Primary Prostate Cancer Cells Research Articles

Silencing of METTL3 effectively hinders invasion and metastasis of prostate cancer cells.

Background: Since primary prostate cancer (PCa) can advance to the life-threatening metastatic PCa, exploring the molecular mechanisms underlying PCa metastasis is crucial for developing the novel targeted preventive strategies for decreasing the mortality of PCa. RNA N6-methyladenosine (m6A) is an emerging regulatory mechanism for gene expression and its specific roles in PCa progression remains elusive.Methods: Western blotting, quantitative real-time PCR and immunohistochemical analyses were used to detect target gene expression in PCa cells in vitro and prostate tissues from patients. RNA immunoprecipitation was conducted to analyze the specific binding of mRNA to the target protein. Migration and invasion assays were used to assess the migratory capacities of cancer cells. The correlation between target gene expression and survival rate of PCa patients was analyzed based the TCGA database.Results: We found that total RNA N6-methyladenosine (m6A) modification levels were markedly upregulated in human PCa tissues due to increased expression of methyltransferase like 3 (METTL3). Further studies revealed that the migratory and invasive capacities of PCa cells were markedly suppressed upon METTL3 knockdown. Mechanistically, METTL3 mediates m6A modification of USP4 mRNA at A2696, and m6A reader protein YTHDF2 binds to and induces degradation of USP4 mRNA by recruiting RNA-binding protein HNRNPD to the mRNA. Decrease of USP4 fails to remove the ubiquitin group from ELAVL1 protein, resulting in a reduction of ELAVL1 protein. Lastly, downregulation of ELAVL1 in turn increases ARHGDIA expression, promoting migration and invasion of PCa cells.Conclusions: Our findings highlight the role of METTL3 in modulating invasion and metastasis of PCa cells, providing insight into promising therapeutic strategies for hindering PCa progressing to deadly metastases.

Identification of AR-V7 downstream genes commonly targeted by AR/AR-V7 and specifically targeted by AR-V7 in castration resistant prostate cancer

Primary prostate cancer (PC) progresses to castration-resistant PC (CRPC) under androgen deprivation therapy, by mechanisms e.g. expression of androgen receptor (AR) splice variant-7 (AR-V7). Here we conducted comprehensive epigenome and transcriptome analyses comparing LNCaP, primary PC cells, and LNCaP95, AR-V7-expressing CRPC cells derived from LNCaP. Of 399 AR-V7 target regions identified through ChIP-seq analysis, 377 could be commonly targeted by hormone-stimulated AR, and 22 were specifically targeted by AR-V7. Among genes neighboring to these AR-V7 target regions, 78 genes were highly expressed in LNCaP95, while AR-V7 knockdown led to significant repression of these genes and suppression of growth of LNCaP95. Of the 78 AR-V7 target genes, 74 were common AR/AR-V7 target genes and 4 were specific AR-V7 target genes; their most suppressed genes by AR-V7 knockdown were NUP210 and SLC3A2, respectively, and underwent subsequent analyses. NUP210 and SLC3A2 were significantly upregulated in clinical CRPC tissues, and their knockdown resulted in significant suppression of cellular growth of LNCaP95 through apoptosis and growth arrest. Collectively, AR-V7 contributes to CRPC proliferation by activating both common AR/AR-V7 target and specific AR-V7 target, e.g. NUP210 and SLC3A2.

LncRNA UCA1 maintains the low-tumorigenic and nonmetastatic status by stabilizing E-cadherin in primary prostate cancer cells.

