inhibitory effects of these compounds against human prostate cancer cell lines PC-3 and DU145 were evaluated. The outcomes indicated a notable trend, with a substantial subset displaying nanomolar range IC50 values against PC-3 and DU145 cells, underscoring their pronounced cytotoxicity. Moreover, a noteworthy distinction surfaces, wherein lactones consistently outperformed their lactam counterparts, further validating their heightened potency for the treatment of prostate cancer. This study contributes significant preclinical evidence substantiating the therapeutic viability of bufadienolides and toad venom as intervention strategies for prostate cancer.

Overcoming statin resistance in prostate cancer cells by targeting the 3-hydroxy-3-methylglutaryl-CoA-reductase

Prostate cancer is the most prevalent malignancy in men. While diagnostic and therapeutic interventions have substantially improved in recent years, disease relapse, treatment resistance, and metastasis remain significant contributors to prostate cancer-related mortality. Therefore, novel therapeutic approaches are needed. Statins are inhibitors of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate pathway which plays an essential role in cholesterol homeostasis. Numerous preclinical studies have provided evidence for the pleiotropic antitumor effects of statins. However, results from clinical studies remain controversial and have shown substantial benefits to even no effects on human malignancies including prostate cancer. Potential statin resistance mechanisms of tumor cells may account for such discrepancies. In our study, we treated human prostate cancer cell lines (PC3, C4–2B, DU-145, LNCaP) with simvastatin, atorvastatin, and rosuvastatin. PC3 cells demonstrated high statin sensitivity, resulting in a significant loss of vitality and clonogenic potential (up to – 70%; p < 0.001) along with an activation of caspases (up to 4-fold; p < 0.001). In contrast, C4–2B and DU-145 cells were statin-resistant. Statin treatment induced a restorative feedback in statin-resistant C4–2B and DU-145 cells through upregulation of the HMGCR gene and protein expression (up to 3-folds; p < 0.01) and its transcription factor sterol-regulatory element binding protein 2 (SREBP-2). This feedback was absent in PC3 cells. Blocking the feedback using HMGCR-specific small-interfering (si)RNA, the SREBP-2 activation inhibitor dipyridamole or the HMGCR degrader SR12813 abolished statin resistance in C4–2B and DU-145 and induced significant activation of caspases by statin treatment (up to 10-fold; p < 0.001). Consistently, long-term treatment with sublethal concentrations of simvastatin established a stable statin resistance of a PC3SIM subclone accompanied by a significant upregulation of both baseline as well as post-statin HMGCR protein (gene expression up to 70-fold; p < 0.001). Importantly, the statin-resistant phenotype of PC3SIM cells was reversible by HMGCR-specific siRNA and dipyridamole. Our investigations reveal a key role of a restorative feedback driven by the HMGCR/SREBP-2 axis in statin resistance mechanisms of prostate cancer cells.

The Role of Adipocytokines and their Receptors in Prostate Cancer: Adiponectin May Protect Against Progression.

Obesity is correlated with an increased risk of developing malignancies, including prostate cancer. Adipocytokines, such as leptin and adiponectin, are a family of hormones derived from adipose tissue that are involved not only in metabolism, but also in the development and progression of various malignancies. However, little is known about their role in prostate cancer. This study aimed to determine how leptin, adiponectin, and their receptors impact the spread of prostate cancer. We first performed immunohistochemical analysis of prostate cancer tissue microarrays to detect leptin, leptin receptor (Ob-R), adiponectin, and adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2). Wound healing assays and western blot analysis were then performed in human prostate cancer cell lines. Immunohistochemistry showed that prostate tissue was not significantly positive for adiponectin. However, its expression tended to decrease according to the International Society of Urological Pathology (ISUP) grade of prostate cancer (p=0.056). In prostate cancer cell lines, administration of the synthetic adiponectin AdipoRon suppressed cell migration as well as the expression of phospho-NF-[Formula: see text]B and cyclooxygenase-2, whereas leptin stimulated these effects. Adiponectin expression tended to be suppressed according to ISUP grade in prostate cancer tissues. In vitro, tumor cell migration was induced by leptin but suppressed by adiponectin. Targeting adipocytokines could be a novel treatment strategy for prostate cancer.

