Advertisement

Transforming Growth Factor Beta-Induced (TGFBI) Is an Anti-Adhesive Protein Regulating the Invasive Growth of Melanoma Cells

  • Pirjo Nummela
    Affiliations
    Department of Pathology, Haartman Institute, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland
    Search for articles by this author
  • Johanna Lammi
    Affiliations
    Research Programs Unit, Molecular Cancer Biology and Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland
    Search for articles by this author
  • Johanna Soikkeli
    Affiliations
    Department of Pathology, Haartman Institute, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland
    Search for articles by this author
  • Olli Saksela
    Affiliations
    Department of Dermatology, Helsinki University Central Hospital, Helsinki, Finland
    Search for articles by this author
  • Pirjo Laakkonen
    Affiliations
    Research Programs Unit, Molecular Cancer Biology and Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland

    Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland
    Search for articles by this author
  • Erkki Hölttä
    Correspondence
    Address reprint requests to Erkki Hölttä, M.D., Ph.D., Department of Pathology, Haartman Institute, P.O. Box 21 (Haartmaninkatu 3), FI-00014 Helsinki, Finland
    Affiliations
    Department of Pathology, Haartman Institute, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland
    Search for articles by this author
Published:February 10, 2012DOI:https://doi.org/10.1016/j.ajpath.2011.12.035
      Melanoma is a malignancy characterized by high invasive/metastatic potential, with no efficient therapy after metastasis. Understanding the molecular mechanisms underlying the invasive/metastatic tendency is therefore important. Our genome-wide gene expression analyses revealed that human melanoma cell lines WM793 and especially WM239 (vertical growth phase and metastatic cells, respectively) overexpress the extracellular matrix (ECM) protein transforming growth factor β induced (TGFBI). In adhesion assays, recombinant TGFBI was strongly anti-adhesive for both melanoma cells and skin fibroblasts. TGFBI further impaired the adhesion of melanoma cells to the adhesive ECM proteins fibronectin, collagen-I, and laminin, known to interact with it. Unexpectedly, WM239 cells migrated/invaded more effectively in three-dimensional collagen-I and Matrigel cultures after knockdown of TGFBI by shRNA expression. However, in the physiological subcutaneous microenvironment in nude mice, after TGFBI knockdown, these cells showed markedly impaired tumor growth and invasive capability; the initially formed small tumors later underwent myxoid degeneration and completely regressed. By contrast, the expanding control tumors showed intense TGFBI staining at the tumor edges, co-localizing with the fibrillar fibronectin/tenascin-C/periostin structures that characteristically surround melanoma cells at invasion fronts. Furthermore, TGFBI was found in similar fibrillar structures in clinical human melanoma metastases as well, co-localizing with fibronectin. These data imply an important role for TGFBI in the ECM deposition and invasive growth of melanoma cells, rendering TGFBI a potential target for therapeutic interventions.
      Given that 90% of deaths from cancer are caused by metastasis, disclosure of the mechanisms behind cancer cell invasion and metastasis would be of utmost importance.
      • Steeg P.S.
      Tumor metastasis: mechanistic insights and clinical challenges.
      • Gupta G.P.
      • Massague J.
      Cancer metastasis: building a framework.
      One of the deadliest malignancies showing a worrisome rise in incidence is malignant melanoma. It affects relatively young people, shows a high tendency for invasion, and has no effective therapy after metastasis. Thus, new therapeutic approaches are urgently needed.
      Extracellular matrix (ECM), a constantly remodeling macromolecular array has emerged as an important regulator of tumor growth. ECM is composed of specialized secreted proteins (such as collagens, laminins, and fibronectins) and proteoglycans, which provide support, anchorage, and cues for the resident cells. Interactions with ECM molecules are important in the regulation of many cellular processes, including cell survival, proliferation, differentiation, cell migration, and cancer cell invasion. In line with this, melanomas, like many other tumors, often show changes in their expression patterns of integrins, the major mediators of cell–ECM contacts, and modify their microenvironment to be more permissive for their growth and dissemination (through secretion of specific ECM molecules and ECM modifying proteases).
      Transforming growth factor β induced (TGFBI; originally known as βig-h3, also keratoepithelin) is a transforming growth factor β (TGF-β1 and TGF-β2)–inducible, secreted ECM protein expressed ubiquitously in various tissues, except brain.
      • Skonier J.
      • Bennett K.
      • Rothwell V.
      • Kosowski S.
      • Plowman G.
      • Wallace P.
      • Edelhoff S.
      • Disteche C.
      • Neubauer M.
      • Marquardt H.
      Beta ig-h3: a transforming growth factor-beta-responsive gene encoding a secreted protein that inhibits cell attachment in vitro and suppresses the growth of CHO cells in nude mice.
      • Zhao Y.L.
      • Piao C.Q.
      • Hei T.K.
      Downregulation of Betaig-h3 gene is causally linked to tumorigenic phenotype in asbestos treated immortalized human bronchial epithelial cells.
      • Ivanov S.V.
      • Ivanova A.V.
      • Salnikow K.
      • Timofeeva O.
      • Subramaniam M.
      • Lerman M.I.
      Two novel VHL targets, TGFBI (BIGH3) and its transactivator KLF10, are up-regulated in renal clear cell carcinoma and other tumors.
      This 68-kDa protein contains an N-terminal secretory signal peptide, a cysteine-rich domain (EMI), four consecutive fasciclin 1 (FAS) repeats, and a C-terminal Arg-Gly-Asp (RGD) integrin-binding motif,
      • Skonier J.
      • Neubauer M.
      • Madisen L.
      • Bennett K.
      • Plowman G.D.
      • Purchio A.F.
      cDNA cloning and sequence analysis of beta ig-h3, a novel gene induced in a human adenocarcinoma cell line after treatment with transforming growth factor-beta.
      and may participate in many processes, including morphogenesis, adhesion/migration, corneal dystrophy, tumorigenesis, wound healing, and inflammation.
      • Thapa N.
      • Lee B.H.
      • Kim I.S.
      TGFBIp/betaig-h3 protein: a versatile matrix molecule induced by TGF-beta.
      TGFBI is most intensively studied in corneal disorders, where mutated TGFBI causes accumulation of cloudy material into the superficial cornea, leading to impaired vision and painful erosions due to poor epithelial adhesion (reviewed by Runager et al.
      • Runager K.
      • Enghild J.J.
      • Klintworth G.K.
      Focus on molecules: transforming growth factor beta induced protein (TGFBIp).
      and Klintworth
      • Klintworth G.K.
      Corneal dystrophies.
      ). Interestingly, several malignant human tumors have been reported to show TGFBI up-regulation, including colorectal,
      • Ivanov S.V.
      • Ivanova A.V.
      • Salnikow K.
      • Timofeeva O.
      • Subramaniam M.
      • Lerman M.I.
      Two novel VHL targets, TGFBI (BIGH3) and its transactivator KLF10, are up-regulated in renal clear cell carcinoma and other tumors.
      • Zhang L.
      • Zhou W.
      • Velculescu V.E.
      • Kern S.E.
      • Hruban R.H.
      • Hamilton S.R.
      • Vogelstein B.
      • Kinzler K.W.
      Gene expression profiles in normal and cancer cells.
      • Kitahara O.
      • Furukawa Y.
      • Tanaka T.
      • Kihara C.
      • Ono K.
      • Yanagawa R.
      • Nita M.E.
      • Takagi T.
      • Nakamura Y.
      • Tsunoda T.
      Alterations of gene expression during colorectal carcinogenesis revealed by cDNA microarrays after laser-capture microdissection of tumor tissues and normal epithelia.
      • Buckhaults P.
      • Rago C.
      • St Croix B.
      • Romans K.E.
      • Saha S.
      • Zhang L.
      • Vogelstein B.
      • Kinzler K.W.
      Secreted and cell surface genes expressed in benign and malignant colorectal tumors.
      • Ma C.
      • Rong Y.
      • Radiloff D.R.
      • Datto M.B.
      • Centeno B.
      • Bao S.
      • Cheng A.W.
      • Lin F.
      • Jiang S.
      • Yeatman T.J.
      • Wang X.F.
      Extracellular matrix protein betaig-h3/TGFBI promotes metastasis of colon cancer by enhancing cell extravasation.
      lung,
      • Sasaki H.
      • Kobayashi Y.
      • Nakashima Y.
      • Moriyama S.
      • Yukiue H.
      • Kaji M.
      • Kiriyama M.
      • Fukai I.
      • Yamakawa Y.
      • Fujii Y.
      Beta IGH3, a TGF-beta inducible gene, is overexpressed in lung cancer.
      and pancreatic cancer,
      • Schneider D.
      • Kleeff J.
      • Berberat P.O.
      • Zhu Z.
      • Korc M.
      • Friess H.
      • Buchler M.W.
      Induction and expression of betaig-h3 in pancreatic cancer cells.
      neuroblastoma,
      • Becker J.
      • Erdlenbruch B.
      • Noskova I.
      • Schramm A.
      • Aumailley M.
      • Schorderet D.F.
      • Schweigerer L.
      Keratoepithelin suppresses the progression of experimental human neuroblastomas.
      glioblastoma,
      • Tso C.L.
      • Shintaku P.
      • Chen J.
      • Liu Q.
      • Liu J.
      • Chen Z.
      • Yoshimoto K.
      • Mischel P.S.
      • Cloughesy T.F.
      • Liau L.M.
      • Nelson S.F.
      Primary glioblastomas express mesenchymal stem-like properties.
      and aggressive sarcomatoid cholangiocarcinoma.
      • Yoo H.J.
      • Yun B.R.
      • Kwon J.H.
      • Ahn H.S.
      • Seol M.A.
      • Lee M.J.
      • Yu G.R.
      • Yu H.C.
      • Hong B.
      • Choi K.
      • Kim D.G.
      Genetic and expression alterations in association with the sarcomatous change of cholangiocarcinoma cells.
      Elevated TGFBI expression has further been related to the aggressiveness of tumors.
      • Sasaki H.
      • Kobayashi Y.
      • Nakashima Y.
      • Moriyama S.
      • Yukiue H.
      • Kaji M.
      • Kiriyama M.
      • Fukai I.
      • Yamakawa Y.
      • Fujii Y.
      Beta IGH3, a TGF-beta inducible gene, is overexpressed in lung cancer.
      • Zajchowski D.A.
      • Bartholdi M.F.
      • Gong Y.
      • Webster L.
      • Liu H.L.
      • Munishkin A.
      • Beauheim C.
      • Harvey S.
      • Ethier S.P.
      • Johnson P.H.
      Identification of gene expression profiles that predict the aggressive behavior of breast cancer cells.
      However, some tumors have been reported to show the opposite, down-regulated expression of TGFBI.
      • Zhao Y.L.
      • Piao C.Q.
      • Hei T.K.
      Downregulation of Betaig-h3 gene is causally linked to tumorigenic phenotype in asbestos treated immortalized human bronchial epithelial cells.
      • Genini M.
      • Schwalbe P.
      • Scholl F.A.
      • Schafer B.W.
      Isolation of genes differentially expressed in human primary myoblasts and embryonal rhabdomyosarcoma.
      Nevertheless, forced expression of TGFBI in hepatoma and colon cancer cells has recently been shown to promote, and, conversely, TGFBI interference to inhibit, cellular invasion in vitro
      • Tang J.
      • Zhou H.W.
      • Jiang J.L.
      • Yang X.M.
      • Li Y.
      • Zhang H.X.
      • Chen Z.N.
      • Guo W.P.
      BetaIg-h3 is involved in the HAb18G/CD147-mediated metastasis process in human hepatoma cells.
      and metastatic spread/extravasation in vivo.
      • Ma C.
      • Rong Y.
      • Radiloff D.R.
      • Datto M.B.
      • Centeno B.
      • Bao S.
      • Cheng A.W.
      • Lin F.
      • Jiang S.
      • Yeatman T.J.
      • Wang X.F.
      Extracellular matrix protein betaig-h3/TGFBI promotes metastasis of colon cancer by enhancing cell extravasation.
      As we found, by global microarray analyses, the melanoma cells from vertical and metastatic growth phases to highly overexpress TGFBI, whose mechanism of action is still poorly understood, we decided to study the function of this molecule in melanoma cell invasion and melanoma progression. Unexpectedly, TGFBI was found to be anti-adhesive for the melanoma cells, and to impair the adhesive contacts of the cells with the ECM molecules fibronectin (FN), collagen-I (COL-I), and laminin (LAM). Most importantly, we show that knockdown of TGFBI expression in metastatic WM239 melanoma cells resulted in dramatic reduction in the tumorigenicity and invasive activity of the cells in nude mice, and complete degeneration and disappearance of the tumor cells.

