title: Tumor-stromal interactions with direct cell contacts enhance motility of non-small cell lung cancer cells through the hedgehog signaling pathway.
abstract: The metastatic potential of non-small cell lung cancer (NSCLC) has been shown to be associated with interactions with the tumor microenvironment, which primarily comprises of cancer-associated fibroblasts (CAFs). Heterotypic cell-cell interactions occur via released signaling molecules and direct physical contact. To investigate the differential contribution of direct cell-cell contact and paracrine signaling factors to NSCLC metastasis, we performed two types of co-cultures: direct co-cultures of the NSCLC cell line H358 with primary cultures of CAFs from patients with resected NSCLC; and indirect co-cultures across a separable membrane. We showed that CAFs more potently induce epithelial-to-mesenchymal transition (EMT) in NSCLC H358 cells through direct contacts than through indirect interactions, as indicated by an elongated and disseminated appearance. Immunocytochemical experiments show that EMT accompanies the expression of mesenchymal cytoskeletal proteins, including vimentin. However, H358 cells proliferate more slowly in direct co-culture than in indirect co-culture. Real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed that H358 cells in direct contact with CAFs up-regulate the expression of the pan-mesenchymal markers α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), transforming growth factor-β (TGFβ) signaling effector SMAD family number-3 (SMAD3), and hedgehog signaling effector GLI family zinc finger-1 (GLI1), compared with the indirect co-culture system. Furthermore, we found that the direct GLI1 transcription targets snail family zinc finger-1 (SNAI1) and SNAI2 are up-regulated, suggesting that the hedgehog signaling pathway is active in direct co-culture. A scratch wound assay showed that direct contact co-culture increases the motility of H358 cells. In conclusion, these findings provide evidence that paracrine factors and direct physical contact between NSCLC cells and CAFs might control the metastatic potential of NSCLC through the hedgehog signaling pathway.
authors.affiliation: Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Republic of Korea. jkimsmc@skku.edu.
authors.forename: Chungyoul
authors.initials: C
authors.lastname: Choe
authors.forename: Yong-Sung
authors.initials: YS
authors.lastname: Shin
authors.forename: Sung-Hyun
authors.initials: SH
authors.lastname: Kim
authors.forename: Mi-Jin
authors.initials: MJ
authors.lastname: Jeon
authors.forename: So-Jung
authors.initials: SJ
authors.lastname: Choi
authors.forename: Jinseon
authors.initials: J
authors.lastname: Lee
authors.forename: Jhingook
authors.initials: J
authors.lastname: Kim
mesh_terms: D001483:Base Sequence; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism / Q000473:pathology*; D002455:Cell Division; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D017931:DNA Primers; D058750:Epithelial-Mesenchymal Transition; D053823:Hedgehog Proteins / Q000378:metabolism*; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology*; D019899:Paracrine Communication; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000473:pathology*
chemical_list: D017931:DNA Primers; D053823:Hedgehog Proteins
keywords: CAFs; EMT; Hedgehog signaling pathway; NFs; SMAD3; direct co-culture; indirect co-culture; lung cancer; proliferation; stromal NSCLC