Progression Of Prostate Cancer Cells Research Articles

Inactivated FABP5 suppresses malignant progression of prostate cancer cells by inhibiting the activation of nuclear fatty acid receptor PPARγ.

Previous study has suggested that the FABP5-PPARγ-signalling transduction pathway gradually replaces the androgen receptor activated pathway in promoting malignant progression of castration-resistant prostate cancer (CRPC) cells. To interfere with this newly discovered FABP5-related signalling pathway, we have produced a highly efficient recombinant FABP5 inhibitor, named dmrFABP5. Treatment with dmrFABP5 significantly supressed the proliferation, migration, invasion and colony formation of the highly malignant prostate cancer cells PC3-M in vitro. To test dmrFABP5's suppressive effect in CRPC, the human PC3-M cells were implanted orthotopically into the prostate gland of immunosuppressed mice to produce tumours. These mice were then treated with dmrFABP5 and produced a highly significant reduction of 100% in metastatic rate and a highly significant reduction of 13-fold in the average size of primary tumours. Immunocytochemial staining showed that the staining intensity of dmrFABP5 treated tumours was reduced by 67%. When tested in vitro, dmrFABP5 suppressed the cancer cells by blocking fatty acid stimulation of PPARγ, and thereby prevented it activating down-stream cancer-promoting or inhibiting cancer-suppressing genes. Our results show that the FABP5 inhibitor dmrFABP5 is a novel molecule for treatment of experimental CRPC and its inhibitory effect is much greater than that produced by SB-FI-26 reported in our previous work.

P66Shc protein through a redox mechanism enhances the progression of prostate cancer cells towards castration-resistance

Prostate cancer (PCa) remains the second leading cause of cancer-related deaths in U.S. men due to the development of the castration-resistant (CR) PCa phenotype. A useful cell model for analysis of the molecular mechanism of PCa progression is required for developing targeted therapies toward CR PCa. In this study, we established a PCa cell progressive model in three separate cell lines, of which androgen-independent (AI) cells were derived from respective androgen-sensitive (AS) cells. Those AI PCa cells obtain the biochemical properties of the clinical CR phenotype, including AR and PSA expression as well as enhanced proliferation and tumorigenicity under androgen-deprived conditions. Thus, those AI cells recapitulate CR PCa and exhibit increased oxidant species levels as well as enhanced signaling of proliferation and survival pathways. H2O2 treatment directly enhanced AS cell growth and migration, which was counteracted by antioxidant N-acetyl cysteine (NAC). We further identified p66Shc protein enhances the production of oxidant species which contributes to phenotypic and cell signaling alterations from AS to AI PCa cells. H2O2-treated LNCaP-AS cells had a similar signaling profile to that of LNCaP-AI or p66Shc subclone cells. Conversely, the oxidant species-driven alterations of LNCaP-AI and p66Shc subclone cell signaling is mitigated by p66Shc knockdown. Moreover, LNCaP-AI cells and p66Shc subclones, but not LNCaP-AS cells, develop xenograft tumors with metastatic nodules, correlating with p66Shc protein levels. Together, the data shows that p66Shc enhances oxidant species production that plays a role in promoting PCa progression to the CR stage.

Transmembrane protein 64 modulates prostate tumor progression by regulating Wnt3a secretion.

Wnt3a is a glycosylated ligand that activates the β-catenin-dependent signaling pathway. Wnt signaling is also important in the prostate tumor microenvironment, and Wnt proteins secreted by the tumor stroma promote resistance to therapy. Bioactive Wnt3a production requires a number of dedicated factors in the secretory cell, but their coordinated functions are not fully understood. We previously reported transmembrane protein 64 (Tmem64) as a novel regulator of the Wnt/β-catenin signaling pathway, which is correlated with β-catenin regulation. In the present study, the role of Tmem64 in prostate cancer cells was investigated by modulating Wnt3a secretion. Overexpression of Tmem64 inhibited Wnt3a secretion and Lef/Tcf-sensitive transcription. By contrast, a Tmem64 mutation deleting the protein's transmembrane region restored Wnt3a secretion. Notably, Tmem64 protein and mRNA in PC3 cells were significantly overexpressed compared with that observed in LNCaP and DU145 cells. In a mouse metastasis model intracardially injected with PC3 cells, Tmem64 expression was downregulated in the metastatic spine and mandible lesions compared with in the primary injection regions. However, Wnt3a was strongly expressed in the metastatic spine and mandible lesions. Collectively, these findings suggest that Tmem64 is involved in the metastatic progression of prostate cancer cells by regulating Wnt3a secretion.

Androgen-responsive lncRNA LINC00304 promotes cell cycle and proliferation via regulating CCNA1.

