LNCaP Sublines Research Articles

Preclinical patient-derived modeling of castration-resistant prostate cancer facilitates individualized assessment of homologous recombination repair deficient disease.

The use of mutation analysis of homologous recombination repair (HRR) genes to estimate PARP-inhibition response may miss a larger proportion of responding patients. Here, we provide preclinical models for castration-resistant prostate cancer (CRPC) that can be used to functionally predict HRR defects. In vitro, CRPC LNCaP sublines revealed an HRR defect and enhanced sensitivity to olaparib and cisplatin due to impaired RAD51 expression and recruitment. Ex vivo-induced castration-resistant tumor slice cultures or tumor slice cultures derived directly from CRPC patients showed increased olaparib- or cisplatin-associated enhancement of residual radiation-induced γH2AX/53BP1 foci. We established patient-derived tumor organoids (PDOs) from CRPC patients. These PDOs are morphologically similar to their primary tumors and genetically clustered with prostate cancer but not with normal prostate or other tumor entities. Using these PDOs, we functionally confirmed the enhanced sensitivity of CRPC patients to olaparib and cisplatin. Moreover, olaparib but not cisplatin significantly decreased the migration rate in CRPC cells. Collectively, we present robust patient-derived preclinical models for CRPC that recapitulate the features of their primary tumors and enable individualized drug screening, allowing translation of treatment sensitivities into tailored clinical therapy recommendations.

RELA is sufficient to mediate interleukin-1 repression of androgen receptor expression and activity in an LNCaP disease progression model.

The androgen receptor (AR) nuclear transcription factor is a therapeutic target for prostate cancer (PCa). Unfortunately, patients can develop resistance to AR-targeted therapies and progress to lethal disease, underscoring the importance of understanding the molecular mechanisms that underlie treatment resistance. Inflammation is implicated in PCa initiation and progression and we have previously reported that the inflammatory cytokine, interleukin-1 (IL-1), represses AR messenger RNA (mRNA) levels and activity in AR-positive (AR+ ) PCa cell lines concomitant with the upregulation of prosurvival biomolecules. Thus, we contend that IL-1 can select for AR-independent, treatment-resistant PCa cells. To begin to explore how IL-1 signaling leads to the repression of AR mRNA levels, we performed comprehensive pathway analysis on our RNA sequencing data from IL-1-treated LNCaP PCa cells. Our pathway analysis predicted nuclear factor kappa B (NF-κB) p65 subunit (RELA), a canonical IL-1 signal transducer, to be significantly active and potentially regulate many genes, including AR. We used small interfering RNA (siRNA) to silence the NF-κB family of transcription factor subunits, RELA, RELB, c-REL, NFKB1, or NFKB2, in IL-1-treated LNCaP, C4-2, and C4-2B PCa cell lines. C4-2 and C4-2B cell lines are castration-resistant LNCaP sublines and represent progression toward metastatic PCa disease, and we have previously shown that IL-1 represses AR mRNA levels in C4-2 and C4-2B cells. siRNA revealed that RELA alone is sufficient to mediate IL-1 repression of AR mRNA and AR activity. Intriguingly, while LNCaP cells are more sensitive to IL-1-mediated repression of AR than C4-2 and C4-2B cells, RELA siRNA led to a more striking derepression of AR mRNA levels and AR activity in C4-2 and C4-2B cells than in LNCaP cells. These data indicate that there are RELA-independent mechanisms that regulate IL-1-mediated AR repression in LNCaP cells and suggest that the switch to RELA-dependent IL-1 repression of AR in C4-2 and C4-2B cells reflects changes in epigenetic and transcriptional programs that evolve during PCa disease progression.

Abstract 1020: p300 and CBP targeting in castration therapy resistant prostate cancer

Abstract p300 and CBP interact with transcription factors and other proteins via their histone acetyltransferase domain (HAT) and a bromodomain (Bd). In prostate cancer, p300 and CBP act as oncogenes and are commonly upregulated. p300 and CBP are valid targets for therapy due to their function as androgen receptor (AR) coactivators and involvement in castration resistance. Revealing downstream pathways and their function in cancer cell proliferation and resistance should further identify potential novel therapeutic targets. The effect of the p300/CBP HAT inhibitor C646 and the Bd inhibitor ICBP-112 on viability was tested. To this end several human PCa cell lines with different AR expression levels and resistance status were used. The resistant cells were derived from DUCaP, LAPC4 and LNCaP cells chronically treated with Enzalutamide (DUCaP ENZA, LAPC4 ENZA and LNCaP ABL ENZA). Resistance was verified by viability assay. Regulation of AR downstream targets was studied by qRT-PCR and Western blot. Gene expression in the different DUCaP and LNCaP sublines after inhibitor treatment was analyzed with RNAseq. Both inhibitors exhibited IC50 values on viability at micromolar concentrations with a significantly lesser effect on androgen-insensitive PC3 cells compared to AR sensitive cells. Significantly increased sensitivity to inhibitors was observed in enzalutamide-resistant DUCaP ENZA, LAPC4 ENZA and LNCaP ABL ENZA cell lines relative to parental cells: respectively 1.83- (*), 2- (*) and 2.7-fold (*) for C646 and not significant, 1.8- (p<0.1) and 1.56-fold (*) for ICBP-112. Moreover, ICBP-112 treatment of DUCaP cells significantly reduced expression of the AR target genes FKBP5, PSA and TMPRSS2 0.6- (**), 0.57- (**) and 0.37-fold (***) respectively. Similar results were obtained with DUCaP ENZA cells 0.46- (*), 0.45- (*) and 0.57-fold (ns) for FKBP5, PSA and TMPRSS2, respectively. Whilst less pronounced, this regulation was also observed at the protein level. Similar results were obtained with C646. RNAseq results suggest multiple changes in sublines after chronic enzalutamide treatment in DUCaP and LNCaP cells. Inhibition of p300 and CBP reduced androgen-, myc- and parts of an EMT-signature, which are likely involved in the development of enzalutamide resistance. Taken together, C646 and ICBP-112 are effective inhibitors of AR dependent PCa. Furthermore, differences between cell lines suggest that the AR background and other factors influence the inhibitor effectivity. A significantly increased effect of the HAT and Bd inhibitors in CRPC suggests an important role for p300/CBP in CRPC. Citation Format: Tobias Furlan, Natalie Sampson, Frédéric R. Santer, Zoran Culig. p300 and CBP targeting in castration therapy resistant 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 1020.

