Vicky Yamamoto

Cleavage of the Wnt Receptor Ryk Regulates Neuronal Differentiation during Cortical Neurogenesis

Ryk is a transmembrane receptor tyrosine kinase (RTK). It functions as a receptor of Wnt proteins required for cell-fate determination, axon guidance, and neurite outgrowth in different organisms; however, the molecular mechanism of Ryk signaling is unknown. Here, we show that Ryk is cleaved, permitting the intracellular C-terminal fragment of Ryk to translocate to the nucleus in response to Wnt3 stimulation. We also show that the cleaved intracellular domain of Ryk is required for Wnt3-induced neuronal differentiation in vitro and in vivo. These results demonstrate an unexpected mechanism of signal transduction for Ryk as a Wnt receptor, in which the intracellular domain itself functions as the transducing molecule to bring extracellular signals from the cell surface into the nucleus, to regulate neural progenitor cell differentiation.

Nanoplatforms for constructing new approaches to cancer treatment, imaging, and drug delivery: What should be the policy?

Nanotechnology is the design and assembly of submicroscopic devices called nanoparticles, which are 1-100 nm in diameter. Nanomedicine is the application of nanotechnology for the diagnosis and treatment of human disease. Disease-specific receptors on the surface of cells provide useful targets for nanoparticles. Because nanoparticles can be engineered from components that (1) recognize disease at the cellular level, (2) are visible on imaging studies, and (3) deliver therapeutic compounds, nanotechnology is well suited for the diagnosis and treatment of a variety of diseases. Nanotechnology will enable earlier detection and treatment of diseases that are best treated in their initial stages, such as cancer. Advances in nanotechnology will also spur the discovery of new methods for delivery of therapeutic compounds, including genes and proteins, to diseased tissue. A myriad of nanostructured drugs with effective site-targeting can be developed by combining a diverse selection of targeting, diagnostic, and therapeutic components. Incorporating immune target specificity with nanostructures introduces a new type of treatment modality, nano-immunochemotherapy, for patients with cancer. In this review, we will discuss the development and potential applications of nanoscale platforms in medical diagnosis and treatment. To impact the care of patients with neurological diseases, advances in nanotechnology will require accelerated translation to the fields of brain mapping, CNS imaging, and nanoneurosurgery. Advances in nanoplatform, nano-imaging, and nano-drug delivery will drive the future development of nanomedicine, personalized medicine, and targeted therapy. We believe that the formation of a science, technology, medicine law-healthcare policy (STML) hub/center, which encourages collaboration among universities, medical centers, US government, industry, patient advocacy groups, charitable foundations, and philanthropists, could significantly facilitate such advancements and contribute to the translation of nanotechnology across medical disciplines.

Infrared thermal imaging: a review of the literature and case report.

Intraoperative Thermal Imaging (ITI) is a novel neuroimaging technique that can potentially locate the margins of primary and metastatic brain tumors. As a result, the additional real-time anatomical and pathophysiological information may significantly contribute to an improved extent of tumor resection. Our objectives in this article are i) to briefly discuss the current status of intraoperative imaging modalities including ITI and ii) to present a case report that evaluates the usefulness of ITI in detection of brain tumor and its margins. In this case report, ITI was used in a patient with a metastatic intracortical melanoma. The thermal profile of the tumor and surrounding normal cerebral cortex were mapped with a ThermaCAM P60 (TCP60) infrared camera by FLIR Systems. The data obtained by TCP60, intra-operatively, revealed a clear demarcation of tumor with significant temperature differences, up to 3.3 degrees C, between the tumor core (36.4 degrees C) and the surrounding normal tissue (33.1 degrees C). Ultrasound and pre-resection MR and CT confirmed the position and size of the metastasis. The volume of the tumor was preoperatively calculated using the CyberKnife software and postoperative volumetric measurement of the tumor residual was calculated by the Gamma Knife software. Our result, along with previously published results of others, suggests that thermal imaging could be used to provide a rapid, non-invasive, and real-time intra-operative imaging.