Abstract 6264: Role of Nuclear ErbB3 localization in regulating ligand-dependent ErbB2-ErbB3 heterodimer activation and downstream targets in prostate cancer

Abstract Objective: Prostate cancer (PCa)-specific nuclear expression of ErbB3, a receptor tyrosine kinase of the epidermal growth factor receptor (EGFR) family, has long been recognized. While an 80kDa nuclear variant of ErbB3 has been identified, full-length 185 kDa ErbB3 also translocates to the nucleus in PCa in a bone microenvironment and androgen status-dependent manner. Here, we studied potential mechanisms by which nuclear ErbB3 may regulate PCa progression. Methods: ErbB3 localization was investigated in vitro (human prostate cancer cell lines, LNCaP, C4-2, 22Rv1) using amiloride or heregulin-1β (ErbB3-specific ligand). ErbB3 expression and subcellular localization were analyzed using immunofluorescent microscopy and subcellular fractionation followed by immunoblot. Physiological readouts included viability, migration and reporter gene assays. Results: Here we show that ErbB3 can occupy a nuclear/perinuclear location in prostate cancer cells. The hormone sensitive PCa line LNCaP expressed higher levels of nuclear ErbB3 compared to castration resistant lines C4-2 and 22Rv1. The diuretic amiloride was previously shown to inhibit ErbB3 internalization by macropinocytosis thus we investigated whether it could also modulate ErbB3 nuclear localization in PCa cells. In LNCaP cells, amiloride dose dependently prevented ErbB3 nuclear transport, whereas in C4-2 and 22Rv1, no such effect was observed. Further analysis revealed that amiloride promoted ErbB3 expression in the membrane, where ErbB3 underwent ligand-dependent phosphorylation, formed ErbB2/ErbB3 dimers, and in turn phosphorylated the downstream targets AKT and ERK1/2 (p42/p44MAPK). On the other hand, EGFR phosphorylation and dimerization were unaffected by amiloride. Our results suggest that ErbB3 is stored in the nuclear region in an inactive form, whereas upon stress induction, including androgen deprivation, ErbB3 is translocated to the plasma membrane, where it is activated by ligand binding, and causes downstream signaling. Conclusion: We previously showed that the EGFR/ErbB2 dual inhibitor lapatinib failed to affect PCa - we now show that this may be due to modulation of nuclear and cytoplasmic ErbB3 expression. Amiloride prevented translocation (and thus expression of) ErbB3 to the nucleus and retained this RTK in the membrane, resulting in the formation of active ErbB2/ErbB3 dimers. Lapatinib was then able to inhibit and inactivate its specific target, ErbB2, thereby inducing significant apoptosis. The above indicates a novel role for nuclear ErbB3 in PCa that can be regulated by the combination of amiloride and lapatinib. Citation Format: Maitreyee K. Jathal, Maria M. Mudryj, Paramita M. Ghosh. Role of Nuclear ErbB3 localization in regulating ligand-dependent ErbB2-ErbB3 heterodimer activation and downstream targets in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6264.

Abstract 4656: Novel mitochondria-based targeting overcomes resistance in prostate cancer