Abstract 2026: Loss of fibroblasts-dependent androgen receptor activation in prostate cancer cells develops castration-resistant prostate cancer

Abstract Well-differentiated prostate cancer (PCa) cells are generally androgen/androgen receptor (AR)-dependent, i.e. AR signaling regulates cell cycle and differentiation. Loss of AR signaling after androgen deprivation therapy (ADT) triggers AR-independent outgrowth, generating poorly differentiated uncontrollable PCa cells. To prevent development and progression of castration-resistant prostate cancer (CRPC), we hypothesize that preservation of AR signaling after ADT is an important target. Tumor stroma surrounding PCa cells is enriched in fibroblasts secreting AR-activating factors, e.g. EGF, IGF1, and IL-6. Thus, we investigated the role of fibroblasts in AR activation of human prostate cancer LNCaP sublines differing in androgen sensitivity under androgen deprivation. Androgen-low-sensitive E9 and F10 cells were obtained from the parental androgen-sensitive LNCaP cell population through use of a limiting dilution method in regular culture condition. In contrast, androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging under hormone-depleted condition. Original fibroblasts pcPrFs (-M5, -M6, -M7) were isolated from needle biopsy samples of patients with PCa. The PCa cells alone or PCa cells plus pcPrF-M5 were grafted beneath the renal capsule of male athymic nude mice, and then mice were castrated on 14 days post transplantation. Tumor volume of E9 cells + pcPrF-M5 became diminished post castration but that of F10 and AIDL cells + pcPrF-M5 was gradually increased even post castration. In E9 cells + pcPrF-M5 tumors, serum PSA became detected on day 21 post castration. Interestingly, serum PSA was gradually increased even in F10 cells inoculated alone. In AIDL tumors with or without pcPrF-M5, serum PSA was not detected because of mutated AR in AIDL cells. Cell growth of E9 and AIDL cells was directly increased by treating with EGF, whereas that in F10 cells was not affected. Phosphorylation of AKT and ERK1/2 in E9 and AIDL cells was also significantly increased by treating with EGF, whereas that in F10 cells was not affected. In the condition of co-cultures with fibroblasts, PSA production was directly increased in E9 cells but not in F10 and AIDL cells. Protein expression of AR splice variant 7 (AR-V7) was only detected in F10 cells. The reduced AR-dependency of PCa cells is an important clinical development because of its association with the cell’ progression to CRPC. Here our results showed that fibroblasts secreting AR-activating factors preserved AR signaling in E9 cells after ADT, indicating that this type of PCa cells can be controlled by ADT. In contrast, we suggest that loss of fibroblasts-dependent AR activation in F10 and AIDL cells may be responsible for development and progression of CRPC. Citation Format: Kenichiro Ishii, Izumi Matsuoka, Takeshi Sasaki, Manabu Kato, Kohei Nishikawa, Hideki Kanda, Yoshifumi Hirokawa, Kazuhiro Iguchi, Kiminobu Arima, Masatoshi Watanabe, Yoshiki Sugimura. Loss of fibroblasts-dependent androgen receptor activation in prostate cancer cells develops castration-resistant 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 2026.

Abstract 4414: Arginine metabolism is modulated by androgen signalling and prostate cancer progression