Functional Impacts of NRXN1 Knockdown on Neurodevelopment in Stem Cell Models

Exonic deletions in NRXN1 have been associated with several neurodevelopmental disorders, including autism, schizophrenia and developmental delay. However, the molecular mechanism by which NRXN1 deletions impact neurodevelopment remains unclear. Here we used human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) as models to investigate the functional impacts of NRXN1 knockdown. We first generated hiPSCs from skin fibroblasts and differentiated them into neural stem cells (NSCs). We reduced NRXN1 expression in NSCs via a controlled shRNAmir-based knockdown system during differentiation, and monitored the transcriptome alteration by RNA-Seq and quantitative PCR at several time points. Interestingly, half reduction of NRXN1 expression resulted in changes of expression levels for the cell adhesion pathway (20 genes, P = 2.8×10−6) and neuron differentiation pathway (13 genes, P = 2.1×10−4), implicating that single-gene perturbation can impact biological networks important for neurodevelopment. Furthermore, astrocyte marker GFAP was significantly reduced in a time dependent manner that correlated with NRXN1 reduction. This observation was reproduced in both hiPSCs and hESCs. In summary, based on in vitro models, NRXN1 deletions impact several biological processes during neurodevelopment, including synaptic adhesion and neuron differentiation. Our study highlights the utility of stem cell models in understanding the functional roles of copy number variations (CNVs) in conferring susceptibility to neurodevelopmental diseases.

Multi-walled carbon nanotube (MWCNT) synthesis, preparation, labeling, and functionalization.

Nanomedicine is a growing field with a great potential for introducing new generation of targeted and personalized drug. Amongst new generation of nano-vectors are carbon nanotubes (CNTs), which can be produced as single or multi-walled. Multi-walled carbon nanotubes (MWCNTs) can be fabricated as biocompatible nanostructures (cylindrical bulky tubes). These structures are currently under investigation for their application in nanomedicine as viable and safe nanovectors for gene and drug delivery. In this chapter, we will provide you with the necessary information to understand the synthesis of MWCNTs, functionalization, PKH26 labeling, RNAi, and DNA loading for in vitro experimentation and in vivo implantation of labeled MWCNT in mice as well as materials used in this experimentation. We used this technique to manipulate microglia as part of a novel application for the brain cancer immunotherapy. Our published data show this is a promising technique for labeling, and gene and drug delivery into microglia.

Iron chelators induce autophagic cell death in multiple myeloma cells

We examined the antineoplastic effects of the iron chelators, deferasirox and deferoxamine in multiple myeloma cell lines as well as primary myeloma cells. These iron chelators showed marked antiproliferative activity as well as cytotoxicity toward myeloma cell lines and deferasirox was cytotoxic to bone marrow plasma cells from myeloma patients. We also demonstrate that autophagy induced by iron deprivation is the dominant mechanism that mediates the cytotoxicity of iron chelators in multiple myeloma. Exposure to iron chelators led to repression of mTOR signaling as evidenced by decreased phosphorylation of its target p70S6 kinase. Iron chelation, in particular with deferasirox has the potential to be readily translated to a clinical trial for multiple myeloma.

Protein Phosphatase 4 and Smek Complex Negatively Regulate Par3 and Promote Neuronal Differentiation of Neural Stem/Progenitor Cells

Neural progenitor cells (NPCs) are multipotent cells that can self-renew and differentiate into neurons and glial cells. However, mechanisms that control their fate decisions are poorly understood. Here, we show that Smek1, a regulatory subunit of the serine/threonine protein phosphatase PP4, promotes neuronal differentiation and suppresses the proliferative capacity of NPCs. We identify the cell polarity protein Par3, a negative regulator of neuronal differentiation, as a Smek1 substrate and demonstrate that Smek1 suppresses its activity. We also show that Smek1, which is predominantly nuclear in NPCs, is excluded from the nucleus during mitosis, allowing it to interact with cortical/cytoplasmic Par3 and mediate its dephosphorylation by the catalytic subunit PP4c. These results identify the PP4/Smek1 complex as a key regulator of neurogenesis.

