Mutations in receptor tyrosine kinase (RTK) growth factor receptors (epidermal growth factor receptor, platelet-derived growth factor receptor, MET and ERBB2), which result in downstream activation ...of the RAS/RAF/MEK/ERK mitogen-activated protein kinase (MAPK) pathway and PI(3)K/Akt pathway, are found in almost all high-grade gliomas and MAPK signaling is necessary for continued glioma maintenance. In addition, BRAF is mutated in the majority of low-grade gliomas and its expression and activity is significantly increased in the majority of high-grade gliomas. Although the importance of RTKs and RAS signaling in glioma development has been shown, the role of BRAF has yet to be characterized. We evaluated the effect of activated BRAF in glioma formation using the retroviral replication-competent avian leukosis virus long terminal repeat, splice acceptor (RCAS)/TVA system to transfer genes encoding activated forms of BRAF, KRas, Akt and Cre to nestin-expressing neural progenitor cells in Ink4a/Arf(lox/lox) mice in vivo. Although expression of activated BRAF alone is not sufficient for tumorigenesis, the combination of activated BRAF and Akt or BRAF with Ink4a/Arf loss is transforming. Interestingly, activated BRAF generates gliomas with characteristics similar to activated KRas in the context of Akt but not Ink4a/Arf loss. Our studies show a role for BRAF activation and signaling in glioma development and as potential target for glioma therapy.
Humans and mice lacking Lrp5 have low BMD. To evaluate whether Lrp5 and Lrp6 interact genetically to control bone or skeletal development, we created mice carrying mutations in both Lrp5 and the ...related gene Lrp6. We found that compound mutants had dose‐dependent deficits in BMD and limb formation, suggesting functional redundancy between these two genes in bone and limb development.
Introduction: Lrp5 and Lrp6 are closely related members of the low density lipoprotein receptor family and are co‐receptors for Wnt ligands. While Lrp5 mutations are associated with low BMD in humans and mice, the role of Lrp6 in bone formation has not been analyzed.
Materials and Methods: To address whether Lrp5 and Lrp6 play complimentary roles in bone and skeletal development, we created mice with mutations in both genes. We inspected limbs of mice from the different genotypic classes of compound mutants to identify abnormalities. DXA and μCT were used to evaluate the effect of mutations in Lrp5 and Lrp6 on BMD and microarchitecture.
Results: Mice heterozygous for mutations in Lrp6 and either heterozygous or homozygous for a mutation in Lrp5 (Lrp6+/−;Lrp5+/− or Lrp6+/−;Lrp5−/−) display limb defects with incomplete penetrance and variable expression. DXA analysis showed that BMD decreased as mice progressively were more deficient in Lrp5 and Lrp6. Lrp6+/−;Lrp5−/− mice were more severely affected than Lrp6+/+;Lrp5−/− mice, whereas Lrp6+/−;Lrp5+/− mice had statistically higher BMD than Lrp6+/+;Lrp5−/− mice and lower BMD compared with wildtype mice and mice heterozygous for either mutation alone.
Conclusions: Lrp6 and Lrp5 genetically interact in limb development in mice. Furthermore, heterozygosity for an inactivating mutation in Lrp6 further reduces BMD in both male and female mice lacking Lrp5.
We report on a new strategy for preparing polymer-nanoparticle composite Faraday rotators for use in magnetic sensing and optical isolation. While most applications of Faraday rotators make use of ...inorganic garnet crystals, these are generally limited by low magneto-optical activity (low Verdet constants), high cost, and/or limited processing options. This has led to an interest in new materials with improved activity and processing characteristics. We have developed a new type of magneto-optical material based on polymer-nanoparticle composites that can be completely prepared by solution processing methods with tunable Verdet constants and device sensitivity. By exchanging native surface ligands on magneto-optically active CoFe
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O
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nanocrystals with polymer compatible ligands, enhanced nanoparticle dispersion in processible polymer matrices was observed at up to 15 wt% inorganic loading. Employing a multilayer polymer film construct, functional Faraday rotator devices were prepared by simple sequential spin-coating of active nanocomposite and protective, barrier cellulose acetate layers. For these assemblies, magneto-optic activity and sensitivity are easily tuned through variation of nanoparticle feed and control of polymer film layers, respectively. These multilayered Faraday rotators show up to a 10-fold enhancement in Verdet constant compared to reference terbium gallium garnets at 1310 nm, opening new possibilities for the fabrication of "plastic garnets" as low cost alternatives to existing inorganic materials for use in the near-IR.
