Prostate Cancer Cell Lines C4-2 Research Articles

Abstract 4386: Androgen hormone signaling regulates the association between the hippo pathway component YAP1 and RNA binding protein NPM1

Abstract Prostate cancer represents a major health concern for men in the United States and globally, disproportionately affecting men of African descent. Despite recent advances, the precise etiology and molecular mechanisms underpinning fatal prostate cancer in humans remain inadequately understood. RNA binding proteins (RBPs) regulate various aspects of RNA biology, and when their regulation is disrupted, they can lead to human diseases, including cancer. However, the comprehensive alterations in RBP-mRNA interactions in prostate cancer have not been explored. In this study, we investigated the dynamics of RBPs and their interactions with polyadenylated mRNA in the castration-sensitive LNCaP and castration-resistant C4-2 prostate cancer cell lines. Using an enhanced RNA interactome capture assay in combination with mass spectrometry, we discovered that LNCaP and C4-2 cells showed distinct RBP-mRNA interaction patterns in steady-state growth conditions and in response to androgen treatment. Additionally, we investigated the interplay between the oncogene Yes-associated protein 1 (YAP1) and the RNA-binding protein nucleophosmin (NPM1). We identified NPM1 as a component of the YAP1 protein complex through mass spectrometry-based proteomics. Our proximity ligation assay demonstrated that androgen signaling could regulate the interaction between YAP1 and NPM1. Also, our GST-pulldown revealed that NPM1 binds to the proline-rich domain of YAP1 in LNCaP cells, while the WW/SH3 domain of YAP1 mediates the interaction with NPM1 in C4-2 cells. Genetic and pharmacological inhibition of NPM1 activity reduced prostate cancer cell growth in culture by modulating cell cycle progression. Furthermore, our computational and immunological analysis of prostate tumor tissues indicated that the upregulation of NPM1 might be associated with the clinical advancement of human prostate cancer. These findings underscore the critical role of the molecular functional connection between YAP1 and RBPs and the global changes in RBP and RNA interaction patterns, in the progression and recurrence of prostate cancer. Citation Format: Abdulrahman M. Dwead, Marwah M. Al-Mathkour, Bekir Cinar. Androgen hormone signaling regulates the association between the hippo pathway component YAP1 and RNA binding protein NPM1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4386.

Photoactivation of pheophorbide-a utilizing 670 nm LEDs elicits cancer suppressive effects in androgen-independent prostate cancer cellular models.

Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer death in men in the USA. Photodynamic therapy (PDT) is a state-of-the-art treatment that combines high selectivity with minor side effects. Pheophorbide-a (Pheo) is a natural pigment with a photosensitizer property. Our study delved into the impact of Pheo alone or Pheo-PDT combination on the androgen-independent metastatic prostate cancer (AIPC) cell lines DU-145 and C4-2. Furthermore, an in-depth examination has been conducted on the photocytotoxicity mechanism of Pheo-PDT in these specific cell lines. In vitro studies were conducted using the AIPC cell lines. DU-145 and C4-2 cells were treated with Pheo at different concentrations for 60 min alone, or Pheo treatment followed by exposure to 670 nm illumination (60 mW/cm2 in 88 s pulses), producing 5 J/cm2 via portable light-emitting diode. Our results show that Pheo-PDT substantially inhibits cell viability, anchorage-independent growth, and migration capacities and induces autophagy and apoptosis via the over-production of reactive oxygen species that mediates endoplasmic reticulum stress in AIPC cell lines. Our study highlights the potential benefits of Pheo-PDT in metastatic hormone-insensitive PCa cell lines. It paves the way for treating localized and locally advanced PCa as a possible candidate for castration-resistant prostate cancer.

Switching mechanism from AR to EGFR signaling via 3-O-sulfated heparan sulfate in castration-resistant prostate cancer

Androgen deprivation therapy is given to suppress prostate cancer growth; however, some cells continue to grow hormone-independently as castration-resistant prostate cancer (CRPC). Sulfated glycosaminoglycans promote ligand binding to receptors as co-receptors, but their role in CRPC remains unknown. Using the human prostate cancer cell line C4-2, which can proliferate in hormone-dependent and hormone-independent conditions, we found that epidermal growth factor (EGF)-activated EGFR–ERK1/2 signaling via 3-O-sulfated heparan sulfate (HS) produced by HS 3-O-sulfotransferase 1 (HS3ST1) is activated in C4-2 cells under hormone depletion. Knockdown of HS3ST1 in C4-2 cells suppressed hormone-independent growth, and inhibited both EGF binding to the cell surface and activation of EGFR–ERK1/2 signaling. Gefitinib, an EGFR inhibitor, significantly suppressed C4-2 cell proliferation and growth of a xenografted C4-2 tumor in castrated mouse. Collectively, our study has revealed a mechanism by which cancer cells switch to hormone-independent growth and identified the key regulator as 3-O-sulfated HS.

Targeting CPT1B as a potential therapeutic strategy in castration-resistant and enzalutamide-resistant prostate cancer.

