Established Prostate Cancer Cell Research Articles

Effect of Acute Exercise on Prostate Cancer Cell Growth

Physical activity is associated with reduced risk of several cancers, including aggressive prostate cancer. The mechanisms mediating the effects are not yet understood; among the candidates are modifications of endogenous hormone levels. Long-term exercise is known to reduce serum levels of growth stimulating hormones. In contrast, the endocrine effects of acute endurance exercise include increased levels of mitogenic factors such as GH and IGF-1. It can be speculated that the elevation of serum growth factors may be detrimental to prostate cancer progression into malignancy. The incentive of the current study is to evaluate the effect of acute exercise serum on prostate cancer cell growth. We designed an exercise intervention where 10 male individuals performed 60 minutes of bicycle exercise at increasing intensity. Serum samples were obtained before (rest serum) and after completed exercise (exercise serum). The established prostate cancer cell line LNCaP was exposed to exercise or rest serum. Exercise serum from 9 out of 10 individuals had a growth inhibitory effect on LNCaP cells. Incubation with pooled exercise serum resulted in a 31% inhibition of LNCaP growth and pre-incubation before subcutaneous injection into SCID mice caused a delay in tumor formation. Serum analyses indicated two possible candidates for the effect; increased levels of IGFBP-1 and reduced levels of EGF. In conclusion, despite the fear of possible detrimental effects of acute exercise serum on tumor cell growth, we show that even the short-term effects seem to add to the overall beneficial influence of exercise on neoplasia.

Abstract 2986: Mechanism of androgen receptor (AR) silencing in small cell prostate carcinoma.

Abstract Small cell prostate cancer (SCPC) is an androgen receptor (AR) negative variant that often emerges in the castration-resistant progression of typical AR-positive prostate adenocarcinomas. Its presence predicts for an aggressive clinical course with poor response to hormonal therapies and a short median survival. Evidence suggests that the emergence of (SCPC) occurs through cellular transdifferentiation. Therefore, we hypothesized that loss of AR gene transcription may depend on epigenetic events. Given the central role of AR in prostate cancer, we reasoned that identifying the mechanisms involved in its downregulation could improve our understanding of how the lethal SCPC subtype arises. Here we investigated the DNA methylation and histone modifications of AR in a group of six prostate tumor xenografts developed from men with CRPC (two AR-positive [MDA PCa 170.4 and MDA PCa 180.30] and four AR-negative SCPC [MDA PCa 144.13, MDA PCa 144.4, MDA PCa 155 and MDA PCa 146.10]) and three established prostate cancer cell lines (one AR-positive [LNCaP] and two AR-negative [PC3 and DU145]). We first evaluated the methylation status of selected CpG sites in the AR promoter-associated CpG island using bisulfite-sequencing followed by pyrosequencing analysis. Except for the cancer cell lines PC-3 and DU145, all other cell lines and xenografts were unmethylated regardless of the AR expression status. Next, we used chromatin immunoprecipitation (ChIP) to evaluate marking of the AR promoter and a putative proximal enhancer by active (H3K4me3 and H3K9ac) and repressive (H3K9me2 and H3K27me3) histone modifications. Marking of the constitutively expressed genes (ACTB and GAPDH) and a repressed gene (HBB) served as control for these experiments. As expected, the only samples with AR marking by H3K4me3 and H3K9ac were the two AR-positive xenografts and LNCaP. Marking by repressive histone modifications was more variable: H3K27me3 was a universal finding in AR-negative samples except for Du-145, and was accompanied by H3K9me2 in two out of four xenografts. H3K27me3 and H3K9me2 also marked the putative enhancer region when AR was repressed. Altogether, these data indicate that promoter DNA methylation of AR is an infrequent finding in prostate cancer, while polycomb protein-based silencing is nearly universal in AR-negative variants. Also, it shows that H3K9me2 promotes reinforcement of silencing. Thus, we propose that reactivation of AR may by achieved in selected cases by inhibiting H3K27me3 alone, while concomitant inhibition of both polycomb and SET domain proteins may be necessary for double-positive H3K27/K9 methylation cases. Given the purported differentiating and tumor suppressor effects of AR, its reactivation in SCPC may be of therapeutic benefit. Citation Format: Brittany N. Kleb, Marcos RH Estecio, Guanglin Wu, Jing-Fang Lu, Christopher J. Logothetis, Sankar Maity, Ana M. Aparicio. Mechanism of androgen receptor (AR) silencing in small cell prostate carcinoma. [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 2986. doi:10.1158/1538-7445.AM2013-2986

