Combinatorial targeting of FGF and ErbB receptors blocks growth and metastatic spread of breast cancer models.

Combinatorial targeting of FGF and ErbB receptors blocks growth and metastatic spread of breast cancer models.

Breast Cancer Res. 2013 Jan 23;15(1):R8

Authors: Issa A, Gill JW, Heideman MR, Sahin O, Wiemann S, Dey JH, Hynes NE

Abstract
ABSTRACT: INTRODUCTION: Targeting receptor tyrosine kinases (RTKs) with kinase inhibitors is a clinically validated anti-cancer approach, however, the effectiveness of individual inhibitors is often short-lived and resistance emerges. Experimental approaches have revealed numerous feed-back loops in tumor cells and that blocking one signaling pathway, be it the receptor or downstream targets, is often not sufficient to cause tumor regression. Alterations in fibroblast growth factor receptor (FGFR) activity have been implicated in breast cancer. Using breast cancer models with autocrine activation of fibroblast growth factor receptors (FGFR), we have examined the impact of targeting FGFRs in vivo with a selective kinase inhibitor in combination with an inhibitor of PI3K/mTOR or with a pan-ErbB inhibitor. METHODS: The 4T1 or 67NR mammary cancer cells are models for basal-like breast cancer and display constitutive FGFR activity. Upon their injection into fat pads of female Balb/c mice both tumor cell lines form tumors; 4T1 tumors, but not 67NR tumors metastasize to lungs. Tumor growth was measured in mice treated with an FGFR inhibitor (dovitinib/TKI258), a PI3K/ mTOR inhibitor (NVP-BEZ235) or with a pan-ErbB inhibitor (AEE788). Inhibitors were administered individually or in combination and lung metastases were quantified in the 4T1 model. To uncover mechanisms underlying inhibitor activity, tumor lysates were examined by western analyses for signaling activity of FGFR/FRS2, ErbB2, the Erk and the PI3K/Akt/mTOR pathways. Tumor sections were examined for proliferation, apoptosis and vessel density using antibodies for P-Histone H3, cleaved Caspase-3 and CD31, respectively. A transcriptome analysis was carried out on tumors treated with dovitinib for different times to identify pathways upregulated by FGFR inhibition. Anti-phosphotyrosine receptor antibody arrays (P-Tyr RTK) were used to screen in an unbiased manner for active receptors in 4T1 tumors. RESULTS: Treatment of 4T1 and 67NR tumor-bearing mice with the combination of dovitinib + NVP-BEZ235 causes tumor stasis. Western analysis of tumor lysates shows strong down-regulation of the FRS2/Erk and PI3K/Akt/mTOR signaling pathways. Examination of tumor sections revealed that the combination treatment results in a significant decrease in proliferation and high numbers of apoptotic cells, in comparison to tumors treated with individual inhibitors. Using P-Tyr RTK arrays, we identified high levels of P-EGFR and P-ErbB2 in 4T1 tumors. Testing AEE788 in the 4T1 and 67NR models revealed that only the combination of dovitinib + AEE788 resulted in blockade of the PI3K/Akt/mTOR pathway, prolonged tumor stasis and in the 4T1 model, a highly significant decrease in lung metastasis. Analyses of the tumor sections revealed that the combination of dovitinib + AEE788 caused a significant decrease in proliferation and high levels of apoptosis. The results show that in vivo these breast cancer models become dependent upon co-activation of FGFR and ErbB receptors for PI3K pathway activity. CONCLUSIONS: The work presented here shows that in the 4T1 and 67NR breast cancer models the combination of dovitinib + NVP-BEZ235 or dovitinib + AEE788 leads to a strong inhibition of tumor growth and, for the 4T1 model, a block in metastatic spread. Only these combinations strongly down-regulate the FGFR/FRS2/Erk and the PI3K/Akt/mTOR signaling pathways and cause high levels of apoptosis. Interestingly, the decrease in mitosis and increase in apoptosis was consistently stronger in the dovitinib + AEE788 treatment-group suggesting that targeting ErbB receptors has broader downstream effects compared to targeting only PI3K/mTOR. In experiments aimed at testing the durability of treatment-response, this combination was also more effective. Considering that sub-classes of human breast tumors co-express ErbB receptors and FGFRs, these results might have implications for targeted therapy.

PMID: 23343422 [PubMed - as supplied by publisher]

ecdysone chir-258 dovitinib

What goes up must come down: transcription factors have their say in making ecdysone pulses.

What goes up must come down: transcription factors have their say in making ecdysone pulses.

Curr Top Dev Biol. 2013;103:35-71

Authors: Ou Q, King-Jones K

Abstract
Insect metamorphosis is one of the most fascinating biological processes in the animal kingdom. The dramatic transition from an immature juvenile to a reproductive adult is under the control of the steroid hormone ecdysone, also known as the insect molting hormone. During Drosophila development, periodic pulses of ecdysone are released from the prothoracic glands, upon which the hormone is rapidly converted in peripheral tissues to its biologically active form, 20-hydroxyecdysone. Each hormone pulse has a unique profile and causes different developmental events, but we only have a rudimentary understanding of how the timing, amplitude, and duration of a given pulse are controlled. A key component involved in the timing of ecdysone pulses is PTTH, a brain-derived neuropeptide. PTTH stimulates ecdysone production through a Ras/Raf/ERK signaling cascade; however, comparatively little is known about the downstream targets of this pathway. In recent years, it has become apparent that transcriptional regulation plays a critical role in regulating the synthesis of ecdysone, but only one transcription factor has a well-defined link to PTTH. Interestingly, many of the ecdysteroidogenic transcription factors were originally characterized as primary response genes in the ecdysone signaling cascade that elicits the biological responses to the hormone in target tissues. To review these developments, we will first provide an overview of the transcription factors that act in the Drosophila ecdysone regulatory hierarchy. We will then discuss the roles of these transcriptional regulators in controlling ecdysone synthesis. In the last section, we will briefly outline transcription factors that likely have roles in regulating ecdysone synthesis but have not been formally identified as downstream effectors of ecdysone.

PMID: 23347515 [PubMed - in process]

c-met inhibitors zm-447439 rad001

Generation of myeloid-derived suppressor cells using prostaglandin E2.

Related Articles

Generation of myeloid-derived suppressor cells using prostaglandin E2.

Transplant Res. 2012;1(1):15

Authors: Obermajer N, Kalinski P

Abstract
ABSTRACT: Myeloid-derived suppressor cells (MDSCs) are natural immunosuppressive cells and endogenous inhibitors of the immune system. We describe a simple and clinically compatible method of generating large numbers of MDSCs using the cultures of peripheral blood-isolated monocytes supplemented with prostaglandin E2 (PGE2). We observed that PGE2 induces endogenous cyclooxygenase (COX)2 expression in cultured monocytes, blocking their differentiation into CD1a+ dendritic cells (DCs) and inducing the expression of indoleamine 2,3-dioxygenase 1, IL-4R?, nitric oxide synthase 2 and IL-10 - typical MDSC-associated suppressive factors. The establishment of a positive feedback loop between PGE2 and COX2, the key regulator of PGE2 synthesis, is both necessary and sufficient to promote the development of CD1a+ DCs to CD14+CD33+CD34+ monocytic MDSCs in granulocyte macrophage colony stimulating factor/IL-4-supplemented monocyte cultures, their stability, production of multiple immunosuppressive mediators and cytotoxic T lymphocyte-suppressive function. In addition to PGE2, selective E-prostanoid receptor (EP)2- and EP4-agonists, but not EP3/1 agonists, also induce the MDSCs development, suggesting that other activators of the EP2/4- and EP2/4-driven signaling pathway (adenylate cyclase/cAMP/PKA/CREB) may be used to promote the development of suppressive cells. Our observations provide a simple method for generating large numbers of MDSCs for the immunotherapy of autoimmune diseases, chronic inflammatory disorders and transplant rejection.

PMID: 23369567 [PubMed - as supplied by publisher]

rad001 ecdysone chir-258

Resistance to DNA-damaging treatment in non-small cell lung cancer tumor-initiating cells involves reduced DNA-PK/ATM activation and diminished cell cycle arrest.

Related Articles

Resistance to DNA-damaging treatment in non-small cell lung cancer tumor-initiating cells involves reduced DNA-PK/ATM activation and diminished cell cycle arrest.

