(B) Statistical evaluation of the unusual nuclei in (A). PTK2 inhibition-induced suffered degrees of ATG3 could actually sensitize cancers cells to DNA-damaging realtors. Our findings fortify the idea that concentrating on PTK2 in conjunction with DNA-damaging realtors is normally a novel technique for cancers therapy. gene expire within the first d after birth with reduced amino acid levels.19 However, it is unknown whether ATG3 is also involved in an autophagy-independent biologic function. PTK2/FAK (protein tyrosine kinase 2) is usually a cytoplasmic protein tyrosine kinase that is overexpressed and activated in several advanced-stage solid cancers.20 It can promote glucose consumption, lipogenesis, and glutamine dependency to promote cancer cell proliferation, motility, and survival.21 Targeting PTK2 in endothelial cells is sufficient to induce tumor cell sensitization to DNA-damaging therapies by downregulating the NFKB/NF-B pathway.22 Small molecule PTK2 inhibitors (PTK2-Is) prevent tumor progression in mice and are being evaluated in clinical trials.23-27 However, the greatest efficacy of PTK2-Is has been observed in combination with other tyrosine kinase inhibitors28,29 or cytotoxic drugs,30,31 but the real mechanism has yet to be fully revealed. In this study, we found that in response to malignancy chemotherapeutic agent treatment, PTK2 induced ATG3 phosphorylation, which led to its significant degradation but was not associated with the induction of autophagy. In addition, PTK2 inhibition caused a sustained level of ATG3, leading to a significant decrease in cell viability. These results implicate ATG3 phosphorylation in the maintenance of cell viability in response to DNA damage and also support the notion that targeting PTK2 in combination with chemotherapy is usually a novel malignancy therapeutic strategy. Results ATG3 is usually degraded during DNA damage treatment ATG proteins have been reported to exert autophagy-independent functions. For example, Ctsb ATG5 expression is usually induced by DNA-damaging brokers and promotes mitotic catastrophe impartial of autophagy.32 Therefore, we examined several ATG protein levels in response to treatment with DNA-damaging drugs. Human colon cancer cell lines HCT116 and LoVo were treated with etoposide for 3?h or cisplatin for 6?h, washed, and incubated with fresh medium. As shown in Physique?1ACD, among the ATG proteins tested, only ATG3 protein levels were gradually decreased after etoposide or cisplatin treatment. To verify whether this phenomenon was cell type-dependent, the levels of ATG proteins were also measured in the cervical malignancy cell collection HeLa and osteosarcoma cell collection U2OS after etoposide treatment. Consistent with the previous results, ATG3 was decreased at the protein level (Physique?S1A-B). Furthermore, we used irradiation (IR) or camptothecin to treat HCT116 cells and found that this is a general phenomenon that occurs in response to DNA damage inducers (Physique?S1C-D). Open in a separate window Physique 1. ATG3 is usually degraded in response to treatment with DNA-damaging drugs. (A) HCT116 cells were treated with DMSO or etoposide (40?M) for 3?h and then incubated with fresh medium for the indicated time. Western blotting was performed to detect different ATG proteins. (B) HCT116 cells were treated with etoposide at numerous concentrations for 3?h and then incubated with fresh medium for 48?h. (C, D) Cisplatin (10?M) (C) or etoposide (40?M) (D) were introduced into HCT116 or LoVo cells, respectively. Cells were then treated as explained in (A). (E, F) HCT116 cells were treated with etoposide (E) or cisplatin (F) as indicated, and then quantitative PCR (qPCR) was used to measure the mRNA levels.The bands were quantified with ImageJ software. specific inhibitor or siRNA, PTK2 (protein tyrosine kinase 2) was confirmed to catalyze the phosphorylation of ATG3 at Y203. Furthermore, a newly recognized function of ATG3 was recognized to be associated with the promotion of DNA damage-induced mitotic catastrophe, in which ATG3 interferes with the function of BAG3, a crucial protein in the mitotic process, by binding. Finally, PTK2 inhibition-induced sustained levels of ATG3 were able to sensitize malignancy cells to DNA-damaging brokers. Our findings strengthen the notion that targeting PTK2 in combination with DNA-damaging brokers is usually a novel strategy for malignancy therapy. gene pass