Metastatic Prostate Cancer Cell Lines Research Articles

Estrogen receptor β regulates AKT activity through up-regulation of INPP4B and inhibits migration of prostate cancer cell line PC-3

Loss of the tumor suppressor, PTEN, is one of the most common findings in prostate cancer (PCa). This loss leads to overactive Akt signaling, which is correlated with increased metastasis and androgen independence. However, another tumor suppressor, inositol-polyphosphate 4-phosphatase type II (INPP4B), can partially compensate for the loss of PTEN. INPP4B is up-regulated by androgens, and this suggests that androgen-deprivation therapy (ADT) would lead to hyperactivity of AKT. However, in the present study, we found that in PCa, samples from men treated with ADT, ERβ, and INPP4B expression were maintained in some samples. To investigate the role of ERβ1 in regulation of INPPB, we engineered the highly metastatic PCa cell line, PC3, to express ERβ1. In these cells, INPP4B was induced by ERβ ligands, and this induction was accompanied by inhibition of Akt activity and reduction in cell migration. These findings reveal that, in the absence of androgens, ERβ1 induces INPP4B to dampen AKT signaling. Since the endogenous ERβ ligand, 3β-Adiol, is lost upon long-term ADT, to obtain the beneficial effects of ERβ1 on AKT signaling, an ERβ agonist should be added along with ADT.

Abstract 5310: Chromatin accessibility landscape and transcriptome of castration resistant prostate cancers reveals novel subtypes and diverse master regulators

Abstract Castration-resistant prostate cancer (CRPC) is a heterogeneous disease with diverse drivers and mechanisms of resistance to androgen receptor (AR) therapy. We generated ATAC-seq and RNA-seq data for twenty-four metastatic human prostate cancer organoids and cell lines. Integration of chromatin accessibility profiles and transcriptomes revealed four subtypes: androgen-receptor(AR)-dependent, neuroendocrine, Wnt-dependent and epithelial mesenchymal transition (EMT). The transcriptomic signatures obtained from these four subtypes enable the classification of 100 metastatic prostate cancer patient samples from Institute Precision Medicine (IPM) and 270 published samples from SU2C study, revealing potential therapeutic vulnerabilities. Furthermore, using novel computational algorithms we constructed regulatory networks and identified the master regulators of each subtype. Currently we're carrying out western blot and quantitative PCR to confirm the subtypes of all prostate cancer models we use, and using drug sensitivity test, CRISPR knockout and cell competition assay to validate the functions of candidates in each subtype. Our study has characterized global chromatin accessibility landscape and transcriptome in the largest number of metastatic prostate cancer models, which revealed novel subtypes and corresponding tumor drivers. Collectively, these organoids, cell lines and matching sequence data provide a resource to the community to study various CRPC models. The molecular classification and corresponding master regulators reveal new drug targets and could potentially guide future therapeutic studies. Citation Format: Fanying Tang, Chen Khuan Wong, Sandra Cohen, Cindy Lee, Minwei Liu, Rohan Bareja, Kenneth Eng, Shaham Beg, Loredana Puca, Cora Sternberg, Juan Miguel Mosquera, Himisha Beltran, Andrea Sboner, Yu Chen, Ekta Khurana. Chromatin accessibility landscape and transcriptome of castration resistant prostate cancers reveals novel subtypes and diverse master regulators [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5310.

TNF-α controls Lipocalin-2 expression in PC-3 prostate cancer cells

Prostate cancer (PCa) is one of the most common and deadly cancers in men worldwide. The surrounding tumor microenvironment (TME) is important in tumor progression, as cytokines and soluble mediators including tumor necrosis factor (TNF-α) or lipocalin-2 (LCN2) can influence tumor growth and formation of metastasis. The exact mechanisms on how these pleiotropic factors affect PCa are still unknown. In this study, we showed for the first time that LCN2 mRNA and protein expression are strongly inducible by TNF-α in the highly metastatic human PCa cell line PC-3. In addition, we observed higher levels of secreted LCN2 in cell culture medium of TNF-α-treated PC-3 cells. We found that different signaling pathways such as p38, NF-κB or JNK were activated shortly after TNF-α treatment. Moreover, the mRNA levels of IL-1β and IL-8 were also significantly increased after 24 h stimulation. Mechanistically, the NF-κB pathway and the JNK signaling axis are directly responsible for LCN2 upregulation. This was shown by the fact that pretreatment with the JNK inhibitors SP600125 or JNK-IN-8 strongly downregulated phosphorylation of c-Jun protein and markedly reduced TNF-α-mediated LCN2 upregulation in PC-3 cells. Likewise, the NF-κB inhibitor QNZ was able to repress TNF-α-induced LCN2 expression in PC-3 cells. Taking into consideration that LCN2 has been described as a tumor promoting factor in PCa, our results indicate that JNK regulates LCN2 expression and unmasks the JNK signaling axis as a possible therapeutic target for patients with PCa.