Cell Death Dis. 2013;4:e478

Authors: Lundholm L, H��g P, Zong D, Juntti T, M�rk B, Lewensohn R, Viktorsson K

Abstract
Increasing evidence suggests that tumor-initiating cells (TICs), also called cancer stem cells, are partly responsible for resistance to DNA-damaging treatment. Here we addressed if such a phenotype may contribute to radio- and cisplatin resistance in non-small cell lung cancer (NSCLC). We showed that four out of eight NSCLC cell lines (H125, A549, H1299 and H23) possess sphere-forming capacity when cultured in stem cell media and three of these display elevated levels of CD133. Indeed, sphere-forming NSCLC cells, hereafter called TICs, showed a reduced apoptotic response and increased survival after irradiation (IR), as compared with the corresponding bulk cell population. Decreased cytotoxicity and apoptotic signaling manifested by diminished poly (ADP-ribose) polymerase (PARP) cleavage and caspase 3 activity was also evident in TICs after cisplatin treatment. Neither radiation nor cisplatin resistance was due to quiescence as H125 TICs proliferated at a rate comparable to bulk cells. However, TICs displayed less pronounced G2 cell cycle arrest and S/G2-phase block after IR and cisplatin, respectively. Additionally, we confirmed a cisplatin-refractory phenotype of H125 TICs in vivo in a mouse xenograft model. We further examined TICs for altered expression or activation of DNA damage repair proteins as a way to explain their increased radio- and/or chemotherapy resistance. Indeed, we found that TICs exhibited increased basal ?H2AX (H2A histone family, member X) expression and diminished DNA damage-induced phosphorylation of DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia-mutated (ATM), Kr�ppel-associated protein 1 (KAP1) and monoubiquitination of Fanconi anemia, complementation group D2 (FANCD2). As a proof of principle, ATM inhibition in bulk cells increased their cisplatin resistance, as demonstrated by reduced PARP cleavage. In conclusion, we show that reduced apoptotic response, altered DNA repair signaling and cell cycle perturbations in NSCLC TICs are possible factors contributing to their therapy resistance, which may be exploited for DNA damage-sensitizing purposes.

PMID: 23370278 [PubMed - as supplied by publisher]

ecdysone chir-258 dovitinib

HPV Episome Stability is Reduced by Aphidicolin and Controlled by DNA Damage Response Pathways.

Related Articles

HPV Episome Stability is Reduced by Aphidicolin and Controlled by DNA Damage Response Pathways.

J Virol. 2013 Jan 30;

Authors: Edwards TG, Helmus MJ, Koeller K, Bashkin JK, Fisher C

Abstract
A highly reproducible Q-PCR assay was used to study the stability of HPV in undifferentiated keratinocytes that maintain viral episomes. Stability refers to the ability of episomes to persist with little copy number variation in cells. In investigating the mechanism of action of PA25, a previously published compound that destabilizes HPV episomes, aphidicolin was also found to markedly decrease episome levels, but via a different pathway than PA25. Since aphidicolin is known to activate DNA damage response (DDR) pathways, effects of inhibitors and siRNAs acting within DDR pathways were investigated. Inhibitors of Chk1 and siRNA directed against ATR significantly reduced viral episomes suggesting that these pathways play a role in maintaining HPV episome stability. Inhibitors of Chk2 and DNA-PK had no effect on episome levels. Pharmacological ATM inhibition had no effect on episome levels, but ATM knockdown by siRNA significantly reduced episomes, suggesting that ATM is playing an important role in HPV episome stability that does not require kinase activity. These results outline two pathways that trigger episome loss from cells and suggest the existence of a little understood mechanism that mediates viral DNA elimination. Together, our results also indicate that HPV episomes have a stability profile that is remarkably similar to that of fragile sites; these similarities are outlined and discussed. This close correspondence may influence the preference of HPV for integration into fragile sites.

PMID: 23365423 [PubMed - as supplied by publisher]

ecdysone chir-258 dovitinib

Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Related Articles

Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Transfusion. 2012 Nov;52(11):2406-13

Authors: Avanzi MP, Chen A, He W, Mitchell WB

Abstract
BACKGROUND: Large-scale in vitro production of platelets (PLTs) from cord blood stem cells is one goal of stem cell research. One step toward this goal will be to produce polyploid megakaryocytes capable of releasing high numbers of PLTs. Megakaryocyte polyploidization requires distinct cytoskeletal and cellular mechanisms, including actin polymerization, myosin activation, microtubule formation, and increased DNA production. In this study we variably combined inhibition of these principal mechanisms of cytokinesis with the goal of driving polyploidization in megakaryocytes.
STUDY DESIGN AND METHODS: Megakaryocytes were derived from umbilical cord blood and cultured with reagents that inhibit distinct mechanisms of cytokinesis: Rho-Rock inhibitor (RRI), Src inhibitor (SI), nicotinamide (NIC), aurora B inhibitor (ABI), and myosin light chain kinase inhibitor (MLCKI). Combinations of reagents were used to determine their interactions and to maximize megakaryocyte ploidy.
RESULTS: Treatment with RRI, NIC, SI, and ABI, but not with MLCKI, increased the final ploidy and RRI was the most effective single reagent. RRI and MLCKI, both inhibitors of MLC activation, resulted in opposite ploidy outcomes. Combinations of reagents also increased ploidy and the use of NIC, SI, and ABI was as effective as RRI alone. Addition of MLCKI to NIC, SI, and ABI reached the highest level of polyploidization.
CONCLUSION: Megakaryocyte polyploidization results from modulation of a combination of distinct cytokinesis pathways. Reagents targeting distinct cytoskeletal pathways produced additive effects in final megakaryocyte ploidy. The RRI, however, showed no additive effect but produced a high final ploidy due to overlapping inhibition of multiple cytokinesis pathways.

PMID: 22612069 [PubMed - indexed for MEDLINE]

c-met inhibitors zm-447439 rad001

Developmental checkpoints and feedback circuits time insect maturation.

Developmental checkpoints and feedback circuits time insect maturation.

Curr Top Dev Biol. 2013;103:1-33

Authors: Rewitz KF, Yamanaka N, O'Connor MB

Abstract
The transition from juvenile to adult is a fundamental process that allows animals to allocate resource toward reproduction after completing a certain amount of growth. In insects, growth to a species-specific target size induces pulses of the steroid hormone ecdysone that triggers metamorphosis and reproductive maturation. The past few years have seen significant progress in understanding the interplay of mechanisms that coordinate timing of ecdysone production and release. These studies show that the neuroendocrine system monitors complex size-related and nutritional signals, as well as external cues, to time production and release of ecdysone. Based on results discussed here, we suggest that developmental progression to adulthood is controlled by checkpoints that regulate the genetic timing program enabling it to adapt to different environmental conditions. These checkpoints utilize a number of signaling pathways to modulate ecdysone production in the prothoracic gland. Release of ecdysone activates an autonomous cascade of both feedforward and feedback signals that determine the duration of the ecdysone pulse at each developmental transitions. Conservation of the genetic mechanisms that coordinate the juvenile-adult transition suggests that insights from the fruit fly Drosophila will provide a framework for future investigation of developmental timing in metazoans.

PMID: 23347514 [PubMed - in process]

rad001 ecdysone chir-258

Everolimus-Eluting Stents Versus Sirolimus- or Paclitaxel-Eluting Stents: Two-Year Results from the Guthrie Health Off-Label Stent (GHOST) Registry.

Related Articles

Everolimus-Eluting Stents Versus Sirolimus- or Paclitaxel-Eluting Stents: Two-Year Results from the Guthrie Health Off-Label Stent (GHOST) Registry.

J Interv Cardiol. 2013 Jan 31;

Authors: Harjai KJ, Kondareddy S, Pinkosky B, Harjai N, Orshaw P, Boura J

Abstract
OBJECTIVES: We sought to compare the safety and effectiveness of everolimus-eluting stents (EES) versus first generation drug-eluting stents (FG-DES; sirolimus-eluting stent [SES] or paclitaxel-eluting stent [PES]). METHODS: In 2,126 patients undergoing percutaneous coronary intervention (PCI), we compared the 2-year incidence of stent thrombosis (ST) and target vessel revascularization (TVR) between the EES versus FG-DES groups. Secondary end-points included all-cause death, myocardial infarction (MI), death or MI, and major adverse cardiovascular events (MACE, including death, MI, ST, or TVR). Further, we evaluated these end-points in 2 propensity-matched subgroups: EES versus SES; EES versus PES. RESULTS: Complete 2-year follow-up was available in 1,911 (90%) patients. Compared to FG-DES, implantation of EES was associated with trends towards lower ST (0.9% vs. 2.8%, P?=?0.068) and TVR (3.8% vs. 7.2%, P?=?0.052), which persisted after adjustment for baseline differences (for ST, adjusted hazard ratio, HR 0.32; 95% confidence interval, 95% CI 0.10-1.02, P?=?0.053; for TVR, HR 0.40; 95% CI 0.22-0.75, P?=?0.004). Compared to SES, EES implantation was associated with lower TVR and a trend towards lower ST. Compared to PES, EES implantation was associated with less ST and TVR and trends towards lower death/MI and MACE. In the EES group, no ST was seen after the first 3 months. CONCLUSIONS: The use of EES compared to FG-DES appears to be associated with reductions in ST and TVR at 2-year follow-up. Improved outcomes with EES are observed in comparison with SES as well as PES.

PMID: 23363439 [PubMed - as supplied by publisher]

rad001 ecdysone chir-258

The anticancer multi-kinase inhibitor dovitinib also targets topoisomerase I and topoisomerase II.

Related Articles

The anticancer multi-kinase inhibitor dovitinib also targets topoisomerase I and topoisomerase II.