Enrichment of Prostate Cancer Stem-Like Cells from Human Prostate Cancer Cell Lines by Culture in Serum-Free Medium and Chemoradiotherapy

The discovery of rare subpopulations of cancer stem cells (CSCs) has created a new focus in cancer research. As CSCs demonstrate resistance to chemoradiation therapy relative to other cancer cells, this allows the enrichment of CSC populations by killing apoptosis-susceptible cancer cells. In this study, three commonly used human prostate cancer (PCa) cell lines (DU145, PC-3 and LNCaP) were examined for their expression of the putative stem cell markers CD133 and CD44 via flow cytometric analysis. Under normal culture conditions, CD133+/CD44+ cells were only present in the DU145 cell line, and comprised only a minor percentage (0.1% ± 0.01%) of the total population. However, the proportion of these CD133+/CD44+ prostate CSCs could be increased in these cell lines via culture in serum-free medium (SFM), or through chemotherapy or radiotherapy. Indeed, after culture in SFM, the proportion of CD133+/CD44+ cells in DU145 and PC-3 had increased to 10.3% and 3.0%, respectively. Moreover, the proportion had increased to 9.8% enriched by chemotherapy and 3.5% by radiotherapy in DU145. Colony-formation tests, cell invasion assays, and tumor xenografts in BALB/c nude mice were used to evaluate the stem cell properties of CD133+/CD44+ PCa cells that were isolated via fluorescence-activated cell sorting (FACS). CD133+/CD44+ cells had an enhanced colony-formation capability and invasive ability in vitro, and displayed greater tumorigenic properties in vivo. These results demonstrate the presence of CD133+/CD44+ prostate CSCs in established PCa cell lines and that populations of these cells can be enriched by culture in SFM or chemoradiotherapy. Finding novel therapies to override chemoradiation resistance in the prostate CSCs is the key to improve long-term results in PCa management.

MicroRNA Regulation of Oncolytic Adenovirus 6 for Selective Treatment of Castration-Resistant Prostate Cancer

Almost all patients with advanced prostate cancer progress to castration-resistant stage with limited treatment options. Oncolytic adenoviruses have been actively pursued as potential agents for cancer treatment. Virtually all clinical trials on oncolytic adenovirus are based on serotype 5. However, viral replication in hepatocytes induces severe liver toxicity and limits its systemic administration for metastatic disease. Moreover, rapid clearance of viral particles injected intravenously further hinders the anticancer efficacy. Adenovirus 6 (Ad6) was previously reported to exhibit less liver toxicity and escape Kupffer cells absorption after systemic administration. To further improve its safety, we generated a novel oncolytic adenovirus Ad6miR, in which four copies of binding sites of a liver-specific microRNA miR122 were incorporated into E1A gene of Ad6. miR122 regulation significantly decreased Ad6 replication in hepatocytes and consequently hepatotoxicity because of the negative regulation of miR122. Cytotoxicity assay using primary or established prostate cancer cell lines showed robust oncolytic activity of Ad6miR. Systemic treatment of established tumors with Ad6miR showed strong antitumor activity, comparable with that of Ad6 or Ad5. Although Ad6 evaded Kupffer cells, its blood clearance rate was as rapid as Ad5. The vast majority of Ad6 particles intravenously injected localized in liver sinusoidal endothelial cells rather than previously reported Kupffer cells. Elevating Ad6miR injection dose increased circulating Ad6miR concentration and its antitumor efficacy. miR122 regulation of Ad6 significantly improves its safety profile after systemic administration, which allows increasing therapeutic doses leading to improved anticancer efficacy of systemic treatment of castration-resistant prostate cancer.

