Protein kinase A-mediated phosphorylation of RhoA on serine 188 triggers the rapid induction of a neuroendocrine-like phenotype in prostate cancer epithelial cells

Whilst androgen ablation therapy is used to treat locally advanced or metastatic forms of prostate cancer, side-effects can include the emergence of an androgen-independent neuroendocrine cell population which is associated with poor prognosis. Here we have examined how cyclic AMP elevation regulates early events in the neuroendocrine differentiation process. We demonstrate that selective activation of protein kinase A is necessary and sufficient for cyclic AMP (cAMP) elevation to rapidly promote a neuroendocrine phenotype in LNCaP cells independent of de novo protein synthesis. Furthermore, the effects of cAMP could be recapitulated by inhibition of RhoA signalling or pharmacological inhibition of Rho kinase. Conversely, expression of constitutively active Gln63Leu-mutated RhoA acted as a dominant-negative inhibitor of cAMP-mediated NE phenotype formation. Consistent with these observations, cAMP elevation triggered the PKA-dependent phosphorylation of RhoA on serine 188, and a non-phosphorylatable Ser188Ala RhoA mutant functioned as a dominant-negative inhibitor of cAMP-mediated neuroendocrine phenotype formation. These results suggest that PKA-mediated inhibition of RhoA via its phosphorylation on serine 188 and the subsequent inhibition of ROCK activity plays a key role in determining initial changes in cellular morphology during LNCaP cell differentiation to a neuroendocrine phenotype. It also raises the possibility that targeted suppression of this pathway to inhibit neuroendocrine cell expansion might be a useful adjuvant to conventional prostate cancer therapy.     

Mitostatin is down-regulated in human prostate cancer and suppresses the invasive phenotype of prostate cancer cells

MITOSTATIN, a novel putative tumor suppressor gene induced by decorin overexpression, is expressed in most normal human tissues but is markedly down-regulated in advanced stages of mammary and bladder carcinomas. Mitostatin negatively affects cell growth, induces cell death and regulates the expression and activation levels of Hsp27. In this study, we demonstrated that ectopic expression of Mitostatin in PC3, DU145, and LNCaP prostate cancer cells not only induced a significant reduction in cell growth, but also inhibited migration and invasion. Moreover, Mitostatin inhibited colony formation in soft-agar of PC3 and LNCaP cells as well as tumorigenicity of LNCaP cells in nude mice. Conversely, targeting endogenous Mitostatin by siRNA and anti-sense strategies in PC3 and DU145 prostate cancer cells enhanced the malignant phenotype in both cell lines. In agreement of these anti-oncogenic roles, we discovered that Mitostatin was absent in ∼35% (n = 124) of prostate tumor samples and its overall reduction was associated with advanced cancer stages. Collectively, our findings indicate that MITOSTATIN may acts as a tumor suppressor gene in prostate cancer and provide a novel cellular and molecular mechanism to be further exploited and deciphered in our understanding of prostate cancer progression.     

Transport of methotrexate into LNCaP human prostate cancer cells

Uptake of methotrexate into the LNCaP human prostate cancer cells was linear for the first 60 min. The initial rate of methotrexate uptake was highest at extracellular pH 4.5 and decreased markedly until pH 7.0 to 8.0. Transport of methotrexate into LNCaP cells showed two components, one saturable -K(m) = 0.13 +/- 0.06 microM and V(max) = 1.20 +/- 0.16 pmol x 45 min(-1) x mg(-1) protein at low concentrations and the other apparently not saturable up to 10 microM. Uptake of methotrexate was inhibited by structural analogs with the K(i) values being 6.53, 12.4, and 85.6 microM for 5-formyltetrahydrofolate, 5-methyltetrahydrofolate, and folic acid, respectively. Uptake of methotrexate into LNCaP cells was not inhibited by the energy poisons in contrast to methotrexate uptake into PC-3 prostate cancer cells. Uptake was inhibited by increasing concentrations of sulfate and phosphate ions and by the organic anions probenecid and DIDS, suggesting that methotrexate may be transported by an anion-exchange mechanism. These results show that methotrexate is transported into LNCaP prostate cancer cells by a carrier-mediated process.     

Transport of methotrexate into PC-3 human prostate cancer cells

Recombinant BmRad51 and BmDmc1, silkworm homologs of the Escherichia coli RecA proteins catalyzing the homologous DNA pairing, were purified from E. coli cells carrying expression vectors. These possessed different enzymatic properties in the joint molecule formation between single-stranded circular DNA and homologous linear double-stranded DNA. The requirement of single-stranded circular DNA for the efficient reaction was twofold higher in BmRad51 than in BmDmc1. Although able to mediate the joint molecule formation independently, a complex of the two enzymes formed prior to single-stranded DNA binding was found to have augmented efficiency of the pairing reaction.     