Long noncoding RNA (lncRNA) plays a vital role in the development of many diseases. The abnormal expression of lncRNA is closely related to the occurrence and development of different kinds of tumors including prostate cancer (PCa). Differentially expressed lncRNA LINC00304 was identified using a publicly available gene expression data set (GSE38241) and quantitative polymerase chain reaction validation. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis were used to predict the molecular function of LINC00304. A lncRNA microarray, bioinformatic analysis, and chromatin immunoprecipitation assay were carried out to verify the upstream androgen receptor (AR) signaling pathway. Subsequently, the function of LINC00304 was observed by a series of in vitro assays. We observed higher expression of LINC00304 in PCa cells and samples compared with normal prostate cells and tissues. Functional analysis of LINC00304 showed it was related to regulating cell cycle process, cellular developmental process, and focal adhesion. Further, we identified androgen-inhibited lncRNA, LINC00304 as a direct target of AR. A series of functional studies revealed that overexpression of LINC00304 could significantly promote cell proliferation and cell cycle progression in PCa cells. We also find that LINC00304 can significantly promote CCNA1 expression in PCa cells. Our results indicate that LINC00304 may represent a new diagnostic and therapeutic biomarker for PCa.

The targeting of MTDH by miR‑145‑5p or miR‑145‑3p is associated with prognosis and regulates the growth and metastasis of prostate cancer cells.

Studies have rarely been conducted on the role of miRNAs in prostate cancer (PCa) cell progression by directly targeting MTDH, at least to the best of our knowledge. Thus, the present study aimed to identify miRNAs closely related with metadherin (MTDH) and to determine their roles in PCa. For this purpose, the expression levels of MTDH in PCa tissues and cell lines were examined by RT-qPCR, immunohistochemistry and western blot analysis. By cell transfection, MTDH was either overexpressed in the normal prostate epithelial cell lines or silenced in tumor cell lines to determine cell viability, invasion and migration. Bioinformatics analysis, RT-qPCR, western blot analysis, dual-luciferase reporter assay and MTT assay were performed to identify direct the target of MTDH and to examine tumor cell viability. Rescue experiments using the PC-3 and LNCaP cells were carried out by MTT assay, scratch wound assay, Transwell assay, RT-qPCR and western blot analysis. Experiments were also conducted using 46 PCa human cancer and adjacent tissues, as wells as on 501 cases of PCa from the TCGA database. It was confirmed that the overexpression of MTDH was associated with a poor prognosis of patients. The overexpression of MTDH was found to promote the viability, invasion and migration of PCa cells. miR-145-5p and miR-145-3p identified from 16 miRNAs were found to be closely related to PCa and to be the targets of MTDH. Both these miRNAs were found to significantly suppress the growth and metastasis of PCa cells by negatively regulating the expression of MTDH. On the whole, the findings of this study demonstrate that MTDH functions as an oncogene in PCa and the inhibition of MTDH by miR-145-5p or miR-145-3p suppressed the growth and metastasis of PCa cells. The miR-145-5p/MTDH and miR-145-3p/MTDH pathways may thus become novel treatment targets for PCa.

Silencing of lncRNA MALAT1 inhibits cell cycle progression via androgen receptor signaling in prostate cancer cells

Prostate cancer is the second common cancer in men with high morbidity and mortality. Androgen receptor (AR) signaling plays a crucial role in occurrence and development of prostate cancer. In this study, we demonstrated that lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) was increased in prostate cancer cells after androgen stimulation, as well as AR. The silencing of MALAT1 inhibited dihydrotestosterone (DHT) administration-induced acceleration of proliferation and cell cycle progression, and increase of AR expression in prostate cancer cells. MALAT1 bound to miR-320b and negatively regulated its expression, and vice versa. AR is a target of miR-320b. The phenotypic changes induced by silencing of MALAT1 were abolished by miR-320b inhibition or AR overexpression. Additionally, MALAT1 knockdown also suppressed the tumorigenesis of prostate cancer cells in nude mice. In summary, the silencing of MALAT1 inactivated AR signaling by sponging miR-320b, and inhibited proliferation and cell cycle progression in prostate cancer cells, suggesting that MALAT1 may be a new target in diagnosis and therapy of prostate cancer in clinic.