Biochem Pharmacol. 2012 Dec 15;84(12):1617-26

Authors: Hasinoff BB, Wu X, Nitiss JL, Kanagasabai R, Yalowich JC

Abstract
Dovitinib (TKI258/CHIR258) is a multi-kinase inhibitor in phase III development for the treatment of several cancers. Dovitinib is a benzimidazole-quinolinone compound that structurally resembles the bisbenzimidazole minor groove binding dye Hoechst 33258. Dovitinib bound to DNA as shown by its ability to increase the DNA melting temperature and by increases in its fluorescence spectrum that occurred upon the addition of DNA. Molecular modeling studies of the docking of dovitinib into an X-ray structure of a Hoechst 33258-DNA complex showed that dovitinib could reasonably be accommodated in the DNA minor groove. Because DNA binders are often topoisomerase I (EC 5.99.1.2) and topoisomerase II (EC 5.99.1.3) inhibitors, the ability of dovitinib to inhibit these DNA processing enzymes was also investigated. Dovitinib inhibited the catalytic decatenation activity of topoisomerase II?. It also inhibited the DNA-independent ATPase activity of yeast topoisomerase II which suggested that it interacted with the ATP binding site. Using isolated human topoisomerase II?, dovitinib stabilized the enzyme-cleavage complex and acted as a topoisomerase II? poison. Dovitinib was also found to be a cellular topoisomerase II poison in human leukemia K562 cells and induced double-strand DNA breaks in K562 cells as evidenced by increased phosphorylation of H2AX. Finally, dovitinib inhibited the topoisomerase I-catalyzed relaxation of plasmid DNA and acted as a cellular topoisomerase I poison. In conclusion, the cell growth inhibitory activity and the anticancer activity of dovitinib may result not only from its ability to inhibit multiple kinases, but also, in part, from its ability to target topoisomerase I and topoisomerase II.

PMID: 23041231 [PubMed - indexed for MEDLINE]

rad001 ecdysone chir-258

Inhibition of titanium particle-induced inflammation by the proteasome inhibitor bortezomib in murine macrophage-like RAW 264.7 cells.

Related Articles

Inhibition of titanium particle-induced inflammation by the proteasome inhibitor bortezomib in murine macrophage-like RAW 264.7 cells.

Inflammation. 2012 Aug;35(4):1411-8

Authors: Mao X, Pan X, Peng X, Cheng T, Zhang X

Abstract
Wear particle-induced inflammatory osteolysis is the major cause of aseptic loosening after total joint replacement. The predominant cell type within periprosthetic tissues is macrophages. We investigate the anti-inflammatory effects of the proteasome inhibitor bortezomib (Bzb) on murine macrophage-like RAW 264.7 cells stimulated with titanium (Ti) particles. RAW 264.7 cells were cultured with 1 nM Bzb and 0.1 mg/ml Ti particles for 48 h; cells without Ti and Bzb or without Bzb were used as negative and loading controls. Results showed that the expression of inflammatory cytokines (TNF-?, IL-1?, IL-6, and IL-10), chemokines [monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 alpha (MIP-1?)], and inflammatory enzymes [inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2)] increased in RAW 264.7 cells cultured with Ti. Bzb treatment significantly reduced the expression of TNF-?, IL-1?, IL-6, MCP-1, MIP-1?, iNOS, and COX-2 and induced the expression of IL-10 in a time-dependent manner. These results suggest that Bzb inhibits Ti-induced inflammation in macrophages, and provide a promising therapeutic target for treating or preventing aseptic loosening.

PMID: 22427154 [PubMed - indexed for MEDLINE]

rad001 ecdysone chir-258

Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Related Articles

Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Transfusion. 2012 Nov;52(11):2406-13

Authors: Avanzi MP, Chen A, He W, Mitchell WB

Abstract
BACKGROUND: Large-scale in vitro production of platelets (PLTs) from cord blood stem cells is one goal of stem cell research. One step toward this goal will be to produce polyploid megakaryocytes capable of releasing high numbers of PLTs. Megakaryocyte polyploidization requires distinct cytoskeletal and cellular mechanisms, including actin polymerization, myosin activation, microtubule formation, and increased DNA production. In this study we variably combined inhibition of these principal mechanisms of cytokinesis with the goal of driving polyploidization in megakaryocytes.
STUDY DESIGN AND METHODS: Megakaryocytes were derived from umbilical cord blood and cultured with reagents that inhibit distinct mechanisms of cytokinesis: Rho-Rock inhibitor (RRI), Src inhibitor (SI), nicotinamide (NIC), aurora B inhibitor (ABI), and myosin light chain kinase inhibitor (MLCKI). Combinations of reagents were used to determine their interactions and to maximize megakaryocyte ploidy.
RESULTS: Treatment with RRI, NIC, SI, and ABI, but not with MLCKI, increased the final ploidy and RRI was the most effective single reagent. RRI and MLCKI, both inhibitors of MLC activation, resulted in opposite ploidy outcomes. Combinations of reagents also increased ploidy and the use of NIC, SI, and ABI was as effective as RRI alone. Addition of MLCKI to NIC, SI, and ABI reached the highest level of polyploidization.
CONCLUSION: Megakaryocyte polyploidization results from modulation of a combination of distinct cytokinesis pathways. Reagents targeting distinct cytoskeletal pathways produced additive effects in final megakaryocyte ploidy. The RRI, however, showed no additive effect but produced a high final ploidy due to overlapping inhibition of multiple cytokinesis pathways.

PMID: 22612069 [PubMed - indexed for MEDLINE]

coxinhibitors c-met inhibitors zm-447439

What goes up must come down: transcription factors have their say in making ecdysone pulses.

What goes up must come down: transcription factors have their say in making ecdysone pulses.

Curr Top Dev Biol. 2013;103:35-71

Authors: Ou Q, King-Jones K

Abstract
Insect metamorphosis is one of the most fascinating biological processes in the animal kingdom. The dramatic transition from an immature juvenile to a reproductive adult is under the control of the steroid hormone ecdysone, also known as the insect molting hormone. During Drosophila development, periodic pulses of ecdysone are released from the prothoracic glands, upon which the hormone is rapidly converted in peripheral tissues to its biologically active form, 20-hydroxyecdysone. Each hormone pulse has a unique profile and causes different developmental events, but we only have a rudimentary understanding of how the timing, amplitude, and duration of a given pulse are controlled. A key component involved in the timing of ecdysone pulses is PTTH, a brain-derived neuropeptide. PTTH stimulates ecdysone production through a Ras/Raf/ERK signaling cascade; however, comparatively little is known about the downstream targets of this pathway. In recent years, it has become apparent that transcriptional regulation plays a critical role in regulating the synthesis of ecdysone, but only one transcription factor has a well-defined link to PTTH. Interestingly, many of the ecdysteroidogenic transcription factors were originally characterized as primary response genes in the ecdysone signaling cascade that elicits the biological responses to the hormone in target tissues. To review these developments, we will first provide an overview of the transcription factors that act in the Drosophila ecdysone regulatory hierarchy. We will then discuss the roles of these transcriptional regulators in controlling ecdysone synthesis. In the last section, we will briefly outline transcription factors that likely have roles in regulating ecdysone synthesis but have not been formally identified as downstream effectors of ecdysone.

PMID: 23347515 [PubMed - in process]

rad001 ecdysone chir-258

Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Related Articles

Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Transfusion. 2012 Nov;52(11):2406-13

Authors: Avanzi MP, Chen A, He W, Mitchell WB

Abstract
BACKGROUND: Large-scale in vitro production of platelets (PLTs) from cord blood stem cells is one goal of stem cell research. One step toward this goal will be to produce polyploid megakaryocytes capable of releasing high numbers of PLTs. Megakaryocyte polyploidization requires distinct cytoskeletal and cellular mechanisms, including actin polymerization, myosin activation, microtubule formation, and increased DNA production. In this study we variably combined inhibition of these principal mechanisms of cytokinesis with the goal of driving polyploidization in megakaryocytes.
STUDY DESIGN AND METHODS: Megakaryocytes were derived from umbilical cord blood and cultured with reagents that inhibit distinct mechanisms of cytokinesis: Rho-Rock inhibitor (RRI), Src inhibitor (SI), nicotinamide (NIC), aurora B inhibitor (ABI), and myosin light chain kinase inhibitor (MLCKI). Combinations of reagents were used to determine their interactions and to maximize megakaryocyte ploidy.
RESULTS: Treatment with RRI, NIC, SI, and ABI, but not with MLCKI, increased the final ploidy and RRI was the most effective single reagent. RRI and MLCKI, both inhibitors of MLC activation, resulted in opposite ploidy outcomes. Combinations of reagents also increased ploidy and the use of NIC, SI, and ABI was as effective as RRI alone. Addition of MLCKI to NIC, SI, and ABI reached the highest level of polyploidization.
CONCLUSION: Megakaryocyte polyploidization results from modulation of a combination of distinct cytokinesis pathways. Reagents targeting distinct cytoskeletal pathways produced additive effects in final megakaryocyte ploidy. The RRI, however, showed no additive effect but produced a high final ploidy due to overlapping inhibition of multiple cytokinesis pathways.

PMID: 22612069 [PubMed - indexed for MEDLINE]

coxinhibitors c-met inhibitors zm-447439

Combinatorial targeting of FGF and ErbB receptors blocks growth and metastatic spread of breast cancer models.

Combinatorial targeting of FGF and ErbB receptors blocks growth and metastatic spread of breast cancer models.