975 ORTHOTOPIC TUMORGRAFTS DERIVED FROM MEN WITH LOCALIZED PROSTATE CANCER REFLECT INITIAL TUMOR PATHOLOGY – A NEW MODEL TO STUDY PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: Animal models that authentically reproduce tumor growth are still sparse in prostate cancer research for several reasons: Whilst orthotopic implantation is technically difficult, most models favor subcutaneous tumor growth that provides little information about natural tumor growth behaviour and tumor stroma interaction. Established prostate cancer cell lines as in vivo xenografts are not able to reflect the variety of tumor specific growth patterns and growth behaviour. Primary cell cultures are difficult to handle and an induction of orthotopic tumors has not been successful yet. Therefore, a tumorgraft model using tumor tissue from prostatectomy specimens was recently developed. METHODS: Balb/c mice were used to graft fresh prostate tumor tissue by surgical orthotopic implantation. Tissue implantation was performed at four inoculation sides in each animal and testosterone propionate was supplemented. Animals were tracked after 12, 16 and 20 weeks by means of 30MHz ultrasound to monitor tumor engraftment. Autopsy, histology and PSA measurements were used to confirm orthotopic tumors after 13, 21 and 28 weeks. RESULTS: To date, tumor implantation was performed in 17 mice. 3 animals were excluded due to early dead or repetitive loss of the testosterone pellet. After successful testosterone supplementation PSA measurement was possible in 12 of 14 mice (86%, range: 0,009 80ng/ml). So far, orthotopic tumor growth was histologically verified in 6 of 14 mice. Ultrasound seems feasible to detect tumors. Of interest, tumorgrafts maintain essential features of the original tumors as tumor growth pattern, PSA expression, androgen receptor expression and high proliferation using human Ki67 immunostaining. CONCLUSIONS: The tumorgrafts resembled histological properties of organ confined prostate cancer. Measurements of serum PSA and three-dimensional ultrasound were useful for monitoring of tumor engraftment and growth. Further studies like comparative genomic hybridization to genetically characterize tumorgrafts are necessary. Besides their role as a bank of tumor samples for repetitive in vitro studies, tumorgrafts display a new approach for an optimized in vivo model of prostate cancer and will allow further investigations on specific components of tumor initiation and progression, including the interactions of prostate cancer cells with the surrounding stroma. Whether this model is also applicable for metastatic progression needs to be further investigated.

Knockdown of β-Catenin Through shRNA Cause a Reversal of EMT and Metastatic Phenotypes Induced by HIF-1α

Obective: Wnt/β-catenin signaling pathway regulates pattern formation during embryogenesis as well as tumor progression. Numbers of studies suggest that this signaling pathway may play an important role in Epithelial-Mesenchymal transition (EMT), however, there was no evidence that Wnt/β-catenin signaling pathway directly controlled the EMT occurrence. Our previous research has successfully proved that overexpression of hypoxia inducible factor-1α (HIF-1α) could induce EMT in LNCaP cells, but not in PC-3. Consistently, the expression of β-catenin protein increased in LNCaP/HIF-1α cells, but not in PC-3/HIF-1α. This study mainly aimed at exploring the essentiality and importance of Wnt/β-catenin signaling pathway in HIF-1α-induced EMT. Methods: Human prostate cancer cells (LNCaP) were stably transfected by recombinant plasmid pcDNA3.1(-)/HIF-1α. The positive clones were selected by G418 and confirmed through western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), and indirect immunofluoesence. Then LNCaP/HIF-1α was transiently transfected with β-catenin shRNA (shRNA1 and shRNA2) and negative shRNA (shRNA-scr). The epithelial markers, mesenchymal markers, and critical proteins in Wnt/β-catenin signaling pathway were separately detected by western blot analysis. Finally, the invasive potency of cells in different transfection group was examined by Matrgel transwell assay. Result: We successfully established prostate cancer cell line LNCaP/HIF-1α and LNCaP/HIF-1α/β-catenin(-). LNCaP/HIF-1α displayed high expression of mesenchymal markers and low expression of epithelial markers. However, compared with LNCaP/HIF-1α, the epithelial marker E-cadherin was increased in LNCaP/HIF-1α/β-catenin(-), whereas the expression of mesenchymal marker N-cadherin, vimentin, MMP-2 were significantly decreased. Inhibition of Wnt signal activity through β-catenin shRNA cause a reversal of EMT induced by HIF-1α in human prostate cancer. Conclusion: Overexpression of HIF-1α stimulates the invasion potency of human prostate carcinoma cells through EMT pathway and Wnt/β-catenin signaling pathway played a vital role in this process. Wnt/β-catenin signaling pathway might be a necessary endogenous signal that directly controlled the EMT occurrence induced by HIF-1α.

