The overall goal of this proposal is to elucidate the mechanisms by which lung tumor exosomes promote an inflammatory response, namely the activation of macrophages, and promote the progression and growth of lung tumors. Our preliminary data indicate that pretreatment of A/J mice with exosomes produced by TC-1 lung tumor cells or exosomes isolated from the lung tissue of NJ mice that have been pretreated with the carcinogen urethane results in more rapid tumor growth and earlier progression of lung carcinomas. Urethane treatment results in the recruitment of the activated form of TRAF2 to the exosomes, and the exosomes with activated TRAF2 are taken up by the bone marrow-derived precursors of macrophages, leading to their maturation and subsequent migration to the lung. The recruitment of the activated TRAF2 to the exosomes requires degradation of the inflammation suppressor protein, CYLD, in the tumor cells;moreover, TRAF2 can mediate ubiquitination of CYLD thereby promoting its degradation. The results of siRNA TRAF2 knockout indicated, however, that TRAF2 is required but is not sufficient for ubiquitination of CYLD. We have now identified two exosomal proteins that interact with TRAF2, Itch and Jabi, which have the potential to enhance the ubiquitination of CYLD in lung tumor cells. We propose to: (1) Confirm the role of lung tumor exosomal TRAF2 in the promotion of urethane-induced lung carcinomas by determining whether knockout of TRAF2 in TC-1 lung tumor cells is sufficient for (0 inhibition of macrophage differentiation and (ii) prevention of TC-1 cell exosome-mediated enhancement of urethane induced lung cancer. (2) Determine if Jabi and itch in the TC-1 exosomes are capable of promoting the differentiation of CD1 1bGr1 cells into activated macrophages. (3) Identification of cells targeted in vivo by exosomes. The data generated should permit the development a highly innovative model of the cellular and molecular basis for exosome-mediated chronic inflammation and promotion of lung tumor growth and suggest novel preventive and therapeutic strategies.
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