Prostate cancer is characterized by aberrant lipid metabolism, including elevated fatty acid oxidation. Carnitine palmitoyltransferase 1B (CPT1B) catalyzes the rate-limiting step of fatty acid oxidation. This study aimed to determine if CPT1B has a critical role in prostate cancer progression and to identify its regulatory mechanism. CPT1B expression data from The Cancer Genome Atlas and Gene Expression Omnibus databases was compared with patient survival data. A tissue microarray was constructed with 60 samples of prostate cancer and immunohistochemically stained for CPT1B. Castration-resistant prostate cancer (CRPC) cell lines 22RV1 and C4-2 in which CPT1B expression had been stably knocked down were established; and cell proliferation, cell cycle distribution, and invasion were investigated by Cell Counting Kit-8 (CCK-8) and colony formation assays, flow cytometry, and Transwell assays, respectively. To examine the impact of androgen receptor (AR) inhibition on CPT1B expression, JASPAR CORE was searched to identify AR-binding sites in CPT1B. Dual luciferase and ChIP assays were performed to confirm CPT1B activity and AR binding, respectively. Differentially expressed genes (DEGs) in prostate cancer underwent gene set enrichment analysis (GSEA). Enzalutamide-resistant C4-2 cells were generated and the mechanism of enzalutamide resistance and downstream signaling pathway changes of CPT1B to C4-2 was explored through CCK-8 test. CPT1B expression was upregulated in human prostate cancer compared with normal prostate tissue and was associated with poor disease-free survival and overall survival. Silencing of CPT1B resulted in downregulated cell proliferation, reduced S-phase distribution, and lower invasive ability, whereas the opposite was observed in CRPC cells overexpressing CPTB1. DEGS in prostate cancer were correlated with G-protein-coupled receptor signaling, molecular transducer activity, and calcium ion binding. AR may regulate CPT1B expression and activity via specific binding sites, as confirmed by dual luciferase and ChIP assays. The CCK-8 experiment demonstrated that CPT1B overexpression in C4-2 cells did not significantly increase the ability of enzalutamide resistance. However, overexpression of CPT1B in C4-2R cells significantly increased the enzalutamide resistance. Upregulation of CPT1B expression increased AKT expression and phosphorylation. CPT1B is upregulated in prostate cancer and is correlated with poor prognosis, indicating its potential as a biomarker. AR inhibits the transcription of CPT1B. In the CRPC cell line, overexpression of CPT1B alone cannot promote enzalutamide resistance, but in the drug-resistant line C4-2R, overexpression of CPT1B can promote the resistance of C4-2R to enzalutamide.

MP16-09 THE ANDROGEN RECEPTOR REPRESSES PROSTATE CANCER INVASION INDUCED BY ONCOMETABOLITE R-2-HYDROXYGLUTARATE THROUGH CIRC51217/MIR-646/TGFβ1/P-SMAD2/3 SIGNALING

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I (MP16)1 Apr 2020MP16-09 THE ANDROGEN RECEPTOR REPRESSES PROSTATE CANCER INVASION INDUCED BY ONCOMETABOLITE R-2-HYDROXYGLUTARATE THROUGH CIRC51217/MIR-646/TGFβ1/P-SMAD2/3 SIGNALING Hua Xu* and Ding-wei Ye Shanghai Hua Xu*Hua Xu* More articles by this author and Ding-wei Ye ShanghaiDing-wei Ye Shanghai More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000841.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: IDH1 (Isocitrate dehydrogenase 1) mutation occurring at codon 132 (R132) in prostate cancer (PCa) is considered as a classifier for a subgroup PCa with accumulation of oncometabolite R-2HG (R-2-hydroxyglutarate). Besides its association with early onset of PCa, little is known about the biological effect of IDH1 mutation and R-2HG in PCa. In this study, we focus on the biological effect of IDH1 R132H mutation, which is the most frequent hotspot mutation, and oncometabolite R-2HG in PCa cell invasion. METHODS: A retrospective cohort of 1012 PCa patients in Fudan University Shanghai Cancer Center were included for next-generation sequencing. IDH1 R132H mutation was discovered as a hot spot mutation for further research. The in vitro study was designed to explore the biological function of IDH1 R132H mutation as well as R-2HG in PCa. Transwell assay was applied to test the cell invasiveness of PC3 and C4-2 PCa cell lines. CircRNA and microRNA candidates were screened based on database and manipulation of their expression was used test their function in the signaling pathway. RESULTS: Based on the next-generation sequencing data from a retrospective cohort of 1012 PCa patients, IDH1 R132H was found to be on the most frequent mutations. We discovered 9 PCa patients (0.9%) carried IDH1 R132H mutation. In the in vitro study, we found that over-expressing mutant IDH1 R132H as well as R-2HG promotes cell invasion in PCa with negative or low expression of androgen receptor (AR). Mechanism dissection revealed that R-2HG can induce circ51217 generation through increasing N6-methyladenosine (m6A) level of TGFβ1 (Transforming growth factor-β 1) mRNA, and in turn sponge miR-646 to further increase TGFβ1 protein expression and TGFβ1/p-Smad2/3 signaling. AR can suppress R-2HG effect at multiple levels, namely repressing TGFβ1 transcription and inhibiting circ51217generation via regulating ADAR2 (Double-stranded RNA-specific editase 1) expression. Preclinical study with in vivo xenograft mouse model also shows that PCa with IDH1 R132H mutation is more invasive in experimental metastasis. CONCLUSIONS: This study demonstrates that IDH1 R132H mutation as well as oncometabolite R-2HG increases cell invasiveness of PCa with negative or low expression of AR through circ51217/miR-646/TGFβ1/p-Smad2/3 signaling. AR can negate R-2HG effect via suppressing transcription of TGFβ1 and generation of circ51217. Source of Funding: National Natural Science Foundation of China (No. 81472377 and 81802570) © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e217-e217 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hua Xu* More articles by this author Ding-wei Ye Shanghai More articles by this author Expand All Advertisement PDF downloadLoading ...