Human prostate cancer stem cells: new features unveiled

Human prostate cancer stem cells: new features unveiled

Evidence of epithelial to mesenchymal transition associated with increased tumorigenic potential in an immortalized normal prostate epithelial cell line

The majority of established human prostate cancer cell lines are derived from metastatic lesions and are already tumorigenic in vivo, therefore immortalized normal prostate cell lines may provide a more relevant model to unveil the mechanisms associated with cancer progression and metastasis. PZ-HPV-7, an immortalized human prostate epithelial cell line was used to generate xenograft tumors in mice. A subline designated HPV-PZ-7T was subsequently derived from the subrenal capsule xenograft of a nude mouse. These cells were further characterized using karyotyping, immunofluorescence, qRT-PCR, Western blotting, and three-dimensional cultures in Matrigel. The PZ-HPV-7 cell line possesses a typical epithelial morphology, expresses basal cell markers, and is capable of forming web-like structures with evidence of budding on Matrigel. PZ-HPV-7 is non-tumorigenic in immunocompromised mice by either subcutaneous injection or subrenal grafting. In contrast, the PZ-HPV-7T cells, derived from a xenograft tumor induced by co-inoculation with matrigel using subrenal grafting, possess a mesenchymal phenotype as well as luminal cell markers and are highly tumorigenic and metastatic in nude mice. Functionally and biochemically, the PZ-HPV-7T subline appears to have undergone an epithelial-to-mesenchymal transition (EMT) from the parental PZ-HPV-7 line. We have developed a novel EMT model using an immortalized normal prostate epithelial cell line and generated a new prostate cancer cell line, PZ-HPV-7T, which may represent an excellent system to study mechanisms associated with prostate cancer progression and metastasis.

Apobec 3G Efficiently Reduces Infectivity of the Human Exogenous Gammaretrovirus XMRV

BackgroundThe human exogenous gammaretrovirus XMRV is thought to be implicated in prostate cancer and chronic fatigue syndrome. Besides pressing epidemiologic questions, the elucidation of the tissue and cell tropism of the virus, as well as its sensitivity to retroviral restriction factors is of fundamental importance. The Apobec3 (A3) proteins, a family of cytidine deaminases, are one important group of host proteins that control primary infection and efficient viral spread.Methodology/Principal FindingsHere we demonstrate that XMRV is resistant to human Apobec 3B, 3C and 3F, while being highly susceptible to the human A3G protein, a factor which is known to confer antiviral activity against most retroviruses. We show that XMRV as well as MoMLV virions package Apobec proteins independent of their specific restriction activity. hA3G was found to be a potent inhibitor of XMRV as well as of MoMLV infectivity. In contrast to MoMLV, XMRV infection can also be partially reduced by low concentrations of mA3. Interestingly, established prostate cancer cell lines, which are highly susceptible to XMRV infection, do not or only weakly express hA3G.ConclusionsOur findings confirm and extend recently published data that show restriction of XMRV infection by hA3G. The results will be of value to explore which cells are infected with XMRV and efficiently support viral spread in vivo. Furthermore, the observation that XMRV infection can be reduced by mA3 is of interest with regard to the current natural reservoir of XMRV infection.

Abstract 1263: Induction of nonsteroidal anti-inflammatory drug-activated gene-1 (Nag-1) by NSAIDs: role of p75NTR

Abstract Epidemiologic studies have shown that patients with long term consumption of certain nonsteroidal anti-inflammatory drugs (NSAIDs) exhibit a reduced incidence of prostate cancer. Treatment of prostrate cancer cell lines with NSAIDs leads to caspase mediated apoptosis. Previously, our laboratory has demonstrated that R-flurbiprofen and Ibuprofen induce the expression of the pro-apoptotic protein p75NTR in PC-3, DU-145 and LNCaP prostate cancer cell lines, with a subsequent decrease in cell survival. The p75NTR is a member of the Tumor Necrosis Factor Receptor superfamily of death receptors. It acts as a tumor suppressor in the normal prostate. The expression of p75NTR is lost as prostate cancer progresses and is minimal in established metastatic prostate cancer cell lines such as PC-3, DU-145, and LNCaP. The observed increase in p75NTR protein due to R-flurbiprofen and Ibuprofen treatment occurs as a result of activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Here we show that the upregulation of p75NTR following treatment with NSAIDs corresponds to a concomitant upregulation of the NSAID activated gene-1 (Nag-1) protein, a pro-apoptotic member of the TGF beta family, in PC-3 cells. Nag-1 has been linked to decreased cell proliferation in many studies, and has been shown to play a pro-apoptotic role in breast, colorectal, gastric and prostate cancer cells. Nag-1 is also known by the names GDF −15, MIC-1, PDF, PLAB and PTGFB. This study also shows that Nag-1 protein induction following NSAID treatment is mediated by the p38 MAPK pathway, and is downstream of the p75NTR induction. This is the first report of a relationship between p75NTR and Nag-1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1263.

