| Objective:Melanoma and hepatocellular carcinoma(HCC)are highly malignant cancers.To study functions of Pin1 in these cancers,we knocked down Pin1 protein by infecting cancer cell lines with lentivirus expressing validated sh RNA and examined its effects on protein substrates,cell behavior and tumor growth using animal model.Furthermore,we examined the anti-cancer activity of Pin1 inhibitor ATRA(all-trans retinoic acid)against melanoma and hepatocellular carcinoma in animal model,providing a rationale for potential clinical application of Pin1 inhibitors in cancer therapy.Methods:1.Effects of Pin1 knockdown on cell line behavior in melanoma and HCC in vitro:Cells were infected by lentivirus expressing Pin1 sh RNA or scrambled sh RNA and selected by drug.Cell morphology was then examined by miscroscopy;cell growth was evaluated by cell growth curve and colony formation assay;cell migration/invation was evaluated by cell monolayer scratch assay and transwell assay.2.Effects of Pin1 knockdown on regulating tumor growth and metastasis in melanoma and HCC in vivo: cancer cells were injected subcutaneously in nude mice and tumor voulume and weight were measured weekly or at the end point;cancer cells were injected through tail vein and the tumor spread to lung were live imaged by luciferase-based assay.3.Downstream factors mediating Pin1’s function in cancer: protein level of Pin1 downstream factors were analyzed by Western-blot;candidate downstream factors were downregualted by specific sh RNA and their effects on cell behaviror were examined by cell growth curve,scratch assay and transwell assay;furthermore,downstream factors were overexpressed to examine whether they could rescue effects of Pin1 knockdown on cells behavior;Pin1’s effects on cancer stem cells were analyzed by FACS using fluorescence conjugated specific protein markers.4.Preclinical study of Pin1 inhibitor ATRA: ATRA’s effects were siminlarly examined as Pin1 knockdown at molecular,cellular and animal levels.Results:1.Pin1 knockdown significantly affects cell behavior in melanoma and HCC in vitro: cancer cells morphology was althered after Pin1 knockdown;growth curve and colony formation assay showed that cell growth was retarded;scratch and transwell assay showed that cell migration/invation was suppressed.2.Tumor growth curve and tumor weight showed that tumor growth was siginificantly suppressed by Pin1 knockdown in melanoma and HCC.Animal live imaging showed that tumor metastasized to lung was significantly suppressed by Pin1 knockdown.3.Protein Western-blot showed that Notch1 is the mostly suppressed protein after Pin1 knockdown in melanoma;Notch1 knockdown showed similar results as Pin1 knockdown at in cancer cells.Notch1 overpression rescued/reversed cell behavior changes after Pin1 knockdown.Melanoma stem cells number was decreased after Pin1 and Notch1 knockdown.In HCC,Pin1 knockdown downregulated mulptiple cancer-driving pathways,therefore suppressed HCC growth.4.Pin1 inhibitor suppressed cancer cells growth and migration/invation,as evalulated by growth curves,scratch assay and transwell assay.FACS assay also showed Pin1 inhibitor treatment decreased cancer stem cells number.In HCC,Pin1 inhibitor ATRA needs to be stabilized by Liarozole to function.In tumor-bearing animal model,ATRA significantly suppressed tumor growth in dose dependent manner.Conclusions:1.Pin1 obviously regulates cancer cells growth and migration/invation in melanoma and HCC in vitro.Pin1 regulates tumorigenesis in melanoma and HCC,and regulates cancer metastasis in melanoma in animal model.Pin1 is a potential target for drug treatment.2.Pin1 functions mainly through regulating Notch1-mediated cancer stem cells in melanoma.Instead,Pin1 regulates multiple cancer-driving pathways in HCC.3.Pin1 inhibitor suppresses cells growth and migration/invation in vitro,decreases cancer stem cells and inhibits tumor growth and metastasis in vivo.Pin1 inhibitor has potential to be further developed into clinical drug. |
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