Regulation of ELL2 stability and polyubiquitination by EAF2 in prostate cancer cells.

Elongation factor for RNA polymerase 2 (ELL2) and ELL associated factor 2 (EAF2) have been reported to have tumor suppressive properties in prostate epithelial cells. We investigated ELL2 expression in human prostate cancer specimens, and ELL2 protein stability and ubiquitination in prostate cancer cells. Immunostaining analysis of human prostate cancer specimens was used to determine ELL2 expression in tumor and normal tissues. ELL2 knockdown in prostate cancer cell lines LNCaP and C4-2 was used to compare proliferation and motility. Deletion and site-directed mutagenesis was used to identify amino acid residues in ELL2 that were important for degradation. ELL2 protein was downregulated in prostate cancer specimens and was up-regulated by androgens in prostate cancer cell lines LNCaP and C4-2. ELL2 knockdown enhanced prostate cancer cell proliferation and motility. ELL2 protein has a short half-life and was stabilized by proteasome inhibitor MG132. Amino acid residues K584 and K599 in ELL2 were important for ELL2 degradation. EAF2 could stabilize ELL2 and inhibited its polyubiquitination. Our findings provide further evidence that ELL2 is a potential tumor suppressor frequently down-regulated in clinical prostate cancer specimens and provides new insights into regulation of ELL2 protein level by polyubiquitination and EAF2 binding.

MiR-204 enhances mitochondrial apoptosis in doxorubicin-treated prostate cancer cells by targeting SIRT1/p53 pathway.

Chemotherapy is important for adjuvant treatment of prostate cancer. However, some cancer cells exhibited low sensitivity to chemotherapeutic agents. We are supposed to sensitize these prostate cancer cells to chemotherapeutic agents such as doxorubicin. Previous reports have suggested that microRNAs (miRNAs) regulate chemosensitivity in various cancers. In the present study, we observed that expression level of miR-204 was decreased in prostate cancer cell lines and patients’ tumors. Furthermore, we found that restore of miR-204 dramatically enhanced the cytotoxicity of doxorubicin (DOX) against prostate cancer cell lines C4-2 and LNCaP carrying wild type (WT) p53. Mechanically, miR-204 in prostate cancer cells targets SIRT1 which is a histone deacetylase, and thus decreasing deacetylation of p53. As the results, acetylated p53 induced by DOX upregulates the expression of Noxa and Puma followed by induction of mitochondrial apoptosis. These data demonstrate that restore of miR-204 in prostate cancer cells enhances the mitochondrial apoptosis induced by doxorubicin by targeting the SIRT1/p53 pathway.

The effect of eleven-nineteen lysine-rich leukemia gene 2 on proliferation of prostate cancer cell C4-2

Objective To observe the level of eleven-nineteen lysine-rich leukemia gene 2 (ELL2) in human prostate cancer tissues and its effect on proliferation of prostate cancer cell C4-2. Methods The expression of ELL2 in human prostate cancer tissues was analyzed by immunohistochemical method, and the relationship between ELL2 and malignancy degree of prostate cancer was evaluated. The human prostate cancer cell line C4-2 was stimulated with synthetic androgen R1881 to detect its response to androgen at different time points. ELL2-small interfering RNA (siRNA) and overexpression of ELL2-bearing plasmid were transfected into C4-2 cells respectively, then cell proliferation was evaluated by methyl thiazol tetrazolium (MTT) assay. The DNA synthesis ability of cells was assessed by 5-Bromo-2’-deoxy-uridine (BrdU) incorporation assay. Results The expression of ELL2 was significantly lower in human prostate cancer than that of benign specimen, and decreased with the increase of malignancy of prostate cancer. The correlation analysis between ELL2 and prostate cancer Gleason score reveal a negative correlation result (r=0.836, P=0.000). The protein expression of ELL2 was significantly increased in 48 h and increased by (38.27±10.11)% (t=7.573, P=0.005) and (58.34±13.70)% (t=8.519, P=0.003) at 24 h and 48 h, respectively. The expression of ELL2 protein in the two RNA interference groups were significantly decreased, and the decreased rate were (56.38±11.26)% (t=10.010, P=0.002) and (65.27±12.10)% (t=10.790, P=0.002), respectively. While the ELL2 protein expression in the overexpression group was significantly increased at (80.23±20.94)% (t=7.662, P=0.005). The proliferation ability of C4-2 cells was significantly increased (39.28±8.52)% (t=11.300, P=0.000) and (48.2±7.87)% (t=15.000, P=0.000) in the two RNA interference groups, respectively, compared with the control group. While its proliferation ability decreased (42.74±10.38)% (t=10.090, P=0.000) in ELL2-bearing plasmid. Transfection of ELL2-specific interfering RNA increased the ability of DNA synthesis, while the transfection of ELL2-bearing plasmid cells had lower DNA synthesis ability. Conclusion ELL2 is lowly expressed in human prostate cancer tissues and negatively correlated with the malignant degree. ELL2 can inhibit the proliferation of prostate cancer cells to a certain extent. Key words: Prostate cancer; Eleven-nineteen lysine-rich leukemia gene 2; Proliferation