Targeting lactate production and efflux in prostate cancer

Prostate cancer (PCa) is the most commonly diagnosed cancer in men worldwide. Screening and management of PCa remain controversial and, therefore, the discovery of novel molecular biomarkers is urgently needed. Alteration in cancer cell metabolism is a recognized hallmark of cancer, whereby cancer cells exhibit high glycolytic rates with subsequent lactate production, regardless of oxygen availability. To maintain the hyperglycolytic phenotype, cancer cells efficiently export lactate through the monocarboxylate transporters MCT1 and MCT4. The impact of inhibiting lactate production/extrusion on PCa cell survival and aggressiveness was investigated in vitro and ex vivo using primary tumor and metastatic PCa cell lines and the chicken embryo chorioallantoic membrane (CAM) model. In this study, we showed the metastatic PCa cell line (DU125) displayed higher expression levels of MCT1/4 isoforms and glycolysis-related markers than the localized prostate tumor-derived cell line (22RV1), indicating these proteins are differentially expressed throughout prostate malignant transformation. Moreover, disruption of lactate export by MCT1/4 silencing resulted in a decrease in PCa cell growth and motility. To support these results, we pharmacological inhibited lactate production (via inhibition of LDH) and release (via inhibition of MCTs) and a reduction in cancer cell growth in vitro and in vivo was observed. In summary, our data provide evidence that MCT1 and MCT4 are important players in prostate neoplastic progression and that inhibition of lactate production/export can be explored as a strategy for PCa treatment.

A promising therapeutic combination for metastatic prostate cancer: Chloroquine as autophagy inhibitor and palladium(II) barbiturate complex

Autophagy is a catabolic process for cells that can provide energy sources and allows cancer cells to evade cell death. Therefore, studies on the combination of autophagy inhibitors with drugs are increasing as a new treatment modality in cancer. Previously, we reported the anti-tumor activity of a Palladium (Pd)(II) complex against different types of cancer in vitro and in vivo. Chloroquine (CQ), the worldwide used anti-malarial drug, has recently been focused as a chemosensitizer in cancer treatment. The aim of this study was to investigate the efficacy of a combined treatment of these agents that work through different mechanisms to provide an effective treatment modality for metastatic prostate cancer that is certainly fatal. Metastatic prostate cancer cell lines (PC-3 and LNCaP) were treated with Pd (II) complex, CQ, and their combination. The combination enhanced apoptosis by increasing phosphatidylserine translocation and pro-apoptotic proteins. Apoptosis was confirmed by the use of apoptosis inhibitor. The formation of acidic vesicular organelles (AVOs) was observed by acridine orange staining in fluorescence microscopy. The Pd (II) complex increased AVOs formation in prostate cancer cells and CQ-pretreatment has potentiated this effect. Importantly, treatment with CQ suppressed the pro-survival function of autophagy, which might have contributed to enhanced cytotoxicity. In addition, PI3K/AKT/mTOR-related protein expressions were altered after the combination of treatments. Our results suggest that combination treatment enhances apoptotic cell death possibly via the inhibition of autophagy, and may therefore be regarded as a novel and better approach for the treatment of metastatic prostate cancer.

Biodistribution and imaging of an hsp90 ligand labelled with 111In and 67Ga for imaging of cell death

Background4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid (GSAO) when conjugated at the γ-glutamyl residue with fluorophores and radio-isotopes is able to image dead and dying cells in vitro and in vivo by binding to intracellular 90-kDa heat shock proteins (hsp90) when cell membrane integrity is compromised. The ability to image cell death has potential clinical impact especially for early treatment response assessment in oncology. This work aims to assess the biodistribution and tumour uptake of diethylene triamine pentaacetic acid GSAO labelled with 111In ([111In]In-DTPA-GSAO) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid GSAO labelled with 67Ga ([67Ga]Ga-DOTA-GSAO) in a murine subcutaneous tumour xenograft model and estimate dosimetry of [67Ga]Ga-DOTA-GSAO.ResultsThere was good tumour uptake of both [111In]In-DTPA-GSAO and [67Ga]Ga-DOTA-GSAO (2.44 ± 0.26% injected activity per gramme of tissue (%IA/g) and 2.75 ± 0.34 %IA/g, respectively) in Balb c nu/nu mice bearing subcutaneous tumour xenografts of a human metastatic prostate cancer cell line (PC3M-luc-c6). Peak tumour uptake occurred at 2.7 h post injection. [111In]In-DTPA-GSAO and [67Ga]Ga-DOTA-GSAO demonstrated increased uptake in the liver (4.40 ± 0.86 %IA/g and 1.72 ± 0.27 %IA/g, respectively), kidneys (16.54 ± 3.86 %IA/g and 8.16 ± 1.33 %IA/g) and spleen (6.44 ± 1.24 %IA/g and 1.85 ± 0.44 %IA/g); however, uptake in these organs was significantly lower with [67Ga]Ga-DOTA-GSAO (p = 0.006, p = 0.017 and p = 0.003, respectively). Uptake of [67Ga]Ga-DOTA-GSAO into tumour was higher than all organs except the kidneys. There was negligible uptake in the other organs. Excretion of [67Ga]Ga-DOTA-GSAO was more rapid than [111In]In-DTPA-GSAO. Estimated effective dose of [67Ga]Ga-DOTA-GSAO for an adult male human was 1.54 × 10− 2 mSv/MBq.Conclusions[67Ga]Ga-DOTA-GSAO demonstrates higher specific uptake in dead and dying cells within tumours and lower uptake in normal organs than [111In]In-DTPA-GSAO. [67Ga]Ga-DOTA-GSAO may be potentially useful for imaging cell death in vivo. Dosimetry estimates for [67Ga]Ga-DOTA-GSAO are acceptable for future human studies. This work also prepares for development of 68Ga GSAO radiopharmaceuticals.

