CaP Cell Lines Research Articles

Benchmark Datasets for Computational Drug Discovery: Pancreatic Cancer and Cardiovascular Disorders as Case Studies

The current issue of the Biomedical Data Journal (BMDJ)on Computational Drug Discovery focuses on the publication of datasets that will allow researchers to investigate deeper computational techniques for drug design and drug repositioning for pancreatic cancer and cardiovascular disorders. Drug repositioning is the task of finding new targets for old drugs and has been in the spotlight for the past few years. The average cost for launching a new drug into the market is estimated to ~1.8 billion dollars. Apart from that, the drugs that make it to the market are very few. Notably, from 1999 to 2008, only 50 compounds were FDA approved in the U.S., out of which 17 were identified as arising from target-based discovery methods. This stresses the importance of drug repositioning in the process of drug development, since it accelerates the process, minimizes the associated costs, and, in parallel, contributes to the prevention of noxious adverse events and toxicological liabilities. Especially for the case studies of pancreatic cancer and cardiovascular disorders, there is a dire need of datasets which would allow researchers to examine drug repositioning opportunities, and certainly could act as an important curating or guiding factor for any future drug design efforts. Given this need, the current issue focuses on datasets in these two cases. The papers in this issue can be divided in three categories: pancreatic cancer dataset, 1 general dataset for computational drug repositioning modelling, 2 and cardiovascular diseases datasets. 3,4,5 The first paper 1 presents a dataset from the pancreatic cancer case study. Raw data obtained from exposing the human pancreatic adenocarcinoma cell line Capan-2 to Gemcitabine and the HSP27-inhibitor BVDU ((E)-5-(2-Bromovinyl)-2′-deoxyuridine, RP101) are described. Differential gene expression was displayed using DNA microarrays. The raw measurements of the pairwise comparison of control (untreated), Gemcitabine-, BVDU-, and Gemcitabine with BVDU - treated probes are released. The second paper 2 introduces a dataset for computational drug repositioning, comprising 54 drug repositioning cases, the information for which was manually compiled, curated and integrated. The new indications for the reported cases have been approved by FDA or EU RUS in the period 1955-2013.

MicroRNA-222 Controls Human Pancreatic Cancer Cell Line Capan-2 Proliferation by P57 Targeting.

Pancreatic cancer (PC) is one of the most common cancers and has a poor prognosis due to late diagnosis and ineffective therapeutic multimodality. MicroRNAs (miRNAs, miRs) are a group of non-coding, small RNAs with active biological activities. In our investigation, human pancreatic cancer cell line Capan-2 were transfected with miR-222 mimics, inhibitors or their negative controls. Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8), EdU incorporation assay and cell cycle determination by flow cytometry. MiR-222 and putative target gene expression levels including p27, p57 and PTEN were determined using quantitative reverse transcription polymerase chain reactions and Western blotting. Our results showed that miR-222 could lead to increased vitality and proliferative rate of Capan-2 cells, and also higher S-phase and lower G1-phase of cell cycle. Further, we found p57 at protein level, but not p27 nor PTEN, was regulated by miR-222 in Capan-2 cells. Finally, we co-transfected miR-222 inhibitor and p57 si-RNA into Capan-2 cells, and found that proliferation-suppressing effects of miR-222 inhibitor on Capan-2 cells could be partially reversed by silencing p57. Our results indicate that miR-222 controls Capan-2 cell proliferation by targeting p57. This study provides a novel idea for developing effective therapeutic strategy for PC patients through inhibiting miR-222.

CRKL protein overexpression enhances cell proliferation and invasion in pancreatic cancer.

CRKL is an adapter protein which is overexpressed in many malignant tumors and plays crucial roles in tumor progression. However, expression pattern and biological roles of CRKL in pancreatic cancer have not been examined. In the present study, we found that CRKL expression in pancreatic cancer specimens was higher than that in normal pancreatic tissues. Colony formation assay and Matrigel invasion assay showed that the overexpression of CRKL in Bxpc3 and Capan2 cell lines with low endogenous expression increased cell proliferation and invasion. Flow cytometry showed that CRKL promoted cell proliferation by facilitating cell cycle. Further analysis of cell cycle- and invasion-related molecules showed that CRKL upregulated cyclin D1, cyclin A, matrix metalloproteinase 2 (MMP2) expression, and phosphorylated extracellular signal (ERK)-regulated kinase. In conclusion, our study demonstrated that CRKL was overexpressed in human pancreatic cancers and contributed to pancreatic cancer cell proliferation and invasion through ERK signaling.

