Quincy A. Quick

α-Actinin 1 and α-actinin 4: Contrasting roles in the survival, motility, and RhoA signaling of astrocytoma cells

Alpha-actinin is a prominent actin filament associated protein for which different isoforms exist. Here, we have examined whether the two highly homologous non-muscle alpha-actinin isoforms 1 and 4 exhibit functional differences in astrocytoma cells. The protein levels of these isoforms were differentially regulated during the development and progression of astrocytomas, as alpha-actinin 1 was higher in astrocytomas compared to normal brains whereas alpha-actinin 4 was elevated in high-grade astrocytomas compared to normal brains and low grade astrocytomas. RNAi demonstrated contrasted contributions of alpha-actinin 1 and 4 to the malignant behavior of U-373, U-87 and A172 astrocytoma cells. While alpha-actinin 1 appeared to favor the expansion of U-373, U-87 and A172 astrocytoma cell populations, alpha-actinin 4 played this role only for U-373 cells. On the other hand, downregulation of alpha-actinin 4, but not 1, reduced cell motility, adhesion, cortical actin, and RhoA levels. Finally, in the three astrocytoma cell lines examined, alpha-actinin 1 and 4 had contrasted biochemical properties as alpha-actinin 4 was significantly more abundant in the actin cytoskeleton than alpha-actinin 1. Collectively, these findings suggest that alpha-actinin 1 and 4 are differentially regulated during the development and progression of astrocytomas because each of these isoforms uniquely contributes to distinct malignant properties of astrocytoma cells.

Inner ear formation during the early larval development of Xenopus laevis

The formation of the eight independent endorgan compartments (sacculus, utricle, horizontal canal, anterior canal, posterior canal, lagena, amphibian papilla, and basilar papilla) of the Xenopus laevis inner ear is illustrated as the otic vesicle develops into a complex labyrinthine structure. The morphology of transverse sections and whole‐mounts of the inner ear was assessed in seven developmental stages (28, 31, 37, 42, 45, 47, 50) using brightfield and laser scanning confocal microscopy. The presence of mechanosensory hair cells in the sensory epithelia was determined by identification of stereociliary bundles in cryosectioned tissue and whole‐mounts of the inner ear labeled with the fluorescent F‐actin probe Alexa‐488 phalloidin. Between stages 28 and 45, the otic vesicle grows in size, stereociliary bundles appear and increase in number, and the pars inferior and pars superior become visible. The initial formation of vestibular compartments with their nascent stereociliary bundles is seen by larval stage 47, and all eight vestibular and auditory compartments with their characteristic sensory fields are present by larval stage 50. Thus, in Xenopus, inner ear compartments are established between stages 45 and 50, a 2‐week period during which the ear quadruples in length in the anteroposterior dimension. The anatomical images presented here demonstrate the morphological changes that occur as the otic vesicle forms the auditory and vestibular endorgans of the inner ear. These images provide a resource for investigations of gene expression patterns in Xenopus during inner ear compartmentalization and morphogenesis. Developmental Dynamics 234:791–801, 2005.

The microtubule inhibiting agent epothilone B antagonizes glioma cell motility associated with reorganization of the actin-binding protein α-actinin 4

Invasion of normal brain tissue by brain tumor cells is a major contributing factor to the recurrence and resistance of clinically diagnosed glioblastomas to therapy (surgery, chemotherapy, radiation). Here, we have assessed the efficacy of the microtubule inhibiting agent epothilone B on glioblastoma cell motility, a prerequisite cellular program of invasive glioblastomas. Using cell migration assays and immunofluorescence techniques we demonstrated that epothilone B abrogated glioblastoma cell motility as a consequence of α-actinin 4 redistristrubiton and the breakdown of cellular structures (leading edge, stress fibers) it is associated with during cell migration. Evaluation of the microtubule actin cross linking factor in glioblastoma cells also revealed epothilone B invoked changes in this cytoskeleton cross linking protein, resembling α-actinin 4 changes in response to epothilone B. We have demonstrated in this study that epothilone B antagonizes glioblastoma cell motility due to the disruption of cytoskeleton binding proteins that aide in preserving the structural organization of the cytoskeleton filamentous network. Furthermore, we provide preclincial evidence that epothilone B effects on glioblastomas are not limited to the impairment of dividing tumors cells but that it also targets migratory and invasive glioblastoma cells, suggesting that this agent has potential clinical benefit due to its ability to target divergent cellular programs in the glioblastoma tumor mass.