Abstract Background: Prostate cancer (PC) is a major contributor to cancer-related mortality in men, with metastatic castration-resistant PC (mCRPC) presenting a significant obstacle due to its resistance to standard treatments. Current therapies, including nonsteroidal antiandrogens and chemotherapy drugs, provide limited effectiveness and are often accompanied by severe side effects. Recently, statins have gained attention as antitumor agents, demonstrating chemopreventive effects on a range of human cancers while maintaining a well-established safety profile. However, the use of statins as standalone treatment is limited by insufficient cancer cell targeting. Methods: We synthesized a DZ-simvastatin (DZ-SIM) conjugate that combines a near-infrared heptamethine carbocyanine dye (DZ) with simvastatin for selective tumor cell targeting. Cancer cell survival was measured by crystal violet and MTT assay. Co-localization of DZ-SIM with mitochondria and lysosome in cells was detected under confocal microscope using Mitotracker and Lysotracker. Cellular and mitochondrial cholesterol levels were detected by LC-MS. Male nude mice were inoculated subcutaneously with 22Rv1-Luc cells as a prostate cancer xenograft mouse model. We used Swiss-PDB and Rosetta algorithms to model the structure of the OATP1B3 channel. Oxygen consumption rate and extracellular acidification rate were measured by Seahorse assay. After treatment with DZ-SIM, differentially expressed genes profiling of prostate cancer cells was performed using RNAseq. Results: Our research demonstrates the conjugate's ability to effectively kill androgen-independent and metastatic PC cells without harming normal cells. Furthermore, DZ-SIM has been shown to kill multiple human PC cell lines, regardless of androgen receptor status or therapeutic resistance. DZ-SIM has been shown to inhibit growth in human prostate cancer cell line xenograft models. NIR fluorescence imaging showed specific localization of DZ-SIM to cancer, but not to normal cells and tissues. It could localize into cancer cells by binding the transmembrane transporter OATP to primarily target subcellular organelle mitochondria, decreasing the OCR and ECAR of cancer cells. DZ-SIM can bind to multiple cytosolic and mitochondrial proteins as detected by NIRF imaging. Cholesterol levels also decrease in whole cells and mitochondria. Ingenuity Pathway Analysis (IPA) of RNAseq showed that the four signaling pathways in the DZ-SIM treatment group are significantly enriched in genes related to cholesterol synthesis. Conclusion: DZ-SIM specifically targets PC cells and their mitochondria, resulting in loss of mitochondrial function and consequent cell death. With a unique subcellular targeting strategy, the conjugate holds the potential to outperform existing chemotherapeutic drugs. Citation Format: Yan Ou, Adrian Lim, Ning Zhai, Mouad Edderkaoui, Stephen Pandol, Ruoxiang Wang, Yi Zhang. Novel mitochondria-based targeting overcomes resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4656.

Abstract 5581: The DACH1 gene, frequently co-deleted with BRCA2 in prostate cancer, governs PARP inhibitor resistance

Abstract Background: Prostate cancer (PCa), the second leading cause of death in American men, includes distinct genetic subtypes with distinct therapeutic vulnerabilities (1,2). The DACH1 gene, in the 13q21.31-q21.33 region, encodes a winged helix/Forkhead DNA-binding protein that governs cell-fate determination that is deleted in ~ 18% of human PCa, where it is associated with poor prognosis (3). Prostate-specific deletion of the Dach1 gene enhanced prostatic intraepithelial neoplasia (PIN). Methods: Analysis of DACH1 gene deletion and expression was conducted from public data bases and human prostate cancer samples. Cells derived from Dach1 gene deletion mice and human prostate cancer cell lines with DACH1 overexpression were analyzed for PARP inhibitor responses and mechanisms of PARPi resistance. Transgenic prostate Oncomice were generated in which the Dach1 gene was deleted in the prostate. Results: Herein, DACH1 was shown to be co-deleted with BRCA2 in each of 8 separate cohorts (N=2,186 patients), in ~3-12% of patients. Dach1 gene deletion or knockdown conferred PARP inhibitor resistance (talazoparib &amp;gt; niraparib &amp;gt; olaparib &amp;gt; rucaparib &amp;gt; veliparib). DACH1 enhanced PARP sensitivity through several mechanisms affecting replication stress, PARP1 binding, inhibiting G9a (reducing H3K9me2), inhibiting CHK1p and CDK1p and inducing expression of snoRNA, specifically the snoRNA known to bind PARP. PARP1 has a BRCT (BRCA1 C-terminus 1) homology domain governing PARP1 dimerization and binding to intact DNA in a complex within a nucleosome. DACH1 binding to PARP1 requires the BRCT domain. Conclusions: There is a correlation between DACH1a-/- and PARPi resistance, correlating with PARPi trapping ability. As reduced Dach1a expression may define a subclass of PCa that warrants specific therapies, testing for DACH1a may be warranted.