      Materials and Methods

      Cell Culture

      Primary human melanocytes (42V and Mela3) and primary human adult skin fibroblasts were isolated and cultured as previously.
      • Kääriäinen E.
      • Nummela P.
      • Soikkeli J.
      • Yin M.
      • Lukk M.
      • Jahkola T.
      • Virolainen S.
      • Ora A.
      • Ukkonen E.
      • Saksela O.
      • Hölttä E.
      Switch to an invasive growth phase in melanoma is associated with tenascin-C, fibronectin, and procollagen-I forming specific channel structures for invasion.
      • Soikkeli J.
      • Podlasz P.
      • Yin M.
      • Nummela P.
      • Jahkola T.
      • Virolainen S.
      • Krogerus L.
      • Heikkilä P.
      • von Smitten K.
      • Saksela O.
      • Hölttä E.
      Metastatic outgrowth encompasses COL-I, FN1, and POSTN up-regulation and assembly to fibrillar networks regulating cell adhesion, migration, and growth.
      The melanoma cell lines, WM793 and WM239, established from a vertical growth phase (VGP) melanoma and a melanoma metastasis, respectively, were kindly provided by Dr. Meenhard Herlyn (Wistar Institute, Philadelphia, PA).

      Microarray Analyses

      Total RNAs were isolated from Mela3, 42V, WM793, and WM239 cells, and analyzed by Affymetrix HG-U133 Set arrays (Santa Clara, CA) as before.
      • Kääriäinen E.
      • Nummela P.
      • Soikkeli J.
      • Yin M.
      • Lukk M.
      • Jahkola T.
      • Virolainen S.
      • Ora A.
      • Ukkonen E.
      • Saksela O.
      • Hölttä E.
      Switch to an invasive growth phase in melanoma is associated with tenascin-C, fibronectin, and procollagen-I forming specific channel structures for invasion.
      • Nummela P.
      • Yin M.
      • Kielosto M.
      • Leaner V.
      • Birrer M.J.
      • Hölttä E.
      Thymosin beta4 is a determinant of the transformed phenotype and invasiveness of S-adenosylmethionine decarboxylase-transfected fibroblasts.
      • Soikkeli J.
      • Lukk M.
      • Nummela P.
      • Virolainen S.
      • Jahkola T.
      • Katainen R.
      • Harju L.
      • Ukkonen E.
      • Saksela O.
      • Hölttä E.
      Systematic search for the best gene expression markers for melanoma micrometastasis detection.
      Total RNAs isolated from WM239 cells transfected with the empty vector, non-target small hairpin RNA (shRNA) vector, and TGFBI shRNA construct G6 were hybridized to Affymetrix Human Gene 1.0 ST arrays by Biomedicum Genomics (Helsinki, Finland). The probesets were summarized by RMA algorithm (RMAExpress 1.0.4, http://rmaexpress.bmbolstad.com). Human melanoma lymph node metastases (n = 13) and normal lymph nodes (n = 11) were analyzed by Affymetrix HG-U133 Set arrays as detailed.
      • Soikkeli J.
      • Podlasz P.
      • Yin M.
      • Nummela P.
      • Jahkola T.
      • Virolainen S.
      • Krogerus L.
      • Heikkilä P.
      • von Smitten K.
      • Saksela O.
      • Hölttä E.
      Metastatic outgrowth encompasses COL-I, FN1, and POSTN up-regulation and assembly to fibrillar networks regulating cell adhesion, migration, and growth.