Long noncoding RNAs (LncRNAs) have emerged as important players in cancer biology. Increasing evidence suggests that LncRNAs are frequently dysregulated in cancer and may function as oncogenes or tumor suppressors. Urothelial carcinoma associated 1 (UCA1), a LncRNA, firstly identified in bladder transitional cell carcinoma, seems to act as an oncogene in many different types of human cancers by promoting cell proliferation and migration. In this study, we revealed a novel biological function of UCA1, which was different from thatreported by previous studies, was responsible for maintaining the low-tumorigenic, nonmetastatic phenotypes in primary prostate epithelial cells. UCA1 could stabilize E-cadherin protein by preventing the interaction between E-cadherin and its E3 ligase MDM2, which suppressed MDM2-mediated ubiquitination and degradation of E-cadherin. In addition, we also found that UCA1 acted as a sponge of miR-296-3p, which targeted E-cadherin gene CDH1 messenger RNA at the posttranscription level. Taken together, these findings demonstrated that UCA1 had a new important role in effectively keeping E-cadherin at a high level through a dual mechanism, which maintained primary prostate cancer cells at the low-tumorigenic and nonmetastatic status.

Abstract 2015: A transcriptionally enhanced biosensor to detect and monitor biopsy-dissociated primary prostate cancer single cell drug responses by bioluminescence microscopy

Abstract Background: In the past decade, the therapeutic landscape of metastatic castration resistant prostate cancer (mCRPC) has been transformed with the introduction of novel pharmaceutical agents including novel antiandrogens (nAA) such as darolutamide, enzalutamide or apalutamide. Currently, the management of mCRPC is mainly based on biomarkers obtained from the patient's blood or bulk tumor samples, which do not take into account for prostate cancer (PCa) cell heterogeneity. Objectives: To develop a bioluminescence imaging technology enabling: 1) the detection of PCa single cells from dissociated biopsies and 2) single cell nAA sensitivity assessment. Methods: The PCA3 promoter (PCa specific) and PSEBC promoter (androgen receptor-driven to monitor response to nAA therapy) have been incorporated in the non-replicating adenoviral multi-promoter integrated two-step transcriptional amplification system (MP-ITSTA) that allows imaging of complementary activities of two different promoters by a single output reporter gene. Thus, PCA3/PSEBC-ITSTA system was designed to monitor androgen receptor (AR) activity specifically within each single PCa cell by bioluminescence microscopy. Prostatic biopsies were dissociated in the presence of collagenase II and DNase for 18h. PCa cell lines or fresh biopsies-dissociated cancer cells were transduced for 72h and then covered with an extracellular matrix gel. Single cell bioluminescence microscopy images were done before and after exposure to DHT or DHT+AA to specifically monitoring therapy response. Changes in cell number and luminescence intensity after AA were normalized to the DHT group to exclude non-specific cell-death due to infection or time spent in culture. Results: PCA3/PSEBC-ITSTA was specific to PCa cell lines. By determining the ratio of AR active cells before and after DHT or DHT+AA treatment in culture, PCA3-Cre-PSEBC-ITSTA was able to determine the AA sensitivity of LNCaP (sensitive), LAPC4 (moderately resistant) and 22Rv1 (resistant) PCa cell lines. Also, our method could detect and monitor AA sensitivity of primary PCa cells harvested from eight radical prostatectomy specimens. PCA3/PSEBC-ITSTA could dynamically quantify AR transcriptional activity during enzalutamide or bicalutamide treatments, in a dose dependent manner, and could unveil tumor AA response heterogeneity. As expected, inhibition of AR activity was stronger with enzalutamide than with bicalutamide. Conclusions: Our bioluminescence microscopy-based biosensor could detect primary PCa cells in culture and it allows dynamic evaluation of AA sensitivity at a single-cell level in a heterogeneous cell population harvested from radical prostatectomy biopsies. We believe our approach opens to a novel field of treatment response predictive tools for cancer treatment decision-making. Citation Format: Audrey Champagne, Pallavi Jain, Bertrand Neveu, Frédéric Pouliot. A transcriptionally enhanced biosensor to detect and monitor biopsy-dissociated primary prostate cancer single cell drug responses by bioluminescence microscopy [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 2015.