Abstract 6348: Unveiling diverse trimeric forms of STEAP1 in human prostate cancer cells: Exploring STEAP1-STEAP2 interdependence for expression and plasma membrane localization

Abstract Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) is an emerging therapeutic target that is a member of the STEAP1-4 family of metalloreductases. We recently reported the preclinical characterization of xaluritamig (AMG 509), a STEAP1 XmAb® 2+1 T-cell engager molecule that is being developed for the treatment of prostate cancer (Nolan-Stevaux et al, 2023). Xaluritamig is designed to preferentially target tumor cells that express high levels of STEAP1. However, prostate tumor cells also express high levels of STEAP2. Here, we characterize the interactions between STEAP1 and STEAP2 in prostate cancer cell lines and the impact to xaluritamig activity. Bulk mRNA as well as single-cell gene expression data (scRNAseq) reveal a strong correlation between STEAP1 and STEAP2 expression in human prostate cancer, suggesting that the STEAP proteins may form both homotrimers and heterotrimers in prostate tumors. To test this idea, we investigated the structural behavior of STEAP1/2 trimers in human prostate cancer cell lines that endogenously express STEAP1 and STEAP2. Using xaluritamig and the parental anti-STEAP1 antibody, we show that STEAP1 primarily forms heterotrimers with STEAP2 in prostate cancer cells but can also assemble as homotrimers in the absence of STEAP2. Both STEAP1/2 heterotrimers and STEAP1 homotrimers can be targeted by xaluritamig, leading to T cell-dependent cellular cytotoxicity. Similarly, we found that anti-STEAP2 antibodies detected STEAP2 homotrimers when STEAP1 expression was ablated in cells, and that both STEAP1/2 heterotrimers and STEAP2 homotrimers were targeted by a STEAP2-specific T-cell engager molecule. We also demonstrate that neither STEAP1 nor STEAP2 significantly influences the mRNA or protein expression of the other. Our data elucidates the versatility of STEAP family members in forming both homotrimers and heterotrimers on the plasma membrane of human prostate cancer cells and the potential for therapeutic targeting with STEAP1/2-directed therapies. Characterizing the capability of existing STEAP1 or STEAP2 targeting therapeutics to bind their respective targets, whether within a homotrimer or heterotrimer, is crucial for a comprehensive understanding of the therapeutic potential of these drugs. Further research will help delineate the mechanism underlying the formation of STEAP1/2 trimers in cancer cells and continue advancing our understanding of this intriguing biological relationship. Citation Format: Cong Li, Lingming Liang, Joonhee Park, Olga Pryshchep, Binnaz Staley, Peng Li, Ben Alba, Xuan Liu, Fei Li, Gregory L. Moore, Matthew J. Bernett, Umesh Muchhal, John Desjarlais, Angela Coxon, Julie M. Bailis. Unveiling diverse trimeric forms of STEAP1 in human prostate cancer cells: Exploring STEAP1-STEAP2 interdependence for expression and plasma membrane localization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6348.

Abstract 7418: LongFuse: Detecting gene fusion transcripts from high throughput long-read single cell RNA sequencing data

Abstract Gene fusion resulted from genomic translocation plays important oncogenic roles including but not limited to gene activation and changing protein coding sequences. Historically, fusion gene analysis has been limited to bulk levels resulting in mixed oncogene activation models. Single cell Nanopore RNA sequencing (scNanoRNAseq) has the unique strength in detecting full-length chimeric transcripts in thousands of single cells in parallel. However, robust computational tools for detecting fusion genes in single cells from scNanoRNAseq data are missing. Here, we developed LongFuse, a fast and accurate tool, to detect fusion genes and their splicing isoforms in single cells from scNanoRNAseq data. LongFuse implements an XOR logic gate algorithm to speed up fusion candidate searching and stringent criteria to eliminate false discoveries due to trans-splicing events and randomly ligated chimeras. We applied LongFUSE onto scNanoRNAseq data of both human prostate cancer cell line, VCaP, and tumor tissues with known TMPRSS2-ERG fusion. Our results showed that both cancer cell line and prostate tumors harbored a mixture of fusion positive and fusion negative tumor cells. Gene expression analysis revealed transcriptional differences between fusion positive and negative cells including genes involved in cancer cell proliferation and invasion programs such as ELL2, ZBTB16, IGF1R, ANK3, MYPOP and PLPP1 genes. LongFUSE also calculates the splicing isoforms of fusion gene transcripts, allowing prediction of protein sequences of fusion gene transcripts. Taken together, our data demonstrated that LongFUSE is a robust computational tool for detecting gene fusions and their splicing isoforms at single cell level from scNanoRNAseq data. Citation Format: Cheng-Kai Shiau, Yueying He, Hsiao-Yun Lin, Lina Lu, Xiao-Dong Lu, Jindan Yu, Ruli Gao. LongFuse: Detecting gene fusion transcripts from high throughput long-read single cell RNA sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7418.

