TOPK Inhibitor Inhibits The Promoting Effect Of Inflammation On Metastatic Colonization

Objective:The final stage of tumor metastasis,metastatic colonization,is the most appropriate stage of targeted therapy.A systemic inflammatory response induced by tumors or other factors can promote metastatic colonization or trigger the growth of dormant micrometastases.In this study,we investigated whether the inhibitor of TOPK(TOPKi)could keep tumor-suppressing function of neutrophils,inhibit their functional conversion to tumor-promoting,and thus prevent tumor metastatic colonization.Methods:(1)Mice were inoculated i.m.and i.v.with tumor cells,the therapeutic effects of different doses of TOPK inhibitor on tumor growth and metastasis and its toxicity were observed.The inhibitory effect of TOPK inhibitor and TOPK knockdown on tumor metastasis was compared by inoculating mice with normal tumor cells followed by TOPK inhibitor treatment,or inoculating mice with tumor cells stably expressing TOPK interference RNA.Air-LPS was used to establish chronic inflammation in mice(Air-LPS-mice).Air-LPS-mice were inoculated with B16F1 cells to observe whether the inflammation could promote tumor metastatic colonization and whether TOPK inhibitor could antagonize that promotion effect of inflammation.(2)The levels of inflammatory cytokines in serum of tumor-bearing mice and Air-LPS-mice were detected by ELISA.Air-LPS-mice were inoculated with B16F1 through tail vein,while anti-Ly6G antibody was injected intraperitoneally to deplete neutrophils in vivo.Then the role of neutrophils in the inflammation-promoted tumor metastasis was confirmed.Neutrophils were isolated from bone marrow or peritoneal cavity of mice by density gradient centrifugation.The animal model of co-inoculation with tumor cells and neutrophils was established to observe the effects of neutrophils from tumor-bearing mice and Air-LPS-mice treated with or without TOPK inhibitor on tumor development.(3)The expression of tumor promotion related genes in neutrophils from Air-LPS-mice treated with TOPK inhibitor was detected by real-time PCR.The gene expression of inflammatory cytokines and their protein level in serum were measured to estimate whether TOPK inhibitor could influence the production of inflammatory cytokines.The pG-CSF/pIL-6(pG/pI6)mice were prepared by intramuscular transfection of the eukaryotic expression vectors of mouse G-CSF and IL-6.The pG/pI6 mice were treated with TOPK inhibitor.Then,BM-PMNs and PC-PMNs were isolated and used for co-inoculation tests to evaluate the relationship between the TOPK inhibitor and functional conversion of neutrophils induced by G-CSF/IL-6.BM-PMNs or PC-PMNs from normal and pG/pI6 mice pretreated with TOPK inhibitor were stimulated by G-CSF/IL-6 or T-sMs.Real-time PCR was used to detect the expression of genes related to tumor promotion function to determine whether TOPK inhibitor antagonized the effect of G-CSF/IL-6 or tumor microenvironment on neutrophils.(4)BM-PMNs and PC-PMNs were isolated from Air-LPS-mice or pG/pI6 mice treated or untreated with TOPK inhibitor.The expression of Trail and Rab27a was detected by real-time PCR to observe the effect TOPK inhibitor on the antitumor functional gene expression of neutrophils.The peritoneal neutrophils were isolated and stimulated with T-sMs to observe whether TOPK inhibitor could make neutrophils primed by G-CSF/IL-6 maintain antitumor activity in tumor microenvironment;the expression of PI3K and p38 MAPK were detected by Western Blot.The activation rate of signal pathway was analyzed.The release rate of MPO and NE was used to represent the degranulation ability of PMNs,and the role TOPK inhibitor played during the activation of PI3K and p38 MAPK signal pathway was studied.(5)After pretreatment with TOPK or STAT3 inhibitor,BM-PMNs of normal mice were stimulated with G-CSF/IL-6;PC-PMNs from normal and Air-LPS inflammatory mice were stimulated with T-sMs,then the expressions of Mmp9,Bv8,Arg,iNos,Trail and Rab27a was detected by Real-time PCR.The effects of TOPK and STAT3 inhibitor on the pro-tumor and antitumor functions of neutrophils were compared.(6)After pretreatment with TOPK or STAT3 inhibitor,normal PC-PMNs were stimulated with G-CSF/IL-6 or T-sMs;BM-PMNs of Air-LPS inflammatory mice and pG/pI6 mice treated with TOPK inhibitor or STAT3 inhibitor;BM-PMNs of normal mice and Air-LPS inflammatory mice were pretreated with TOPK inhibitor or STAT3 inhibitor and then treated with G-CSF/IL-6;the expressions of p-STAT3,STAT3,p-TOPK and TOPK protein were detected by Western blot,or the mRNA level of Stat3 or Topk was detected by real-time PCR.The effects of TOPK and STAT3 on each other’s activation and production were observed.