away within the first d after birth with reduced amino acid levels.19 However, it is unknown whether ATG3 is also involved in an autophagy-independent biologic function. PTK2/FAK (protein tyrosine kinase 2) is usually a cytoplasmic protein tyrosine kinase that is overexpressed and activated in several advanced-stage solid cancers.20 It can promote glucose consumption, lipogenesis, and glutamine dependency to promote cancer cell proliferation, motility, and survival.21 Targeting PTK2 in endothelial cells is sufficient to induce tumor cell sensitization to DNA-damaging therapies by downregulating the NFKB/NF-B pathway.22 Small molecule PTK2 inhibitors (PTK2-Is) prevent tumor progression in mice and are being evaluated in clinical trials.23-27 However, the greatest efficacy of PTK2-Is has been observed in combination with other tyrosine kinase inhibitors28,29 or cytotoxic drugs,30,31 but the real mechanism has yet to be fully revealed. In this study, we found that in response to malignancy chemotherapeutic agent treatment, PTK2 induced ATG3 phosphorylation, which led to its significant degradation but was not associated with the induction of autophagy. In addition, PTK2 inhibition caused a sustained level of ATG3, leading to a significant decrease in cell viability. These results implicate ATG3 phosphorylation in the maintenance of cell viability in response to DNA damage and also support the notion that targeting PTK2 in combination with chemotherapy is usually (Z)-9-Propenyladenine a novel malignancy therapeutic strategy. Results ATG3 is usually degraded during DNA damage treatment ATG proteins have been reported to exert autophagy-independent functions. For example, ATG5 expression is usually induced by DNA-damaging brokers and promotes mitotic (Z)-9-Propenyladenine catastrophe impartial of autophagy.32 Therefore, we examined several ATG protein levels in response to treatment with DNA-damaging drugs. Human colon cancer cell lines HCT116 and LoVo were treated with etoposide for 3?h or cisplatin for 6?h, washed, and incubated with fresh medium. As shown in Physique?1ACD, among the ATG proteins tested, only ATG3 protein levels were gradually decreased after etoposide or cisplatin treatment. To verify whether this phenomenon was cell type-dependent, the levels of ATG proteins were also measured in the cervical malignancy cell collection HeLa and osteosarcoma cell collection U2OS after etoposide treatment. Consistent with the previous results, ATG3 was decreased at the protein level (Physique?S1A-B). Furthermore, we used irradiation (IR) or camptothecin to treat HCT116 cells and found that this is a general phenomenon that occurs in response to DNA damage inducers (Physique?S1C-D). Open in a separate window Physique 1. ATG3 is usually degraded in response to treatment with DNA-damaging drugs. (A) HCT116 cells were treated with DMSO or etoposide (40?M) for 3?h and then incubated with fresh medium for the indicated time. Western blotting was performed to detect different ATG proteins. (Z)-9-Propenyladenine (B) HCT116 cells were treated with etoposide at numerous (Z)-9-Propenyladenine concentrations for 3?h and then incubated with fresh medium for 48?h. (C, D) Cisplatin (10?M) (C) or etoposide (40?M) (D) were introduced into HCT116 or LoVo cells, respectively. Cells were then treated as explained in (A). (E, F) HCT116 cells were treated with etoposide (E) or cisplatin (F) as indicated, and then quantitative PCR (qPCR) was used to measure the mRNA levels of mRNA levels (Physique?1ECF), protein degradation might be responsible for the decrease in ATG3 protein levels in response to DNA-damaging drug treatment. To determine the pathways involved in the ATG3 degradation, we pretreated HCT116 cells with a panel of inhibitors that included the proteasome inhibitor MG132 and the lysosome inhibitor chloroquine (CHQ). Treatment with MG132 significantly blocked the etoposide-induced reduction of ATG3 levels, but CHQ experienced no such effect on ATG3 degradation (Physique?1G), suggesting that ATG3 is degraded by the proteasomal pathway in response to etoposide treatment. Next, to determine whether ubiquitination is required for ATG3 degradation, an HA-ubiquitin plasmid was transfected into HCT116 cells; ATG3 ubiquitination was detected by co-IP with an anti-ATG3 antibody followed by immunoblotting with an anti-HA antibody. As shown in Physique?1H, ATG3-conjugated ubiquitin after etoposide treatment.