Abstract 1634: Cyclin D1 induces Akt1 activity in RB deficient prostate cancer

Abstract Background: Prostate cancer (PCa), the second leading cause of death in American men, is a genetically heterogeneous disease. The aberrant activation of AKT is one of the most frequent alterations in human cancers, and an elevated level of phosphorylated AKT at Ser473 site (pSer473) is associated with metastatic cancers (1). In prostate cancer (PCa), PI3K/AKT activation induces tumorigenesis (2), androgen receptor (AR)-mediated signaling, enzalutamide resistance and stemness, synergizing with other pathways including the notch intracellular domain (NICD). The cyclin D1 gene (CCND1), which is overexpressed in PCa, conveys canonical function by encoding the regulatory subunit of a holoenzyme that phosphorylates the retinoblastoma gene (RB) product, and non-canonical functions through phosphorylating other substrates and transcriptional activities. Cyclin D1 expression augments growth of some castrate-resistant PCa (CRPC) and clinical trials are targeting the cyclin D1 related CDK kinase activity in pRB+ PCa. Several Rb-independent mechanisms of action have been described (3). In recent studies the effect of CDK inhibitors was similar in patients with Rb+ vs. Rb− PCa (4). Whether Rb expression is required for the antiproliferative effects of CKD4/6 inhibition in PCa remains to be established. Methods: The kinetics of Akt1 induction by mitogens was assessed using the phosphorylation of the dichromic fluorescent (DCF) dye substrate LS456. We used cyclin D1 shRNA to show endogenous cyclin D1 augmented AKT1 signaling in response to mitogens. Results: Cyclin D1 reintroduction into cyclin D1−/− cells augmented Akt1 phosphorylation, requiring the cyclin D1 K112 residue. CDK inhibitors reduced Akt activity in RB+ and RB− human prostate cancer cell lines. Prostate-specific epithelial cell deletion of cyclin D1 (probasin-CRE-cyclin D1fl/fl) reduced prostate epithelial cell Akt1 activity assessed by immunohistochemical (IHC) staining. pAktSer473 was reduced in cell nuclei and the cytoplasm of both epithelial cells and stromal fibroblasts without a significant change in the abundance of Akt1. The downstream targets of Akt signaling (pTSC2Ser939, and pFKHRSer319) and ARSer213P were reduced in both epithelial cells and stromal fibroblasts. Genetic deletion of both epithelial cell and stromal cyclin D1 further reduced Akt1 activity, prostate epithelial cell proliferation, and markers of cancer stem cells. Proteomic analysis of cyclin D1-expressing stroma identified heterotypic chemokine signals that augment PCa AKT activity. Conclusions: Cyclin D1 induces Akt1 activity in both RB+ and RB− PCa cells which may contribute to tumor progression and therapy resistance. Reference: (1) Chen YL, Law PY, Loh HH. Curr Med Chem Anticancer Agents 2005;5:575-892. (2) Stoyanova T, et al. Proc Natl Acad Sci U S A 2013;110:20111-63. (3) Chen K, et al. Cell Rep 2020;32:1081514. (4) de Kouchkovsky I, et al. Clin Cancer Res 2022;28:1531-9 Citation Format: Xuanmao Jiao, Anthony W. Ashton, Zhiping Li, Ritika Harish, Danni Li, Gordon Robertson, Duanwen Shen, Xiaoming Ju, Ke Chen, Wei Zhang, Samuel Archilefu, Kenneth A. Iczkowski, Hallgeir Rui, Beatrice Knudsen, Paul Timpson, Max Nobis, Richard G. Pestell. Cyclin D1 induces Akt1 activity in RB deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1634.