Abstract 2802: Establishing and optimizing treatment-naïve prostate cancer patient-derived organoids culture: Implications for personalized medicine

Abstract Background: Prostate Cancer (PCa) is a heterogeneous disease. Currently available in vitro and in vivo cancer models fail to recapitulate the spectrum of clinical and biological challenges; accurate models for translational research remain a significant unmet need. Objective: The aim of this work is to employ fresh tissue specimens from a treatment-naïve cohort of patients with prostate cancer to optimize and establish 3D patient-derived organoids as an in vitro disease model for PCa progression and drug response. In addition, acknowledging the difficulty in establishing primary PCa cell lines, our aim was to generate novel PCa patient-derived cell lines. Methods: Fresh specimens were collected from treatment-naïve patients with prostate cancer during radical prostatectomy (one unaffected and one tumor). Briefly, fresh tissue samples were digested enzymatically, and the resulting cell suspensions were plated in a 3D environment that employs Matrigel as an extracellular matrix. At first, a cocktail of 12 factors, previously recognized, was used to establish organoids. Subsequently, we have optimized the recipe to a minimal 5-factors media for both organoids and cells. Organoids and the corresponding tissue specimens were characterized using immunofluorescent analysis and immunohistochemistry. Furthermore, patient-derived organoids were employed for the assessment of drug response. In addition, PCa patient-derived cell lines were generated and their culture conditions were optimized. These cells were further characterized using immunofluorescent analysis. Results: Fresh tissues were successfully established as organoids and as 2D cells using the same culture media that contains 12 original factors. The presence of prostate luminal (Cytokeratin 8, Androgen Receptor, and Prostate Specific Antigen) and basal (Cytokeratin 5 and P63) epithelial lineages was confirmed by immunohistochemical and immunofluorescent analyses. Notably, we have identified five essential components (NAC, NOG, A83, B27, and Nicotinamide) that support the establishment of organoids culture in a less expensive, more efficient manner. Moreover, we optimized the culture and maintenance of patient-derived 2D cells by plating on collagen type I and we have reduced the medium requirement to include EGF only as an essential component. Importantly, the results showed differential drug and radiation response between patient samples. Conclusion: This study provides a repertoire of novel patient-derived organoids and cells from a unique cohort of treatment-naïve patients, as our results demonstrate that we succeeded in delineating the essential components needed to grow prostate organoids and primary cells with a high success rate and long-term maintenance in culture. We hope that we can use this platform in the future to predict treatment response in a personalized fashion leading to better management of the disease. Citation Format: Katia Cheaito, Hisham Bahmad, Alissar Monzer, Farah Ballout, HIba Msheik, Ola Hadadeh, Christelle Dagher, Nour Saheb, Ayman Tawil, Marwan El-Sabban, Albert El-Hajj, Deborah Mukherji, Wassim G. Abou-Kheir. Establishing and optimizing treatment-naïve prostate cancer patient-derived organoids culture: Implications for personalized medicine [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 2802.

Abstract 3645: Epigenetic regulation of oncogenic transcription mediated by aberrantly activated enhancers in prostate cancer