Identification of Novel Prognosis and Prediction Markers in Advanced Prostate Cancer Tissues Based on Quantitative Proteomics.

Prostate cancer (PCa) is the most frequent cancer found in males worldwide, and its mortality rate is increasing every year. However, there are no known molecular markers for advanced or aggressive PCa, and there is an urgent clinical need for biomarkers that can be used for prognosis and prediction of PCa. Mass spectrometry-based proteomics was used to identify new biomarkers in tissues obtained from patients with PCa who were diagnosed with T2, T3, or metastatic PCa in regional lymph nodes. Among 1,904 proteins identified in the prostate tissues, 344 differentially expressed proteins were defined, of which 124 were up-regulated and 216 were down-regulated. Subsequently, based on the results of partial least squares discriminant analysis and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, we proposed that spermidine synthase (SRM), nucleolar and coiled-body phosphoprotein 1 (NOLC1), and prostacyclin synthase (PTGIS) represent new protein biomarkers for diagnosis of advanced PCa. These proteomics results were verified by immunoblot assays in metastatic PCa cell lines and by indirect enzyme-linked immunosorbent assay in prostate specimens. SRM was significantly increased depending on the cancer stage, confirming the possibility of using SRM as a biomarker for prognosis and prediction of advanced PCa.

Anticancer and antimicrobial effects of novel ciprofloxacin fatty acids conjugates

Ciprofloxacin (CP) has a confirmed cytotoxic action on some cancerous cells, but in high, non-pharmacological concentrations. Considering features of natural fatty acids, such as biocompatibility, biodegradability and their increased cellular uptake by cancer cells, it seems that combining them with a drug could improve its bioavailability, and thus cytotoxicity. Therefore, the aim of this study was coupling of CP with saturated and unsaturated fatty acids, and evaluation of their cytotoxicity, apoptosis-inducing effects and inhibition of IL-6 release in human primary (SW480) and metastatic (SW620) colon cancer, metastatic prostate cancer (PC3) and normal (HaCaT) cell lines. The PC3 cell line was the most sensitive to the presence of the obtained conjugates. The value of IC50 for oleic acid conjugate (4) was 7.7 μM, and it was 12 times lower than for CP alone (101.4 μM). The studied derivatives induced late apoptosis in all cancer cell lines, but not in normal cells. The most potent apoptosis inducer was conjugate 4, that resulted in the highest percentage of PC3 cells in late apoptosis (81.5% ± 3.9), followed by elaidic acid amide 5 (75% ± 4.8). The strongest pro-apoptic effects on SW480 cells were demonstrated by conjugates of DHA (8) and sorbic (2) acids, whereas in SW620 cell lines, compounds 2 and 5 appeared to be the most effective. To establish the mechanism of cytotoxic action of derivatives 2, 4, 5, the level of interleukin-6 (IL-6) was measured. The compounds with the highest cytotoxic potential significantly decreased the release of IL-6 by cancer cells. Additionally, all conjugates were evaluated for their in vitro antimicrobial activity. Short chain amides - crotonic (1) and sorbic (2) - were the most active against Staphyloccoci. The second-mentioned amide has shown both strong antistaphylococcal and antitumor properties.

