Marie E. Beckner

Glycolytic glioma cells with active glycogen synthase are sensitive to PTEN and inhibitors of PI3K and gluconeogenesis

Increased glycolysis is characteristic of malignancy. Previously, with a mitochondrial inhibitor, we demonstrated that glycolytic ATP production was sufficient to support migration of melanoma cells. Recently, we found that glycolytic enzymes were abundant and some were increased in pseudopodia formed by U87 glioma (astrocytoma) cells. In this study, we examined cell migration, adhesion (a step in migration), and Matrigel invasion of U87 and LN229 glioma cells when their mitochondria were inhibited with sodium azide or limited by 1% O2. Cell migration, adhesion, and invasion were comparable, with and without mitochondrial inhibition. Upon discovering that glycolysis alone can support glioma cell migration, unique features of glucose metabolism in astrocytic cells were investigated. The ability of astrocytic cells to remove lactate, the inhibitor of glycolysis, via gluconeogenesis and incorporation into glycogen led to consideration of supportive genetic mutations. Loss of phosphatase and tensin homolog (PTEN) releases glycogenesis from constitutive inhibition by glycogen synthase kinase-3 (GSK3). We hypothesize that glycolysis in gliomas can support invasive migration, especially when aided by loss of PTENs regulation on the phosphatidylinositol-3 kinase (PI3K)/Akt pathway leading to inhibition of GSK3. Migration of PTEN-mutated U87 cells was studied for release of extracellular lactic acid and support by gluconeogenesis, loss of PTEN, and active PI3K. Lactic acid levels plateaued and phosphorylation changes confirmed activation of the PI3K/Akt pathway and glycogen synthase when cells relied only on glycolysis. Glycolytic U87 cell migration and phosphorylation of GSK3 were inhibited by PTEN transfection. Glycolytic migration was also suppressed by inhibiting PI3K and gluconeogenesis with wortmannin and metformin, respectively. These findings confirm that glycolytic glioma cells can migrate invasively and that the loss of PTEN is supportive, with activated glycogenic potential included among the relevant downstream effects.

Proteomic characterization of harvested pseudopodia with differential gel electrophoresis and specific antibodies

Malignant gliomas (astrocytomas) are lethal tumors that invade the brain. Invasive cell migration is initiated by extension of pseudopodia into interstitial spaces. In this study, U87 glioma cells formed pseudopodia in vitro as cells pushed through 3 μm pores of polycarbonate membranes. Harvesting pseudopodia in a novel two-step method provided material for proteomic analysis. Differences in the protein profiles of pseudopodia and whole cells were found using differential gel electrophoresis (DIGE) and immunoblotting. Proteins from two-dimensional (2D) gels with MRs of 20–100 kDa and pIs of 3.0–10.0 were identified by peptide mass fingerprinting analysis using mass spectrometry. For DIGE, lysates of pseudopodia and whole cells were each labeled with electrophilic forms of fluorescent dyes, Cy3 or Cy5, and analyzed as mixtures. Analysis was repeated with reciprocal labeling. Differences in protein distributions were detected by manual inspection and computer analysis. Topographical digital maps of the scanned gels were used for algorithmic spot matching, normalization of background, quantifying spot differences, and elimination of artifacts. Pseudopodial proteins in Coomassie-stained 2D gels included isoforms of glycolytic enzymes as the largest group, seven of 24 proteins. Peptide mass fingerprint analysis of DIGE gels demonstrated increased isoforms of annexin (Anx) I, AnxII, enolase, pyruvate kinase, and aldolase, and decreased mitochondrial manganese superoxide dismutase and transketolase in pseudopodia. Specific antibodies showed restricted immunoreactivity of the hepatocyte growth factor (HGF) α chain to pseudopodia, indicating localization of its active form. Met (the HGF receptor), actin, and total AnxI were increased in pseudopodial lysates on immunoblots. Increased constituents of the pseudopodial proteome in glioma cells, identified in this study as actin, HGF, Met, and isoforms of AnxI, AnxII, and several glycolytic enzymes, represent therapeutic targets to consider for suppression of tumor invasion.

Giant cell tumor of the skull: a case report and review of the literature

BACKGROUND Giant cell tumors are benign lesions that typically occur at the epiphyses of long bones that typically present with pain or swelling. Most data on giant cell tumors in the skull consist of case reports, and many large series of giant cell tumors have no examples in the skull. METHODS We report a case of giant cell tumor of the skull and review the literature on these lesions. RESULTS A 24-year-old woman presented with localized tenderness and mild swelling over the left inferior parietal and occipital bones. She was neurologically intact with a nonmobile, tender, palpable mass over the left subocciptal area. A computed tomography (CT) scan showed a radiolucent, expansile, lytic lesion involving the left occipital bone. The patient underwent a left occipital craniectomy with resection of the bone and epidural mass. Permanent histopathologic sections and immunostains revealed a giant cell tumor. CONCLUSIONS Giant cell tumors are generally benign, locally aggressive lesions for which surgical excision is the treatment of choice. This report contributes to the scarce literature on these tumors in the skull.