Abstract 4036: The microRNA profile of prostate carcinoma obtained by deep sequencing reveals potential miRNA targets such as myosin VI

Abstract Background: Prostate cancer is a leading cause of tumor mortality. In order to identify and characterize the underlying molecular mechanisms, we performed microRNA (miRNA) profiling of primary prostate cancers and non-cancer prostate tissue. Furthermore, using database analysis, we sought to identify regulatory targets of aberrantly expressed miRNAs. Material and Methods: We performed comparative miRNA expression profiling in ten prostate cancer specimens and ten non-cancer prostate tissue samples using deep sequencing of cDNA libraries. The deregulation of selected miRNAs was validated in established prostate cancer cell lines by Northern blotting as well as in 26 paired samples of prostate cancer tissue and adjacent normal tissue by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The regulative capabilities of selected miRNAs on potential target genes were analyzed in vitro using reporter gene constructs and transfection of miRNA expression vectors Results: Using deep sequencing we found that 16 miRNAs were up-regulated more than 1.5-fold and 17 miRNAs were down regulated more than 1.5-fold. The most pronounced deregulation was noted for miRNAs miR-375 and miR-200c which showed an up-regulation of more than 4.5-fold and miR-143 and miR-145 which were down regulated 4-fold each. The differential expression of these miRNAs could be confirmed by Northern blot analysis of established prostate cancer cell lines. By analyzing 26 primary prostate cancer samples by qRT-PCR we could detect a significant deregulation of the miRNAs miR-375, miR-143 and miR-145 (p<0.05, paired students T-test). Independent target prediction databases (TargetScan, PicTar) indicate, that the 3′ untranslated region (3′UTR) of the Myosin VI gene is a regulative target for both miR-143 and miR-145. This prediction could be confirmed using a luciferase reporter gene vector containing the Myosin VI 3′UTR. Furthermore, transient expression of miR-143 and miR-145 reduced the amount of Myosin VI protein by 20% and 48% respectively. Conclusion: Our findings indicate that specific miRNAs are reproducibly found to be deregulated in different cohorts of prostate cancer specimens using independent experimental methods. This is the first report to show that miRNAs miR-143 and miR-145 are capable of negatively regulating the protein expression of Myosin VI. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4036.

The MicroRNA Profile of Prostate Carcinoma Obtained by Deep Sequencing

Prostate cancer is a leading cause of tumor mortality. To characterize the underlying molecular mechanisms, we have compared the microRNA (miRNA) profile of primary prostate cancers and noncancer prostate tissues using deep sequencing. MiRNAs are small noncoding RNAs of 21 to 25 nucleotides that regulate gene expression through the inhibition of protein synthesis. We find that 33 miRNAs were upregulated or downregulated >1.5-fold. The deregulation of selected miRNAs was confirmed by both Northern blotting and quantitative reverse transcription-PCR in established prostate cancer cell lines and clinical tissue samples. A computational search indicated the 3'-untranslated region (UTR) of the mRNA for myosin VI (MYO6) as a potential target for both miR-143 and miR-145, the expression of which was reduced in the tumor tissues. Upregulation of myosin VI in prostate cancer was previously shown by immunohistochemistry. The level of MYO6 mRNA was significantly induced in all primary tumor tissues compared with the nontumor tissue from the same patient. This finding was matched to the upregulation of myosin VI in established prostate cancer cell lines. In luciferase reporter analysis, we find a significant negative regulatory effect on the MYO6 3'UTR by both miR-143 and miR-145. Mutation of the potential binding sites for miR-143 and miR-145 in the MYO6 3'UTR resulted in a loss of responsiveness to the corresponding miRNA. Our data indicate that miR-143 and miR-145 are involved in the regulation of MYO6 expression and possibly in the development of prostate cancer.