Silencing TTK expression inhibits the proliferation and progression of prostate cancer

PurposeThe main objective of our study was to explore changes in the expression levels of differentially expressed genes associated with prostate cancer progression and to design a series of experiments to verify the function of differentially expressed genes. MethodThe transcriptome datas of 499 cases of prostate cancer patients was downloaded from TCGA database. Differential genes associated with Gleason score were selected and filtered out by p < 0.05 and spearman coefficient >0.3. KEGG signaling pathway was enriched by differentially expressed genes, and TTK was selected as the research object. The expression of TTK was tested in prostate cancer tissues and prostate cancer cell lines. The changes of biological behavior of prostate cancer cell lines were verified after TTK was knocked out by siRNA and tumorigenic effect of TTK was verified by shRNA in vivo experiments. ResultThe expression of TTK was positively correlated with Gleason score of prostate cancer, and the expression of protein and mRNA in metastatic prostate cancer cell lines was higher than that in non-metastatic prostate cancer cell lines. Vitro biological experiments showed that TTK gene knockout could inhibit the proliferation, invasion and migration of PC3 and DU145 cells, and promote cell apoptosis. In vivo experiments showed that TTK knockout inhibited tumorigenesis in mice. It was found that the expression of CDK2 and CCNE1 decreased after TTK was knocked out. ConclusionOur results suggest that TTK is a gene associated with malignancy of PCa and could be a novel therapeutic target for clinical application.

Effective targeting of RNA polymerase I in treatment-resistant prostate cancer.

Advanced prostate cancers depend on protein synthesis for continued survival and accelerated rates of metabolism for growth. RNA polymerase I (Pol I) is the enzyme responsible for ribosomal RNA (rRNA) transcription and a rate-limiting step for ribosome biogenesis. We have shown using a specific and sensitive RNA probe for the 45S rRNA precursor that rRNA synthesis is increased in prostate adenocarcinoma compared to nonmalignant epithelium. We have introduced a first-in-class Pol I inhibitor, BMH-21, that targets cancer cells of multiple origins, and holds potential for clinical translation. The effect of BMH-21 was tested in prostate cancer cell lines and in prostate cancer xenograft and mouse genetic models. We show that BMH-21 inhibits Pol I transcription in metastatic, castration-resistant, and enzalutamide treatment-resistant prostate cancer cell lines. The genetic abrogation of Pol I effectively blocks the growth of prostate cancer cells. Silencing of p53, a pathway activated downstream of Pol I, does not diminish this effect. We find that BMH-21 significantly inhibited tumor growth and reduced the Ki67 proliferation index in an enzalutamide-resistant xenograft tumor model. A decrease in 45S rRNA synthesis demonstrated on-target activity. Furthermore, the Pol I inhibitor significantly inhibited tumor growth and pathology in an aggressive genetically modified Hoxb13-MYC|Hoxb13-Cre|Ptenfl/fl (BMPC) mouse prostate cancer model. Taken together, BMH-21 is a novel promising molecule for the treatment of castration-resistant prostate cancer.

Betaglycan drives the mesenchymal stromal cell osteogenic program and prostate cancer-induced osteogenesis.

Bone metastatic prostate cancer provokes extensive osteogenesis by driving the recruitment and osteoblastic differentiation of mesenchymal stromal cells (MSCs). The resulting lesions greatly contribute to patient morbidity and mortality, underscoring the need for defining how prostate metastases subvert the MSC-osteoblast differentiation program. To gain insights into this process we profiled the effects of co-culture of primary MSCs with validated bone metastatic prostate cancer cell line models. These analyses revealed a cast of shared differentially induced genes in MSC, including betaglycan, a co-receptor for TGFβ. Betaglycan has not been studied in the context of bone metastatic disease previously. Here we report that loss of betaglycan in MSC is sufficient to augment TGFβ signaling, proliferation and migration, and completely blocks the MSC-osteoblast differentiation program. Further, betaglycan was revealed as necessary for prostate cancer-induced osteogenesis in vivo. Mechanistically, gene expression analysis revealed betaglycan controls the expression of a large repertoire of genes in MSCs, and that betaglycan loss provokes >60-fold increase in the expression of Wnt5a that plays important roles in stemness. In accord with the increased Wnt5a levels, there was a marked induction of canonical Wnt signaling in betaglycan ablated MSCs, and the addition of recombinant Wnt5a to MSCs was sufficient to impair osteogenic differentiation. Finally, the addition of Wnt5a neutralizing antibody was sufficient to induce the expression of osteogenic genes in betaglycan-ablated MSCs. Collectively, these findings suggest a betaglycan-Wnt5a circuit represents an attractive vulnerability to ameliorate prostate cancer-induced osteogenesis.