Androgen receptor represses the neuroendocrine transdifferentiation process in prostate cancer cells

Androgen-ablation therapy is an effective method for treating prostate cancer. However, prostate tumors that survive long-term androgen-ablation therapy are classified as androgen-independent as they proliferate in the absence of androgens, and they tend to be enriched for neuroendocrine (NE) cells. Androgen withdrawal causes androgen-dependent prostate cancer cells to adopt a pronounced NE phenotype, suggesting that androgen receptor (AR) represses an intrinsic NE transdifferentiation process in prostate cancer cells. In this report we show that short interfering RNA-induced AR silencing induced a NE phenotype that manifested itself in the growth of dendritic-like processes in both the androgen-dependent LNCaP and androgen-independent LNCaP-AI human prostate cancer cells. Western blot analysis revealed that neuronal-specific enolase, a marker of the neuronal lineage, was increased by AR knockdown in LNCaP cells. The expression levels of the neuronal-specific cytoskeletal proteins beta-tubulin III, nestin, and glial acidic fibrillary protein were also characterized in AR knockdown cells. Most interestingly, AR silencing induced beta-tubulin III expression in LNCaP cells, while AR knockdown increased glial acidic fibrillary protein levels in both LNCaP and LNCaP-AI cells. Lastly, AR silencing reduced the proliferative capacity of LNCaP and LNCaP-AI cells. Our data demonstrate that AR actively represses an intrinsic NE transdifferentiation process in androgen-responsive prostate cancer cells and suggest a potential link between AR inactivation and the increased frequency of NE cells in androgen-independent tumors.     

Aldehyde dehydrogenase activity selects for the holoclone phenotype in prostate cancer cells

Aldehyde dehydrogenase 1 (ALDH) activity is considered to be a marker of cancer stem cells (CSCs) in many tumour models, since these cells are more proliferative and tumourigenic than ALDH^Lo cells in experimental models. However it is unclear whether all CSC-like cells are within the ALDH^Hi population, or whether all ALDH^Hi cells are highly proliferative and tumourigenic. The ability to establish a stem cell hierarchy in vitro, whereby sub-populations of cells have differing proliferative and differentiation capacities, is an alternate indication of the presence of stem cell-like populations within cell lines. In this study, we have examined the interaction between ALDH status and the ability to establish a stem cell hierarchy in PC3 prostate cancer cells. We demonstrate that PC3 cells contain a stem cell hierarchy, and isolation of ALDH^Hi cells enriches for the most primitive holoclone population, however holoclone formation is not restricted to ALDH^Hi cells. In addition, we show that ALDH activity undergoes phenotypic plasticity, since the ALDH^Lo population can develop ALDH^Hi populations comparable to parental cells within 2 weeks in culture. Furthermore, we show that the majority of ALDH^Hi cells are found within the least primitive paraclone population, which is circumvented by culturing PC3 cells as spheroids in defined medium favouring stem cell characteristics. Although ALDH^Hi status enriches for holoclone formation, this activity may be mediated by a minority of ALDH^Hi cells.     

Uptake of 5-methyltetrahydrofolate into PC-3 human prostate cancer cells is carrier-mediated

Uptake of 5-methyltetrahydrofolate into the PC-3 human prostate cancer cells was linear for the first 60 min. There was no difference in the initial rate of uptake in cells incubated in folate-free medium for 24 or 48 hr compared to control cells grown in folate-containing medium. The initial rate of 5-methyltetrahydrofolate uptake showed little dependence on extracellular pH and it was independent of extracellular sodium ions. Transport of 5-methyltetrahydrofolate into PC-3 cells was saturable - K(m) = 0.74 micro M and V(max) = 7.78 nmol/10(9)cells/min and these kinetic constants were not different in cells incubated for 24 hr in folate-free medium (K(m) = 0.80 +/- 0.22, V(max) = 8.52 +/- 0.50; P = 0.09, N = 3). Uptake of 5-methyltetrahydrofolate was inhibited by structural analogs with the K(i) values being 0.50, 1.79, and 31.8 micro M for 5-formyltetrahydrofolate, methotrexate, and folic acid, respectively. Uptake of 5-methyltetrahydrofolate was inhibited by the energy poisons, sodium cyanide, sodium arsenate, p-chloromercuriphenylsulfonate, and sodium azide. Uptake was inhibited by increasing concentrations of sulfate and phosphate ions, suggesting that 5-methyltetrahydrofolate may be transported by an anion-exchange mechanism. These results show that 5-methyltetrahydrofolate is transported into PC-3 prostate cancer cells by a carrier-mediated process.     