Breast Cancer Res. 2013 Jan 23;15(1):R8

Authors: Issa A, Gill JW, Heideman MR, Sahin O, Wiemann S, Dey JH, Hynes NE

Abstract
ABSTRACT: INTRODUCTION: Targeting receptor tyrosine kinases (RTKs) with kinase inhibitors is a clinically validated anti-cancer approach, however, the effectiveness of individual inhibitors is often short-lived and resistance emerges. Experimental approaches have revealed numerous feed-back loops in tumor cells and that blocking one signaling pathway, be it the receptor or downstream targets, is often not sufficient to cause tumor regression. Alterations in fibroblast growth factor receptor (FGFR) activity have been implicated in breast cancer. Using breast cancer models with autocrine activation of fibroblast growth factor receptors (FGFR), we have examined the impact of targeting FGFRs in vivo with a selective kinase inhibitor in combination with an inhibitor of PI3K/mTOR or with a pan-ErbB inhibitor. METHODS: The 4T1 or 67NR mammary cancer cells are models for basal-like breast cancer and display constitutive FGFR activity. Upon their injection into fat pads of female Balb/c mice both tumor cell lines form tumors; 4T1 tumors, but not 67NR tumors metastasize to lungs. Tumor growth was measured in mice treated with an FGFR inhibitor (dovitinib/TKI258), a PI3K/ mTOR inhibitor (NVP-BEZ235) or with a pan-ErbB inhibitor (AEE788). Inhibitors were administered individually or in combination and lung metastases were quantified in the 4T1 model. To uncover mechanisms underlying inhibitor activity, tumor lysates were examined by western analyses for signaling activity of FGFR/FRS2, ErbB2, the Erk and the PI3K/Akt/mTOR pathways. Tumor sections were examined for proliferation, apoptosis and vessel density using antibodies for P-Histone H3, cleaved Caspase-3 and CD31, respectively. A transcriptome analysis was carried out on tumors treated with dovitinib for different times to identify pathways upregulated by FGFR inhibition. Anti-phosphotyrosine receptor antibody arrays (P-Tyr RTK) were used to screen in an unbiased manner for active receptors in 4T1 tumors. RESULTS: Treatment of 4T1 and 67NR tumor-bearing mice with the combination of dovitinib + NVP-BEZ235 causes tumor stasis. Western analysis of tumor lysates shows strong down-regulation of the FRS2/Erk and PI3K/Akt/mTOR signaling pathways. Examination of tumor sections revealed that the combination treatment results in a significant decrease in proliferation and high numbers of apoptotic cells, in comparison to tumors treated with individual inhibitors. Using P-Tyr RTK arrays, we identified high levels of P-EGFR and P-ErbB2 in 4T1 tumors. Testing AEE788 in the 4T1 and 67NR models revealed that only the combination of dovitinib + AEE788 resulted in blockade of the PI3K/Akt/mTOR pathway, prolonged tumor stasis and in the 4T1 model, a highly significant decrease in lung metastasis. Analyses of the tumor sections revealed that the combination of dovitinib + AEE788 caused a significant decrease in proliferation and high levels of apoptosis. The results show that in vivo these breast cancer models become dependent upon co-activation of FGFR and ErbB receptors for PI3K pathway activity. CONCLUSIONS: The work presented here shows that in the 4T1 and 67NR breast cancer models the combination of dovitinib + NVP-BEZ235 or dovitinib + AEE788 leads to a strong inhibition of tumor growth and, for the 4T1 model, a block in metastatic spread. Only these combinations strongly down-regulate the FGFR/FRS2/Erk and the PI3K/Akt/mTOR signaling pathways and cause high levels of apoptosis. Interestingly, the decrease in mitosis and increase in apoptosis was consistently stronger in the dovitinib + AEE788 treatment-group suggesting that targeting ErbB receptors has broader downstream effects compared to targeting only PI3K/mTOR. In experiments aimed at testing the durability of treatment-response, this combination was also more effective. Considering that sub-classes of human breast tumors co-express ErbB receptors and FGFRs, these results might have implications for targeted therapy.

PMID: 23343422 [PubMed - as supplied by publisher]

dna-pk coxinhibitors c-met inhibitors

Combinatorial targeting of FGF and ErbB receptors blocks growth and metastatic spread of breast cancer models.

Combinatorial targeting of FGF and ErbB receptors blocks growth and metastatic spread of breast cancer models.

Breast Cancer Res. 2013 Jan 23;15(1):R8

Authors: Issa A, Gill JW, Heideman MR, Sahin O, Wiemann S, Dey JH, Hynes NE

Abstract
ABSTRACT: INTRODUCTION: Targeting receptor tyrosine kinases (RTKs) with kinase inhibitors is a clinically validated anti-cancer approach, however, the effectiveness of individual inhibitors is often short-lived and resistance emerges. Experimental approaches have revealed numerous feed-back loops in tumor cells and that blocking one signaling pathway, be it the receptor or downstream targets, is often not sufficient to cause tumor regression. Alterations in fibroblast growth factor receptor (FGFR) activity have been implicated in breast cancer. Using breast cancer models with autocrine activation of fibroblast growth factor receptors (FGFR), we have examined the impact of targeting FGFRs in vivo with a selective kinase inhibitor in combination with an inhibitor of PI3K/mTOR or with a pan-ErbB inhibitor. METHODS: The 4T1 or 67NR mammary cancer cells are models for basal-like breast cancer and display constitutive FGFR activity. Upon their injection into fat pads of female Balb/c mice both tumor cell lines form tumors; 4T1 tumors, but not 67NR tumors metastasize to lungs. Tumor growth was measured in mice treated with an FGFR inhibitor (dovitinib/TKI258), a PI3K/ mTOR inhibitor (NVP-BEZ235) or with a pan-ErbB inhibitor (AEE788). Inhibitors were administered individually or in combination and lung metastases were quantified in the 4T1 model. To uncover mechanisms underlying inhibitor activity, tumor lysates were examined by western analyses for signaling activity of FGFR/FRS2, ErbB2, the Erk and the PI3K/Akt/mTOR pathways. Tumor sections were examined for proliferation, apoptosis and vessel density using antibodies for P-Histone H3, cleaved Caspase-3 and CD31, respectively. A transcriptome analysis was carried out on tumors treated with dovitinib for different times to identify pathways upregulated by FGFR inhibition. Anti-phosphotyrosine receptor antibody arrays (P-Tyr RTK) were used to screen in an unbiased manner for active receptors in 4T1 tumors. RESULTS: Treatment of 4T1 and 67NR tumor-bearing mice with the combination of dovitinib + NVP-BEZ235 causes tumor stasis. Western analysis of tumor lysates shows strong down-regulation of the FRS2/Erk and PI3K/Akt/mTOR signaling pathways. Examination of tumor sections revealed that the combination treatment results in a significant decrease in proliferation and high numbers of apoptotic cells, in comparison to tumors treated with individual inhibitors. Using P-Tyr RTK arrays, we identified high levels of P-EGFR and P-ErbB2 in 4T1 tumors. Testing AEE788 in the 4T1 and 67NR models revealed that only the combination of dovitinib + AEE788 resulted in blockade of the PI3K/Akt/mTOR pathway, prolonged tumor stasis and in the 4T1 model, a highly significant decrease in lung metastasis. Analyses of the tumor sections revealed that the combination of dovitinib + AEE788 caused a significant decrease in proliferation and high levels of apoptosis. The results show that in vivo these breast cancer models become dependent upon co-activation of FGFR and ErbB receptors for PI3K pathway activity. CONCLUSIONS: The work presented here shows that in the 4T1 and 67NR breast cancer models the combination of dovitinib + NVP-BEZ235 or dovitinib + AEE788 leads to a strong inhibition of tumor growth and, for the 4T1 model, a block in metastatic spread. Only these combinations strongly down-regulate the FGFR/FRS2/Erk and the PI3K/Akt/mTOR signaling pathways and cause high levels of apoptosis. Interestingly, the decrease in mitosis and increase in apoptosis was consistently stronger in the dovitinib + AEE788 treatment-group suggesting that targeting ErbB receptors has broader downstream effects compared to targeting only PI3K/mTOR. In experiments aimed at testing the durability of treatment-response, this combination was also more effective. Considering that sub-classes of human breast tumors co-express ErbB receptors and FGFRs, these results might have implications for targeted therapy.

PMID: 23343422 [PubMed - as supplied by publisher]

ecdysone chir-258 dovitinib

What goes up must come down: transcription factors have their say in making ecdysone pulses.

What goes up must come down: transcription factors have their say in making ecdysone pulses.

Curr Top Dev Biol. 2013;103:35-71

Authors: Ou Q, King-Jones K

Abstract
Insect metamorphosis is one of the most fascinating biological processes in the animal kingdom. The dramatic transition from an immature juvenile to a reproductive adult is under the control of the steroid hormone ecdysone, also known as the insect molting hormone. During Drosophila development, periodic pulses of ecdysone are released from the prothoracic glands, upon which the hormone is rapidly converted in peripheral tissues to its biologically active form, 20-hydroxyecdysone. Each hormone pulse has a unique profile and causes different developmental events, but we only have a rudimentary understanding of how the timing, amplitude, and duration of a given pulse are controlled. A key component involved in the timing of ecdysone pulses is PTTH, a brain-derived neuropeptide. PTTH stimulates ecdysone production through a Ras/Raf/ERK signaling cascade; however, comparatively little is known about the downstream targets of this pathway. In recent years, it has become apparent that transcriptional regulation plays a critical role in regulating the synthesis of ecdysone, but only one transcription factor has a well-defined link to PTTH. Interestingly, many of the ecdysteroidogenic transcription factors were originally characterized as primary response genes in the ecdysone signaling cascade that elicits the biological responses to the hormone in target tissues. To review these developments, we will first provide an overview of the transcription factors that act in the Drosophila ecdysone regulatory hierarchy. We will then discuss the roles of these transcriptional regulators in controlling ecdysone synthesis. In the last section, we will briefly outline transcription factors that likely have roles in regulating ecdysone synthesis but have not been formally identified as downstream effectors of ecdysone.