Abstract Since androgen receptor (AR) is a master transcription factor of prostate gland in both normal development and carcinogenesis, androgen deprivation therapy (ADT) is quite effective for primary prostate cancer (PC) even in advanced disease. During ADT, however, PC tumors generally acquire resistance through oncogenic activation independent from androgen-AR signaling, and progress to lethal castration-resistant PC (CRPC). Transcription factors (TFs) such as AR regulate gene expression through binding regulatory elements of genome called as enhancers. Aberrantly activated enhancers regulating oncogenes could be one of the mechanisms of cancer therapy resistance. In this study, to clarify how aberrant enhancer activation contribute to genesis of CRPC, we performed comprehensive transcriptome and epigenome analyses by RNA-seq and ChIP-seq for H3K4me1, H3K4me3, H3K27ac, H3K79me2, BRD4, AR, FOXA1 and RNA polymerase 2 (RNApol2). Using androgen dependent PC cells LNCaP and its derivative CRPC cells LNCaP95, we extracted BRD4(+)/AR(+)/FOXA1(+) enhancers and super enhancers (SEs) defined by cluster of H3K27ac regardless of BRD4, AR, FOXA1 binding. SE neighboring genes were mostly targeted by BRD4(+)/AR(+)/FOXA1(+) enhancers in LNCaP (57.5%), but markedly less in LNCaP95 (25.3%), suggesting a shift from AR-dependent to AR-independent regulation. In contrast, SE neighboring genes were mostly epigenetically modified with H3K79me2 in both LNCaP (77.4%) and LNCaP95 (76.2%). In addition, the mRNA expression levels of H3K79methyltansferase DOT1L was upregulated in CRPC clinical tissues. While BRD4 inhibition was quite effective for suppressing cell growth in LNCaP, but less effective in LNCaP95, DOT1L inhibition was markedly effective to both cells, inducing marked cell cycle arrest and apoptosis. Furthermore, precise analyses of RNApol2 behavior and transcriptome alteration revealed that BRD4 and DOT1L regulated different genes in a different manner. Mechanistically, while BRD4 inhibition suppressed AR target genes through reducing initiation of transcriptional elongation, DOT1L inhibition suppressed SE neighboring genes through reducing termination of transcriptional elongation. In conclusion, our results indicated that BRD4 and DOT1L played critical role in regulating transcription mediated by aberrantly activated enhancers, and could be targeted as an epigenetic therapeutic strategy for CRPC. Citation Format: Hiroaki Sato, Masahiro Sugiura, Manato Kanesaka, Atsushi Okabe, Masaki Fukuyo, Shinichi Sakamoto, Akira Komiya, Tomohiko Ichikawa, Atsushi Kaneda. Epigenetic regulation of oncogenic transcription mediated by aberrantly activated enhancers in prostate cancer [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 3645.

SETD1A Promotes Proliferation of Castration-Resistant Prostate Cancer Cells via FOXM1 Transcription.

Androgen deprivation therapy eventually leads to the development of castration-resistant prostate cancer (CRPC). Here, we demonstrate for the first time that the histone H3K4 methyltransferase SETD1A is a major regulator for the proliferation of metastatic CRPC (mCRPC). The expression of SETD1A was significantly correlated with the survival rate of patients with prostate cancer. SETD1A, which is expressed at a higher level in mCRPC than in primary prostate cancer cells, promotes the expression of FOXM1, a gene encoding a cell proliferation-specific transcription factor. SETD1A is recruited to the promoter region of FOXM1 (forkhead box M1) upon binding to E2F1, a protein that regulates the transcription of FOXM1 and contributes to the trimethylation of H3K4 in the FOXM1 promoter region. In addition, SETD1A is essential for the expression of stem cell factor (e.g., OCT4, octamer-binding transcription factor 4) and stem cell formation in mCRPC, suggesting the importance of SETD1A expression in mCRPC tumor formation. Notably, poor prognosis is associated with high expression of the SETD1A–FOXM1 pair in clinical data sets. Therefore, our study suggests that SETD1A plays an important role in the proliferation of mCRPC by regulating FOXM1 transcription.

Comparison of the Gene Expression Profiles of Human Primary Prostate Epithelial Cells and DU145 Prostate Cancer Cells

Comparison of the Gene Expression Profiles of Human Primary Prostate Epithelial Cells and DU145 Prostate Cancer Cells

Comparison of the Gene Expression Profiles of Human Primary Prostate Epithelial Cells and DU145 Prostate Cancer Cells

Prostate cancer remains the second leading cause of cancerassociated deaths of men in the United States...

Intraoperative 68Ga-PSMA Cerenkov Luminescence Imaging for Surgical Margins in Radical Prostatectomy: A Feasibility Study.

