AMPK/SIRT1 signaling through p38MAPK mediates Interleukin-6 induced neuroendocrine differentiation of LNCaP prostate cancer cells

Neuroendocrine Prostate Cancer (NEPC) is an aggressive form of androgen independent prostate cancer (AIPC), correlated with therapeutic resistance. Interleukin (IL)-6 promotes proliferation and neuroendocrine differentiation (NED) of androgen dependent LNCaP cells. We treated LNCaP cells with IL-6 and observed for in vitro NED of cells and also expression of NE markers βIII tubulin, neuron-specific enolase (NSE) and chromogranin A (ChA). Here we investigated the proteins and/or pathways involved in NED of LNCaP cells induced by IL-6 and characterized their role in NED of PCa cells. We found that the altered proteins modulated AMPK signaling pathway in NE cells. Remarkably, IL-6 induces NED of LNCaP cells through activation of AMPK and SIRT1 and also both of these are co-regulated while playing a predominant role in NED of LNCaP cells. Of the few requirements of AMPK-SIRT1 activation, increased eNOS is essential for NED by elevating Nitric oxide (NO) levels. Pleiotropic effects of NO ultimately regulate p38MAPK in IL-6 induced NED. Hence, IL-6 induced AMPK-SIRT1 activation eventually transfers its activation signals through p38MAPK for advancing NED of LNCaP cells. Moreover, inactivation of p38MAPK with specific inhibitor (SB203580) attenuated IL-6 induced NED of LNCaP cells. Therefore, IL-6 promotes NED of PCa cells via AMPK/SIRT1/p38MAPK signaling. Finally, targeting AMPK-SIRT1 or p38MAPK in androgen independent PC3 cells with neuroendocrine features reversed their neuroendocrine characteristics. Taken together these novel findings reveal that targeting p38MAPK mitigated NED of PCa cells, and thus it can be a favorable target to overcome progression of NEPC.     

Phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathway is essential for neuroendocrine differentiation of prostate cancer

Hormonal therapy of prostate cancer, by inhibiting androgen production and/or androgen function, is the treatment of choice for advanced prostate cancer. Although most patients respond initially, the effect is only temporary, and the tumor cells will resume proliferation in an androgen-deprived environment. The mechanism for androgen-independent proliferation of cancer cells is unclear. Hormonal therapy induces neuroendocrine differentiation of prostate cancer cells, which is hypothesized to contribute to tumor recurrence by a paracrine mechanism. We studied signal transduction pathways of neuroendocrine differentiation in LNCaP cells after androgen withdrawal, and we showed that both the phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathway and ERK are activated, but only the former is required for neuroendocrine differentiation. A constitutively active AKT promotes neuroendocrine differentiation and a dominant negative AKT inhibits it. Activation of AKT by IGF-1 leads to neuroendocrine differentiation, and neuroendocrine differentiation induced by epinephrine requires AKT activation. We also show that the AKT pathway is likely responsible for neuroendocrine differentiation in DU145, an androgen-independent prostate cancer cell line. Therefore, our study demonstrated a novel function of the AKT pathway in prostate cancer progression and identified potential targets that may be explored for the treatment of androgen-independent cancer.     

Basic helix-loop-helix transcription factors regulate the neuroendocrine differentiation of fetal mouse pulmonary epithelium

To clarify the mechanisms that regulate neuroendocrine differentiation of fetal lung epithelia, we have studied the expression of the mammalian homologs of achaete-scute complex (Mash1) (Ascl1 - Mouse Genome Informatics); hairy and enhancer of split1 (Hes1); and the expression of Notch/Notch-ligand system in the fetal and adult mouse lungs, and in the lungs of Mash1- or Hes1-deficient mice. Immunohistochemical studies revealed that Mash1-positive cells seemed to belong to pulmonary neuroendocrine cells (PNEC) and their precursors. In mice deficient for Mash1, no PNEC were detected. Hes1-positive cells belong to non-neuroendocrine cells. In the mice deficient in Hes1, in which Mash1 mRNA was upregulated, PNEC appeared precociously, and the number of PNEC was markedly increased. NeuroD (Neurod1 - Mouse Genome Informatics) expression in the lung was detected in the adult, and was enhanced in the fetal lungs of Hes1-null mice. Expression of Notch1, Notch2, Notch3 and Notch4 mRNAs in the mouse lung increased with age, and Notch1 mRNA was expressed in a Hes1-dependent manner. Notch1, Notch2 and Notch3 were immunohistochemically detected in non-neuroendocrine cells. Moreover, analyses of the lungs from the gene-targeted mice suggested that expression of Delta-like 1 (Dll1 - Mouse Genome Informatics) mRNA depends on Mash1. Thus, the neuroendocrine differentiation depends on basic helix-loop-helix factors, and Notch/Notch-ligand pathways may be involved in determining the cell differentiation fate in fetal airway epithelium.     

