Henrick Horita

Autophagy regulates selective HMGB1 release in tumor cells that are destined to die.

Macroautophagy (hereafter referred to as autophagy) can increase or decrease the amount of cell death in response to various stimuli. To test whether autophagy also controls the characteristics associated with dying cells, we studied tumor cell killing by epidermal growth factor receptor-targeted diphtheria toxin (DT-EGF). DT-EGF kills epithelial and glioblastoma tumor cells with similar efficiency but by different mechanisms that depend on whether the cells activate autophagy when treated with the drug. Dying cells in which autophagy is induced selectively release the immune modulator high-mobility group B1 (HMGB1) without causing lysis of the cell membrane and classical necrosis. Conversely, cells that are killed by DT-EGF where autophagy is blocked, activate caspases but retain HMGB1. These data suggest that it may be feasible to manipulate the immunogenicity of dying cells by increasing or decreasing autophagy.

SDF-1α Induction in Mature Smooth Muscle Cells by Inactivation of PTEN Is a Critical Mediator of Exacerbated Injury-Induced Neointima Formation

Objective—PTEN inactivation selectively in smooth muscle cells (SMC) initiates multiple downstream events driving neointima formation, including SMC cytokine/chemokine production, in particular stromal cell-derived factor-1&agr; (SDF-1&agr;). We investigated the effects of SDF-1&agr; on resident SMC and bone marrow–derived cells and in mediating neointima formation. Methods and Results—Inducible, SMC-specific PTEN knockout mice (PTEN iKO) were bred to floxed-stop ROSA26-&bgr;-galactosidase (&bgr;Gal) mice to fate-map mature SMC in response to injury; mice received wild-type green fluorescent protein–labeled bone marrow to track recruitment. Following wire-induced femoral artery injury, &bgr;Gal(+) SMC accumulated in the intima and adventitia. Compared with wild-type, PTEN iKO mice exhibited massive neointima formation, increased replicating intimal and medial &bgr;Gal(+)SMC, and enhanced vascular recruitment of bone marrow cells following injury. Inhibiting SDF-1&agr; blocked these events and reversed enhanced neointima formation observed in PTEN iKO mice. Most recruited green fluorescent protein(+) cells stained positive for macrophage markers but not SMC markers. SMC-macrophage interactions resulted in a persistent SMC inflammatory phenotype that was dependent on SMC PTEN and SDF-1&agr; expression. Conclusion—Resident SMC play a multifaceted role in neointima formation by contributing the majority of neointimal cells, regulating recruitment of inflammatory cells, and contributing to adventitial remodeling. The SMC PTEN-SDF-1&agr; axis is a critical regulator of these events.

Inactivation of the tumour suppressor, PTEN, in smooth muscle promotes a pro-inflammatory phenotype and enhances neointima formation

AIMS Phosphatase and tensin homolog (PTEN) is implicated as a negative regulator of vascular smooth muscle cell (SMC) proliferation and injury-induced vascular remodelling. We tested if selective depletion of PTEN only in SMC is sufficient to promote SMC phenotypic modulation, cytokine production, and enhanced neointima formation. METHODS AND RESULTS Smooth muscle marker expression and induction of pro-inflammatory cytokines were compared in cultured SMC expressing control or PTEN-specific shRNA. Compared with controls, PTEN-deficient SMC exhibited increased phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signalling and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) activity, reduced expression of SM markers (SM-alpha-actin and calponin), and increased production of stromal cell-derived factor-1alpha (SDF-1alpha), monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), and chemokine (C-X-C motif) ligand 1 (KC/CXCL1) under basal conditions. PI3K/Akt or mTOR inhibition reversed repression of SM marker expression, whereas PI3K/Akt or NF-kappaB inhibition blocked cytokine induction mediated by PTEN depletion. Carotid ligation in mice with genetic reduction of PTEN specifically in SMC (SMC-specific PTEN heterozygotes) resulted in enhanced neointima formation, increased SMC hyperplasia, reduced SM-alpha-actin and calponin expression, and increased NF-kappaB and cytokine expression compared with wild-types. Lesion formation in SMC-specific heterozygotes was similar to lesion formation in global PTEN heterozygotes, indicating that inactivation of PTEN exclusively in SMC is sufficient to induce considerable increases in neointima formation. CONCLUSION PTEN activation specifically in SMC is a common upstream regulator of multiple downstream events involved in pathological vascular remodelling, including proliferation, de-differentiation, and production of multiple cytokines.