Abstract Background: Metabolic reprogramming has been described as one of the hallmarks of cancer. A pathway of promise in prostate cancer management is arginine (Arg) metabolism. Phase 2 clinical trials are underway to study arginine deprivation as a treatment of prostate cancer. However, heterogeneous response has been noted and further studies are needed to better identify prostate tumors most reliant on Arg metabolism. The aim of this study was to investigate if Arg metabolism was associated with androgen signalling and prostate cancer progression from androgen dependent (AD) to androgen independent (AI) phenotype. Methods: The LNCaP prostate cancer cell line was used. Four sets of conditions were tested: LNCaP control, LNCaP in the presence of dihydrotestosterone (DHT), LNCaP with MDV3100 (an AR inhibitor), and a subline of LNCaP (CSS90) which has become AI. MTT viability assay was used to assess cell viability. The Seahorse XF bioenergetics analyzer was used to measure metabolic oxygen consumption. Gas Chromatography and Mass Spectroscopy (GC/MS) was used for metabolomics characterization. Results: LNCaP cells at a baseline are Arg-dependent. Arg starvation led to a 63% decrease in LNCaP cell viability (p<0.0001) in 24hrs. By contrast, the AI CSS90 only demonstrated a 23% decrease in cell viability when depleted of Arg (p<0.01 with Arg control, p<0.001 between LNCaP and CSS90) demonstrating AD cells were much more dependent on exogenous Arg. Enhanced AR signaling with DHT further sensitized cells to arginine depletion with a 73% decrease, and low level AR inhibition decreased response to arginine depletion with only a 21% decrease in cell viability (p<0.001 for all changes). Metabolic characterization by GC/MS demonstrated a 33.8 fold increase in buildup of an arginine metabolism intermediate, ornithine in the CSS90 cells vs LNCaP (p<0.0001) reflecting compensatory ability of the CSS90 cells to endogenously manage Arg metabolism. Modulation of androgen signaling with DHT and MDV3100 demonstrated similar increased reliance of exogenous Arg for AD cells with increased androgen signaling with a 2.2 fold increase in Arg dependent oxygen consumption with DHT and 95% drop with MDV (p<0.0001 for both). Conclusions: Arginine is potentially an important aspect of prostate cancer metabolism. Arg targeted therapies may best be suited towards earlier stage tumors and Arg metabolism characterization may have potential as a biomarker of progression to AI prostate cancer. Citation Format: De-Xue Fu, Hubert Huang, Jee-Hoon Song, Min Xu, Lucy Liu, Mohamad Afnan Khan, Krish Chandrasekaran, Arif Hussain, Ganesh Sriram, Mohummad M. Siddiqui. Arginine metabolism is modulated by androgen signalling and prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4414. doi:10.1158/1538-7445.AM2017-4414

Crosstalk between RON and androgen receptor signaling in the development of castration resistant prostate cancer.

Castrate-resistant prostate cancer (CRPC) is the fatal form of prostate cancer. Although reactivation of androgen receptor (AR) occurs following androgen deprivation, the precise mechanism involved is unclear. Here we show that the receptor tyrosine kinase, RON alters mechanical properties of cells to influence epithelial to mesenchymal transition and functions as a transcription factor to differentially regulate AR signaling. RON inhibits AR activation and subset of AR-regulated transcripts in androgen responsive LNCaP cells. However in C4-2B, a castrate-resistant sub-line of LNCaP and AR-negative androgen independent DU145 cells, RON activates subset of AR-regulated transcripts. Expression of AR in PC-3 cells leads to activation of RON under androgen deprivation but not under androgen proficient conditions implicating a role for RON in androgen independence. Consistently, RON expression is significantly elevated in castrate resistant prostate tumors. Taken together our results suggest that RON activation could aid in promoting androgen independence and that inhibition of RON in combination with AR antagonist(s) merits serious consideration as a therapeutic option during hormone deprivation therapy.

Muscadine grape skin extract can antagonize Snail-cathepsin L-mediated invasion, migration and osteoclastogenesis in prostate and breast cancer cells.

To develop new and effective chemopreventive agents against bone metastasis, we assessed the effects of muscadine grape skin extract (MSKE), whose main bioactive component is anthocyanin, on bone turnover, using prostate and breast cancer cell models overexpressing Snail transcription factor. MSKE has been shown previously to promote apoptosis in prostate cancer cells without affecting normal prostate epithelial cells. Snail is overexpressed in prostate and breast cancer, and is associated with increased invasion, migration and bone turnover/osteoclastogenesis. Cathepsin L (CatL) is a cysteine cathepsin protease that is overexpressed in cancer and involved in bone turnover. Snail overexpression in prostate (LNCaP, ARCaP-E) and breast (MCF-7) cancer cells led to increased CatL expression/activity and phosphorylated STAT-3 (pSTAT-3), compared to Neo vector controls, while the reverse was observed in C4-2 (the aggressive subline of LNCaP) cells with Snail knockdown. Moreover, CatL expression was higher in prostate and breast tumor tissue compared to normal tissue. MSKE decreased Snail and pSTAT3 expression, and abrogated Snail-mediated CatL activity, migration and invasion. Additionally, Snail overexpression promoted osteoclastogenesis, which was significantly inhibited by the MSKE as effectively as Z-FY-CHO, a CatL-specific inhibitor, or osteoprotegerin, a receptor activator of nuclear factor kappa B ligand (RANKL) antagonist. Overall, these novel findings suggest that Snail regulation of CatL may occur via STAT-3 signaling and can be antagonized by MSKE, leading to decreased cell invasion, migration and bone turnover. Therefore, inhibition using a natural product such as MSKE could potentially be a promising bioactive compound for bone metastatic cancer.

Abstract C57: Snail transcription factor can regulate cathepsin L activity in prostate carcinomas

