A Crosstalk Between Castration-Resistant Prostate Cancer Cells, M2 Macrophages, and NK Cells: Role of the ATM-PI3K/AKT-PD-L1 Pathway.

Castration-resistant prostate cancer (CRPC) in males is associated with a poor prognosis and a higher risk of treatment-related adverse effects, with high mortality among cancers globally. It is thus imperative to explore novel potential molecules with dual therapeutic and biomarker functions. Based on the recent research findings, the expression levels of ataxia telangiectasia mutant kinase (ATM) in prostate cancer (PC) tissues collected from CRPC patients were higher than hormone-dependent PC patients. Using CRPC cell lines (C4-2 and CWR22Rv1), the transwell chamber experiments revealed ATM promoted macrophage recruitment in CRPC cells in vitro via C-X-C motif chemokine ligand 12 (CXCL12). Further in vitro investigations demonstrated that polarized macrophages prevented NK cell recruitment and reduced the immunocidal activity of NK cells against CRPC cell lines. Moreover, ATM boosted programmed death receptor ligand 1 (PD-L1) expression while inhibiting NK group 2D (NKG2D) ligand expression in selected cell lines via PI3K/AKT signaling pathway. The in vivo investigations revealed ATM induced proliferation of CRPC and macrophage recruitment, while the NK cell recruitment was found to suppress ATM expression and CRPC proliferation. In conclusion, it could be demonstrated that inhibiting ATM increased the susceptibility of CRPC to NK cell inhibitors by dampening the CXCL12 and PI3K/AKT-PD-L1 pathways, thereby offering a novel and individualized treatment protocol for treating CRPC.

Kinetochore protein MAD1 participates in the DNA damage response through ataxia-telangiectasia mutated kinase-mediated phosphorylation and enhanced interaction with KU80.

Objective: Mitotic arrest-deficient protein 1 (MAD1) is a kinetochore protein essential for the mitotic spindle checkpoint. Proteomic studies have indicated that MAD1 is a component of the DNA damage response (DDR) pathway. However, whether and how MAD1 might be directly involved in the DDR is largely unknown. Methods: We ectopically expressed the wild type, or a phosphorylation-site--mutated form of MAD1 in MAD1 knockdown cells to look for complementation effects. We used the comet assay, colony formation assay, immunofluorescence staining, and flow cytometry to assess the DDR, radiosensitivity, and the G2/M checkpoint. We employed co-immunoprecipitation followed by mass spectrometry to identify MAD1 interacting proteins. Data were analyzed using the unpaired Student's t-test. Results: We showed that MAD1 was required for an optimal DDR, as knocking down MAD1 resulted in impaired DNA repair and hypersensitivity to ionizing radiation (IR). We found that IR-induced serine 214 phosphorylation was ataxia-telangiectasia mutated (ATM) kinase-dependent. Mutation of serine 214 to alanine failed to rescue the phenotypes of MAD1 knockdown cells in response to IR. Using mass spectrometry, we identified a protein complex mediated by MAD1 serine 214 phosphorylation in response to IR. Among them, we showed that KU80 was a key protein that displayed enhanced interaction with MAD1 after DNA damage. Finally, we showed that MAD1 interaction with KU80 required serine 214 phosphorylation, and it was essential for activation of DNA protein kinases catalytic subunit (DNA-PKcs). Conclusions: MAD1 serine 214 phosphorylation mediated by ATM kinase in response to IR was required for the interaction with KU80 and activation of DNA-PKCs.

Human Kinase/Phosphatase-Wide RNAi Screening Identified Checkpoint Kinase 2 as a Cellular Factor Facilitating Japanese Encephalitis Virus Infection.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes acute encephalitis in humans with high mortality. Not much is known about the interactions between viral and cellular factors that regulate JEV infection. By using a kinase/phosphatase-wide RNAi screening approach, we identified a cell cycle-regulating molecule, checkpoint kinase 2 (CHK2), that plays a role in regulating JEV replication. JEV infection induced G1 arrest and activated CHK2. Inactivation of CHK2 and its upstream ataxia-telangiectasia mutated kinase in JEV-infected cells by using inhibitors reduced virus replication. Likewise, JEV replication was significantly decreased by knockdown of CHK2 expression with shRNA-producing lentiviral transduction. We identified CHK2 as a cellular factor participating in JEV replication, for a new strategy in addressing JEV infection.