KMT2D Deficiency Enhances The Anti-cancer Activity Of L48H37 In Pancreatic Cancer And Its Mechanisms

Background and purpose: 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 have also been elucidated.However,the relationship between these two factors in the treatment of pancreatic cancer remains unknown.Our working is to determine the anti-cancer effects of L48H37 in PDAC and the role of KMT2 D on its therapeutic efficacy.Methods: The viability and proliferation of primary(PANC-1 and Mia Pa Ca-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-PE/7-AAD,JC-1,DCFH-DA and PI respectively,and flow cytometric acquisition.The protein and m RNA levels of relevant factors was analyzed using Western blotting,immunofluorescence and real time-quantitative PCR.In vitro migration was assessed by the wound healing assay.Lentiviral-mediated knockdown of KMT2 D was combined with L48H37,and changes in apoptosis and associated signal transduction proteins were detected by the above experimental techniques.The in situ expression of KMT2 D in human PDAC and paired adjacent normal tissues were determined by immunohistochemistry(IHC).In vivo tumor xenografts were established by injecting nude mice with PDAC cells.Bioinformatics analyses were also conducted using public databases such as GEO,TCGA,and Oncomine.Results: L48H37 inhibited the proliferation and induced apoptosis in human pancreatic cancer metastatic cell lines in a dose-and timedependent manner,along with reducing MMP,increasing ROS levels,arresting cell cycle at the G2/M stages and activating the endoplasmic reticulum(ER)stress-associated PERK/EIF2A/ATF4/CHOP signaling pathway.Knocking down activating transcription factor 4(ATF4)significantly upregulated KMT2 D in the PDAC cells,along with decreasing L48H37-induced apoptosis.Furthermore,silencing KMT2D in the L48H37-treated cells significantly augmented apoptosis and the ER stress pathway,indicating that KMT2 D depletion is essential for the anti-neoplastic effects of L48H37.The subcutaneous xenografts of mice formed by KMT2 D knockdown PDAC cells significantly reduced tumor burden after combined use of L48H37 compared to control knockdown.We also identified differentially expressed genes(DEGs)in KMT2 D knockdown and control knockdown PDAC cell lines,which were categorized into the regulating exogenous apoptotic signal transduction and the homeostasis in multicellular organisms in terms of function and pathway enrichment results.KMT2 D high-and low-expressing PDAC patients from the TCGA database showed similar survival rates,but higher KMT2 D expression was found to be associated with poor tumor grading in clinical and pathological analysis.Furthermore,the level of KMT2 D methylation was not correlated with its m RNA level,but hypermethylation status of KMT2 D was associated with the history of chronic pancreatitis.Conclusions: L48H37 exerts a potent anti-cancer effect in PDAC,which is augmented by KMT2 D deficiency.The expression of KMT2 D was higher in PDAC tissues and the patients with higher expression of KMT2 D had poor tumor grading.The hypermethylation status of KMT2D was correlated with the history of chronic pancreatitis.