Dual Targeting SHP2 And EphA2 Synergistically Inhibits Colorectal Cancer

ObjectsPatients with metastatic colorectal cancer(m CRC)are eligible for targeted therapy.However,the clinical application of anti-EGFR agents represented by Cetuximab are limited by mutations of downstream signals especially KRAS and BRAF,which occur in about 60%CRC.SHP2 inhibitors are reported to be effective in KRAS or BRAF mutant CRC with limited potency.Feedback activation of Eph A2 can be seen in multiple cell lines.We assume that dual inhibition of SHP2 and Eph A2 may exert synergistic effect in terms of treating CRC.Therefore,we conducted the following research:(1)Dynamic observation of p Eph A2(S897)following inhibition of SHP2 and verification of feedback activation of ERK-RSK1-Eph A2(S897)in CRC cell lines harboring KRAS or BRAF mutation;synergistic lethality of dual inhibition on CRC cells proven by proliferation assays(2)Pathways by which dual targeting SHP2 and Eph A2 exerts synergistic lethality on CRC cells and identification of pivotal downstream effectors(3)Analysis of prognostic value of p SHP2 and AURKA(downstream effector)expression in CRC patientsMethodsThe experimental methods are described below:(1)Six CRC cell lines harboring KRAS or BRAF mutation were treated with SHP2 inhibitor RMC-4550(10μM)and lysed after indicated points in time.The activity of p Eph A2(S897)and upstream candidates ERK,RSK1 and AKT was detected with immunoblot using corresponding antibodies against total proteins and phosphorylated counterparts.ERK-RSK1 was validated as upstream regulator of Eph A2 ligand-independent activity by treating CRC cells with ERK inhibitor and immunoblot assay.Then the anti-proliferation effect of Eph A2 inhibitor on KRAS or BRAF mutated CRC cells was verified by CCK-8 proliferation assay.The synergistic effect of dual inhibition of SHP2 and Eph A2 was examined by CCK-8 and colony formation assays.(2)CRC cell lines harboring KRAS or BRAF mutation were treated with SHP2 inhibitor RMC-4550(5μM),Eph A2 inhibitor ALW-Ⅱ-41-27(300n M)or the combination for 12 or 24 hours and lysed to detect the changes of ERK and AKT and their phosphorylated forms.Transcriptome dataset GSE120208 was adopted for pathway enrichment analyses GO and GSEA to identify pathways affected after knockdown of EPHA2.Downstream candidates AURKA and TPX2 were detected using samples indicated above by immunoblot and corresponding antibodies.(3)Formalin-fixed paraffin-embedded(FFPE)samples of CRC were adopted for immunohistochemistry stain to detect abundance of p SHP2(Y542)and AURKA.The stain of targeted proteins on every slide was scored.Patients’ clinicopathological information and follow-up data were collected for survival analyses to find the relationship between targeted protein abundance and survival.Results(1)We found the universal feedback activation of ERK-RSK1-Eph A2(S897)axis following SHP2 inhibition,suggesting the mechanism of insensitivity of CRC to SHP2 inhibitor treatment.Inhibition of ERK instead of AKT resulted in the suppression of RSK1 and Eph A2.Also,the dose response effect of Eph A2 inhibitor on CRC cell lines was independent of KRAS or BRAF status.The synergistic lethality of dual inhibition of SHP2 and Eph A2 was tested by proliferation assays.(2)Eph A2 inhibition alone resulted in suppressed AKT,while with combined SHP2 inhibition,more complete suppression was observed.Pathway enrichment analyses showed that pathways involving cell cycle and G2/M transition were downregulated with the knockdown of EPHA2 while immunity related pathways upregulated.AURKA and its direct upstream regulator TPX2 were downregulated transcriptionally following Eph A2 inhibition,suggesting they were both regulated by Eph A2 transcriptionally.Dual inhibition of SHP2 and AURKA resulted in similar effect on CRC cell lines harboring KRAS or BRAF mutation.(3)We detected p SHP2(Y542)and AURKA abundance in CRC FFPE samples with IHC stain and found no significant correlation between IHC score and clinicopathological information including primary location,histological grade,TNM stage,serum CEA and serum CA199.High IHC scores of p SHP2(Y542)and AURKA were both indicators of poor OS and DFS,and the prognosis of samples with coexisting high IHC scores of p SHP2(Y542)and AURKA was even worse,while accounting for a small proportion of patients.Conclusions(1)Targeted inhibition of SHP2 can induce feedback activation of ERK-RSK1-Eph A2 axis.Dual inhibition of SHP and Eph A2 resulted in synergistic lethality in CRC cell lines harboring KRAS or BRAF mutation.(2)Inhibition of Eph A2 was accompanied with downregulation of cell cycle pathways and upregulation of interferon response.Dual inhibition of SHP and Eph A2 suppressed AKT by blocking both RTKs effectors and AURKA-AKT circulation.Dual inhibition of SHP and AURKA exerted similar effect on CRC cell lines with Dual inhibition of SHP and Eph A2.(3)High p SHP2(Y542)and AURKA IHC scores were prognostic indicators of poor OS and DFS.Coexisting high IHC scores of p SHP2(Y542)and AURKA defined a small subgroup of CRC with the worst survival.