Abstract Prostate cancer (PCa) is the most frequently diagnosed cancer in men, the second leading cause of male cancer deaths in the U.S, and also presents the greatest racial disparity of any cancer in the U.S. The incidence and mortality of PCa is higher in African-American men (AA), compared to other ethnic groups. Snail transcription factor expression is increased in prostate cancer and associated with epithelial-mesenchymal transition (EMT), whereby epithelial cancer cells expressing epithelial markers such as E-cadherin transition into spindle-shaped mesenchymal cells expressing markers such as vimentin. Snail can turn on proteases like MMP-9 to increase degradation of basement membrane and extracellular matrix proteins prior to cancer cell invasion and migration. We have preliminary data showing that muscadine grape skin extract (MSKE), derived from muscadine grape (Vitis rotundifolia), a common red grape used to produce red wine can decrease Snail expression in prostate cancer cells. Cathepsin L is a cysteine cathepsin that is overexpressed in prostate cancer and involved in the repression of E-cadherin, a hallmark of EMT. We investigated whether there could be a link between Snail and Cathepsin L. We hypothesize that the Snail expression may be increased in African American tissue as compared to other races and that Snail can induce the increased activity of Cathepsin L which can be antagonized by MSKE. Initially, we examined the expression of Snail and Cathepsin L in prostate cancer cells by Western blot analysis and Cathepsin L Activity via Zymography. The cell lines utilized in this study are LNCaP, C4-2 (the aggressive subline of LNCaP), mesenchymal ARCaP (ARCaP-M) and epithelial ARCaP (ARCaP-E). Preliminary results suggest that Snail expression and Cathepsin L activity is high in prostate cancer cells compared to normal prostate epithelial cells. We also obtained human prostate cancer tissue from the Bahamas which we are currently staining for Snail and Cathepsin L, to compare to staining from tissue microarrays. We have observed so far that there is both Snail and Cathepsin L expression in the tissue samples. We then utilitized LNCaP and ARCaP-E prostate cancer cells stably transfected with an empty Neo vector (LNCaP Neo; ARCaP Neo) or constituitively active Snail cDNA (LNCaP Snail; ARCaP Snail) that have been shown previously to represent an EMT model, to examine Cathepsin L and Stat-3 activity. We observed that Snail overexpression led to increased Cathepsin L and Stat-3 activity compared to cells with the Neo vector. Additionally ARCaP-E cells overexpresing Snail, C4-2, or ARCaP-M cells treated with MSKE (5ug/mL and 20ug/mL) for a period of 72hrs led to decreased Cathepsin L and Stat-3 activity. Furthermore, MSKE reverted EMT as shown by increased E-cadherein expression, decreased vimentin expression and decrease in cell migration. Taken together, these findings suggest that Snail my increase Cathepsin L activity via the Stat-3 signaling pathway in prostate cancer cells and MSKE could potentially be a promising bioactive compound for human cancer prevention and therapy. Citation Format: Liza Burton, Peri Nappagan, Basil Smith, Manu Platt, Camille Ragin, Valerie Odero-Marah. Snail transcription factor can regulate cathepsin L activity in prostate carcinomas. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C57. doi:10.1158/1538-7755.DISP13-C57

Difference in Protein Expression Profile and Chemotherapy Drugs Response of Different Progression Stages of LNCaP Sublines and Other Human Prostate Cancer Cells

Androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, 80-90% of the patients who receive androgen ablation therapy ultimately develop recurrent tumors in 12-33 months after treatment with a median overall survival time of 1-2 years after relapse. LNCaP is a commonly used cell line established from a human lymph node metastatic lesion of prostatic adenocarcinoma. We previously established two relapsed androgen receptor (AR)-rich androgen-independent LNCaP sublines 104-R1 (androgen depleted for 12 months) and 104-R2 cells (androgen depleted for 24 months) from AR-positive androgen-dependent LNCaP 104-S cells. LNCaP 104-R1 and 104-R2 mimics the AR-positive hormone-refractory relapsed tumors in patients receiving androgen ablation therapy. Androgen treatment stimulates proliferation of 104-S cells, but causes growth inhibition and G1 cell cycle arrest in 104-R1 and 104-R2 cells. We investigated the protein expression profile difference between LNCaP 104-S vs. LNCaP 104-R1, 104-R2, PC-3, and DU-145 cells as well as examined the sensitivity of these prostate cancer cells to different chemotherapy drugs and small molecule inhibitors. Compared to 104-S cells, 104-R1 and 104-R2 cells express higher protein levels of AR, PSA, c-Myc, Skp2, BCL-2, P53, p-MDM2 S166, Rb, and p-Rb S807/811. The 104-R1 and 104-R2 cells express higher ratio of p-Akt S473/Akt, p-EGFR/EGFR, and p-Src/Src, but lower ratio of p-ERK/ERK than 104-S cells. PC-3 and DU-145 cells express higher c-Myc, Skp2, Akt, Akt1, and phospho-EGFR but less phospho-Akt and phospho-ERK. Overexpression of Skp2 increased resistance of LNCaP cells to chemotherapy drugs. Paclitaxel, androgen, and inhibitors for PI3K/Akt, EGFR, Src, or Bcl-2 seem to be potential choices for treatment of advanced prostate cancers. Our study provides rationale for targeting Akt, EGFR, Src, Bcl-2, and AR signaling as a treatment for AR-positive relapsed prostate tumors after hormone therapy.

