Progression Of Prostate Cancer Cells Research Articles

DSCAM-AS1 promotes the development of prostate cancer

PurposeThe purpose of this study was to investigate the role of lncRNA DSCAM-AS1 in prostate cancer to find new therapeutic targets and promote the research progress of prostate cancer.MethodsRT-qPCR was used to detect DSCAM-AS1 expression in prostate cancer tissues, normal tissues, human normal prostate epithelial cells (RWPE), and four prostate cancer cell lines. The clinical and prognostic role of DSCAM-AS1 was evaluated by the Kaplan–Meier curve and chi-square test. Secondly, a dual luciferase reporter gene assay was used to study the regulatory mechanism between miR-338-3p and DSCAM-AS1. Finally, the roles of DSCAM-AS1 and miR-338-3p in prostate cancer cell proliferation and metastasis were explored by CCK-8 and Transwell assays.ResultsIt was found that DSCAM-AS1 upregulation could serve as a warning of deterioration and poor prognosis in prostate cancer patients, and that knockdown of DSCAM-AS1 expression inhibited the progression of prostate cancer cells. In addition, miR-338-3p, a target of DSCAM-AS1, was found to be down-regulated in prostate cancer cells and miR-338-3p knockdown could reverse the inhibitory effect of DSCAM-AS1 silencing on prostate cancer.ConclusionDSCAM-AS1 is up-regulated in prostate cancer and regulates the progression of prostate cancer cells by targeting miR-338-3p.

Abstract 3238: Therapeutic targeting of the genome guardian sirtuins in metastatic prostate cancer

Abstract Introduction: The effective treatment paradigm for the lethal form of metastatic prostate cancer (mPCa) remains challenging. Almost all mPCas eventually develops therapy resistance against androgen receptor (AR) inhibitors (e.g., Enzalutamide, Abiraterone) or to poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors (Olaparib, Niraparib, Talazoparib) (HRRd) when used as monotherapy. Although the patients in the ARSi+PARPi combination produced significantly longer radiologic progression-free survival (rPFS), a substantial number had attained therapy resistance by the end of the study tenure. So, a newer therapy is urgently needed. In recent years, a new targetable therapeutics development domain has been unfolding with an expanding understanding of the role of SIRTuins in prostate cancer cell survival, invasion, and progression to an aggressive metastatic state. Here, we will investigate the therapeutic efficacy of SIRT1/7 inhibitor (SIRT1/7i) and determine synergy with DNA-damaging PARP inhibitor combinations through a battery of cell and molecular assays using various prostate cancer cellular models. Methods: We have treated the mPCa cell lines 22Rv1, LAPC4, PC3 and LnCAP as well as prostate epithelial cell line RWPE1 with SIRT1 inhibitor SIRT1-IN-2 (MedChemExpress, HY-146689), SIRT7 inhibitor 97491 (MedChemExpress, HY-135899) and AR inhibitor Enzalutamide individually as well as in combination of PARP inhibitor Olaparib. The treatment of the SIRT1 and SIRT7 monotherapy arm was 72 hours long, and combination therapy continued for 7 days. The cell viability was assessed with CellTiter-Glo® 2.0 Cell Viability Assay (Promega). The cell cycle progression and proliferation analysis were done using flow cytometry through Propidium iodide staining and AnnexinV-FITC apoptosis detection chemistry. Cell survival was orthogonally assessed using colony counting assays. We adopt RAD51 foci counting assays and COMET assays for evaluating treatment-induced genome instability. Results: The preliminary result suggested that the SIRT1i+Olaparib and SIRT7i+Olaparib combination significantly sensitized the mPCa cell lines 22Rv1, PC3, and LnCAP for cell death compared to the prostate epithelial cell RWPE1. Upon the combination treatment, the mPCa cell lines showed elevated apoptotic cell death, reconfirmed through colony counting assay. Conclusion: Therapeutic combination treatment of SIRT1 and SIRT7 inhibition with Olaparib could be an effective treatment option for aggressive metastatic prostate cancer. Citation Format: Subhajit Giri. Therapeutic targeting of the genome guardian sirtuins in metastatic 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 3238.

N6-methyladenosine-modified circular RNA family with sequence similarity 126, member A affects cholesterol synthesis and malignant progression of prostate cancer cells by targeting microRNA-505-3p to mediate calnexin.