Abstract C61: The PI3K pathway inhibition in prostate cancer cells is enhanced by dual targeting with the mTOR inhibitor RAD001 and a mTOR/PI3K inhibitor leading to decreased cell proliferation and increased autophagy

Abstract Introduction: Metastatic prostate cancer patients often become resistant to standard treatments such as androgen ablation therapy and docetaxel treatment and novel treatments are needed. Loss of PTEN and subsequent activation of the PI3K pathway is common in patients with advanced prostate cancer. However, use of mTOR inhibitors such as rapamycin analogues as single agents has had limited clinical impact and the identification of a potent therapeutic strategy for PI3K inhibition remains a critical question. Experimental procedures: We have used gene expression data generated from metastatic tumors collected from patients with advanced prostate cancer both before and after treatment with RAD001 along with in vitro experiments in established prostate cancer cell lines to determine the impact of TORC1 inhibition, PI3K kinase inhibition, and dual inhibition on PI3K pathway signaling and cancer cell kinetics. Summary: Treatment with RAD001 decreased tumor levels of pS6 but failed to result in a single clinical response. Gene set enrichment analysis of metastatic castration-resistant prostate cancer biopsies prior to and following treatment with RAD001 was associated with increased PI3K activity following treatment. Analysis of the activity of the downstream components of the PI3K/AKT pathway following RAD001 treatment showed reduced pS6 levels, had minimal impact on p4EBP1 levels, and increased phosphorylation of AKT1. Cytotoxic synergy is observed when RAD001 is combined with the PI3K inhibitor, LY294002 and dual PI3K/mTOR kinase inhibitors. These combinations result in more profound reduction of TORC1 activity resulting in decreased cell proliferation and increased autophagy but a continued persistence of pAKT. Reverse phase proteomics supports increased AKT activity in an androgen-receptor positive prostate cancer cell line treated with the combination as multiple AKT targets experience increased phosphorylation. Conclusion: TORC1 inhibition results in an upregulation of PI3K activity and therapeutic combinations that inhibit both TORC1 and PI3K demonstrate cytotoxic synergy. The signaling manifestations of these combinations are a profound inhibition of TORC1 activity together with persistent phosphorylation and activity of AKT. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C61.

Invasive prostate cancer cells are tumor initiating cells that have a stem cell-like genomic signature

Development of metastasis is a leading cause of cancer-induced death. Acquisition of an invasive tumor cell phenotype suggests loss of cell adhesion and basement membrane breakdown during a process termed epithelial-to-mesenchymal transition (EMT). Recently, cancer stem cells (CSC) were discovered to mediate solid tumor initiation and progression. Prostate CSCs are a subpopulation of CD44(+) cells within the tumor that give rise to differentiated tumor cells and also self-renew. Using both primary and established prostate cancer cell lines, we tested the assumption that CSCs are more invasive. The ability of unsorted cells and CD44-positive and -negative subpopulations to undergo Matrigel invasion and EMT was evaluated, and the gene expression profiles of these cells were analyzed by microarray and a subset confirmed using QRT-PCR. Our data reveal that a subpopulation of CD44(+) CSC-like cells invade Matrigel through an EMT, while in contrast, CD44(-) cells are non-invasive. Furthermore, the genomic profile of the invasive cells closely resembles that of CD44(+)CD24(-) prostate CSCs and shows evidence for increased Hedgehog signaling. Finally, invasive cells from DU145 and primary prostate cancer cells are more tumorigenic in NOD/SCID mice compared with non-invasive cells. Our data strongly suggest that basement membrane invasion, an early and necessary step in metastasis development, is mediated by these potential cancer stem cells.

