PLA2G16 is a mutant p53/KLF5 transcriptional target and promotes glycolysis of pancreatic cancer

Abstract

PLA2G16 is a member of the phospholipase family that catalyses the generation of lysophosphatidic acids (LPAs) and free fatty acids (FFAs) from phosphatidic acid. In the current study, we explored the functional role of PLA2G16 in pancreatic adenocarcinoma (PAAD) and the genetic/epigenetic alterations leading to its dysregulation. Bioinformatic analysis was performed using data from The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx) and the Human Protein Atlas (HPA). Then, PANC-1 and MIA-PaCa-2 cells harbouring TP53 mutations were used for cellular and animal studies. Results showed that PL2G16 expression was significantly up-regulated in PAAD tissue and was associated with unfavourable survival. PLA2G16 inhibition suppressed pancreatic cell growth in vitro and in vivo and also inhibited aerobic glycolysis. Bioinformatic analysis indicated that KLF5 was positively correlated with PLA2G16 expression in PAAD tumours with TP53 mutation. TP53 or KLF5 inhibition significantly reduced PLA2G16 expression at both mRNA and protein levels. Dual-luciferase and chromatin Immunoprecipitation-quantitative polymerase chain reaction assays showed that KLF5 directly bound to the PLA2G16 promoter and activated its transcription. Co-immunoprecipitation assay indicated that mutant p53 had a physical interaction with KLF5. Inhibition of mutant p53 impaired the transcriptional activating effects of KLF5. In PAAD cases in TCGA, PLA2G16 expression was positively correlated with its copy number (Pearson's r = 0.51, P < 0.001), but was strongly and negatively correlated with the methylation level of cg09518969 (Pearson's r = -0.64, P < 0.001), a 5'-cytosine-phosphodiester bond-guanine-3' site within its gene locus. In conclusion, this study revealed a novel mutant p53/KLF5-PLA2G16 regulatory axis on tumour growth and glycolysis in PAAD.

Keywords: KLF5; PLA2G16; glycolysis; mutant p53; pancreatic cancer.

FIGURE 1

Aberrant PLA2G16 expression was associated with unfavourable survival of pancreatic adenocarcinoma (PAAD). A and B, Schematic diagram (A) and violin chart (B) showing the exonic and intronic structure of PLA2G16 transcripts and the expression of the protein‐coding transcripts in primary PAAD cases in The Cancer Genome Atlas (TCGA) and normal pancreas in GTEx. C and D, Comparison of PLA2G16 mRNA expression in different grades of PAAD (C) and between the cases with or without historical chronic pancreatitis (D) in TCGA. E, Representative images of IHC staining of PLA2G16 in normal pancreas and PAAD tissues. Image credit: Human Protein Atlas, from https://www.proteinatlas.org/ENSG00000176485‐PLA2G16/tissue/pancreas and https://www.proteinatlas.org/ENSG00000176485‐PLA2G16/pathology/pancreatic+cancer#ihc. F, Representative images of IHC staining of PLA2G16 in normal pancreas, pancreatitis and PAAD in human tissue array. PBS was used as negative control. Fibrosarcoma tissue was used as positive control. G and H, Summary of PLA2G16 protein expression score of 10 PAAD cases examined in the Human Protein Atlas (G) and 16 PAAD cases in the tissue array (H). I‐K, K‐M survival analysis of progression‐free survival (PFS) (I), disease‐specific survival (DSS) (J) and overall survival (OS) (K) in PAAD cases extracted from TCGA Pan‐Cancer, by median PLA2G16 expression separation

FIGURE 2

PLA2G16 enhances pancreatic cell growth in vitro and in vivo. A and B, quantitative real‐time PCR (qRT‐PCR) (A) and Western blot (B) analysis of PLA2G16 expression in PANC‐1 and MIA‐PaCa‐2 cells 48 h (qRT‐PCR)/72 h (Western blot) after lentiviral‐mediated PLA2G16 knockdown. C and D, cell count kit assay of the proliferation of PANC‐1 (C) and MIA‐PaCa‐2 (D) cells with or without PLA2G16 knockdown. E and F, Representative image (up) and quantification (down) of colony formation of PANC‐1 (E) and MIA‐PaCa‐2 (F) cells with or without PLA2G16 knockdown. G‐I, Representative image (G) and quantification (H, I) of flow cytometric analysis of apoptotic PANC‐1 and MIA‐PaCa‐2 cells 48 h after lentiviral‐mediated PLA2G16 knockdown. J and K, Representative images (J) of xenograft tumour developed by MIA‐PaCa‐2 cells with or without PLA2G16 knockdown and the corresponding tumour growth curve (K)

FIGURE 3

PLA2G16 enhances glycolysis in pancreatic cancer cells. A, Group stratification for single‐gene gene set enrichment analysis in primary pancreatic adenocarcinoma from The Cancer Genome Atlas (TCGA) Pan‐Cancer and summary of gene set enrichment in the high PLA2G16 expression group. B‐E, Measurement of extracellular acidification rates (ECAR) (B, C) and oxygen consumption rate (OCR) (D, E) in PANC‐1 (B, D) and MIA‐PaCa‐2 (C, E) cells with or without PLA2G16 knockdown. F and G, Measurement of lactate production (F) and glucose uptake (G) in PANC‐1 and MIA‐PaCa‐2 cells with or without PLA2G16 knockdown. Intracellular glucose levels were measured and normalized based on protein concentration. FCCP, carbonyl cyanide 4‐(trifluoromethoxy) phenylhydrazone