Prostate cancer (PCa) is the most commonly diagnosed malignancy in men. In tumor biology, n6-methyladenosine (m6A) can mediate the production of circular RNAs (circRNAs). This study focused on the mechanism of m6A-modified circRNA family with sequence similarity 126, member A (FAM126A) in PCa. Cell counting kit-8 assay, colony formation assay, 5-ethynyl-2'-deoxyuridine assay, transwell assay, and xenograft mouse models were applied to study the role of circFAM126A in PCa cell growth and tumor metastasis, and cellular triglyceride and cholesterol levels were measured to assess cholesterol synthesis. RNA immunoprecipitation, RNA pull-down, luciferase reporter gene assay, and western blot were adopted to explore the underlying molecular mechanism. Data showed that circFAM126A was upregulated in PCa and promoted PCa progression in vitro. m6A modification of circFAM126A enhanced transcriptional stability. CircFAM126A targeted microRNA (miR)-505-3p to mediate calnexin (CANX). Up-regulating miR-505-3p or inhibiting CANX suppressed cholesterol synthesis and malignant progression in PCa cells. Overexpressing CANX suppressed the inhibitory effect of circFAM126A silencing or miR-505-3p upregulation on PCa cells. Our current findings provide a new therapeutic strategy for the treatment of PCa.

The role of polyethylenimine-functionalized gold nanoclusters carrying plasmid CMTM5 in impeding the malignant progression of prostate cancer cells by promoting EGFR endocytosis.

In this research, polyethylenimine-functionalized gold nanoclusters (PEI-AuNCs) were synthesized for the delivery of plasmid CMTM5 (pCMTM5) to prostate cancer (PCa) cells, with the objective of elucidating the mechanism underlying its anticancer efficacy. The PEI-AuNCs loaded with pCMTM5 (PEI-AuNCs@pCMTM5) tumor-targeting drug delivery system was established. Subsequently, both the obtained PEI-AuNCs and PEI-AuNCs@pCMTM5 underwent characterization through a transmission electron microscope (TEM) and dynamic light scattering (DLS). Employing RT-qPCR, western blot, flow cytometry, immunofluorescence, and co-immunoprecipitation (co-IP) assays, the consequences of CMTM5 overexpression on the expression of EGFR were investigated. Moreover, the influence of PEI-AuNCs@pCMTM5 on PC-3 cells was assessed through CCK-8, wound healing assay, and Transwell experiments. As a result, the PEI-AuNCs and PEI-AuNCs@pCMTM5 were presented as uniformly dispersed spherical with stable particle sizes and positive charges, showcasing favorable dispersion within the solution. In comparison to Lip2000, the PEI-AuNCs demonstrated superior transfection efficiency and lower cellular toxicity. Following the overexpression of CMTM5, the proliferative capacity of PC-3 cells was markedly suppressed, while both migratory and invasive abilities exhibited noteworthy reduction, with the efficacy of PEI-AuNCs@pCMTM5 consistently outperforming that of free pCMTM5. Subsequent mechanistic investigations unveiled that CMTM5 does not directly inhibit the synthesis of EGFR or facilitate its degradation, but rather influences the endocytic process of EGFR. In conclusion, the PEI-AuNCs nano-delivery system exhibits good biocompatibility and efficaciously conveys pCMTM5 to PCa cells. Crucially, pCMTM5 does not directly interact with EGFR, and CMTM5 governs the malignant progression of PC3 cells by promoting EGFR endocytosis.

FABP5 can substitute for androgen receptor in malignant progression of prostate cancer cells.