Abstract 4005: CD44 isoform variant 6 is associated with prostate cancer progression, metastasis and chemo-/radio-resistance via PI3K/Akt/mTOR and Wnt/β-catenin signaling pathways in vitro

Abstract Aim: The role of CD44 variant 6 (CD44v6) in prostate cancer (CaP) metastasis and progression remains unclear. The aim of this study was 1) to examine the expression of CD44v6 in metastatic CaP cell lines, and normal, benign hyperplastic, and CaP tissues in different stages and lymph node metastases; 2) to investigate the role of CD44v6 in CaP progression, metastasis and therapy resistance as well as underlying pathways in vitro for a potential therapeutic target. Methods: Immunohistochemical/immunocytochemical analysis using an anti-CD44V6 monoclonal antibody (MAb) was performed on metastatic CaP cell lines and paraffin embedded specimens from patients (n=10) who underwent radical prostatectomy (RP), lymph node metastases (n=10), benign prostatic hyperplasia (BPH) (n=10), and normal prostate tissues (n=10). The immunostaining results from CaP cell lines were further confirmed by Western blot. The functional roles of CD44v6 in CaP metastasis and chemo- and radio-sensitivity were examined by knocking down (KD) CD44v6 gene using siRNAs in PC-3M, DU145 and LNCaP CaP cells, respectively and then performing cell invasion, colony formation, proliferation rate, sphere formation, chemo- and radio-sensitivity assays. Results: The expression of CD44v6 was found moderately to strongly positive in androgen-responsive (LNCaP, LNCaP-LN3) and androgen-nonresponsive (PC-3, PC-3M, DU145 and LNCaP-C4B2) metastatic CaP cell lines. The expression of CD44v6 was found negative in normal prostate and BPH tissues, and moderately to strongly positive primary CaP tissues and lymph node metastases. Three CD44v6-KD cell lines showed notable decreases in functions of invasion, colony formation, proliferation, sphere formation, and an increase in chemodrug and radiation sensitivity, compared to the parental cell lines, respectively. Obvious down-regulation of PI3K/Akt/mTOR and Wnt/β-catenin signaling pathways were observed in three CD44v6-KD cell lines by Western Blot. Conclusions: CD44v6 is a biomarker involved in CaP progression and metastasis, associated with CaP chemo-/radio-resistance via PI3K/Akt/mTOR and Wnt/β-catenin signaling pathways and could be an ideal potential therapeutic target for the treatment of castrate-resistant prostate cancer (CRPC). The in vivo study of CD44v6 functions in CaP animal models is ongoing in our laboratory. Citation Format: Jie Ni, Paul Cozzi, Jingli Hao, Julia Beretov, Lei Chang, Wei Duan, Warick Delprado, Peter Graham, Joseph Bucci, John Kearsley, Yong Li. CD44 isoform variant 6 is associated with prostate cancer progression, metastasis and chemo-/radio-resistance via PI3K/Akt/mTOR and Wnt/β-catenin signaling pathways in vitro. [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 4005. doi:10.1158/1538-7445.AM2014-4005

Abstract 171: Adipose-derived mesenchymal stem cell (ADSC) has the differentiation capacity toward cancer associated fibroblast (CAF) and reproduce the morphology of the clinical tumor stroma

Abstract [Objectives] Crosstalk between cancer cells and Cancer-Associated Fibroblast (CAF) plays a crucial role that comprises 3D organization of solid cancers. It seems general understanding that the main origin of CAF is circulating bone marrow derived Mesenchymal Stem Cell (BM-MSC), though locally pre-existing resident MSC of adipose tissue (AD-MSC) should partly contribute to CAF formation. The role of CAF and MSC to tumorigenesis remains controversial,since some studies reported cancer stimulating effect by externally added MSC or its potential differentiating into CAF, while others showed its suppressive effect. The capability of MSC's innate tropism for cancer, which enables us to apply it for the cellular delivery of anti-cancer molecules, has attracted much attention. One of the key issues should be whether externally added MSC would accepted as a member of cancer 3D organization, and would contribute to the cancer morphology. We tried to reveal whether AD-MSC, which is much convenient source as engineered MSC than BM-MSC, stimulates or inhibit tumor growth, and how MSC contribute to tumor stromal morphogenesis. [Materials & Methods] Experiment: To evaluate the growth advantage by adding MSC, and how MSC contribute to tumor stromal morphogenesis, two differently-originated ADSC cell lines transfected with GFP was mixed with human pancreatic-cancer cell line (Capan-1) at a rate of 3×106 /5×106 , and inoculated into the back subcutaneous resion of BALB/cAJcl-nu/nu mice. The tumor volume was calculated at Day4, Day7 and Day10 to assess the growth advantage of tumor mixed with ADSC. The mice was sacrificed at Day 10 and the histology of the resected tumor was evaluated with fluorescence microscopy and immunostaining procedure with anti-GFP antibody. [Results] Capan-1 formed significantly larger subcutaneous tumor by adding with both two AD-MSC cell lines(352mm3,265mm3) compared to control (70mm3) at Day10. In the tumor mixed with one ADSC cell line, cancer cells formed ductal stractures and contained fiber rich stroma with GFP positive fibroblasts among cancer ducts, mimicked the similar distribution to that in clinical solid cancer specimen. However, in the graft with the other ADSC cell line, cancer cells presented cord-like structures or deregulated cell proliferation, not formed ductal stractures. Though GFP positive cells could be observed, they thinly and rondamly distributed in the stroma. [Discussion] Both two AD-MSC plays promotive role in tumor growth, and one of which strongly contribute to the tumor stromal morphogenesis. Our AD-MSC could be regarded as a case which obviously differentiated into CAF, and would be a good candidate source for forecomming CAF therapy. Further studies revealing CAFs specific signature would helpful for providing stable AD-MSC source. Citation Format: Yuki Inagaki, Tatsuya Oda, Tomohiro Kurokawa, Ryoichi Miyamoto, Yasuyuki Kida, Nobuhiro Ohkohchi. Adipose-derived mesenchymal stem cell (ADSC) has the differentiation capacity toward cancer associated fibroblast (CAF) and reproduce the morphology of the clinical tumor stroma. [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 171. doi:10.1158/1538-7445.AM2014-171

