PKM2 promotes tumor angiogenesis by regulating HIF-1α through NF-κB activation.

BACKGROUND: Initially identified as a molecule that regulates the final step of glycolysis, the M2 isoform of pyruvate kinase (PKM2) was recently reported to have a central role in the metabolic reprogramming of cancer cells as well as participating in cell cycle progression and gene transcription. Despite intensive efforts, the intricate molecular mechanisms through which PKM2 regulates tumor progression remain elusive. METHODS: The proliferation and apoptosis of various pancreatic cancer cells using lentiviral-mediated PKM2 abrogation were assessed in vitro via Western blot and flow cytometric assay while the in vivo experiments involved tumor xenograft on chicken chorionallantoic membranes and immunohistochemistry on human tissue specimens. In order to decipher the molecular mechanism of HIF-1α and p65/RelA regulation by PKM2 in cancer cells cultivated in hypoxic atmosphere or normoxia we involved various biochemical assays such as Western blotting, immunoprecipitation, reporter gene assay and ELISA. RESULTS: Strong expression of PKM2 was observed in 68 % of human pancreatic adenocarcinoma specimens and almost all analyzed pancreatic cancer cell lines. Abrogation of PKM2 resulted in impaired proliferation and augmented apoptosis in vitro as well as impaired tumor growth and decreased blood vessel formation in vivo. Furthermore, deletion of PKM2 negatively impacted hypoxia-induced HIF-1α accumulation and promoter activity ultimately resulting in impaired secretion of VEGF. CONCLUSIONS: Our study suggests that in hypoxic pancreatic tumors PKM2 interferes both with NF-κB/p65 and HIF-1α activation that ultimately triggers VEGF-A secretion and subsequent blood vessel formation.

The small conductance calcium-activated potassium channel 3 (SK3) is a molecular target for Edelfosine to reduce the invasive potential of urothelial carcinoma cells.

Metastasis is the survival-determining factor in urothelial carcinoma (UC) of the urinary bladder. The small conductance calcium-activated potassium channel 3 (SK3) enhances tumor cell invasion in breast cancer and malignant melanoma. Since Edelfosine, a glycerophospholipid with antitumoral properties, effectively inhibits SK3 channel activity, our goal was to evaluate SK3 as a potential molecular target to inhibit the gain of an invasive phenotype in UC. SK3 protein expression was analyzed in 208 tissue samples and UC cell lines. Effects of Edelfosine on SK3 expression and intracellular calcium levels as well as on cell morphology, cell survival and proliferation were assessed using immunoblotting, potentiometric fluorescence microscopy, and clonogenic/cell survival assay; furthermore, we analyzed the effect of Edelfosine and SK3 RNAi knockdown on tumor cell migration and invasion in vitro and in vivo. We found that SK3 is strongly expressed in muscle-invasive UC and in the RT112 cellular tumor model. Higher concentrations of Edelfosine have a strong antitumoral effect on UC cells, while 1 µM effectively inhibits migration/invasion of UC cells in vitro and in vivo comparable to the SK3 knockdown phenotype. Taken together, our results show strong expression of SK3 in muscle-invasive UC, consistent with the postulated role of the protein in tumor cell invasion. Edelfosine is able to effectively inhibit migration and invasion of UC cells in vitro and in vivo in an SK3-dependent way, pointing towards a possible role for Edelfosine as an antiinvasive drug to effectively inhibit UC cell invasion and metastasis.

High-resolution MRI analysis of breast cancer xenograft on the chick chorioallantoic membrane.