Fatty acid‑binding protein 5 (FABP5) and androgen receptor (AR) are critical promoters of prostate cancer. In the present study, the effects of knocking out the FABP5 or AR genes on malignant characteristics of prostate cancer cells were investigated, and changes in the expression of certain key proteins in the FABP5 (or AR)‑peroxisome proliferator activated receptor‑γ (PPARγ)‑vascular endothelial growth factor (VEGF) signaling pathway were monitored. The results obtained showed that FABP5‑ or AR‑knockout (KO) led to a marked suppression of the malignant characteristics of the cells, in part, through disrupting this signaling pathway. Moreover, FABP5 and AR are able to interact with each other to regulate this pathway, with FABP5 controlling the dominant AR splicing variant 7 (ARV7), and AR, in return, regulates the expression of FABP5. Comparisons of the RNA profiles revealed the existence of numerous differentially expressed genes (DEGs) comparing between the parental and the FABP5‑ or AR‑KO cells. The six most abundant changes in DEGs were found to be attributable to the transition from androgen‑responsive to androgen‑unresponsive, castration‑resistant prostate cancer (CRPC) cells. These findings have provided novel insights into the complex molecular pathogenesis of CRPC cells, and have demonstrated that interactions between FABP5 and AR contribute to the transition of prostate cancer cells to an androgen‑independent state. Moreover, gene enrichment analysis revealed that the most highly enriched biological processes associated with the DEGs included those responsive to fatty acids, cholesterol and sterol biosynthesis, as well as to lipid and fatty acid transportation. Since these pathways regulated by FABP5 or AR may be crucial in terms of transducing signals for cancer cell progression, targeting FABP5, AR and their associated pathways, rather than AR alone, may provide a new avenue for the development of therapeutic strategies geared towards suppressing the malignant progression to CRPC cells.

Extracellular Mechanical Stimuli Alters the Metastatic Progression of Prostate Cancer Cells within 3D Tissue Matrix.

This study aimed to understand extracellular mechanical stimuli's effect on prostate cancer cells' metastatic progression within a three-dimensional (3D) bone-like microenvironment. In this study, a mechanical loading platform, EQUicycler, has been employed to create physiologically relevant static and cyclic mechanical stimuli to a prostate cancer cell (PC-3)-embedded 3D tissue matrix. Three mechanical stimuli conditions were applied: control (no loading), cyclic (1% strain at 1 Hz), and static mechanical stimuli (1% strain). The changes in prostate cancer cells' cytoskeletal reorganization, polarity (elongation index), proliferation, expression level of N-Cadherin (metastasis-associated gene), and migratory potential within the 3D collagen structures were assessed upon mechanical stimuli. The results have shown that static mechanical stimuli increased the metastasis progression factors, including cell elongation (p < 0.001), cellular F-actin accumulation (p < 0.001), actin polymerization (p < 0.001), N-Cadherin gene expression, and invasion capacity of PC-3 cells within a bone-like microenvironment compared to its cyclic and control loading counterparts. This study established a novel system for studying metastatic cancer cells within bone and enables the creation of biomimetic in vitro models for cancer research and mechanobiology.

Dauricine regulates prostate cancer progression by inhibiting PI3K/AKT-dependent M2 polarization of macrophages

M2 type tumor-associated macrophages, an essential component of the tumor microenvironment (TME), have been proved to contribute to tumor metastasis. Dauricine (Dau) has recently received widespread attention due to its multiple targets and low price. However, the effect of Dau on macrophage polarization of TME remains unclear. In this study, we investigated the effect of Dau on prostate cancer (PCa) metastasis and specifically its correlation to macrophage polarization. Our results showed that Dau efficiently suppressed M2 polarization of macrophages induced by interleukin (IL) −4 and IL-13. Mechanistically, Dau inhibited the activity of PI3K/AKT signaling pathway, which subsequently suppressed macrophage differentiation to M2 type. Importantly, our study indicated that Dau decreased the release of chitinase 3-like protein 1 (CHI3L1) from M2 macrophages, which ultimately inhibited the M2 macrophage-mediated progression of PCa cells in vitro and in vivo. Taken together, our data demonstrated that Dau suppressed M2 polarization of macrophages via downregulation of the PI3K/AKT signaling pathway, in turn, preventing proliferation, epithelial–mesenchymal transition, migration, and invasion of PCa cells. Thus, this study reveals a previously unrecognized function of Dau in inhibition of PCa progression via intervention in M2 polarization of macrophages.

Comprehensively analysis of splicing factors to construct prognosis prediction classifier in prostate cancer.