Abstract 370: The role of DLX1 as a biomarker and novel target in poor prognostic prostate cancer

Abstract Introduction: Previous work from our lab has identified a molecularly distinct subgroup of primary prostate cancers with biology similar to metastatic prostate cancers. These “met-like” cancers can be identified by a 70 gene classifier and are statistically more likely to result in distant metastases than their “non-met like” counterparts. This study aimed to identify exploitable biology common to both “met-like” primary tumours and metastatic prostate cancer. Experimental procedures: Differential gene expression and Ingenuity pathway analysis (IPA) was used to identify nodal genes upregulated in both “met-like” primary prostate cancers and metastatic prostate cancers. siRNA screening was performed on 40 nodal genes in a panel of normal, primary and metastatic prostate cancers. Cell viability was assessed using cell titer-glo luminescent Assay. Relative levels of apoptosis were assessed using PARP, caspase-8 and caspase-3 cleavage Western Blots. siRNA mediated knockdowns were confirmed by Western Blot and rtPCR. Results: We have identified upregulation of the homeobox transcription factor DLX1 in “met-like” primary prostate cancers and metastatic prostate cancers. High levels of DLX1 expression were found to be independently predicative of biochemical recurrence and metastatic disease in primary prostate cancers. DLX1 knockdown resulted in significantly decreased cell viability in primary and metastatic prostate cancer cell lines with minimal reduction of viability in normal prostate cells. Additionally DLX1 knockdown was shown to result in increased PARP, caspase-8 and caspase-3 cleavage providing evidence of apoptotic cell death. Furthermore knockdown of DLX1 resulted in significantly increased transcript levels of the pro-apoptotic BH3 protein BIK, suggesting its involvement in the observed apoptotic cell death. Conclusion: DLX1 has previously been identified as a marker of prostate cancer metastasis with increased expression being observed in both primary and metastatic prostate cancers. However we have observed increased DLX1 expression in a subset of primary prostate cancers that developed metastatic diseased compared to primary tumors that did not metastasise. The elucidation of apoptotic mechanisms following DLX1 knockdown may uncover potential exploitable biology that may be used for the treatment of prostate cancer. Citation Format: Nicholas Forsythe, Cathal McKinney, Nuala McCabe, Richard D. Kennedy. The role of DLX1 as a biomarker and novel target in poor prognostic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 370.

Abstract 1913: Role of glypican 1 in regulating fibroblast activation in the prostate cancer tumor microenvironment

Abstract Cancer is a heterogeneous collection of neoplastic cells, surrounded by non-malignant, stromal cells, and extracellular matrix (ECM), among other constituents. Cross-talk between tumor and stromal cells forms a dynamic tumor microenvironment (TME) that mediates cell growth, proliferation, and metastasis. The TME can inhibit cancer growth and proliferation during the initial stages of carcinogenesis; however, cancer cells can evade these inhibitory signals, and reshape the TME, making it more amenable to their own growth. This gives rise to cancer-associated stromal cells, including cancer-associated fibroblasts (CAFs). CAFs further modulate the TME by regulating the release of growth signals, cell death, ECM remodeling, angiogenesis, and epithelial-mesenchymal transition. Glypican-1 (GPC1) is a cell surface heparan sulfate proteoglycan (HSPG) that has both pro-mitogenic and pro-metastatic activity. GPC1’s role in regulating fibroblasts and their transformation into CAFs has not been investigated. We studied the ability of prostate cancer cells to alter the TME in vitro by exposing human bone fibroblasts (HS-5 cells) to tumor conditioned media (TCM) isolated from a human neuroendocrine (DU-145) and bone (PC-3) metastatic prostate cancer cell lines and assessed changes in cell morphology and cytotoxicity (using MTT). Exposure of HS-5 cells to TCM from DU-145 and PC-3 cells altered cellular morphology to a more mesenchymal phenotype similar to that seen in active myofibroblasts. TCM also decreased MTT staining in a time-dependent manner. We also determined the effect of GPC1 knockdown (KD) in HS-5 cells on cell morphology, migration and cytotoxicity. Interestingly, we saw phenotypic switching of GPC1 KD HS-5 cells to a myofibroblast like morphology akin to that when treated with TCM. There was no change in cytotoxicity in GPC1 KD HS-5 cells as compared to control cells. However, GPC1 KD enhanced the migration of HS-5 cells in wound healing assay. Migration was also increased in GPC1 KD HS-5 cells upon treatment with TCM. GPC1 KD also increased HS-5 cell contractility significantly. Collectively, these data support the hypothesis that GPC1 plays an inhibitory role in the TME of prostate cancer by regulating the activation of fibroblasts. Citation Format: Sukhneeraj P. Kaur, Hee K. Lee, Robert D. Arnold, Brian S. Cummings. Role of glypican 1 in regulating fibroblast activation in the prostate cancer tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1913.