Our objective was to assess the feasibility and accuracy of Cerenkov luminescence imaging (CLI) for assessment of surgical margins intraoperatively during radical prostatectomy. Methods: A single-center feasibility study included 10 patients with high-risk primary prostate cancer (PC). 68Ga-prostate-specific membrane antigen (PSMA) PET/CT scans were performed followed by radical prostatectomy and intraoperative CLI of the excised prostate. In addition to imaging the intact prostate, in the first 2 patients the prostate gland was incised and imaged with CLI to visualize the primary tumor. We compared the tumor margin status on CLI to postoperative histopathology. Measured CLI intensities were determined as tumor-to-background ratio. Results: Tumor cells were successfully detected on the incised prostate CLI images as confirmed by histopathology. Three of 10 men had histopathologically positive surgical margins (PSMs), and 2 of 3 PSMs were accurately detected on CLI. Overall, 25 (72%) of 35 regions of interest proved to visualize a tumor signal according to standard histopathology. The median tumor radiance in these areas was 11,301 photons/s/cm2/sr (range, 3,328-25,428 photons/s/cm2/sr), and median tumor-to-background ratio was 4.2 (range, 2.1-11.6). False-positive signals were seen mainly at the prostate base, with PC cells overlaid by benign tissue. PSMA immunohistochemistry revealed strong PSMA staining of benign gland tissue, which impacts measured activities. Conclusion: This feasibility showed that 68Ga-PSMA CLI is a new intraoperative imaging technique capable of imaging the entire specimen's surface to detect PC tissue at the resection margin. Further optimization of the CLI protocol, or the use of lower-energy imaging tracers such as 18F-PSMA, is required to reduce false-positives. A larger study will be performed to assess diagnostic performance.

SMAD3/SP1 complex-mediated constitutive active loop between lncRNA PCAT7 and TGF-β signaling promotes prostate cancer bone metastasis.

Bone metastasis is associated with cancer‐related death in patients with prostate cancer (PCa). Long noncoding RNAs (lncRNAs) play critical roles in tumor progression of PCa. Nevertheless, the biological function of lncRNAs in PCa bone metastasis remains unclear. PCAT7 was identified as a bone metastasis‐related lncRNA via analyzing TCGA dataset. Meanwhile, PCAT7 was found to be elevated in primary PCa tissues with bone metastasis and associated with bone metastasis status and poor prognosis of patients with PCa. Functionally, our results reveal that PCAT7 overexpression promotes PCa bone metastasis in vivo, as well as migration, invasion, and EMT of PCa cells in vitro; on the contrary, PCAT7 knockdown has an inverse effect. Mechanistically, PCAT7 activates TGF‐β/SMAD signaling by upregulating TGFBR1 expression via sponging miR‐324‐5p. In turn, TGF‐β signaling forms a positive feedback loop with PCAT7 via SMAD3/SP1 complex‐induced PCAT7 upregulation. Finally, the clinical positive correlation between PCAT7 and TGFBR1 and TGF‐β signaling activity, and the negative association with miR‐324‐5p are further demonstrated in PCa tissues and clinical primary PCa cells. This study reveals a novel mechanism that is responsible for the constitutive activation of TGF‐β signaling in PCa bone metastasis, implying that PCAT7 can act as a potential therapeutic target against bone metastasis of PCa via disrupting the constitutive active loop between PCAT7 and TGF‐β signaling.

Primary prostate cancer educates bone stroma through exosomal pyruvate kinase M2 to promote bone metastasis.

Prostate cancer (PCa) metastasizes selectively to bone through unknown mechanisms. In the current study, we identified exosome-mediated transfer of pyruvate kinase M2 (PKM2) from PCa cells into bone marrow stromal cells (BMSCs) as a novel mechanism through which primary tumor-derived exosomes promote premetastatic niche formation. We found that PKM2 up-regulates BMSC CXCL12 production in a HIF-1α-dependent fashion, which subsequently enhances PCa seeding and growth in the bone marrow. Furthermore, serum-derived exosomes from patients with either primary PCa or PCa metastasis, as opposed to healthy men, reveal that increased exosome PKM2 expression is associated with metastasis, suggesting clinical relevance of exosome PKM2 in PCa. Targeting the exosome-induced CXCL12 axis diminished exosome-mediated bone metastasis. In summary, primary PCa cells educate the bone marrow to create a premetastatic niche through primary PCa exosome-mediated transfer of PKM2 into BMSCs and subsequent up-regulation of CXCL12. This novel mechanism indicates the potential for exosome PKM2 as a biomarker and suggests therapeutic targets for PCa bone metastasis.

