Fig 1: Mutant p53 regulates LPA signaling through ACP6. (a) Lysophosphatidic acid (LPA) is produced by autotaxin (ATX) from lysophosphatidylcholine (LPC). Lysophosphatidic acid phosphatase type 6 (ACP6) hydrolyzes LPA to monoacylglycerol (MAG) and inorganic phosphate (Pi). (b) Representative ACP6 immunohistochemistry (IHC) on patient tissues (n = 7, scale bars: 20 and 100 µm) (c) qRT-PCR of ACP6 in patient samples: ovarian tumors (OvTu; n = 18 patients) and normal fallopian tube tissues (nFT; n = 12 patients). (d) ACP6 protein expression in wild-type (wt) p53 and R273H p53 mutant FTEC. (e) Representative immunofluorescence (IF) of ACP6 (red) and p53 (pantropic antibody; green) in tumor and adjacent fallopian tube tissue. (f) Immunofluorescence of an HGSOC harboring a R248Q p53 mutation. Analysis of ACP6 (red) and p53 (green). (g) ACP6 promoter activity in wild-type (wt) p53 FTEC and R175H, R249S and R273H p53 mutant FTEC as assessed by dual luciferase assay (n = 3, two-way analysis of variance). For (a–g): ***P < 0.001, ****P < 0.0001. Error bars are SEM.
Fig 2: Overexpression of ACP6 suppresses metastasis. Functional assays investigating the effect of ACP6 knockdown or overexpression in HeyA8 cells that were transduced with lentiviral constructs—shACP6, shCTRL, pLX304-ACP6 (ACP6) or empty pLX304 vector (Ctrl)—to downregulate or overexpress ACP6, respectively: (a) in vitro proliferation (72 hours, n = 10), (b) in vitro adhesion (30 minutes, n = 3), (c) in vitro migration (4 hours, n = 4), (d) in vitro invasion (8 hours, n = 4) and (e) in vivo tumorigenesis (3 weeks, n = 5). (f) Representative Ki-67 immunohistochemistry (IHC) of mouse omental tissue sections (from e) and quantification of positive cells (n = 4 slides per group, scale bar: 50 µm). (g) Kaplan-Meier overall survival plot for serous ovarian cancer patients finds that low expression of ACP6 is associated with a poor overall survival (40.4 months versus 48 months median overall survival for high ACP6 expression). ACP6 expression is significantly reduced in (h) breast and (i) endometrial cancer patients harboring TP53 mutations compared to those that maintain wild-type TP53. (j) Mutant p53 transcriptionally decreases ACP6 expression, leading to LPA accumulation that contributes to a more aggressive ovarian cancer cell phenotype. Monoacylglycerol (MAG). For (a–j): *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars are SEM.
Fig 3: Downregulation of ACP6 regulates LPA signaling. (a) Immunoblot analysis and quantification of p-paxillin (Y118) and p-FAK (Y397) normalized to total protein signal in wild-type p53 primary FTEC transfected with an siACP6 or siCTRL construct (n = 3). (b) Immunoblot analysis and quantification of focal adhesion proteins in p53 FTEC stably expressing R273H and transiently transfected with ACP6 or a control construct (n = 3). (c) Treatment of wild-type (wt) p53 FTEC transiently transfected with siACP6 construct followed by treatment with 2 different concentrations of Ki16425 for 1 hr. Numbers above membranes indicate relative quantification of phosphorylation. For (a–c): *P < 0.05, **P < 0.01. Error bars are SEM.
Supplier Page from DNASU for ACP6 (Homo sapiens) in pLX304 (Gateway V5-tagged lentiviral expression vector)