MiR-490-3p modulates the progression of prostate cancer through regulating histone deacetylase 2.

microRNAs (miRNAs) were regarded as critical participators for human cancers progression including prostate cancer (PCa) and have the potential to be used as treatment targets for cancers. Herein, we validated a tumor-suppressive miRNA, miR-490-3p, which may suppress PCa progression. Histone deacetylase 2 (HDAC2) is a protein that aberrantly expressed in several cancers. However, the role of HDAC2 in the progression of PCa has not been fully elucidated. Expression of miR-490-3p and HDAC2 in PCa was investigated. The effects of miR-490-3p or HDAC2 expression on PCa cell behaviors were analyzed. Association between miR-490-3p and HDAC2 was analyzed by luciferase activity reporter assay and Western blot assay. We demonstrated that miR-490-3p functioned as a tumor-suppressive role in PCa progression. We found miR-490-3p expression was decreased in PCa cell lines. Down-regulation of miR-490-3p promoted the growth, migration, invasion but inhibited apoptosis of PCa cells. HDAC2 was validated as a direct target of miR-490-3p and promoted the progression of PCa cells. Further studies showed that HDAC2 could reverse the effects of miR-490-3p on growth, migration, invasion and apoptosis of PCa cells. Our data highlighted the key role of miR-490-3p in the progression of PCa. Thus, miR-490-3p may be a novel cancer-specific therapeutic.

MicroRNA-30e inhibits adhesion, migration, invasion and cell cycle progression of prostate cancer cells via inhibition of the activation of the MAPK signaling pathway by downregulating CHRM3.

Prostate cancer (PCa) testing is currently based on measurement of serum prostate-specific antigen levels and digital rectal examination, which are limited by a low predictive value and the adverse effects associated with overdiagnosis and overtreatment. Recent studies have reported that the abnormal expression of microRNAs (miRNAs) is associated with the mechanism underlying the development of PCa. Thus, the aim of the present study was to investigate the effects of miR-30e and its target gene, M3 muscarinic acetylcholine receptor (CHRM3), on the adhesion, migration, invasion and cell cycle distribution of PCa cells via the mitogen-activated protein kinase (MAPK) signaling pathway. The differentially expressed genes were screened in the Gene Expression Omnibus database from a gene expression microarray (GSE55945) of PCa. PCa tissues and adjacent tissues were collected from patients with PCa. The PC-3 and DU145 human PCa cell lines were treated with activator, inhibitor and siRNAs. The effects of miR-30e on cell adhesion, migration, invasion and cell cycle distribution with the involvement of CHRM3 and the MAPK signaling pathway were investigated. The bioinformatics results demonstrated that the CHRM3 gene and the MAKP signaling pathway were involved in the progression of PCa, and has-miR-30e was selected for further study. The levels of miR-30e were significantly downregulated, while the levels of CHRM3 were obviously upregulated in PCa. CHRM3 was verified as a target gene of miR-30e. Upregulation of miR-30e and downregulation of CHRM3 decreased the levels of p-P38, p-extracellular signal-regulated kinase, p-c-Jun N-terminal kinase, p-c-fos and p-c-JUN, cell adhesion, migration and invasion ability, and the number of cells in the S phase, while they increased the number of cells in the G0 and G1 phases. The findings of the present study suggest that miR-30e inhibited the adhesion, migration, invasion and cell cycle entry of PCa cells by suppressing the activation of the MAPK signaling pathway and inhibiting CHRM3 expression. Thus, miR-30e may serve as a candidate target for the treatment of PCa.

Long noncoding RNA FOXC2-AS1 facilitates the proliferation and progression of prostate cancer via targeting miR-1253/EZH2

The vital roles of long noncoding RNAs (lncRNAs) in the cancers have been evidenced. However, there are still numerous unsolved queries for the molecular mechanism. This study tries to investigate the role of lncRNA FOXC2-AS1 in the human prostate cancer tumorigenesis. Results stated that lncRNA FOXC2-AS1 was ectopically up-regulated in prostate cancer tissue and cells. The over-expression of FOXC2-AS1 indicates the poor prognosis of prostate cancer patients. Functionally, the gain- and loss-of-functional experiments revealed that FOXC2-AS1 promoted the proliferation and tumor growth of prostate cancer cells in vitro and in vivo. Mechanically, we found that miR-1253 targeted FOXC2-AS1 at the 3′‑untranslated regions (UTR), which in turn bind the EZH2 mRNA 3-UTR. Luciferase reporter assay and rescue experiment confirmed the FOXC2-AS1/miR-1253/EZH2 pathway. In conclusion, we confirmed that lncRNA FOXC2-AS1 accelerated the tumor progression of prostate cancer cells by regulating the proliferation and tumor growth through miR-1253/EZH2 axis.

