Epstein-Barr virus-encoded miR-BART11-3p modulates the DUSP6-MAPK axis to promote gastric cancer cell proliferation and metastasis.

Epstein-Barr virus (EBV)-encoded miRNAs within the BamHI-A rightward transcript (BART) region are abundantly expressed in EBV-associated gastric cancer (EBVaGC), suggesting that they play roles in tumorigenesis. However, how these viral miRNAs contribute to the development of EBVaGC remains largely obscure. In this study, we found that EBV-encoded miR-BART11-3p targets 3' -UTR of dual-specificity phosphatase 6 (DUSP6) mRNA to upregulate ERK phosphorylation and downregulate JNK and p38 phosphorylation. By doing so, miR-BART11-3p promotes gastric cancer (GC) cell proliferation, migration, and invasion in vitro, and facilitates tumor growth in vivo. Restoration of DUSP6 expression reverses the tumor-promoting activity of miR-BART11-3p in AGS GC cells. Consistently, knockdown of DUSP6 ablates the antitumor effects of miR-BART11-3p inhibitors in EBV-positive GC cells. Furthermore, blocking ERK phosphorylation with trametinib inhibited the proliferation, migration, and invasion of miR-BART11-3p-expressing AGS cells. Administration of a miR-BART11-3p antagomir reduced the growth of EBV-positive xenograft tumors. Together, these findings reveal a novel mechanism by which EBV dysregulates MAPK pathways through an EBV-encoded microRNA to promote the development and progression of EBVaGC, which may be harnessed to develop new therapeutics to treat EBVaGC. IMPORTANCE The Epstein-Barr virus (EBV) is the first human tumor virus found to encode miRNAs, which within the BART region have been detected abundantly in EBV-associated gastric cancer (EBVaGC) and play various roles in promoting tumorigenesis. In our study, we observed that EBV-miR-BART11-3p promotes cell proliferation and induces migration and invasion in GC. Interestingly, we showed that miR-BART11-3p upregulates p-ERK and downregulates p-JNK and p-p38 by directly targeting 3'-UTR of dual-specificity phosphatase 6 (DUSP6). Restoration of DUSP6 rescues the effects generated by miR-BART11-3p in GC cells, and blocking ERK phosphorylation with Trametinib augments JNK and p38 phosphorylation and inhibits the effects of miR-BART11-3p-expressing AGS cells, suggesting that miR-BART11-3p promotes cell proliferation, migration, and invasion by modulating DUSP6-MAPK axis in EBVaGC. The findings presented in this study provide new mechanisms into the tumorigenesis in EBVaGC and new avenues for the development of therapeutic strategies to combat EBVaGC targeting miR-BART11-3p or phospho-ERK.

DNAJC10 maintains survival and self-renewal of leukemia stem cells through PERK branch of the unfolded protein response.

Leukemia stem cells (LSC) require frequent adaptation to maintain their self-renewal ability in the face of longer exposure to cell-intrinsic and cell-extrinsic stresses. However, the mechanisms by which LSC maintain their leukemogenic activities, and how individual LSC respond to stress, remain poorly understood. Here, we found that DNAJC10, a member of HSP40 family, was frequently up-regulated in various types of acute myeloid leukemia (AML) and in LSC-enriched cells. Deficiency of DNAJC10 leads to a dramatic increase in the apoptosis of both human leukemia cell lines and LSC-enriched populations. Although DNAJC10 is not required for normal hematopoiesis, deficiency of Dnajc10 significantly abrogated AML development and suppressed self-renewal of LSC in the MLL-AF9-induced murine leukemia model. Mechanistically, inhibition of DNAJC10 specifically induces endoplasmic reticulum stress and promotes activation of PERK-EIF2α-ATF4 branch of unfolded protein response (UPR). Blocking PERK by GSK2606414 (PERKi) or shRNA rescued the loss of function of DNAJC10 both in vitro and in vivo. Importantly, deficiency of DNAJC10 increased sensitivity of AML cells to daunorubicin (DNR) and cytarabine (Ara-C). These data revealed that DNAJC10 functions as an oncogene in MLL-AF9-induced AML via regulation of the PERK branch of the UPR. DNAJC10 may be an ideal therapeutic target for eliminating LSC, and improving the effectiveness of DNR and Ara-C.

Establishment and characterization of two Epstein-Barr virus-positive gastric cancer cell lines with epitheliotropic M81 strain undergoing distinct viral and altered cellular expression profiles.

Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is a distinct subtype of gastric cancer (GC) distinguished by the presence of the EBV genome and limited viral gene expression within malignant epithelial cells. EBV infection is generally thought to be a relatively late event following atrophic gastritis in carcinogenesis, which implies the heterogeneity of EBVaGC. To facilitate the study of the role of EBV in EBVaGC, we established two EBV-positive GC cell lines (AGS-EBV and HGC27-EBV) with an epitheliotropic EBV strain M81 and characterized viral and cellular gene expression profiles in comparison to SNU719, a naturally derived EBV-positive GC cell line. Like SNU719, AGS-EBV and HGC27-EBV stably maintained their EBV genomes and expressed EBV-encoded small RNAs and nuclear antigen EBNA1. Comprehensive analysis of the expression of EBV-encoded miRNAs within the BamHI-A region rightward transcript region, and the transcripts of EBV latent and lytic genes in cell lines, as well as xenografts, reveals that AGS-EBV and HGC27-EBV cells undergo distinct viral expression profiles. A very small fraction of AGS-EBV and SNU719 cells can spontaneously produce infectious progeny virions, while HGC27-EBV does not. AGS-EBV (both M81 and Akata) cells largely mimic SNU719 cells in viral gene expression profiles, and altered cellular functions and pathways perturbed by EBV infection. Phylogenetic analysis of the EBV genome shows both M81 and Akata EBV strains are closely related to clinical EBVaGC isolates. Taken together, these two newly established EBV-positive GC cell lines can serve as models to further investigate the role of EBV in different contexts of gastric carcinogenesis and identify novel therapeutics against EBVaGC.

CLDN4 promotes growth of acute myeloid leukemia cells via regulating AKT and ERK1/2 signaling.

Acute myeloid leukemia (AML) is the most common acute leukemia affecting adults. The tight junction protein CLDN4 is closely related to the development of various epithelial cell carcinomas. However, whether CLDN4 contributes to AML development remains unclear. For the first time, we found that expression of CLDN4 is aberrantly up-regulated in AML cells. Knockdown of CLDN4 expression resulted in a dramatic decreased cell growth, elevated apoptosis of AML cells. Further, we revealed that knockdown of CLDN4 inhibits mRNA expression of PIK3R3 and MAP2K2, thus suppresses activation of AKT and ERK1/2. More importantly, activating AKT branch by SC79 partially compromised CLDN4 knockdown induced cell viability inhibition. In addition, we found that higher expression of CLDN4 is connected to worse survival and is an independent indicator of shorter disease free survival (DFS) in AML patients. Together, our results indicate that CLDN4 contributes to AML pathogenesis, and suggests that targeting CLDN4 is a promising option for AML treatment.

Overexpression of Fgf8 in the epidermis inhibits hair follicle development.

The hair follicle is a classical model for studying epithelial-mesenchymal interactions. Given the critical role of fibroblast growth factor 8 (Fgf8) in embryonic development, we generated a mouse model that overexpresses Fgf8 specifically in the epidermis. Interestingly, these mutant mice exhibited stunted, smaller bodies and severe hypotrichosis. Histological analysis showed that the hair follicles in the mutants were arrested at stage 2 of hair development. The density of hair follicles in the mutant mice was also lower compared to that in the control mice. Overexpression of Fgf8 inhibited the proliferation of epidermal cells and simultaneously promoted apoptosis, leading to the arrest of hair follicle development. Further analysis showed that sonic hedgehog (Shh) and bone morphogenetic protein 4 (Bmp4) were downregulated and upregulated, respectively. To summarize, our study demonstrates that FGF signalling plays an important role in the regulation of hair follicle development.

Kaempferol inhibits non-homologous end joining repair via regulating Ku80 stability in glioma cancer.

BACKGROUND: Targeting DNA damage response and DNA repair proficiency of cancers is an important anticancer strategy. Kaempferol (Kae), a natural flavonoid, displays potent antitumor properties in some cancers. However, the precise underlying mechanism of Kae regulates DNA repair system are poorly understood. PURPOSE: We aim to evaluate the efficacy of Kae in the treatment of human glioma as well as the molecular mechanism regarding DNA repair. STUDY DESIGN: Effects of Kae on glioma cells were detected using CCK-8 and EdU labeling assays. The molecular mechanism of Kae on glioma was determined using RNAseq. The inhibition effects of Kae on DNA repair were verified using Immunoprecipitation, immunofluorescence, and pimEJ5-GFP report assays. For in vivo study, orthotopic xenograft models were established and treated with Kae or vehicle. Glioma development was monitored by bioluminescence imaging, Magnetic Resonance Imaging (MRI), and brain sections Hematoxylin/Eosin (HE) staining. Immunohistochemical (IHC) analysis was used to detect expression of Ku80, Ki67 and γH2AX in engrafted glioma tissue. RESULTS: We found that Kae remarkably inhibits viability of glioma cells and decreases its proliferation. Mechanistically, Kae regulates multiple functional pathways associated with cancer, including non-homologous end joining (NHEJ) repair. Further studies revealed that Kae inhibits release of Ku80 from the double-strand breaks (DSBs) sites via reducing ubiquitylation and degradation of Ku80. Therefore, Kae significantly suppresses NHEJ repair and induces accumulation of DSBs in glioma cells. Moreover, Kae displays a dramatic inhibition effects on glioma growth in an orthotopic transplantation model. These data demonstrate that Kae can induce deubiquitination of Ku80, suppress NHEJ repair and inhibit glioma growth. CONCLUSION: Our findings indicate that inhibiting release of Ku80 from the DSBs by Kae may be a potential effective approach for glioma treatment.