Distinct and specific roles of AKT1 and AKT2 in androgen-sensitive and androgen-independent prostate cancer cells

AKT isoforms are expressed in prostate cancer and their expression and localization have different associations with clinical characteristics. However, the distinct roles of the AKT isoforms in prostate cancer cells are largely unknown. In the present study, we demonstrate distinct roles for AKT1 and AKT2 in cell growth and migration. Ablation of AKT1 and AKT2 decreased the proliferation of the androgen-independent cell line PC-3, although by different mechanisms. AKT1 ablation induced loss of cell adhesion and subsequent apoptosis. AKT2 (but not AKT1) ablation promoted cell cycle arrest at G0/G1, associated with downregulation of cyclin D, CDK6 and CDK2, and upregulation and cytoplasmic-to-nuclear redistribution of p27. The increase of p27 protein levels was due to more gene transcription and an increase in protein stability. The increased stability of p27 was induced by delocalisation of Skp2 and a lower level of p27 phosphorylation at Thr187. AKT1 and AKT2 ablation inhibited and stimulated PC-3 cell migration, respectively. An AKT isoform-specific function could be associated with its subcellular localization. We found that AKT1 and AKT2 were mainly localised in the cytoplasm and nucleus, respectively. In androgen-sensitive cell line LNCaP, the ablation of AKT1 or AKT2 caused apoptosis but in androgen-independent LNCaP sublines, the effect of AKT1 ablation was lower; whereas no changes were observed after AKT2 ablation. Taken together, our data show that AKT1 and AKT2 have non-redundant roles in the regulation of PC-3 cell proliferation and migration. These could be explained by their subcellular localization and/or the specific regulation of downstream effectors. Furthermore, contribution of AKT isoforms to the progression of prostate cancer may change from an androgen-sensitive to a hormone-refractory stage. These findings may help design new targeted strategies for inhibiting AKT isoforms in prostate cancer.

Does changing androgen receptor status during prostate cancer development impact upon cholesterol homeostasis?

BackgroundRecent evidence associates prostate cancer with high cholesterol levels, with cholesterol being an important raw material for cell-growth. Within the cell, cholesterol homeostasis is maintained by two master transcription factors: sterol-regulatory element-binding protein 2 (SREBP-2) and liver X receptor (LXR). We previously showed that the androgen receptor, a major player in prostate cell physiology, toggles these transcription factors to promote cholesterol accumulation. Given that prostate cancer therapy targets the androgen receptor, selecting for cells with altered androgen receptor activity, how would this affect SREBP-2 and LXR activity? Using a novel prostate cancer progression model, we explored how this crosstalk between the androgen receptor and cholesterol homeostasis changes during prostate cancer development.Methodology/Principal FindingsFirstly, we characterised our progression model, which involved 1) culturing LNCaP cells at physiological testosterone levels to generate androgen-tolerant LNCaP-305 cells, and 2) culturing LNCaP-305 with the anti-androgen casodex to generate castration-resistant LNCaP-364 cells. This progression was accompanied by upregulated androgen receptor expression, typically seen clinically, and a reduction in androgen receptor activity. Although this influenced how SREBP-2 and LXR target genes responded to androgen treatment, cellular cholesterol levels and their response to changing sterol status was similar in all LNCaP sub-lines.Conclusion/SignificanceOverall cholesterol homeostasis is unaffected by changing androgen receptor activity in prostate cancer cells. This does not negate the relationship between androgens and cholesterol homeostasis, but rather suggests that other factors compensate for altered androgen receptor activity. Given that cholesterol regulation is maintained during progression, this supports the growing idea that cholesterol metabolism is a suitable target for prostate cancer.

The Essential Role of Giα2 in Prostate Cancer Cell Migration

Cell- and receptor-specific regulation of cell migration by Gi/oα-proteins remains unknown in prostate cancer cells. In the present study, oxytocin (OXT) receptor was detected at the protein level in total cell lysates from C81 (an androgen-independent subline of LNCaP), DU145 and PC3 prostate cancer cells, but not in immortalized normal prostate luminal epithelial cells (RWPE1), and OXT-induced migration of PC3 cells. This effect of OXT has been shown to be mediated by Gi/oα-dependent signaling. Accordingly, OXT inhibited forskolin-induced luciferase activity in PC3 cells that were transfected with a luciferase reporter for cyclic AMP activity. Although mRNAs for all three Giα isoforms were present in PC3 cells, Giα2 was the most abundant isoform that was detected at the protein level. Pertussis toxin (PTx) inhibited the OXT-induced migration of PC3 cells. Ectopic expression of the PTx-resistant Giα2-C352G, but not wild-type Giα2, abolished this effect of PTx on OXT-induced cell migration. The Giα2-targeting siRNA was shown to specifically reduce Giα2 mRNA and protein in prostate cancer cells. The Giα2-targeting siRNA eliminated OXT-induced migration of PC3 cells. These data suggest that Giα2 plays an important role in the effects of OXT on PC3 cell migration. The Giα2-targeting siRNA also inhibited EGF-induced migration of PC3 and DU145 cells. Expression of the siRNA-resistant Giα2, but not wild type Giα2, restored the effects of EGF in PC3 cells transfected with the Giα2-targeting siRNA. In conclusion, Giα2 plays an essential role in OXT and EGF signaling to induce prostate cancer cell migration.

PAGE4 Positivity Is Associated with Attenuated AR Signaling and Predicts Patient Survival in Hormone-Naive Prostate Cancer

Aberrant activation of the androgen receptor (AR) plays a key role during prostate cancer (PCa) development and progression to castration-resistant prostate cancer (CR-PCa) after androgen deprivation therapy, the mainstay systemic treatment for PCa. New strategies to abrogate AR activity and biomarkers that predict aggressive tumor behavior are essential for improved therapeutic intervention. PCa tissue microarrays herein reveal that prostate-associated gene 4 (PAGE4), an X-linked cancer/testis antigen, is highly up-regulated in the epithelium of preneoplastic lesions compared with benign epithelium, but subsequently decreases with tumor progression. We show that AR signaling is attenuated in PAGE4-expressing cells both in vitro and in vivo, most likely via impaired androgen-induced AR nuclear translocation and subsequently reduced AR protein stabilization and phosphorylation at serines 81 and 213. Consistently, epithelial PAGE4 protein levels inversely correlated with AR activation status in hormone-naive and CR-PCa clinical specimens. Moreover, PAGE4 impaired the development of CR-PCa xenografts, and strong PAGE4 immunoreactivity independently predicted favorable patient survival in hormone-naive PCa. Collectively, these data suggest that dysregulation of epithelial PAGE4 modulates AR signaling, thereby promoting progression to advanced lethal PCa and highlight the potential value of PAGE4 as a prognostic and therapeutic target.