Roles and Potential Clinical Applications of Intermediate Filament Proteins in Brain Tumors

Intermediate filament (IF) proteins are cytoplasmic and nuclear cytoskeletal proteins. Of the ~70 IF proteins, nearly 12 are found in the nervous system, where their expression is largely cell-type specific. Astrocytes express glial fibrillary acidic protein (GFAP), whereas different neuron types contain neurofilament proteins, α-internexin, or peripherin. These proteins are often downregulated in brain cancer. In addition, brain cancer cells may also contain vimentin, nestin, and synemin, which are the IF proteins found in neural progenitor cells. In different brain tumor types, the expression of nestin, vimentin, and α-internexin appears to correlate with the clinical outcome. Experimental investigations have also demonstrated that IF proteins have distinct roles in specific brain tumor cell behaviors: nestin, for instance, is important for the proliferation of glioma cells, whereas synemin also affect their mobility. The mechanisms responsible for these effects involve the interaction of IF proteins with specific signaling pathways. Synemin, for instance, positively regulates glioma cell proliferation by antagonizing protein phosphatase 2A. Further evidence for the potential of IF proteins as therapeutic targets derives from animal models showing the influence of IF proteins on tumor growth. Nestin downregulation, for instance, dramatically reduced intracerebral glioma growth. Selective targeted therapies of IFs to date primarily include gene therapy approaches using nestin or GFAP gene promoters to drive transgene expression into glioma cells. Although attempts to identify small molecules specifically antagonizing IF proteins have been unsuccessful to date, it is anticipated that the identification of such compounds will be instrumental in expanding therapeutic approaches for brain tumors.

Violacein induces p44/42 mitogen-activated protein kinase-mediated solid tumor cell death and inhibits tumor cell migration.

Microbial secondary metabolites have emerged as alternative novel drugs for the treatment of human cancers. Violacein, a purple pigment produced by Chromobacterium violaceum, was investigated in the present study for its anti-tumor properties in tumor cell lines. Clinically applicable concentrations of violacein were demonstrated to inhibit the proliferative capacity of tumor cell lines according to a crystal violet proliferation assay. The underlying mechanism was the promotion of apoptotic cell death, as indicated by poly(ADP ribose) polymerase cleavage and p44/42 mitogen-activated protein kinase signaling determined by western blot analysis. Collectively, this provided mechanistic evidence that violacein elicits extracellular-signal regulated kinase-induced apoptosis via the intrinsic pathway. The anti-malignant properties of violacein in the present study were further demonstrated by its inhibitory effects on brain tumor cell migration, specifically glioblastomas, one of the most invasive and therapeutically resistant neoplasms in the clinic. Additionally, solid tumors examined in the present study displayed differential cellular responses and sensitivities to violacein as observed by morphologically induced cellular changes that contributed to its anti-migratory properties. In conclusion, violacein is a novel natural product with the potential to kill several types of human tumor cell lines, as well as prevent disease recurrence by antagonizing cellular processes that contribute to metastatic invasion.

Caspase-dependent signaling underlies glioblastoma cell death in response to the fungal metabolite, fusarochromanone

Fungal metabolites continue to show promise as a viable class of anticancer agents. In the present study, we investigated the efficacy of the fungal metabolite, fusarochromanone (FC101), for its antitumor activities in glioblastomas, which have a median survival of less than two years and a poor clinical response to surgical resection, radiation therapy and chemotherapy. Using clinically applicable doses, we demonstrated that FC101 induced glioblastoma apoptotic cell death via caspase dependent signaling, as indicated by the cleavage of poly(ADP-ribose) polymerase, glioblastoma (PARP). FC101 also induced differential reactive oxygen species (ROS) levels in glioblastoma cells, contrasting a defined role of oxidative stress in apoptotic cell death observed with other fungal metabolites. Furthermore, the antitumorigenic effects of FC101 on tumor cell migration were assessed. Cell migration assays revealed that FC101 significantly reduced the migratory capacity of glioblastomas, which are incredibly invasive tumors. Taken together, the present study establishes FC101 as a candidate anticancer agent for the cooperative treatment of glioblastomas.

