Amino acid influx via LAT1 regulates iron demand and sensitivity to PPMX-T003 of aggressive natural killer cell leukemia.

Aggressive natural killer cell leukemia (ANKL) is a rare hematological malignancy with a fulminant clinical course. Our previous study revealed that ANKL cells proliferate predominantly in the liver sinusoids and strongly depend on transferrin supplementation. In addition, we demonstrated that liver-resident ANKL cells are sensitive to PPMX-T003, an anti-human transferrin receptor 1 inhibitory antibody, whereas spleen-resident ANKL cells are resistant to transferrin receptor 1 inhibition. However, the microenvironmental factors that regulate the iron dependency of ANKL cells remain unclear. In this study, we first revealed that the anti-neoplastic effect of PPMX-T003 was characterized by DNA double-strand breaks in a DNA replication-dependent manner, similar to conventional cytotoxic agents. We also found that the influx of extracellular amino acids via LAT1 stimulated sensitivity to PPMX-T003. Taken together, we discovered that the amount of extracellular amino acid influx through LAT1 was the key environmental factor determining the iron dependency of ANKL cells via adjustment of their mTOR/Myc activity, which provides a good explanation for the different sensitivity to PPMX-T003 between liver- and spleen-resident ANKL cells, as the liver sinusoid contains abundant amino acids absorbed from the gut.

Anti-HBV drug entecavir ameliorates DSS-induced colitis through PD-L1 induction.

PD-L1-mediated signaling is one of the major processes that regulate local inflammatory responses in the gut. To date, protective effects against colitis through direct Fc-fused PD-L1 administration or indirect PD-L1 induction by probiotics have been reported. We have previously shown that the anti-HBV drug entecavir (ETV) induces PD-L1 expression in human hepatocytes. In the present study, we investigated whether ETV induces PD-L1 expression in intestinal cells and provides a protective effect against DSS-induced colitis. ETV induced PD-L1 expression in epithelial cells, rather than T and B cells, improving the symptoms of colitis. In the mechanistic analysis, Th17 cell differentiation was inhibited and B cell infiltration into the lamina propria was reduced. In addition, PD-L1 expression was positively correlated with Foxp3 or CSF1-R. In conclusion, ETV upregulated PD-L1 expression in epithelial cells and ameliorated inflammation in DSS-induced colitis. These results suggest that ETV may be a potential therapeutic agent as a PD-L1 enhancer for the treatment of human IBD.

DNA Damage Triggers the Nuclear Accumulation of RASSF6 Tumor Suppressor Protein via CDK9 and BAF53 To Regulate p53 Target Gene Transcription.

RASSF6, a member of the tumor suppressor Ras-association domain family (RASSF) proteins, regulates cell cycle arrest and apoptosis via p53 and plays a tumor suppressor role. We previously reported that RASSF6 blocks MDM2-mediated p53 degradation and enhances p53 expression. In this study, we demonstrated that RASSF6 has nuclear localization and nuclear export signals and that DNA damage triggers the nuclear accumulation of RASSF6. We found that RASSF6 directly binds to BAF53, the component of SWI/SNF complex. DNA damage induces CDK9-mediated phosphorylation of BAF53, which enhances the interaction with RASSF6 and increases the amount of RASSF6 in the nucleus. Subsequently, RASSF6 augments the interaction between BAF53 and BAF60a, another component of the SWI/SNF complex, and further promotes the interaction of BAF53 and BAF60a with p53. BAF53 silencing or BAF60a silencing attenuates RASSF6-mediated p53 target gene transcription and apoptosis. Thus, RASSF6 is involved in the regulation of DNA damage-induced complex formation, including BAF53, BAF60a, and p53.

The RAS-interacting chaperone UNC119 drives the RASSF6-MDM2-p53 axis and antagonizes RAS-mediated malignant transformation.