Androgen deprivation causes truncation of the C‐terminal region of androgen receptor in human prostate cancer LNCaP cells

The androgen receptor (AR) acts as a ligand-dependent transcription factor, whereas mutant AR lacking the C-terminal ligand-binding domain functions in a ligand-independent manner. In the present study we report that the C-terminal truncated AR, which we named AR-NH1 (the N-terminal fragment of AR cleaved in the neighborhood of helix 1 of the ligand-binding domain), is produced in LNCaP prostatic carcinoma cells. The AR-NH1 of ~90 kDa was observed in an androgen-independent LNCaP subline and was further accumulated by the proteasome inhibitor MG132. MG132 treatment caused the accumulation of AR-NH1 even in parent LNCaP cells. AR-NH1 was produced in the absence of ligand or in the presence of the AR antagonist bicalutamide, whereas AR agonists suppressed its production. AR-NH1 was detected with different AR antibodies recognizing amino acid residues 1-20 and 300-316 and was also generated from exogenous AR. Both siRNA-mediated AR knockdown and treatment with a serine protease inhibitor (4-(2-aminoethyl)-benzenesulfonyl fluoride) reduced AR-NH1 levels. According to the predicted cleavage site (between amino acid residues 660-685) and its nuclear localization, it is assumed that AR-NH1 functions as a constitutively active transcription factor. These data suggest that AR-NH1 is produced under hormone therapy and contributes to the development of castration-resistant prostate cancer due to its ligand-independent transcriptional activity.

788 ABERRANT ACTIVATION OF PARACRINE LOOP BETWEEN PROSTATE CANCER CELLS AND CARCINOMA-ASSOCIATED FIBROBLASTS BY OVEREXPRESSION OF STROMAL INTERLEUKIN-6

You have accessJournal of UrologyProstate Cancer: Basic Research IV1 Apr 2012788 ABERRANT ACTIVATION OF PARACRINE LOOP BETWEEN PROSTATE CANCER CELLS AND CARCINOMA-ASSOCIATED FIBROBLASTS BY OVEREXPRESSION OF STROMAL INTERLEUKIN-6 Kenichiro Ishii, Yoichi Iwamoto, Manabu Kato, Kazuhiro Iguchi, Atsushi Mizokami, Kiminobu Arima, Taizo Shiraishi, and Yoshiki Sugimura Kenichiro IshiiKenichiro Ishii Tsu, Japan More articles by this author , Yoichi IwamotoYoichi Iwamoto Tsu, Japan More articles by this author , Manabu KatoManabu Kato Tsu, Japan More articles by this author , Kazuhiro IguchiKazuhiro Iguchi Gifu, Japan More articles by this author , Atsushi MizokamiAtsushi Mizokami Ishikawa, Japan More articles by this author , Kiminobu ArimaKiminobu Arima Tsu, Japan More articles by this author , Taizo ShiraishiTaizo Shiraishi Tsu, Japan More articles by this author , and Yoshiki SugimuraYoshiki Sugimura Tsu, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.876AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In the tumor microenvironment, activation of tumor-stromal interactions is considered to play a critical role in the promotion of tumorigenesis. During primary cancer progression, cancer cells generate the carcinoma-associated fibroblasts (CAFs) in adjacent normal fibroblasts, and then CAFs support the proliferation and invasion of adjacent cancer cells in a paracrine fashion. The serum level of interleukin-6 (IL-6) is a significant prognostic factor for prostate cancer (PCa) patients with disseminated diseases, suggesting positive roles of IL-6 in the aberrant activation of paracrine loop between PCa cells and CAFs. In this study, we investigated the relationship between responsiveness of PCa cells to stromal IL-6 and cellular AR signaling. METHODS The androgen-low-sensitive E9 cells were obtained from androgen-sensitive LNCaP cell population by limiting dilution method in regular culture condition. In contrast, the androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging in hormone-depleted condition. PCaSC-8 and PCaSC-9 cells were primary cultured from different human PCa specimens. RESULTS IL-6 and VEGF protein levels in both PCaSC-8 and PCaSC-9 cells were generally higher than normal human prostate stromal cells PrSC. In contrast to stromal cells, no IL-6 secretion from LNCaP, E9, and AIDL was detected. IL-6 treatment (10 ng/mL) suppressed cell proliferation of LNCaP and E9 accompanied with changes of cell morphology, i.e. induction of neuroendocrine-like differentiation. Induction of PSA secretion was observed only in IL-6-treated LNCaP. VEGF secretion was significantly increased in both IL-6-treated LNCaP and AIDL. A selective alpha1-adrenoceptor antagonist naftopidil (10 μM) but not tamsulosin suppressed IL-6-induced VEGF secretion in AIDL. CONCLUSIONS In this study, we have shown the evidence that secretions of IL-6 and VEGF from primary cultured CAFs derived PCa patients are generally higher than normal fibroblasts. Our results suggest that overexpression of stromal IL-6 might increase VEGF secretion from adjacent PCa cells in a paracrine fashion. Using LNCaP sublines, E9 and AIDL, differing in androgen-sensitivity, we demonstrated here that IL-6-induced VEGF secretion from PCa cells might be through cellular AR signaling-independent pathway. Identifying mechanisms underlying the aberrant activation of paracrine loop between PCa cells and CAFs is an initial step toward designing CAF-targeted therapy for treatment of PCa. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e322-e323 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kenichiro Ishii Tsu, Japan More articles by this author Yoichi Iwamoto Tsu, Japan More articles by this author Manabu Kato Tsu, Japan More articles by this author Kazuhiro Iguchi Gifu, Japan More articles by this author Atsushi Mizokami Ishikawa, Japan More articles by this author Kiminobu Arima Tsu, Japan More articles by this author Taizo Shiraishi Tsu, Japan More articles by this author Yoshiki Sugimura Tsu, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract B33: Flexibility of the exome of the LNCaP cell line(s): Differences in heterozygocity and heterogeneity

