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Metastasis involves the interaction of the tumor, immune and endothelial cells. Cell death proteins, such as inhibitors of apoptosis proteins (IAPs), are critical players in survival, inflammation and permeability. Whether the use of Smac mimetics, which target cIAP1/2 for degradation would affect metastasis is unknown. We show Smac mimetics reduced metastasis due to the loss of cIAP1 but not cIAP2 in experimental metastasis models. The endothelial compartment rather than the immune cells was responsible for reduction of extravasation upon loss of cIAP1. Loss of cIAP1 in primary endothelial cells did not lead to cell death but resulted in an unresponsive endothelium barrier to permeability factors causing a reduction in tumor cell extravasation. Unexpectedly, the co-loss of TNFR1 and cIAP1 restored the tumor load. We were surprised to find lymphotoxin alpha (LTA), and not TNF, secreted by the tumor cells was critical for the extravasation. Using TCGA data, we found high levels of LTA mRNA expression correlated with decreased survival in kidney carcinoma and associated with advance disease stage. Our data suggest that Smac mimetics, targeting cIAP1/2, may reduce metastasis to the lung through a LTA/TNFR mechanism by altering the endothelial barrier and inhibiting the ability of tumor cells to extravasate.

Simple Summary The ability of tumor cells to spread from one site to another within a patient is a critical factor in cancer survival. Tumor cell migration or metastasis is a complex process, which involves several stages. In this study, we specifically examine the stage in which the tumor cell must exit the blood stream. We find that the loss of cIAP1, a member of the inhibitors of apoptosis protein family (IAPs), alters the ability of tumor cells to exit the blood vessel or extravasate. Endothelial cell viability did not appear to be affected in the absence of cIAP1. Instead, we identified that the loss of cIAP1 hampers the response of endothelial cells to signals from the tumor cells to change shape and allow for migration through the endothelial barrier. Abstract In this study, we determined whether Smac mimetics play a role in metastasis, specifically in circulation, tumor extravasation and growth in a metastatic site. Reports suggest inducing the degradation of IAPs through use of Smac mimetics, alters the ability of the tumor cell to metastasize. However, a role for the immune or stromal compartment in affecting the ability of tumor cells to metastasize upon loss of IAPs has not been defined. To address this open question, we utilized syngeneic tumor models in a late-stage model of metastasis. Loss of cIAP1 in the endothelial compartment, rather than depletion of cIAP2 or absence of cIAP1 in the hematopoietic compartment, caused reduction of tumor load in the lung. Our results underline the involvement of the endothelium in hindering tumor cell extravasation upon loss of cIAP1, in contrast to the immune compartment. Endothelial specific depletion of cIAP1 did not lead to cell death but resulted in an unresponsive endothelium barrier to permeability factors causing a decrease in tumor cell extravasation. Surprisingly, lymphotoxin alpha (LTA), and not TNF, secreted by the tumor cells, was critical for the extravasation. Using TCGA, we found high LTA mRNA expression correlated with decreased survival in kidney carcinoma and associated with advanced disease stage. Our data suggest that Smac mimetics, targeting cIAP1/2, reduce metastasis to the lung by inhibiting tumor cell extravasation.

SummaryRecent data suggests that LPS stimulation can trigger inflammasome activation through a TNFR2/TNF/TNFR1 mediated loop in xiap−/− macrophages. Yet, the direct role TNFR2-specific activation plays in the absence of XIAP is unknown. We found TNFR2-specific activation lead to cell death in xiap−/− myeloid cells, particularly in the absence the RING domain. RIPK1/TAK1 kinase activity downstream of TNFR2 resulted in a TNF/TNFR1 cell death independent of necroptosis. TNFR2-specific activation lead to a similar inflammatory NF-kB driven transcriptional profile as TNFR1 activation with the exception of up-regulation of NLRP3 and caspase-11. Activation and up-regulation of the canonical inflammasome was mediated by RIPK1 kinase activity and ROS production. While both RIPK1 kinase activity and ROS production reduced cell death as well as release of IL-1β, the release of IL-18 was not reduced to basal levels. This study supports, targeting TNFR2 specifically to reduce IL-18 release in XIAP deficient (XLP-2) patients.

Necroptosis is an inflammatory form of programmed cell death requiring receptor-interacting protein kinase 1, 3 (RIPK1, RIPK3) and mixed lineage kinase domain-like protein (MLKL). The kinase of RIPK3 phosphorylates MLKL causing MLKL to form a pore-like structure, allowing intracellular contents to release and cell death to occur. Alternatively, RIPK1 and RIPK3 have been shown to regulate cytokine production directly influencing inflammatory immune infiltrates. Recent data suggest that necroptosis may contribute to the malignant transformation of tumor cells in vivo and we asked whether necroptosis may have a role in the tumor microenvironment altering the ability of the tumor to grow or metastasize. To determine if necroptosis in the tumor microenvironment could promote inflammation alone or by initiating necroptosis and thereby influencing growth or metastasis of tumors, we utilized a syngeneic tumor model of metastasis. Loss of RIPK3 in the tumor microenvironment reduced the number of tumor nodules in the lung by 46%. Loss of the kinase activity in RIPK1, a member of the necrosome also reduced tumor nodules in the lung by 38%. However, the loss of kinase activity in RIPK3 or the loss of MLKL only marginally altered the ability of tumor cells to form in the lung. Using bone marrow chimeras, the decrease in tumor nodules in the Ripk3−/− appeared to be due to the stromal compartment rather than the hematopoietic compartment. Transmigration assays showed decreased ability of tumor cells to transmigrate through the vascular endothelial layer, which correlated with decreased permeability in the Ripk3−/− mice after tumor injection. In response to permeability factors, such as vascular endothelial growth factor, RIPK3 null endothelial cells showed decreased p38/HSP27 activation. Taken together, our results suggest an alternative function for RIPK1/RIPK3 in vascular permeability leading to decreased number of metastasis.