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.
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.
Mitogen-activated protein kinase (MAPK) signaling regulates fundamental cellular functions including proliferation, differentiation, and survival. We have demonstrated previously that inhibiting MAPK ...signaling induces apoptosis in melanoma cells but not in normal melanocytes, suggesting that the MAPK pathway propagates essential survival signals in melanoma cells. Here, we report that the 90-kDa ribosomal S6 kinase (RSK), a downstream effector in the MAPK signaling cascade, phosphorylates and inactivates the Bcl-2 homology 3-only proapoptotic protein Bad, thereby mediating a MAPK-dependent tumor-specific survival signal in melanoma cells. The MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK)/RSK MAPK signaling module is constitutively hyperactivated, and Bad is maintained in its inactive state by phosphorylation at Ser(75) in a MEK/ERK/RSK-dependent manner in melanoma cells. In contrast, in normal melanocytes, Bad is highly phosphorylated at multiple residues (Ser(75), Ser(99), and Ser(118)) in a MAPK pathway-independent manner. Importantly, ectopic expression of a constitutively activated RSK mutant abrogates Bad activation and renders melanoma cells resistant to apoptosis induced by a MEK inhibitor. Furthermore, overexpressing alanine-substituted (S75A) Bad further sensitizes melanoma cells to MEK inhibitor-induced apoptosis. Our results suggest that the MAPK pathway mediates melanoma-specific survival signaling by differentially regulating RSK-mediated phosphorylation of the proapoptotic protein Bad and may present potentially selective therapeutic targets for the treatment of melanomas.
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.
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.
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.
Molecular imaging relies on the development of sensitive and specific probes coupled with imaging hardware and software to provide information about the molecular status of a disease and its response ...to therapy, which are important aspects of disease management. As genomic and proteomic information from a variety of cardiovascular diseases becomes available, new cellular and molecular targets will provide an imaging readout of fundamental disease processes. A review of the development and application of several cardiovascular probes is presented here. Strategies for labeling cells with superparamagnetic iron oxide nanoparticles enable monitoring of the delivery of stem cell therapies. Small molecules and biologics (e.g., proteins and antibodies) with high affinity and specificity for cell surface receptors or cellular proteins as well as enzyme substrates or inhibitors may be labeled with single-photon-emitting or positron-emitting isotopes for nuclear molecular imaging applications. Labeling of bispecific antibodies with single-photon-emitting isotopes coupled with a pretargeting strategy may be used to enhance signal accumulation in small lesions. Emerging nanomaterials will provide platforms that have various sizes and structures and that may be used to develop multimeric, multimodal molecular imaging agents to probe one or more targets simultaneously. These platforms may be chemically manipulated to afford molecules with specific targeting and clearance properties. These examples of molecular imaging probes are characteristic of the multidisciplinary nature of the extraction of advanced biochemical information that will enhance diagnostic evaluation and drug development and predict clinical outcomes, fulfilling the promise of personalized medicine and improved patient care.
In Parkinson disease (PD), the benefit of levodopa therapy becomes less marked over time, perhaps because degeneration of nigrostrial neurons causes progressive loss of aromatic l-amino acid ...decarboxylase (AADC), the enzyme that converts levodopa into dopamine. In a primate model of PD, intrastriatal infusion of an adeno-associated viral type 2 vector containing the human AADC gene (AAV-hAADC) results in robust response to low-dose levodopa without the side effects associated with higher doses. These data prompted a clinical trial.
Patients with moderately advanced PD received bilateral intraputaminal infusion of AAV-hAADC vector. Low-dose and high-dose cohorts (5 patients in each) were studied using standardized clinical rating scales at baseline and 6 months. PET scans using the AADC tracer (18)Ffluoro-L-m-tyrosine (FMT) were performed as a measure of gene expression.
The gene therapy was well tolerated, but 1 symptomatic and 2 asymptomatic intracranial hemorrhages followed the operative procedure. Total and motor rating scales improved in both cohorts. Motor diaries also showed increased on-time and reduced off-time without increased "on" time dyskinesia. At 6 months, FMT PET showed a 30% increase of putaminal uptake in the low-dose cohort and a 75% increase in the high-dose cohort.
This study provides class IV evidence that bilateral intrastriatal infusion of adeno-associated viral type 2 vector containing the human AADC gene improves mean scores on the Unified Parkinson's Disease Rating Scale by approximately 30% in the on and off states, but the surgical procedure may be associated with an increased risk of intracranial hemorrhage and self-limited headache.
Abstract RNA interference (RNAi) has recently emerged as a promising antiviral technique in vertebrates. Although most studies have used exogenous short interfering RNAs (siRNAs) to inhibit viral ...replication, vectors expressing short hairpin RNAs (shRNA-mirs) in the context of a modified endogenous micro-RNA (miRNA) are more efficient and are practical for in vivo delivery. In this study, replication competent retroviral vectors were designed to deliver shRNA-mirs targeting subgroup B avian leukosis virus (ALV), the most effective of which reduced expression of protein targets by as much as 90% in cultured avian cells. Cells expressing shRNA-mirs targeting the tvb receptor sequence or the viral env(B) sequence significantly inhibited ALV(B) replication. This study demonstrates efficient antiviral RNAi in avian cells using shRNA-mirs expressed from pol II promoters, including an inducible promoter, allowing for the regulation of the antiviral effect by doxycycline.
Aberrant activation of rat sarcoma (Ras) signaling contributes to the development of a variety of human cancers, including gliomas. To determine the dependence of high-grade gliomas on continued Ras ...signaling, we developed a doxycycline-regulated Kirsten Ras (KRas) glioma mouse model. We previously demonstrated that KRas is required for the maintenance of glioblastoma multiforme tumors arising in the context of activated Akt signaling in vivo; inhibition of KRas expression resulted in apoptotic tumor regression and significantly increased survival. We utilized a well-established glioma mouse model to determine the reliance of gliomas on continued KRas signaling in the context of Ink4a/Arf deficiency, a common occurrence in human gliomas. Despite the dependency of primary gliomas on continued KRas signaling, a significant percentage of tumors progressed to a KRas-independent state in the absence of Ink4a/Arf expression, demonstrating that these tumor suppressors play a critical role in the suppression of glioma recurrence. While even advanced stages of gliomas may remain dependent upon KRas signaling for maintenance and growth, our findings demonstrate that loss of Ink4a/Arf facilitates the acquisition of oncogene independence and tumor recurrence. Furthermore, reactivation of the Ras mitogen-activated protein kinase pathway in the absence of virally delivered KRas expression is a common mechanism of recurrence in this context.
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