PMID: 23347515 [PubMed - in process]

coxinhibitors c-met inhibitors zm-447439

Impact of Epidermal Growth Factor Receptor and KRAS Mutations on Clinical Outcome in Resected Non-Small Cell Lung Cancer Patients.

Impact of Epidermal Growth Factor Receptor and KRAS Mutations on Clinical Outcome in Resected Non-Small Cell Lung Cancer Patients.

Am J Clin Oncol. 2013 Jan 24;

Authors: Ragusa M, Vannucci J, Ludovini V, Bianconi F, Treggiari S, Tofanetti FR, Flacco A, Colella R, Sidoni A, Crin� L, Puma F

Abstract
OBJECTIVES:: Surgery yields best results for non-small cell lung cancer (NSCLC) patients. Epidermal growth factor receptor (EGFR) and its downstream factor Kirsten rat sarcoma viral oncogene homolog (KRAS) are variably mutated in NSCLC. Such mutations predict clinical response to tyrosine kinase inhibitors. This study evaluated incidence and correlation of EGFR and KRAS mutations with clinicopathologic parameters and outcome in resected stage I to III NSCLC. METHODS:: We analyzed the clinical characteristics and outcome data for 230 patients who underwent resection at our institution for stage I to III NSCLC. The tumors were assessed for both EGFR (exons 18 to 21) and KRAS (exons 2 and 3) mutations by nested polymerase chain reaction and sequenced in both sense and antisense direction. Kaplan-Meier estimates of overall survival and disease-free survival were calculated for clinical and biological variables using Cox model. RESULTS:: EGFR and KRAS mutations were detected in 22 (9.6%) and 39 (16.9%) patients, respectively. In the whole population, both EGFR and KRAS mutations were significantly correlated with adenocarcinoma (ADC). Overall, EGFR mutations were more frequent in women (P<0.0001) and in nonsmokers (P<0.0001). In the ADC/BAC group, KRAS mutations were more frequent in man (P<0.02) and EGFR mutations (exon 19 deletion and L858R) demonstrated a tendency towards worse disease-free survival (P=0.056). No difference in outcome was seen between patients harboring KRAS mutations compared with KRAS wild type. CONCLUSIONS:: EGFR and KRAS mutations are frequent in ADCs and are not prognostic factors for survival. EGFR mutations could be used to identify patients suitable for adjuvant treatment with targeted therapy resulting in potentially improved outcomes.

PMID: 23357969 [PubMed - as supplied by publisher]

ecdysone chir-258 dovitinib

Warburg effect and translocation-induced genomic instability: two yeast models for cancer cells.

Warburg effect and translocation-induced genomic instability: two yeast models for cancer cells.

Front Oncol. 2012;2:212

Authors: Tosato V, Gr�ning NM, Breitenbach M, Arnak R, Ralser M, Bruschi CV

Abstract
Yeast has been established as an efficient model system to study biological principles underpinning human health. In this review we focus on yeast models covering two aspects of cancer formation and progression (i) the activity of pyruvate kinase (PK), which recapitulates metabolic features of cancer cells, including the Warburg effect, and (ii) chromosome bridge-induced translocation (BIT) mimiking genome instability in cancer. Saccharomyces cerevisiae is an excellent model to study cancer cell metabolism, as exponentially growing yeast cells exhibit many metabolic similarities with rapidly proliferating cancer cells. The metabolic reconfiguration includes an increase in glucose uptake and fermentation, at the expense of respiration and oxidative phosphorylation (the Warburg effect), and involves a broad reconfiguration of nucleotide and amino acid metabolism. Both in yeast and humans, the regulation of this process seems to have a central player, PK, which is up-regulated in cancer, and to occur mostly on a post-transcriptional and post-translational basis. Furthermore, BIT allows to generate selectable translocation-derived recombinants ("translocants"), between any two desired chromosomal locations, in wild-type yeast strains transformed with a linear DNA cassette carrying a selectable marker flanked by two DNA sequences homologous to different chromosomes. Using the BIT system, targeted non-reciprocal translocations in mitosis are easily inducible. An extensive collection of different yeast translocants exhibiting genome instability and aberrant phenotypes similar to cancer cells has been produced and subjected to analysis. In this review, we hence provide an overview upon two yeast cancer models, and extrapolate general principles for mimicking human disease mechanisms in yeast.

PMID: 23346549 [PubMed - in process]

chir-258 dovitinib dna-pk

Inhibition of pancreatic intraepithelial neoplasia progression to carcinoma by nitric oxide-releasing aspirin in p48(Cre/+)-LSL-Kras(G12D/+) mice.

Related Articles

Inhibition of pancreatic intraepithelial neoplasia progression to carcinoma by nitric oxide-releasing aspirin in p48(Cre/+)-LSL-Kras(G12D/+) mice.

Neoplasia. 2012 Sep;14(9):778-87

Authors: Rao CV, Mohammed A, Janakiram NB, Li Q, Ritchie RL, Lightfoot S, Vibhudutta A, Steele VE

Abstract
Nitric oxide-releasing aspirin (NO-aspirin) represents a novel class of promising chemopreventive agents. Unlike conventional nonsteroidal anti-inflammatory drugs, NO-aspirin seems to be free of adverse effects while retaining the beneficial activities of its parent compound. The effect of NO-aspirin on pancreatic carcinogenesis was investigated by assessing the development of precursor pancreatic lesions and adenocarcinomas in Kras(G12D/+) transgenic mice that recapitulate human pancreatic cancer progression. Six-week-old male p48(Cre/+)-LSL-Kras(G12D/+) transgenic mice (20 per group) were fed diets containing 0, 1000, or 2000 ppm NO-aspirin. The development of pancreatic tumors was monitored by positron emission tomography imaging. All mice were killed at the age of 41 weeks and assessed for pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC) and for molecular changes in the tumors. Our results reveal that NO-aspirin at 1000 and 2000 ppm significantly suppressed pancreatic tumor weights, PDAC incidence, and carcinoma in situ (PanIN-3 lesions). The degree of inhibition of PanIN-3 and carcinoma was more pronounced with NO-aspirin at 1000 ppm (58.8% and 48%, respectively) than with 2000 ppm (47% and 20%, respectively). NO-aspirin at 1000 ppm significantly inhibited the spread of carcinoma in the pancreas (?97%; P < .0001). Decreased expression of cyclooxygenase (COX; with ?42% inhibition of total COX activity), inducible nitric oxide synthase, proliferating cell nuclear antigen, Bcl-2, cyclin D1, and ?-catenin was observed, with induction of p21, p38, and p53 in the pancreas of NO-aspirin-treated mice. These results suggest that low-dose NO-aspirin possesses inhibitory activity against pancreatic carcinogenesis by modulating multiple molecular targets.

PMID: 23019409 [PubMed - indexed for MEDLINE]

chir-258 dovitinib dna-pk

Mitochondrial Aldehyde Dehydrogenase Obliterates Endoplasmic Reticulum Stress-Induced Cardiac Contractile Dysfunction via Correction of Autophagy.

Related Articles

Mitochondrial Aldehyde Dehydrogenase Obliterates Endoplasmic Reticulum Stress-Induced Cardiac Contractile Dysfunction via Correction of Autophagy.

Biochim Biophys Acta. 2013 Jan 23;

Authors: Zhang B, Zhang Y, La Cour KH, Richmond KL, Wang X, Ren J

Abstract
ER stress triggers myocardial contractile dysfunction while effective therapeutic regimen is still lacking. Mitochondrial aldehyde dehydrogenase (ALDH2), an essential mitochondrial enzyme governing mitochondrial and cardiac function, displays distinct beneficial effect on the heart. This study was designed to evaluate the effect of ALDH2 on ER stress-induced cardiac anomalies and the underlying mechanism involved with a special focus on autophagy. WT and ALDH2 transgenic mice were subjected to the ER stress inducer thapsigargin (1mg/kg, i.p., 48hrs). Echocardiographic, cardiomyocyte contractile and intracellular Ca(2+) properties as well as myocardial histology, autophagy and autophagy regulatory proteins were evaluated. ER stress led to compromised echocardiographic indices (elevated LVESD, reduced fractional shortening and cardiac output), cardiomyocyte contractile and intracellular Ca(2+) properties and cell survival, associated with upregulated autophagy, dampened phosphorylation of Akt and its downstream signal molecules TSC2 and mTOR, the effects of which were alleviated or mitigated by ALDH2. Thapsigargin promoted ER stress proteins Gadd153 and GRP78 without altering cardiomyocyte size and interstitial fibrosis, the effects of which were unaffected by ALDH2. Treatment with thapsigargin in vitro mimicked in vivo ER stress-induced cardiomyocyte contractile anomalies including depressed peak shortening and maximal velocity of shortening/relengthening as well as prolonged relengthening duration, the effect of which was abrogated by the autophagy inhibitor 3-methyladenine and the ALDH2 activator Alda-1. Interestingly, Alda-1-induced beneficial effect against ER stress was obliterated by autophagy inducer rapamycin, Akt inhibitor AktI and mTOR inhibitor RAD001. These data suggest a beneficial role of ALDH2 against ER stress-induced cardiac anomalies possibly through autophagy reduction.