EGF-activated PI3K/Akt signalling coordinates leucine uptake by regulating LAT3 expression in prostate cancer

BackgroundGrowth factors, such as EGF, activate the PI3K/Akt/mTORC1 signalling pathway, which regulates a distinct program of protein synthesis leading to cell growth. This pathway relies on mTORC1 sensing sufficient levels of intracellular amino acids, such as leucine, which are required for mTORC1 activation. However, it is currently unknown whether there is a direct link between these external growth signals and intracellular amino acid levels. In primary prostate cancer cells, intracellular leucine levels are regulated by L-type amino acid transporter 3 (LAT3/SLC43A1), and we therefore investigated whether LAT3 is regulated by growth factor signalling.MethodsTo investigate how PI3K/Akt signalling regulates leucine transport, prostate cancer cells were treated with different PI3K/Akt inhibitors, or stable knock down of LAT3 by shRNA, followed by analysis of leucine uptake, western blotting, immunofluorescent staining and proximity ligation assay.ResultsInhibition of PI3K/Akt signalling significantly reduced leucine transport in LNCaP and PC-3 human prostate cancer cell lines, while growth factor addition significantly increased leucine uptake. These effects appeared to be mediated by LAT3 transport, as LAT3 knockdown blocked leucine uptake, and was not rescued by growth factor activation or further inhibited by signalling pathway inhibition. We further demonstrated that EGF significantly increased LAT3 protein levels when Akt was phosphorylated, and that Akt and LAT3 co-localised on the plasma membrane in EGF-activated LNCaP cells. These effects were likely due to stabilisation of LAT3 protein levels on the plasma membrane, with EGF treatment preventing ubiquitin-mediated LAT3 degradation.ConclusionGrowth factor-activated PI3K/Akt signalling pathway regulates leucine transport through LAT3 in prostate cancer cell lines. These data support a direct link between growth factor and amino acid uptake, providing a mechanism by which the cells rapidly coordinate amino acid uptake for cell growth.

Human SP-D Acts as an Innate Immune Surveillance Molecule Against Androgen-Responsive and Androgen-Resistant Prostate Cancer Cells.

Surfactant Protein D (SP-D), a pattern recognition innate immune molecule, has been implicated in the immune surveillance against cancer. A recent report showed an association of decreased SP-D expression in human prostate adenocarcinoma with an increased Gleason score and severity. In the present study, the SP-D expression was evaluated in primary prostate epithelial cells (PrEC) and prostate cancer cell lines. LNCaP, an androgen dependent prostate cancer cell line, exhibited significantly lower mRNA and protein levels of SP-D than PrEC and the androgen independent cell lines (PC3 and DU145). A recombinant fragment of human SP-D, rfhSP-D, showed a dose and time dependent binding to prostate cancer cells via its carbohydrate recognition domain. This study, for the first time, provides evidence of significant and specific cell death of tumor cells in rfhSP-D treated explants as well as primary tumor cells isolated from tissue biopsies of metatstatic prostate cancer patients. Viability of PrEC was not altered by rfhSP-D. Treated LNCaP (p53+/+) and PC3 (p53 −/−) cells exhibited reduced cell viability in a dose and time dependent manner and were arrested in G2/M and G1/G0 phase of the cell cycle, respectively. rfhSP-D treated LNCaP cells showed a significant upregulation of p53 whereas a significant downregulation of pAkt was observed in both PC3 and LNCaP cell lines. The rfhSP-D-induced apoptosis signaling cascade involved upregulation of Bax:Bcl2 ratio, cytochrome c and cleaved products of caspase 7. The study concludes that rfhSP-D induces apoptosis in prostate tumor explants as well as in androgen dependent and independent prostate cancer cells via p53 and pAkt pathways.

