Defective actin accumulation impairs human natural killer cell function in patients with dedicator of cytokinesis 8 deficiency.

BACKGROUND: Dedicator of cytokinesis 8 (DOCK8) mutations are responsible for a rare primary combined immunodeficiency syndrome associated with severe cutaneous viral infections, increased IgE levels, autoimmunity, and malignancy. Natural killer (NK) cells are essential for tumor surveillance and defense against virally infected cells. NK cell function relies on Wiskott-Aldrich syndrome protein for filamentous actin (F-actin) accumulation at the lytic NK cell immunologic synapse. DOCK8 activates cell division cycle 42, which, together with Wiskott-Aldrich syndrome protein, coordinates F-actin reorganization. Although abnormalities in T- and B-cell function have been described in DOCK8-deficient patients, the role of NK cells in this disease is unclear. OBJECTIVES: We sought to understand the role of DOCK8 in NK cell function to determine whether NK cell abnormalities explain the pathogenesis of the clinical syndrome of DOCK8 deficiency. METHODS: A cohort of DOCK8-deficient patients was assembled, and patients' NK cells, as well as NK cell lines with stably reduced DOCK8 expression, were studied. NK cell cytotoxicity, F-actin content, and lytic immunologic synapse formation were measured. RESULTS: DOCK8-deficient patients' NK cells and DOCK8 knockdown cell lines all had decreased NK cell cytotoxicity, which could not be restored after IL-2 stimulation. Importantly, DOCK8 deficiency impaired F-actin accumulation at the lytic immunologic synapse without affecting overall NK cell F-actin content. CONCLUSIONS: DOCK8 deficiency results in severely impaired NK cell function because of an inability to form a mature lytic immunologic synapse through targeted synaptic F-actin accumulation. This defect might underlie and explain important attributes of the DOCK8 deficiency clinical syndrome, including the unusual susceptibility to viral infection and malignancy.

Conditional expression of the mutant Ki-rasG12C allele results in formation of benign lung adenomas: development of a novel mouse lung tumor model.

To determine the effects of expression of mutant Ki-ras on lung tumorigenesis, we developed a bitransgenic mouse model that expresses the human Ki-ras(G12C) allele in alveolar type II and/or Clara cells in a tetracycline-inducible, lung-specific manner. Expression of Ki-ras(G12C) caused multiple, small lung tumors over a 12-month time period. Although tumor multiplicity increased upon continued Ki-ras expression, most lung lesions were hyperplasias or well-differentiated adenomas. This is in contrast to the more severe phenotypes observed in other transgenic mouse models in which different mutant Ki-ras alleles were expressed in the lung. Expression of Ki-ras(G12C) was associated with a 2-fold increase in the activation of the Ras and Ral signaling pathways and increased phosphorylation of Ras downstream effectors, including Erk, p90 ribosomal S6 kinase, ribosomal S6 protein, p38 and MAPKAPK-2. In contrast, expression of the transgene had no effect on the activation of the JNK and Akt signaling pathways. Withdrawal of doxycycline for 1 month resulted in almost a complete absence of proliferative pulmonary lesions, suggesting tumor regression in the absence of Ki-ras expression. Mutant Ki-ras(G12C) expression was sufficient for initial lung tumor transformation, required for maintenance of tumor phenotype, and induced transformation of lung epithelial cells by the activation of multiple effector pathways. These results describe a novel mouse lung tumor model demonstrating benign tumor development in the absence of tumor progression, which will provide a new tool for understanding the early stages of lung tumor pathogenesis.

Tuberous sclerosis-associated neoplasms express activated p42/44 mitogen-activated protein (MAP) kinase, and inhibition of MAP kinase signaling results in decreased in vivo tumor growth.

PURPOSE: Tuberous sclerosis (TS) is a common autosomal disorder attributable to inactivation of the tumor suppressor genes tuberin and hamartin. To determine whether mitogen-activated protein (MAP) kinase signaling plays a role in the pathogenesis of TS, we stained human TS-associated neoplasms with antibodies directed against activated MAP kinase, and observed high-level expression. EXPERIMENTAL DESIGN: To determine whether MAP kinase is functionally important for the development of neoplasia in TS, we established a murine model of TS-associated neoplasia (Tsc2Ang1 cells) from a tumor arising in a mouse heterozygous for tuberin. Tsc2Ang1 cells demonstrate tumorigenesis in vivo and high-level expression of activated MAP kinase in vitro. The functionality of MAP kinase signaling was assessed by inactivating MAP kinase using a dominant-negative MAP kinase kinase in tsc2ang1 cells and assessing the effect of this intervention on in vivo tumorigenicity and production of the potent angiogenic factor vascular endothelial growth factor (VEGF). RESULTS: Human TS-related neoplasms demonstrate high-level expression of activated MAP kinase, as does a tumor arising in a mouse heterozygous for tuberin. The inhibition of MAP kinase signaling by the introduction of a dominant-negative MAP kinase kinase leads to the inhibition of tumor growth in vivo and decreased production of VEGF. CONCLUSIONS: MAP kinase is activated in TS-related neoplasia in mice and humans. Inhibition of MAP kinase leads to decreased tumor growth in vivo. Pharmacological inhibition of MAP kinase may be a therapeutic target in the prevention and treatment of TS-related tumors.