      RT-PCR Analyses

      The expression levels of TGFBI, TGF-β1, TGF-β2, TGF-β3, thymosin β4, integrin β1, talin, and ACTB (β-actin) were analyzed by reverse transcription–PCR (RT-PCR) analyses as described previously
      • Nummela P.
      • Yin M.
      • Kielosto M.
      • Leaner V.
      • Birrer M.J.
      • Hölttä E.
      Thymosin beta4 is a determinant of the transformed phenotype and invasiveness of S-adenosylmethionine decarboxylase-transfected fibroblasts.
      • Soikkeli J.
      • Lukk M.
      • Nummela P.
      • Virolainen S.
      • Jahkola T.
      • Katainen R.
      • Harju L.
      • Ukkonen E.
      • Saksela O.
      • Hölttä E.
      Systematic search for the best gene expression markers for melanoma micrometastasis detection.
      using 1 μg of total RNA; PCR variables are detailed in Table 1. The primers and PCR conditions for ACTB were the same as previously.
      • Soikkeli J.
      • Lukk M.
      • Nummela P.
      • Virolainen S.
      • Jahkola T.
      • Katainen R.
      • Harju L.
      • Ukkonen E.
      • Saksela O.
      • Hölttä E.
      Systematic search for the best gene expression markers for melanoma micrometastasis detection.
      All of the primers were from Sigma-Aldrich (St. Louis, MO). PCR products were resolved in 2% agarose gels and stained with GelStar Nucleic Acid Gel Stain (Cambrex Bio Science Rockland, Rockland, ME). The resulting bands were visualized under UV light and documented with Alpha Imager HP and AlphaEaseFC Software, version 5.0.1 (Alpha Innotech, San Leandro, CA).
      Table 1Primer Sequences and PCR Conditions in Study
      Gene name (Gene ID)SequenceAnnealing (°C)Cycles
      Numbers of cycles were optimized to be in the linear range.
      TGFBI (NM_000358)F 5′-TGTGTGCTGAAGCCATCGTT-3′6125
      R 5′-ACATGGACCACGCCATTTGT-3′
      TGF-β1 (BC022242)F 5′-ACAATTCCTGGCGATACCTCAGCA-3′6125
      R 5′-TCTTCTCCGTGGAGCTGAAGCAAT-3′
      TGF-β2 (BC096235)F 5′-AAACAAGAGCAGAAGGCGAATGGC-3′6130
      R 5′-CTCCAGCACAGAAGTTGGCATTGT-3′
      TGF-β3 (BC018503)F 5′-TGGACTTCGGCCACATCAAGAAGA-3′6140
      R 5′-TCTCCATTGGGCTGAAAGGTGTGA-3′
      TMSB4X (BT007090)F 5′-TGTCTGACAAACCCGATATGGCT-3′5735
      R 5′-GCCTGCTTGCTTCTCCTGTTCAAT-3′
      ITGB1 (NM_002211)F 5′-TGCGAGTGTGGTGTCTGTAAGTGT-3′5630
      R 5′-TGACCACAGTTGTTACGGCACTCT-3′
      TLN1 (AF177198)F 5′-AGGAAGCATGTGGACCTTTGGAGA-3′6130
      R 5′-TGGCAGCCAGAAGAAGTTTGCTTG-3′
      F, forward; R, reverse.
      low asterisk Numbers of cycles were optimized to be in the linear range.

      Western Blot Analyses of Secreted Proteins

      Cells were grown to 85% to 90% confluence, rinsed, and cultured in the serum-free medium for 15 to 16 hours. The conditioned media were collected and concentrated as described
      • Ravanko K.
      • Järvinen K.
      • Helin J.
      • Kalkkinen N.
      • Hölttä E.
      Cysteine cathepsins are central contributors of invasion by cultured adenosylmethionine decarboxylase-transformed rodent fibroblasts.
      using Amicon Ultracel-10 centrifugal filters (MWCO 10,000; Millipore, Bedford, MA), and equal amounts of proteins (10 μg) in 1x Laemmli sample buffer were resolved by 10% SDS–polyacrylamide gel electrophoresis gels. After transfer onto nitrocellulose membranes (Bio-Rad, Hercules, CA), the membranes were stained with Ponceau S to ascertain equal loading, and were immunoblotted for specific proteins.
      • Kielosto M.
      • Nummela P.
      • Katainen R.
      • Leaner V.
      • Birrer M.J.
      • Hölttä E.
      Reversible regulation of the transformed phenotype of ornithine decarboxylase- and ras-overexpressing cells by dominant-negative mutants of c-Jun.

      Adhesion/Spreading Assays

      Cell-binding assays were performed on 96-well plates
      • Soikkeli J.
      • Podlasz P.
      • Yin M.
      • Nummela P.
      • Jahkola T.
      • Virolainen S.
      • Krogerus L.
      • Heikkilä P.
      • von Smitten K.
      • Saksela O.
      • Hölttä E.
      Metastatic outgrowth encompasses COL-I, FN1, and POSTN up-regulation and assembly to fibrillar networks regulating cell adhesion, migration, and growth.
      coated with 5 or 10 μg/mL (volume, 100 μl) recombinant human βIG-H3 (TGFBI; R&D Systems, Minneapolis, MN), alone or combined (1:1) with human cellular fibronectin (cFN; US Biological, Swampscott, MA), human plasma fibronectin (pFN; Chemicon International, Temecula, CA), rat tail COL-I (BD Biosciences, Franklin Lakes, NJ), human collagen type IV (COL-IV; BD Biosciences), mouse LAM (Roche Applied Science, Mannheim, Germany), or recombinant human vitronectin (VTN; R&D Systems). Control wells were coated with fatty acid–free, low-endotoxin BSA (Sigma-Aldrich). The cells were trypsinized (0.1% trypsin), washed, and allowed to recover for 20 to 30 minutes. Then, 20,000 or 25,000 cells (in 100 μl of RPMI 1640) were added onto coated wells and allowed to attach for 90 minutes (±15 minutes) at 37°C, after which they were photographed (at ×100 magnification) using an Olympus IX71 microscope and Olympus DP70 camera (Tokyo, Japan). The percentage of attached and spread cells was calculated from three photographs taken per well. All of the experiments were repeated at least three times, with two or three replicas each time.

      Transfection Experiments to Knock Down TGFBI Expression

      Empty lentiviral pLKO.1 vector, the Expression Arrest TRC shRNA target set for NM_000358 (TGFBI), (Thermo Fisher Scientific, Huntsville, AL), and non-target shRNA control vector (Sigma-Aldrich) were transfected into WM239 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Stable cell lines were generated by puromycin selection (InvivoGen, San Diego, CA; 0.5 μg/mL added 48 hours after the transfection, later raised to 1 or 2 μg/mL) and cylinder cloning (nine TGFBI shRNA clones). Two selected TGFBI shRNA clones, 239 G5B and 239 G6C (bearing TGFBI shRNA constructs G5 and G6, respectively), as well as a non-target shRNA clone 239 nt3A and an empty vector clone 239 pLKO.1, were used in the following experiments.

      Three-Dimensional Migration/Invasion Assays

      Infiltrative growth of WM239 cells transfected with no vector, empty vector, non-target shRNA control vector, or the TGFBI shRNA vectors was studied in three-dimensional (3D) COL-I or Matrigel matrices as described.
      • Nummela P.
      • Yin M.
      • Kielosto M.
      • Leaner V.
      • Birrer M.J.
      • Hölttä E.
      Thymosin beta4 is a determinant of the transformed phenotype and invasiveness of S-adenosylmethionine decarboxylase-transfected fibroblasts.
      • Ravanko K.
      • Järvinen K.
      • Helin J.
      • Kalkkinen N.
      • Hölttä E.
      Cysteine cathepsins are central contributors of invasion by cultured adenosylmethionine decarboxylase-transformed rodent fibroblasts.
      • Kielosto M.
      • Nummela P.
      • Katainen R.
      • Leaner V.
      • Birrer M.J.
      • Hölttä E.
      Reversible regulation of the transformed phenotype of ornithine decarboxylase- and ras-overexpressing cells by dominant-negative mutants of c-Jun.
      In brief, 30,000 melanoma cells were cast between two thick layers of polymerized COL-I solution (BD Biosciences; 2 mg/mL polymerized according to the instructions of the manufacturer) or growth factor–reduced Matrigel (BD Biosciences; lower gel 1:3 dilution, upper gel 1:4 dilution), overlaid with RPMI 1640 + 10% FBS, and followed up daily by microscopy for 6 days. The growth medium was changed after 3 days of growth. The assay was repeated more than three times with two replicas each time.