Splicing factors (SFs) are proteins that control the alternative splicing (AS) of RNAs, which have been recognized as new cancer hallmarks. Their dysregulation has been found to be involved in many biological processes of cancer, such as carcinogenesis, proliferation, metastasis and senescence. Dysregulation of SFs has been demonstrated to contribute to the progression of prostate cancer (PCa). However, a comprehensive analysis of the prognosis value of SFs in PCa is limited. In this work, we systematically analysed 393 SFs to deeply characterize the expression patterns, clinical relevance and biological functions of SFs in PCa. We identified 53 survival-related SFs that can stratify PCa into two de nove molecular subtypes with distinct mRNA expression and AS-event expression patterns and displayed significant differences in pathway activity and clinical outcomes. An SF-based classifier was established using LASSO-COX regression with six key SFs (BCAS1, LSM3, DHX16, NOVA2, RBM47 and SNRPN), which showed promising prognosis-prediction performance with a receiver operating characteristic (ROC) >0.700 in both the training and testing datasets, as well as in three external PCa cohorts (DKFZ, GSE70769 and GSE21035). CRISPR/CAS9 screening data and cell-level functional analysis suggested that LSM3 and DHX16 are essential factors for the proliferation and cell cycle progression in PCa cells. This study proposes that SFs and AS events are potential multidimensional biomarkers for the diagnosis, prognosis and treatment of PCa.

Characterization of the TRPV6 calcium channel-specific phenotype by RNA-seq in castration-resistant human prostate cancer cells.

Background: Transient receptor potential vanilloid subfamily member 6 (TRPV6), a highly calcium-selective channel, has been shown to play a significant role in calcium homeostasis and to participate both in vitro and in vivo in growth, cell survival, and drug resistance of prostate cancer. Its role and the corresponding calcium-dependent pathways were mainly studied in hormone-dependent human prostate cancer cell lines, often used as a model of early-stage prostate cancers. The goal of the present study was to describe the TRPV6-specific phenotype and signaling pathways it is involved in, using castration-resistant prostate cancer cell lines. Methods: RNA sequencing (RNA-seq) was used to study the gene expression impacted by TRPV6 using PC3Mtrpv6-/- versus PC3Mtrpv6+/+ and its derivative PC3M-luc-C6trpv6+/+ cell line in its native and TRPV6 overexpressed form. In addition to the whole-cell RNA sequencing, immunoblotting, quantitative PCR, and calcium imaging were used to validate trpv6 gene status and functional consequences, in both trpv6 -/- and TRPV6 overexpression cell lines. Results: trpv6 -/- status was validated using both immunoblotting and quantitative PCR, and the functional consequences of either trpv6 gene deletion or TRPV6 overexpression were shown using calcium imaging. RNA-seq analysis demonstrated that the calcium channel TRPV6, being a crucial player of calcium signaling, significantly impacts the expression of genes involved in cancer progression, such as cell cycle regulation, chemotaxis, migration, invasion, apoptosis, ferroptosis as well as drug resistance, and extracellular matrix (ECM) re-organization. Conclusion: Our data suggest that the trpv6 gene is involved in and regulates multiple pathways related to tumor progression and drug resistance in castration-resistant prostate cancer cells.

Crosstalk between Microtubule Stabilizing Agents and Prostate Cancer.

A variety of microtubule-stabilizing cytotoxic agents (MSA) with diverse chemical scaffolds have been discovered from marine sponges, microorganisms, and plants. Two MSAs, docetaxel and cabazitaxel, are the exclusive chemotherapeutics that convey a survival benefit in patients with castration-resistant prostate cancer (CRPC). Additional MSAs have been investigated for their potential in treating prostate cancer in both clinical and preclinical settings. Independent of promoting mitotic arrest, MSAs can suppress the nuclear accumulation of androgen receptor (AR), which is the driving force for prostate cancer cell growth and progression. The alternative mechanism not only helps to better understand the clinical efficacy of docetaxel and cabazitaxel for AR-driven CRPC but also provides an avenue to seek better treatments for various forms of prostate cancer. The dual mechanisms of action enable MSAs to suppress AR-null prostate cancer cell proliferation by cell mitosis pathway and to interfere with the AR signaling pathway in AR positive cells. MSA chemotherapeutics, being administered alone or in combination with other therapeutics, may serve as the optimal therapeutic option for patients with either castration-sensitive or castration-resistant prostate cancer. This review provides an overview of the anti-prostate cancer profiles (including preclinical and clinical studies, and clinical use) of diverse MSAs, as well as the mechanism of action.

Protein Levels of Anti-Apoptotic Mcl-1 and the Deubiquitinase USP9x Are Cooperatively Upregulated during Prostate Cancer Progression and Limit Response of Prostate Cancer Cells to Radiotherapy.