Activation of c-MET induces a stem-like phenotype in human prostate cancer

Prostate cancer consists of secretory cells and a population of immature cells. The function of immature cells and their mutual relation with secretory cells are still poorly understood. Immature cells either have a hierarchical relation to secretory cells (stem cell model) or represent an inducible population emerging upon appropriate stimulation of differentiated cells. Hepatocyte Growth Factor (HGF) receptor c-MET is specifically expressed in immature prostate cells. Our objective is to determine the role of immature cells in prostate cancer by analysis of the HGF/c-MET pathway.Gene-expression profiling of DU145 prostate cancer cells stimulated with HGF revealed induction of a molecular signature associated with stem cells, characterized by up-regulation of CD49b, CD49f, CD44 and SOX9, and down-regulation of CD24 ('stem-like signature'). We confirmed the acquisition of a stem-like phenotype by quantitative PCR, FACS analysis and Western blotting. Further, HGF led to activation of the stem cell related Notch pathway by up-regulation of its ligands Jagged-1 and Delta-like 4. Small molecules SU11274 and PHA665752 targeting c-MET activity were both able to block the molecular and biologic effects of HGF. Knock-down of c-MET by shRNA infection resulted in significant reduction and delay of orthotopic tumour-formation in male NMRI mice. Immunohistochemical analysis in prostatectomies revealed significant enrichment of c-MET positive cells at the invasive front, and demonstrated co-expression of c-MET with stem-like markers CD49b and CD49f.In conclusion, activation of c-MET in prostate cancer cells induced a stem-like phenotype, indicating a dynamic relation between differentiated and stem-like cells in this malignancy. Its mediation of efficient tumour-formation in vivo and predominant receptor expression at the invasive front implicate that c-MET regulates tumour infiltration in surrounding tissues putatively by acquisition of a stem-like phenotype.     

Induction of invasive phenotype by Casodex in hormone-sensitive prostate cancer cells

The cellular mechanisms of anti-androgen-induced tumor regression have not been investigated in great detail. We have compared the induction of cell death in the androgen-dependent, non-invasive LNCaP prostate cancer cell line by Casodex and TNF-. Both agents induce a dose and time-dependent decrease in cell viability in vitro. However, Casodex does not induce classical DNA fragmentation to oligonucleosomes typically induced by TNF-, but rather induces cleavage to form intermediate 60 kb DNA fragments. RT-PCR based analysis demonstrates that in LNCaP cells Casodex coordinately alters the expression of steady-state level of mRNAs of several matrix metalloproteases and their cognate inhibitors (most notably MMP2 and TIMP1). Zymography and reverse zymography confirm that the ratio of metalloprotease(s) to inhibitor(s) is altered in favor of activation of the proteases. In a small percentage of the treated LNCaP cells, the activation of the extracellular matrix (ECM)-proteases by Casodex also induces an invasive phenotype. The acquisition of an invasive phenotype is not seen when LNCaP cells are treated with TNF-, and is not seen when the LNCaP cells are treated with both compounds simultaneously, suggesting that the phenomenon may be specific to particular classes of compounds. These observations have significant implications in the treatment of prostate cancer, since the appearance of a more aggressive phenotype following treatment is clearly undesirable.     

Proteomic analysis of cancer stem cells in human prostate cancer cells

Results from recent studies support the hypothesis that cancer stem cells (CSCs) are responsible for tumor initiation and formation. Here, we applied a proteome profiling approach to investigate the mechanisms of CSCs and to identify potential biomarkers in the prostate cancer cell line DU145. Using MACS, the DU145 prostate cancer cell line was isolated into CD44+ or CD44− cells. In sphere culture, CD44+ cells possessed stem cell characteristics and highly expressed genes known to be important in stem cell maintenance. In addition, they showed strong tumorigenic potential in the clonogenic assay and soft agar colony formation assay. We then analyzed and identified proteins that were differentially expressed between CD44+ and CD44− using two-dimensional gel electrophoresis and LC-MS/MS. Cofilin and Annexin A5, which are associated with proliferation or metastasis in cancer, were found to be positively correlated with CD44 expression. These results provide information that will be important to the development of new cancer diagnostic tools and understanding the mechanisms of CSCs although a more detailed study is necessary to investigate the roles of Cofilin and Annexin A5 in CSCs.     