      Transwell Migration Assay

      Migration of the cells was additionally evaluated with COL-I-coated Falcon cell culture inserts (BD Biosciences) as described elsewhere,
      • Soikkeli J.
      • Podlasz P.
      • Yin M.
      • Nummela P.
      • Jahkola T.
      • Virolainen S.
      • Krogerus L.
      • Heikkilä P.
      • von Smitten K.
      • Saksela O.
      • Hölttä E.
      Metastatic outgrowth encompasses COL-I, FN1, and POSTN up-regulation and assembly to fibrillar networks regulating cell adhesion, migration, and growth.
      using 45 μg/mL of COL-I (BD Biosciences) for coating of the both sides of the membrane, 30,000 cells, and 10% FBS as a chemoattractant in the lower chamber. Migration of the cells through the membrane was visualized by staining with 0.5% Crystal Violet after fixation with 3.5% paraformaldehyde. Cells from the upper surface of the membrane were carefully wiped out, and the chambers were photographed. Migrated cells were quantified by counting five microscopic fields from each chamber (at ×100 magnification). Of these, the average number of cells per field was calculated. The assay was repeated three times with two replicas each time.

      Immunofluorescence Staining

      Cells were grown overnight on glass coverslips, fixed with 3.5% paraformaldehyde, and immunostained
      • Nummela P.
      • Yin M.
      • Kielosto M.
      • Leaner V.
      • Birrer M.J.
      • Hölttä E.
      Thymosin beta4 is a determinant of the transformed phenotype and invasiveness of S-adenosylmethionine decarboxylase-transfected fibroblasts.
      using Alexa Fluor 594–conjugated phalloidin (Molecular Probes, Leiden, the Netherlands) or specific antibodies detailed in Table 2. The secondary antibodies used were Alexa Fluor 488–conjugated anti-mouse and anti-rabbit IgG antibodies (both from Invitrogen), and fluorescein isothiocyanate–conjugated anti-goat IgG antibody (DAKO, Glostrup, Denmark). Vectashield H-1200 mounting medium containing DAPI (Vector Laboratories, Burlingame, CA) was used for mounting. Fluorescence images were taken with a Zeiss Axiophot2 epifluorescence microscope (Carl Zeiss, Oberkochen, Germany) with a 63× (N.A: 1.25, oil) objective, QImaging Retiga 4000R digital camera, and QCapture Pro 6 software (both from QImaging, Surrey, BC, Canada).
      Table 2Antibodies Used in Study
      TargetClone/codeSourceSpecificityMethodManufacturer
      COL-Iab6308Mouseh, r, b, mkIFAbcam (Cambridge, UK)
      FNBP8036RabbithIFAcris Antibodies (Herford, Germany)
      NCL-FIBMousehIHCNovocastra (Newcastle Upon Tyne, UK)
      A0245RabbithIHCDakoCytomation (Glostrup, Denmark)
      ITGAVAV1MousehIFMillipore (Bedford, MA)
      ITGB1P5D2MousehIFSanta Cruz Biotechnology (Santa Cruz, CA)
      MCAM/CD146ab75769Rabbith, m, rIHCAbcam
      MCM7EP1974YRabbith, m, rIHCEpitomics (Burlingame, CA)
      P-FAK (Y397)ab4803Rabbith, mIFAbcam
      POSTNRD181045050Rabbith, r, c, mIHCBioVendor (Heidelberg, Germany)
      S100Z0311Rabbith, m, r, bIHCDakoCytomation
      TGFBI10188-1-APRabbith, mWB, IHCProtein Tech Group (Chicago, IL)
      TLNTD77MousehIFMillipore
      TN-CBC-24MousehIHCAbcam
      α-TubulinDM1AMousebrIFAbcam
      β-Tubulinab21057GoathIFAbcam
      VCL7F9Mouseb, h, m, mk, rbIFMillipore
      VTN8E6(LJ8)MousehIFMillipore
      b, bovine; br, broad range; c, chicken; h, human; IF, immunofluorescence staining; IHC, immunohistochemistry; m, mouse; mk, monkey; r, rat; rb, rabbit; W, Western blotting.

      Tumorigenicity Assays and Histological Analyses

      Animal protocols were approved by the Experimental Animal Committee of Provincial Government of Southern Finland. TGFBI shRNA construct G6-bearing WM239 cells and non-target shRNA-bearing control cells were injected (3 × 106 cells in 75 μl of Opti-MEM I [ Invitrogen] with or without 75 μl of growth factor–reduced Matrigel) subcutaneously into the lower abdominal regions of 7-week-old female BALB/c nu/nu mice, five to six animals per group (one inoculation per animal). Tumor growth was monitored two to three times per week using a caliper (measuring in three dimensions), and the mice were euthanized at day 30 (the first experiment) or later (on tumor reaching the ethical limits; the second experiment). The tumors, lungs, livers, and axillar lymph nodes were removed, fixed with formalin, and embedded in paraffin or frozen in liquid nitrogen (two tumors per group). For histological analyses, 5-μm sections were cut from the paraffin blocks and stained with H&E, Herovici's stain (for young and mature COL-I/III), and Alcian blue–periodic acid-Schiff (PAS) stain (for acidic and neutral mucopolysaccharides) according to routine procedures or immunostained for the ECM proteins TGFBI, FN, tenascin-C (TN-C), and periostin (POSTN), melanoma cell markers S100 and melanoma cell adhesion molecule (MCAM), and a DNA replication marker minichromosome maintenance complex component 7 (MCM7) essentially as described previously
      • Kääriäinen E.
      • Nummela P.
      • Soikkeli J.
      • Yin M.
      • Lukk M.
      • Jahkola T.
      • Virolainen S.
      • Ora A.
      • Ukkonen E.
      • Saksela O.
      • Hölttä E.
      Switch to an invasive growth phase in melanoma is associated with tenascin-C, fibronectin, and procollagen-I forming specific channel structures for invasion.
      • Kielosto M.
      • Nummela P.
      • Järvinen K.
      • Yin M.
      • Hölttä E.
      Identification of integrins alpha6 and beta7 as c-Jun- and transformation-relevant genes in highly invasive fibrosarcoma cells.
      using microwaving (TGFBI, POSTN, S100, MCAM, and MCM7) or enzymatic (FN and TN-C) antigen retrieval and 3-amino-9-ethylcarbazole (AEC; Sigma-Aldrich) as a chromogen. Human melanoma lymph node metastases (n = 12) were obtained by surgical excision at Helsinki University Central Hospital and were immunostained for TGFBI and FN as above. Protocols for taking the human tissue specimens were approved by the Ethics Committees of Helsinki University Central Hospital.

      Statistical Analysis

      Values are presented as means ± SD. Significance between tumor growth rates was determined by Student's t-test.

      Results

      TGFBI Is Up-Regulated in Human Melanoma Cells and Skin Fibroblasts

      In genome-wide microarray analyses of VGP (WM793) and metastatic (WM239) melanoma cells (see Supplemental Table S1 at http://ajp.amjpathol.org), both cell lines, especially the metastatic one,
      • Hsu M.Y.
      • Elder D.E.
      • Herlyn M.
      Melanoma: the Wistar melanoma (WM) cell lines.
      • Cruz-Munoz W.
      • Man S.
      • Xu P.
      • Kerbel R.S.
      Development of a preclinical model of spontaneous human melanoma central nervous system metastasis.
      showed highly up-regulated expression of TGFBI, as compared with normal melanocytes (42V and Mela3). Notably, the up-regulation of TGFBI was the second highest increase in the metastatic WM239 cells, superseded only by the matrix metallopeptidase 1 (MMP1; see Supplemental Table S1 at http://ajp.amjpathol.org). As MMP1 is already highly up-regulated in the early VGP WM793 cells, it may be associated with the acquisition of the invasive VGP phenotype (Blackburn et al.
      • Blackburn J.S.
      • Liu I.
      • Coon C.I.
      • Brinckerhoff C.E.
      A matrix metalloproteinase-1/protease activated receptor-1 signaling axis promotes melanoma invasion and metastasis.
      and references therein) rather than the metastatic phenotype, which, in turn, may be true for TGFBI. By RT-PCR analyses, we verified the TGFBI up-regulation in both melanoma cell lines (Figure 1A). These cells also express the TGFBI's inducers TGF-β1 and TGF-β2 (Figure 1A), but not TGF-β3 (data not shown). In addition, TGFBI, TGF-β1, and TGF-β2 expression was also detected in normal human skin fibroblasts (Figure 1A). Furthermore, of the melanoma cell lines, the metastatic WM239 cells, especially, were found to secrete high amounts of the TGFBI protein (Figure 1B).
      Figure thumbnail gr1
      Figure 1Melanoma cells express transforming growth factor β induced (TGFBI) and its inducers TGF-β1 and TGF-β2. A: mRNA expression levels of TGFBI, TGF-β1, and TGF-β2 in normal melanocytes (Mela 3 and 42V), vertical growth phase melanoma cell line (WM793), metastatic melanoma cell line (WM239), and human adult skin fibroblasts as analyzed by RT-PCR. β-Actin (ACTB) was used as a loading control. B: TGFBI secretion by 42V, WM793, and WM239 cells analyzed by Western blotting of concentrated conditioned media (10 μg protein).

