Non-canonical WNT5A signaling up-regulates the expression of the tumor suppressor 15-PGDH and induces differentiation of colon cancer cells - PubMed (original) (raw)
Non-canonical WNT5A signaling up-regulates the expression of the tumor suppressor 15-PGDH and induces differentiation of colon cancer cells
Lubna M Mehdawi et al. Mol Oncol. 2016 Nov.
Abstract
The tumor suppressor 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme in prostaglandin E2 catabolism and is down-regulated in colorectal cancer (CRC) tissue. Canonical Wnt signaling is frequently elevated in colon cancers and has been shown to down-regulate 15-PGDH expression. Therefore, we have in the current study investigated if the non-canonical ligand WNT5A relates to increased expression of 15-PGDH in colon cancer cells. In the same cohort of patients, we demonstrated a parallel and significant loss of 15-PGDH and WNT5A protein expression in CRC tissues compared with matched normal colon tissues. Furthermore, patients with low 15-PGDH/WNT5A expression in their tumors showed reduced survival compared with patients with high 15-PGDH/WNT5A expression. To investigate if WNT5A signaling directly affects 15-PGDH expression, we performed in vitro analyses of colon cancer cells (HT-29 and Caco-2). Both cell lines, when treated with recombinant WNT5A (rWNT5A) or Foxy-5, a WNT5A-mimicking peptide, responded by increasing their expression of 15-PGDH mRNA and protein. Our investigations showed that rWNT5A and Foxy-5 induced this increased expression of 15-PGDH through reduced β-catenin signaling as well as increased JNK/AP-1 signaling in colon cancer cells. WNT5A signaling also induced increased 15-PGDH expression in a breast cancer cell line both in vitro and in vivo. In agreement, WNT5A signaling also increased the expression of the differentiation markers sucrose-isomaltase and mucin-2 in colon cancer cells. Our results show that WNT5A signaling regulates 15-PGDH expression, thus uncovering a novel mechanism by which WNT5A acts as a tumor suppressor and suggests that increased 15-PGDH expression could be used as an indicator of a positive response to Foxy-5 in patients treated with this WNT5A agonist.
Keywords: 15-PGDH; Colon cancer; JNK; WNT-5A; β-Catenin.
Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Figures
Figure 1
Expression of 15‐PGDH and WNT5A in colorectal carcinomas and in matched normal control tissue. A, The images shown are representative immunohistochemical stainings of 15‐PGDH protein expression in colorectal carcinoma tissue and matched normal controls. The intensity of 15‐PGDH protein staining was scored and the results from this scoring are presented in the graph. B, The images shown are representative immunohistochemical stainings of WNT5A protein expression in colorectal carcinoma tissue and matched normal controls. The intensity of WNT5A protein staining was scored and the results from this scoring are presented in the graph. The results are presented as mean ± standard error of the mean (SEM); ***P < 0.001.
Figure 2
Kaplan–Meier survival curves of colorectal cancer patients with different levels of 15‐PGDH and WNT5A protein in their tumor tissue. A and C, Representative immunohistochemical images of low and high levels of 15‐PGDH and WNT5A expression in the tumor tissue from colon cancer patients. B, Kaplan–Meier survival curves of colorectal cancer patients with low or high levels of 15‐PGDH expression. D, Kaplan–Meier survival curves of colorectal cancer patients with low or high levels of WNT5A expression. E, Kaplan–Meier survival curves of colorectal cancer patients with low or high expression levels of both 15‐PGDH and WNT5A. Cumulative survival is shown in months, and the differences between the groups were assessed using log‐rank testing.
Figure 3
The effect of rWNT5A and Foxy‐5 on the expression of 15‐PGDH in colon cancer cells. A, Western blot analyses and subsequent densitometric evaluations of 15‐PGDH protein expression in HT‐29 colon cancer cells not stimulated (Cnt) or stimulated with rWNT5A (400 ng/ml) for 24, 48 or 72 h. B, Western blot analyses and subsequent densitometric evaluations of 15‐PGDH protein expression in HT‐29 colon cancer cells not stimulated (Cnt) or stimulated with Foxy‐5 (100 μM) for 24, 48 or 72 h. The images shown in A, and B, are representative western blots of 15‐PGDH and re‐probed blots for β‐actin to ensure equal loading. The diagrams outline the results of the densitometric analyses expressed as 15‐PGDH/β‐actin ratio. C and D, QPCR analyses of 15‐PGDH mRNA levels in HT‐29 cells not stimulated (Cnt) or stimulated with C, 400 ng/ml rWNT5A or D, 100 μM Foxy‐5 for 6, 9, 12, 18, 24, 48 or 72 h. E, Analyses of 15‐PGDH protein expression in Caco‐2 colon cancer cells not stimulated (Cnt) or stimulated with either 400 ng/ml rWNT5A or 100 μM Foxy‐5 for 24 h. The images shown are a representative western blot of 15‐PGDH and a re‐probed blot for β‐actin to ensure equal loading. The diagram outlines the results of the densitometric analyses expressed as 15‐PGDH/β‐actin ratio. The data are calculated as percentage of untreated control cells and given as mean ± standard error of mean (SEM) of at least five separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 4
