Regulatory Mechanism And Anti-tumor Function Of Novel Inhibitors Of The Wnt And CXCL12/CXCR4 Signaling Pathways

[Background and significance]Cancer is a major threat to human health.Many malignant tumors exhibit fast growth,high metastasis rate and strong heterogeneous.It is of great significance to develop targeted therapy-based precision therapy against cancer.Abnormal activation of Wnt signaling is closely related to the occurrence and development of cancer.Targeted inhibition of Wnt signaling pathway is considered as a potential strategy for anti-tumor therapy.Although Wnt pathway has become an important target for the development of anti-tumor drugs,there is no approved therapeutic drugs targeting Wnt signaling pathway in clinical.Therefore,the discovery of novel Wnt pathway inhibitors with high efficacy and low-toxicity will pave the way for developing anti-tumor drugs.Tumor metastasis is the leading cause of cancer patients’ death.Chemokine(C-X-C motif)receptor 4(CXCR4)is upregulated in cancer tissues from patients and its high expression level is associated with poor prognosis in multiple cancers.Activation of CXCL12/CXCR4 signaling pathway promotes tumor metastasis.Interestingly,activation of CXCL12/CXCR4 enhances the accumulation of β-catenin in the Wnt signaling pathway.Furthermore,growing evidence suggests that inhibition of CXCR4 promotes efficacy of chemotherapy and tumor immunotherapy,and reduces the tumors metastasis.Given that the effect of single drug treatment in many tumors is not effective,the combination of multi-targeted treatment against tumors has attracted much attention.Therefore,it is of great significance to evaluate the anti-tumor effect of combined inhibition of Wnt and CXCL12/CXCR4 signaling pathways[Objective]This study has three aims:(1)to discover effective and low-toxic small molecule inhibitors of Wnt signaling pathway for the treatment of Wnt-activated tumors,and to understand how these inhibitors regulate Wnt signaling;(2)to identify effective CXCR4 inhibitors for the intervention of tumor metastasis;(3)to clarify the anti-tumor effect of a Wnt inhibitor combined with an CXCR4 inhibitor in tumor cells with Wnt activation and high expression of CXCR4.[Methods]We used the fluorescence report detection system to screen high-efficient small molecule inhibitors of Wnt signaling pathway,and investigated their action in both autocrine and paracrine Wnt activation pathways.Furthermore,we used Wnt-activated tumor cells,including teratoma PA-1 cells with continuous autocrine Wnt ligand protein and human pancreatic cancer AsPC-1 cells with mutated Rnf43,to clarify the potential targets of Wnt inhibitors in vitro.Established tumor xenograft mouse models to evaluate the effect of these Wnt inhibitors on tumor growth and Wnt signaling pathway in vivo.In addition,we screened for CXCR4 antagonists with high-affinity binding to CXCR4 based on the competitive binding assay.We further evaluated their inhibitory effects on migration and invasion of metastatic breast cancer MDA-MB-231 cells through wound healing experiment and transwell invasion experiment in vitro.We established mouse model of tumor metastasis by tail intravenous injection of tumor cells and further assessed the effect of a potent CXCR4 antagonist on tumor metastasis in vivo.At last,we assessed the anti-tumor effect of a Wnt inhibitor combined with a CXCR4 antagonist on the invasion and colony formation of tumor cells with Wnt activation and high expression of CXCR4.[Results](1)We identified PP327 as a potent small molecule inhibitor of Wnt signaling pathway.PP327 inhibits both autocrine and paracrine Wnt activation pathways PP327 blocks the PORCN-mediated Wnt protein secretion,and shows stronger inhibitory effect on Wnt signaling at the cellular level compared with LGK974,which is a PORCN inhibitor under phase II clinical trial.(2)PP327 effectively inhibited Wnt signaling pathway activation both in vivo and in vitro,evidenced by reduced levels of LRP6 and Dvl2 phosphorylation and the decreased expression of Wnt target gene Axin2.(3)Within the tolerated dose range,PP327 exerts significant anti-tumor activity in Wnt-activated cancers including self-activated teratomas and Rnf43-mutated pancreatic cancer.(4)We also developed an effective CXCR4 antagonist CXC188,which significantly reduced the invasion and migration of tumor cells in vitro.(5)CXC188 effectively inhibits tumor cell metastasis to the lung in mice and displays no obvious side effect in an effective dose.(6)In tumor cells with Wnt activation and high expression of CXCR4,the combination of PP327 and CXC188 leads to stronger inhibition of the invasion and clonal growth in tumor cells compared with the single agent.[Conclusion]In summary,we discovered PP327 as a potent inhibitor of Wnt signaling pathway by preventing PORCN-mediated Wnt protein secretion.PP327 has effective anti-tumor activity in vitro and in vivo against multiple models of Wnt-activated tumors.Moreover,we identified an effective oral CXCR4 antagonist CXC188,which potently represses tumor cell invasion and migration in vitro,and tumor metastasis in vivo.Most importantly,the combination of PP327 and CXC188 exerts synergistic anti-tumor effect.