IAP Antagonists Enhance Apoptotic Response to Enzalutamide in Castration-Resistant Prostate Cancer Cells via Autocrine TNF-α Signaling.

Castration-resistant prostate cancer (CRPC) remains incurable and identifying effective treatments continues to present a clinical challenge. Although treatment with enzalutamide, a second generation androgen receptor (AR) antagonist, prolongs survival in prostate cancer patients, responses can be limited by intrinsic resistance or acquired resistance. A potential mechanism of resistance to androgen axis inhibition is evasion of apoptosis. Inhibitor of apoptosis proteins (IAPs) are found to be overexpressed in prostate cancer and function to block apoptosis and promote survival signaling. Novel, small-molecule IAP antagonists, such as AEG40995, are emerging as a strategy to induce apoptosis and increase therapeutic response in cancer. Human prostate cancer cell lines LNCaP and C4-2 were treated with enzalutamide with or without addition of IAP antagonist AEG40995 and proliferation and survival were determined by MTS and clonogenic assay. Western blot was used to evaluate IAP protein expression changes and PARP-1 cleavage was assessed as indication of apoptosis. Flow cytometry was performed to analyze apoptosis in treated cells. Caspase activity was determined by luminescence assay. Quantitative real-time PCR and immunometric ELISA was used to assess TNF-α (transcript and protein levels, respectively) in response to treatment. In this study, we demonstrate that IAP antagonist AEG40995 exhibits minimal effects on prostate cancer cell proliferation or survival, but rapidly degrades cIAP1 protein. Combination treatment with enzalutamide demonstrates that AEG40995 increases apoptosis and reduces proliferation and clonogenic survival in cell line models of prostate cancer. Mechanistically, we demonstrate that apoptosis in response to enzalutamide and IAP antagonist requires activation of caspase-8, suggesting extrinsic/death receptor apoptosis signaling. Assessment of TNF-α in response to combination treatment with enzalutamide and AEG40995 reveals increased mRNA expression and autocrine protein secretion. Blocking TNF-α signaling abrogates the apoptotic response demonstrating that TNF-α plays a critical role in executing cell death in response to this drug combination. These findings suggest that IAP antagonists can increase sensitivity and amplify the caspase-mediated apoptotic response to enzalutamide through TNF-α signaling mechanisms. Combination with an IAP antagonist increases enzalutamide sensitivity, lowers the apoptotic threshold and may combat drug resistance in patients with prostate cancer. Prostate 77:866-877, 2017. © 2017 Wiley Periodicals, Inc.

Upregulation of long non-coding RNA PlncRNA-1 promotes proliferation and induces epithelial-mesenchymal transition in prostate cancer.

ObjectiveTo confirm that PlncRNA-1 regulates the cell cycle in prostate cancer cells and induces epithelial-mesenchymal transition (EMT) in prostate cancer through the TGF-β1 pathway.ResultsPlncRNA-1 and TGF-β1 expression levels were significantly higher in prostate cancer tissues than in normal prostate tissues (P < 0.05) and were significantly positively correlated. TGF-β1, N-cadherin and Cyclin-D1 were downregulated and E-Cadherin was upregulated in LNCAP cells after silencing of PlncRNA-1, as determined by real-time PCR and Western blot. TGF-β1, N-cadherin and Cyclin-D1 were upregulated and E-cadherin was downregulated in C4-2 cells, as determined by real-time PCR and Western blot. Overexpression of PlncRNA-1 in C4-2 cells was observed when TGF-β1 inhibitor LY2109761 was added. Western blot analysis showed that compared with their expression when TGF-β1 inhibitor LY2109761 was not added, N-Cadherin and CyclinD1 expression decreased and E-Cadherin expression increased. Transwell results showed that the invasive ability of C4-2 cells was enhanced after overexpression of PlncRNA-1, and the invasion ability was decreased after addition of TGF-β1 inhibitor LY2109761. The cell cycle was blocked by overexpression of PlncRNA-1 in C4-2 and by the addition of TGF-β1 inhibitor LY2109761, as determined by flow cytometry. In vitro experiments showed that PlncRNA-1 can regulate the growth of prostate cancer cells and EMT through the TGF-β1 pathway. In vivo experiments also confirmed the above results. Tumor growth was significantly blocked by overexpressing PlncRNA-1 in C4-2 cells and by the TGF-β1 inhibitor LY2109761 in animal experiments.Materials and MethodsThe expression levels of PlncRNA-1 and TGF-β1 were analyzed in 19 prostate cancer tissue samples and in adjacent normal tissue samples, 4 Pca cell lines, including LNCaP, C4-2, DU145, and PC3, and 1 normal prostate epithelial cell line RWPE-1. LNCAP cells were divided into the LNCAP control group and the LNCAP-PlncRNA-1-siRNA group. Cells from the prostate cancer cell line C4-2 were divided into the C4-2 control group and the C4-2-PlncRNA-1 experimental group. Changes in TGF-β1, E-cadherin and N-cadherin were detected by qPCR and Western Blot assay after silencing and overexpression of PlncRNA-1. The cell cycle, cell invasion, and levels of Cyclin-D1, E-Cadherin, and N-Cadherin were observed after adding TGF-β1 inhibitor LY2109761 in the C4-2-PlncRNA-1 group. The effects of TGF-β1 inhibitor LY2109761 on the tumorigenicity of C4-2 cells after overexpression of PlncRNA-1 was investigated in vivo.ConclusionsPlncRNA-1 is an oncogene that regulates the cell cycle, cyclin-D1 and EMT in prostate cancer cells through the TGF-β1 pathway.