Angiotensin 1\u20137 modulates molecular and cellular processes central to the pathogenesis of prostate cancer

Angiotensin 1–7 (Ang1–7) is an endogenous bioactive component of the renin-angiotensin system (RAS). In addition to its cardiovascular properties, its anti-proliferative and anti-angiogenic traits are believed to play important roles in carcinogenesis. The present study examines the influence of Ang1–7 on processes associated with development and progression of prostate cancer cells. Our findings indicate that while Ang1–7 (1 nM; 48 h) can effectively reduce cell proliferation in DU-145, it can induce a significant decrease in the expression of MKI67 in LNCaP. In both cell lines we also observed a reduction in colony size in soft agar assay. A various changes in gene expression were noted after exposure to Ang1–7: those of anti- and pro-apoptotic agents and the NF-kB family of transcription factors, as well as mesenchymal cell markers and vascular endothelial growth factor A (VEGFA). In addition, Ang1–7 was found to modulate cell adhesion and matrix metallopeptidase (MMP) activity. Changes were also observed in the levels of angiotensin receptors and sex steroid hormone receptors. Ang1–7 reduced the levels of estrogen receptor alpha gene (ESR1) and increased the expression of estrogen receptor beta gene (ESR2) in all prostate cancer cells; it also up-regulated androgen receptor (AR) expression in androgen-sensitive cells but contradictory effect was observed in androgen- irresponsive cell lines. In summary, the results confirm the existence of complex network between the various elements of the local RAS and the molecular and cellular mechanisms of prostate cancerogenesis. The response of cancer cells to Ang1–7 appears to vary dependently on the dose and time of incubation as well as the aggressiveness and the hormonal status of cells.

Abstract B027: CD117 tyrosine kinase activation drives prostate cancer aggressiveness

Abstract Tyrosine kinase receptors have been shown to drive prostate cancer progression and metastasis. In particular, CD117/c-kit expression is upregulated during tumor progression with the highest levels being expressed in bone metastases. In addition, the numbers of circulating CD117+ tumor cells were higher in advanced prostate cancer patients. The presence of these cells in patients’ circulation after radical prostatectomy was also associated with biochemical recurrence. Further, the expression of CD117 can be upregulated on prostate cancer cells after repeated culturing in the bone microenvironment. These data indicated that CD117 expression and activation may be associated with prostate cancer aggressiveness. LNCaP-C4-2 cells were sorted into CD117+ and negative cell populations. Proliferation, Matrigel invasion, sphere formation, and gene expression were compared in the two cell populations in the presence and absence of the ligand stem cell factor (SCF) or CD117 and Akt inhibitors. To track cells during coculture and competition experiments, sorted cells were infected to express mCherry (CD117+) or ZsGreen (negative). Using these cells, competition was tracked using an IncuCyte ZOOM live cell imager for in vitro studies and an IVIS imager for in vivo xenograft tumor initiation studies. To better visualize transendothelial migration, a 3D microfluidic metastasis-on-a-chip was developed to track tumor cell migration through the ECM and across an endothelial cell layer. Here, we examine the activation of CD117 in prostate cancer cell progression and migration. CD117 expression was associated with increased proliferation, migration, and EMT marker expression. Interestingly, treatment with the CD117 ligand stem cell factor (SCF) reduced proliferation but further enhanced invasion, indicating that CD117 activation may drive metastasis. CD117 activation stimulated Akt expression and drove the cancer stem cell phenotype as demonstrated by increased Oct4 and Sox2 expression. CD117 expression was associated with increased expression of cancer progression and EMT signaling pathways. Using live cell imaging, competition between CD117+ and negative cells was visualized in proliferation, scratch healing, invasion, transendothelial migration, and sphere formation. Xenograft models also demonstrated competition in vivo with CD117 expression increasing tumor initiation. Intravital imaging and sectioning of tumors demonstrated the localization and composition of coinjected cells. Using 3D microfluidics, we have modeled the competition between CD117+ and negative cell populations during transendothelial migration. CD117 expression on prostate cancer cells drives a more aggressive cell phenotype and may be involved in metastasis. Further, CD117+ cells represent a possible cancer stem cell population. Citation Format: Lihong Shi, Brittni Foster, Koran Harris, Aleksander Skardal, Bethany A. Kerr. CD117 tyrosine kinase activation drives prostate cancer aggressiveness [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B027.