Blockade of Angio-Associated Migratory Cell Protein Inhibits Smooth Muscle Cell Migration and Neointima Formation in Accelerated Atherosclerosis

OBJECTIVES The aim of this study was to elucidate the role of angio-associated migratory cell protein (AAMP) for the migration of vascular smooth muscle cells (SMCs) and for the development of neointimal hyperplasia after vascular injury. BACKGROUND Although AAMP has been shown to participate in angiogenesis and cancerogenesis and is predominantly expressed in cells with a migratory phenotype, involvement of AAMP during neointima (NI) formation after arterial injury has not been analyzed previously. METHODS The AAMP content in SMCs was examined using 2-photon laser-scanning microscopy and subcellular fractioning. Migratory potential of SMCs transiently transfected with AAMP expression vectors, transfected with small interfering ribonucleic acid (siRNA), or treated with antirecombinant angio-associated migratory cell protein-antibody (anti-rAAMP-ab) was examined using transwell migration chamber assays. Expression of AAMP was determined in the atherogenic apolipoprotein E knockout (apoE(-/-)) mouse model and in the porcine coronary restenosis model by immunohistochemistry and by Western blot. ApoE(-/-) mice were treated intraperitoneally with anti-rAAMP-ab, and wire-injured carotid arteries were examined. RESULTS Angio-associated migratory cell protein is localized in the membrane of SMCs, and its expression is enhanced in NI-derived SMCs. The AAMP overexpression increases, while both treatment with anti-rAAMP-ab and transfection with siRNA decreases SMC migration. Knockdown of AAMP decreases RhoA activity in the membrane fraction of SMCs. The AAMP expression by SMCs is enhanced in both animal models. Anti-rAAMP-ab reduces neointimal SMC density at 1 week and NI formation at 4 weeks in apoE(-/-) mice without affecting proliferation of SMCs. CONCLUSIONS These data reveal an important functional role of AAMP in the migration of SMCs, identifying AAMP as a potential target to limit lesion formation after injury.

Intracranial extramedullary hematopoiesis associated with pilocytic astrocytoma: a case report

Intracranial EMH is only occasionally found in primary brain tumors (mostly hemangioblastomas) and, to our knowledge, this is the first case of EMH associated with an astrocytoma. Intracranial extramedullary hematopoiesis (EMH) is described in a 29-year-old man with a recurrent pilocytic astrocytoma in the tectal region. Special stains confirmed the identities of erythroid, myeloid and megakaryocytic cells. The patient had no evidence of a predisposing bone marrow disorder or systemic EMH. Although the presence of multinucleated and blastic cells associated with a low-grade brain neoplasm is unusual, recognition of hematopoietic lineages allows EMH to be readily identified. Another tumor resection after a year of follow-up confirmed the absence of malignant progression in this recurrent astrocytoma. The small number of cases describing intracranial EMH in the absence of systemic hematologic abnormalities are correlated with the findings in this case. The low incidence of intracranial EMH indicates that cells with hematopoietic potential are seldom exposed to a supportive microenvironment within the central nervous system. However, intracranial EMH should be included as a potential, ancillary diagnosis when considering brain lesions. This may be particularly true if medical therapies involving growth factors or stem cells are found to promote hematopoiesis.

Albumin marks pseudopodia of astrocytoma cells responding to hepatocyte growth factor or serum

It is well accepted that dysfunction in the blood brain barrier (BBB) allows permeation of albumin from the bloodstream into astrocytic brain tumors, especially glioblastomas, the most aggressive astrocytomas. In vitro, bovine serum albumin (BSA) aids functional cell assays by maintaining cytokines and growth factors in solution and delivering its cargo of fatty acids. Earlier, we showed that BSA was prominent in lysates prepared from pseudopodia formed by U87 astrocytoma cells. The present studies investigated the association of albumin with pseudopodia formed by U87 and LN229 astrocytoma cells. With hepatocyte growth factor (HGF) stimulation, cell migration was enhanced and BSA, especially its dimerized form, was prominent in pseudopodia compared to unmigrated cells on one-dimensional gels and immunoblots. When lysates were equalized for levels of glyceraldehyde-3-phosphate dehydrogenase, the rise for BSA levels in pseudopodia vs migrated cells was comparable or greater than levels noted for established pseudopodial proteins, β-actin and ezrin. The increase for dimerized BSA in pseudopodia compared to unmigrated cells was greater than the rise in levels of β-actin, ezrin, HGF, and phosphorylated Met when pseudopodia were harvested from filters with 1 μm pores using either cell line. Fluorescein (F)-labeled BSA co-localized with HGF on actin-rich cellular protrusions and with CM-DiI labeled pseudopodial plasma membranes. The F-BSA highlighted small, individual pseudopodial profiles more so than complex pseudopodial networks (reticulopodia) or unmigrated cells. Labeled human serum albumin also decorated pseudopodia preferentially. Albumins association with pseudopodia may help to explain its selective accumulation in astrocytomas in vivo. The leaky BBB permits serum albumin to enter the microenvironment of astrocytomas thus allowing their invasive cells contact with serum albumin as a source of fatty acids that would be useful for remodeling cell membranes in pseudopodia. Thus, albumin potentially aids and marks invasion as it accumulates in these tumors.

Molecular Techniques to Detect Disease and Response to Therapy: Minimal Residual Disease

Evaluation for the presence of neoplastic cells or karyotypic abnormalities has traditionally been performed to monitor therapeutic response of hematolymphoid neoplasms. The application of multicolor flow cytometry and nucleic acid amplification techniques has extended evaluable markers and lowered limits of detection, thus leading to the term “minimal residual disease” (MRD). The ability to monitor MRD has, in turn, led to new concepts in the definition of disease “remission” and early relapse and opportunities for personalized therapy. This chapter focuses on the principles of the molecular assessment of MRD.