The chick chorioallantoic membrane (CAM) model has been successfully used to study angiogenesis, cancer progression and its pharmacological treatment, tumor pharmacokinetics, and properties of novel nanomaterials. MRI is an attractive technique for non-invasive and longitudinal monitoring of physiological processes and tumor growth. This study proposes an age-adapted cooling regime for immobilization of the chick embryo, enabling high-resolution MRI of the embryo and the CAM tumor xenograft. 64 chick embryos were enrolled in this study. The novel immobilization and imaging protocol was optimized in 29 embryos. From d7 to d18 immobilization of the embryo up to 90 min was achieved by cooling at 4 °C pre-imaging, with cooling times adapted to age. Its application to tumor growth monitoring was evaluated in 15 embryos after xenotransplantation of human MDA-MB-231 breast cancer cells on CAM. Tumor volumes were monitored from d4 to d9 after grafting (d11 to d16 after incubation) applying a T2 -weighted multislice RARE sequence. At d9 after grafting, the tumors were collected and compared with the MRI-derived data by histology and weight measurements. Additional imaging methods comprising DWI, T2 mapping, and the bio-distribution of contrast agents were tested at d9 after grafting in 20 further embryos. With the adaptive cooling regime, motion artifacts could be completely avoided for up to 90 min scan time, enabling high-resolution in ovo imaging. Excellent anatomical details could be obtained in the embryo and tumors. Tumor volumes could be quantified over time. The results prove the feasibility of high-resolution MRI for longitudinal tumor and organ growth monitoring. The suggested method is promising for future applications such as testing tailored and/or targeted treatment strategies, longitudinal monitoring of tumor development, analysis of therapeutic efficacies of drugs, or assessment of tumor pharmacokinetics. The method provides an alternative to animal experimentation.

Thioredoxin-1 promotes anti-inflammatory macrophages of the M2 phenotype and antagonizes atherosclerosis.

OBJECTIVE: Oxidative stress is believed to play a key role in cardiovascular disorders. Thioredoxin (Trx) is an oxidative stress-limiting protein with anti-inflammatory and antiapoptotic properties. Here, we analyzed whether Trx-1 might exert atheroprotective effects by promoting macrophage differentiation into the M2 anti-inflammatory phenotype. METHODS AND RESULTS: Trx-1 at 1 µg/mL induced downregulation of p16(INK4a) and significantly promoted the polarization of anti-inflammatory M2 macrophages in macrophages exposed to interleukin (IL)-4 at 15 ng/mL or IL-4/IL-13 (10 ng/mL each) in vitro, as evidenced by the expression of the CD206 and IL-10 markers. In addition, Trx-1 induced downregulation of nuclear translocation of activator protein-1 and Ref-1, and significantly reduced the lipopolysaccharide-induced differentiation of inflammatory M1 macrophages, as indicated by the decreased expression of the M1 cytokines, tumor necrosis factor-α and monocyte chemoattractant protein-1. Consistently, Trx-1 administered to hyperlipoproteinemic ApoE2.Ki mice at 30 µg/30 g body weight challenged either with lipopolysaccharide at 30 µg/30 g body weight or with IL-4 at 500 ng/30 g body weight significantly induced the M2 phenotype while inhibiting differentiation of macrophages into the M1 phenotype in liver and thymus. ApoE2.Ki mice challenged once weekly with lipopolysaccharide for 5 weeks developed severe atherosclerotic lesions enriched with macrophages expressing predominantly M1 over M2 markers. In contrast, however, daily injections of Trx-1 shifted the phenotype pattern of lesional macrophages in these animals to predominantly M2 over M1, and the aortic lesion area was significantly reduced (from 100%±18% to 62.8%±9.8%; n=8; P<0.01). Consistently, Trx-1 colocalized with M2 but not with M1 macrophage markers in human atherosclerotic vessel specimens. CONCLUSIONS: The ability of Trx-1 to promote differentiation of macrophages into an alternative, anti-inflammatory phenotype may explain its protective effects in cardiovascular diseases. These data provide novel insight into the link between oxidative stress and cardiovascular diseases.

Plasmin triggers chemotaxis of monocyte-derived dendritic cells through an Akt2-dependent pathway and promotes a T-helper type-1 response.