Analysis of the Molecular Networks in Androgen Dependent and Independent Prostate Cancer Revealed Fragile and Robust Subsystems

Androgen ablation therapy is currently the primary treatment for metastatic prostate cancer. Unfortunately, in nearly all cases, androgen ablation fails to permanently arrest cancer progression. As androgens like testosterone are withdrawn, prostate cancer cells lose their androgen sensitivity and begin to proliferate without hormone growth factors. In this study, we constructed and analyzed a mathematical model of the integration between hormone growth factor signaling, androgen receptor activation, and the expression of cyclin D and Prostate-Specific Antigen in human LNCaP prostate adenocarcinoma cells. The objective of the study was to investigate which signaling systems were important in the loss of androgen dependence. The model was formulated as a set of ordinary differential equations which described 212 species and 384 interactions, including both the mRNA and protein levels for key species. An ensemble approach was chosen to constrain model parameters and to estimate the impact of parametric uncertainty on model predictions. Model parameters were identified using 14 steady-state and dynamic LNCaP data sets taken from literature sources. Alterations in the rate of Prostatic Acid Phosphatase expression was sufficient to capture varying levels of androgen dependence. Analysis of the model provided insight into the importance of network components as a function of androgen dependence. The importance of androgen receptor availability and the MAPK/Akt signaling axes was independent of androgen status. Interestingly, androgen receptor availability was important even in androgen-independent LNCaP cells. Translation became progressively more important in androgen-independent LNCaP cells. Further analysis suggested a positive synergy between the MAPK and Akt signaling axes and the translation of key proliferative markers like cyclin D in androgen-independent cells. Taken together, the results support the targeting of both the Akt and MAPK pathways. Moreover, the analysis suggested that direct targeting of the translational machinery, specifically eIF4E, could be efficacious in androgen-independent prostate cancers.     

Oscillations in notch signaling regulate maintenance of neural progenitors

Expression of the Notch effector gene Hes1 is required for maintenance of neural progenitors in the embryonic brain, but persistent and high levels of Hes1 expression inhibit proliferation and differentiation of these cells. Here, by using a real-time imaging method, we found that Hes1 expression dynamically oscillates in neural progenitors. Furthermore, sustained overexpression of Hes1 downregulates expression of proneural genes, Notch ligands, and cell cycle regulators, suggesting that their proper expression depends on Hes1 oscillation. Surprisingly, the proneural gene Neurogenin2 (Ngn2) and the Notch ligand Delta-like1 (Dll1) are also expressed in an oscillatory manner by neural progenitors, and inhibition of Notch signaling, a condition known to induce neuronal differentiation, leads to downregulation of Hes1 and sustained upregulation of Ngn2 and Dll1. These results suggest that Hes1 oscillation regulates Ngn2 and Dll1 oscillations, which in turn lead to maintenance of neural progenitors by mutual activation of Notch signaling.     

A Notch-dependent molecular circuitry initiates pancreatic endocrine and ductal cell differentiation

In the pancreas, Notch signaling is thought to prevent cell differentiation, thereby maintaining progenitors in an undifferentiated state. Here, we show that Notch renders progenitors competent to differentiate into ductal and endocrine cells by inducing activators of cell differentiation. Notch signaling promotes the expression of Sox9, which cell-autonomously activates the pro-endocrine gene Ngn3. However, at high Notch activity endocrine differentiation is blocked, as Notch also induces expression of the Ngn3 repressor Hes1. At the transition from high to intermediate Notch activity, only Sox9, but not Hes1, is maintained, thus de-repressing Ngn3 and initiating endocrine differentiation. In the absence of Sox9 activity, endocrine and ductal cells fail to differentiate, resulting in polycystic ducts devoid of primary cilia. Although Sox9 is required for Ngn3 induction, endocrine differentiation necessitates subsequent Sox9 downregulation and evasion from Notch activity via cell-autonomous repression of Sox9 by Ngn3. If high Notch levels are maintained, endocrine progenitors retain Sox9 and undergo ductal fate conversion. Taken together, our findings establish a novel role for Notch in initiating both ductal and endocrine development and reveal that Notch does not function in an on-off mode, but that a gradient of Notch activity produces distinct cellular states during pancreas development.     