The critical role of nanoparticle dispersion on Faraday rotator activity was studied, revealing new routes for fabricating "plastic garnets" as low cost alternatives to existing inorganic materials for optical isolation and magnetic sensing.
Heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) is a ligand for the EGF receptor (EGFR), one of the most commonly amplified receptor tyrosine kinases (RTKs) in glioblastoma ...(GBM). While HBEGF has been found to be expressed in a subset of malignant gliomas, its sufficiency for glioma initiation has not been evaluated. In this study, we demonstrate that HBEGF can initiate GBM in mice in the context of Ink4a/Arf and Pten loss, and that these tumors are similar to the classical GBM subtype observed in patients. Isogenic astrocytes from these mice showed activation not only of Egfr but also the RTK Axl in response to HBEGF stimulation. Deletion of either Egfr or Axl decreased the tumorigenic properties of HBEGF-transformed cells; however, only EGFR was able to rescue the phenotype in cells lacking both RTKs indicating that Egfr is required for activation of Axl in this context. Silencing of HBEGF in vivo resulted in tumor regression and significantly increased survival, suggesting that HBEGF may be a clinically relevant target.
The RAS/RAF mitogen-activated protein kinase pathway (MAPK) is highly active in many tumor types including the majority of high-grade gliomas and expression of activated RAS or RAF in neural ...progenitor cells combined with either AKT activation or Ink4a/Arf loss leads to the development of high-grade gliomas in vivo. This strongly suggests that this pathway is necessary for glioma formation and maintenance. To further define the role of this pathway in the development of high-grade gliomas, we used the established RCAS/TVA glioma mouse model to test the ability of activated MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK), a RAF effector, to induce tumors in vivo in the context of activated AKT or Ink4a/Arf loss. Although expression of activated MEK alone in neural progenitor cells is not sufficient for tumorigenesis, the combination of activated MEK and AKT or MEK with Ink4a/Arf loss is transforming. The data reveal that activation of the classical RAS/MAPK pathway, which is mediated through MEK, leads to the development of high-grade gliomas in vivo and suggest that MEK may be a relevant target for glioma therapy. To test this, we treated both mouse and human glioma cells with the MEK inhibitor PD0325901. Although this treatment induced apoptosis in a significant percentage of the cells, the effect was enhanced by combined treatment with the phosphatidylinositol 3-kinase (PI3K)/mTOR inhibitor NVP-BEZ235. Our results demonstrate that combined inhibition of MEK and PI3K/mTOR is a rational strategy for the treatment of high-grade gliomas and may be an effective adjuvant therapy for this disease.
The development of targeted inhibitors, vemurafenib and dabrafenib, has led to improved clinical outcome for melanoma patients with BRAF
mutations. Although the initial response to these inhibitors ...can be dramatic, sometimes causing complete tumor regression, the majority of melanomas eventually become resistant. Mitogen-activated protein kinase kinase (MEK) mutations are found in primary melanomas and frequently reported in BRAF melanomas that develop resistance to targeted therapy; however, melanoma is a molecularly heterogeneous cancer, and which mutations are drivers and which are passengers remains to be determined. In this study, we demonstrate that in BRAF
melanoma cell lines, activating MEK mutations drive resistance and contribute to suboptimal growth of melanoma cells following the withdrawal of BRAF inhibition. In this manner, the cells are drug-addicted, suggesting that melanoma cells evolve a 'just right' level of mitogen-activated protein kinase signaling and the additive effects of MEK and BRAF mutations are counterproductive. We also used a novel mouse model of melanoma to demonstrate that several of these MEK mutants promote the development, growth and maintenance of melanoma in vivo in the context of Cdkn2a and Pten loss. By utilizing a genetic approach to control mutant MEK expression in vivo, we were able to induce tumor regression and significantly increase survival; however, after a long latency, all tumors subsequently became resistant. These data suggest that resistance to BRAF or MEK inhibitors is probably inevitable, and novel therapeutic approaches are needed to target dormant tumors.