Abstract B076: Osteoclasts influence androgen-related gene expression in prostate cancer cells

Abstract Background: The majority of progressive prostate cancer will develop metastases in the skeleton. In bone environment, the regulated destruction of bone matrix by osteoclasts is closely connected to the formation of new bone by osteoblasts. Although prostate cancer often forms sclerotic bone lesions, evidence from recent research show that prostate cancer cells also produce factors to promote osteoclastic action. However, little is known on how osteoclasts regulate the prostate cancer growth in bone. The aim of the present study was to investigate the role of osteoclasts in the crosstalk of bone microenvironment and prostate cancer cells. Methods: The interactions between prostate cancer cells and osteoclasts were studied in a coculture model using the transwell permeable supports system. Two castration-resistant prostate cancer cell lines (LNCaP-19 and PC3) were used together with osteoclasts that were transformed from the RAW 264.7 cells by stimulation with RANKL. After 48 hours coculturing, analysis on gene expression level was carried out with qRT-PCR. Results: The results show that osteoclasst directly influence gene expression patterns in prostate cancer cells, affecting genes generally involved in tumor progression, mainly in the osteolytic cell line PC3. In LNCaP-19 cells, which are sensitive to androgens, osteoclast stimulation upregulated expression of androgen receptor (AR), as well as the AR splice-variant ARV7. In contrast, in androgen-insensitive PC3 cells no such changes in AR and AR-V7 mRNA were detected. Instead, the expression of the steroidogenic enzymes CYP11A1 (converting cholesterol into pregnenolone) and CYP19A1 (converting testosterone into estrogen) were increased by osteoclast stimulation. In addition, osteoclast stimulation differentially affects expression of genes related to bone formation in the tumor cells. LNCaP-19, which gives osteoblastic lesions in mouse tibia, responds with increased expression of genes related to TGF-b signaling, osteogenesis, and bone matrix formation. In contrast, the osteolytic cell line PC3 downregulates expression of osteogenic genes in response to osteoclast stimulation. Conclusion: This study shows that there is a direct crosstalk between osteoclasts and prostate cancer cells, where the osteoclasts influence gene expression related to androgen signaling, steroidogenesis, and osteogenesis in different ways based on the androgen and bone-related characteristics of the prostate cancer cells. Since upregulated AR and induced ARV7 expression have been shown to assist the progression of castration-resistant prostate cancer cells, inhibiting this crosstalk by targeting osteoclasts may improve efficacy of androgen-targeting drugs. Citation Format: Junchi Huang, Jan-Erik Damber, Karin Welen. Osteoclasts influence androgen-related gene expression in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B076.

Astragaloside attenuates the progression of prostate cancer cells through endoplasmic reticulum stress pathways.

Astragaloside (As) has been demonstrated extensively to serve roles in a variety of tumor types, including glioma, lung cancer, colorectal cancer, breast cancer and cervical cancer, and has therefore been widely used in Traditional Chinese Medicine. To the best of our knowledge, the present study was the first to investigate the efficacy of the Traditional Chinese Medicine astragaloside on tumor growth and the apoptosis of prostate cancer cells. In addition, further investigation into the underlying molecular mechanisms via the endoplasmic reticulum (ER) stress pathway was also performed. In the present study, the human prostate cancer DU-145 cell line was employed as an experimental model in vitro and cells were divided into five treatment groups: Dimethyl sulfoxide (DMSO) group (control), low-dose astragaloside group (L-As; 20 nmol/l), moderate-dose astragaloside group (M-As; 50 nmol/l), high-dose astragaloside group (H-As, 100 nmol/l) and ER stress suppressor group (tauroursodeoxycholic acid; TUDCA). The proliferative ability and apoptosis rate of the DU-145 cells were detected via Cell Counting kit-8 methods and flow cytometry, respectively. Furthermore, the ER stress factors [binding immunoglobulin protein (BiP), C/EBP homologous protein (CHOP) and caspase-12] were assessed through reverse transcription polymerase chain reaction. Additionally, the protein expression levels of inositol-requiring enzyme 1 (IRE1), phosphorylated protein kinase R-like ER kinase (p-PERK), iron-regulated transcriptional activator Aft (AFT)4 and AFT6 were measured detected by western blot analysis. Administration of As significantly reduced the cell viability and promoted apoptosis (P<0.05) in a dose-dependent manner. Expression of ER-stressed genes BiP, CHOP and caspase-12 mRNA was increased by As administration, while TUDCA treatment led to a lower mRNA expression of these genes, compared with the control group. Results of western blot analysis indicated that the protein expression of IRE1, AFT4 and AFT6 was upregulated in the H-As group, and that the ratio of p-PERK/PERK was also higher than in the other groups. The administration of As demonstrated significant therapeutic effects on the proliferation of prostate cancer cells, as well as the expression of related proteins and genes. The results of the present study suggested future clinical potential of As for the treatment of prostate cancer.