Abstract 912: P21-activated kinase 4 (PAK4) as a predictive marker for gemcitabine in pancreatic cancer cell line

Abstract Pancreatic cancer is a highly lethal disease. Although gemcitabine is activelyused in pancreatic cancer, modest activity and relativeresistanceare revealed.P21-activated kinases(PAKs) are involved in cytoskeletal reorganization, gene transcription, cellproliferation and survival, and oncogenic transformation.The gene amplification and protein overexpression of PAK1 and PAK4were reported in pancreatic cancer, which were associated with K-ras mutation, MUC13 and Smad4. Therefore, we hypothesized that PAKs could predict the drug-sensitivity of gemcitabine in pancreatic cancer and could be a therapeutic target. Using cell viability assay of human pancreatic cancer cell lines (Capan1, Capan2, MIA-paca2, PANC1, Aspc1, SNU213, SNU410) for gemcitabine, Capan-2, PANC-1 and SNU-410 cell lines was identifiedas resistant cell lines for gemcitabine. Through western blots of molecules related with down-steam pathway and drug-sensitivity, higher expression of PAK4 and lower expression of human equilibrative nucleoside transporter 1 (hENT1) were associated with the resistance for gemcitabine. PAK4 down-regulation through siRNA induced the up-regulation of hENT1, which is well-known as predictive marker for gemcitabine activity. In resistance cell lines (Capan-2, PANC-1, SNU-410), down-regulation of PAK4 through siRNArestored the drug-sensitivity for gemcitabine. In conclusion, PAK4 can be a potential predictive marker and therapeutic target in gemcitabine chemotherapy for pancreatic cancer. Citation Format: Sung Ung Moon, Jin Won Kim, Ji Hea Sung, Mi Hyun Kang, Hyun Chang, Jeong Ok Lee, Yu Jung Kim, Keun Wook Lee, Jee Hyun Kim, Soo Mee Bang, Jong Seok Lee. P21-activated kinase 4 (PAK4) as a predictive marker for gemcitabine in pancreatic cancer cell line. [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 912. doi:10.1158/1538-7445.AM2014-912

Abstract 327: Targeting autophagy overcomes enzalutamide resistance in castrate-resistant prostate cancer cells and improves therapeutic response in a xenograft model

Abstract Androgen deprivation or treatment with the androgen receptor signaling inhibitor (ARSI), Enzalutamide (MDV-3100, ENZA) or biclutamide induces autophagy in androgen-dependent and castration resistant CaP (CRPC) cell lines. This autophagic process is mediated by AMPK activation and the suppression of mTOR through Raptor phosphorylation (Serine 792). Using autophagy modulators such as clomipramine (CMI) or metformin in combination with ENZA drastically reduces the cell survival in the colony formation assay. Metformin in combination with ENZA causes AR degradation, which is inhibited by proteasome inhibitor MG132. Transcriptome deep sequencing of parental and ENZA resistant C4-2B cells was carried out to examine differential gene expression pattern that may be related to their ability to survive under constant high exposure to ARSI. In-vivo studies with mice orthotopically implanted with ENZA resistant cells demonstrates that the combination of ENZA and autophagy modulators, CMI or metformin significantly reduces tumor growth when compared to control groups (p<0.005). Blocking autophagy pharmacologically or genetically significantly impairs prostate cancer cell survival in- vitro and in-vivo, implying the therapeutics potential of autophagy inhibitors in the antiandrogen resistance setting.This work is supported in part by Stand Up To Cancer - Prostate Cancer Foundation–Prostate Dream Team Translational Cancer Research Grant. This research grant is made possible by the generous support of the Movember Foundation. Stand Up To Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research. Citation Format: Joy C. Yang, Hao G. Nguyen, Hsing-Jien Kung, Xu-Bao Shi, Derya Tilki, Ralph W. deVere White, Allen C. Gao, Christopher P. Evans. Targeting autophagy overcomes enzalutamide resistance in castrate-resistant prostate cancer cells and improves therapeutic response in a xenograft model. [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 327. doi:10.1158/1538-7445.AM2014-327

Abstract 189: Stromal primary cultures from advanced human prostate cancer obtained from needle biopsies revealed distinctive gene expression pattern and functionality