OBJECTIVE: Dendritic cells (DC) accumulate in atherosclerotic arteries where they can modulate atherogenesis. We investigated whether plasmin might alter the function of human DC. METHODS AND RESULTS: Stimulation of monocyte-derived DC with plasmin elicited a time-dependent actin polymerization and chemotaxis comparable to that triggered by the standard chemoattractant formyl-methionyl-leucyl-phenylalanine. Plasmin triggered rapid activation of Akt and mitogen-activated protein kinases, followed by phosphorylation of the regulatory myosin light chain and chemotaxis. For the chemotactic DC migration, the activation of Akt and p38 and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases were indispensable, as shown by pharmacological inhibitors. DC express Akt1 and Akt2, but not Akt3. However, in DC, plasmin activates exclusively Akt2 via a p38 mitogen-activated protein kinase-dependent pathway. Accordingly, knockdown of Akt2 with short-hairpin RNA, but not of Akt1, blocked the plasmin-induced extracellular signal-regulated kinase 1/2 activation and the chemotactic response. Moreover, plasmin-stimulated DC induced polarization of CD4(+) T cells toward the interferon-gamma-producing, proinflammatory Th1 phenotype. Consistent with a role for DC and adaptive immune response in atherogenesis, we demonstrate DC in human atherosclerotic vessels and show that plasmin is abundant in human atherosclerotic lesions, where it colocalizes with DC. CONCLUSIONS: Plasmin generation in the atherosclerotic vessel wall might contribute to accumulation of DC, activation of the adaptive immune response, and aggravation of atherosclerosis.

Protein kinase D2 is a crucial regulator of tumour cell-endothelial cell communication in gastrointestinal tumours.

BACKGROUND: Tumour angiogenesis is crucially dependent on the communication between the tumour and the associated endothelium. Protein kinase D (PKD) isoenzymes mediate vascular endothelial growth factor-A (VEGF-A) induced endothelial cell proliferation and migration and are also highly expressed in various tumours. AIM: To examine the role of PKDs for tumour proliferation and angiogenesis selectively in pancreatic and gastric tumours and in tumour-associated endothelium in vitro and in vivo. METHODS: PKD2 expression in human tumours was determined by immunohistochemistry. The effect of PKD2 depletion in endothelial cells by siRNAs was examined in sprouting assays, the chorioallantois model (CAM) and tumour xenografts. In murine endothelium in vivo PKD2 was knocked-down by splice switching oligonucleotides. Human PKD2 was depleted in xenografts by siRNAs and PKD2-miRs. PKD2 activation by hypoxia and its role for hypoxia-induced NR4/TR3- and VEGF-A promoter activity, expression and secretion was investigated in cell lines. RESULTS: PKD2 is expressed in gastrointestinal tumours and in the tumour-associated endothelium. Tumour growth and angiogenesis in the CAM and in tumour xenografts require PKD expression in endothelial cells. Conversely, hypoxia activates PKD2 in pancreatic cancer cells and PKD2 was identified as the major mediator of hypoxia-stimulated VEGF-A promoter activity, expression and secretion in tumour cells. PKD2 depletion in pancreatic tumours inhibited tumour-driven blood vessel formation and tumour growth in the CAM and in orthotopic pancreatic cancer xenografts. CONCLUSION: PKD2 regulates hypoxia-induced VEGF-A expression/secretion by tumour cells and VEGF-A stimulated blood vessel formation. PKD2 is a novel, essential mediator of tumour cell-endothelial cell communication and a promising therapeutic target to inhibit angiogenesis in gastrointestinal cancers.

A New CYP2E1 Inhibitor, 12-Imidazolyl-1-dodecanol, Represents a Potential Treatment for Hepatocellular Carcinoma.