Abstract The LNCaP cell line is a frequently used model for basic and preclinical studies of human prostate adenocarcinoma. We used next-generation sequencing to identify Single Nucleotide Variants (SNVs) and indels in the exome from this cell line. Rigorous filtering of more than 25 million reads and elimination of known SNPs led to a list of 1,886 SNVs that cause an amino acid change and a list of 51 indels. Due to the lack of the normal genome of the patient from which the LNCaP line was derived, we can't determine all SNPs. Indeed, the number of SNVs remains surprisingly high, certainly if we compare it with the number of variants that has recently been reported to occur in prostate cancer samples. The known LNCaP point mutation in the AR gene and the deletion in the PTEN gene were present, and over 40 new SNVs and SNPS have been confirmed with genomic PCR and conventional Sanger sequencing. We used open-source software to predict which of the SNVs most likely affect protein functions. An application of Endavour software incorporated three predictive software packages to calculate the effects of the variations on protein structure, as well as data annotations, protein-protein interactions, LNCaP expression levels according to published gene array datasets, and a PubMed search linking the changed protein with prostate cancer. This has led to a prioritization of the SNVs. While the top 9 SNVs reside in protein kinase genes, several others are likely to affect the function of proteins that are involved in the androgen response. We also checked whether there are SNVs present in 115 genes that have been reported in the literature to be linked with the development of prostate cancer. In this group, we detected 14 SNVs (APC, AR, ARAF, CDH1, CDK4, CHEK2, ERBB2, MSH6, MYC, NF1, NOTCH1, PLXNB1, SMO and TERT) and 12 SNPs. While none of the 14 SNV were present in other prostate cancer cell lines (DU145, PC-3, 22Rv1, and VCaP) as expected, some of the SNPs were. Moreover, for most SNVs and SNPS, two alternative bases were detected in the LNCaP genome. For each of the cancer gene SNVs and SNPs, both variants could be detected in the LNCaP cDNA, indicating that in those cases the wild type and the mutated gene are expressed simultaniously. Nine of the fourteen variants that we detected in the exome of our LNCaP cells were found back in the genomes of LNCaP cells from other laboratories as well as in monoclonal sublines, like LNCaP-TR2 and C4-2B. This indicates that LNCaP cells are heterozygous rather than heterogenous for these SNVs. Surprisingly, four of the thirteen variants that we tested for (CDH1, CDK4, NOTCH1 and PLXNB1) were absent in the genome of some of the LNCaP sublines. Hence, there are few, but important genetic variations between LNCaP cell lines used in different laboratories. This indicates some genetic flexibility, which might explain why cell lines in different laboratories react differently. In conclusion, our data show that the LNCaP cell line has a surprising degree of flexibility in its exome sequence. It is unknown yet whether the same is true for other cell lines. We speculate that part of the LNCaP flexibility arises from differences in selective forces due to the many variations in LNCaP subculturing protocols and media between different laboratories. Citation Format: Lien Spans, Zeynep Kalender, Stein Aerts, Frank Claessens. Flexibility of the exome of the LNCaP cell line(s): Differences in heterozygocity and heterogeneity [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B33.

LNCaP prostate cancer cells with autocrine interleukin‐6 expression are resistant to IL‐6‐induced neuroendocrine differentiation due to increased expression of suppressors of cytokine signaling

Neuroendocrine differentiation (NED) is one of the mechanisms underlying development of castration-resistant prostate cancer (CRPC). In this study, we investigated IL-6-induced NED in two LNCaP sublines. LNCaP-S17, an LNCaP subline that secretes IL-6, and LNCaP-C3, a control subline that does not express IL-6, were analyzed for IL-6-induced NED, activation of JAK2 and STAT3 pathways, and expression of IL-6/IL-6R signaling proteins and downstream target genes. IL-6 did not induce NED in LNCaP-S17 cells, even though IL-6 induced NED in LNCaP-C3 cells. IL-6 activated JAK2 and STAT3 pathways in LNCaP-C3 cells but not in LNCaP-S17 cells. IL-6 did not activate ERK1/2, AKT, or NF-κB pathways in either cell line. Both LNCaP-C3 and LNCaP-S17 cell lines expressed IL-6R, gp130, and TYK2 at almost the same levels and did not express JAK1 or JAK3. The basal level of JAK2 expression was slightly higher in LNCaP-C3 cells than in LNCaP-S17 cells. Two suppressors of cytokine signaling, SOCS7 and cytokine-inducible SH2 protein (CIS), were expressed constitutively at higher levels in LNCaP-S17 cells than in LNCaP-C3 cells, while SOCS1 to SOCS6 were expressed at approximately the same levels. Using siRNA to knockdown SOCS7 and CIS expression in LNCaP-S17 cells led to increased phosphorylation of STAT3 upon IL-6 stimulation. LNCaP-S17 cells are resistant to exogenous IL-6-induced NED due to increased levels of CIS/SOCS7 that block activation of JAK2-STAT3 pathways.