Abstract 128: Effects of NSAIDs on gene expression of small leucine-rich proteoglycans in prostate cancer cells

Abstract Background: Malignant transformation has been shown to be favored by the interaction between cancer cells and the tumor microenvironment, with a special role attributed to molecular components present in both compartments, such as proteoglycans, including Small Leucine-Rich Proteoglycans (SLRPs). SLRPs are increasingly recognized as powerful signaling molecules involved in cancer growth and inflammation. Recent studies suggest that, among other cancers, prostate cancer (PCa) undergoes changes in the expression patterns of these components of the extracellular matrix to promote their survival, growth, and spread. Growing evidence links inflammation to initiation and progression to advanced metastatic disease of PCa and other human cancers. Non-steroidal anti-inflammatory drugs (NSAIDs) have been associated with reduced risk of several cancers, including PCa. The protective effect of NSAIDs has been attributed to inhibition of cancer metastasis. The aim of this work was to determine the expression profiles of SLRPs in a highly metastatic prostate cancer cell line (PC-3) compared to a non-tumorigenic cell line (PWR-1E) and to identify the effects of NSAIDs in the expression patterns of these molecular components. Methods: Differences in gene expression were determined by qRT-PCR for a panel of 15 SLRPs (ASPN, ECM2, LUM, PRELP, KERA, OMD, EPYC, OPTC, CHAD, NYX, PODN, OGN, BGN, FMOD, and DCN). Alterations in the expression of these genes in response to treatment with NSAIDs (ibuprofen and nimesulide) were also determined. Results: Compared to non-tumorigenic PWR-1E cells, highly metastatic PC-3 cells showed higher expression levels of seven genes encoding SLRPs (FMOD, BGN, LUM, ASPN, ECM2, PRELP and EPYC). Of the remaining eight genes, DCN was not differentially expressed while PODN, NYX, KERA, OMD, OPTC, OGN and CHAD had not detectable expression in either PC-3 or PWR-1E cells. Treatment of PC-3 cells with ibuprofen and nimesulide affected the expression pattern of genes encoding SLRPs. Ibuprofen treatment increased the expression of PRELP and BGN and decreased the expression of EPYC and ECM2. On the other hand, nimesulide treatment resulted in increased expression of PRELP and decreased expression of LUM, EPYC and ECM2. Conclusions: highly metastatic PC-3 cells had increased expression levels of SLRPs compared to non-tumorigenic PWR-1E cells. NSAIDs (Nimesulide and ibuprofen) changed the expression pattern of SLRPGs in metastatic PC-3 cells. Pharmacological effects of NSAIDs in prostate cancer warrants further investigation at the clinical settings. Additional studies are required to determine the clinical usefulness of NSAIDs in prostate cancer. Citation Format: Niradiz Reyes, Juan Rebollo, Jan Geliebter. Effects of NSAIDs on gene expression of small leucine-rich proteoglycans in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 128.

Abstract 1022: The role of dopamine signaling in prostate cancer disparities

Abstract Prostate cancer is the second leading cause of cancer-related deaths among men in western countries. Majority of prostate tumors often evolves as metastatic castration-resistant prostate cancer (CRPC) following hormonal therapy. Metastatic prostate cancer is the major cause of mortalities from this disease. African-American (AA) men have the highest incidence and mortality rate compared to any other race such as Caucasian American (CA). Despite recent advances, the cellular and molecular mechanisms that contribute to this disparity are unknown, obstructing the development of an effective therapy for this disease. Genetic and epigenetic studies have indicated that the biology of prostate cancer in AA men is substantially different from CA. Here, we investigate the dopamine (DA) signaling to better understand the mechanism of prostate cancer disparity. Our analysis of the prostate cancer public data sets showed that the dopamine receptor (DR) genes were amplified in up to 18% prostate cancer clinical cases. We have demonstrated that the levels of DR transcripts were significantly higher in the metastatic prostate cancer cell lines with African genetic ancestry than CA counters. We also demonstrate that the efficacy of pimozide, a potent DRD inhibitor and an FDA-approved antipsychotic, on AA cells was significantly higher than CA cells. In addition, we have found that L-dopa, an immediate precursor of DA differentially regulates the growth of AA and CA prostate cancer cells in a dose dependent manner. We also found that carbidopa, a potent inhibitor of the dopa decarboxylase, suppressed prostate cancer cell growth by L-dopa and showed that carbidopa re-sensitized CRPC cell line to enzalutamide, a potent inhibitor of androgen receptor (AR) signaling. These observations suggest that DA/DR signaling plays a critical role in prostate cancer disparity, making the DA/DR-AR axis a viable therapeutic target to reduce disparities in cancer mortality. Citation Format: Isaiah R. Pickett, Ava M. Boston, Marwah M. Almathkour, Camille Ragin, Mehmet A. Bilen, Adeboye O. Osunkoya, Bekir Cinar. The role of dopamine signaling in prostate cancer disparities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1022.

