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

Abstract

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 histone-lysine 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 KMT2D-knockdown 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.

Keywords: Bioinformatics; Curcumin analog; Histone methyltransferase 2D; Pancreatic neoplasms; Therapeutic effects.

Figure 1

L48H37 inhibits proliferation and promotes apoptosis in pancreatic cancer cells. A-D: Percentage of viable SW1990, ASPC-1, PANC-1 and MIA PaCa₂ cells incubated with different concentrations of L48H37 (1.25, 2.5, 5, 10, 20 and 40 μmol/L) or DMSO (negative control) for 24, 48 and 72 h. The IC₅₀ values in the different cell lines are shown; E: Representative pictures of colonies from SW1990 and ASPC-1 cells treated with increasing concentrations of L48H37 for 24 h; F-G: SW1990 and Aspc-1 cells were treated with various concentrations of L48H37 for 24 h. Cell apoptosis was detected by flow cytometry. Histogram illustrating the rate of apoptosis cells. Data were expressed as mean ± SEM; F: ^bP < 0.01 vs L48H37 (0 μmol/L) group. ^dP < 0.01 vs L48H37 (10 μmol/L) group. ^fP < 0.01 vs L48H37 (15 μmol/L) group; G: ^bP < 0.01 vs L48H37 (0 μmol/L) group; ^dP < 0.01 vs L48H37 (5 μmol/L) group. ^fP < 0.01 vs L48H37 (10 μmol/L) group; H-I: Western blotting showing expression levels of Cleaved caspase3, Bcl-2 and Bax proteins in SW1990 and Aspc-1 cells following treatment with DMSO or L48H37 for 12 h. Grayscale values of Bcl-2 and Bax were measured relative to β-actin, and the ratio of Bcl-2/Bax expression was calculated. Data were expressed as mean ± SEM; H: ^bP < 0.01 vs L48H37 (0 μmol/L) group. ^dP < 0.01 vs L48H37 (10 μmol/L) group. ^fP < 0.01 vs L48H37 (15 μmol/L) group; I: ^bP < 0.01 vs L48H37 (0 μmol/L) group. ^dP < 0.01 vs L48H37 (5 μmol/L) group. ^fP < 0.01 vs L48H37 (10 μmol/L) group.

Figure 2

L48H37 alters mitochondrial membrane potential, reactive oxygen species levels, cell cycle profile arrest and endoplasmic reticulum stress pathway in pancreatic ductal adenocarcinoma cells. A, B: Representative images and FACS plots of SW1990 cells treated with 5, 10 and 15 μmol/L L48H37 and stained with JC-1 probe (200× magnification); C, E: Reactive oxygen species (ROS) generation induced by L48H37 within 12 h was measured in SW1990 and Aspc-1 cells by staining with DCFH-DA (25 μmol/L) for 30 min. ROS level was acquired by flow cytometry. Histogram showing the DCFH-DA geometric mean in cells treated with different L48H37 concentrations. Data were expressed as mean ± SEM; D: ^bP < 0.01 vs DMSO group. ^dP < 0.01 vs L48H37 (10 μmol/L) group; E: ^bP < 0.01 vs DMSO group. ^dP < 0.01 vs L48H37 (5 μM) group. ^eP < 0.05 vs L48H37 (10 μmol/L) group; F-H: SW1990 and Aspc-1 cells were harvested 24 h with L48H37 or DMSO, and then cycle distribution was assessed by Propidium Iodide staining. Histogram illustrating the rate of G2/M phase cells. Data were expressed as mean ± SEM; G: ^bP < 0.01 vs DMSO group. ^dP < 0.01 vs L48H37 (10 μmol/L) group. ^fP < 0.01 vs L48H37 (15 μmol/L) group; H: ^bP < 0.01 vs DMSO group. ^dP < 0.01 vs L48H37 (5 μmol/L) group; I-K: SW1990 cells were treated with L48H37 for the indicated times or treated with various concentrations of L48H37 or DMSO. The protein levels of p-PERK, PERK, p-EIF2α, EIF2α, ATF4, CHOP, cleaved caspase-9, and cleaved caspase-8 were analyzed by Western blot. β-actin was used as an internal control.

Figure 3

ATF-4 knockdown attenuates L48H37-induced apoptosis and upregulates KMT2D. A, B: KMT2D mRNA and protein levels in the control and ATF-4-knockdown SW1990 and ASPC-1 cells treated with 15 µM L48H37 for 12 h. β-actin was used as an internal control. Data were expressed as mean ± SEM. ^bP < 0.01 vs siCTRL + DMSO group. ^dP < 0.01 vs siCTRL + L48H37 group; C: Representative IF image showing KMT2D expression in control and ATF4-knockdown SW1990 cells treated with L48H37; D: FACS plot showing apoptosis in control and ATF4 knockdown SW1990 cells treated with L48H37; E, F: Western blotting showing levels of p-PERK, PERK, p-EIF2α, EIF2α, ATF4 and CHOP in control and ATF4 knockdown SW1990 cells treated with L48H37. β-actin was used as an internal control. PERK: Protein kinase RNA-like endoplasmic reticulum kinase; EIF2A: Eukaryotic initiation factor 2α; ATF-4: Activating transcription factor-4; CHOP: Enhancer-binding protein-homologous protein.