      TGFBI Is an Anti-Adhesive Protein for Melanoma Cells and Skin Fibroblasts

      To elucidate the function(s) of TGFBI in melanoma cell invasion, we first analyzed the binding/spreading of WM793 and WM239 cells onto surfaces coated with TGFBI, pFN, cFN, COL-I, COL-IV, LAM, or VTN. As a negative control we used BSA. Notably, neither of the melanoma cell lines bound to TGFBI (showing even less binding than BSA), whereas both cell lines bound effectively to pFN, cFN, COL-I, COL-IV, and VTN (91% to 93% of WM793 and 72% to 88% of WM239 cells; Figure 2). To LAM, these two cell lines showed differential binding: WM239 cells bound only poorly (10%), whereas WM793 cells bound well (83%) (Figure 2). Besides the melanoma cells, a strong anti-adhesive effect for TGFBI was detected with normal human adult skin fibroblasts (see Supplemental Figure S1 at http://ajp.amjpathol.org), which also showed high TGFBI expression (Figure 1A). When TGFBI was combined (1:1) with the adhesive ECM molecules cFN, pFN, COL-I, or COL-IV, the morphology of the adhered cells was markedly changed from spread to more spindle-shaped and round cells (cFN and COL-I results are shown in Figure 3A, upper and middle rows, and 3B), implying less adhesive contacts with the coated surface. When combined with the adhesion molecule VTN, TGFBI showed only a minor, if any, anti-adhesive effect (Figure 3A, lower row, and 3B).
      Figure thumbnail gr2
      Figure 2Recombinant human TGFBI functions as an anti-adhesive molecule for melanoma cells. Percentage of adhered and spread WM793 and WM239 cells in 96 wells coated with BSA (control), plasma fibronectin (pFN), cellular fibronectin (cFN), collagen-I (COL-I), collagen-IV (COL-IV), laminin (LAM), vitronectin (VTN), and TGFBI (10 μg/mL). Cells (20,000/well) were allowed to attach for 90 minutes (±15 minutes), followed by acquisition of three images per well, and calculation of the percentage of bound cells. The graph shows a representative experiment from three individual experiments (with two or three replicas) with mean values ± SD.
      Figure thumbnail gr3
      Figure 3TGFBI impairs melanoma cell adhesion to fibronectin and collagen but not to vitronectin. A: Morphology of WM239 cells bound to fibronectin (cFN), collagen (COL-I), and vitronectin (VTN), and to the same proteins mixed with BSA or the recombinant human TGFBI. Original magnification, ×200. B: Quantification of the percentage (±SD) of adhered/spread cells calculated from the experiment shown in A (two wells of each, three images from each well). Similar results were obtained using WM793 cells.

      TGFBI Knockdown Leads to More Effective Migration of Melanoma Cells in Vitro

      To study the functional significance of TGFBI, we knocked down its expression in the metastatic WM239 cells using TGFBI shRNA expression (Figure 4A and see Supplemental Figure S2 at http://ajp.amjpathol.org). When the WM239 TGFBI-knockdown (TGFBI-KD) cells were cultured in 3D COL-I cultures, they attached and dispersed much more readily than the control WM239 cells transfected with no vector, empty vector, or the non-target shRNA control vector (Figure 4B). Similar increased migration of TGFBI-KD cells was observed with COL-I-coated transwell chambers (see Supplemental Figure S3 at http://ajp.amjpathol.org). Further, when cultured in 3D Matrigel, the TGFBI-KD cells initiated invasion earlier and formed larger invasive colonies than the control cells during the 6-day follow-up (Figure 4C).
      Figure thumbnail gr4
      Figure 4TGFBI knockdown cells (TGFBI-KD) show increased motility and dispersion in 3D cultures in vitro. A: Secreted TGFBI protein from WM239 cells transfected with no vector, non-target shRNA control vector, and TGFBI shRNA vectors (clones G5B and G6C) analyzed by Western blotting of concentrated conditioned media (10 μg protein). (For verification of the media samples containing comparable amounts of total proteins, see at http://ajp.amjpathol.org). Growth of control cells and TGFBI-KD cells in 3D COL-I gel (B) and 3D Matrigel matrix (C) after 1 and 2 days, respectively. Original magnification, ×100.

      TGFBI-KD Cells Have Pseudopodia-Like Structures Showing Highly Concentrated Actin Filaments Co-Localizing with Integrin β1 and Talin

      Because cytoskeleton plays an important role in cell adhesion and migration, we next immunostained TGFBI-KD cells for F-actin, and α- and β-tubulins. WM239 cells transfected with the non-target shRNA vector served as control (Figure 5, A–D). Immunostaining of the tubulins showed no marked difference (data not shown); interestingly, however, the KD cells showed highly concentrated actin filaments at pseudopodia-like structures (Figure 5, E, G, I, and K). In additional double stainings, integrin β1 and talin immunostaining co-localized with F-actin in these areas (Figure 5, E and F and G and H), whereas vinculin staining did not co-localize with the pseudopodia-like structures, but showed a staining pattern similar to that in the control cells (Figure 5, I and J, and see Supplemental Figure S4 at http://ajp.amjpathol.org). In addition, neither phosphorylated focal adhesion kinase (P-FAK; Figure 5L and see Supplemental Figure S4 at http://ajp.amjpathol.org), integrin αV, FN, COL-I, or VTN (data not shown) staining was similarly concentrated in those areas (although P-FAK with multiple spot-like adhesion sites showed some co-localization at a few sites).
      Figure thumbnail gr5
      Figure 5TGFBI-KD cells show areas of highly concentrated actin filaments co-localizing with integrin β1 and talin. WM239 cells transfected with the non-target shRNA control vector (nt3A, A–D) and TGFBI shRNA vector G6C (E–L), stained for actin (ACT)/phalloidin (red; A, C, E, G, I, K), integrin β1 (ITGB1; green; B, F), talin (TLN; green; D, H), vinculin (VCL; green; J), and phosphorylated (Y397) focal adhesion kinase (P-FAK; green; L). Arrows show representative areas of concentrated actin filaments co-localizing with ITGB1 and TLN. Original magnification, ×630.

      Expression of the Actin-Sequestering Molecule Thymosin β4 Is Decreased in TGFBI-KD Cells

      Because TGFBI-KD cells showed altered localization of actin filaments, we next studied the levels of the actin monomer sequestering protein thymosin β4 (regulating actin dynamics) in these cells. By RT-PCR analyses, thymosin β4 expression was markedly reduced in TGFBI-KD cells, whereas no marked change was detected in the integrin β1, talin, or β-actin expression levels (Figure 6). For more holistic analysis of the gene expression changes in TGFBI-KD cells, we performed microarray analysis with Affymetrix Human Gene 1.0 ST arrays. The number of up-regulated genes (twofold or greater) as compared to WM239 cells transfected with a non-target shRNA control vector was as high as 960, and that of down-regulated ones 1205 (to be published elsewhere). The microarray results verified the down-regulation of thymosin β4 (folds 20.6, 16.4, 15.6, and 13.8 with different probe sets), the fold change being the seventh highest. No marked change in the integrin β1, talin, or β-actin expression at the mRNA level was detected with this method either (except for a slight decrease in β-actin).
      Figure thumbnail gr6
      Figure 6TGFBI-KD cells show decreased mRNA expression of the actin sequestering molecule thymosin β4. mRNA expression levels of thymosin β4 (TMSB4X), integrin β1 (ITGB1), talin (TLN1), and β-actin (ACTB) in WM239 cells transfected with no vector, non-target shRNA control vector, and TGFBI shRNA vectors (clones G5B and G6C) analyzed by RT-PCR. Note that the expression of ITGB1 stays stable (serving as a loading control), whereas ACTB shows a slight decrease (consistent with microarray results).