Abstract 5171: GRP78 as a potential modulator of cell adhesion markers in metastatic prostate cancer and multiple myeloma

Abstract Background: Glucose regulated protein 78 (GRP78) is a resident chaperone of the endoplasmic reticulum and a master regulator of the unfolded protein response under physiological and pathological cell stress conditions. GRP78 is overexpressed in many cancers, regulating a variety of signaling pathways associated with tumor initiation, proliferation, adhesion and invasion which contributes to metastatic spread. GRP78 can also regulate cell survival and apoptotic pathways to alter responsiveness to anticancer drugs. Tumors that reside in or metastasize to the bone and bone marrow (BM) space can develop pro-survival signals through their direct adhesive interactions with stromal elements of this niche thereby resisting the cytotoxic effects of drug treatment. In this study, we report a direct correlation between GRP78 and the adhesion molecule N-cadherin (N-cad), known to play a critical role in the adhesive interactions of multiple myeloma and metastatic prostate cancer with the bone microenvironment. Methods: N-cad expression levels (transcription and protein) were evaluated upon siRNA mediated silencing of GRP78 in the MM.1S multiple myeloma and the PC3 metastatic prostate cancer cell lines. Furthermore, we evaluated the effects of GRP78 knockdown (KD) on epithelial-mesenchymal (EMT) transition markers, morphological changes and adhesion of PC3 cells. Results: We demonstrate that GRP78 KD: 1) induced the concomitant downregulation of N-cad in both PC3 and MM.1S cells, 2) resulted in significant decreases of both N-cad and E-cad protein levels in PC3 cells, 3) induced TGF-β1 and Snail-2 expression, potentially accounting for the observed downregulation of E-cad. Our data also suggests that N-cad regulation via GRP78 KD supersedes the effects of TGF-β1; i.e., N-cad was downregulated in spite of the fact that TGF-β1 expression was significantly increased upon GRP78 KD. Conclusion: This work implicates GRP78 as a modulator of cell adhesion markers in MM and PCa. Our results may have clinical implications underscoring GRP78 as a potential therapeutic target to reduce the adhesive nature of metastatic tumors to the bone niche. Citation Format: Christopher Cultrara, David Sabatino, Jenny Zilberberg. GRP78 as a potential modulator of cell adhesion markers in metastatic prostate cancer and multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5171.

Abstract 2960: NCX4040, a nitric oxide (NO) donating form of aspirin as a potential treatment for prostate cancer

Abstract Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to be beneficial in preventing various types of cancers including prostate cancer (PCa). Of all the NSAIDs in use, Aspirin appears to be one of the promising anticancer agents for PCa management. However, experimental and epidemiological studies indicated that larger doses of Aspirin and long term supplementation is required to achieve anticancer effect with accompanied side effects. Therefore, our study focused on evaluating Aspirin derivative, namely NCX4040 which is a NO releasing form of Aspirin as a potent alternative for PCa treatment while minimizing the side effects. In the present study, we assessed the anticancer effects of NCX4040 on PC-3, a bone metastatic PCa cell line employing various in-vitro cellular assays. In our preliminary studies, we compared the effects of NCX4040 with that of Aspirin and NO releasing compound DETA/NO on PC-3 cells using MTT as read out. These results showed that NCX4040 is highly potent in inhibiting the viability of PC-3 cells when compared to Aspirin or DETA/NO treatments. Hence, further studies were focused on characterizing the anticancer properties as well as the underlying anticancer mechanism of NCX4040. Of note, our results showed that NCX4040 robustly inhibited the colony forming ability of PC-3 cells in-vitro implicating its antitumorigenic potential. In addition to inhibiting colony formation of PC-3 cells, NCX4040 also suppressed the Transwell matrigel invasion of PC-3 cells suggesting the antimetastatic effects of NCX4040. The observed antimetastatic effect of NCX4040 in PC-3 PCa cells appears to be via targeting actin cytoskeleton as confirmed by confocal microscopic analysis using phalloidin stain. Following these findings, we performed cell cycle analysis to determine whether NCX4040 enforces cell cycle arrest in PC-3 cells. These results showed that NCX4040 induced robust G0 cell cycle arrest in a dose dependent manner. Moreover, flow cytometry analysis using Annexin V and PI staining revealed that NCX4040 induced potent apoptotic cell death in PC-3 cells. To delineate the underlying anticancer mechanism of NCX4040, PC-3 cells treated with NCX4040 was subjected to apoptotic protein array analysis. Results of this study inferred that NCX4040 down regulated the expression of key anti-apoptotic proteins such as Survivin, XIAP and IAPs. In conclusion, our study suggests that NCX4040 is a promising alternative agent for PCa treatment. Citation Format: SOMAIAH CHINNAPAKA, Gnanasekar Munirathinam. NCX4040, a nitric oxide (NO) donating form of aspirin as a potential treatment for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2960.