Stem Cell Characteristics in Prostate Cancer Cell Lines

BackgroundRecent studies indicate the presence of a small, stem-like cell population in several human cancers that is crucial for the tumour (re)population. ObjectiveSix established prostate cancer (PCa) cell lines—DU145, DuCaP, LAPC-4, 22Rv1, LNCaP, and PC-3—were examined for their stem cell properties in vitro. Design, settings, and participantsThe colony-forming efficiency and self-renewal ability of morphologically distinguishable holoclones and paraclones were tested with low-density plating and serial passaging. Expression of the putative stem cell marker CD133 and breast cancer resistance protein (BCRP) was examined with flow cytometry, and immunohistochemical stainings were made for CD133, α2-integrin, nestin, BCRP, cytokeratin 5 (CK5), and cytokeratin 18 (CK18). Results and limitationsFive out of six cell lines formed clear holo-, mero-, and paraclones. Unlike paraclones, we can maintain DU145 holoclones in culture for several passages, which is indicative of self-renewal ability. Using fluorescence-activated cell sorting (FACS) analysis only in DU145 cells, a small fraction (0.01%) of CD133+ cells was detected. CD133+ cells; however, like DU145 BCRP+ (0.15%) cells, they were not more clonogenic, and they did not show more holoclone formation than the marker-negative cells or unselected cells. Immunohistochemistry revealed α2-integrin and BCRP as potential stem cell markers and CK5 with the combination of CK18 to distinguish transient amplifying cells. ConclusionsThese results indicate the possible presence of stem-like cells in several established PCa cell lines. CD133 selection does not enrich for stem-like cells in PCa cell lines.

Editorial Comment on: Stem Cell Characteristics in Prostate Cancer Cell Lines

Development of the prostate is thought to originate fromprostate stem cells that also give rise to characteristic regeneration of the gland after castration-induced involution [1]. According to the concept of cancer stem cells, prostate cancer is correspondingly initiated from a small number of malignant stem-like cells that are able to propagate the rest of the tumor-cell population [1–3]. Being resistant to present treatment modalities, they are able to reproduce the tumor, even if the majority of tumor cells are eliminated by the treatment. Identification and characterization of the putative cancer stem cells would thus be crucial for developing methods for eradication of these cells. Presently, there is no consensus on the markers for prostate, prostate cancer stem, or progenitor cells, but the candidates include CD133 (prominin), CD44, a2-integrin, breast cancer resistance protein (BCRP), c-kit, and others. Tumor cells isolated from human prostate cancer on the basis of expressing these markers have been able to reproduce tumor-like tissue in immune-deficient mice, suggesting that they could be cancer-initiating stem cells. These cells, however, are rare in tumors; therefore, it is very difficult to investigate them. In the present study, Pfeiffer and Schalken [4] asked whether established prostate cancer cell lines contain such cells. By determining colony-forming ability, differential morphology of colonies, and serial passaging, the authors show that most prostate cell lines contain stemlike cells with a high capacity for self-renewal. These cells also expressed a2-integrin and BCRP, but, interestingly, CD133 did not seem to differentiate them from nonclonogenic tumor cells, as it does in tumor tissue [2,3]. Additionally, despite continuous growth capacity, the PC3 line did not seem to include stem-like cells according to the criteria used. These results suggest that most prostate cancer cell lines do contain stem-like cells as well as the progeny of more differentiated cells, although their characteristics may differ from those in tumors. It is necessary in the future to verify the ‘‘stemness’’ of these cells in vivo in tumor models because the stem-cell niche-forming stromal cells are the vital partner of stem cells [5].

Androgen-independent prostate cancer cells acquire the complete steroidogenic potential of synthesizing testosterone from cholesterol

The proliferation and differentiation of normal prostate epithelial cells depends upon the action of androgens produced by the testis. Prostate cancers retain the ability to respond to androgens in the initial stages of cancer development, but progressively become independent of exogenous androgens in advanced stages of the disease while maintaining the expression of functional androgen receptor (AR). In the present study, we have determined the potential of prostate cancer cells to synthesize androgens from cholesterol which may be involved in intracrine regulation of AR in advanced stages of the disease. Established androgen-independent prostate cancer cell lines, PC3 and DU145 cells, expressed mRNA and proteins for scavenger receptor type B1 (SRB1), steroidogenic acute regulatory (StAR) protein, cytochrome P450 cholesterol side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and other enzymes involved in androgen biosynthesis. Expression of all these proteins and enzymes was significantly higher in the androgen-independent derivative of LNCaP prostate cancer cells (C81) than in the androgen-dependent cell line (C33). In serum-free cultures, the androgen-independent C81 cells secreted approximately 5-fold higher testosterone than C33 cells as determined in the conditioned media by immunoassays. These cells could also directly convert radioactive cholesterol into testosterone which was identified by thin layer chromatography. These results for the first time show that prostate cancer cells in advanced stages of the disease could synthesize androgens from cholesterol and hence are not dependent upon testicular and/or adrenal androgens.