Figure 4

KMT2D knockdown enhances apoptosis in pancreatic ductal adenocarcinoma cells. A-C: Immunoblot showing KMT2D and H3K4me1 protein levels in SW1990 and ASPC-1 cells treated with 15 µM L48H37 for 8 h. β-actin and Histone H3 were used as an internal control. The KMT2D mRNA levels were measured by RT-qPCR 24 h later. Data were expressed as mean ± SEM. ^aP < 0.05 vs DMSO group; D, E: KMT2D mRNA and protein levels in control and KMT2D-knockdown SW1990 and ASPC-1 cells treated with L48H37 (5, 10 and 15 μmol/L) or DMSO for three consecutive days, and percentage of viable cells. Data were expressed as mean ± SEM. ^aP < 0.05 vs shCTRL group at the same time point. ^bP < 0.01 vs shCTRL group at the same time point; F: Apoptosis rates in the control and KMT2D-knockdown SW1990 and ASPC-1 cells; G, H: Western blotting showing levels of p-PERK, PERK, p-EIF2α, EIF2α, ATF4 and CHOP in the control and KMT2D-knockdown SW1990 and ASPC-1 cells. β-actin was used as an internal control; I, J: shKMT2D or shCTRL SW1990 cells were harvested 24 h with L48H37, then cycle distribution was assessed by Propidium Iodide staining. Histogram showing the percentage of control and KMT2D knockdown SW1990 and ASPC-1 cells in the G0/G1, S, and G2/M phases. Data were expressed as mean ± SEM; J: ^bP < 0.01 vs shCTRL group in the same cell cycle. ^dP < 0.01 vs shKMT2D group in the same cell cycle. PERK: Protein kinase RNA-like endoplasmic reticulum kinase; EIF2A: Eukaryotic initiation factor 2α; ATF-4: Activating transcription factor-4; CHOP: Enhancer-binding protein-homologous protein.

Figure 5

KMT2D knockdown augments L48H37-induced apoptosis and blocks migration in vitro. A, B: Venn diagram showing overlapping DEGs in KMT2D knockdown Colo357 and SUIT-2 cells. Upregulated genes are shown in blue and red, and the downregulated in yellow and green in the Colo357 and SUIT-2 cells, respectively; C, D: Functional analysis of the overlapping DEGs; E, F: Graph showing the number of manually tracked migrating control and KMT2D knockdown SW1990 cells treated with L48H37.

Figure 6

KMT2D knockdown synergizes with L48H37 to inhibit pancreatic ductal adenocarcinoma xenograft growth in vivo. A: The gross image of resected tumors; B: Effect of 5 mg/kg L48H37 treatment on wet tumor weight in relatively independent four sub-groups (shCTRL group, shKMT2D group, shCTRL + 5 mg/kg L48H37 group and shKMT2D + 5 mg/kg L48H37 group). Data were expressed as mean ± SEM. ^bP < 0.01 vs shCTRL group. ^cP < 0.05 vs shKMT2D group. ^eP < 0.05 vs shCTRL + 5 mg/kg L48H37 group; C: Tumor volumes recorded at different follow-up times in the above four sub-groups. Data were expressed as mean ± SEM. P < 0.01 (shCTRL group vs shKMT2D group; shCTRL group vs shCTRL + 5 mg/kg L48H37 group; shCTRL group vs shKMT2D + 5 mg/kg L48H37 group; shKMT2D group vs shKMT2D + 5 mg/kg L48H37 group; shCTRL + 5 mg/kg L48H37 group vs shKMT2D + 5 mg/kg L48H37 group).

Figure 7

The prognostic value of the KMT2D in pancreatic ductal adenocarcinoma. A-D: Representative immunohistochemistry images showing KMT2D or H3K4me1 expression in PDAC tissues and paired normal tissues (200× and 400× magnification). The histogram shows the percentage of positively-stained area as calculated by Fiji. Data were expressed as mean ± SEM. ^bP < 0.01 vs shCTRL group; E, F: Box-plot graph showing KMT2D expression in tumor and normal tissues from GEPIA and Oncomine databases; G, H: Kaplan-Meier curves showing overall survival and disease-free survival of TCGA database patients based on KMT2D expression.