KMT2D deficiency enhances the anti-cancer activity of L48H37 in pancreatic ductal adenocarcinoma

摘要

BACKGROUND Novel therapeutic strategies are urgently needed for patients with a delayed diagnosis of pancreatic ductal adenocarcinoma(PDAC)in order to improve their chances of survival.Recent studies have shown potent anti-neoplastic effects of curcumin and its analogues.In addition,the role of histone methyltransferases on cancer therapeutics has also been elucidated.However,the relationship between these two factors in the treatment of pancreatic cancer remains unknown.Our working hypothesis was that L48H37,a novel curcumin analog,has better efficacy in pancreatic cancer cell growth inhibition in the absence of histonelysine N-methyltransferase 2D(KMT2D).AIM To determine the anti-cancer effects of L48H37 in PDAC,and the role of KMT2D on its therapeutic efficacy.METHODS The viability and proliferation of primary(PANC-1 and MIA PaCa-2)and metastatic(SW1990 and ASPC-1)PDAC cell lines treated with L48H37 was determined by CCK8 and colony formation assay.Apoptosis,mitochondrial membrane potential(MMP),reactive oxygen species(ROS)levels,and cell cycle profile were determined by staining the cells with Annexin-V/7-AAD,JC-1,DCFH-DA,and PI respectively,as well as flow cytometric acquisition.In vitro migration was assessed by the wound healing assay.The protein and mRNA levels of relevant factors were analyzed using Western blotting,immunofluorescence and real time-quantitative PCR.The in situ expression of KMT2D in both human PDAC and paired adjacent normal tissues was determined by immunohistochemistry.In vivo tumor xenografts were established by injecting nude mice with PDAC cells.Bioinformatics analyses were also conducted using gene expression databases and TCGA.RESULTS L48H37 inhibited the proliferation and induced apoptosis in SW1990 and ASPC-1 cells in a dose-and time-dependent manner,while also reducing MMP,increasing ROS levels,arresting cell cycle at the G2/M stages and activating the endoplasmic reticulum(ER)stress-associated protein kinase RNA-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/activating transcription factor 4(ATF4)/CHOP signaling pathway.Knocking down ATF4 significantly upregulated KMT2D in PDAC cells,and also decreased L48H37-induced apoptosis.Furthermore,silencing KMT2D in L48H37-treated cells significantly augmented apoptosis and the ER stress pathway,indicating that KMT2D depletion is essential for the anti-neoplastic effects of L48H37.Administering L48H37 to mice bearing tumors derived from control or KMT2Dknockdown PDAC cells significantly decreased the tumor burden.We also identified several differentially expressed genes in PDAC cell lines expressing very low levels of KMT2D that were functionally categorized into the extrinsic apoptotic signaling pathway.The KMT2D high-and low-expressing PDAC patients from the TCGA database showed similar survival rates,but higher KMT2D expression was associated with poor tumor grade in clinical and pathological analyses.CONCLUSION L48H37 exerts a potent anti-cancer effect in PDAC,which is augmented by KMT2D deficiency.