Abstract Introduction: Although several pieces of data indicate a critical role of the carcinoma associated stroma (CAS) in the progression of prostate cancer (CaP), little is known about the specific mechanisms by which CAS contributes to the evolution of the castration-recurrent phenotype in locally advanced/metastatic CaP patients. The main challenge in this area has been the ability to obtain primary cultures of human prostate stroma cells (HPSCs) from locally advanced or metastatic CaP, because therapeutic surgery is not a feasible option for these patients. The aim of this study was to standardize a methodology to obtain primary cultures of HPSCs from from needle biopsies of the primary tumor of advanced CaP and to investigate whether HPSCs from different histological/pathological origins have distinct transcriptional programs. Material and Methods: Primary cultures of HPSCs were obtained from explants of needle biopsies from the peripheral zone of primary tumor of advanced/metastatic CaP patients (T3/T4) or from peripheral zone from patients without evidence of neoplasia (BAS). HPSCs were characterized using gene expression profile (Illumina), qPCR, immunocytochemistry and western blot. The phenotypic alterations were analyzed using the stroma cell markers calponin, vimentin, pro-collagen and α-smooth muscle actin. Migration and invasion assays using co-cultures of stroma-epithelium were utilize to evaluate the functional role of CAS. Results: Analysis of primary cultures of HPSCs from benign and locally advanced and/or metastatic CaP tissue explants revealed that CAS are a heterogeneous population of cells, typified by the increment of myofibroblasts/fibroblasts ratio and collagen production. The proliferation rate and doubling time was significantly higher in CAS compared to BAS. Analysis of gene expression profile revealed that CAS exhibited a unique transcriptional program, up-regulating genes involved in cell proliferation migration and cell adhesion. Furthermore, migration and invasion assays using co-cultures of stroma-epithelium demonstrated that HPSCs from CAS, but not BAS, contributed to the invasive potential of CaP cell lines. Discussion: Our studies demonstrated that CAS from the primary tumor of advanced/metastatic CaP patients was intrinsically different from the stroma associated to non-invasive CaP. Accordingly, primary cultures of HPSCs from primary tumors of metastatic patients provide an invaluable tool for modeling the effect of perturbation of the prostate tissue microenvironment during the progression of CaP. Citation Format: Javier Cerda-Infante, Paola F. Conejeros, Romina A. Coronado, Ignacio F. San Francisco, Gary J. Smith, Alejandro S. Godoy, Viviana P. Montecinos. Stromal primary cultures from advanced human prostate cancer obtained from needle biopsies revealed distinctive gene expression pattern and functionality. [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 189. doi:10.1158/1538-7445.AM2014-189

Pioglitazone inhibits the proliferation and metastasis of human pancreatic cancer cells.

Proliferator-activated receptor-γ (PPAR-γ) is a nuclear receptor that acts as a transcription factor in several types of tissue. PPAR-γ ligands are known to inhibit numerous cancer cell processes, including pancreatic cancer cell proliferation through terminal differentiation. Previous studies concerning the inhibitory effect of PPAR-γ ligands derived from thiazolidinediones (TZDs) on the metastatic potential of cancer cells have been reported. The present study aimed to investigate whether pioglitazone, a prescription TZD class drug and a ligand of PPAR-γ, inhibits the proliferation and metastasis of pancreatic cancer cells. The inhibitory effect of pioglitazone on the proliferation of the Capan-1, Aspc-1, BxPC-3, PANC-1 and MIApaCa-2 pancreatic cancer cell lines was analyzed. Alterations in carcinoembryonic antigen (CEA), interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2) mRNA expression levels subsequent to pioglitazone treatment were examined in BxPC-3 cells by quantitative reverse transcription polymerase chain reaction. In addition, whether the oral administration of pioglitazone prevents tumorigenesis and spontaneous BxPC-3 cell lymph node and lung metastases was investigated using a rectal xenograft model. Pioglitazone treatment resulted in the inhibition of proliferation in all five pancreatic cancer cell lines in vitro. Pioglitazone induced CEA mRNA expression, suppressed IL-8 and COX-2 mRNA expression in vitro, and inhibited BxPC-3 xenograft growth. Pioglitazone also reduced BxPC-3 cell lymph node and lung metastasis in the rectal xenograft model. These results suggest that pioglitazone treatment inhibited the proliferation and metastasis of pancreatic cancer cells through the induction of differentiation and the inhibition of angiogenesis-associated protein expression.