Ca2+ Selective Host Rotaxane Is Highly Toxic Against Prostate Cancer Cells.

New therapies are needed to eradicate androgen resistant, prostate cancer. Prostate cancer usually metastasizes to bone where the concentration of calcium is high, making Ca2+ a promising toxin. Ionophores can deliver metal cations into cells, but are currently too toxic for human use. We synthesized a new rotaxane (CEHR2) that contains a benzyl 15-crown-5 ether as a blocking group to efficiently bind Ca2+. CEHR2 transfers Ca2+ from an aqueous solution into CHCl3 to greater extent than alkali metal cations and Mg2+. It also transfers Ca2+ to a greater extent than CEHR1, which is a rotaxane with an 18-crown-6 ether as a blocking group. CEHR2 was more toxic against the prostate cancer cell lines PC-3, 22Rv1, and C4-2 than CEHR1. This project demonstrates that crown ether rotaxanes can be designed to bind a targeted metal cation, and this selective cation association can result in enhanced toxicity.

Synthesis, Anticancer Activity and Computational SAR Analysis of Acylsulfonylpiperazines Derivatives

A series of 1-acyl-4-sulfonylpiperazine derivatives has been prepared. The antiproliferative effect of these compounds was evaluated in vitro against human prostate cancer cell line C4-2, several among them exhibited interesting growth inhibitory against this particular cell line. Finally, a molecular modeling study was employed to analyze the structure/activity relationships (SAR) of these novel compounds..

Abstract 3571: Targeted suppression of inhibitor of apoptosis proteins amplifies apoptosis and improves response to enzalutamide in prostate cancer

Abstract Prostate cancer is the most common malignancy and the 2nd leading cause of cancer death in men worldwide. First-line treatment includes androgen deprivation therapy in addition to androgen receptor (AR) antagonists that work to block androgen signaling. Although most patients respond initially to hormone deprivation therapy, responses are not durable and the disease progresses to a lethal stage, termed castrate resistant prostate cancer (CRPC). Interestingly, most CRPCs are still dependent on AR signaling for growth and survival, therefore more potent AR-antagonists such as enzalutamide have recently been FDA approved for treatment of CRPC. However, response rates to enzalutamide can be limited by emergence of acquired resistance or intrinsic resistance. A universal mechanism of resistance to cytotoxic therapies is upregulation of anti-apoptotic programs that function to override death signals and permit survival of the tumor cell. In prostate cancer, overexpression of the Inhibitor of Apoptosis (IAP) proteins cIAP1, cIAP2, and XIAP has been demonstrated in progression to CRPC. Additionally, aberrant expression of these proteins is reported to contribute to anti-androgen resistance. Therefore, to achieve more robust responses to AR inhibition with enzalutamide, we targeted IAP proteins in order to amplify the apoptotic signal and increase cell death in prostate cancer cells. Using a small-molecule IAP inhibitor, we evaluated response to enzalutamide upon IAP inhibition in prostate cancer cell lines LNCAP and C4-2. Here we demonstrate greater proliferation inhibition, reduced cell survival and synergistic induction of apoptosis signaling in response to combined AR antagonism and IAP inhibition. Furthermore, we demonstrate depletion of cIAP1 protein expression upon enzalutamide treatment indicating a possible role for AR in regulating anti-apoptotic pathways. Taken together these results demonstrate that IAP proteins may be a critical survival pathway in CRPC and antagonism of these proteins through small-molecule inhibition can amplify apoptosis and increase cell death in response to enzalutamide. Targeting a critical survival pathway can lower the apoptotic threshold providing a possible therapeutic opportunity to treat and prevent resistance to enzalutamide and increase benefit to patients with this lethal form of prostate cancer. Citation Format: Amanda Pilling, Ok Hwang, Clara Hwang. Targeted suppression of inhibitor of apoptosis proteins amplifies apoptosis and improves response to enzalutamide in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3571.