Cytochrome P450 2E1 (CYP2E1) is a key target protein in the development of alcoholic and nonalcoholic fatty liver disease (FLD). The pathophysiological correlate is the massive production of reactive oxygen species. The role of CYP2E1 in the development of hepatocellular carcinoma (HCC), the final complication of FLD, remains controversial. Specifically, CYP2E1 has not yet been defined as a molecular target for HCC therapy. In addition, a CYP2E1-specific drug has not been developed. We have already shown that our newly developed CYP2E1 inhibitor 12-imidazolyl-1-dodecanol (I-ol) was therapeutically effective against alcoholic and nonalcoholic steatohepatitis. In this study, we investigated the effect of I-ol on HCC tumorigenesis and whether I-ol could serve as a possible treatment option for terminal-stage FLD. I-ol exerted a very highly significant antitumour effect against hepatocellular HepG2 cells. Cell viability was reduced in a dose-dependent manner, with only the highest doses causing a cytotoxic effect associated with caspase 3/7 activation. Comparable results were obtained for the model colorectal adenocarcinoma cell line, DLD-1, whose tumorigenesis is also associated with CYP2E1. Transcriptome analyses showed a clear effect of I-ol on apoptosis and cell-cycle regulation, with the increased expression of p27Kip1 being particularly noticeable. These observations were confirmed at the protein level for HepG2 and DLD-1 cells grafted on a chorioallantoic membrane. Cell-cycle analysis showed a complete loss of proliferating cells with a simultaneous increase in S-phase arrest beginning at a threshold dose of 30 µM. I-ol also reduced xenograft tumour growth in nude mice. This antitumour effect was not associated with tumour cachexia. I-ol was not toxic to healthy tissues or organs. This study demonstrates for the first time the therapeutic effect of the specific CYP2E1 inhibitor I-ol on the tumorigenesis of HCC. Our findings imply that I-ol can potentially be applied therapeutically on patients at the final stage of FLD.

Specific induction of migration and invasion of pancreatic carcinoma cells by RhoC, which differs from RhoA in its localisation and activity.

RhoA and RhoC are highly related Rho GTPases, but differentially control cellular behaviour. We combined molecular, cellular, and biochemical experiments to characterise differences between these highly similar GTPases. Our findings demonstrate that enhanced expression of RhoC results in a striking increase in the migration and invasion of pancreatic carcinoma cells, whereas forced expression of RhoA decreases these actions. These isoform-specific functions correlate with differences in the cellular activity of RhoA and RhoC in human cells, with RhoC being more active than RhoA in activity assays and serum-response factor-dependent gene transcription. Subcellular localisation studies revealed that RhoC is predominantly localised in the membrane-containing fraction, whereas RhoA is mainly localised in the cytoplasmic fraction. These differences are not mediated by a different interaction with RhoGDIs. In vitro GTP/GDP binding analyses demonstrate different affinity of RhoC for GTP[S] and faster intrinsic and guanine nucleotide exchange factor (GEF)-stimulated GDP/GTP exchange rates compared to RhoA. Moreover, the catalytic domains of SopE and Dbs are efficacious GEFs for RhoC. mRNA expression of RhoC is markedly enhanced in advanced pancreatic cancer stages, and thus the differences discovered between RhoA and RhoC might provide explanations for their different influences on cell migration and tumour invasion.

Antiinflammatory and antiatherogenic effects of the NF-kappaB inhibitor acetyl-11-keto-beta-boswellic acid in LPS-challenged ApoE-/- mice.