PMID: 23354068 [PubMed - as supplied by publisher]

chir-258 dovitinib dna-pk

Combinatorial targeting of FGF and ErbB receptors blocks growth and metastatic spread of breast cancer models.

Combinatorial targeting of FGF and ErbB receptors blocks growth and metastatic spread of breast cancer models.

Breast Cancer Res. 2013 Jan 23;15(1):R8

Authors: Issa A, Gill JW, Heideman MR, Sahin O, Wiemann S, Dey JH, Hynes NE

Abstract
ABSTRACT: INTRODUCTION: Targeting receptor tyrosine kinases (RTKs) with kinase inhibitors is a clinically validated anti-cancer approach, however, the effectiveness of individual inhibitors is often short-lived and resistance emerges. Experimental approaches have revealed numerous feed-back loops in tumor cells and that blocking one signaling pathway, be it the receptor or downstream targets, is often not sufficient to cause tumor regression. Alterations in fibroblast growth factor receptor (FGFR) activity have been implicated in breast cancer. Using breast cancer models with autocrine activation of fibroblast growth factor receptors (FGFR), we have examined the impact of targeting FGFRs in vivo with a selective kinase inhibitor in combination with an inhibitor of PI3K/mTOR or with a pan-ErbB inhibitor. METHODS: The 4T1 or 67NR mammary cancer cells are models for basal-like breast cancer and display constitutive FGFR activity. Upon their injection into fat pads of female Balb/c mice both tumor cell lines form tumors; 4T1 tumors, but not 67NR tumors metastasize to lungs. Tumor growth was measured in mice treated with an FGFR inhibitor (dovitinib/TKI258), a PI3K/ mTOR inhibitor (NVP-BEZ235) or with a pan-ErbB inhibitor (AEE788). Inhibitors were administered individually or in combination and lung metastases were quantified in the 4T1 model. To uncover mechanisms underlying inhibitor activity, tumor lysates were examined by western analyses for signaling activity of FGFR/FRS2, ErbB2, the Erk and the PI3K/Akt/mTOR pathways. Tumor sections were examined for proliferation, apoptosis and vessel density using antibodies for P-Histone H3, cleaved Caspase-3 and CD31, respectively. A transcriptome analysis was carried out on tumors treated with dovitinib for different times to identify pathways upregulated by FGFR inhibition. Anti-phosphotyrosine receptor antibody arrays (P-Tyr RTK) were used to screen in an unbiased manner for active receptors in 4T1 tumors. RESULTS: Treatment of 4T1 and 67NR tumor-bearing mice with the combination of dovitinib + NVP-BEZ235 causes tumor stasis. Western analysis of tumor lysates shows strong down-regulation of the FRS2/Erk and PI3K/Akt/mTOR signaling pathways. Examination of tumor sections revealed that the combination treatment results in a significant decrease in proliferation and high numbers of apoptotic cells, in comparison to tumors treated with individual inhibitors. Using P-Tyr RTK arrays, we identified high levels of P-EGFR and P-ErbB2 in 4T1 tumors. Testing AEE788 in the 4T1 and 67NR models revealed that only the combination of dovitinib + AEE788 resulted in blockade of the PI3K/Akt/mTOR pathway, prolonged tumor stasis and in the 4T1 model, a highly significant decrease in lung metastasis. Analyses of the tumor sections revealed that the combination of dovitinib + AEE788 caused a significant decrease in proliferation and high levels of apoptosis. The results show that in vivo these breast cancer models become dependent upon co-activation of FGFR and ErbB receptors for PI3K pathway activity. CONCLUSIONS: The work presented here shows that in the 4T1 and 67NR breast cancer models the combination of dovitinib + NVP-BEZ235 or dovitinib + AEE788 leads to a strong inhibition of tumor growth and, for the 4T1 model, a block in metastatic spread. Only these combinations strongly down-regulate the FGFR/FRS2/Erk and the PI3K/Akt/mTOR signaling pathways and cause high levels of apoptosis. Interestingly, the decrease in mitosis and increase in apoptosis was consistently stronger in the dovitinib + AEE788 treatment-group suggesting that targeting ErbB receptors has broader downstream effects compared to targeting only PI3K/mTOR. In experiments aimed at testing the durability of treatment-response, this combination was also more effective. Considering that sub-classes of human breast tumors co-express ErbB receptors and FGFRs, these results might have implications for targeted therapy.

PMID: 23343422 [PubMed - as supplied by publisher]

c-met inhibitors zm-447439 rad001

A Phase II Study of Bevacizumab and Everolimus as Treatment for Refractory Metastatic Renal Cell Carcinoma.

Related Articles

A Phase II Study of Bevacizumab and Everolimus as Treatment for Refractory Metastatic Renal Cell Carcinoma.

Clin Genitourin Cancer. 2013 Jan 24;

Authors: Harshman LC, Barbeau S, McMillian A, Srinivas S

Abstract
BACKGROUND: Agents that inhibit the vascular endothelial growth factor (VEGF) and mammalian target of rapamycin (mTOR) pathways in metastatic renal cell carcinoma (mRCC) prolong progression-free survival (PFS), but durable complete responses are rare. Combinations of these cytostatic therapies have great potential to improve efficacy and to escape tumoral resistance mechanisms, but supra-additive toxicity is a valid concern. We investigated whether horizontal blockade with the combination of bevacizumab, a monoclonal antibody to VEGF-A, and of everolimus, an oral mTOR inhibitor, improved PFS in patients with clear cell mRCC who had received prior VEGF blockade. METHODS: In this phase II investigator-initiated study, 10 of 30 planned patients were enrolled. Bevacizumab 10 mg/kg was administered intravenously every 14 days. Everolimus was orally dosed at 10 mg daily. The patients were treated until disease progression or unacceptable toxicity. The primary endpoint was PFS. RESULTS: The median age was 55 years. The majority of patients were white men with an Eastern Cooperative Oncology Group performance status of 1 (80%) and intermediate risk disease by Memorial Sloan-Kettering Cancer Center criteria (70%). All the patients had received 1 prior VEGF inhibitor. The median PFS in the 10 evaluable patients was 5.1 months, which was less than the expected historical control of bevacizumab monotherapy at 6 months. The median overall survival was 21 months. The best response was a partial response in 1 patient and stable disease in 9. Forty percent of the patients were discontinued from the study due to toxicity. CONCLUSIONS: In our experience, the combination of bevacizumab and everolimus was toxic. The efficacy achieved did not support its combined use over sequential administration. Ongoing randomized studies will definitively evaluate the combination's efficacy and tolerability.

PMID: 23352238 [PubMed - as supplied by publisher]

zm-447439 rad001 ecdysone

Developmental checkpoints and feedback circuits time insect maturation.

Developmental checkpoints and feedback circuits time insect maturation.

Curr Top Dev Biol. 2013;103:1-33

Authors: Rewitz KF, Yamanaka N, O'Connor MB

Abstract
The transition from juvenile to adult is a fundamental process that allows animals to allocate resource toward reproduction after completing a certain amount of growth. In insects, growth to a species-specific target size induces pulses of the steroid hormone ecdysone that triggers metamorphosis and reproductive maturation. The past few years have seen significant progress in understanding the interplay of mechanisms that coordinate timing of ecdysone production and release. These studies show that the neuroendocrine system monitors complex size-related and nutritional signals, as well as external cues, to time production and release of ecdysone. Based on results discussed here, we suggest that developmental progression to adulthood is controlled by checkpoints that regulate the genetic timing program enabling it to adapt to different environmental conditions. These checkpoints utilize a number of signaling pathways to modulate ecdysone production in the prothoracic gland. Release of ecdysone activates an autonomous cascade of both feedforward and feedback signals that determine the duration of the ecdysone pulse at each developmental transitions. Conservation of the genetic mechanisms that coordinate the juvenile-adult transition suggests that insights from the fruit fly Drosophila will provide a framework for future investigation of developmental timing in metazoans.

PMID: 23347514 [PubMed - in process]

chir-258 dovitinib dna-pk

Warburg effect and translocation-induced genomic instability: two yeast models for cancer cells.

Warburg effect and translocation-induced genomic instability: two yeast models for cancer cells.

Front Oncol. 2012;2:212

Authors: Tosato V, Gr�ning NM, Breitenbach M, Arnak R, Ralser M, Bruschi CV

Abstract
Yeast has been established as an efficient model system to study biological principles underpinning human health. In this review we focus on yeast models covering two aspects of cancer formation and progression (i) the activity of pyruvate kinase (PK), which recapitulates metabolic features of cancer cells, including the Warburg effect, and (ii) chromosome bridge-induced translocation (BIT) mimiking genome instability in cancer. Saccharomyces cerevisiae is an excellent model to study cancer cell metabolism, as exponentially growing yeast cells exhibit many metabolic similarities with rapidly proliferating cancer cells. The metabolic reconfiguration includes an increase in glucose uptake and fermentation, at the expense of respiration and oxidative phosphorylation (the Warburg effect), and involves a broad reconfiguration of nucleotide and amino acid metabolism. Both in yeast and humans, the regulation of this process seems to have a central player, PK, which is up-regulated in cancer, and to occur mostly on a post-transcriptional and post-translational basis. Furthermore, BIT allows to generate selectable translocation-derived recombinants ("translocants"), between any two desired chromosomal locations, in wild-type yeast strains transformed with a linear DNA cassette carrying a selectable marker flanked by two DNA sequences homologous to different chromosomes. Using the BIT system, targeted non-reciprocal translocations in mitosis are easily inducible. An extensive collection of different yeast translocants exhibiting genome instability and aberrant phenotypes similar to cancer cells has been produced and subjected to analysis. In this review, we hence provide an overview upon two yeast cancer models, and extrapolate general principles for mimicking human disease mechanisms in yeast.