Abstract 2467: Role of PCA3 in prostate cancer cell proliferation and progression

Abstract Background PCA3 is a long non-coding RNA used as an important biomarker in early prostate-cancer diagnosis but its biological functions are not understood. The aim of the study is to investigate the potential role of PCA3 in prostate cancer (PCa) initiation, growth and progression. Methods Human genomic DNA was used as templates for cloning of PCA3 gene. Primers were designed and polymerase chain reactions were performed. The 3 exons of PCA3 were cloned and confirmed by sequencing. To determine the impact of PCA3 and its individual exon in PCa cell growth and survival, we over-expressed them respectively in PC-3 and DU145 cell lines via transduction with lentiviral vector. qPCR was performed to examine the expression level of PCA3. To investigate the effects of PCA3 on prostate cancer cells, cell proliferation assay was performed by using trepan blue staining. Colony formation assays were performed to measure the clonogenic capacity of the cells after PCA3 overexpression and treatments with docetaxel. Results and conclusions Sequencing of PCA3 gene amplified from human tumor cells did not find mutations when compared to RefSeq. We overexpressed PCA3 in prostate cancer cells and found that increased PCA3 expression did not have significant impacts on DU145 or PC-3 cell proliferation. However, PCA3 exon 3 did increase the clonogenic capacity of DU145 cells when compared to the controls. DU145 cells with increased PCA3 exon 3 also presented increased resistance to docetaxel. No significance was observed in clonogenic capacity in transduced PC-3 cells, but PCA3 increased the motility of PC-3 cells. Our studies suggest that PCA3 can modulate prostate cancer cell clonogenic capacity, docetaxel resistance, and motility. Further studies are ongoing to determine the mechanisms involved. Citation Format: Bethanie Russel, Jiuhui Wang, Yuanqin Zhang, Daotai Nie. Role of PCA3 in prostate cancer cell proliferation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2467.

Abstract LB-315: CD117 tyrosine kinase activation drives prostate cancer aggressiveness

Abstract Tyrosine kinase receptors drive prostate cancer progression and metastasis. In particular, CD117/c-kit expression is upregulated during tumor progression with the highest levels being expressed in bone metastases. In addition, the numbers of circulating CD117+ tumor cells were higher in advanced prostate cancer patients. The presence of these cells in patients' circulation after radical prostatectomy was also associated with biochemical recurrence. Further, the expression of CD117 can be upregulated on prostate cancer cells after repeated culturing in the bone microenvironment. These data indicated that CD117 expression and activation may be associated with prostate cancer aggressiveness. LNCaP-C4-2 cells were sorted into CD117+ and negative cell populations. Proliferation, matrigel invasion, sphere formation, and gene expression were compared in the two cell populations in the presence and absence of the ligand stem cell factor (SCF) or CD117 and Akt inhibitors. To track cells during coculture and competition experiments, sorted cells were infected to express mCherry (CD117+) or ZsGreen (negative). Using these cells, competition was tracked using an IncuCyte ZOOM live cell imager for in vitro studies and an IVIS imager for in vivo xenograft tumor initiation studies. To better visualize transendothelial migration, a 3D microfluidic metastasis-on-a-chip was developed to track tumor cell migration through the ECM and across an endothelial cell layer. Here, we examine the activation of CD117 in prostate cancer cell progression and migration. CD117 expression was associated with increased proliferation, migration, and EMT marker expression. Interestingly, treatment with the CD117 ligand stem cell factor (SCF) reduced proliferation but further enhanced invasion indicating that CD117 activation may drive metastasis. CD117 activation stimulated Akt expression and drove the cancer stem cell phenotype as demonstrated by increased Oct4 and Sox2 expression. CD117 expression was associated with increased expression of cancer progression and EMT signaling pathways. Using live cell imaging, competition between CD117+ and negative cells was visualized in proliferation, scratch healing, invasion, trans-endothelial migration, and sphere formation. Xenograft models also demonstrated competition in vivo with CD117 expression increasing tumor initiation. Intravital imaging and sectioning of tumors demonstrated the localization and composition of co-injected cells. Using 3D microfluidics, we have modeled the competition between CD117+ and negative cell populations during transendothelial migration. CD117 expression on prostate cancer cells drives a more aggressive cell phenotype and may be involved in metastasis. Further, CD117+ cells represent a possible cancer stem cell population. Citation Format: Lihong Shi, Koran Harris, Brittni Foster, Aleksander Skardal, Bethany Kerr. CD117 tyrosine kinase activation drives prostate cancer aggressiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-315.