MiR-451 suppresses the growth, migration, and invasion of prostate cancer cells by targeting macrophage migration inhibitory factor

Background: Metastasis is the most common cause of death of prostate cancer patients. Identification of key regulators in prostate cancer metastasis is essential for improved management of the disease. Accumulation of evidence suggests that miRNAs play important roles in cancer metastasis. Here, we investigated the role of miR-451 in prostate cancer progression and metastasis. Methods: In this study, we performed reverse transcription-quantitative PCR analysis to examine the expression of miR-451 in clinical prostate cancer samples and cell lines. TargetScan Release 7.0 programs, luciferase reporter assay, and a rescue experiment were used to validate the direct target of miR-451. The protein expression of the miR-451 target was detected by Western blot. MTS cell viability assay, transwell migration assay, and modified Boyden chamber invasion assay were used to demonstrate the effects of increased miR-451 expression on prostate cancer cell growth, migration and invasiveness. Results: We identified the decreased expression of miR-451 in clinical metastatic prostate cancer compared to localized primary prostate cancer and benign prostate. Consistently, we also observed the decreased expression of miR-451 in highly metastatic prostate cancer cell lines (C4-2 and PC3M) compared to their low metastatic counterparts (LNCaP and PC3). Furthermore, we confirmed the macrophage migration inhibitory factor (MIF) is a direct target of miR-451 in prostate cancer and demonstrated a negative correlation between miR-451 and MIF expression in clinical prostate cancer samples. Functional studies showed increased miR-451 expression significantly suppressed cell growth, migration and invasiveness via targeting MIF. The effect of increased miR-451 expression can be reversed by overexpressing MIF in prostate cancer cells. Conclusions: These findings suggest that miR-451 suppresses prostate cancer cell growth, migration and metastasis, which provides insight into the development of novel therapeutic approaches for prostate cancer treatment.

MiR-451 suppresses the growth, migration, and invasion of prostate cancer cells by targeting macrophage migration inhibitory factor

BackgroundMetastasis is the most common cause of death of prostate cancer patients. Identification of key regulators in prostate cancer metastasis is essential for improved management of the disease. Accumulation of evidence suggests that miRNAs play important roles in cancer metastasis. Here, we investigated the role of miR-451 in prostate cancer progression and metastasis.MethodsIn this study, we performed reverse transcription-quantitative PCR analysis to examine the expression of miR-451 in clinical prostate cancer samples and cell lines. TargetScan Release 7.0 programs, luciferase reporter assay, and a rescue experiment were used to validate the direct target of miR-451. The protein expression of the miR-451 target was detected by Western blot. MTS cell viability assay, transwell migration assay, and modified Boyden chamber invasion assay were used to demonstrate the effects of increased miR-451 expression on prostate cancer cell growth, migration and invasiveness.ResultsWe identified the decreased expression of miR-451 in clinical metastatic prostate cancer compared to localized primary prostate cancer and benign prostate. Consistently, we also observed the decreased expression of miR-451 in highly metastatic prostate cancer cell lines (C4-2 and PC3M) compared to their low metastatic counterparts (LNCaP and PC3). Furthermore, we confirmed the macrophage migration inhibitory factor (MIF) is a direct target of miR-451 in prostate cancer and demonstrated a negative correlation between miR-451 and MIF expression in clinical prostate cancer samples. Functional studies showed increased miR-451 expression significantly suppressed cell growth, migration and invasiveness via targeting MIF. The effect of increased miR-451 expression can be reversed by overexpressing MIF in prostate cancer cells.ConclusionsThese findings suggest that miR-451 suppresses prostate cancer cell growth, migration and metastasis, which provides insight into the development of novel therapeutic approaches for prostate cancer treatment.

Metastatic prostate cancer cells are highly sensitive to 3-bromopyruvic acid

Aims3-Bromopyruvate (3-BP), an alkylating agent and a glycolytic inhibitor, is a promising anticancer agent, which can be efficient also against multidrug-resistant cancer cells. The aim of this study was to examine how 3-BP affects the survival and mobility of rat (MAT-LyLu and AT-2) and human (DU-145 and PC-3) metastatic prostate cancer cell lines. Main methodsCytotoxicity was estimated with Neutral Red. Cell mobility was analyzed by time-lapse microscopic monitoring of trajectories of individual cells at 5-min intervals for 6h. ATP was estimated with luciferin/luciferase and glutathione (GSH) with o-phthalaldehyde. Actin cytoskeleton was visualized with phalloidin conjugated with Atto-488. Key findingsAll metastatic prostate cell lines studied were very sensitive to 3-BP (IC50 of 4–26μM). 3-Bromopyruvate drastically reduced cell movement even at concentrations of 5–10μM after 1h treatment. This compound depleted also cellular ATP and GSH, and disrupted actin cytoskeleton. SignificanceThe data obtained suggest that 3-BP can potentially be useful for treatment of metastatic prostate cancer and, especially, be efficient in limiting metastasis.