P15 : Androgen receptor protein expression influences freeze sensitivity of prostate cancer cells

A major challenge in prostate cancer (CaP) treatment is evident due to molecular-based transformations resulting in the loss of response to and/or expression of the androgen receptor phenotype. It is generally recognized that many CaPs transform from androgen-dependent/sensitive (AS) to androgen-independent/insensitive (AI) phenotypes with age. Further, is now appreciated that androgen independent CaP is relatively refractory to in situ therapies. Recently, the presumption that freezing serves as a ubiquitous lethal stress in prostate cancer (CaP) has been challenged by anecdotal clinical experience and experimental evidence demonstrating time–temperature related cell death dependencies. As such, in this study we investigate the difference in freeze sensitivity between AI and AS CaP cell lines to determine the impact of CaP AR responsiveness on cryoablation efficacy. We hypothesized that molecular changes resulting in a more aggressive phenotype following androgen ablation treatments is responsible, in part, for the observed increase in cell tolerance to cryotherapy. A series of studies were designed to identify the cellular mechanisms contributing to variations in freeze response. Survival estimates of AS and AI CaP cell lines following freezing exposure were determined using cell viability assays and fluorescent microscopy. Cell death and stress pathway involvement was detailed through molecular analysis and inhibition studies of select key proteins. LNCaP (passaged multiple times) cells demonstrated a phenotypic shift to freeze resistance that matched the freeze response of AI PC-3 cells along with a transformation to androgen independence, while PC-3 cells stably expressing a functional androgen receptor matched the freezing sensitivity of the AS LNCaP cell line. Importantly, AI cell lines survived and recovered freezing exposure to temperatures to −40 °C, whereas AS cell lines exposed to temperatures colder than −20 °C did not survive. Cell death assays using an apoptotic inhibitor revealed that apoptosis was significantly increased in both AS cell lines following freezing compared to AI counterparts. These results were further confirmed through caspase activity assays and apoptotic fluorescent probes. Protein analysis indicated the androgen receptor is manipulated post-freezing exposure. Finally, AKT inhibition significantly increased the freezing sensitivity of AI CaP cells. These observations are the first to describe a phenotypic variation in CaP cellular response to freezing. Our study demonstrates that AS cancers are far more sensitive to a freezing insult, which might support an interpretation that a younger patient pool may prove to be ideal candidates for cryoablation as a primary therapy. Specifically, CaP cryoablation may prove most effective in CaPs that have not yet progressed to a more resistant AI phenotype. In addition, a further understanding of the role of the AR in freezing responses may increase options for neo-adjunctive treatments targeting the AR signaling pathway.

A genome-wide RNAi screen identifies FOXO4 as a metastasis-suppressor through counteracting PI3K/AKT signal pathway in prostate cancer.

Activation of the PI3K/AKT signal pathway is a known driving force for the progression to castration-recurrent prostate cancer (CR-CaP), which constitutes the major lethal phenotype of CaP. Here, we identify using a genomic shRNA screen the PI3K/AKT-inactivating downstream target, FOXO4, as a potential CaP metastasis suppressor. FOXO4 protein levels inversely correlate with the invasive potential of a panel of human CaP cell lines, with decreased mRNA levels correlating with increased incidence of clinical metastasis. Knockdown (KD) of FOXO4 in human LNCaP cells causes increased invasion in vitro and lymph node (LN) metastasis in vivo without affecting indices of proliferation or apoptosis. Increased Matrigel invasiveness was found by KD of FOXO1 but not FOXO3. Comparison of differentially expressed genes affected by FOXO4-KD in LNCaP cells in culture, in primary tumors and in LN metastases identified a panel of upregulated genes, including PIP, CAMK2N1, PLA2G16 and PGC, which, if knocked down by siRNA, could decrease the increased invasiveness associated with FOXO4 deficiency. Although only some of these genes encode FOXO promoter binding sites, they are all RUNX2-inducible, and RUNX2 binding to the PIP promoter is increased in FOXO4-KD cells. Indeed, the forced expression of FOXO4 reversed the increased invasiveness of LNCaP/shFOXO4 cells; the forced expression of FOXO4 did not alter RUNX2 protein levels, yet it decreased RUNX2 binding to the PIP promoter, resulting in PIP downregulation. Finally, there was a correlation between FOXO4, but not FOXO1 or FOXO3, downregulation and decreased metastasis-free survival in human CaP patients. Our data strongly suggest that increased PI3K/AKT-mediated metastatic invasiveness in CaP is associated with FOXO4 loss, and that mechanisms to induce FOXO4 re-expression might suppress CaP metastatic aggressiveness.

The negative interplay between Aurora A/B and BRCA1/2 controls cancer cell growth and tumorigenesis via distinct regulation of cell cycle progression, cytokinesis, and tetraploidy.

It is well known that the activation of Aurora A/B (Aur A/B) or inactivation of BRCA1/2 induces tumor formation. Others and we have reported that the mutual suppression between Aur A/B and BRCA1/2 may manipulate cancer cell growth and tumorigenesis, however, the interactive regulation and mechanism between these molecules are still elusive. In this study, by consecutive silencing of Aur A/B or/and BRCA1/2 with specific shRNAs, we showed that, in BRCA2-deficient pancreatic cancer cell line Capan-1 and in ovarian cancer cell line OVCA433, Aur A/B and BRCA1/2 inversely regulated the expression of each other likely through proteasome-mediated proteolysis but not through gene transcription. Aur A/B and BRCA1/2 conversely regulated cell cycle progression mainly through control of p53 and cyclin A. Moreover, the disruption of Aur A/B blocked abnormal cytokinesis and decreased cell multinuclearity and chromosome tetraploidy, whereas the deprivation of BRCA1/2 promoted the abnormal cytokinesis and enhanced the cell multinuclearity and tetraploidy. Furthermore, we showed by animal assays that the depletion of Aur A/B inhibited tumor growth of both cell lines, while the knockdown of BRCA1/2 promoted the tumor growth. However, the concurrent silencing of Aur A/B and BRCA1/2 diminished the effects of these molecules on the regulation of cell cycle, cytokinesis, and tetraploidy, leading to the burdened tumor sizes similar to those induced by scrambled shRNA-treated control cells. In summary, our study revealed that the negative interplay between Aur A/B and BRCA1/2 inversely controls the cell proliferation, cell cycle progression, cell multinuclearity, and tetraploidization to modulate tumorigenesis.

