The absence of LPA receptor 2 reduces the tumorigenesis by ApcMin mutation in the intestine

Abstract

Lysophosphatidic acid (LPA) is a lipid mediator that mediates several effects that promote cancer progress. The LPA receptor type 2 (LPA(2)) expression is often elevated in several types of cancers, including colorectal cancer (CRC). In this study, we investigated the role of LPA(2) in the development of intestinal adenomas by comparing Apc(Min/+) mice with Apc(Min/+)/Lpar2(-/-) mice. There were 50% fewer intestinal adenomas in Apc(Min/+)/Lpar2(-/-) mice than Apc(Min/+) mice. Smaller-size adenomas (<1 mm) were found at higher frequencies in Apc(Min/+)/Lpar2(-/-) mice compared with Apc(Min/+) mice at the two age groups examined. The expression level of LPA(2) correlated with increased size of intestinal adenomas. Reduced tumor multiplicity and size in Apc(Min/+)/Lpar2(-/-) mice correlated with decreased proliferation of intestinal epithelial cells. Apc(Min/+)/Lpar2(-/-) mice showed an increased level of apoptosis, suggesting that LPA(2)-mediated signaling stimulates intestinal tumor development and progress by regulating both cell proliferation and survival. In addition, the expression levels of Krüpple-like factor 5 (KLF5), β-catenin, cyclin D1, c-Myc, and hypoxia-inducible factor-1α (HIF-1α) were significantly altered in Apc(Min/+)/Lpar2(-/-) mice compared with Apc(Min/+) mice. In vitro studies using HCT116 cells showed that LPA induced cyclin D1, c-Myc, and HIF-1α expression, which was attenuated by knockdown of LPA(2). In summary, intestinal tumor initiated by Apc mutations is altered by LPA(2)-mediated signaling, which regulates tumor growth and survival by altering multiple targets.

Fig. 1.

Intestinal adenomas in Apc^Min/+ [mice in which familial adenomatous polyposis (FAP) is modeled by the multiple intestinal neoplasia]/Lpar2^−/− [founder C57BL/6 mice heterozygous for the lysophosphatidic acid receptor type 2 (LPA₂) allele] mice. Mice were killed at the age of 15 or 21 wk, and the no. of adenomas of the two age groups was counted. A: comparison of the no. of intestinal adenomas/animal in two age groups of Apc^Min/+ and Apc^Min/+/Lpar2^−/− mice. *P < 0.01. B: comparison of the no. of colonic adenomas/mouse. *P < 0.01. C: graph showing the distribution of adenomas by adenoma size in Apc^Min/+ and Apc^Min/+/Lpar2^−/− mice at the age of 15 or 21 wk.

Fig. 2.

The expression of lysophosphatidic acid (LPA) receptors in intestinal adenomas of Apc^Min/+ mice. Adenomas were isolated under a dissecting microscope and grouped according to their sizes. As controls, the healthy ileal epithelial cells from wild-type (WT) and Apc^Min/+ mice were isolated and processed. Total RNA was prepared for quantitative RT-PCR to determine the mRNA levels of LPA receptors. A: expression levels of LPA receptor mRNAs are shown. The mRNA level of each LPA receptor was normalized to the 18S mRNA level of the same sample. The relative expression levels are expressed as fold changes relative to the LPA₂ mRNA level in the intestinal epithelia of WT (=Lpar2^+/+) mice; n = 6. B: the expression levels of LPA₂ mRNA in WT and adenomas of Apc^Min/+ mice are shown. LPA₂ mRNA expression level was normalized to 18S mRNA expression; n = 6. *P < 0.01 between two groups of adenomas.

Fig. 3.

Cell proliferation and apoptosis in Apc^Min/+ and Apc^Min/+/Lpar2^−/− mice. A: intestinal sections from WT and Lpar2^−/− mice were immunolabeled using the antibodies against Ki67. Scale bar: 200 μm. B: intestinal sections from Apc^Min/+ and Apc^Min/+/Lpar2^−/− mice were immunolabeled using an antibody against cleaved caspase-3. Left, intestinal sections of normal-appearing ileum of 21-wk-old mice. Right, adenomatous lesions. Red arrows indicate cleaved caspase-3-positive cells. Scale bar (in blue): 30 μm. C: intestinal sections from Apc^Min/+ and Apc^Min/+/Lpar2^−/− mice were immunolabeled using an anti-Ki67 antibody. Left, intestinal sections of normal-appearing ileum of 21-wk-old mice. Right, adenomatous lesions. Scale bar (in red): 200 μm. Representative images are shown. D: no. of cells positive for cleaved caspase-3 in adenomas is shown; 5–6 microscopic views/section and 10 sections from 3 animals of each genotype were examined. *P < 0.01 by paired 2-tailed t-test.