Acute Myeloid Leukemia-Targeted Toxin Activates Both Apoptotic and Necroptotic Death Mechanisms

Background Acute myelogenous leukemia (AML) is the second most common leukemia with approximately 13,410 new cases and 8,990 deaths annually in the United States. A novel fusion toxin treatment, diphtheria toxin GM-CSF (DT-GMCSF) has been shown to selectively eliminate leukemic repopulating cells that are critical for the formation of AML. We previously showed that DT-GMCSF treatment of U937 cells, an AML cell line, causes activation of caspases and the induction of apoptosis. Methods and Findings In this study we further investigate the mechanisms of cell death induced by DT-GMCSF and show that, in addition to the activation of caspase-dependent apoptosis, DT-GMCSF also kills AML cells by simultaneously activating caspase-independent necroptosis. These mechanisms depend on the ability of the targeted toxin to inhibit protein synthesis, and are not affected by the receptor that is targeted or the mechanism through which protein synthesis is blocked. Conclusions We conclude that fusion toxin proteins may be effective for treating AML cells whether or not they are defective in apoptosis.

Vascular Smooth Muscle Cell–Derived Transforming Growth Factor-β Promotes Maturation of Activated, Neointima Lesion–Like Macrophages

Objective—To define the contribution of vascular smooth muscle cell (SMC)–derived factors to macrophage phenotypic modulation in the setting of vascular injury. Approach and Results—By flow cytometry, macrophages (M4) were the predominant myeloid cell type recruited to wire-injured femoral arteries, in mouse, compared with neutrophils or eosinophils. Recruited macrophages from injured vessels exhibited a distinct expression profile relative to circulating mononuclear cells (peripheral blood monocytes; increased: interleukin-6, interleukin-10, interleukin-12b, CC chemokine receptor [CCR]3, CCR7, tumor necrosis factor-&agr;, inducible nitric oxide synthase, arginase 1; decreased: interleukin-12a, matrix metalloproteinase [MMP]9). This phenotype was recapitulated in vitro by maturing rat bone marrow cells in the presence of macrophage-colony stimulating factor and 20% conditioned media from cultured rat SMC (sM&phgr;) compared with maturation in macrophage-colony stimulating factor alone (M0). Recombinant transforming growth factor (TGF)-&bgr;1 recapitulated the effect of SMC conditioned media. Macrophage maturation studies performed in the presence of a pan-TGF-&bgr; neutralizing antibody, a TGF-&bgr; receptor inhibitor, or conditioned media from TGF-&bgr;–depleted SMCs confirmed that the SMC-derived factor responsible for macrophage activation was TGF-&bgr;. Finally, the effect of SMC-mediated macrophage activation on SMC biology was assessed. SMCs cocultured with sM&phgr; exhibited increased rates of proliferation relative to SMCs cultured alone or with M0 macrophages. Conclusions—SMC-derived TGF-&bgr; modulates the phenotype of maturing macrophages in vitro, recapitulating the phenotype found in vascular lesions in vivo. SMC-modulated macrophages induce SMC activation to a greater extent than control macrophages.

Nuclear PTEN functions as an essential regulator of SRF-dependent transcription to control smooth muscle differentiation.

