Abstract
Lysophosphatidic acid (LPA) is enriched in the serum and malignant effusion of cancer patients and plays a key role in tumorigenesis and metastasis. LPA-activated mesenchymal stem cells promote tumorigenic potentials of cancer cells through a paracrine mechanism. LPA-conditioned medium (LPA CM) from human adipose tissue-derived mesenchymal stem cells (hASCs) elicited adhesion and proliferation of A549 human lung adenocarcinoma cells. To identify proteins involved in the LPA-stimulated paracrine functions of hASCs, we analyzed the LPA CM using liquid-chromatography tandem mass spectrometry-based shotgun proteomics. We identified βig-h3, an extracellular matrix protein that is implicated in tumorigenesis and metastasis, as an LPA-induced secreted protein in hASCs. LPA-induced βig-h3 expression was abrogated by pretreating hASCs with the LPA receptor(1/3) inhibitor Ki16425 or small interfering RNA-mediated silencing of endogenous LPA(1). LPA-induced βig-h3 expression was blocked by treating the cells with the Rho kinase inhibitor Y27632, implying that LPA-induced βig-h3 expression is mediated by the LPA(1)- Rho kinase pathway. Immunodepletion or siRNA-mediated silencing of βig-h3 abrogated LPA CM-stimulated adhesion and proliferation of A549 cells, whereas retroviral overexpression of βig-h3 in hASCs potentiated it. Furthermore, recombinant βig-h3 protein stimulated the proliferation and adhesion of A549 human lung adenocarcinoma cells. These results suggest that hASC-derived βig-h3 plays a key role in tumorigenesis by stimulating the adhesion and proliferation of cancer cells and it can be applicable as a biomarker and therapeutic target for lung cancer.
Highlights
From the ‡Medical Research Center for Ischemic Tissue Regeneration and Medical Research Institute, §Department of Physiology, Pusan National University, Yangsan 626-870, Gyeongsangnam-do, Republic of Korea; ¶Research Institute of Convergence Biomedical Science and Technology, Pusan National University Yangsan Hospital, Yangsan 626-770, Gyeongsangnam-do, Republic of Korea; ʈNovaCell Technology Inc., Pohang, Republic of Korea, **Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, 101 Dongindong, Jung-gu, Daegu 700-422, Republic of Korea
These results suggest that tumorigenesis and metastasis of carcinoma cells are acquired by paracrine signals from Mesenchymal stem cells (MSCs) within the tumor-associated stroma
We have reported that periostin is secreted from human adipose tissue-derived mesenchymal stem cells in response to Lysophosphatidic acid (LPA) treatment, and the recombinant periostin protein stimulates the adhesion and migration of epithelial ovarian cancer cells [24]. ig-h3 was originally identified as transforming growth factor-1 (TGF-1)induced protein in A549 human adenocarcinoma cells [25]. ig-h3 is normally expressed in fibroblasts, keratinocytes, and muscle cells (26 –28)
Summary
Materials—␣-Minimum essential medium, trypsin, and fetal bovine serum were purchased from Invitrogen (Carlsbad, CA, http://www.invitrogen.com). 1-Oleoyl-sn-glycero-3-phosphate (LPA), Ki16425, SB431542, fatty acid-free bovine serum albumin (BSA), anti-␣-SMA antibody, and Protein A agarose were from Sigma-Aldrich (St. Louis, MO, http:// www.sigmaaldrich.com). The cells were briefly rinsed twice with PBS and incubated with 15 ml of ␣-minimum essential medium in the absence or presence of 10 M LPA for 48 h before collecting media. Cell Adhesion Assay—Ninety-six-well microculture plates (Falcon, Becton-Dickinson, Mountain View, CA) were incubated with recombinant ig-h3 proteins or conditioned medium from hASCs at 37 °C for 1 h and blocked with PBS containing 0.2% BSA for 1 h at 37 °C. After incubation of hASCs in serum-free medium containing siRNAs for 4 h, the cells were cultured in growth medium for 24 h, and the expression levels of LPA1 and GAPDH were determined by reverse transcription-polymerase chain reaction analysis. Group differences were assessed with two-way analysis of variance (ANOVA), followed by post hoc comparisons tested with Scheffe’s method
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