Spliceosome component SF3B1 as novel prognostic biomarker and therapeutic target for prostate cancer

Prostate cancer (PCa) is one of the most common cancers types among men. Development and progression of PCa is associated with aberrant expression of oncogenic splicing-variants (eg, AR-v7), suggesting that dysregulation of the splicing process might represent a potential actionable target for PCa. Expression levels (mRNA and protein) of SF3B1, one of the main components of the splicing machinery, were analyzed in different cohorts of PCa patients (clinically localized [n = 84], highly aggressive PCa [n = 42], and TCGA dataset [n = 497]). Functional and mechanistic assays were performed in response to pladienolide-B in nontumor and tumor-derived prostate cells. Our results revealed that SF3B1 was overexpressed in PCa tissues and its levels were associated with clinically relevant PCa-aggressive features (eg, metastasis/AR-v7 expression). Moreover, inhibition of SF3B1 activity by pladienolide-B reduced functional parameters of aggressiveness (proliferation/migration/tumorspheres-formation/apoptosis) in PCa cell lines, irrespective of AR-v7 expression, and reduced viability of primary PCa cells. Antitumor actions of pladienolide-B involved: (1) inhibition of PI3K/AKT and JNK signaling pathways, (2) modulation of tumor markers and splicing variants (AR-v7/In1-ghrelin), and (3) regulation of key components of mRNA homeostasis-associated machineries (spliceosome/SURF/EJC). Altogether, our results demonstrated that SF3B1 is overexpressed and associated with malignant features in PCa, and its inhibition reduces PCa aggressiveness, suggesting that SF3B1 could represent a novel prognostic biomarker and a therapeutic target in PCa.

Abstract 2653: Usefulness of plasma exosomes to characterize hypoxic phenotype in primary prostate tumors

Abstract Hypoxia in primary prostate cancer (PCa) selects aggressive clones and causes treatment failure. However, there are several clinical challenges in measuring hypoxia in PCa. For example, MRI needs sophisticated instruments and highly trained personnel; needle electrodes could cause bleeding; and pimonidazole and immuno-histochemistry (IHC) for hypoxia biomarkers require surgical removal of tumor. Hence, novel non-invasive measures are needed to assess hypoxia. Exosomes are nano-vesicles (40-150 nm in size) secreted by cells for intercellular communication and are present in all biofluids loaded with unique cargo that could predict the cell of their origin and cellular physiologic state. Our primary goal here was to use exosomes to non-invasively assess hypoxia in PCa. We employed ultracentrifugation, mass spectrometry (MS), immunogold labeling, electron microscopy, PET, IHC, xenograft and immune-pull down methods. First, we isolated exosomes from the conditioned media of African American PCa MDA PCa 2b and E006AA-hT cells cultured under normoxia (21% O2) and hypoxia (1% O2) by ultracentrifugation. Next, through a novel method involving exosomal surface shaving and LC-MS/MS, we identified 13 unique proteins present only on the surface of exosomes secreted by PCa cells under hypoxia. Among the 13 unique proteins, Annexin A6 expression was highly correlated with 44 hypoxia signature genes in TCGA-PRAD. Next, Annexin A6 expression was confirmed on the surface of exosomes isolated from the blood of nude mice implanted with human PCa 22Rv1 xenografts. Hypoxia in xenografts was confirmed by F18MISO-PET imaging, and pimonidazole staining in xenograft tissues. Next, we pull-down Annexin A6+ exosomes using biotin-tagged human Annexin A6 antibody and streptavidin tagged agarose resin, and analyzed proteins loading by MS. Interestingly, Annexin A6+ exosomes showed a hypoxia signature as 35/172 loaded proteins were hypoxia and HIF1α regulated. We next isolated Annexin A6+ exosomes from the blood of African American and Caucasian PCa patients (n=5 each) with high grade disease (Gleason score >7) and confirmed Annexin A6 expression on their surface. Importantly, for similar Gleason score disease, Annexin A6+ exosomes concentration was 2.6 times higher in African American patients, and could be associated with disease aggressiveness. Together, these results suggest that exosomes could be useful to non-invasively assess hypoxia in PCa. Citation Format: Gatikrushna Panigrahi, Yixin Su, Leslimar Rios-Colon, Taylor Peak, Kiran Sai, Prakash Praharaj, Jingyun Lee, Cristina Furdui, Ashok Hemal, Deepak Kumar, Gagan Deep. Usefulness of plasma exosomes to characterize hypoxic phenotype in primary prostate tumors [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 2653.

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.