Abstract 3202: Investigating the induction of ferroptosis by nano drug delivery system

Abstract Prostate cancer is the second leading cause of cancer-related death in U.S. men. Genomic and clinical associations have identified loss of RB function as a dominant mechanism driving prostate cancer lethality. We recently demonstrated that ferroptosis induction in vivo by ferroptosis inducers blocks RB1-deficient prostate tumor growth and metastasis and leads to improved survival of the mice. As all ferroptosis inducers are newly discovered, there is a great need to improve their therapeutic potential through different avenues, including innovative drug delivery systems. Poly Lactic-co-glycolic acid (PLGA) is an FDA-approved biodegradable polymer. Due to their strong biocompatibility and consistent release, PLGA-based nanotherapeutics are an attractive carrier for small molecule drugs. This study aims to further improve the efficacy of ferroptosis induction against lethal RB1-deficient prostate cancer through PLGA-based ferroptosis-induced nanoparticles (NPs). The PLGA NPs encapsulating JKE-1674 were synthesized using the solvent evaporation method. The NPs were characterized using dynamic light scattering (DLS) and scanning electron microscopy (SEM) to assess the physicochemical properties of the NPs. The cell-killing efficacy of free-form or PLGA NP encapsulated JKE-1674 was then evaluated in vitro. Our analyses showed that the particle size and polydispersity index (PDI) of JKE-1674 PLGA NPs were 160.8 nm and 0.049, respectively. SEM images of JKE-1674 PLGA NPs further validated the DLS data. These findings suggest that encapsulation of JKE-1674 within the PLGA NPs results in a stable and biocompatible formation. Critically, JKE-1674 encapsulated by PLGA NPs is as potent as JKE-1674 in its free form to induce ferroptosis and associated lipid peroxidation in various human prostate cancer cell lines. This study presents the first effort to develop and validate PLGA-based ferroptosis-induced nanotherapeutics. Based on our in vitro data, we will test the in vivo efficacy of JKE-1674 PLGA NPs against lethal RB1 deficient prostate cancer. Citation Format: Eden M. Jacob, Sarah Y. Kim, Parul Sirohi, Mu-En Wang, Meredith L. Davis, Alyssa R. Bawcom, Ashutosh Chilkoti, Mark R. Wiesner, Jiaoti Huang, Ming Chen. Investigating the induction of ferroptosis by nano drug delivery system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3202.

Abstract 470: AR-induced miRNA124-3p.2 suppresses the metastasis-promoting factor CCL2

Abstract Objective: The standard treatment for advanced prostate cancer is androgen deprivation therapy that blocks androgen receptor (AR) signaling, but this therapy becomes ineffective within a few years, resulting in castration-resistant prostate cancer. We have previously shown that blockade of AR signaling induces CCL2 and activates cancer cells through an autocrine mechanism, but why AR blockade induces CCL2 secretion has not been elucidated. Micro RNAs (miRs) are endogenous non-coding RNAs that regulate the translation of various proteins. In this study, we investigated whether miRs are involved in CCL2 induction. Subjects and Methods: We used miRNA PCR Plate Array and Target Scan (https://www.targetscan.org) to search for miRs that are controlled by AR and bind to and repress the CCL2 gene. We knocked down the AR of human prostate cancer cell lines LNCaP and C4-2B with siRNA (siAR) and examined the expression of target miRs and CCL2. siAR-transfected LNCaP and C4-2B were transfected with target miR mimic and CCL2 expression was examined. siAR-transfected LNCaP, C4-2B, and C4-2B were transfected with target miR mimic and CCL2 expression was examined. siAR-transfected LNCaP and C4-2B were transfected with target miR mimic and CCL2 expression was examined. The target miR mimic was introduced into them, and the changes in migration ability were evaluated. Results: miR-124-3p.2 was extracted as a candidate from miRNA PCR Plate Array and Target Scan. miR-124-3p.2 expression was upregulated in both LNCaP and C4-2B when DHT was added and downregulated when CCL2 was added. In siAR LNCaP and C4-2B, the expression of CCL2 was decreased when miR-124-3p.2 mimic was introduced. Furthermore, in siAR-transfected LNCaP, C4-2B, the introduction of miR-124-3p.2 mimic suppressed the migration. Conclusion: AR suppresses CCL2 via miR124-3p.2. miR124-3p.2-mediated regulation of CCL2 may be a new therapeutic target for advanced prostate cancer. Citation Format: Shuhei Aoyama, Kouji Izumi, Atsushi Mizokami. AR-induced miRNA124-3p.2 suppresses the metastasis-promoting factor CCL2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 470.