Radiotherapy constitutes an important therapeutic option for prostate cancer. However, prostate cancer cells often acquire resistance during cancer progression, limiting the cytotoxic effects of radiotherapy. Among factors regulating sensitivity to radiotherapy are members of the Bcl-2 protein family, known to regulate apoptosis at the mitochondrial level. Here, we analyzed the role of anti-apoptotic Mcl-1 and USP9x, a deubiquitinase stabilizing Mcl-1 protein levels, in prostate cancer progression and response to radiotherapy. Changes in Mcl-1 and USP9x levels during prostate cancer progression were determined by immunohistochemistry. Neutralization of Mcl-1 and USP9x was achieved by siRNA-mediated knockdown. We analyzed Mcl-1 stability after translational inhibition by cycloheximide. Cell death was determined by flow cytometry using an exclusion assay of mitochondrial membrane potential-sensitive dye. Changes in the clonogenic potential were examined by colony formation assay. Protein levels of Mcl-1 and USP9x increased during prostate cancer progression, and high protein levels correlated with advanced prostate cancer stages. The stability of Mcl-1 reflected Mcl-1 protein levels in LNCaP and PC3 prostate cancer cells. Moreover, radiotherapy itself affected Mcl-1 protein turnover in prostate cancer cells. Particularly in LNCaP cells, the knockdown of USP9x expression reduced Mcl-1 protein levels and increased sensitivity to radiotherapy. Posttranslational regulation of protein stability was often responsible for high protein levels of Mcl-1. Moreover, we demonstrated that deubiquitinase USP9x as a factor regulating Mcl-1 levels in prostate cancer cells, thus limiting cytotoxic response to radiotherapy.

Abstract LB157: Cereblon inhibits prostate cancer progression and metastasis by negatively regulating 6PGD

Abstract As the third enzyme of the pentose phosphate pathway (PPP), abnormally elevated levels of 6-phosphogluconate dehydrogenase (6PGD) have been documented in various human cancers. We demonstrate that reduced cereblon (CRBN) protein expression is the underlying mechanism of elevated 6PGD expression in metastatic prostate cancer cells. We establish 6PGD as a new endogenous substrate for CRBN by demonstrating that it interacts directly with CRBN and is ubiquitinated by CRL4CRBN. In addition, CRBN negatively regulates prostate cancer cell progression and metastasis, as abnormally high 6PGD, in the absence of sufficient CRBN, enhances the metastatic potential of prostate cancer in vitro and in vivo. Our findings show convincingly that carbohydrate metabolism regulated by 6PGD is linked to prostate cancer metastasis via CRBN. Based on these data, we propose that the 6PGD-CRBN axis may be a suitable target for further research into new therapeutics for mitigating prostate cancer metastasis. Citation Format: Koushik Guchhait, Hyeon Seung Yoon, Seungheon Shin, Hyun-Su An, Hye Seung Nam, Francisco D. Yanqui-Rivera, Samara M. Oña, Miguel Á. Mendez, Seokjae Park, Eun-Kyoung Kim, Jong Yeon Hwang, Jee-Young Han, Doo Yong Chung, Daeho Park, Su-Geun Yang, Chul-Seung Park, Steve K. Cho. Cereblon inhibits prostate cancer progression and metastasis by negatively regulating 6PGD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB157.

Abstract 2881: Investigating cholesterol transfer between macrophages and prostate cancer cells

Abstract Background: Androgens stimulate androgen receptor (AR) signaling, driving prostate cancer (PC) cell proliferation and progression. Androgen deprivation therapy (ADT) effectively treats primary disease, but 20-30% of patients relapse after ~5 years, resulting in castration-resistant prostate cancer (CRPC), which has limited treatment options. We have previously shown in a murine model of CRPC that macrophages promote AR signaling in CRPC and that depleting tumor macrophages extends survival in response to ADT with Lupron. This correlates with reduced androgen levels in macrophage-depleted tumors and the ability of macrophages to transfer cholesterol, the anabolic precursor of androgens, to prostate cancer cells. However, the mechanism by which this transfer occurs, and whether cholesterol exchange mediates resistance to ADT, is currently unknown. Methods: To assess cholesterol transfer from macrophages to cancer cells, we established a culture system involving prostate cancer cell lines and either the RAW264.7 macrophage cell line or bone marrow-derived macrophages (BMDMs). Macrophages were pre-loaded with cholesterol using fluorescently labeled low-density lipoprotein (LDL) and were co-cultured or placed in a transwell system with tumor cells for 24 hours. Cholesterol transfer was then measured by flow cytometry. Results: Fluorescent signal from LDL was detected in 50-70% of tumor cells after 24 hours of co-culture. Cell contact was required for cholesterol transfer, as no fluorescence was detected in prostate cancer cells during transwell experiments. Macrophages were also able to internalize and transfer acetylated LDL, which is poorly taken up by tumor cells compared to unmodified LDL. Macrophage polarization with interleukin-4 did not impact cholesterol transfer and uptake of acetylated LDL occurred independently of the scavenger receptor CD36. The scavenger receptor SR-B1, which is often upregulated in prostate cancer cells, was dispensable for transfer, as knocking out SR-B1 via CRISPR/Cas9 in cancer cell lines did not reduce LDL uptake. Conclusion: Cholesterol transfer between macrophages and prostate cancer cells occurs in a contact-dependent manner, independently of the scavenger receptors most commonly associated with cholesterol accumulation. Citation Format: Olabisi Osunmakinde, Brian Ruffell. Investigating cholesterol transfer between macrophages and prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2881.

