A novel viral mechanism for dysregulation of beta-catenin in Kaposi's sarcoma-associated herpesvirus latency.

The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is expressed in all KSHV-associated tumors, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). We found that beta-catenin is overexpressed in both PEL cells and KS tissue. Introduction of anti-LANA small interfering RNA (siRNA) into PEL cells eliminated beta-catenin accumulation; LANA itself upregulated expression of beta-catenin in transfected cells. LANA stabilizes beta-catenin by binding to the negative regulator GSK-3beta, causing a cell cycle-dependent nuclear accumulation of GSK-3beta. The LANA C terminus contains sequences similar to the GSK-3beta-binding domain of Axin. Disruption of this region resulted in a mutant LANA that failed to re-localize GSK-3beta or stabilize beta-catenin. The importance of this pathway to KSHV-driven cell proliferation was highlighted by the observation that LANA, but not mutant LANA, stimulates entry into S phase. Redistribution of GSK-3beta can therefore be a source of beta-catenin dysregulation in human cancers.

Mesenchymal stem cells from human fat engineered to secrete BMP4 are nononcogenic, suppress brain cancer, and prolong survival.

PURPOSE: Glioblastoma is the most common adult primary malignant intracranial cancer. It is associated with poor outcomes because of its invasiveness and resistance to multimodal therapies. Human adipose-derived mesenchymal stem cells (hAMSC) are a potential treatment because of their tumor tropism, ease of isolation, and ability to be engineered. In addition, bone morphogenetic protein 4 (BMP4) has tumor-suppressive effects on glioblastoma and glioblastoma brain tumor-initiating cells (BTIC), but is difficult to deliver to brain tumors. We sought to engineer BMP4-secreting hAMSCs (hAMSCs-BMP4) and evaluate their therapeutic potential on glioblastoma. EXPERIMENTAL DESIGN: The reciprocal effects of hAMSCs on primary human BTIC proliferation, differentiation, and migration were evaluated in vitro. The safety of hAMSC use was evaluated in vivo by intracranial coinjections of hAMSCs and BTICs in nude mice. The therapeutic effects of hAMSCs and hAMSCs-BMP4 on the proliferation and migration of glioblastoma cells as well as the differentiation of BTICs, and survival of glioblastoma-bearing mice were evaluated by intracardiac injection of these cells into an in vivo intracranial glioblastoma murine model. RESULTS: hAMSCs-BMP4 targeted both the glioblastoma tumor bulk and migratory glioblastoma cells, as well as induced differentiation of BTICs, decreased proliferation, and reduced the migratory capacity of glioblastomas in vitro and in vivo. In addition, hAMSCs-BMP4 significantly prolonged survival in a murine model of glioblastoma. We also demonstrate that the use of hAMSCs in vivo is safe. CONCLUSIONS: Both unmodified and engineered hAMSCs are nononcogenic and effective against glioblastoma, and hAMSCs-BMP4 are a promising cell-based treatment option for glioblastoma.

Antiparasitic mebendazole shows survival benefit in 2 preclinical models of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and aggressive brain cancer, and despite treatment advances, patient prognosis remains poor. During routine animal studies, we serendipitously observed that fenbendazole, a benzimidazole antihelminthic used to treat pinworm infection, inhibited brain tumor engraftment. Subsequent in vitro and in vivo experiments with benzimidazoles identified mebendazole as the more promising drug for GBM therapy. In GBM cell lines, mebendazole displayed cytotoxicity, with half-maximal inhibitory concentrations ranging from 0.1 to 0.3 µM. Mebendazole disrupted microtubule formation in GBM cells, and in vitro activity was correlated with reduced tubulin polymerization. Subsequently, we showed that mebendazole significantly extended mean survival up to 63% in syngeneic and xenograft orthotopic mouse glioma models. Mebendazole has been approved by the US Food and Drug Administration for parasitic infections, has a long track-record of safe human use, and was effective in our animal models with doses documented as safe in humans. Our findings indicate that mebendazole is a possible novel anti-brain tumor therapeutic that could be further tested in clinical trials.

Connexin43 expression levels influence intercellular coupling and cell proliferation of native murine cardiac fibroblasts.

Little is known about connexin expression and function in murine cardiac fibroblasts. The authors isolated native ventricular fibroblasts from adult mice and determined that although they expressed both connexin43 (Cx43) and connexin45 (Cx45), the relative abundance of Cx45 was greater than that of Cx43 in fibroblasts compared to myocytes, and the electrophoretic mobility of both Cx43 and Cx45 differed in fibroblasts and in myocytes. Increasing Cx43 expression by adenoviral infection increased intercellular coupling, whereas decreasing Cx43 expression by genetic ablation decreased coupling. Interestingly, increasing Cx43 expression reduced fibroblast proliferation, whereas decreasing Cx43 expression increased proliferation. These data demonstrate that native fibroblasts isolated from the mouse heart exhibit intercellular coupling via gap junctions containing both Cx43 and Cx45. Fibroblast proliferation is inversely related to the expression level of Cx43. Thus, connexin expression and remodeling is likely to alter fibroblast function, maintenance of the extracellular matrix, and ventricular remodeling in both normal and diseased hearts.

Lytic replication-associated protein (RAP) encoded by Kaposi sarcoma-associated herpesvirus causes p21CIP-1-mediated G1 cell cycle arrest through CCAAT/enhancer-binding protein-alpha.

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic DNA virus that causes Kaposi sarcoma and AIDS-related primary effusion lymphoma (PEL). Here we show that KSHV lytic cycle replication in PEL cells induces G(1) cell cycle arrest, presumably to facilitate the progression of viral DNA replication. Expression of a KSHV-encoded early lytic protein referred to as RAP or K8 is induced within 12-24 h after the onset of lytic cycle induction in host PEL cells, and coincides with increased levels of both the endogenous C/EBPalpha and p21(CIP-1) proteins in the nucleus of the same cells. The KSHV RAP protein binds to C/EBPalpha in vitro and stimulates C/EBPalpha-induced expression from both the C/EBPalpha and p21 promoters in cotransfected cells. A recombinant adenovirus expressing the RAP protein induced the expression of both the C/EBPalpha and p21 proteins in primary human fibroblasts, and flow cytometric analysis revealed a dramatic inhibition of G(1) to S cell cycle progression in the same cells. All of these effects were abolished in cells that lack C/EBPalpha or by deletion of the basic/leucine zipper region in RAP that interacts with C/EBPalpha. Therefore, C/EBPalpha is essential for the p21-mediated inhibition of G(1) to S-phase progression by RAP in KSHV-infected host cells.