Mutations in the Wnt co-receptor LRP5 alter bone mass in humans, but the mechanisms responsible for Wnts actions in bone are unclear. To investigate the role of the classical Wnt signaling pathway in ...osteogenesis, we generated mice lacking the β-catenin or adenomatous polyposis coli (Apc) genes in osteoblasts. Loss of β-catenin produced severe osteopenia with striking increases in osteoclasts, whereas constitutive activation of β-catenin in the conditional Apc mutants resulted in dramatically increased bone deposition and a disappearance of osteoclasts. In vitro, osteoblasts lacking the β-catenin gene exhibited impaired maturation and mineralization with elevated expression of the osteoclast differentiation factor, receptor activated by nuclear factor-κB ligand (RANKL), and diminished expression of the RANKL decoy receptor, osteoprotegerin. By contrast, Apc-deficient osteoblasts matured normally but demonstrated decreased expression of RANKL and increased osteoprotegerin. These findings suggest that Wnt/β-catenin signaling in osteoblasts coordinates postnatal bone acquisition by controlling the differentiation and activity of both osteoblasts and osteoclasts.
Blood lipids are important risk factors for coronary artery disease (CAD). Here we perform an exome-wide association study by genotyping 12,685 Chinese, using a custom Illumina HumanExome BeadChip, ...to identify additional loci influencing lipid levels. Single-variant association analysis on 65,671 single nucleotide polymorphisms reveals 19 loci associated with lipids at exome-wide significance (P<2.69 × 10(-7)), including three Asian-specific coding variants in known genes (CETP p.Asp459Gly, PCSK9 p.Arg93Cys and LDLR p.Arg257Trp). Furthermore, missense variants at two novel loci-PNPLA3 p.Ile148Met and PKD1L3 p.Thr429Ser-also influence levels of triglycerides and low-density lipoprotein cholesterol, respectively. Another novel gene, TEAD2, is found to be associated with high-density lipoprotein cholesterol through gene-based association analysis. Most of these newly identified coding variants show suggestive association (P<0.05) with CAD. These findings demonstrate that exome-wide genotyping on samples of non-European ancestry can identify additional population-specific possible causal variants, shedding light on novel lipid biology and CAD.
RAS genes are mutated in approximately 30% of all human cancers. Interestingly, there exists a strong bias in favor of mutation of only one of the three major RAS genes in tumors of different ...cellular origins. NRAS mutations occur in approximately 20% of human melanomas, whereas HRAS and KRAS mutations are rare in this disease. To define the mechanism(s) responsible for this preference in melanocytes, we compared the transformation efficiencies of mutant NRAS and KRAS in immortal, non-transformed Ink4a/Arf-deficient melanocytes. NRAS mutation leads to increased cellular proliferation and is potently tumorigenic. In contrast, KRAS mutation does not enhance melanocyte proliferation and is only weakly tumorigenic on its own. Although both NRAS and KRAS activate mitogen-activated protein kinase signaling, only NRAS enhances MYC activity in these cells. Our data suggest that the activity of specific RAS isoforms is context-dependent and provide a possible explanation for the prevalence of NRAS mutations in melanoma. In addition, understanding this mechanism will have important implications for cancer therapies targeting RAS pathways.
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