Growth factors in human prostate cancer cells: implications for an improved treatment of prostate cancer

It has been previously shown that estrogens may exert their action on human breast cancer cells through coordinated control of secreted growth factors which act in an autocrine and paracrine fashion. Growth stimulation of the androgen receptor negative prostate carcinoma cell line DU-145 by dihydrotestosterone in the presence of the androgen-responsive human prostate carcinoma cell line LNCaP now indicates that androgens may regulate growth of prostate carcinoma cells through related mechanisms. A variety of androgen-regulated growth modulatory activities with autocrine and paracrine potential can be detected in conditioned media from LNCaP cells partially purified by ion exchange chromatography. Androgen-induced growth of LNCaP cells is partially inhibited by the polyanions suramin and dextran sulfates which antagonize growth factor action. These data suggest the existence of at least two different mechanisms of growth regulation by androgen which can be distinguished by their different sensitivity to growth factor inhibitory agents. We conclude that the combination of antipeptidergic substances and androgen withdrawal would represent a new and promising strategy for treatment of human prostate cancer.     

Chronic hypoxia promotes an aggressive phenotype in rat prostate cancer cells

In general, tumors cells that are resistant to apoptosis and increase angiogenesis are a result of the hypoxic responses contributing to the malignant phenotype. In this study, we developed a chronic hypoxic cell model (HMLL), by incubating the prostate cancer MatLyLu cells in a hypoxic chamber (1% O₂) over 3 weeks. Surviving cells were selected through each cell passage and were grown in the hypoxic condition up to 8 weeks. This strategy resulted in survival of only 5% of the cells. The surviving hypoxic cells displayed a greater stimulation on hypoxic adaptive response, including a greater expression of glucose transporter1 (Glut1) and VEGF secretion. In addition, higher invasion activity was observed in the chronic hypoxic HMLL cells as compared to MatLyLu cells exposed to acute hypoxia (1% O₂, 5 h) using the matrigel assay. To further examine the role of HIF-1α in tumor progression, both MatLyLu and HMLL cells were transfected with dominant-negative form of HIF-1α (DNHIF-1α). The Matrigel invasion activity induced by chronic hypoxia was significantly attenuated by DNHIF-1α. These results suggest that signaling pathways leading to hypoxic response may be differentially regulated in chronic hypoxic cells and acute hypoxic cells. Chronic hypoxia may play a greater role than acute hypoxia in promoting the aggressive phenotype of tumor cells. This observation mimics the clinical scenario where tumor cells following treatment with radiation are subjected to hypoxic conditions. The reemergence of tumor following treatment usually results in tumor cells that are more aggressive and metastatic.     

Chronic hypoxia promotes an aggressive phenotype in rat prostate cancer cells

In general, tumors cells that are resistant to apoptosis and increase angiogenesis are a result of the hypoxic responses contributing to the malignant phenotype. In this study, we developed a chronic hypoxic cell model (HMLL), by incubating the prostate cancer MatLyLu cells in a hypoxic chamber (1% O(2)) over 3 weeks. Surviving cells were selected through each cell passage and were grown in the hypoxic condition up to 8 weeks. This strategy resulted in survival of only 5% of the cells. The surviving hypoxic cells displayed a greater stimulation on hypoxic adaptive response, including a greater expression of glucose transporter1 (Glut1) and VEGF secretion. In addition, higher invasion activity was observed in the chronic hypoxic HMLL cells as compared to MatLyLu cells exposed to acute hypoxia (1% O(2), 5 h) using the matrigel assay. To further examine the role of HIF-1alpha in tumor progression, both MatLyLu and HMLL cells were transfected with dominant-negative form of HIF-1alpha (DNHIF-1alpha). The Matrigel invasion activity induced by chronic hypoxia was significantly attenuated by DNHIF-1alpha. These results suggest that signaling pathways leading to hypoxic response may be differentially regulated in chronic hypoxic cells and acute hypoxic cells. Chronic hypoxia may play a greater role than acute hypoxia in promoting the aggressive phenotype of tumor cells. This observation mimics the clinical scenario where tumor cells following treatment with radiation are subjected to hypoxic conditions. The reemergence of tumor following treatment usually results in tumor cells that are more aggressive and metastatic.     

In vitro transdifferentiation of adult pancreatic acinar cells into insulin-expressing cells

Despite a recent breakthrough in human islet transplantation for treating diabetes mellitus, the limited availability of insulin-producing tissue is still a major obstacle. Here, we studied whether adult pancreatic acinar cells have the potential to transdifferentiate into islet or beta cells. Pancreatic acini were isolated from 7- to 8-weeks-old male Sprague-Dawley rats and cultured in suspension. Within 1 week, most of the acinar cells lost amylase expression and converted to cells with a duct cell phenotype. Insulin-positive cells were also observed, mainly at the periphery of the acini-derived spheroids. Insulin gene and protein expression was increased. Presence of a few insulin-positive cells coexpressing cytokeratins suggests that a spontaneous acinar to ductal cell transdifferentiation process was further going on towards beta cells. This study provides the first evidence that adult pancreatic acinar cells could be differentiated into insulin-expressing cells in vitro.     