OBJECTIVE: In this article, we studied the effect of acetyl-11-keto-beta-boswellic acid (AKbetaBA), a natural inhibitor of the proinflammatory transcription factor NF-kappaB on the development of atherosclerotic lesions in apolipoprotein E-deficient (apoE-/-) mice. METHODS AND RESULTS: Atherosclerotic lesions were induced by weekly LPS injection in apoE-/- mice. LPS alone increased atherosclerotic lesion size by approximately 100%, and treatment with AKbetaBA significantly reduced it by approximately 50%. Moreover, the activity of NF-kappaB was also reduced in the atherosclerotic plaques of LPS-injected apoE-/- mice treated with AKbetaBA. As a consequence, AKbetaBA treatment led to a significant downregulation of several NF-kappaB-dependent genes such as MCP-1, MCP-3, IL-1alpha, MIP-2, VEGF, and TF. By contrast, AKbetaBA did not affect the plasma concentrations of triglycerides, total cholesterol, antioxidized LDL antibodies, and various subsets of lymphocyte-derived cytokines. Moreover, AKbetaBA potently inhibited the IkappaB kinase (IKK) activity immunoprecipitated from LPS-stimulated mouse macrophages and mononuclear cells leading to decreased phosphorylation of IkappaB alpha and inhibition of p65/NF-kappaB activation. Comparable AKbetaBA-mediated inhibition was also observed in LPS-stimulated human macrophages. CONCLUSIONS: The inhibition of NF-kappaB activity by plant resins from species of the Boswellia family might represent an alternative for classical medicine treatments for chronic inflammatory diseases such as atherosclerosis.

Oncogenic K-Ras down-regulates Rac1 and RhoA activity and enhances migration and invasion of pancreatic carcinoma cells through activation of p38.

Activating mutations in the K-ras gene are genetic alterations frequently found in human carcinomas, particularly in pancreatic adenocarcinomas. Mutation of the K-ras gene is thought to be an early and important event in pancreatic tumor initiation, but the precise role of the mutant K-Ras proteins in neoplastic progression is still unknown. In the present study, we have characterized the influence of oncogenic K-Ras on the phenotype and on the signal transduction of epitheloid PANC-1 pancreatic carcinoma cells by generating PANC-1 cell clones, which stably express EGFP(enhanced green fluorescent protein)-K-Ras (V12). EGFP-K-Ras (V12)-expressing cells exhibited a more fibroblastoid cellular phenotype with irregular cell shape and disorganized cytokeratin filaments. Moreover, these cells showed a marked enhancement of their migratory and invasive properties. Stable expression of EGFP-K-Ras (V12) down-regulated the activity of Rac1 and RhoA, resulting in reduced subcortical actin filaments and stress fibers, which might contribute to the epithelial dedifferentiation. Characterization of the activity of mitogen-activated protein kinases revealed that EGFP-K-Ras (V12) enhanced the activity of p38, but did not affect the activities of the Raf/MEK/ERK cascade and JNK. While inhibition of either MEK or JNK activity had no effect on EGFP-K-Ras (V12)-induced migration, inhibition of p38 activity markedly reduced EGFP-K-Ras (V12)-induced migration. Collectively, the results suggest that oncogenic K-Ras enhances the malignant phenotype and identify the mitogen-activated protein kinase p38 as a target to inhibit oncogenic K-Ras-induced pancreatic tumor cell migration.

Baseline MAPK signaling activity confers intrinsic radioresistance to KRAS-mutant colorectal carcinoma cells by rapid upregulation of heterogeneous nuclear ribonucleoprotein K (hnRNP K).

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is overexpressed in malignant tumors and involved in DNA damage response upon ionizing radiation (IR). Here, we investigate its role in radioresistance of colorectal carcinoma (CRC) and evaluate a pharmacological approach to enhance CRC radiosensitivity via downregulation of hnRNP K. We show that hnRNP K is overexpressed in CRC tissue specimens and upregulated in response to IR in vitro, which occurs faster in KRAS-mutant CRC cells. HnRNP K knockdown impairs cell survival, cell cycle progression and KRAS-dependent radioresistance and increases apoptosis. Using the chicken chorioallantoic membrane assay, a decrease in xenograft tumor growth and radioresistance upon hnRNP K depletion could be verified in vivo, and comparable effects were achieved by suppression of hnRNP K expression using the MEK inhibitor MEK162 (Binimetinib). In summary, KRAS-mutant CRC shows intrinsic radioresistance along with rapid upregulation of hnRNP K in response to IR that can effectively be targeted by MEK inhibition. Our results point towards a possible use of MAPK pathway inhibitors to decrease radioresistance of KRAS-mutant CRC via downregulation of hnRNP K.