GnRH-Gemcitabine Conjugates for the Treatment of Androgen-Independent Prostate Cancer: Pharmacokinetic Enhancements Combined with Targeted Drug Delivery

Gemcitabine, a drug with established efficacy against a number of solid tumors, has therapeutic limitations due to its rapid metabolic inactivation. The aim of this study was the development of an innovative strategy to produce a metabolically stable analogue of gemcitabine that could also be selectively delivered to prostate cancer (CaP) cells based on cell surface expression of the Gonadotropin Releasing Hormone-Receptor (GnRH-R). The synthesis and evaluation of conjugated molecules, consisting of gemcitabine linked to a GnRH agonist, is presented along with results in androgen-independent prostate cancer models. NMR and ligand binding assays were employed to verify conservation of microenvironments responsible for binding of novel GnRH-gemcitabine conjugates to the GnRH-R. In vitro cytotoxicity, cellular uptake, and metabolite formation of the conjugates were examined in CaP cell lines. Selected conjugates were efficacious in the in vitro assays with one of them, namely, GSG, displaying high antiproliferative activity in CaP cell lines along with significant metabolic and pharmacokinetic advantages in comparison to gemcitabine. Finally, treatment of GnRH-R positive xenografted mice with GSG showed a significant advantage in tumor growth inhibition when compared to gemcitabine.

Methylthioadenosine phosphorylase is required for prostate cancer growth in vitro and in vivo (766.2)

Methylthioadenosine phosphorylase (MTAP) helps to sustain methionine metabolism, which, either directly or indirectly, plays a significant role in polyamine synthesis, DNA synthesis, and DNA methylation. MTAP is one of the most frequently deleted genes in human cancers, possibly contributing to genetic and epigenetic damage. Because prostate cells have an exceptionally high level of polyamine biosynthesis impinging on methionine metabolism, we investigated MTAP status in human prostate cancer (CaP). In contrast to many other types of cancer, MTAP was never deleted in CaP, as assessed in a sample of 13 cell lines by PCR and 75 CaP patients at different stages of the disease by tissue microarray analysis. In the context of folate deficiency, which stresses methionine metabolism, transcriptional up regulation of MTAP allowed CaP cells to adapt to the metabolic stress in order to provide sufficiency of key metabolites as assessed by HPLC analysis. We therefore hypothesized that MTAP may play an essential role in sustaining CaP growth. We treated a panel of CaP cell lines with a specific MTAP inhibitor. Consistent with MTAP’s importance in prostate cells’ metabolism, we found that the inhibitor was very effective in preventing CaP growth in vitro, with an IC50 between 0.7 and 1.4 µM. Finally, MTAP knock down in LNCaP cells prevented tumor establishment in nude mice. We conclude that MTAP plays a pivotal role in CaP growth and that, because of its importance specific to prostate cells, it could be a novel, ideal target for CaP therapy.Grant Funding Source: AICR American Institute for Cancer Research