PMID: 23346549 [PubMed - in process]

dovitinib dna-pk coxinhibitors

Warburg effect and translocation-induced genomic instability: two yeast models for cancer cells.

Warburg effect and translocation-induced genomic instability: two yeast models for cancer cells.

Front Oncol. 2012;2:212

Authors: Tosato V, Gr�ning NM, Breitenbach M, Arnak R, Ralser M, Bruschi CV

Abstract
Yeast has been established as an efficient model system to study biological principles underpinning human health. In this review we focus on yeast models covering two aspects of cancer formation and progression (i) the activity of pyruvate kinase (PK), which recapitulates metabolic features of cancer cells, including the Warburg effect, and (ii) chromosome bridge-induced translocation (BIT) mimiking genome instability in cancer. Saccharomyces cerevisiae is an excellent model to study cancer cell metabolism, as exponentially growing yeast cells exhibit many metabolic similarities with rapidly proliferating cancer cells. The metabolic reconfiguration includes an increase in glucose uptake and fermentation, at the expense of respiration and oxidative phosphorylation (the Warburg effect), and involves a broad reconfiguration of nucleotide and amino acid metabolism. Both in yeast and humans, the regulation of this process seems to have a central player, PK, which is up-regulated in cancer, and to occur mostly on a post-transcriptional and post-translational basis. Furthermore, BIT allows to generate selectable translocation-derived recombinants ("translocants"), between any two desired chromosomal locations, in wild-type yeast strains transformed with a linear DNA cassette carrying a selectable marker flanked by two DNA sequences homologous to different chromosomes. Using the BIT system, targeted non-reciprocal translocations in mitosis are easily inducible. An extensive collection of different yeast translocants exhibiting genome instability and aberrant phenotypes similar to cancer cells has been produced and subjected to analysis. In this review, we hence provide an overview upon two yeast cancer models, and extrapolate general principles for mimicking human disease mechanisms in yeast.

PMID: 23346549 [PubMed - in process]

c-met inhibitors zm-447439 rad001

Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Related Articles

Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Transfusion. 2012 Nov;52(11):2406-13

Authors: Avanzi MP, Chen A, He W, Mitchell WB

Abstract
BACKGROUND: Large-scale in vitro production of platelets (PLTs) from cord blood stem cells is one goal of stem cell research. One step toward this goal will be to produce polyploid megakaryocytes capable of releasing high numbers of PLTs. Megakaryocyte polyploidization requires distinct cytoskeletal and cellular mechanisms, including actin polymerization, myosin activation, microtubule formation, and increased DNA production. In this study we variably combined inhibition of these principal mechanisms of cytokinesis with the goal of driving polyploidization in megakaryocytes.
STUDY DESIGN AND METHODS: Megakaryocytes were derived from umbilical cord blood and cultured with reagents that inhibit distinct mechanisms of cytokinesis: Rho-Rock inhibitor (RRI), Src inhibitor (SI), nicotinamide (NIC), aurora B inhibitor (ABI), and myosin light chain kinase inhibitor (MLCKI). Combinations of reagents were used to determine their interactions and to maximize megakaryocyte ploidy.
RESULTS: Treatment with RRI, NIC, SI, and ABI, but not with MLCKI, increased the final ploidy and RRI was the most effective single reagent. RRI and MLCKI, both inhibitors of MLC activation, resulted in opposite ploidy outcomes. Combinations of reagents also increased ploidy and the use of NIC, SI, and ABI was as effective as RRI alone. Addition of MLCKI to NIC, SI, and ABI reached the highest level of polyploidization.
CONCLUSION: Megakaryocyte polyploidization results from modulation of a combination of distinct cytokinesis pathways. Reagents targeting distinct cytoskeletal pathways produced additive effects in final megakaryocyte ploidy. The RRI, however, showed no additive effect but produced a high final ploidy due to overlapping inhibition of multiple cytokinesis pathways.

PMID: 22612069 [PubMed - indexed for MEDLINE]

zm-447439 rad001

Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Related Articles

Optimizing megakaryocyte polyploidization by targeting multiple pathways of cytokinesis.

Transfusion. 2012 Nov;52(11):2406-13

Authors: Avanzi MP, Chen A, He W, Mitchell WB

Abstract
BACKGROUND: Large-scale in vitro production of platelets (PLTs) from cord blood stem cells is one goal of stem cell research. One step toward this goal will be to produce polyploid megakaryocytes capable of releasing high numbers of PLTs. Megakaryocyte polyploidization requires distinct cytoskeletal and cellular mechanisms, including actin polymerization, myosin activation, microtubule formation, and increased DNA production. In this study we variably combined inhibition of these principal mechanisms of cytokinesis with the goal of driving polyploidization in megakaryocytes.
STUDY DESIGN AND METHODS: Megakaryocytes were derived from umbilical cord blood and cultured with reagents that inhibit distinct mechanisms of cytokinesis: Rho-Rock inhibitor (RRI), Src inhibitor (SI), nicotinamide (NIC), aurora B inhibitor (ABI), and myosin light chain kinase inhibitor (MLCKI). Combinations of reagents were used to determine their interactions and to maximize megakaryocyte ploidy.
RESULTS: Treatment with RRI, NIC, SI, and ABI, but not with MLCKI, increased the final ploidy and RRI was the most effective single reagent. RRI and MLCKI, both inhibitors of MLC activation, resulted in opposite ploidy outcomes. Combinations of reagents also increased ploidy and the use of NIC, SI, and ABI was as effective as RRI alone. Addition of MLCKI to NIC, SI, and ABI reached the highest level of polyploidization.
CONCLUSION: Megakaryocyte polyploidization results from modulation of a combination of distinct cytokinesis pathways. Reagents targeting distinct cytoskeletal pathways produced additive effects in final megakaryocyte ploidy. The RRI, however, showed no additive effect but produced a high final ploidy due to overlapping inhibition of multiple cytokinesis pathways.

PMID: 22612069 [PubMed - indexed for MEDLINE]

dna-pk coxinhibitors c-met inhibitors

Enhanced NF?B Activity Impairs Vascular Function through PARP-1, SP-1 and COX2-Dependent Mechanisms in Type 2 Diabetes.

Enhanced NF?B Activity Impairs Vascular Function through PARP-1, SP-1 and COX2-Dependent Mechanisms in Type 2 Diabetes.

Diabetes. 2013 Jan 24;

Authors: Kassan M, Choi SK, Galan M, Bishop A, Umezawa K, Trebak M, Belmadani S, Matrougui K

Abstract
Type 2 diabetes (T2D) is associated with vascular dysfunction. We hypothesized that increased nuclear factor kappa B (NF?B) signaling contributes to vascular dysfunction in T2D. We have treated type 2 diabetic (db(-)/db(-)) and control (db(-)/db(+)) mice with two NF?B inhibitors (DHMEQ, 6mg/kg, twice a week and IKK-NBD peptide, 500 ?g/kg/day) for four weeks. Pressure-induced myogenic tone (MT) was significantly potentiated, while endothelium dependent relaxation (EDR) was impaired in small coronary arterioles (CA) and mesenteric resistance artery (MRA) from diabetic mice compared to control. Interestingly, diabetic mice treated with NF?B inhibitors significantly reduced MT potentiation and improved EDR. Importantly, vascular function was also rescued in db(-)/db(-p50NF?B-/-) and db(-)/db(-PARP-1-/-) double knockout mice compared to db(-)/db(-) mice. Additionally, the acute in vitro down regulation of NF?B-p65 using p65NF?B shRNA lentivirus in arteries from db(-)/db(-) mice also improved vascular function. The NF?B inhibition did not affect blood glucose level and body weight. The RNA levels for Sp-1 and eNOS phosphorylation were decreased, while p65NF?B phosphorylation, cleaved PARP-1 and COX-2 expression were increased in arteries from diabetic mice, which were restored after NF?B inhibition and in db(-)/db(-p50NF?B-/-) and db(-)/db(-PARP-1-/-) mice.In the present study, we provided evidence that enhanced NF?B activity impairs vascular function by PARP-1, Sp-1 and COX-2-dependent mechanisms in male type 2 diabetic mice. Therefore, NF?B could be a potential target to overcome diabetes-induced vascular dysfunction.

PMID: 23349490 [PubMed - as supplied by publisher]

coxinhibitors c-met inhibitors zm-447439

The Blocking of c-Met Signaling Induces Apoptosis through the Increase of p53 Protein in Lung Cancer.

The Blocking of c-Met Signaling Induces Apoptosis through the Increase of p53 Protein in Lung Cancer.