Vascular disease progression is associated with marked changes in vascular smooth muscle cell (SMC) phenotype and function. SMC contractile gene expression and, thus differentiation, is under direct transcriptional control by the transcription factor, serum response factor (SRF); however, the mechanisms dynamically regulating SMC phenotype are not fully defined. Here we report that the lipid and protein phosphatase, PTEN, has a novel role in the nucleus by functioning as an indispensible regulator with SRF to maintain the differentiated SM phenotype. PTEN interacts with the N-terminal domain of SRF and PTEN–SRF interaction promotes SRF binding to essential promoter elements in SM-specific genes. Factors inducing phenotypic switching promote loss of nuclear PTEN through nucleo-cytoplasmic translocation resulting in reduced myogenically active SRF, but enhanced SRF activity on target genes involved in proliferation. Overall decreased expression of PTEN was observed in intimal SMCs of human atherosclerotic lesions underlying the potential clinical importance of these findings.

ESE-1 is Required to Maintain the Transformed Phenotype of MCF-7 and ZR-75-1 Human Breast Cancer Cells

Background: ETS factors comprise a large transcription factor family known to play a significant role in cellular development, differentiation, and transformation. The human Epithelial Specific Ets factor-1, ESE-1, is particularly relevant in breast cancer. Specifically, increased mRNA expression of ESE-1 and the Her2/neu proto- oncogene are correlated in breast cancer, and activation of the Her2/Neu receptor induces ESE-1 gene transcription. Stable expression of ESE-1 initiated transformation of ESE-1-negative MCF-12A immortalized human mammary epithelial cells, leading to increased migration, invasion and anchorage independent growth. However, little is known about ESE-1 protein expression and its role in maintaining the transformed phenotype in human breast cancer cell lines. Results: Here, we used an anti-ESE-1 mouse monoclonal antibody in Western blot and immunofluorescent cell analyses to show that ESE-1 is expressed as a nuclear protein in MCF-7, T47D and ZR-75-1 transformed, tumorigenic mammary epithelial cell lines, and that it is not expressed in transformed MDA-MB-231 and nontransformed MCF-10A and MCF-12A cells. In addition, specific knockdown of endogenous ESE-1 in the human breast carcinoma ZR-75-1 and MCF-7 cell lines decreased colony formation and anchorage independent growth. Mechanistically, ESE-1 knockdown decreased cellular proliferation, but had no effect on apoptosis. Conclusions: These results establish that the knockdown of a single ETS factor, ESE-1, is sufficient to reverse the transformed phenotype in breast cancer and demonstrate that ESE-1 is required for cellular proliferation. Thus, ESE-1 plays a key role in maintaining the transformed phenotype in breast cancer, providing a novel single-point target for therapy.

EGFR-targeted diphtheria toxin stimulates TRAIL killing of glioblastoma cells by depleting anti-apoptotic proteins

Current treatments for Glioblastoma multiforme (GBM) involve surgery, radiotherapy, and cytotoxic chemotherapy; however, these treatments are not effective and there is an urgent need for better treatments. We investigated GBM cell killing by a novel drug combination involving DT-EGF, an Epidermal Growth Factor Receptor-targeted bacterial toxin, and Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL) or antibodies that activate the TRAIL receptors DR4 and DR5. DT-EGF kills GBM cells by a non apoptotic mechanism whereas TRAIL kills by inducing apoptosis. GBM cells treated with DT-EGF and TRAIL were killed in a synergistic fashion in vitro and the combination was more effective than either treatment alone in vivo. Tumor cell death with the combination occurred by caspase activation and apoptosis due to DT-EGF positively regulating TRAIL killing by depleting FLIP, a selective inhibitor of TRAIL receptor-induced apoptosis. These data provide a mechanism-based rationale for combining targeted toxins and TRAIL receptor agonists to treat GBM.

Acute myeloid leukemia-targeted toxins kill tumor cells by cell type-specific mechanisms and synergize with TRAIL to allow manipulation of the extent and mechanism of tumor cell death

Acute myeloid leukemia-targeted toxins kill tumor cells by cell type-specific mechanisms and synergize with TRAIL to allow manipulation of the extent and mechanism of tumor cell death