Abstract 3609: Interaction of interleukin-6/8/18 with prostate cancer cell secreted growth factors are crucial for osteoblast proliferation and differentiation

Abstract There is currently no effective therapy for bone metastatic prostate cancer, which is the main cause of mortality in people with prostate cancer. Poor prognosis of bone metastasis has limitations in current therapeutics mainly due to the complexity of microenvironment. Our focus was on the interactions between prostate cancer cells and the bone microenvironment, including osteoclasts and osteoblasts in regard to growth factors and cytokines, as the bone microenvironment plays a significant role in this process. The present study shows the downstream effects of Stavudine (KB-S); a known antiviral compound along with 5-fluoro-1-((1R,4R)-4-((tetrahydro-2H-pyran-2-yl)oxy)cyclopent-2-en-1-yl)pyrimidine-2,4(1H,3H)-dione (KB-G) and 1-((1R,4R)-4-hydroxycyclopent-2-en-1-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (KB-H) on bone metastatic prostate cancer. Effects of KB-G/H and S on HEK-293 control cells were determined as moderate under tested conditions. A series of KB-G/H and S (10, 25, 50, 75 and 100 µM) was given for a 4-day period (with 24 h intervals) and the prostate cancer cell population was determined at the end of the 4-day treatment period. Preliminary results indicated that all 3 compounds significantly decreased DU-145 and PC-3 cell population compared to the effects on HEK-293 cells. Results suggested that all 3 compounds can decrease the prostate cancer cell progression by enhancing apoptosis. Our data also suggested that all 3 compounds downregulated interleukin (IL)-18, IL-8, IL-6 and C-X-C motif ligand-1(CXCL-1) in prostate cancer cells (PC-3 and DU-145). A human osteoblast cell line (hFOB 1.19) is being used to study the effect of cytokines in relation to transforming growth factor β (TGFβ), insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF) that are secreted by prostate cancer cells. In-vitro migration/invasion assays, immunoprecipitation (IP), immunofluorescence microscope, cytokine profile array, real time qPCR and Western blot techniques are being used in this study to further analyze role of cytokines and growth factors on osteoblast differentiation. Our preliminary results suggest that all 3 compounds can be used to disrupt the main steps of prostate cancer bone metastasis. Results suggested that KB-G/H and S can be targeted to develop customized, tailored therapies for bone metastatic prostate cancer which merit further research. Citation Format: Aaron Jeshua Todman, Wishrawana S. Ratnayake, Luke Lajmi, Sloan Breedy, Kirpal Bisht, Mildred Acevedo-Duncan. Interaction of interleukin-6/8/18 with prostate cancer cell secreted growth factors are crucial for osteoblast proliferation and differentiation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3609.

Mitotic phosphorylation of Tau/MAPT modulates cell cycle progression in prostate cancer cells