miR-221 通过作用DVL2 影响前列腺癌细胞系的侵袭功能*

目的:评价miR-221在前列腺癌细胞系中表达的变化对其神经内分泌样转化及其侵袭功能的影响。方法:以Northern blot检测LNCaP,LNCaP-AI两种前列腺癌细胞系中7种microRNA的表达变化;细胞转染法检测在雄激素剥夺环境中LNCaP和LNCaP-AI细胞系中miR-221的作用;CCK-8法检测细胞在不同阶段的生长增殖水平;Transwell法检测转染细胞的侵袭能力;qRT-PCR和Western blot检测转染的细胞中神经元特异性烯醇化酶(NSE)及dishevelled-2(DVL2)表达的变化。结果:与雄激素依赖性前列腺癌(ADPC)的细胞系LNCaP相比,miR-221在雄激素非依赖性前列腺癌(AIPC)的细胞系LNCaP-AI中明显高表达。通过转染使miR-221在LNCaP细胞系中高表达可促进细胞的NSE表达,加速其神经内分泌样分化。而在LNCaP-AI细胞系中下调miR-221水平则会升高靶基因DVL2的表达水平,并增强LNCaP-AI细胞的迁移和侵袭能力。结论:该实验证实在AIPC和ADPC细胞系中miR-221存在表达差异。miR-221可促进前列腺癌细胞的神经内分泌样转化,这可能是导致前列腺癌雄激素非依赖转化的重要原因。MiR-221可通过作用DVL2调节晚期前列腺癌细胞的转移和侵袭。     

Androgen dependent regulation of protein kinase A subunits in prostate cancer cells

Neuroendocrine (NE) cells may play a role in prostate cancer progression. Both androgen deprivation and cAMP are well known inducers of NE differentiation (NED) in the prostate. Gene-expression profiling of LNCaP cells, incubated in androgen stripped medium, showed that the Cbeta isoform of PKA is up-regulated during NE differentiation. Furthermore, by using semi-quantitative RT-PCR and immunoblotting analysis, we observed that the Cbeta splice variants are differentially regulated during this process. Whereas the Cbeta2 splice variant is down-regulated in growth arrested LNCaP cells, the Cbeta1, Cbeta3 and Cbeta4 variants, as well as the RIIbeta subunit of PKA, are induced in NE-like LNCaP cells. The opposite effect of Cbeta expression could be mimicked by androgen stimulation, implying the Cbeta gene of PKA as a putative new target gene for the androgen receptor in prostate cancer. Moreover, to investigate expression of PKA subunits during prostate cancer progression, we did immunoblotting of several prostatic cell lines and normal and tumor tissue from prostate cancer patients. Interestingly, multiple Cbeta subunits were also observed in human prostate specimens, and the Cbeta2 variant was up-regulated in tumor cells. In conclusion, it seems that the Cbeta isoforms play different roles in proliferation and differentiation and could therefore be potential markers for prostate cancer progression.     

The Raf/MEK/extracellular signal-regulated kinase 1/2 pathway can mediate growth inhibitory and differentiation signaling via androgen receptor downregulation in prostate cancer cells

Upregulated ERK1/2 activity is correlated with androgen receptor (AR) downregulation in certain prostate cancer (PCa) that exhibits androgen deprivation-induced neuroendocrine differentiation, but its functional relevance requires elucidation. We found that sustained ERK1/2 activation using active Raf or MEK1/2 mutants is sufficient to induce AR downregulation at mRNA and protein levels in LNCaP. Downregulation of AR protein, but not mRNA, was blocked by proteasome inhibitors, MG132 and bortezomib, indicating that the pathway regulation is mediated at multiple points. Ectopic expression of a constitutively active AR inhibited Raf/MEK/ERK-mediated regulation of the differentiation markers, neuron-specific enolase and neutral endopeptidase, and the cyclin-dependent kinase inhibitors, p16^INK4A and p21^CIP1, but not Rb phosphorylation and E2F1 expression, indicating that AR has a specific role in the pathway-mediated differentiation and growth inhibitory signaling. However, despite the sufficient role of Raf/MEK/ERK, its inhibition using U0126 or ERK1/2 knockdown could not block androgen deprivation-induced AR downregulation in an LNCaP neuroendocrine differentiation model, suggesting that additional signaling pathways are involved in the regulation. We additionally report that sustained Raf/MEK/ERK activity can downregulate full length as well as hormone binding domain-deficient AR isoforms in androgen-refractory C4-2 and CWR22Rv1, but not in LAPC4 and MDA-PCa-2b. Our study demonstrates a novel role of the Raf/MEK/ERK pathway in regulating AR expression in certain PCa types and provides an insight into PCa responses to its aberrant activation.     