Entwicklung eines dreidimensionalen Prostatakarzinomzellkulturmodells

Much prostate cancer research is based on cell culture results. Recent genomic studies found major differences between primary prostate cancer tissue and established prostate cancer cell lines, which calls into question the clinical relevance of study results based on cell cultures.Using primary cultures of prostate cancer cells from prostatectomy specimens seems to be a reasonable solution, but primary cell cultures are much more difficult to establish. In this study, a primary cell culture model was combined with an invasion assay. With this combination it was possible not only to select invasive cell clones from the primary culture but also to culture these cells in a three-dimensional model, forming spheroids. A further characterization of this cell population was done by comparative genomic hybridization, showing numerous genetic alterations. The presented cell culture model offers, for the first time, an opportunity to isolate invasive growing cells from primary prostate cancer tissue and cultivate these cells for further analyses.

The p38 MAPK Pathway Mediates Aryl Propionic Acid–Induced Messenger RNA Stability of p75NTR in Prostate Cancer Cells

The p75(NTR) acts as a tumor suppressor in the prostate, but its expression is lost as prostate cancer progresses and is minimal in established prostate cancer cell lines such as PC-3, DU-145, and LNCaP. Previously, we showed that treatment with R-flurbiprofen or ibuprofen induced p75(NTR) expression in PC-3 and DU-145 cells leading to p75(NTR)-mediated decreased survival. Here, we investigate the mechanism by which these drugs induce p75(NTR) expression. We show that the observed increase in p75(NTR) protein due to R-flurbiprofen and ibuprofen treatment was accompanied by an increase in p75(NTR) mRNA, and this increase in mRNA was the result of increased mRNA stability and not by an up-regulation of transcription. In addition, we show that treatment with R-flurbiprofen or ibuprofen led to sustained activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Furthermore, inhibition of the p38 MAPK pathway with the p38 MAPK-specific inhibitor SB202190 or by small interfering RNA (siRNA) knockdown of p38 MAPK protein prevented induction of p75(NTR) by R-flurbiprofen and ibuprofen. We also observed that siRNA knockdown of MAPK-activated protein kinase (MK)-2 and MK3, the kinases downstream of p38 MAPK that are responsible for the mRNA stabilizing effects of the p38 MAPK pathway, also prevented an induction of p75(NTR) by R-flurbiprofen and ibuprofen. Finally, we identify the RNA stabilizing protein HuR and the posttranscriptional regulator eukaryotic translation initiation factor 4E as two possible mechanisms by which the p38 MAPK pathway may increase p75(NTR) expression. Collectively, the data suggest that R-flurbiprofen and ibuprofen induce p75(NTR) expression by increased mRNA stability that is mediated through the p38 MAPK pathway.

Lack of Demonstrable Autocrine Hedgehog Signaling in Human Prostate Cancer Cell Lines

Several recent reports highlighted the role of hedgehog signaling in prostate cancer. However, the relative contributions of autocrine and paracrine hedgehog signaling to tumor growth and progression are unclear. Efforts to model autocrine signaling for drug development have been hampered by conflicting reports of the presence or absence of autocrine signaling in established human prostate cancer cell lines. We comprehensively characterized the expression of hedgehog pathway genes in the 3 prostate cancer cell lines LNCaP, PC3 and 22RV1 (American Type Culture Collection, Manassas, Virginia). We also examined their response to Shh ligand and to the hedgehog pathway inhibitor cyclopamine (Toronto Research Chemicals, Toronto, Ontario, Canada). Expression of hedgehog ligand, patched and Gli1 in all 3 cell lines was lower than the expression level in normal human prostate tissue. All 3 cell lines showed hedgehog target gene activation when transfected with an activated form of Gli2 but none showed a detectable transcriptional response to hedgehog ligand or to transfection with an activated form of smoothened. Furthermore, treatment with the hedgehog pathway inhibitor cyclopamine did not inhibit hedgehog target gene expression in any of the 3 prostate cancer cell lines, although cyclopamine inhibited proliferation in culture. LNCaP, PC3 and 22RV1 show no evidence of autocrine signaling by ligand dependent mechanisms and cyclopamine mediated inhibition of growth in culture occurs without of any discernible effect on canonical hedgehog pathway activity.