PD32-02 NOVEL PI3K/P110BETA-SPECIFIC INHIBITORS ELIMINATE ENZALUTAMIDE RESISTANCE IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) III1 Apr 2016PD32-02 NOVEL PI3K/P110BETA-SPECIFIC INHIBITORS ELIMINATE ENZALUTAMIDE RESISTANCE IN PROSTATE CANCER Marcus Austenfeld, Chenchen He, Yifen Wang, Qingting Hu, Brantley Thrasher, and Benyi Li Marcus AustenfeldMarcus Austenfeld More articles by this author , Chenchen HeChenchen He More articles by this author , Yifen WangYifen Wang More articles by this author , Qingting HuQingting Hu More articles by this author , Brantley ThrasherBrantley Thrasher More articles by this author , and Benyi LiBenyi Li More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.678AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES We and others have demonstrated that the class IA PI3K isoform p110beta is critical in prostate cancer development and progression, indicating p110beta as a bona fide target for prostate cancer therapy. Our recent work consistently shows that prostate cancer cell-specific delivery of nano-micellar TGX221 completely blocked prostate cancer growth in vivo, suggesting that TGX221-based therapy is feasible for further development. Unfortunately, the small chemical TGX221 is not soluble in water, hampering its clinical development. To solve this problem, we recently synthesized a panel of TGX221 analogs with increased water solubility. In this study, we tested these novel inhibitors (TGX221 analogs) in multiple enzalutamide-resistant prostate cancer cell lines for their activity in androgen receptor (AR) signaling and cancer cell growth. METHODS Prostate cancer cell lines 22RV1, VCaP and C4-2 were utilized. AR transactivation was evaluated with the ARE-LUC luciferase reporter assay and PSA mRNA level was assessed with real-time RT-PCR. Cancer cell growth was assessed with NCI-approved Sulforhodamine B-based assay. PI3K activation, AR protein levels and cell apoptosis were evaluated in Western blot assays. RESULTS Our novel TGX221 analogs, shown in Figure 1 with water solubility, are much more water soluble (47-99 mM in water) than TGX221, which can only be dissolved in organic solution. Most importantly, these analogs retained a similar inhibitory effect as TGX221 on cell proliferation, AR transactivation and PI3K/AKT activation in prostate cancer cells. Addition of our TGX221 analogs significantly enhanced the enzalutamide-induced anti-cancer effect in LNCaP and LAPC4 cells already sensitive to enzalutamide treatment, as evaluated by cell proliferation and AR transactivation assays. Moreover, these novel TGX221 analogs also sensitized resistant 22RV1 cells to enzalutamide treatment with increased apoptotic cell death. CONCLUSIONS These data strongly suggest that our novel TGX221 analogs possess great potential to be developed as feasible anti-cancer agents for castration-resistant prostate cancers. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e761 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Marcus Austenfeld More articles by this author Chenchen He More articles by this author Yifen Wang More articles by this author Qingting Hu More articles by this author Brantley Thrasher More articles by this author Benyi Li More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Over-expression of lipocalin 2 promotes cell migration and invasion through activating ERK signaling to increase SLUG expression in prostate cancer.

Metastasis is the primary cause of prostate cancer (PCa) lethality and poses a huge clinical obstacle. Lipocalin 2 (LCN2), a member of the lipocalin family, is aberrantly expressed in some human cancers and has been implicated in the progression of some tumors. However, the role of LCN2 in the metastatic capacity of prostate cancer (PCa) is poorly understood. LCN2 expression was examined by RT-qPCR and/or immunoblotting in human prostate tissue specimens and prostate cancer cell lines LNCaP, C4-2, 22RV1, PC3, DU-145, and PC3MM2. LCN2 protein level in human serum samples was determined by ELISA. Lentiviruses-mediated over-expression of LCN2 and knockdown of LCN2 was conducted to evaluate the role of LCN2 in cell migratory and invasive capacities of prostate cancer cells. Cell migration and invasion was examined by transwell chamber assay. Knockdown of SLUG by lentivirus was performed to investigate its role in LCN2-promoted cell migration and invasion in vitro (22RV1 cell line) and metastasis in vivo (tail vein metastasis assay in nude mice). Role of ERK signaling in LCN2-mediated up-regulation of SLUG was assayed by using ERK inhibitor U0126. We confirmed that LCN2 levels were correlated positively with invasive prostate cancer in human tissue and serum samples, and were also consistently associated with the invasive capacity of prostate cancer cell lines. The over-expression of LCN2 in 22RV1 cells (not highly invasive) promoted the epithelial-mesenchymal transition (EMT), increasing cell motility and invasiveness, while the knockdown of LCN2 in PC3 cells (highly invasive) inhibited EMT, decreasing cell motility and invasiveness. Among the multiple EMT transcription factors, LCN2 specifically induces the expression of SLUG, which was shown here to be required for the LCN2-induced increase in the invasive capacity of prostate cancer cells both in vitro and in vivo. Mechanistically, LCN2 promoted SLUG expression via activating ERK signaling pathway. LCN2 plays an important role in promoting cell migration and invasion of prostate cancer by inducing EMT through the ERK/SLUG axis. Therefore, targeted inhibition of LCN2 may represent a therapeutic strategy to prevent the metastasis of prostate cancer.