Fig. 4.

Krüpple-like factor 5 (KLF5) and β-catenin expression in Apc^Min/+/Lpar2^−/− mice compared with Apc^Min/+ mice. Intestinal sections of 21-wk-old Apc^Min/+ and Apc^Min/+/Lpar2^−/− mice were stained for KLF5 (A) and β-catenin (B). Left, intestinal sections of normal-appearing ileum of 21-wk-old mice. Right, adenomatous lesions. Scale bars: 200 μm (in red) for KLF5 and 50 μm (in green) for β-catenin. C: equal amounts of mouse intestinal lysates were analyzed by Western blotting for the expression levels of β-catenin. For Apc^Min/+ and Apc^Min/+/Lpar2^−/− mice, lysates were prepared from isolated adenomas. β-Actin expression was used as a loading control. Relative changes in β-catenin expression are indicated below the immunoblot. Representative images from 4 separate experiments are shown.

Fig. 5.

Reduced levels of cyclin D1 and c-Myc in adenomas of Apc^Min/+/Lpar2^−/− mice. Immunohistochemical labeling of cyclin D1 (A) and c-Myc (B) was performed on paraffin-embedded intestinal section of Apc^Min/+ and Apc^Min/+/Lpar2^−/− mice. Left and right, representative images of normal-appearing ileum and adenomatous lesion of 21-wk-old mice, respectively. Scale bar: 200 μm. Representative images from 5 separate experiments are shown. C: equal amounts of mouse intestinal lysates were analyzed by Western blotting for the expression levels of cyclin D1 and c-Myc. β-Actin expression was used as a loading control. The relative changes in cyclin D1 and c-Myc expression are indicated. D: HCT116 cells transfected with control-RNA interference (RNAi) or LPA₂-RNAi were treated with 1 μM LPA for 8 or 24 h, and the expression levels of cyclin D1 and c-Myc were determined by Western immunobotting. β-Actin expression was used as a loading control. Representative results from 4 independent experiments are shown.

Fig. 6.

Reduced hypoxia in Apc^Min/+/Lpar2^−/− mice. A: immunohistochemical labeling of hypoxia-inducible factor-1α (HIF-1α) was performed on paraffin-embedded intestinal sections of Apc^Min/+ and Apc^Min/+/Lpar2^−/− mice. Left and right, representative images of normal-appearing ileum and adenomatous lesions from 21-wk-old mice, respectively. Scale bar: 50 μm. B: HIF-1α expression in total intestinal mucosal lysates was determined by Western blotting. β-Actin expression was used as a loading control. Relative changes in HIF-1α expression are indicated. C: 3 individual tumors (T1, T2, and T3) from Apc^Min/+ or Apc^Min/+/Lpar2^−/− mice were isolated, and HIF-1α and glucose transporter 1 (GLUT1) expression in each tumor was determined. Relative changes in protein expression are indicated. D: the effect of LPA on HIF-1α expression in HCT116 cells was determined. Cells were treated with 1 μM LPA or carrier under normoxic or hypoxic conditions for 16 h. E: HCT116 cells transfected with control-RNAi or LPA₂-RNAi were incubated with 1 or 10 μM LPA for 16 h. HIF-1α and β-actin protein expression levels were determined. A representative Western blot from 3 independent experiments is shown.

Fig. 7.

Altered oxygen gradient in Apc^Min/+/Lpar2^−/− mice. A: mice were treated with pimonidazole (60 mg/kg ip) 3 h before death. Immunohistochemical labeling of antibody against protein adducts of reductively activated 2-nitroimidazoles was performed on paraffin-embedded intestinal sections of Apc^Min/+ and Apc^Min/+/Lpar2^−/− mice. Left, intestinal sections of normal-appearing ileum of 21-wk-old mice. Right, adenomatous lesions. Scale bar: 200 μm. B: intestinal sections of WT mice treated with PBS or pimonidazole were immunolabeled as described in materials and methods. PBS-treated mice showed no apparent staining with Hypoxyprobe-1 PAb2627 antibodies. Scale bar: 200 μm.