Multiple roles of Notch signaling in the regulation of epidermal development

Recent studies have shown that Notch signaling plays an important role in epidermal development, but the underlying molecular mechanisms remain unclear. Here, by integrating loss- and gain-of-function studies of Notch receptors and Hes1, we describe molecular information about the role of Notch signaling in epidermal development. We show that Notch signaling determines spinous cell fate and induces terminal differentiation by a mechanism independent of Hes1, but Hes1 is required for maintenance of the immature state of spinous cells. Notch signaling induces Ascl2 expression to promote terminal differentiation, while simultaneously repressing Ascl2 through Hes1 to inhibit premature terminal differentiation. Despite the critical role of Hes1 in epidermal development, Hes1 null epidermis transplanted to adult mice showed no obvious defects, suggesting that this role of Hes1 may be restricted to developmental stages. Overall, we conclude that Notch signaling orchestrates the balance between differentiation and immature programs in suprabasal cells during epidermal development.     

The PPARγ ligand ciglitazone regulates androgen receptor activation differently in androgen-dependent versus androgen-independent human prostate cancer cells

The androgen receptor (AR) regulates growth and progression of androgen-dependent as well as androgen-independent prostate cancer cells. Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have been reported to reduce AR activation in androgen-dependent LNCaP prostate cancer cells. To determine whether PPARγ ligands are equally effective at inhibiting AR activity in androgen-independent prostate cancer, we examined the effect of the PPARγ ligands ciglitazone and rosiglitazone on C4-2 cells, an androgen- independent derivative of the LNCaP cell line. Luciferase-based reporter assays and Western blot analysis demonstrated that PPARγ ligand reduced dihydrotestosterone (DHT)-induced increases in AR activity in LNCaP cells. However, in C4-2 cells, these compounds increased DHT-induced AR driven luciferase activity. In addition, ciglitazone did not significantly alter DHT-mediated increases in prostate specific antigen (PSA) protein or mRNA levels within C4-2 cells. siRNA-based experiments demonstrated that the ciglitazone-induced regulation of AR activity observed in C4-2 cells was dependent on the presence of PPARγ. Furthermore, overexpression of the AR corepressor cyclin D1 inhibited the ability of ciglitazone to induce AR luciferase activity in C4-2 cells. Thus, our data suggest that both PPARγ and cyclin D1 levels influence the ability of ciglitazone to differentially regulate AR signaling in androgen-independent C4-2 prostate cancer cells.     

Up-regulation of Bcl-2 is required for the progression of prostate cancer cells from an androgen-dependent to an androgen-independent growth stage

Bcl-2 is an anti-apoptotic oncoprotein and its protein levels are inversely correlated with prognosis in many cancers.However, the role of Bcl-2 in the progression of prostate cancer is not clear. Here we report that Bcl-2 is required for theprogression of LNCaP prostate cancer cells from an androgen-dependent to an androgen-independent growth stage. ThemRNA and protein levels of Bcl-2 are significantly increased in androgen-independent prostate cancer cells. shRNA-medi-ated gene silencing of Bcl-2 in androgen-independent prostate cancer cells promotes UV-induced apoptosis and suppressesthe growth of prostate tumors in vivo. Growing androgen-dependent cells under androgen-deprivation conditions resultsin formation of androgen-independent colonies; and the transition from androgen-dependent to androgen-independentgrowth is blocked by ectopic expression of the Bcl-2 antagonist Bax or Bcl-2 shRNA. Thus, our results demonstratethat Bcl-2 is not only critical for the survival of androgen-independent prostate cancer cells, but is also required for theprogression of prostate cancer cells from an androgen-dependent to an androgen-independent growth stage.