MP49-11 THE RHOA EFFECTOR PROTEIN KINASE N1 AS A TARGET FOR SELECTIVE ANDROGEN DEPRIVATION THERAPY

You have accessJournal of UrologyProstate Cancer: Basic Research IV1 Apr 2014MP49-11 THE RHOA EFFECTOR PROTEIN KINASE N1 AS A TARGET FOR SELECTIVE ANDROGEN DEPRIVATION THERAPY Adam DePriest, Kelly Duncan, Elena Pop, James Mohler, and Hannelore Heemers Adam DePriestAdam DePriest More articles by this author , Kelly DuncanKelly Duncan More articles by this author , Elena PopElena Pop More articles by this author , James MohlerJames Mohler More articles by this author , and Hannelore HeemersHannelore Heemers More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1112AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail Introduction and Objectives The androgen receptor (AR) is the principal target for treatment of advanced prostate cancer (CaP). Androgen deprivation therapy (ADT) inhibits systemic AR action, but is not curative and causes severe side effects. The development of more effective and selective forms of ADT requires insights in the molecular mechanisms by which AR governs CaP progression. Our group has identified a novel mode of androgen action in which effects of androgens are mediated by Serum Response Factor (SRF). This mechanism controls a very small fraction of androgen action that is associated with aggressive CaP behavior. Androgen control over SRF action is achieved via androgen activation of the small GTPase RhoA. Here, the signal transduction that conveys androgen-responsiveness from RhoA to SRF, and its potential for selective inhibition of SRF-mediated androgen action, is identified. Methods An siRNA screen was performed to identify the RhoA effector protein(s) that mediates signaling from androgen-activated RhoA to SRF. qRT-PCR, custom oligoarray, promoter-reporter transfection and Western blotting approaches were used to study androgen-responsiveness of Androgen Response Element (ARE)-driven AR target genes or CArG box-driven SRF target genes. Androgen-stimulated (AS) and castration-recurrent (CR) CaP cells were treated with siRNAs targeting the RhoA effector Protein Kinase N1 (PKN1) or the PKN1 inhibitor lestaurtinib. Validation studies involved CaP cell lines that (over)express wild-type RhoA, dominant-active RhoA, dominant-negative RhoA, wild-type PKN1, or PKN1 forms mutated in its kinase domain or RhoA-interacting regions. Results The RhoA effector PKN1 was isolated as key mediator of androgen-responsive SRF action. PKN1 is overexpressed in CaP, where its overexpression has been correlated with CaP dedifferentiation. Loss of PKN1 decreased the androgen-responsiveness of SRF target genes, but affected androgen regulation of only 12 of 452 (or 2.6%) of ARE-driven genes. PKN1-dependent androgen regulation of SRF target genes was maintained in AS and CR CaP cells and in CaP cells that overexpress wild-type or dominant-active RhoA. Mutation of PKN1’s kinase domain or RhoA interaction sites attenuated androgen regulation of SRF target genes. PKN1 inhibitor lestaurtinib preferentially impeded androgen regulation of SRF target genes over that of ARE-driven genes. Conclusions PKN1 inhibitors may represent more selective forms of ADT that affect only androgen-dependent events that underlie CaP progression. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e506 Peer Review Report Advertisement Copyright & Permissions© 2014MetricsAuthor Information Adam DePriest More articles by this author Kelly Duncan More articles by this author Elena Pop More articles by this author James Mohler More articles by this author Hannelore Heemers More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

CARMA3 is upregulated in human pancreatic carcinoma, and its depletion inhibits tumor proliferation, migration, and invasion

Elevated CARMA3 expression has been reported to be involved in tumor progression of several cancer types. In the present study, we examined the expression pattern of CARMA3 protein and its biological roles in human pancreatic carcinoma. Using immunohistochemistry, we checked CARMA3 protein expression in 95 pancreatic ductal carcinoma specimens. We found that CARMA3 was overexpressed in 34 of 95 (35.8 %) specimens. A significant association was observed between CARMA3 overexpression with histological grade (p=0.0099) and nodal status (p=0.0126). To further explore its biological roles, we knocked down CARMA3 expression in CAPAN2 cell line using small interfering RNA (siRNA). MTT growth assay, wound healing assay, and Transwell assay showed that CARMA3 depletion inhibited cell proliferation, migration, and invasion. We also showed that CARMA3 depletion inhibited EGF-induced nuclear factor-kappaB (NF-κB) activation and its target genes' expression. The effect of CARMA3 depletion on NF-κB signaling was significantly reduced in Bcl10-depleted cells. In conclusion, CARMA3 is overexpressed in pancreatic cancer and regulates malignant cell growth, invasion, and NF-κB signaling, which was dependent on its association with Bcl10.

Resveratrol plays dual roles in pancreatic cancer cells

Although the potential anticancer effect of resveratrol (RSV) on pancreatic cancer has been reported, its mechanism was not fully understood. The role of vascular endothelial growth factor B (VEGF-B) in cancer remains controversial. Herein, we aimed to examine whether the anticancer effect of RSV was related to the VEGF-B. The effect of RSV on pancreatic cancer cell line (capan-2 cells) was evaluated by CCK-8 assay, Hoechst 33342 staining, and flow cytometry. The mRNA level of VEGF-B was measured by real-time PCR. VEGF-B expression was knockdown by small interfering RNA (siRNA).The protein levels of VEGF-B, glycogen synthase kinase-3 beta (GSK-3β), and Bax were measured by Western blot. Resveratrol treatment inhibited tumor growth, induced apoptosis, and up-regulated Bax expression in capan-2 cells. The mRNA and protein levels of VEGF-B were up-regulated after RSV treatment. However, VEGF-B siRNA treatment increased the apoptotic rate, and inhibited tumor activator GSK-3β, while Bax expression was not affected. The combination of RSV and VEGF-B siRNA showed significantly higher apoptotic rate in comparison with RSV or VEGF-B siRNA mono-treatment group. Resveratrol plays dual roles in pancreatic cancer: as a tumor suppressor via the up-regulation of Bax; as a tumor activator via the up-regulation of VEGF-B; and the anticancer effect of RSV is much stronger than the cancer promotion effect. The combination of RSV with pharmacological inhibitor of VEGF-B might, therefore, be a promising modality for clinical pancreatic cancer therapy.