      TGFBI-KD Cells Show Dramatically Impaired Tumor Growth in Mice

      To study the effect of TGFBI knockdown on melanoma cell growth/invasion in a more complex, physiological microenvironment, we first injected TGFBI-KD cells (3 × 106 cells) in growth factor–reduced Matrigel subcutaneously into nude mice. Interestingly, initially the TGFBI-KD cells showed somewhat accelerated tumor growth during the first week (P < 0.001 at day 7), but later on the tumors showed remarkable growth retardation and shrinkage as compared with the tumors induced by the control, non-target shRNA–transfected cells (P < 0.001 at day 30) (Figure 7A). One TGFBI-KD–induced tumor was even completely regressed during the follow-up. Analyses of the tumors by H&E (Figure 7B, upper row) and S100 immunostaining (for melanoma cells; see Supplemental Figure S5, A–C at http://ajp.amjpathol.org) further revealed a very dramatic decrease in tumor cell density in the TGFBI-KD–induced tumors, especially at the tumor periphery (see Supplemental Figure S5C at http://ajp.amjpathol.org). In line with this, the mitotic figures were also extremely rare in the TGFBI-KD-induced tumors and most of them were aberrant ones (data not shown). Furthermore, immunostaining for the DNA replication marker MCM7 showed an extensive reduction in tumor cell proliferation in the TGFBI-KD–induced tumors as compared to the control tumors (see Supplemental Figure S6 at http://ajp.amjpathol.org). Notably, the TGFBI-KD–induced tumors were also unable to invade through the muscle tissue, in contrast to the control tumors (Figure 7B, upper row).
      Figure thumbnail gr7
      Figure 7TGFBI knockdown reduces the tumorigenicity of melanoma cells in nude mice. A: Growth curves of the tumors induced by the non-target shRNA–expressing control cells and TGFBI-KD cells in nude mice. Cells (3 × 106) mixed with Matrigel were subcutaneously injected into the lower abdominal regions of the mice, and tumor growth was monitored by measuring the tumor volume with a caliper two to three times per week, starting at day 5 postimplantation. B: Histochemical analysis of the control tumors (left column) and TGFBI-KD–induced tumors (right column) with H&E staining (top row), TGFBI immunostaining (middle row), and FN immunostaining (bottom row). Insets in lower row show negative controls. Note that the invasive control tumors have infiltrated into the striated muscle (m, red in H&E). Original magnification, ×200.
      To exclude the possibility that the co-injected Matrigel might have contributed to the tumorigenicity results, and to follow the regression of TGFBI-KD-induced tumors for a longer time, we repeated the in vivo experiment without Matrigel. Again, TGFBI-KD cells grew better during the first days (although to a much smaller size than with Matrigel), and then started to regress (see Supplemental Figure S7 at http://ajp.amjpathol.org). Strikingly, after a follow-up of 25 days, all of the TGFBI-KD-induced tumors had disappeared. Three of the six TGFBI-KD–inoculated mice were further followed up for up to 2 months, with no sign of tumor relapse. As all of the TGFBI-KD-induced tumors in this experiment disappeared completely, tissues from the first tumorigenicity experiment (inoculation of the cells with Matrigel) were used for subsequent histochemical comparisons.
      In immunohistochemical analyses of ECM molecules, the control tumors showed intense TGFBI staining at the edges/invasion fronts of the infiltrating tumors (Figure 7B, left column middle row, and see Supplemental Figure S8 A at http://ajp.amjpathol.org), whereas the small, non-infiltrative TGFBI-KD–induced tumors showed either no or only faint, occasional TGFBI staining at the center of the tumors (Figure 7B, right column middle row, and data not shown). It is interesting to note that the degenerating TGFBI-KD melanoma cells neither appeared to activate/recruit host stromal cells to produce TGFBI (for compensation), as the TGFBI antibody used should detect both human and mouse proteins. Intriguingly, in the control tumors, TGFBI staining revealed similar fibrillar structures around the invading melanoma cells that we have previously reported for FN, TN-C, procollagen-I (PCOL-I), and POSTN immunostainings of clinical human melanoma specimens.
      • Kääriäinen E.
      • Nummela P.
      • Soikkeli J.
      • Yin M.
      • Lukk M.
      • Jahkola T.
      • Virolainen S.
      • Ora A.
      • Ukkonen E.
      • Saksela O.
      • Hölttä E.
      Switch to an invasive growth phase in melanoma is associated with tenascin-C, fibronectin, and procollagen-I forming specific channel structures for invasion.
      • Soikkeli J.
      • Podlasz P.
      • Yin M.
      • Nummela P.
      • Jahkola T.
      • Virolainen S.
      • Krogerus L.
      • Heikkilä P.
      • von Smitten K.
      • Saksela O.
      • Hölttä E.
      Metastatic outgrowth encompasses COL-I, FN1, and POSTN up-regulation and assembly to fibrillar networks regulating cell adhesion, migration, and growth.
      Moreover, staining of the murine control tumors for FN, TN-C, and POSTN revealed fibrillar structures around the melanoma cells in these tissues as well, being most prominent at the growing tumor edges (Figure 7B, left column lower row, and see Supplemental Figure S8, B–D at http://ajp.amjpathol.org). In marked contrast to this, the TGFBI-KD-induced tumors were devoid of these fibrillar structures and showed only disorganized, amorphous staining patterns (as shown for FN in Figure 7B, right column lower row).
      Additional analyses of the tumors by Herovici's stain (see Supplemental Figure S5 D–F at http://ajp.amjpathol.org) and Alcian blue–PAS (see Supplemental Figure S5, G–I at http://ajp.amjpathol.org) revealed TGFBI-KD–induced tumors to contain fragmented COL-I/III fibers (especially at tumor edges; see Supplemental Figure S5F at http://ajp.amjpathol.org) and abundant PAS-positive, neutral mucoid material, resembling the extensive myxoid degeneration seen in gastrointestinal stromal tumors (GISTs) after treatment with the tyrosinase kinase inhibitor Glivec.
      • Joensuu H.
      • Roberts P.J.
      • Sarlomo-Rikala M.
      • Andersson L.C.
      • Tervahartiala P.
      • Tuveson D.
      • Silberman S.
      • Capdeville R.
      • Dimitrijevic S.
      • Druker B.
      • Demetri G.D.
      Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor.
      Indeed, it seems that in TGFBI-KD–induced tumors, also the tumor cells themselves undergo myxoid degeneration, in addition to the connective tissue cells (the classical definition).

      TGFBI Is Localized in Fibrillar Structures Surrounding Melanoma Cells in Clinical Human Melanoma Metastases

      To see the potential clinical relevance of the above findings, we finally immunostained TGFBI in human melanoma lymph node metastases (n = 12). Also, in these clinical specimens, TGFBI was found in fibrillar strand and ring structures surrounding melanoma cell nests (Figure 8, A and B). TGFBI was further found to co-localize with FN in these structures (Figure 8, C–H).
      Figure thumbnail gr8
      Figure 8Immunostaining of human melanoma lymph node metastases with antibodies specific to TGFBI and FN. A and B: TGFBI staining of two metastasis specimens. Original magnification: ×100 (A); ×400 (B). C–H: TGFBI (left column) and FN (right column) staining of two more metastasis specimens. Magnification: ×100 (top row); ×200 (middle and bottom rows).