Inhibition of clonogenic tumor growth: a novel function of Smac contributing to its antitumor activity.

While second mitochondria derived activator of caspase (Smac) has been described to sensitize for apoptosis, its effect on cell viability in the absence of apoptotic stimuli has remained unclear. Here, we report that Smac inhibits clonogenic tumor growth by blocking random migration and proliferation and by enhancing apoptosis in a cell density and cell type dependent manner in SH-EP neuroblastoma cells. Inhibition of clonogenic survival by overexpression of full-length or processed Smac strictly depended on low cell density, and was reversible by replatement at high density. We discovered that Smac inhibits cell motility and random migration at low cell density. In addition, Smac enhanced apoptosis and inhibited protein, but not mRNA expression of XIAP, survivin and other short-lived proteins (FLIP, p21), indicating that Smac may globally inhibit protein expression. Also, Smac inhibited proliferation and increased polynucleation with no evidence for polyploidy, cell cycle arrest or senescence indicating that Smac impaired cell division. Interestingly, inhibition of clonogenic capacity by Smac occurred independent of its apoptosis promoting activity. By demonstrating that Smac restrains clonogenic tumor growth, our findings may have important implications for control of tumor growth and/or its metastatic spread. Thus, Smac agonists may be useful in cancer therapy, for example, for tumor control in minimal residual disease. Oncogene (2005) 24, 7190-7202. doi:10.1038/sj.onc.1208876; published online 8 August 2005.

Activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase is required for glial cell line-derived neurotrophic factor-induced migration and invasion of pancreatic carcinoma cells.

Pancreatic carcinoma cells exhibit a pronounced tendency to invade along and into intra- and extrapancreatic nerves, even at early stages of the disease. The neurotrophic factor glial cell line-derived neurotrophic factor (GDNF) has been shown to promote pancreatic cancer cell invasion. Here, we demonstrate that pancreatic carcinoma cell lines, such as PANC-1, expressed the RET and GDNF family receptor alpha receptor components for GDNF and that primary pancreatic tumor samples, derived from carcinomas with regional lymph node metastasis, exhibited marked expression of the mRNA encoding the RET51 isoform. Moreover, GDNF was an efficacious and potent chemoattractant for pancreatic carcinoma cells as examined in in vitro and in vivo model systems. Treatment of PANC-1 cells with GDNF resulted in activation of the monomeric GTPases N-Ras, Rac1, and RhoA, in activation of the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK) and in activation of the phosphatidylinositol 3-kinase/Akt pathway. Both inhibition of the Ras-Raf-MEK (mitogen-activated protein/ERK kinase)-ERK cascade by either stable expression of dominant-negative H-Ras(N17) or addition of the MEK1 inhibitor PD98059 as well as inhibition of the phosphatidylinositol 3-kinase pathway by LY294002 prevented GDNF-induced migration and invasion of PANC-1 cells. These results demonstrate that pancreatic tumor cell migration and possibly perineural invasion in response to GDNF is critically controlled by activation of the Ras-Raf-MEK-ERK and the phosphatidylinositol 3-kinase pathway.

Tbx3 fosters pancreatic cancer growth by increased angiogenesis and activin/nodal-dependent induction of stemness.

