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 A/J 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 ubiquination 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 CSN5, which have the potential to enhance the ubiquitination of CYLD in lung tumor cells. Our recent data indicating that CYLD is not packed in exosomes isolated from the peripheral blood of lung cancer patients although it is present in exosomes from healthy volunteers indicate the potential clinical relevance of this model that directly links the effects of a carcinogen with an inflammatory response and promotion of tumor development. 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 (i) inhibition of macrophage differentiation and (ii) prevention of lung tumor exosome-mediated enhancement of urethane induced lung cancer. (2) Determine if other exosomal proteins that interact with CYLD participate in the ubiquitination of CYLD in TC-1 tumor cells and determine if the elimination of these proteins stabilizes exosomal CYLD and suppresses exosomal TRAF2 activation resulting in the inhibition of macrophage activation and prevention of tumor growth. (3) Determine if macrophages pulsed with lung tumor exosomes isolated from lung cancer patients promote lung tumor growth in a NOD-SCID mouse model. The data generated should permit the development of a highly innovative model of the cellular and molecular basis for exosome-mediated chronic inflammation and promotion of tumor growth and indicate novel preventive and therapeutic strategies.
The proposed research is highly relevant to characterizing the association of lung tumor exosomes mediated inflammatory responses and lung tumor growth in that it is focused on characterizing the molecular mechanisms underlying the ability of tumor exosomes to both activate macrophages and promote tumor growth. The central hypothesis is that exosomes produced by lung tumor cells, or lung tumor tissue are taken up by bone marrow myeloid precursor cells. These cells further differentiate into macrophages and migrate into the lung, promote the progression of urethane- induced lung cancer.