WNT5A signaling modulates 15‐PGDH expression via different mechanisms A, Western blot analyses and densitometric evaluations of phosphorylated JNK (p‐JNK) expression in HT‐29 colon cancer cells not stimulated (Cnt) or stimulated with 100 μM Foxy‐5 for 0.5, 1, 2, or 24 h. The images shown are a representative western blot of p‐JNK and re‐probed blots for total JNK and β‐actin to ensure equal loading. The diagram outlines the results of the densitometric analyses expressed as p‐JNK/total JNK ratio. B, Luciferase activity in HT‐29 cells transfected with a 15‐PGDH promoter construct for 24 h, incubated for 30 min in the absence or presence of the JNK inhibitor1 (JNKI1, 10 μM) and, finally, either not stimulated (Cnt) or stimulated with rWNT5A (400 ng/ml) or Foxy‐5 (100 μM) for 24 h in the absence or presence of the JNKI1. C, 15‐PGDH mRNA expression in HT‐29 cells incubated for 30 min in the absence or presence of JNKI1 (10 μM) and then either not stimulated (Cnt) or stimulated with either 400 ng/ml rWNT5A or 100 μM Foxy‐5 for 24 h in the absence or presence of JNKI1 (10 μM). D, Western blot analyses of nuclear fractions from HT‐29 cells not stimulated (Cnt) or stimulated with either 400 ng/ml rWNT5A or 100 μM Foxy‐5 for 24 h. The images shown are western blots of phosphorylated AP‐1 (p‐AP‐1; re‐probed for total AP‐1) and c‐Myc (re‐probed for Lamin‐B), the re‐probing were performed to ensure equal loading. E and F, Western blot analyses and subsequent densitometric evaluations of active β‐catenin and phosphorylated β‐catenin protein expressions in HT‐29 cells not stimulated (Cnt) or stimulated with rWNT5A (400 ng/ml) or Foxy‐5 (100 μM) for 24 h. The images shown are representative western blots that were re‐probed for total β‐catenin and finally also for β‐actin to ensure equal loading. The diagrams in E and F outline the results of the densitometric analyses. G, Western blot analyses and subsequent densitometric evaluations of 15‐PGDH protein expression in HT‐29 cells not stimulated (Cnt) or stimulated with 400 ng/ml rWNT5A for 24 h in the absence or presence of the XAV‐939 (10 μM) and H, the same blots analyzed in G were also re‐probed for active β‐catenin and total β‐catenin. The images shown in G and H are representative western blots and all blots were re‐probed for β‐actin to ensure equal loading. I, Luciferase activity in HT‐29 cells transfected with a 15‐PGDH promoter construct for 24 h, incubated for 30 min in the absence or presence of the β‐catenin activator CHIR‐99021 (1.25 μM) and, finally, either not stimulated (Cnt) or stimulated with rWNT5A (400 ng/ml) for 24 h in the absence or presence of CHIR‐99021. J, Cyclin D1 mRNA expression in HT‐29 cells not stimulated (Cnt) or stimulated with 100 μM Foxy‐5 in the absence or presence of the β‐catenin activator CHIR‐99021 (1.25 μM) for 48 h. The data are normalized against untreated controls, and the results are presented as mean ± standard error of the mean (SEM) of at least five separate experiments; *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
The effect of rWNT5A and Foxy‐5 on the expression of the 15‐PGDH protein in breast cancer cells. A, Western blot analyses and subsequent densitometric evaluations of 15‐PGDH protein expression in MDA‐MB‐468 breast cancer cells not stimulated (Cnt) or stimulated with either rWNT5A (400 ng/ml) or Foxy‐5 (100 μM) for 24 h. The images shown are a representative western blot of 15‐PGDH that was re‐probed for β‐actin to ensure equal loading. The diagram outlines the results of the densitometric analyses and the results are presented as a percentage of untreated controls. The results are presented as mean ± standard error of the mean (SEM) of at least four separate experiments; *P < 0.05. B and C, Effect of WNT5A on tumor growth. Volumes and weights of tumors derived from MDA‐MB‐468 cells either transfected with an empty vector (EV) or a WNT5A expressing vector (WNT5A). Volumes and weights were determined at day 24 when the experiments were terminated. D, Representative images from in vivo experiments showing the expression of 15‐PGDH in tumors derived from MDA‐MB‐468 cells transfected with either an empty vector (EV; left panel) or a WNT5A expressing vector (WNT5A; right panel) at day 24. The accumulated 15‐PGDH data are presented as percentage of cells with strong 15‐PGDH expression (Nsr). The animal results are given as means ± standard error of the mean (SEM) for 18 tumors derived from 468(EV) cells and 18 tumors derived from 468(WNT5A) cells; *P < 0.05, ***P < 0.001.
Figure 6
WNT5A signaling induces differentiation of colon cancer cells. A, Western blot analyses and subsequent densitometric evaluations of sucrase‐isomaltase (SI) expression in HT‐29 colon cancer cells not stimulated (Cnt) or stimulated with 400 ng/ml rWNT5A for 24, 48, or 72 h. The images shown are a representative western blot of SI that was re‐probed for β‐actin to ensure equal loading. The diagram outlines the results of the densitometric analyses. B, Western blot analyses and subsequent densitometric evaluations of SI expression in Caco‐2 colon cancer cells not stimulated or stimulated with either rWNT5A (400 ng/ml) or Foxy‐5 (100 μM) for 24 h. The images shown are a representative western blot of SI that was re‐probed for β‐actin to ensure equal loading. The diagram outlines the results of the densitometric analyses. C and D, QPCR analyses of SI mRNA and mucin‐2 mRNA levels in HT‐29 colon cancer cells incubated for 30 min in the absence or presence of JNKl1 (10 μM) or the β‐catenin activator CHIR‐99021 (1.25 μM) and either not stimulated (Cnt) or stimulated with rWNT5A (400 ng/ml) or Foxy‐5 (100 μM) for C, 48 h and D, 72 h. The accumulated data are calculated as the percentage of untreated controls and the results are presented as mean ± standard error of the mean (SEM) of at least four separate experiments; *P < 0.05, **P < 0.01, ***P < 0.001.
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