Cell fate decisions and pluripotency, but also malignancy depend on networks of key transcriptional regulators. The T-box transcription factor TBX3 has been implicated in the regulation of embryonic stem cell self-renewal and cardiogenesis. We have recently discovered that forced TBX3 expression in embryonic stem cells promotes mesendoderm specification directly by activating key lineage specification factors and indirectly by enhancing paracrine NODAL signalling. Interestingly, aberrant TBX3 expression is associated with breast cancer and melanoma formation. In other cancers, loss of TBX3 expression is associated with a more aggressive phenotype e.g. in gastric and cervical cancer. The precise function of TBX3 in pancreatic ductal adenocarcinoma remains to be determined. In the current study we provide conclusive evidence for TBX3 overexpression in pancreatic cancer samples as compared to healthy tissue. While proliferation remains unaltered, forced TBX3 expression strongly increases migration and invasion, but also angiogenesis in vitro and in vivo. Finally, we describe the TBX3-dependency of cancer stem cells that perpetuate themselves through an autocrine TBX3-ACTIVIN/NODAL signalling loop to sustain stemness. Thus, TBX3 is a new key player among pluripotency-related genes driving cancer formation.

High-performance liquid chromatographic determination of acetyl-11-keto-alpha-boswellic acid, a novel pentacyclic triterpenoid, in plasma using a fluorinated stationary phase and photodiode array detection: application in pharmacokinetic studies.

A rapid, sensitive and selective HPLC separation with photodiode array detection was developed for the analysis of the novel pentacyclic triterpenoid acetyl-11-keto-alpha-boswellic acid. Complete baseline separation of acetyl-11-keto-alpha-boswellic acid from the corresponding isomer acetyl-11-keto-beta-boswellic acid was achieved on a fluorinated stationary phase. The standard curve was linear from 0.98 nmol/l to 196 nmol/l acetyl-11-keto-alpha-boswellic acid. The compound was isolated from chick embryonic plasma using extraction on diatomaceous earth with an overall average extraction yield of 82%. This method was applied in a kinetic study on the chick chorioallantoic membrane model (CAM) and showed unequivocal separation between acetyl-11-keto-alpha-boswellic acid and acetyl-11-keto-beta-boswellic acid unachievable so far.

A time frame permissive for Protein Kinase D2 activity to direct angiogenesis in mouse embryonic stem cells.

The protein kinase D isoenzymes PKD1/2/3 are prominent downstream targets of PKCs (Protein Kinase Cs) and phospholipase D in various biological systems. Recently, we identified PKD isoforms as novel mediators of tumour cell-endothelial cell communication, tumour cell motility and metastasis. Although PKD isoforms have been implicated in physiological/tumour angiogenesis, a role of PKDs during embryonic development, vasculogenesis and angiogenesis still remains elusive. We investigated the role of PKDs in germ layer segregation and subsequent vasculogenesis and angiogenesis using mouse embryonic stem cells (ESCs). We show that mouse ESCs predominantly express PKD2 followed by PKD3 while PKD1 displays negligible levels. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated at the time of germ layer segregation. Time-restricted PKD2-activation limits mesendoderm formation and subsequent cardiovasculogenesis during early differentiation while leading to branching angiogenesis during late differentiation. In line, PKD2 loss-of-function analyses showed induction of mesendodermal differentiation in expense of the neuroectodermal germ layer. Our in vivo findings demonstrate that embryoid bodies transplanted on chicken chorioallantoic membrane induced an angiogenic response indicating that timed overexpression of PKD2 from day 4 onwards leads to augmented angiogenesis in differentiating ESCs. Taken together, our results describe novel and time-dependent facets of PKD2 during early cell fate determination.

Stilbene induced inhibition of androgen receptor dimerization: implications for AR and ARΔLBD-signalling in human prostate cancer cells.