      Discussion

      In this study, we show that the ECM protein TGFBI plays an important role in the invasive growth of melanoma cells, which is a prerequisite for metastasis. Our microarray analyses showed that the WM793 and WM239 melanoma cells exhibit up-regulated expression of TGFBI. In accordance with this, an analysis of multiple melanoma microarray studies
      • Hoek K.S.
      DNA microarray analyses of melanoma gene expression: a decade in the mines.
      has revealed TGFBI up-regulation in several other melanoma cell lines. Interestingly, the metastatic WM239 cells showed a 4.0-fold increase in TGFBI mRNA expression and even higher increase in the amount of secreted TGFBI protein as compared to the VGP WM793 cells. Similarly, Ma et al
      • Ma C.
      • Rong Y.
      • Radiloff D.R.
      • Datto M.B.
      • Centeno B.
      • Bao S.
      • Cheng A.W.
      • Lin F.
      • Jiang S.
      • Yeatman T.J.
      • Wang X.F.
      Extracellular matrix protein betaig-h3/TGFBI promotes metastasis of colon cancer by enhancing cell extravasation.
      have reported a lymph node metastasis-derived colon cancer cell line to secrete more TGFBI than the primary tumor-derived cell line. In addition, a few other studies have related TGFBI up-regulation to increased aggressiveness.
      • Sasaki H.
      • Kobayashi Y.
      • Nakashima Y.
      • Moriyama S.
      • Yukiue H.
      • Kaji M.
      • Kiriyama M.
      • Fukai I.
      • Yamakawa Y.
      • Fujii Y.
      Beta IGH3, a TGF-beta inducible gene, is overexpressed in lung cancer.
      • Zajchowski D.A.
      • Bartholdi M.F.
      • Gong Y.
      • Webster L.
      • Liu H.L.
      • Munishkin A.
      • Beauheim C.
      • Harvey S.
      • Ethier S.P.
      • Johnson P.H.
      Identification of gene expression profiles that predict the aggressive behavior of breast cancer cells.
      • Tang J.
      • Zhou H.W.
      • Jiang J.L.
      • Yang X.M.
      • Li Y.
      • Zhang H.X.
      • Chen Z.N.
      • Guo W.P.
      BetaIg-h3 is involved in the HAb18G/CD147-mediated metastasis process in human hepatoma cells.
      Considering the underlying mechanism for the increase in TGFBI, it is of note that melanoma cells have been found to exhibit constitutive TGF-β expression/signaling both in vitro and in vivo (as in Soikkeli et al
      • Soikkeli J.
      • Podlasz P.
      • Yin M.
      • Nummela P.
      • Jahkola T.
      • Virolainen S.
      • Krogerus L.
      • Heikkilä P.
      • von Smitten K.
      • Saksela O.
      • Hölttä E.
      Metastatic outgrowth encompasses COL-I, FN1, and POSTN up-regulation and assembly to fibrillar networks regulating cell adhesion, migration, and growth.
      and Javelaud et al
      • Javelaud D.
      • Alexaki V.I.
      • Mauviel A.
      Transforming growth factor-beta in cutaneous melanoma.
      and this study), which points to a potential autocrine mechanism contributing to TGFBI up-regulation in melanoma cells.
      When studying the effect of TGFBI on cell adhesion and spreading, we found, surprisingly, that TGFBI was even less adhesive than BSA for the melanoma cell lines, and for the human skin fibroblasts. Furthermore, when combined with known adhesive ECM proteins, TGFBI clearly diminished cell adhesion to cFN and pFN, as well as to COL-I and -IV. These data strongly suggest an anti-adhesive function for TGFBI in these cells. In previous studies, TGFBI (or enhanced expression of TGFBI) has been reported both to inhibit
      • Skonier J.
      • Bennett K.
      • Rothwell V.
      • Kosowski S.
      • Plowman G.
      • Wallace P.
      • Edelhoff S.
      • Disteche C.
      • Neubauer M.
      • Marquardt H.
      Beta ig-h3: a transforming growth factor-beta-responsive gene encoding a secreted protein that inhibits cell attachment in vitro and suppresses the growth of CHO cells in nude mice.
      • Becker J.
      • Erdlenbruch B.
      • Noskova I.
      • Schramm A.
      • Aumailley M.
      • Schorderet D.F.
      • Schweigerer L.
      Keratoepithelin suppresses the progression of experimental human neuroblastomas.
      • Shelton L.
      • Troilo D.
      • Lerner M.R.
      • Gusev Y.
      • Brackett D.J.
      • Rada J.S.
      Microarray analysis of choroid/RPE gene expression in marmoset eyes undergoing changes in ocular growth and refraction.
      • Shelton L.
      • Summers-Rada J.
      TGFBIp inhibits the attachment of human scleral fibroblasts to collagen type I.
      and to promote
      • LeBaron R.G.
      • Bezverkov K.I.
      • Zimber M.P.
      • Pavelec R.
      • Skonier J.
      • Purchio A.F.
      Beta IG-H3, a novel secretory protein inducible by transforming growth factor-beta, is present in normal skin and promotes the adhesion and spreading of dermal fibroblasts in vitro.
      • Hashimoto K.
      • Noshiro M.
      • Ohno S.
      • Kawamoto T.
      • Satakeda H.
      • Akagawa Y.
      • Nakashima K.
      • Okimura A.
      • Ishida H.
      • Okamoto T.
      • Pan H.
      • Shen M.
      • Yan W.
      • Kato Y.
      Characterization of a cartilage-derived 66-kDa protein (RGD-CAP/beta ig-h3) that binds to collagen.
      • Ohno S.
      • Noshiro M.
      • Makihira S.
      • Kawamoto T.
      • Shen M.
      • Yan W.
      • Kawashima-Ohya Y.
      • Fujimoto K.
      • Tanne K.
      • Kato Y.
      RGD-CAP ((beta)ig-h3) enhances the spreading of chondrocytes and fibroblasts via integrin alpha(1)beta(1).
      • Kim J.E.
      • Kim S.J.
      • Lee B.H.
      • Park R.W.
      • Kim K.S.
      • Kim I.S.
      Identification of motifs for cell adhesion within the repeated domains of transforming growth factor-beta-induced gene, betaig-h3.
      • Billings P.C.
      • Whitbeck J.C.
      • Adams C.S.
      • Abrams W.R.
      • Cohen A.J.
      • Engelsberg B.N.
      • Howard P.S.
      • Rosenbloom J.
      The transforming growth factor-beta-inducible matrix protein (beta)ig-h3 interacts with fibronectin.
      • Nam J.O.
      • Kim J.E.
      • Jeong H.W.
      • Lee S.J.
      • Lee B.H.
      • Choi J.Y.
      • Park R.W.
      • Park J.Y.
      • Kim I.S.
      Identification of the alphavbeta3 integrin-interacting motif of betaig-h3 and its anti-angiogenic effect.
      • Reinboth B.
      • Thomas J.
      • Hanssen E.
      • Gibson M.A.
      Beta ig-h3 interacts directly with biglycan and decorin, promotes collagen VI aggregation, and participates in ternary complexing with these macromolecules.
      the adhesion of various cell types. In addition, some studies have reported differential effects of TGFBI on adhesion of different cells.
      • Shelton L.
      • Summers-Rada J.
      TGFBIp inhibits the attachment of human scleral fibroblasts to collagen type I.
      • Ohno S.
      • Noshiro M.
      • Makihira S.
      • Kawamoto T.
      • Shen M.
      • Yan W.
      • Kawashima-Ohya Y.
      • Fujimoto K.
      • Tanne K.
      • Kato Y.
      RGD-CAP ((beta)ig-h3) enhances the spreading of chondrocytes and fibroblasts via integrin alpha(1)beta(1).
      • Ferguson J.W.
      • Mikesh M.F.
      • Wheeler E.F.
      • LeBaron R.G.
      Developmental expression patterns of Beta-ig (betaIG-H3) and its function as a cell adhesion protein.
      As the adhesion differences in the latter reports have been observed with the same assay protocols, they cannot originate from variations in the experimental procedures or the form of the protein used (purified/recombinant mammalian/recombinant bacterial), but likely depend on specific properties of the cell types (eg, cell surface receptors) and the microenvironmental context. Physiologically, this kind of cell type-specific action of TGFBI may be important for tissue development and repair. It is also worth noting here that in the corneal dystrophy patients studied, the cornea seems to be the only affected tissue, even if the TGFBI mutation is present in all of the cells of an affected individual.
      • Klintworth G.K.
      Corneal dystrophies.
      • Kim J.E.
      • Park R.W.
      • Choi J.Y.
      • Bae Y.C.
      • Kim K.S.
      • Joo C.K.
      • Kim I.S.
      Molecular properties of wild-type and mutant betaIG-H3 proteins.
      In support of the idea of cell surface/microenvironmental molecules interplaying with TGFBI, TGFBI has indeed been shown to bind to several ECM molecules, such as COL-I, -II, -IV, and -VI, FN, LAM, proteoglycans (such as decorin and biglycan), and POSTN, a molecule showing a structure highly similar to that of TGFBI itself.
      • Hashimoto K.
      • Noshiro M.
      • Ohno S.
      • Kawamoto T.
      • Satakeda H.
      • Akagawa Y.