PO-283 Characterisation of hTERT-immortalised prostate-derived stromal and epithelial cells: an authentic in vitro model for tumour microenvironment studies

IntroductionTumour development begins with mutational changes to the genetic makeup of a cell; tumour progression is not solely determined by the mutated cell, but also by the tumour’s microenvironment. Prostate cancer, a leading cancer diagnosed in men, has been determined to be highly influenced by its surrounding stroma, particularly fibroblasts. It has been demonstrated that cancer-associated prostate fibroblasts (CAFs) differ from normal-associated prostate fibroblasts (NAFs). However, human prostate cancer model systems have focused largely on prostate cancer epithelial cells. Currently, a need exists for a more physiologically relevant human cell model system to study prostate cancer progression within the context of its tumour microenvironment.Material and methodsIn this study, we characterised three prostate-derived cells: CAFs, NAFs, and prostate epithelial cells (PrEs); all three lines were immortalised by human telomerase reverse transcriptase (hTERT) alone and selected with puromycin. The selected clones grew continuously in culture for more than 40 population doublings in our hands.Results and discussionsOur data shows that the hTERT-immortalised CAFs proliferate faster than the NAFs; in addition, both CAFs and NAFs express fibroblast markers such as TE7 and alpha smooth muscle actin (a-SMA), while neither cell line expresses epithelial markers such as CK14. Both CAFs and NAFs also express elevated levels of a-SMA upon TGF-b stimulation. All three prostate-derived cells weakly express the prostate specific marker AR, and show similar markers staining after long time passaging. Importantly, conditioned media collected from CAFs promotes tumour cell growth better than NAF conditioned media.ConclusionIn conclusion, CAFs, NAFs, and immortalised PrEs may provide a very valuable model system for the study of prostate cancer cell progression and tumour microenvironment studies.

PO-316 Genome-wide gene expression analysis of a murine model of prostate cancer cell progression: towards identification of high-potential therapeutic targets

IntroductionProstate cancer (PC) is the most frequently diagnosed cancer among men and the second leading cause of male cancer-related deaths worldwide. The poor prognosis of PC is largely due to late diagnosis of the disease when it has progressed to advanced stages marked by androgen-independence. We construe that these direly needed advances are limited by our poor understanding of early events in the progression of PC and that would thus represent ideal targets for early intervention. To begin to fill this void, we interrogated molecular ‘oncophenotypes’ that embody the transition of PC from an androgen-dependent (AD) to –independent (AI) state.Material and methodsWe have previously established AD and AI murine PC cell lines, PLum-AD and PLum-AI, respectively, which recapitulate primary and progressive PC morphologically and genetically. We statistically surveyed global gene expression in these cell lines by microarray analysis. Differential profiles were functionally interrogated by pathways, gene set enrichment and topological gene-gene network analyses – features built in the commercially available software Ingenuity Pathways Analysis.Results and discussionsGene expression analysis of PLum-AD and PLum-AI transcriptomes (n=3 each), revealed 723 differentially expressed genes (392 upregulated and 331 downregulated) in PLum-AI compared with PLum-AD cells. Gene set analysis demonstrated enrichment of biological functions and pathways in PLum-AI cells that are central to tumour aggressiveness including epithelial-to-mesenchymal transition (EMT), cell migration, and cell invasion. Further analysis demonstrated that the p38 mitogen activated protein kinase (MAPK) was predicted to be significantly activated in the PLum-AI cells. Conversely, gene sets that previously showed to be associated with favourable response to the p38 inhibitor SB203580 were attenuated in the PLum-AI cells, suggesting that these aggressive cells may be therapeutically vulnerable to p38 inhibition. Network analysis also alluded to activation of other signalling networks particularly those associated with enhanced EMT, inflammation and immune function/response.ConclusionOur pilot genome-wide gene expression analysis of a murine model of PC progression identified molecular oncophenotypes (e.g. inflammation and EMT) that denote this progression and that comprise high-potential targets (e.g. p38) for early treatment of PC. Efforts are underway to functionally assess the potential roles of these genes as therapeutic targets for PC progression.

Abstract 2115: An authenticated in vitro model for prostate microenvironment studies utilizing prostate epithelial cells and stromal-derived cells