Synergistic co-targeting of prostate-specific membrane antigen and androgen receptor in prostate cancer.

Antibody-drug conjugates (ADCs) are an emerging class of cancer therapies that have demonstrated favorable activity both as single agents and as components of combination regimens. Phase 2 testing of an ADC targeting prostate-specific membrane antigen (PSMA) in advanced prostate cancer has shown antitumor activity. The present study examined PSMA ADC used in combination with potent antiandrogens (enzalutamide and abiraterone) and other compounds. Antiproliferative activity and expression of PSMA, prostate-specific antigen and androgen receptor were evaluated in the prostate cancer cell lines LNCaP and C4-2. Cells were tested for susceptibility to antiandrogens or other inhibitors, used alone and in combination with PSMA ADC. Potential drug synergy or antagonism was evaluated using the Bliss independence method. Enzalutamide and abiraterone demonstrated robust, statistically significant synergy when combined with PSMA ADC. Largely additive activity was observed between the antiandrogens and the individual components of the ADC (free drug and unmodified antibody). Rapamycin also synergized with PSMA ADC in certain settings. Synergy was linked in part to upregulation of PSMA expression. In androgen-dependent LNCaP cells, enzalutamide and abiraterone each inhibited proliferation, upregulated PSMA expression, and synergized with PSMA ADC. In androgen-independent C4-2 cells, enzalutamide and abiraterone showed no measurable antiproliferative activity on their own but increased PSMA expression and synergized with PSMA ADC nonetheless. PSMA expression increased progressively over 3 weeks with enzalutamide and returned to baseline levels 1 week after enzalutamide removal. The findings support exploration of clinical treatment regimens that combine potent antiandrogens and PSMA-targeted therapies for prostate cancer.

Abstract LB-190: Soluble E-Cadherin promotes cell invasion but not proliferation in prostate cancer

Abstract Metastasis is responsible for the majority of prostate cancer-related deaths. Despite progress in early detection and management, there remains no effective, long-term cure for metastatic prostate cancer, partially due to the unclear molecular mechanism of cancer cell invasion and metastasis. Clinical reports discovered that soluble E-cadherin (sE-Cad, the cleaved ectodomain of human E-Cadherin protein) was elevated in body fluids and serum of patients with a variety of cancers, and its serum levels have been shown to correlate positively with metastatic cancers and disease invasiveness. However, little is known about the role of sE-Cad in metastasis, or the molecular mechanism of its abnormal accumulation in cancerous microenvironment. Our lab generated C4-2 prostate cancer cell lines which would secret synthetic sE-Cad by Doxycycline induction. We found that increased sE-Cad in the conditioned medium promotes cell invasion, but not cell proliferation. We analyzed the protein subcellular localizations in different cell compartments (membrane, cytoskeleton, nucleus and cytoplasm) and observed increased binding of synthetic sE-Cad to membrane E-Cadherin, indicating that sE-Cad could promote cell motility by interacting with membrane proteins and possibly disrupting the cell-cell adhesion. This work is supported by Wake Forest University institutional funds to KC Balaji and by NIH grant CA079448 to X Fang. Citation Format: Xiaolan Fang, K. C. Balaji, Bita NickKholgh, Kenneth Gyabaah. Soluble E-Cadherin promotes cell invasion but not proliferation in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-190. doi:10.1158/1538-7445.AM2014-LB-190

Combination AZD5363 with Enzalutamide Significantly Delays Enzalutamide-resistant Prostate Cancer in Preclinical Models

The phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt (PI3K/Akt) pathway is a key pathway activated in castrate-resistant prostate cancer (CRPC). This preclinical study evaluates targeting of Akt with AZD5363 alone and in combination with enzalutamide (ENZ) to prevent and delay resistance. Our results demonstrate AZD5363 has significant proapoptotic, antiproliferative activity as monotherapy in ENZ-resistant cell lines in vitro and significantly decreased tumour growth in ENZ-resistant xenograft. The combination of AZD5363 and ENZ showed synergistic decreases in cell proliferation and induced cell-cycle arrest and apoptosis in prostate cancer cell lines LNCaP and C4-2. Notably, the combination of AZD5363 and ENZ resulted in an impressive regression of castrate-resistant LNCaP xenograft tumours without any recurrence demonstrated, whereas progression occurred with both monotherapies. Serum prostate-specific antigen (PSA) levels were also continuously suppressed, and nadir PSA levels were lower in the combination arm compared to ENZ alone. Combination AZD5363 and ENZ at time of castration similarly resulted in significant regression of tumours, with greater relative suppression of PSA compared to when administered to castrate-resistant xenografts. In summary, combination AZD5363 and ENZ significantly delays the development of ENZ resistance in preclinical models through synergistic increases in apoptosis and cell cycle arrest. Our results also suggest greater efficacy may be seen with earlier combination treatment. This study provides preclinical data to support evaluation of combination targeting of the PI3K/Akt pathway and the androgen-receptor axis in the clinic using AZD5363 and ENZ, respectively. Patient summaryTargeting of the Akt and androgen receptor pathways with AZD5363 and enzalutamide, respectively, significantly delayed the development of enzalutamide-resistant prostate cancer through increased apoptosis and cell cycle arrest. This preclinical synergy provides a strong rationale for clinical evaluation of this combination.