Overcoming radioresistance in head and neck squamous cell carcinoma.

Radiation therapy plays an essential role in the treatment of head and neck squamous cell carcinoma (HNSCC), yet therapeutic efficacy is hindered by treatment-associated toxicity and tumor recurrence. In comparison to other cancers, innovation has proved challenging, with the epidermal growth factor receptor (EGFR) antibody cetuximab being the only new radiosensitizing agent approved by the FDA in over half a century. This review examines the physiological mechanisms that contribute to radioresistance in HNSCC as well as preclinical and clinical data regarding novel radiosensitizing agents, with an emphasis on those with highest translational promise.

The effect of human papillomavirus on DNA repair in head and neck squamous cell carcinoma

Much of the current literature regarding the molecular pathophysiology of human papillomavirus (HPV) in head and neck squamous cell carcinoma (HNSCC) has focused on the viruss effect on cell cycle modulation and cell proliferation. A second mechanism of pathogenicity employed by HPV, dysregulation of cellular DNA repair processes, has been more sparsely studied. The purpose of this review is to describe current understanding about the effect of HPV on DNA repair in HNSCC, taking cues from cervical cancer literature. HPV affects DNA-damage response pathways by interacting with many proteins, including ATM, ATR, MRN, γ-H2AX, Chk1, Chk2, p53, BRCA1, BRCA2, RAD51, Rb-related proteins 107 and 130, Tip60, and p16INK4A. Further elucidation of these pathways could lead to development of targeted therapies and improvement of current treatment protocols.

Wnt Signaling Pathway Inhibition Enhances Efficacy of Cetuximab

Objectives: 1) Investigate whether Wnt pathway inhibition via a small molecule inhibitor, ICG-001, enhances cetuximab efficacy in head and neck squamous cell carcinoma (HNSCC). 2) Elucidate the mechanism of cetuximab sensitization and increased cell death that results from ICG-001/cetuximab cotreatment. Methods: HNSCC cell line SCC-15 was grown for 24 hours then incubated with: ICG-001, cetuximab, or both for 48-72 hrs. Cells grown in media alone served as control. Cell proliferation was analyzed using WST-1 and clonogenic assays. The mechanism of ICG-001/cetuximab interaction was investigated using reverse transcription polymerase chain reaction (RT-PCR), Western blot, immunohistochemistry, and TUNEL staining. Results: SCC-15 cells treated with ICG-001/cetuximab demonstrated significantly decreased viability (19.9%) as determined by WST-1 assay compared to control (100%), cetuximab (90.3%), or ICG-001 (35.1%) treatment (P < 0.05). Additionally, clonogenic assay demonstrated that combination ICG-001/cetuximab treatment led to significantly decreased cell proliferation (2.6%) compared to control (100%) or monotherapy (cetuximab 94.3%; ICG-001 20.2%; P < 0.05). ICG-001 treatment resulted in increased epidermal growth factor receptor (EGFR) expression, as demonstrated by Western blot, RT-PCR, and immunohistochemistry. TUNEL-positive cells were shown to be significantly increased in both ICG-001 and ICG-001/cetuximab treatment groups. Additionally, combination therapy increased levels of cleaved caspase-3, a marker of apoptosis. Conclusions: ICG-001/cetuximab cotreatment results in cetuximab sensitization and increased cell death in HNSCC in vitro. Increased EGFR expression and pathway dependence leading to cetuximab vulnerability, along with increased apoptosis, play a major role in this observed effect. ICG-001/cetuximab cotreatment represents a potential novel therapy in HNSCC management which may offer superior outcomes with fewer side effects compared to existing first-line treatment.