PurposeTau/MAPT (microtubule associated protein tau) protein is actively studied for the pathologic consequences of its aberrant proteostasis in central nervous system leading to neurodegenerative diseases. Besides its ability to generate insoluble toxic oligomers, Tau homeostasis has attracted attention for its involvement in the formation of the mitotic spindle. This evidence, in association with the description of Tau expression in extra-neuronal tissues, and mainly in cancer tissues, constitutes the rationale for a more in-depth investigation of Tau role also in neoplastic diseases.MethodsIn our study, we investigated the expression of phosphorylated Tau in prostate cancer cell lines with particular focus on the residue Thr231 present in microtubule binding domain.ResultsThe analysis of prostate cancer cells synchronized with nocodazole demonstrated that the expression of Tau protein phosphorylated at residue Thr231 is restricted to G2/M cell cycle phase. The phosphorylated form was unable to bind tubulin and it does not localize on mitotic spindle. As demonstrated by the use of specific inhibitors, the phosphorylation status of Tau is under the direct control of cdk5 and PP2A, while cdk1 activation was able to exert an indirect control. These mechanisms were also active in cells treated with docetaxel, where counteracting the expression of the dephosphorylated form, by kinase inhibition or protein silencing, determined resistance to drug toxicity.ConclusionsWe hypothesize that phosphorylation status of Tau is a key marker for G2/M phase in prostate cancer cells and that the forced modulation of Tau phosphorylation can interfere with the capacity of cell to efficiently progress through G2/M phase.

The inflammatory macrophages repress the growth of bone metastatic human prostate cancer cells via TNF-α and IL-6 signaling: Involvement of cell signaling regulator regucalcin

Macrophages in the cancer microenvironments may play a regulatory role in the progression and metastasis of prostate cancer cells. However, the crosstalk between macrophages and prostate cancer cells is poorly understood. This study elucidates whether inflammatory macrophages regulate the proliferation and death of human prostate cancer cells in vitro. The RAW264.7 mouse macrophages were cocultured with PC-3 or DU-145 wild-type cells by using a Transwell chamber in vitro. RAW264.7 cells were cocultured with PC-3 or DU-145 cells in the presence of lipopolysaccharide (LPS). This coculturing blocked the proliferation and accelerated the death of cancer cells. Interestingly, cancer cell proliferation was repressed and death was promoted by the addition of the conditioned medium obtained from RAW264.7 cells treated with LPS. Culturing with LPS mostly augmented the production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the culture medium of RAW264.7 cells. The effects of the conditioned medium on the proliferation and death of PC-3 or DU-145 cells were blocked by NF-κB or STAT3 signaling inhibitors. Moreover, the effects of the conditioned medium on the proliferation and death of prostate cancer cells were not expressed in regucalcin-overexpressing cancer cells that diminish the levels of NF-κB p65 and STAT3. Culturing with extracellular TNF-α, IL-6, or regucalcin triggered inhibition of the proliferation of PC-3 wild-type cells. The levels of regucalcin in PC-3 cells were elevated by TNF-α or IL-6 stimulation. This study demonstrates that inflammatory macrophages triggered the loss of prostate cancer cells via the signaling process of NF-κB, STAT3, or regucalcin.

Interstitial fluid flow contributes to prostate cancer invasion and migration to bone; study conducted using a novel horizontal flow bioreactor

Prostate cancer bone metastasis is the leading cause of cancer-related mortality in men in the United States, causing severe damage to skeletal tissue. The treatment of advanced-stage prostate cancer is always challenging due to limited drug treatment options, resulting in low survival rates. There is a scarcity of knowledge regarding the mechanisms associated with the effects of biomechanical cues by the interstitial fluid flow on prostate cancer cell growth and migration. We have designed a novel bioreactor system to demonstrate the impact of interstitial fluid flow on the migration of prostate cancer cells to the bone during extravasation. First, we demonstrated that a high flow rate induces apoptosis in PC3 cells via TGF-β1 mediated signaling; thus, physiological flow rate conditions are optimum for cell growth. Next, to understand the role of interstitial fluid flow in prostate cancer migration, we evaluated the migration rate of cells under static and dynamic conditions in the presence or absence of bone. We report that CXCR4 levels were not significantly changed under static and dynamic conditions, indicating that CXCR4 activation in PC3 cells is not influenced by flow conditions but by the bone, where CXCR4 levels were upregulated. The bone-upregulated CXCR4 levels led to increased MMP-9 levels resulting in a high migration rate in the presence of bone. In addition, upregulated levels of α v β 3 integrins under fluid flow conditions contributed to an overall increase in the migration rate of PC3 cells. Overall, this study demonstrates the potential role of interstitial fluid flow in prostate cancer invasion. Understanding the critical role of interstitial fluid flow in promoting prostate cancer cell progression will enhance current therapies for advanced-stage prostate cancer and provide improved treatment options for patients.

