Downregulation of lipolysis-stimulated lipoprotein receptor promotes cell invasion via claudin-1-mediated matrix metalloproteinases in human endometrial cancer

Abstract

Lipolysis-stimulated lipoprotein receptor (LSR) is a novel molecule present at tricellular contacts which recruits tricellulin (TRIC), a molecular component of tricellular tight junctions (tTJs). LSR and TRIC are colocalized with the bicellular tight junction (bTJ) protein claudin (CLDN)-1-based tight junction strands at tricellular corners. Knockdown of LSR in normal epithelial cells affects tTJ formation and the epithelial barrier function. In cancer cells knockdown of LSR has been demonstrated to increase cell invasion. However, the detailed mechanisms of how the downregulation of LSR enhances cell invasion in cancer remain unclear. In the present study, knockdown of LSR by small interfering RNA (siRNA) in Sawano human endometrial adenocarcinoma cells induced cell invasion. In LSR-knockdown Sawano cells, upregulation of CLDN-1 protein, which contributes to the cell invasion via matrix metalloproteinases (MMPs), was observed compared with the control group by western blotting and immunostaining. Knockdown of LSR significantly induced Sp1 transcription factor activity in the CLDN-1 promoter region. In LSR-knockdown Sawano cells, DNA microarray analysis demonstrated that MMP-1, MMP-2 and MMP-10 mRNA levels were increased, and the protein levels of membrane-type 1-MMP, MMP-2, MMP-9 and MMP-10 were shown to be increased on western blots. Knockdown of CLDN-1 with siRNA prevented the upregulation of cell invasion induced by the knockdown of LSR in Sawano cells. On the invasive front of human endometrial carcinoma tissue samples, a decrease in LSR and increase in CLDN-1 protein levels were observed using immunohistochemical methods. In conclusion, the results indicate that the downregulation of LSR promotes cell invasion of human endometrial cancer via CLDN-1 mediation of MMPs. This mechanism is important for studying the association of tTJs with the cellular invasion of cancer.

Keywords: Sp1 transcription factor; cell invasion; claudin-1; endometrial cancer; lipolysis-stimulated lipoprotein receptor; matrix metalloproteinases.

Figure 1.

(A) Matrigel invasion assay of Sawano cells transfected with three sets of siRNA, A, B and C, LSR. Scale bar, 100 µm. The results are presented below as a histogram. Control vs. siRNA, **P<0.01. (B) Western blotting for LSR, TRIC, OCLN, CLDN-1, −3, −4 and −7 in Sawano cells transfected with three sets of siRNA against LSR. The results for the expression of LSR and CLDN-1 are presented as a histogram. Control vs. siRNA, *P<0.05, **P<0.01. (C) Immunostaining of LSR and CLDN-1 in Sawano cells transfected with siRNA-C against LSR. Scale bar, 20 µm. (D) Sp1 transcription factor activity in Sawano cells transfected with siRNA-C against LSR. NC vs. Sp1 control, **P<0.01; Sp1 control vs. Sp1 siRNA, ^##P<0.01. siRNA, small interfering RNA; LSR, lipolysis-stimulated lipoprotein receptor; TRIC, tricellulin; OCLN, occludin; CLDN, claudin; NC, negative control.

Figure 2.

(A) Western blotting for LSR, CLDN-1, MT1-MMP, MMP-2, MMP-9 and MMP-10 in Sawano cells transfected with three sets of siRNA against LSR. The results are presented below as a histogram. (B) Western blotting for LSR and CLDN-1 in Sawano cells transfected with or without siRNAs against LSR and CLDN-1. The results are presented as a histogram. (C) Immunostaining for LSR and CLDN-1 in Sawano cells transfected with or without siRNAs against LSR and CLDN-1. Scale bar, 20 µm. LSR, lipolysis-stimulated lipoprotein receptor; CLDN, claudin; MT1, membrane-type 1; MMP, metalloproteinase; siRNA, small interfering RNA.

Figure 3.

(A) Matrigel invasion assay of Sawano cells transfected with or without siRNAs of lipolysis-stimulated lipoprotein receptor (LSR) and claudin-1 (CLDN-1). Scale bar, 200 µm. The results are presented below as a histogram. Control vs. siRNA-LSR, **P<0.01; siRNA-LSR vs. siRNAs-LSR/CLDN-1, ^##P<0.01. (B) H&E and immunohistochemical staining for LSR and CLDN-1 in human endometrial carcinoma (arrowheads indicate the invasive front; arrows indicate the gland-like structure). Scale bar, 100 µm. siRNA, small interfering RNA; LSR, lipolysis-stimulated lipoprotein receptor; CLDN, claudin; H&E, hematoxylin and eosin.