Abstract 2984: Prostate cancer associated glycosylated RNAs: Who are you and where are you hiding

Abstract Glycosylated RNA (glycoRNA) is an emerging field. Carbohydrate modifications are vital for biomolecule (proteins and lipids) stability, transport, and localization. Glycosylation influences cell functions like differentiation, apoptosis, and immune response, potentially impacting human disease. The Bertozzi lab initially discovered that RNAs can carry sugar modifications when they metabolically labeled mammalian cultured cells using bioorthogonal chemistry methods normally employed for glycosylated protein and lipid enrichment. Our lab wanted to investigate the translational potential of glycoRNAs and determine if these modifications could be developed as novel clinical targets for aggressive prostate cancer. (PCa). As advanced forms of PCa often lead to poor prognoses, it is imperative that better screening biomarkers and therapeutic treatment options are developed to increase patient survivorship. We predicted that glycoRNA expression would correlate with malignancy, metastatic status, and hormone sensitivity in prostate cells. Our lab used the sialic acid metabolic precursor Ac4ManNAz and click chemistry to label a panel of human prostate cancer cell lines representing the spectrum of prostate disease followed by rigorous RNA isolation procedures and northern blot analysis. We found that human prostate cell lines with no (RWPE-1) or low malignancy (LNCaP) expressed higher levels of glycoRNA belonging to the small &amp;lt;200 nt (but not large) noncoding RNA class compared to aggressive, metastatic PCa cell lines. Our glycosidase studies and lectin northern blot analysis indicated that RNA carries both N-linked and O-linked modifications. Based on these findings, we hypothesized that glycosylated RNA plays an important role in maintaining prostate homeostasis and in cancer protection. To better profile these glycoRNAs, we aimed to: 1) Test a wider range of metabolic labeling regents to characterize the glycoRNA populations using a panel of human prostate cell lines; 2) Perform cellular fractionation studies to determine localization of glycoRNA in prostate cells; and 3) Explore glycoRNAs in a genetic animal model. These studies focused on LNCaP PCa cell lines, which express high levels of glycoRNA. We found that small glycoRNA expression was noted when LNCaP cells were metabolically labeled with Ac4GalNAz (predominantly O-linked) and Ac4ManNAz (predominantly N-linked), verifying glycoRNA heterogeneity. Cellular fractionation studies indicated glycoRNA localization was enriched in the membrane fraction of LNCaP cells using these metabolic labels. Furthermore, we verified that C. elegans grown on Ac4GalNAz plates expressed small glycoRNA, indicating biological conservation. We are now characterizing these glycoRNAs using RNA sequencing and LC-MS/MS glycomics. Our findings will provide novel insights into cancer-associated glycoRNA, leading to improved clinical tools for PCa. Citation Format: Samantha McGuire, Esther Jones, Spencer Moen, Aurora Esquela-Kerscher. Prostate cancer associated glycosylated RNAs: Who are you and where are you hiding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2984.