Cancer Res Treat. 2012 Dec;44(4):251-61

Authors: Jung HY, Joo HJ, Park JK, Kim YH

Abstract
PURPOSE: c-Met is an attractive potential target for novel therapeutic inhibition of human cancer, and c-Met tyrosine kinase inhibitors (TKIs) are effective growth inhibitors of various malignancies. However, their mechanisms in anticancer effects are not clear. In the present study, we investigated the possibility that blocking c-Met signaling induces p53-mediated growth inhibition in lung cancer.
MATERIALS AND METHODS: The growth inhibitory effects of c-Met TKI (SU11274) on lung cancer cells and a xenograft model were assessed using the MTT assay, flow cytometry, and terminal deoxyribonucleotide transferase-mediated nick-end labeling staining. The role of p53 protein in the sensitivity of c-Met TKI (SU11274) was examined by Western blot analysis and immunohistochemistry.
RESULTS: SU11274 significantly induced apoptosis in A549 cells with wild-type p53, compared with that in Calu-1 cells with null-type p53. SU11274 increased p53 protein by enhancing the stability of p53 protein. Increased p53 protein by SU11274 induced up-regulation of Bax and PUMA expression and down-regulation of Bcl-2 expression, subsequently activating caspase 3. In p53 knock-out and knock-in systems, we confirmed that SU11274 caused apoptosis through the p53-mediated apoptotic pathway. Likewise, in the A549 xenograft model, SU11274 effectively shrank tumor volume and induced apoptosis via increased p53 protein expression. Blocking c-Met signaling increased the level of p53 protein.
CONCLUSION: Our finding suggested that p53 plays an important role in SU11274-induced apoptosis, and p53 status seems to be related to the sensitivity to SU11274 in lung cancer.

PMID: 23341789 [PubMed - in process]

rad001 ecdysone chir-258

Developmental checkpoints and feedback circuits time insect maturation.

Developmental checkpoints and feedback circuits time insect maturation.

Curr Top Dev Biol. 2013;103:1-33

Authors: Rewitz KF, Yamanaka N, O'Connor MB

Abstract
The transition from juvenile to adult is a fundamental process that allows animals to allocate resource toward reproduction after completing a certain amount of growth. In insects, growth to a species-specific target size induces pulses of the steroid hormone ecdysone that triggers metamorphosis and reproductive maturation. The past few years have seen significant progress in understanding the interplay of mechanisms that coordinate timing of ecdysone production and release. These studies show that the neuroendocrine system monitors complex size-related and nutritional signals, as well as external cues, to time production and release of ecdysone. Based on results discussed here, we suggest that developmental progression to adulthood is controlled by checkpoints that regulate the genetic timing program enabling it to adapt to different environmental conditions. These checkpoints utilize a number of signaling pathways to modulate ecdysone production in the prothoracic gland. Release of ecdysone activates an autonomous cascade of both feedforward and feedback signals that determine the duration of the ecdysone pulse at each developmental transitions. Conservation of the genetic mechanisms that coordinate the juvenile-adult transition suggests that insights from the fruit fly Drosophila will provide a framework for future investigation of developmental timing in metazoans.

PMID: 23347514 [PubMed - in process]

chir-258 dovitinib dna-pk

Exposure to Extremely Low-Frequency Electromagnetic Fields Modulates Na(+) Currents in Rat Cerebellar Granule Cells through Increase of AA/PGE(2) and EP Receptor-Mediated cAMP/PKA Pathway.

Exposure to Extremely Low-Frequency Electromagnetic Fields Modulates Na(+) Currents in Rat Cerebellar Granule Cells through Increase of AA/PGE(2) and EP Receptor-Mediated cAMP/PKA Pathway.

PLoS One. 2013;8(1):e54376

Authors: He YL, Liu DD, Fang YJ, Zhan XQ, Yao JJ, Mei YA

Abstract
Although the modulation of Ca(2+) channel activity by extremely low-frequency electromagnetic fields (ELF-EMF) has been studied previously, few reports have addressed the effects of such fields on the activity of voltage-activated Na(+) channels (Na(v)). Here, we investigated the effects of ELF-EMF on Na(v) activity in rat cerebellar granule cells (GCs). Our results reveal that exposing cerebellar GCs to ELF-EMF for 10-60 min significantly increased Na(v) currents (I(Na)) by 30-125% in a time- and intensity-dependent manner. The Na(v) channel steady-state activation curve, but not the steady-state inactivation curve, was significantly shifted (by 5.2 mV) towards hyperpolarization by ELF-EMF stimulation. This phenomenon is similar to the effect of intracellular application of arachidonic acid (AA) and prostaglandin E(2) (PGE(2)) on I(Na) in cerebellar GCs. Increases in intracellular AA, PGE(2) and phosphorylated PKA levels in cerebellar GCs were observed following ELF-EMF exposure. Western blottings indicated that the Na(V) 1.2 protein on the cerebellar GCs membrane was increased, the total expression levels of Na(V) 1.2 protein were not affected after exposure to ELF-EMF. Cyclooxygenase inhibitors and PGE(2) receptor (EP) antagonists were able to eliminate this ELF-EMF-induced increase in phosphorylated PKA and I(Na). In addition, ELF-EMF exposure significantly enhanced the activity of PLA(2) in cerebellar GCs but did not affect COX-1 or COX-2 activity. Together, these data demonstrate for the first time that neuronal I(Na) is significantly increased by ELF-EMF exposure via a cPLA2 AA PGE(2) EP receptors PKA signaling pathway.

PMID: 23349866 [PubMed - in process]

dna-pk coxinhibitors c-met inhibitors

A Comparison of Ku0063794, a Dual mTORC1 and mTORC2 Inhibitor, and Temsirolimus in Preclinical Renal Cell Carcinoma Models.

A Comparison of Ku0063794, a Dual mTORC1 and mTORC2 Inhibitor, and Temsirolimus in Preclinical Renal Cell Carcinoma Models.

PLoS One. 2013;8(1):e54918

Authors: Zhang H, Berel D, Wang Y, Li P, Bhowmick NA, Figlin RA, Kim HL

Abstract
Rapamycin analogs, temsirolimus and everolimus, are approved for the treatment of advance renal cell carcinoma (RCC). Currently approved agents inhibit mechanistic target of rapamycin (mTOR) complex 1 (mTORC1). However, the mTOR kinase exists in two distinct multiprotein complexes, mTORC1 and mTORC2, and both complexes may be critical regulators of cell metabolism, growth and proliferation. Furthermore, it has been proposed that drug resistance develops due to compensatory activation of mTORC2 signaling during treatment with temsirolimus or everolimus. We evaluated Ku0063794, which is a small molecule that inhibits both mTOR complexes. Ku0063794 was compared to temsirolimus in preclinical models for renal cell carcinoma. Ku0063794 was effective in inhibiting the phosphorylation of signaling proteins downstream of both mTORC1 and mTORC2, including p70 S6K, 4E-BP1 and Akt. Ku0063794 was more effective than temsirolimus in decreasing the viability and growth of RCC cell lines, Caki-1 and 786-O, in vitro by inducing cell cycle arrest and autophagy, but not apoptosis. However, in a xenograft model there was no difference in the inhibition of tumor growth by Ku0063794 or temsirolimus. A potential explanation is that temsirolimus has additional effects on the tumor microenvironment. Consistent with this possibility, temsirolimus, but not Ku0063794, decreased tumor angiogenesis in vivo, and decreased the viability of HUVEC (Human Umbilical Vein Endothelial Cells) cells in vitro at pharmacologically relevant concentrations. Furthermore, expression levels of VEGF and PDGF were lower in Caki-1 and 786-O cells treated with temsirolimus than cells treated with Ku0063794.

PMID: 23349989 [PubMed - in process]

dovitinib dna-pk coxinhibitors

Role of mammalian target of rapamycin inhibitor in the treatment of metastatic epithelioid angiomyolipoma: A case report.

Role of mammalian target of rapamycin inhibitor in the treatment of metastatic epithelioid angiomyolipoma: A case report.

Int J Urol. 2013 Jan 24;

Authors: Kohno J, Matsui Y, Yamasaki T, Shibasaki N, Kamba T, Yoshimura K, Sumiyoshi S, Mikami Y, Ogawa O

Abstract
Epithelioid angiomyolipoma has malignant potential; however, no effective therapy has been established for advanced cases. A 50-year-old woman with a history of right nephrectomy for epithelioid angiomyolipoma was referred to our institution. Computed tomography and magnetic resonance imaging showed multiple tumors in her lung, liver and pelvic cavity. The liver and pelvic tumor specimens obtained by needle biopsy confirmed the diagnosis of epithelioid angiomyolipoma recurrence. The patient was treated with everolimus (10?mg/day). Three months later, pulmonary lesions disappeared; liver and pelvic tumors significantly shrank in size, but the pelvic tumor gradually enlarged again. We carried out surgical resection of the residual liver and pelvic cavity tumors. Although the mammalian target of rapamycin inhibitor seems to be effective for treating epithelioid angiomyolipoma, its long-term effects remain unknown. Thus, aggressive administration of a multidisciplinary treatment including molecular target therapy and surgical resection is required to improve the prognosis of epithelioid angiomyolipoma.

PMID: 23347205 [PubMed - as supplied by publisher]

ecdysone chir-258 dovitinib