ADAM22 Promotes Chemo-induced Metastasis And Chemoresistance In Gastric Cancer Through Interaction With Integrin β1

【Background】 Gastric cancer is the most common gastrointestinal malignancy in China and chemotherapy plays pivotal role in multidisciplinary management of it.The gastroenterologists have always been confused by the paradox that while chemotherapy is effective in reducing the tumor burden,it only slightly prolongs patients’ life span.This is mostly due to multidrug resistance which subsequently leads to tumor progression.It was reported that chemotherapy might promote metastasis by inducing EMT of tumor cells,remodeling tumor microenvironment and enriching cancer stem cells,in addition to their tumoricidal effect.So far,the effect of chemotherapy on gastric cancer metastasis is not well studied.Secretome is an important branch of proteomics that focuses on secreted and shed membrane-bound protein.The application of secretome to cancer research has facilitated the discovery of novel tumor biomarkers and provided a new technology for elucidating mechanisms underlying tumorigenesis and progression.【Objectives】To screen potential candidates that promote the metastatic capability of multidrug resistant gastric cancer cells and clarify the underlying mechanism.【Methods】 1.Transwell assay and the tail vein injection lung metastasis assay were employed to evaluate the in vitro and in vivo metastatic potential of chemoresistant and parental gastric cancer cells.Non-label quantitative mass spectrum was applied to screen differentially secreted protein in the serum free conditioned medium of SGC7901/ADR,SGC7901/VCR and SGC7901 cells,and the screening results was overlapped with a previously acquired m RNA profiling result.q RT-PCR and Western blot were used to validate the screening results in gastric and several other kinds of drug resistant cancer cell lines.The clinical significance of the potential candidate was evaluated by checking a public database.The induction of potential candidate expression by chemotherapeutics was examined using q RT-PCR and immunohistochemistry in cell lines and patient-derived gastric cancer xenografts(PDX)respectively.2.ADAM22 expression in SGC7901/ADR and SGC7901/VCR was silenced using specific si RNAs and the metastatic capability was measured using Transwell assay and tail vein injection lung metastasis assay.3.q RT-PCR and Western blot were used to detect classical EMT markers.The in vitro adhesion ability was measured on Fibronectin or Collagen Ⅰ coated plates and the formation of focal adhesion and F-actin fiber was detected using immunofluorescence.The retention of metastatic tumor cells in the lung was measured by tail vein injection of fluorescently labelled cells.The in vitro transendothelial assay and the Matrigel-on-Top three dimensional culture were used to evaluate the extravasation,propagation as well as FLPs formation capability of disseminated cancer cells.4.Total and phosphorylated FAK and paxillin was measured using standard Western blot protocol and the Integrin β1(ITGB1)activating epitope was measured under nonreducing condition.Active Rac1 and Cdc42 were enriched using the GST-pulldown assay and detected with standard Western blot.The alteration cell adhesion,migration and metastasis capability were detected using the in vitro adhesion assay,transwell assay and the tail vein injection assay after stimulatory ITGB1 antibody was added to ADAM22-silenced cells.The interaction between ADAM22 and ITGB1 was checked using immunoprecipitation.Endogenous ADAM22 was silenced using a si RNA targeting the 5’-UTR of ADAM22 m RNA and rescue of cell adhesion,migration and 3D propagation by wild type and Disintegrin domain-deleted ADAM22 were measured as described above.5.IC50 of various chemotherapeutics towards ADAM22,Rac1 or Cdc42-silenced SGC7901/ADR and SGC7901/VCR cells were measured and calculated using the MTT assay.Alteration of HIF1α and ABCB1 m RNA and the encoded protein levels in ADAM22,Rac1 or Cdc42-silenced SGC7901/ADR and SGC7901/VCR cells were detected using q RT-PCR and Western blot.6.The expression of ADAM22 and HUTS4 in primary and/or metastatic gastric cancer tissues were detected using immunohistochemistry.Nonparametric statistical analysis was applied to analyze ADAM22 expression in primary and metastatic gastric cancer tissue.Spearman correlation between the staining intensity of ADAM22 and HUTS4 in primary gastric cancer tissue was performed.Kaplan-Meier survival analysis and Cox multivariate regression was used to uncover the clinical significance of ADAM22 and HUTS4 expression in gastric cancer tissue.