Dual PI3K/mTOR Inhibitor NVP-BEZ235 Sensitizes Docetaxel in Castration Resistant Prostate Cancer

Effective therapeutic strategies that can achieve long-term improvement in patients with castration resistant prostate cancer are urgently needed. We recently reported that the activated PI3K/Akt/mTOR signaling pathway induced by docetaxel explains resistance to docetaxel in castration resistant prostate cancer. In this study we explored the efficacy of NVP-BEZ235, a dual PI3K and mTORC1/2 inhibitor, for docetaxel resistant castration resistant prostate cancer. We used the 2 human castration resistant prostate cancer cell lines C4-2 and C4-2AT6. At our laboratory C4-2AT6 cells were established from C4-2 under androgen ablated treatment for 6 months. We investigated the efficacy of NVP-BEZ235 monotherapy and NVP-BEZ235 combined with docetaxel in vitro and in vivo. Increased phosphorylated Akt in C4-2AT6 cells was significantly inhibited by NVP-BEZ235 in a dose and time dependent manner. WST cell proliferation assay results in C4-2AT6 cells revealed that combined administration of NVP-BEZ235 and docetaxel had significant, synergistically greater cytotoxicity than NVP-BEZ235 or docetaxel monotherapy. Combined NVP-BEZ235 (40 mg/kg) and docetaxel (4 mg/kg) in vivo in a castrated mouse xenograft model inhibited C4-2AT6 tumor growth to a greater degree than in the monotherapy groups. Also, NVP-BEZ235 showed significant efficacy with docetaxel at a low concentration in vivo, suggesting that NVP-BEZ235 effectively decreased resistance to docetaxel. Results suggest that inhibition of the PI3K/Akt/mTOR signaling pathway by NVP-BEZ235 can overcome docetaxel resistance in human castration resistant prostate cancer. Our findings provide a molecular basis for the clinical use of combined administration of NVP-BEZ235 and docetaxel in patients with castration resistant prostate cancer.

Abstract 3801: Androgen Receptor non-genomic regulation of prostate tumor cell invasion and metastasis through Src signaling and Matriptase .

Abstract Prostate cancer is the leading cause of cancer deaths in men here in the U.S. and over 240,000 new cases are predicted to be diagnosed this year. Initially, tumors arising in this gland rely on the androgen receptor (AR) and its ligand, dihydrotestosterone (DHT) for growth and survival. At this stage, androgen deprivation therapy (ADT) can lead to tumor regression. Unfortunately, patients typically relapse within two to three years and tumors no longer respond to ADT; this stage is termed castration-resistant prostate cancer (CRPC) for which there is no cure. At this stage, AR expression is either increased or mutationally activated and non-responsive to physiological levels of androgen. Several reports have shown that AR has both genomic signaling within nucleus and non-genomic signaling within the cytoplasm. Using prostate tumor cell line PC-3, expressing wild-type AR (PC3-AR), we reported that AR genomic signaling leads to an increase in integrin α6β1 transcription and expression, which in turn increases Bcl-xL expression, a mechanism that regulates prostate cancer cell survival. However, a role for AR and integrin α6β1 in prostate cancer metastasis has not been examined. In addition to promoting cell survival, integrins are critically important for cell movement and can activate Src. We observed that in the absence of androgen, PC3-AR cells have increased Src activity as well as enhanced cell migration and invasion relative to cells that do not expressing AR. Androgen responsive prostate cancer cell lines LNCaP, VCaP and C42 which express AR endogenously, have enhanced invasion and Src activity once stimulated with androgen. We found that knock-down of AR decreased Src activity in PC3-AR cells, as well as in LNCaP and C42 cells. We also found that inhibiting the expression of either AR or Src blocked the ability of cells to invade through matrix in vitro. However, neither AR nor Src possess the ability to degrade ECM proteins. Thus, we hypothesize that AR activates Src in a non-genomic fashion to promote a metastatic phenotype by the regulation of a downstream protease. To test our hypothesis, we set out to determine how AR/Src signaling controls the expression and/or activity of a protease that can degrade proteins in the ECM to promote metastasis. Our data shows that the protease downstream of AR/Src signaling is matriptase, a transmembrane serine protease that has been recently reported to cleave laminin. We observe matriptase cellular expression levels decrease in androgen responsive cell lines once stimulated with androgen as early as twenty minutes. This is suggestive that this is regulated through a non-genomic signaling mechanism. Also seen in these androgen stimulated cells within twenty minutes is that matriptase is shed and found within the media at higher levels relative to the media of non-stimulated cells. Citation Format: Jelani C. Zarif, Laura E. Lamb, Cindy K. Miranti. Androgen Receptor non-genomic regulation of prostate tumor cell invasion and metastasis through Src signaling and Matriptase . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3801. doi:10.1158/1538-7445.AM2013-3801