1′-Acetoxychavicol acetate promotes caspase 3-activated glioblastoma cell death by overcoming enhanced cytokine expression

The brain consumes ∼20% of the oxygen utilized in the human body, meaning that brain tumors are vulnerable to paradoxical physiological effects from free radical generation. In the present study, 1′-acetoxychavicol acetate (ACA), a naturally derived antioxidant that inhibits xanthine oxidase, was evaluated for its role as an anti-tumorigenic agent in glioblastomas. The study revealed that ACA inhibited glioblastoma cell proliferation as a consequence of promoting apoptotic cell death by enhancing caspase 3 activity. It was also shown that ACA impaired the migratory ability of glioblastoma cells by decreasing their adhesive properties. Additionally, ACA increased the protein expression levels of the pro-survival signaling cytokines, IL-6 and IL-1α, established cell protectors and survival molecules in brain tumors. Together, these results demonstrate that, despite enhanced expression of compensatory signaling molecules that contribute to tumor cell survival, ACA is an effective pro-apoptotic inducing agent in glioblastomas.

Microtubule-Actin Crosslinking Factor 1 and Plakins as Therapeutic Drug Targets

Plakins are a family of seven cytoskeletal cross-linker proteins (microtubule-actin crosslinking factor 1 (MACF), bullous pemphigoid antigen (BPAG1) desmoplakin, envoplakin, periplakin, plectin, epiplakin) that network the three major filaments that comprise the cytoskeleton. Plakins have been found to be involved in disorders and diseases of the skin, heart, nervous system, and cancer that are attributed to autoimmune responses and genetic alterations of these macromolecules. Despite their role and involvement across a spectrum of several diseases, there are no current drugs or pharmacological agents that specifically target the members of this protein family. On the contrary, microtubules have traditionally been targeted by microtubule inhibiting agents, used for the treatment of diseases such as cancer, in spite of the deleterious toxicities associated with their clinical utility. The Research Collaboratory for Structural Bioinformatics (RCSB) was used here to identify therapeutic drugs targeting the plakin proteins, particularly the spectraplakins MACF1 and BPAG1, which contain microtubule-binding domains. RCSB analysis revealed that plakin proteins had 329 ligands, of which more than 50% were MACF1 and BPAG1 ligands and 10 were documented, clinically or experimentally, to have several therapeutic applications as anticancer, anti-inflammatory, and antibiotic agents.

Abstract 4750: Characterization of the cytoskeletal protein MACF1 in lung cancer

Genetic variation between cancer patients with the same tumor and intratumor genetic heterogeneity have broadened the complexity of factors that contribute to clinical therapeutic resistance and poor response outcomes. This has made it imperative to identity atypical expressed outliers that influence the maintenance and progression of cancers. We have identified and investigated the spectraplakin protein, Microtubule Actin Cross-Linking Factor 1 (MACF1) which functions as an actin-microfilament and microtubule crosslinker for its role in lung cancers. Genomic data generated by the Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) indicate that the MACF1 gene is mutated and/or amplified in ∼15% of squamous lung cancers and 25% of lung cancers, respectively. Further examination with immunohistochemistry expression analysis revealed that MACF1 is highly expressed in several lung cancer subtypes, particularly adenocarcinomas and squamous cell carcinomas, as compared to normal lung tissue. Additionally, functional studies showed that suppression of MACF1 with RNA interference significantly impaired the reproductive capacity of these solid tumors. Taken together, MACF1 represents a unique target with genetic alterations and diagnostic biomarker potential in lung cancers, the leading cause of cancer deaths in the United States. Citation Format: Najlaa Afghani, Quincy A. Quick. Characterization of the cytoskeletal protein MACF1 in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4750.

Auditory organs in Xenopus laevis and Xenopus tropicalis

The genus Xenopus is comprised of over 20 genetically divergent species that occupy aquatic habitats such as lakes, rivers, and swamps south of the African Sahara. Like other anurans, advertisement calls are important signals for reproductive behavior and Xenopus have a vocal apparatus adapted for underwater sound production. The tetraploid Xenopus laevis is a well‐established model organism for cell and developmental biology. Xenopus tropicalis, a diploid member of the Silurana group, is a newer Xenopus model for molecular genetics that is suited for transgenic studies. Both species are being used for investigations of inner ear organogenesis. Results presented here use light and confocal microscopy to examine the structure of the auditory organs of X. laevis and X. tropicalis. Images gathered from sectioned tissue stained with hematoxylin/eosin or microdissected organs labeled with Alexa 488 phalloidin (Molecular Probes) and propidium iodide illustrate the organization and innervation of the sensory epi...