Inhibition of Glycogen Synthase Kinase-3β Counteracts Ligand-Independent Activity of the Androgen Receptor in Castration Resistant Prostate Cancer

In order to generate genomic signals, the androgen receptor (AR) has to be transported into the nucleus upon androgenic stimuli. However, there is evidence from in vitro experiments that in castration-resistant prostate cancer (CRPC) cells the AR is able to translocate into the nucleus in a ligand-independent manner. The recent finding that inhibition of the glycogen-synthase-kinase 3β (GSK-3β) induces a rapid nuclear export of the AR in androgen-stimulated prostate cancer cells prompted us to analyze the effects of a GSK-3β inhibition in the castration-resistant LNCaP sublines C4-2 and LNCaP-SSR. Both cell lines exhibit high levels of nuclear AR in the absence of androgenic stimuli. Exposure of these cells to the maleimide SB216763, a potent GSK-3β inhibitor, resulted in a rapid nuclear export of the AR even under androgen-deprived conditions. Moreover, the ability of C4-2 and LNCaP-SSR cells to grow in the absence of androgens was diminished after pharmacological inhibition of GSK-3β in vitro. The ability of SB216763 to modulate AR signalling and function in CRPC in vivo was additionally demonstrated in a modified chick chorioallantoic membrane xenograft assay after systemic delivery of SB216763. Our data suggest that inhibition of GSK-3β helps target the AR for export from the nucleus thereby diminishing the effects of mislocated AR in CRPC cells. Therefore, inhibition of GSK-3β could be an interesting new strategy for the treatment of CRPC.

Androgen receptor coregulators NOCR1, TIF2, and ARA70 may account for the hydroxyflutamide insensitivity of prostate cancer cells

Prostate cancer cells can switch from an androgen-dependent state to an androgen-independent state after a continuous androgen ablation therapy. However, the molecular mechanisms underlying this switch are still unclear. Therefore, we explored the change in androgen receptor (AR)-related gene expression during this transition in a novel cell model. Prostate cancer cells were continuously treated with competitive androgen receptor inhibitor hydroxyflutamide for 1.5 years, which yielded an flutamide-insensitive LNCaP subline, LNCaP-flu, as confirmed by MTT assays, flow cytometry, and electron microscopy. We analyzed the differences in gene expression in LNCaP-flu cells and LNCaP cells using gene chips and follow-up RT-PCR. Over 2,428 genes were differentially expressed between these cell lines: 1,194 were down-regulated and 1,234 were up-regulated. Three genes in particular were considered related to the androgen-dependent transition: NCOR1, TIF2 (NCOA2), and ARA70 (NCOA4). There were no apparent changes in expression of the androgen receptor or prostate-specific antigen. ARs and associated coregulators play a central role in the flutamide-insensitive transition of prostate cancer cells. Although AR expression does not change during this transition, the change in AR coregulators may be a critical factor in the development of antiandrogen insensitivity.

Naftopidil, a Selective α1-Adrenoceptor Antagonist, Suppresses Human Prostate Tumor Growth by Altering Interactions between Tumor Cells and Stroma

In prostate cancer, tumor-stroma interactions play a critical role in the promotion of tumorigenesis, and thus the prevention of those interactions is a promising target to suppress tumor growth. Several studies demonstrated that alpha(1)-adrenoceptor (α(1)-AR) antagonists, therapeutic drugs for benign prostatic hyperplasia, have growth inhibitory effects on human prostate cancer (PCa) cells through induction of apoptosis or G(1) cell-cycle arrest. However, their direct actions on stromal cells surrounding cancer cells have not yet been elucidated. In this study, we investigated the effects of subtype-selective α(1)-AR antagonists (naftopidil, tamsulosin, and silodosin) on prostate tumor growth with a focus on the role of stroma, using commercially available fibroblast cells (PrSC). Tumorigenic studies in vivo showed significant reductions in tumor growth when E9 cells (an androgen low-sensitive LNCaP subline) grafted with PrSC were treated with naftopidil. In in vitro analyses, naftopidil and silodosin showed antiproliferative effects on PCa cells regardless of androgen sensitivity and α(1)-AR subtype expression. In PrSC, a strong growth inhibitory effect was observed with naftopidil but not silodosin. Flow cytometric analysis revealed that naftopidil, but not silodosin, induced G(1) cell-cycle arrest in both PCa cells and PrSC. In naftopidil-treated PrSC, total interleukin-6 protein was significantly reduced with increased suppression of cell proliferation. Silodosin induced weak early apoptosis only in PCa cells. These findings demonstrated that naftopidil strongly suppressed cell proliferation of stromal cells, resulting in decreased tumorigenic soluble factor, suggesting that naftopidil might be effective in preventing stromal support of tumor cells.