BACKGROUND: Advanced castration resistant prostate cancer (CRPC) is often characterized by an increase of C-terminally truncated, constitutively active androgen receptor (AR) variants. Due to the absence of a ligand binding domain located in the AR-C-terminus, these receptor variants (also termed ARΔLBD) are unable to respond to all classical forms of endocrine treatments like surgical/chemical castration and/or application of anti-androgens. METHODOLOGY: In this study we tested the effects of the naturally occurring stilbene resveratrol (RSV) and (E)-4-(2, 6-Difluorostyryl)-N, N-dimethylaniline, a fluorinated dialkylaminostilbene (FIDAS) on AR- and ARΔLBD in prostate cancer cells. The ability of the compounds to modulate transcriptional activity of AR and the ARΔLBD-variant Q640X was shown by reporter gene assays. Expression of endogenous AR and ARΔLBD mRNA and protein levels were determined by qRT-PCR and Western Blot. Nuclear translocation of AR-molecules was analyzed by fluorescence microscopy. AR and ARΔLBD/Q640X homo-/heterodimer formation was assessed by mammalian two hybrid assays. Biological activity of both compounds in vivo was demonstrated using a chick chorioallantoic membrane xenograft assay. RESULTS: The stilbenes RSV and FIDAS were able to significantly diminish AR and Q640X-signalling. Successful inhibition of the Q640X suggests that RSV and FIDAS are not interfering with the AR-ligand binding domain like all currently available anti-hormonal drugs. Repression of AR and Q640X-signalling by RSV and FIDAS in prostate cancer cells was caused by an inhibition of the AR and/or Q640X-dimerization. Although systemic bioavailability of both stilbenes is very low, both compounds were also able to downregulate tumor growth and AR-signalling in vivo. CONCLUSION: RSV and FIDAS are able to inhibit the dimerization of AR and ARΔLBD molecules suggesting that stilbenes might serve as lead compounds for a novel generation of AR-inhibitors.

Active targeting of mesoporous silica drug carriers enhances γ-secretase inhibitor efficacy in an in vivo model for breast cancer.

AIM: In this article, we use an alternative cancer model for the evaluation of nanotherapy, and assess the impact of surface functionalization and active targeting of mesoporous silica nanoparticles (MSNPs) on therapeutic efficacy in vivo. MATERIALS & METHODS: We used the chorioallantoic membrane xenograft assay to investigate the biodistribution and therapeutic efficacy of folate versus polyethyleneimine-functionalized γ-secretase inhibitor-loaded MSNPs in breast and prostate tumor models. RESULTS: γ-secretase inhibitor-loaded MSNPs inhibited tumor growth in breast and prostate cancer xenografts. Folate conjugation improved the therapeutic outcome in folic acid receptor-positive breast cancer, but not in prostate cancer lacking the receptor. CONCLUSION: The results demonstrate that therapeutic efficacy is linked to cellular uptake of MSNPs as opposed to tumor accumulation, and show that MSNP-based delivery of γ-secretase inhibitors is therapeutically effective in both breast and prostate cancer. In this article, we present a model system for a medium-to-high throughput, cost-effective, quantitative evaluation of nanoparticulate drug carriers.

HSP90 supports tumor growth and angiogenesis through PRKD2 protein stabilization.

The kinase PRKD2 (protein kinase D) is a crucial regulator of tumor cell-endothelial cell communication in gastrointestinal tumors and glioblastomas, but its mechanistic contributions to malignant development are not understood. Here, we report that the oncogenic chaperone HSP90 binds to and stabilizes PRKD2 in human cancer cells. Pharmacologic inhibition of HSP90 with structurally divergent small molecules currently in clinical development triggered proteasome-dependent degradation of PRKD2, augmenting apoptosis in human cancer cells of various tissue origins. Conversely, ectopic expression of PRKD2 protected cancer cells from the apoptotic effects of HSP90 abrogation, restoring blood vessel formation in two preclinical models of solid tumors. Mechanistic studies revealed that PRKD2 is essential for hypoxia-induced accumulation of hypoxia-inducible factor-1α (HIF1α) and activation of NF-κB in tumor cells. Notably, ectopic expression of PRKD2 was able to partially restore HIF1α and secreted VEGF-A levels in hypoxic cancer cells treated with HSP90 inhibitors. Taken together, our findings indicate that signals from hypoxia and HSP90 pathways are interconnected and funneled by PRKD2 into the NF-κB/VEGF-A signaling axis to promote tumor angiogenesis and tumor growth.