The gene encoding the proto-oncogene GTPase RAS is frequently mutated in human cancers. Mutated RAS proteins trigger antiapoptotic and cell-proliferative signals and lead to oncogenesis. However, RAS also induces apoptosis and senescence, which may contribute to the eradication of cells with RAS mutations. We previously reported that Ras association domain family member 6 (RASSF6) binds MDM2 and stabilizes the tumor suppressor p53 and that the active form of KRAS promotes the interaction between RASSF6 and MDM2. We also reported that Unc-119 lipid-binding chaperone (UNC119A), a chaperone of myristoylated proteins, interacts with RASSF6 and regulates RASSF6-mediated apoptosis. In this study, using several human cancer cell lines, quantitative RT-PCR, RNAi-based gene silencing, and immunoprecipitation/-fluorescence and cell biology assays, we report that UNC119A interacts with the active form of KRAS and that the C-terminal modification of KRAS is required for this interaction. We also noted that the hydrophobic pocket of UNC119A, which binds the myristoylated peptides, is not involved in the interaction. We observed that UNC119A promotes the binding of KRAS to RASSF6, enhances the interaction between RASSF6 and MDM2, and induces apoptosis. Conversely, UNC119A silencing promoted soft-agar colony formation, migration, and invasiveness in KRAS-mutated cancer cells. We conclude that UNC119A promotes KRAS-mediated p53-dependent apoptosis via RASSF6 and may play a tumor-suppressive role in cells with KRAS mutations.

UNC119 is a binding partner of tumor suppressor Ras-association domain family 6 and induces apoptosis and cell cycle arrest by MDM2 and p53.

Ras-association domain family 6 (RASSF6) is a tumor suppressor that interacts with MDM2 and stabilizes p53. Caenorhabditis elegans unc-119 encodes a protein that is required for normal development of the nervous system. Humans have 2 unc-119 homologues, UNC119 and UNC119B. We have identified UNC119 as a RASSF6-interacting protein. UNC119 promotes the interaction between RASSF6 and MDM2 and stabilizes p53. Thus, UNC119 induces apoptosis by RASSF6 and p53. UNC119 depletion impairs DNA repair after DNA damage and results in polyploid cell generation. These findings support that UNC119 is a regulator of the RASSF6-MDM2-p53 axis and functions as a tumor suppressor.

Domain analysis of Ras-association domain family member 6 upon interaction with MDM2.

The tumor suppressor Ras-association domain family member 6 (RASSF6) has Ras-association domain (RA) and Salvador/RASSF/Hippo domain (SARAH). RASSF6 antagonizes MDM2, stabilizes p53, and induces apoptosis and cell cycle arrest. We previously demonstrated the interaction between RASSF6 and MDM2, but did not determine how both proteins interact with each other. We have shown here that N-terminal, RA, and SARAH domains of RASSF6 interact with MDM2 at distinct regions. RA binds to the RING-finger region of MDM2 and stabilizes p53. SARAH binds RA and blocks the interaction between RA and MDM2. RA overexpression induces p53-dependent apoptosis and senescence. In the presence of active KRas, the interaction between RA and MDM2 is recovered. In this way, RA and SARAH play an important role in Ras-mediated regulation of p53.

Dynamics and plasticity of the epithelial to mesenchymal transition induced by miR-200 family inhibition.

Whereas miR-200 family is known to be involved in the epithelial-to-mesenchymal transition (EMT), a crucial biological process observed in normal and pathological contexts, it has been largely unclear how far the functional levels of these tiny RNAs alone can propagate the molecular events to accomplish this process within several days. By developing a potent inhibitor of miR-200 family members (TuD-141/200c), the expression of which is strictly regulatable by the Tet (tetracycline)-On system, we found using a human colorectal cell line, HCT116, that several direct gene target mRNAs (Zeb1/Zeb2, ESRP1, FN1and FHOD1) of miR-200 family were elevated with distinct kinetics. Prompt induction of the transcriptional suppressors, Zeb1/Zeb2 in turn reduced the expression levels of miR-200c/-141 locus, EpCAM, ESRP1 and E-Cad. The loss of ESRP1 subsequently switched the splicing isoforms of CD44 and p120 catenin mRNAs to mesenchymal type. Importantly, within 9 days after the release from the inhibition of miR-200 family, all of the expression changes in the 14 genes observed in this study returned to their original levels in the epithelial cells. This suggests that the inherent epithelial plasticity is supported by a weak retention of key regulatory gene expression in either the epithelial or mesenchymal states through epigenetic regulation.