(7)The models of minimal dormant metastases were established through inoculation i.v.with non-metastatic tumor cells stimulated by TGF-β1/H2O2/HOCI(T/H/H),and then the Air-LPS inflammation were induced in the same mice,to observe whether the persistent inflammatory state could promote the formation of macro metastatic nodules,the role of neutrophils in the promoting process,and whether the TOPK inhibitor could inhibit this process or not.Results:(1)TOPK inhibitor showed dose-dependent tumor suppressing effect.The high dose(100 mg/kg body weight)had the best therapeutic effect,but the side-effects was obvious because the mice weight of that group was decreased significantly.Compared with muscle inoculation,the inhibitory effect of low dose(1 mg/kg body weight)on tumor metastasis was more obvious.The metastasis-depressing effect of TOPK inhibitor was superior to TOPK knockdown.Compared with normal mice,the number of lung nodules formed by B16F1 cells in the Air-LPS inflammatory mice was significantly increased,and tumor cells through extravasation in these mice did not increase;it had the best efficacy of metastatic colonization prevention that TOPK inhibitor was utilized from the onset of inflammation.(2)The serum levels of G-CSF,IL-6 and IL-1β were increased in tumor-bearing mice and Air-LPS inflammatory mice.Anti-LY6G could remove neutrophils effectively in vivo,tumor metastases could not form on the lung surface of the Air-LPS inflammatory mice whose neutrophils were eliminated totally.The BM-PMNs and PC-PMNs from normal mice suppressed tumor growth;the neutrophils from tumor-bearing mice and Air-LPS inflammatory mice promoted tumor growth,whereas the neutrophils from these two kinds of mice treated with TOPK inhibitor suppressed tumor too.(3)The expression of Mmp-9,Bv8,Arg and iNos gene in neutrophils of Air-LPS inflammatory mice was increased,and the expression of these genes was decreased after TOPK inhibitor treatment.There was no significant difference in G-CSF and IL-6 between TOPK inhibitor and non-treated group.BM-PMNs and PC-PMNs from pG/pI6 mice promoted tumor growth;the neutrophils from tumor-bearing mice and Air-LPS inflammatory mice promoted tumor growth,whereas the neutrophils from this kind of mice treated with TOPK inhibitor transformed into tumor growth inhibition.The expression of Mmp-9,Bv8,Argand iNos genes of normal BM-PMNs and PC-PMNs stimulated with G-CSF/IL-6 or T-sMs was increased,and pretreatment with TOPK inhibitor could significantly inhibited the increment of these four genes’ levels.The expression of Mmp-9,Bv8,Arg and iNos gene was further increased in PC-PMNs from pG/pI6 mice by T-sMs stimulation,but their expression was inhibited in the pG/pI6 mice with TOPK inhibitor treatment.(4)BM-PMNs in Air-LPS inflammatory mice,BM-PMNs and PC-PMNs in pG/pI6 mice,their Trail and Rab27a were lower,while treatment of TOPK inhibitor on these mice made the two genes restored to normal mouse levels.The PC-PMNs from pG/pI6 mice with TOPK inhibitor treatment,their Trail and Rab27a were increased after T-sMs stimulation.The activation of PI3K and p38 MAPK signaling pathways was inefficient in the PC-PMNs in Air-LPS inflammatory mice and pG/pI6 mice,with reduced release of MPO and NE.After TOPK inhibitor treatment,the phosphorylation levels of PI3K and P38 MAPK were the same as the normal mouse,while MPO and NE release increased.(5)Normal mice’s BM-PMNs,compared with stimulated by G-CSF/IL-6 only,the expression of Mmp-9,Bv8,Arg and iNos was decreased in TOPK inhibitor or STAT3 inhibitor pretreated group,and the expression of Trail and Rab27a was increased.PC-PMNs from normal mice and Air-LPS inflammatory mice,compared with T-sMs stimulation only,the expression of Mmp-9,Bv8,Arg and iNos in TOPK inhibitor or STAT3 inhibitor pretreatment group was significantly decreased,and the expression of Trail and Rab27a was increased.(6)G-CSF/IL-6 or T-sMs made the phosphorylation levels of p-STAT3 and p-TOPK protein increase,TOPK inhibitor or STAT3 inhibitor had no effect on p-STAT3 or p-TOPK respectively.In the BM-PMNs of Air-LPS inflammatory mice and pG/pI6 mice the levels of p-STAT3,STAT3,p-TOPK and TOPK were increased,while the expression of Stat3 and Topk genes was increased too.TOPK inhibitor therapy only inhibited the STAT3’s production;STAT3 inhibitor only inhibited TOPK’s production.G-CSF/IL-6 stimulated the expression of Stat3 and Topk in the BM-PMNs of normal mice and Air-LPS inflammatory mice,and the expression of Stat3 was inhibited by TOPK inhibitor pretreatment,STAT3 inhibitor also inhibited Topk expression.(7)B16F0 stimulated by TGF-β1/H2O2/HOC1(T/H/H)only formed minimal dormant metastases in normal mice,but visible metastatic nodules in Air-LPS inflammatory and EET mice.After exposure to TOPK inhibitor treatment,or removal of neutrophils by anti-LY6G in Air-LPS inflammatory mice could not make minimal dormant metastases develop into visible nodules.Conclusion:Inflammation promotes metastasis,in particular,promotes the formation of metastatic colonization,which can activate tumor minimal dormant metastases to develop into macro metastatic foci.In this process,the role of tumor promoting neutrophils induced by tumor or other inflammatory microenvironment was essential.TOPK inhibitors act on neutrophils in tumor or other inflammatory microenvironment,antagonize the induction of tumor or G-CSF/IL6,block functional transformation to tumor-promotion and maintain their function of tumor suppressor by inhibiting the expression of Mmp9,Bv8,Arg,iNos and boosting the anti-tumor function-related gene such as Trail and Rab27a,activating PI3K and p38 MAPK signaling pathway thoroughly which enhance the degranulation effect of neutrophils,and reducing the production of STAT3 simultaneously.So targeting TOPK of neutrophils can effectively prevent tumor metastatic colonization.