Abstract 4493: Protein structure inspired drug discovery

Abstract Introduction: Drug action is mediated by a small molecule therapeutic binding to a specific structural motif on a protein. As structure determines function, it follows that unique structural motifs are enriched for sites of unique function and high therapeutic target value. We hypothesized that protein structure constitutes high value primary information that can drive the discovery of novel therapeutic agents. Method: We developed a unique physics-based protein structure analysis platform scaled to run on a supercomputer, allowing us to consider dynamic protein flexibility and the influence of solvent upon protein structure, and to probe large protein libraries as a primary source of structure information that can support up-front structure-based screens. We developed and then probed a high resolution protein x-ray crystallographic library, created and curated by us. From a 60 million compound library, we conducted virtual screens for docking to identified sites. 8-day colony formation assays were conducted on a panel of 8 human breast, prostate, lung and colon cancer cell lines, as well as 8- and 14-day trilineage hematopoietic colony formation assays on human cord blood stem cells. Findings: From the first 180 deposited protein structures, we screened for unique protein surface structure. Structures were deemed unique if they were not otherwise known to be associated with any particular functional roles and were not the binding site of known therapeutics. Further selection included those that were clefts and possessed physical characteristics compatible with binding small chemicals whose properties have been linked to effective therapeutics. For each of 8 sites on 6 different proteins, a suite of analytics probed docking solutions from the compound library, generating a ranked list of 100 compounds for each site. Based on factors inclusive of availability and low potential for toxicity, compounds were acquired and used in in-parallel selection and de-selection functional assays. Cancer cell growth inhibition was a positive selection criterion, with additional ranking based on greater potency and broader efficacy. Bone marrow toxicity is limiting for most drugs, and inhibition of bone marrow stem cell growth was as a deselection criterion. Multiple compounds clustered around 2 sites on 2 different proteins. One compound, Dxr2-017, exhibited very high relative efficacy against colon and breast cancer cells. Evaluation of Dxr2-017 in the NCI-60 panel corroborated these findings, and showed even higher efficacy against melanoma. We demonstrated that Dxr2-017 inhibited melanoma growth with low nanomolar efficacy, while concentrations of over 2300-fold higher had only limited inhibitory effects on bone marrow stem cells. Conclusions: This study provides proof-of-principle that protein structure constitutes high value primary information that can support identification of novel therapeutics. This approach is widely applicable and expandable. Citation Format: Fangfang Qiao, T. Andrew Binkowski, Wayne Anderson, Weining Chen, Gray Schiltz, Karl Scheidt, Amarnath Natarajan, Raymond Bergan. Protein structure inspired drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4493.

Abstract 5539: PRDM16 is an intrinsic regulator of dormancy in bone disseminated prostate cancer cells

Abstract Metastatic prostate cancer (PCa) frequently occurs in the bone and can remain inactive for extended periods after primary tumor treatment. Understanding the intricate molecular mechanisms that govern this phenomenon could open the door to novel therapies, potentially reducing mortality rates. To address this, we developed a novel protocol to induce cellular dormancy in PCa cell lines (RM1, 22Rv1, LAPC4) in vitro. We noted that dormant cells exhibited distinctive clustering and could be reactivated when exposed to serum even after 21 days of stasis. The dormancy program was validated by multiple assays including cell cycle analysis, membrane dye retention, and Edu+ incorporation. RNASeq analysis showed significant transcriptional changes during dormancy induction, with 8 genes commonly upregulated across multiple human and mouse PCa cell lines, of which the transcription factor, PRDM16. We focused on PRDM16 since, 1) it is a known regulator of hematopoietic stem cell quiescence, 2) our emerging data points to bone morphogenetic protein-7 (BMP7), a well-known exogenous mediator of dormancy as an inducer of PRDM16 expression and 3) clinically, PRDM16 is suppressed in primary PCa and active metastases. First, we validated the upregulation of PRDM16 during dormancy in vitro. Silencing PRDM16 led to impaired dormancy phenotype and cell death, while its overexpression significantly mitigated proliferation rates and promoted survival. In vivo, we employed an intra-iliac immunocompetent model (RM1 &amp; C57BL6J) and demonstrated that overexpressing PRDM16 in active RM1 cells resulted in significant reduction of metastasis formation and Ki67 expression concurrent with extended survival when injected in vivo compared to control cells. Prostate cancer cells with PRDM16 over-expression were detected in bone sections as solitary or small clusters (&amp;lt;20 cells) demonstrating an inverse relationship between PRDM16 expression and metastatic outbreak. In defining the targets via which PRDM16 controls dormancy entry, we focused on RAR related orphan receptor C (RORC) based on bioinformatic analysis and publicly available PRDM16 ChIP-Seq. Our data demonstrate that pharmacological inhibition of RORC using GSK805 promoted the apoptosis of dormant PCa cells specifically when compared to their actively growing counterparts. In conclusion, our studies have revealed the critical role of PRDM16 in promoting PCa cell dormancy, in part via upregulating RORC. Translationally, clinically approved RORC inhibitors for non-cancerous conditions (psoriasis) have been developed. We expect, based on our results, that these inhibitors could be repurposed for the eradication of dormant PCa cells in patients with advanced disease. Citation Format: Mostafa M. Nasr, Tao Li, Ryan T. Bishop, Jeremy S. Frieling, Conor C. Lynch. PRDM16 is an intrinsic regulator of dormancy in bone disseminated prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5539.