FIGURE 4

Both mutant p53 and KLF5 increase PLA2G16 expression in pancreatic cancer. A, A heatmap showing TP53 mutation and PLA2G16 expression in pancreatic adenocarcinoma (PAAD) cases in The Cancer Genome Atlas (TCGA). B, A violin plot chart comparing PLA2G16expression between PAAD cases with or without TP53 mutations. C, Flow chart showing the screening process to identify PLA2G16 correlated TFs in TP53 mutant PAAD cases. D and E, Comparison of KLF5 mRNA expression between normal pancreas in GTEx and PAAD in TCGA (D) and between PAAD cases with or without TP53 mutations (E). F, Representative images of KLF5 protein expression in PAAD tissue. Image credit: Human Protein Atlas, from: https://www.proteinatlas.org/ENSG00000102554‐KLF5/pathology/pancreatic+cancer. G and H, Plot charts showing the correlation between PLA2G16 and KLF5 mRNA expression in TP53 mutant (G) and TP53 wild‐type (H) PAAD cases. I and J, K‐M survival analysis of PFS (I) and DSS (J) in PAAD cases in TCGA Pan‐Cancer, by median KLF5 expression separation. K and L, Quantitative real‐time PCR (qRT‐PCR) analysis of PLA2G16 mRNA expression in PANC‐1 and MIA‐PaCa‐2 cells 48 h after lentiviral‐mediated TP53 (K) or KLF5 (L) inhibition. M, Western blot analysis of p53, KLF5 and PLA2G16 protein expression 72 h after lentiviral‐mediated TP53 (up) or KLF5 (down) inhibition. N and O, qRT‐PCR (M) and Western blot assay (O) of PLA2G16 expression in PANC‐1 and MIA‐PaCa‐2 cells with TP53 and KLF5 inhibition (shTP53#2 and shKLF5#1) separately or in combination

FIGURE 5

Mutant p53 enhances KLF5‐induced PLA2G16 transcriptional activation. A, Predicted binding sites of KLF5 in the promoter region of PLA2G16. B, The promoter activity of the PLA2G16 gene was measured using a dual‐luciferase reporter assay. PANC‐1 and MIA‐PaCa‐2 cells were transfected with pGL3‐basic or reporter constructs carrying different lengths of the 5ʹ‐flanking region of the PLA2G16 promoter as indicated. C‐E, KLF5 depletion reduced the activity of the PLA2G16 promoter. PANC‐1 and MIA‐PaCa‐2 cells with or without lentiviral‐mediated KLF5 inhibition were transfected reporter constructs carrying pGL3‐(−1303/+239) (C), pGL3‐(−400/+239) (D) and pGL3‐(−100/+239) (E). 24 h later, luciferase activity was determined. F, Western blot assay of KLF5 expression 48 h after lentiviral‐mediated overexpression. G‐J, 24 h after infection with lenti‐KLF5 or vector, PANC‐1 and MIA‐PaCa‐2 cells were transfected reporter constructs carrying pGL3‐(−1303/+239) (G‐H) or mutant‐pGL3‐(−1303/+239) with mutant sequences (C to A) of the four binding sites (I, J). 24 h later, luciferase activity was determined. K, Schematic image showing the location of the designed primer sets for ChIP‐quantitative polymerase chain reaction (qPCR) assay, by anti‐KLF5 immunoprecipitation. L‐O, ChIP‐qPCR assays were performed using anti‐KLF5 (L, M) or anti‐TP53 (N, O) and control IgG antibodies in PANC‐1 (L, N) and MIA‐PaCa‐2 (M, O) cells. Fold enrichment of the indicated PLA2G16 promoter segments was calculated. P, Co‐IP assay to explore the potential binding between KLF5 and p53 in MIA‐PaCa‐2 and PANC‐1 cells. Q and R, ChIP‐qPCR assays were performed using anti‐KLF5 in MIA‐PaCa‐2 (Q) and PANC‐1 (R) cells with or without TP53 inhibition. Fold enrichment of the indicated regions of the PLA2G16 promoter was calculated. *P < .05; **P < .01; ***P < .001

FIGURE 6

PLA2G16 up‐regulation was also associated with gene‐level copy amplification and hypomethylation. A, A heatmap showing PLA2G16 expression, copy number alteration and methylation level of nine 5’‐cytosine‐phosphodiester bond‐guanine‐3’ sites in pancreatic adenocarcinoma (PAAD) cases in The Cancer Genome Atlas (TCGA). B, A plot chart showing the correlation between PLA2G16 expression and its copy number in 176 PAAD cases in TCGA. C, Comparison of PLA2G16 expression copy number between TP53 wild‐type and mutant PAAD cases. D, A plot chart showing the correlation between PLA2G16 expression and the β‐value of cg09518969 in 177 PAAD cases in TCGA. E, Comparison of cg09518969 methylation value between TP53 wild‐type and mutant PAAD cases