3D porous chitosan-alginate scaffold stiffness promotes differential responses in prostate cancer cell lines

Prostate cancer (PCa) is a leading cause of death for men worldwide. Most PCa patients die from metastasis and bone is the most common metastatic site. Three dimensional (3D) porous chitosan-alginate (CA) scaffolds were developed for bone tissue engineering and demonstrated for culture of cancer cells and enrichment of cancer stem cells. However, only a single scaffold composition was studied. Three compositions of 3D porous CA scaffolds (2, 4, and 6 wt%) were used to investigate the effect of scaffold stiffness on PCa cell response with PC-3, C4-2B, and 22Rv1 cell lines. The PC-3 cells formed cell clusters while the C4-2B and 22Rv1 cells formed multicellular spheroids. The three cell lines demonstrated stiffness independent cell growth and expressed phenotypic PCa biomarkers. The osteoblastic PCa lines C4-2B and 22Rv1 mineralized in basal media, while the osteolytic PC-3 line did not, demonstrating that CA scaffold cultures revealed differences in PCa phenotypes. The CA scaffolds are a 3D culture platform that supports PCa growth and phenotypic expression with adjustable scaffold stiffness to mimic stages of metastatic progression. Further investigation of the scaffolds for co-culture of PCa cells with fibroblasts and primary PCa cell culture should be conducted to develop a platform for screening chemotherapies.

JQ1, a BET-bromodomain inhibitor, inhibits human cancer growth and suppresses PD-L1 expression.

Most traditional cytotoxic chemotherapeutic agents have poor aqueous solubility and significant toxicity. Hence, there is a need to develop molecule-targeted drugs. Programmed death-ligand 1 (PD-L1) is associated with the prognosis of several cancer types, and blockade of PD-1/PD-L1 signaling increases the amplitude of anti-tumor immunity. In the present study, we investigated the effects of JQ1, a bromodomain and extraterminal-bromodomain inhibitor, on cell growth, and messenger RNA (mRNA) and protein levels of PD-L1 in renal cell carcinoma primary culture cells, and prostate, liver, and lung cancer cell lines. The results of the cell counting kit-8 assay suggested that JQ1 inhibits cell growth in a dose-dependent manner. The mRNA and protein levels of PD-L1 decreased in the primary culture of JQ1-treated renal carcinoma, prostate cancer, liver cancer, and lung cancer cell lines. In addition, the mRNA level of PD-L2 also decreased in the JQ1-treated cells. Overall, JQ1 might be a potential anti-tumor agent.

Auriculasin sensitizes primary prostate cancer cells to TRAIL-mediated apoptosis through up-regulation of the DR5-dependent pathway

Primary prostate cancer cells frequently develop resistance toward chemotherapy as well as most chemotherapeutics have been reported to induce undesirable cytotoxicity in normal cells. In this study, we performed sensitizing activity analysis of auriculasin (AC) to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in RC-58T/h/SA#4 primary prostate cancer cells without significant cytotoxicity in RWPE-1 prostate epithelial cells. Combined treatment with AC and TRAIL at optimal concentrations resulted in tumor-specific apoptotic cell death in RC-58T/h/SA#4 cells, characterized by DNA fragmentation, accumulation of apoptotic cell population, and nuclear condensation. Compared to single treatment with AC or TRAIL, co-treatment with AC and TRAIL significantly increased expression of Bax, cleaved PARP, AIF, endo G, and cytochrome c but decreased expression of phosphorylation of AKT and mammalian target of rapamycin (mTOR), phosphoinositide 3-kinase (PI3K), Bcl-2 and caspases-9, -8, -3, and -10. The sensitizing effect of AC to TRAIL was well correlated with inhibition of death receptor 5 (DR5) CHOP, and p53 expression. Moreover, pre-treatment with a chimeric blocking antibody for DR5 effectively reduced AC-TRAIL-induced cell death and apoptosis-related protein expression. These results suggest that non-toxic concentrations of AC sensitize TRAIL-resistant primary prostate cancer cells to TRAIL-mediated apoptosis via up-regulation of DR5 and downstream signaling pathways.