Arachidonic acid induction of Rho-mediated transendothelial migration in prostate cancer

Background:Bone metastases in prostate cancer (CaP) result in CaP-related morbidity/mortality. The omega-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) and lipophilic statins affect metastasis-like behaviour in CaP cells, regulating the critical metastatic step of CaP migration to the bone marrow stroma.Methods:Microscopic analysis and measurement of adhesion and invasion of CaP cells through bone marrow endothelial cells (BMEC) was undertaken with AA stimulation and/or simvastatin (SIM) treatment. Amoeboid characteristics of PC-3, PC3-GFP and DU-145 were analysed by western blotting and Rho assays.Results:The CaP cell lines PC-3, PC3-GFP and DU-145 share the ability to migrate across a BMEC layer. Specific amoeboid inhibition decreased transendothelial migration (TEM). AA stimulates amoeboid characteristics, driven by Rho signalling. Selective knock-down of components of the Rho pathway (RhoA, RhoC, Rho-associated protein kinase 1 (ROCK1) and ROCK2) showed that Rho signalling is crucial to TEM. Functions of these components were analysed, regarding adhesion to BMEC, migration in 2D and the induction of the amoeboid phenotype by AA. TEM was reduced by SIM treatment of PC3-GFP and DU-145, which inhibited Rho pathway signalling.Conclusions:AA-induced TEM is mediated by the induction of a Rho-driven amoeboid phenotype. Inhibition of this cell migratory process may be an important therapeutic target in high-risk CaP.

CD44 variant 6 is associated with prostate cancer metastasis and chemo‐/radioresistance

Prostate cancer (CaP) is the second leading malignancy in older men in Western countries. The role of CD44 variant 6 (CD44v6) in CaP progression and therapeutic resistance is still uncertain. Here, we investigated the roles of CD44v6 in CaP metastasis and chemo/radioresistance. Expression of CD44v6 in metastatic CaP cell lines, human primary CaP tissues and lymph node metastases was assessed using immunofluorescence and immunohistochemistry, respectively. Knock down (KD) of CD44v6 was performed in PC-3M, DU145, and LNCaP cells using small interfering RNA (siRNA), and confirmed by confocal microscope, Western blot and quantitative real time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by proliferation and colony formation assays. The adhesive ability and invasive potential were assessed using a hyaluronic acid (HA) adhesion and a matrigel chamber assay, respectively. Tumorigenesis potential and chemo-/radiosensitivity were measured by a sphere formation assay and a colony assay, respectively. Over-expression of CD44v6 was found in primary CaP tissues and lymph node metastases including cancer cells and surrounding stromal cells. KD of CD44v6 suppressed CaP proliferative, invasive and adhesive abilities, reduced sphere formation, enhanced chemo-/radiosensitivity, and down-regulated epithelial-mesenchymal transition (EMT), PI3K/Akt/mTOR, and Wnt/β-catenin signaling pathway proteins in vitro. Our findings demonstrate that CD44v6 is an important cancer stem cell-like marker associated with CaP proliferation, invasion, adhesion, metastasis, chemo-/radioresistance, and the induction of EMT as well as the activation PI3K/Akt/mTOR and Wnt signaling pathways, suggesting that CD44v6 is a novel therapeutic target to sensitize CaP cells to chemo/radiotherapy.

Prostate cancer bone metastases acquire resistance to androgen deprivation via WNT5A-mediated BMP-6 induction

Background:Androgen ablation is the first-line therapy for patients with metastatic prostate cancer (CaP). However, castration resistance will eventually emerge. In the present study, we have investigated the role of bone morphogenetic protein-6 (BMP-6) in the development of castration-resistant prostate cancer (CRPC) in the context of bone metastases.Methods:We initially investigated the clinical course of 158 men with advanced CaP who were treated with primary androgen deprivation therapy. To elucidate the underlying mechanism of CRPC in the context of bone metastases, we examined the impact of bone stromal cells on CaP in the absence of androgens using a co-culture model.Results:In the 158 patients, we found that the median time to prostate-specific antigen progression was significantly shorter when bone metastases were present (14 months (95% CI, 10.2–17.8 months) vs 57 months (95% CI, 19.4–94.6 months)). These results suggest that bone–tumour interactions may accelerate castration resistance. Consistent with this hypothesis, in vitro co-cultures demonstrated that CaP cells proliferated under an androgen-depleted condition when incubated with bone stromal cells. Mechanistically, gene expression analysis using quantitative polymerase chain reaction arrays showed a dramatic induction of BMP-6 by CaP cell lines in the presence of bone stromal cells. Further studies revealed that WNT5A derived from bone stromal cells induced the expression of BMP-6 by CaP cells; BMP-6 in turn stimulated cellular proliferation of CaP cells in an androgen-deprived media via a physical interaction between Smad5 and β-catenin. Intracellularly, WNT5A increased BMP-6 expression via protein kinase C/NF-κB pathway in CaP cell lines.Conclusions:These observations suggest that bone–CaP interaction leads to castration resistance via WNT5A/BMP-6 loop.