【Results】 1.The metastatic assay showed that chemoresistant gastric cancer cell displayed increased both in vitro and in vivo metastatic capability and intriguingly more and larger metastatic nodules were observed in lungs of mice received chemoresistant cell injection.The secretome analysis identified 91 proteins that concomitantly increased in the conditioned medium of SGC7901/ADR and SGC7901/VCR cells,of which 23 also displayed increased m RNA expression.q RT-PCR and Western blot results showed that ADAM22 expression was elevated not only in chemoresistant gastric cancer cells but also in chemoresistant leukemia and head and neck squamous cell carcinoma cells.Searching the prognosis information of ADAM22 expression in the Kaplan-Meier plot database showed that higher ADAM22 expression associated with shorter overall survival.q RT-PCR and immunohistochemistry results showed that ADAM22 expression can be induced by various chemotherapeutics in both gastric cancer cell lines and patient-derived xenograft tissues.2.Transwell and tail vein injection assay showed that inhibition of ADAM22 expression in SGC7901/ADR and SGC7901/VCR cells severely impaired their in vitro migration and in vivo metastasis potential.Less lung metastatic nodules and smaller metastatic nodule size were observed in the ADAM22-silenced cell injected mice.3.Classical EMT marker E-cadherin and Vimentin expression was not affected by ADAM22 inhibition.Adhesion ability of chemoresistant cells to extracellular matrix protein Fibronectin and Collagen Ⅰ as well as cell retention in the lung was reduced after downregulation of ADAM22 expression.Immunofluorescent staining results showed the focal adhesion and F-actin fiber formation was inhibited in the ADAM22-silenced SGC7901/ADR and SGC7901/VCR cells.Meanwhile,reduced transendothelial,Mo T propagation and FLPs formation were observed after ADAM22 downregulation in chemoresistant cells.4.Reduced phosphorylation of FAK and Paxillin were observed in ADAM22-silenced chemoresistant cells.GST-pulldown and Western blot results showed that the activity of Rho GTPase Rac1 and Cdc42 was decreased after ADAM22 inhibition using si RNA.The activity of ITGB1 was reduced as indicated by Western blot result recognized by the anti-HUTS4 epitope antibody in ADAM22 silenced cells.Activation of ITGB1 using a stimulatory antibody reversed the reduced adhesion and metastatic potential of chemoresistant cells resulting from ADAM22 downregulation.Co-immunoprecipitation results showed that ITGB1 interacted with wild type but not Disintegrin-deleted ADAM22.Echoing the co-immunoprecipitation result,only wild type ADAM22 rescued the reduced adhesion,migration and 3D propagation capability of SGC7901/ADR and SGC7901/VCR cell after inhibition of endogenous ADAM22 expression.7.Downregulation ADAM22,Rac1 or Cdc42 increased sensitivity of SGC87901/ADR and SGC7901 towards various chemotherapeutics as indicated by the IC50 value.q RT-PCR and Western blot results detected reduced expression of HIF1α and ABCB1 as well as the encoded protein.8.Immunohistochemistry staining results showed that ADAM22 expression was increased in the metastatic gastric cancer tissue than the primary cancer tissue.The expression of HUTS4 in gastric cancer tissue positively correlated with that of ADAM22.Kaplan-Meier survival analysis showed that high ADAM22 or HUTS4 expression correlated with poor prognosis.Multi-variate analysis using the Cox regression model showed that ADAM22/HUTS4 expression,tumor N staging and age served as independent poor prognosis indicators.【Conclusion】In the present study we showed that chemotherapeutics induced ADAM22 expression in gastric cancer cells which promoted metastasis through interacting with ITGB1 and increasing cell adhesion,transendothelial and propagation capability.This pathway also contributed to the multidrug resistance phenotype of gastric cancer cell via upregulating HIF1α and ABCB1 expression.This study provided evidence for the linkage between chemoresistance and metastasis with opportunity for intervention.