title: Activation of Transforming Growth Factor Beta 1 Signaling in Gastric Cancer-associated Fibroblasts Increases Their Motility, via Expression of Rhomboid 5 Homolog 2, and Ability to Induce Invasiveness of Gastric Cancer Cells.
abstract: Fibroblasts that interact with cancer cells are called cancer-associated fibroblasts (CAFs), which promote progression of different tumor types. We investigated the characteristics and functions of CAFs in diffuse-type gastric cancers (DGCs) by analyzing features of their genome and gene expression patterns.
  
  
  
  We isolated CAFs and adjacent non-cancer fibroblasts (NFs) from 110 gastric cancer (GC) tissues from patients who underwent gastrectomy in Japan from 2008 through 2016. Cells were identified using specific markers of various cell types by immunoblot and flow cytometry. We selected pairs of CAFs and NFs for whole-exome and RNA sequencing analyses, and compared expression of specific genes using quantitative reverse transcription PCR. Protein levels and phosphorylation were compared by immunoblot and immunofluorescence analyses. Rhomboid 5 homolog 2 (RHBDF2) was overexpressed from a transgene in fibroblasts or knocked down using small interfering RNAs. Motility and invasiveness of isolated fibroblasts and GC cell lines (AGS, KATOIII, MKN45, NUGC3, NUGC4, OCUM-2MD3 and OCUM-12 cell lines) were quantified by real-time imaging analyses. We analyzed 7 independent sets of DNA microarray data from patients with GC and associated expression levels of specific genes with patient survival times. Nude mice were given injections of OCUM-2MD3 in the stomach wall; tumors and metastases were collected and analyzed by immunohistochemistry.
  
  
  
  Many of the genes with increased expression in CAFs compared with NFs were associated with transforming growth factor beta 1 (TGFB1) activity. When CAFs were cultured in extracellular matrix, they became more motile than NFs; DGC cells incubated with CAFs were also more motile and invasive in vitro than DGC cells not incubated with CAFs. When injected into nude mice, CAF-incubated DGC cells invaded a greater number of lymphatic vessels than NF-incubated DGC cells. We identified RHBDF2 as a gene overexpressed in CAFs compared with NFs. Knockdown of RHBDF2 in CAFs reduced their elongation and motility in response to TGFB1, whereas overexpression of RHBDF2 in NFs increased their motility in extracellular matrix. RHBDF2 appeared to regulate oncogenic and non-canonical TGFB1 signaling. Knockdown of RHBDF2 in CAFs reduced cleavage of the TGFB receptor 1 (TGFBR1) by ADAM metallopeptidase domain 17 (ADAM17 or TACE) and reduced expression of genes that regulate motility. Incubation of NFs with in interleukin 1 alpha (IL1A), IL1B or tumor necrosis factor, secreted by DGCs, increased fibroblast expression of RHBDF2. Simultaneous high expression of these cytokines in GC samples was associated with shorter survival times of patients.
  
  
  
  In CAFs isolated from human DGCs, we observed increased expression of RHBDF2, which regulates TGFB1 signaling. Expression of RHBDF2 in fibroblasts is induced by inflammatory cytokines (such as IL1A, IL1B, and tumor necrosis factor) secreted by DGCs. RHBDF2 promotes cleavage of TGFBR1 by activating TACE and motility of CAFs in response to TGFB1. These highly motile CAFs induce DGCs to invade extracellular matrix and lymphatic vessels in nude mice.
authors.affiliation: Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore; Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan; International Research Center of Medical Sciences (IRCMS), Kumamoto University, Kumamoto, Japan.
authors.forename: Takatsugu
authors.initials: T
authors.lastname: Ishimoto
authors.affiliation: Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan; International Research Center of Medical Sciences (IRCMS), Kumamoto University, Kumamoto, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Miyake
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Tannistha
authors.initials: T
authors.lastname: Nandi
authors.affiliation: Department of Surgical Oncology, Osaka City University Graduate School of Medicine, Osaka, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Yashiro
authors.affiliation: Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, Tokyo, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Onishi
authors.affiliation: Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore.
authors.forename: Kie Kyon
authors.initials: KK
authors.lastname: Huang
authors.affiliation: Genome Institute of Singapore, Singapore.
authors.forename: Suling Joyce
authors.initials: SJ
authors.lastname: Lin
authors.affiliation: Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore.
authors.forename: Ramnarayanan
authors.initials: R
authors.lastname: Kalpana
authors.affiliation: Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore.
authors.forename: Su Ting
authors.initials: ST
authors.lastname: Tay
authors.affiliation: Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Suzuki
authors.affiliation: Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore.
authors.forename: Byoung Chul
authors.initials: BC
authors.lastname: Cho
authors.affiliation: Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kuroda
authors.affiliation: Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan; International Research Center of Medical Sciences (IRCMS), Kumamoto University, Kumamoto, Japan.
authors.forename: Kota
authors.initials: K
authors.lastname: Arima
authors.affiliation: Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Izumi
authors.affiliation: Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Iwatsuki
authors.affiliation: Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan.
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Baba
authors.affiliation: Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Oki
authors.affiliation: Department of Gastroenterological Surgery, Cancer Institute Hospital of Japanese Foundation for Cancer Research, Tokyo, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, Tokyo, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Saya
authors.affiliation: Department of Surgical Oncology, Osaka City University Graduate School of Medicine, Osaka, Japan.
authors.forename: Kosei
authors.initials: K
authors.lastname: Hirakawa
authors.affiliation: Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan. Electronic address: hdobaba@kumamoto-u.ac.jp.
authors.forename: Hideo
authors.initials: H
authors.lastname: Baba
authors.affiliation: Program in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore; Genome Institute of Singapore, Singapore. Electronic address: gmstanp@duke-nus.edu.sg.
authors.forename: Patrick
authors.initials: P
authors.lastname: Tan
mesh_terms: D000072198:ADAM17 Protein / Q000378:metabolism; D000818:Animals; D000072645:Cancer-Associated Fibroblasts / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D018920:Coculture Techniques; D059472:Exome; D005109:Extracellular Matrix; D005260:Female; D015870:Gene Expression; D006801:Humans; D053582:Interleukin-1alpha / Q000494:pharmacology; D053583:Interleukin-1beta / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008875:Middle Aged; D009361:Neoplasm Invasiveness / Q000235:genetics; D009368:Neoplasm Transplantation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D017423:Sequence Analysis, RNA; D015398:Signal Transduction / Q000235:genetics; D013274:Stomach Neoplasms / Q000737:chemistry / Q000378:metabolism* / Q000473:pathology; D015996:Survival Rate; D059467:Transcriptome; D053773:Transforming Growth Factor beta1 / Q000235:genetics* / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D002352:Carrier Proteins; D053582:Interleukin-1alpha; D053583:Interleukin-1beta; D047908:Intracellular Signaling Peptides and Proteins; C580845:RHBDF2 protein, human; D018125:Receptors, Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; D017346:Protein Serine-Threonine Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625841:TGFBR1 protein, human; C000625842:Tgfbr1 protein, mouse; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human
keywords: Inflammation; Stomach Cancer; Tumor Cells; Tumor Stroma