Abstract Prostate cancer remains one of the most common cancers diagnosed in men and one of the leading causes of cancer death in men. Tumor development and progression have been shown to be highly influenced not simply by the genetic makeup of a cell, but by its surrounding stroma, particularly fibroblasts. It has been demonstrated that prostate cancer-associated fibroblasts (CAFs, which are located marginal to the prostate tumor), differ from prostate normal-associated fibroblast (NAFs, which are located distal to the prostate tumor), on their contribution to tumor progression. However, human prostate cancer in-vitro model systems have focused largely on prostate cancer epithelial cells exclusively. A need exists for a more physiologically relevant human cell model system to study prostate cancer progression within the context of its tumor microenvironment. In this study, we utilized prostate cancer-associated fibroblasts (CAFs), prostate normal-associated fibroblasts (NAFs) and normal prostate epithelial (PrE) cells; all three lines were immortalized by hTERT (human telomerase reverse transcriptase) alone and they were continuously passaged for at least 15 passages without any indications of a decrease in growth rate. All cell lines express appropriate specific cell lineage markers for either fibroblasts or epithelial cells. Fibroblasts expressed TE7 and alpha smooth muscle actin (a-SMA), while prostate epithelial cells expressed cytokeratin 5, low levels of prostate specific antigen (PSA) and high levels of p63 throughout their continuous passage; all characteristics in accord with their primary cell counterparts. Next, cell proliferation was measured for various prostate-derived epithelial cells under the influence of CAFs and NAFs cells. Normal prostate epithelial cell proliferation was inhibited and produced a visible morphological change in the cells in the presence of CAFs or CAF-conditioned medium. Meanwhile, the effects of stromal cells on prostate cancer cells was cell line dependent, demonstrating both promotion and inhibition of growth of selected cancer cells. Surprisingly, both CAFs and NAFs promoted cancer cell growth, but CAFs promoted a greater increase in cell proliferation than NAFs in some cancer cell lines. This study demonstrates that these three hTERT immortalized cells from human prostate are a valuable model system for the study of prostate cancer cell progression and tumor micro environment studies. Citation Format: Luis G. Rodriguez, Russell E. McDaniel, Xiangshan Zhao, Chaozhong Zou. An authenticated in vitro model for prostate microenvironment studies utilizing prostate epithelial cells and stromal-derived cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2115.

Plagiochiline A Inhibits Cytokinetic Abscission and Induces Cell Death.

We previously reported on the isolation and biological activities of plagiochiline A (1), a 2,3-secoaromadendrane-type sesquiterpenoid from the Peruvian medicinal plant, Plagiochila disticha. This compound was found to have antiproliferative effects on a variety of solid tumor cell lines, as well as several leukemia cell lines. Other researchers have also noted the cytotoxicity of plagiochiline A (isolated from different plant species), but there are no prior reports regarding the mechanism for this bioactivity. Here, we have evaluated the effects of plagiochiline A on cell cycle progression in DU145 prostate cancer cells. A cell cycle analysis indicated that plagiochiline A caused a significant increase in the percentage of cells in the G2/M phase when compared with control cells. When cells were stained and observed by fluorescence microscopy to examine progress through the mitotic phase, we found a significant increase in the proportion of cells with features of late cytokinesis (cells connected by intercellular bridges) in the plagiochiline A-treated samples. These results suggest that plagiochiline A inhibits cell division by preventing completion of cytokinesis, particularly at the final abscission stage. We also determined that plagiochiline A reduces DU145 cell survival in clonogenic assays and that it induces substantial cell death in these cells.

Non-canonical activation of hedgehog in prostate cancer cells mediated by the interaction of transcriptionally active androgen receptor proteins with Gli3

Hedgehog (Hh) is an oncogenic signaling pathway that regulates the activity of Gli transcription factors. Canonical Hh is a Smoothened- (Smo-) driven process that alters the post-translational processing of Gli2/Gli3 proteins. Though evidence supports a role for Gli action in prostate cancer (PCa) cell growth and progression, there is little indication that Smo is involved. Here we describe a non-canonical means for activation of Gli transcription in PCa cells mediated by the binding of transcriptionally-active androgen receptors (ARs) to Gli3. Androgens stimulated reporter expression from a Gli-dependent promoter in a variety of AR + PCa cells and this activity was suppressed by an anti-androgen, Enz, or by AR knockdown. Androgens also upregulated expression of endogenous Gli-dependent genes. This activity was associated with increased intranuclear binding of Gli3 to AR that was antagonized by Enz. Fine mapping of the AR binding domain on Gli2 showed that AR recognizes the Gli protein processing domain (PPD) in the C-terminus. Mutations in the arginine-/serine repeat elements of the Gli2 PPD involved in phosphorylation and ubiquitinylation blocked the binding to AR. β-TrCP, a ubiquitin ligase that recognizes the Gli PPD, competed with AR for binding to this site. AR binding to Gli3 suppressed its proteolytic processing to the Gli3 repressor form (Gli3R) whereas AR knockdown increased Gli3R. Both full-length and truncated ARs were able to activate Gli transcription. Finally, we found that an ARbinding decoy polypeptide derived from the Gli2 C-terminus can compete with Gli3 for binding to AR. Exogenous overexpression of this decoy suppressed Gli transcriptional activity in PCa cells. Collectively, this work identifies a novel pathway for non-canonical activation of Hh signaling in PCa cells and identifies a means for interference that may have clinical relevance for PCa patients.