Differentiation of human fetal mesenchymal stem cells into cells with an oligodendrocyte phenotype

The potential of mesenchymal stem cells (MSC) to differentiate into neural lineages has raised the possibility of autologous cell transplantation as a therapy for neurodegenerative diseases. We have identified a population of circulating human fetal mesenchymal stem cells (hfMSC) that are highly proliferative and can readily differentiate into mesodermal lineages such as bone, cartilage, fat and muscle. Here, we demonstrate for the first time that primary hfMSC can differentiate into cells with an oligodendrocyte phenotype both in vitro and in vivo. By exposing hfMSC to neuronal conditioned medium or by introducing the pro-oligodendrocyte gene, Olig-2, hfMSC adopted an oligodendrocyte-like morphology, expressed oligodendrocyte markers and appeared to mature appropriately in culture. Importantly we also demonstrate the differentiation of a clonal population of hfMSC into both mesodermal (bone) and ectodermal (oligodendrocyte) lineages. In the developing murine brain transplanted hfMSC integrated into the parenchyma but oligodendrocyte differentiation of these naïve hfMSC was very low. However, the proportion of cells expressing oligodendrocyte markers increased significantly (from 0.2% to 4%) by pre-exposing the cells to differentiation medium in vitro prior to transplantation. Importantly, the process of in vivo differentiation occurred without cell fusion. These findings suggest that hfMSC may provide a potential source of oligodendrocytes for study and potential therapy.     

Glyoxalase 1 sustains the metastatic phenotype of prostate cancer cells via EMT control

Metastasis is the primary cause of death in prostate cancer (PCa) patients. Effective therapeutic intervention in metastatic PCa is undermined by our poor understanding of its molecular aetiology. Defining the mechanisms underlying PCa metastasis may lead to insights into how to decrease morbidity and mortality in this disease. Glyoxalase 1 (Glo1) is the detoxification enzyme of methylglyoxal (MG), a potent precursor of advanced glycation end products (AGEs). Hydroimidazolone (MG‐H1) and argpyrimidine (AP) are AGEs originating from MG‐mediated post‐translational modification of proteins at arginine residues. AP is involved in the control of epithelial to mesenchymal transition (EMT), a crucial determinant of cancer metastasis and invasion, whose regulation mechanisms in malignant cells are still emerging. Here, we uncover a novel mechanism linking Glo1 to the maintenance of the metastatic phenotype of PCa cells by controlling EMT by engaging the tumour suppressor miR‐101, MG‐H1‐AP and TGF‐β1/Smad signalling. Moreover, circulating levels of Glo1, miR‐101, MG‐H1‐AP and TGF‐β1 in patients with metastatic compared with non‐metastatic PCa support our in vitro results, demonstrating their clinical relevance. We suggest that Glo1, together with miR‐101, might be potential therapeutic targets for metastatic PCa, possibly by metformin administration.     

DNA damage phenotype and prostate cancer risk

The capacity of an individual to process DNA damage is considered a crucial factor in carcinogenesis. The comet assay is a phenotypic measure of the combined effects of sensitivity to a mutagen exposure and repair capacity. In this paper, we evaluate the association of the DNA repair kinetics, as measured by the comet assay, with prostate cancer risk. In a pilot study of 55 men with prostate cancer, 53 men without the disease, and 71 men free of cancer at biopsy, we investigated the association of DNA damage with prostate cancer risk at early (0-15 min) and later (15-45 min) stages following gamma-radiation exposure. Although residual damage within 45 min was the same for all groups (65% of DNA in comet tail disappeared), prostate cancer cases had a slower first phase (38% vs. 41%) and faster second phase (27% vs. 22%) of the repair response compared to controls. When subjects were categorized into quartiles, according to efficiency of repairing DNA damage, high repair-efficiency within the first 15 min after exposure was not associated with prostate cancer risk while higher at the 15-45 min period was associated with increased risk (OR for highest-to-lowest quartiles=3.24, 95% CI=0.98-10.66, p-trend=0.04). Despite limited sample size, our data suggest that DNA repair kinetics marginally differ between prostate cancer cases and controls. This small difference could be associated with differential responses to DNA damage among susceptible individuals.