Hsa_circ_0063329 inhibits prostate cancer growth and metastasis by modulating the miR-605-5p/tgif2 axis

ABSTRACT Circular RNAs play crucial regulatory roles in the progression of various cancers. Nevertheless, the underlying mechanisms of circRNAs in prostate cancer (PCa) proliferation and metastasis remain largely uncertain. Here, we performed circRNA microarray analyses to identify differentially expressed circRNAs in a normal prostate epithelial cell line and PCa cell lines. We found that hsa_circ_0063329 was significantly downregulated in PCa. A series of in vitro and in vivo functional assays showed that overexpression of hsa_circ_0063329 inhibits PCa cell progression, while silencing of hsa_circ_0063329 achieved the opposite effects. Mechanistically, bioinformatics analysis, RNA pulldown, RNA-seq and dual-luciferase reporter assays demonstrated that hsa_circ_0063329 exerts its effect by sponging miR-605-5p to derepress TG-interacting factor 2 (TGIF2) and inactivate the TGF-β pathway. In conclusion, hsa_circ_0063329 inhibits the proliferation and metastasis of PCa via modulation of the miR-605-5p/TGIF2 axis, and targeting hsa_circ_0063329 may provide a promising treatment strategy for aggressive PCa.

GLIDR promotes the aggressiveness progression of prostate cancer cells by sponging miR-128–3p

Glioblastoma downregulated RNA (GLIDR) is a newly discovered long non-coding RNA (lncRNA) that its increased expression indicates a poor prognosis of prostate cancer (PCa). However, the effect of GLIDR on PCa cells is not clear. Our study investigated the role and molecular mechanism of GLIDR in PCa cells. The results showed that GLIDR expression levels were higher in PCa samples and cells than in control. GLIDR could regulate the invasive potential, epithelial-to-mesenchymal transition (EMT) and proliferation in PC-3 and LnCaP cells. Besides, GLIDR could weaken the inhibitory effects of miR-128-3p on invasion, EMT and proliferation in PCa cells. Western blotting proved that miR-128-3p affected the expression of EMT markers, such as E-cadherin, Snail and N-cadherin, and GLIDR could reversed the effects of miR-128-3p on the expression levels of EMT markers in PCa cells. In addition, knockdown of miR-128-3p stimulated the invasion, EMT, and proliferation in PCa cells, whereas these effects were reversed when GLIDR expression was knocked down. GLIDR knockdown inhibited the invasion, EMT, and proliferation in PCa cells, and GLIDR was shown to sponge miR-128-3p. Together, these results highlight GLIDR as a potential therapeutic target for the PCa treatment.

Molecular mechanisms on how FABP5 inhibitors promote apoptosis-induction sensitivity of prostate cancer cells.

Previous work showed that FABP5 inhibitors suppressed the malignant progression of prostate cancer cells, and this suppression might be achieved partially by promoting apoptosis. But the mechanisms involved were not known. Here, we investigated the effect of inhibitors on apoptosis and studied the relevant mechanisms. WtrFABP5 significantly reduced apoptotic cells in 22Rv1 and PC3 by 18% and 42%, respectively. In contrast, the chemical inhibitor SB-FI-26 produced significant increases in percentages of apoptotic cells in 22Rv1 and PC3 by 18.8% (±4.1) and 4.6% (±1.1), respectively. The bio- inhibitor dmrFABP5 also did so by 23.1% (±2.4) and 15.8% (±3.0), respectively, in these cell lines. Both FABP5 inhibitors significantly reduced the levels of the phosphorylated nuclear fatty acid receptor PPARγ, indicating that these inhibitors promoted apoptosis-induction sensitivity of the cancer cells by suppressing the biological activity of PPARγ. Thus, the phosphorylated PPARγ levels were reduced by FABP5 inhibitors, the levels of the phosphorylated AKT and activated nuclear factor kapper B (NFκB) were coordinately altered by additions of the inhibitors. These changes eventually led to the increased levels of cleaved caspase-9 and cleaved caspase-3; and thus, increase in the percentage of cells undergoing apoptosis. In untreated prostate cancer cells, increased FABP5 suppressed the apoptosis by increasing the biological activity of PPARγ, which, in turn, led to a reduced apoptosis by interfering with the AKTor NFκB signaling pathway. Our results suggested that the FABP5 inhibitors enhanced the apoptosis-induction of prostate cancer cells by reversing the biological effect of FABP5 and its related pathway.