Molecular Mechanism Of Molecules Released From Damaged Tumor Cells Promoting Tumor Cell Metastasis And Suppressing The Inhibitory Effect Of αvβ3Targeted Agent

The damaged or injury tumor cells produced by chemotherapy or radiotherapyreleased DAMPs, many of these molecules are endogenous TLR4ligand. It has beenreported that TLR4expressed not only on immune cells but also on many types oftumor cells, the activation of TLR4on tumor cell could promotes tumor growth,proliferation and evading immune surveilliance. However, it is still not clear whetherthe TLR4ligand released by damaged tumor cells influence the tumor invasivemetastasis and the mechanism of this effect.In the present study, we used DTC-Ms (molecules from damaged tumor cells) toinvestigate the effect of TLR4ligand on tumor cell metastasis. The resultsdemonstrated that DTC-Ms promoted tumor cell invasive migration.To verify whetherthe promoting-effect was mediated by TLR4ligand, we further downregulated theTLR4on tumor cell and found that DTC-Ms promoted tumor invasion throughactivating TLR4signaling pathway. DTC-Ms enhanced tumor cell adhesion abilitythrough upregulating the expression of integrin αvβ3and increasing its activity. Thenwe investigated whether DTC-Ms influenced the effect of CH50, a peptide whichinhibits tumor metastasis through targeting integrin αvβ3. The results indicated thatDTC-Ms attenuated the inhibitory effect of CH50.Triggering of TLR4could activate several signaling pathways in cells, includingNF-κB, TRIF, PI3K, and p38MAPK pathways. To ascertain which signal pathwaywas crucial for the effect of TLR4signaling in tumor cell, we investigated the effectof TLR4ligand in presence of QNZ (NF-κB inhibitor), resveratrol (TRIF inhibitor), wortmannin (PI3K inhibitor), and SB203580(p38MAPK inhibitor). QNZ completelyabrogated the promoting effect of DTC-Ms, whereas other inhibitors only slightlyinfluenced its effect, suggesting that the activation of NF-κB was crucial for thepromoting effect of TLR4ligand on tumor cell invasive migration. In addition, QNZelinilated the promotig-effect of DTC-Ms on expression and activity of αvβ3andabrogated the antagonizing-effect of DTC-Ms on CH50. All of these results suggestthat TLR4ligand released by damaged tumor cell play an active role in tumormetastasis by activating TLR4/NF-κB siganing pathway and NF-κB inhibitor couldabrogate the promoting-effect of TLR4ligand.Survival of tumor cells in blood circulation is crucial in tumor cell metastaticspread. Prolonging the survival of disseminating tumor cells in the circulation directlyincreases metastasis. Next, we investigated whether the TLR4ligand releasd bydamaged tumor cells could influence the tumor cell survival in blood circulation. Ourresults indicate that DTC-Ms enhance tumor cell anti-apoptosis ability and reduce theapoptosis of tumor cells in blood, increase the expression of Bcl-xL and inhibite theexpression of Bax in tumor cell, and NF-κB inhibitor abrogate the effect of DTC-Ms.we further found that DTC-Ms enhanced tumor cell proliferation capability afterextavasated into new organs and resulted in more information of colonies in the lung.Moreover, DTC-Ms eliminated the CH50inhibitory effect on tumor metastasis. Takentogether, these results indicate that TLR4ligand increases tumor cell anti-apoptosisability to promote tumor cell survival and extravasation in blood and antagonizes thetherapeutic effect of CH50by increasing the apoptosis-resistance and proliferation oftumor cells after migrating into target tissue.The above data suggest that NF-κB inhibitor abrogates the promoting-effect ofDTC-Ms on tumor cell metastasis and survival, we then investiaged whether NF-κBinhibitor could synergistically enhance the anti-metastasis effect of CH50.Considering the safety and practical clinical application, we used proteasome inhibitors, bortezomib to inhibit NF-κB activity. The results of in vivo experimentsindicate that combination of bortezomib and CH50eliminated TLR4ligand effect andeffectively inhibited tumor metastasis and prolonged the mice survival.In our study, we investigated the impact of TLR4ligand released by damagedtumor cells on metastasis and tried to explore the underlying mechanisms and thepotential therapeutic target to eliminate the effect of TLR4signaling. Our resultsshowed that TLR4ligand enhanced the tumor cell anti-apoptosis ability and survivalin blood circulation through TLR4/NF-κB signaling pathway, promoted theexpression and activity of αvβ3, antagonized the inhibitory effect of CH50. NF-κBinhibitor abolished the negative effect of TLR4ligand on CH50treatment, suggestingthat NF-κB inhibitor potentiates the efficacy of αvβ3integrin targeting drugs and thetreatment of αvβ3integrin antagonist with NF-κB inhibitor effectively prevents tumormetastasis.Our study provides effective therpy strategy for clinical targeted tumortherpy.