      • Nakashima K.
      • Okimura A.
      • Ishida H.
      • Okamoto T.
      • Pan H.
      • Shen M.
      • Yan W.
      • Kato Y.
      Characterization of a cartilage-derived 66-kDa protein (RGD-CAP/beta ig-h3) that binds to collagen.
      • Billings P.C.
      • Whitbeck J.C.
      • Adams C.S.
      • Abrams W.R.
      • Cohen A.J.
      • Engelsberg B.N.
      • Howard P.S.
      • Rosenbloom J.
      The transforming growth factor-beta-inducible matrix protein (beta)ig-h3 interacts with fibronectin.
      • Reinboth B.
      • Thomas J.
      • Hanssen E.
      • Gibson M.A.
      Beta ig-h3 interacts directly with biglycan and decorin, promotes collagen VI aggregation, and participates in ternary complexing with these macromolecules.
      • Kim J.E.
      • Park R.W.
      • Choi J.Y.
      • Bae Y.C.
      • Kim K.S.
      • Joo C.K.
      • Kim I.S.
      Molecular properties of wild-type and mutant betaIG-H3 proteins.
      • Kim B.Y.
      • Olzmann J.A.
      • Choi S.I.
      • Ahn S.Y.
      • Kim T.I.
      • Cho H.S.
      • Suh H.
      • Kim E.K.
      Corneal dystrophy-associated R124H mutation disrupts TGFBI interaction with Periostin and causes mislocalization to the lysosome.
      Our cellular adhesion studies with TGFBI and various ECM protein coatings thus nicely fit with the in vitro interaction studies of these molecules. Of further note, TGFBI has also been shown to interact with various integrins, including α1β1,
      • Ohno S.
      • Noshiro M.
      • Makihira S.
      • Kawamoto T.
      • Shen M.
      • Yan W.
      • Kawashima-Ohya Y.
      • Fujimoto K.
      • Tanne K.
      • Kato Y.
      RGD-CAP ((beta)ig-h3) enhances the spreading of chondrocytes and fibroblasts via integrin alpha(1)beta(1).
      α3β1,
      • Kim J.E.
      • Kim S.J.
      • Lee B.H.
      • Park R.W.
      • Kim K.S.
      • Kim I.S.
      Identification of motifs for cell adhesion within the repeated domains of transforming growth factor-beta-induced gene, betaig-h3.
      αvβ5,
      • Ma C.
      • Rong Y.
      • Radiloff D.R.
      • Datto M.B.
      • Centeno B.
      • Bao S.
      • Cheng A.W.
      • Lin F.
      • Jiang S.
      • Yeatman T.J.
      • Wang X.F.
      Extracellular matrix protein betaig-h3/TGFBI promotes metastasis of colon cancer by enhancing cell extravasation.
      • Shelton L.
      • Summers-Rada J.
      TGFBIp inhibits the attachment of human scleral fibroblasts to collagen type I.
      α6β4,
      • Kim M.O.
      • Yun S.J.
      • Kim I.S.
      • Sohn S.
      • Lee E.H.
      Transforming growth factor-beta-inducible gene-h3 (beta(ig)-h3) promotes cell adhesion of human astrocytoma cells in vitro: implication of alpha6beta4 integrin.
      αvβ3,
      • Shelton L.
      • Summers-Rada J.
      TGFBIp inhibits the attachment of human scleral fibroblasts to collagen type I.
      • Nam J.O.
      • Kim J.E.
      • Jeong H.W.
      • Lee S.J.
      • Lee B.H.
      • Choi J.Y.
      • Park R.W.
      • Park J.Y.
      • Kim I.S.
      Identification of the alphavbeta3 integrin-interacting motif of betaig-h3 and its anti-angiogenic effect.
      • Thapa N.
      • Kang K.B.
      • Kim I.S.
      Beta ig-h3 mediates osteoblast adhesion and inhibits differentiation.
      • Nam E.J.
      • Sa K.H.
      • You D.W.
      • Cho J.H.
      • Seo J.S.
      • Han S.W.
      • Park J.Y.
      • Kim S.I.
      • Kyung H.S.
      • Kim I.S.
      • Kang Y.M.
      Up-regulated transforming growth factor beta-inducible gene h3 in rheumatoid arthritis mediates adhesion and migration of synoviocytes through alpha v beta3 integrin: regulation by cytokines.
      • Ahmed A.A.
      • Mills A.D.
      • Ibrahim A.E.
      • Temple J.
      • Blenkiron C.
      • Vias M.
      • Massie C.E.
      • Iyer N.G.
      • McGeoch A.
      • Crawford R.
      • Nicke B.
      • Downward J.
      • Swanton C.
      • Bell S.D.
      • Earl H.M.
      • Laskey R.A.
      • Caldas C.
      • Brenton J.D.
      The extracellular matrix protein TGFBI induces microtubule stabilization and sensitizes ovarian cancers to paclitaxel.
      and αMβ2.
      • Kim H.J.
      • Kim I.S.
      Transforming growth factor-beta-induced gene product, as a novel ligand of integrin alphaMbeta2, promotes monocytes adhesion, migration and chemotaxis.
      As cells may secrete various ECM molecules and express different sets of integrins on their surface, the cell type may indeed have an influence to the binding and effects of TGFBI.
      In functional studies of TGFBI in melanoma cell invasion/spreading, we used shRNA technology to knock down TGFBI expression in the metastatic WM239 cells, and, surprisingly, found that the resulting TGFBI-KD cells showed increased motility and invasiveness in 3D COL-I and Matrigel cultures (followed up for 6 days). Much of this may, however, be due to the more rapid attachment of TGFBI-KD cells, as noticed in adhesion/spreading assays (data not shown), and subsequent more rapid initiation of the migration/invasion process. Of particular interest, our phalloidin stainings revealed cellular extensions/adhesion sites with highly concentrated actin filament bundles at the edges of the migratory TGFBI-KD cells. Moreover, double-labeling of the cells showed co-localization of these actin filament conglomerates with intense integrin β1 and talin immunostainings. Integrin β1, a subunit of cell surface integrin receptors dimerizing with more than 10 integrin α subunits, can mediate cell attachment to many ECM molecules, including COLs, LAMs, FN, and VTN. Talin, a cytoskeletal protein, in turn, interacts directly with the cytoplasmic tail of integrin β subunits, thereby linking cell surface integrin heterodimer to the actin cytoskeleton, leading to integrin activation.
      • Ziegler W.H.
      • Gingras A.R.
      • Critchley D.R.
      • Emsley J.
      Integrin connections to the cytoskeleton through talin and vinculin.
      Hence, talin co-localizes with activated integrins. As these areas of strong actin filaments and activated integrin β1 were not found in TGFBI overexpressing cells, TGFBI might well function anti-adhesively by blocking the binding of integrin β1 to its adhesive ECM ligands, thereby preventing/modulating integrin activation. In addition to the concentrated actin filaments, we found a down-regulated mRNA expression of the actin sequestering molecule thymosin β4 in TGFBI-KD cells. Thymosin β4 sequesters monomeric actin (G-actin) by binding it 1:1, thereby maintaining a large pool of actin monomers from which G-actin can easily be released for actin polymerization.
      • Mannherz H.G.
      • Hannappel E.
      The beta-thymosins: intracellular and extracellular activities of a versatile actin binding protein family.
      Down-regulation of thymosin β4 in TGFBI-KD cells might thus be associated with a pronounced shift toward F-actin in the equilibrium between G- and F-actin. These data suggest an important role for TGFBI in the regulation of integrin signaling and dynamics of the actin cytoskeleton.
      Most importantly, the TGFBI-KD cells showed greatly impaired tumorigenicity in vivo after subcutaneous injection into nude mice. During the first week of tumor formation, the cells appeared to behave consistently with the in vitro studies ie, started to grow, but later on, their growth became seriously impaired, and the tumors shrank and even completely regressed. In 2D-cultures in vitro, TGFBI-KD cells instead showed somewhat increased growth rate (data not shown), demonstrating that the results obtained with culturing cells in the presence of serum-containing media on plastic Petri dishes should be interpreted with caution. As TGFBI interacts with so many ECM proteins and integrin receptors, the discrepancy between the in vitro and in vivo results could originate from the increased complexity of tissue microenvironment and/or heterotypic signaling between the different cell types. Considering this, it is apparent that also more advanced 3D-model systems (eg, organotypic cultures for long-term follow-up) have to be developed to more faithfully reflect the situation in vivo. Furthermore, the relative abundances/balance of TGFBI and the other ECM proteins (pre-existing or newly synthesized) are likely to be of importance for the outcome of the cellular responses. As TGFBI-KD cells first initiated growth but then regressed, it is possible that down-regulation of TGFBI is advantageous for the cell attachment to the pre-existing matrix (initiation of growth), but that later on TGFBI production is needed for the deposition of a new, organized growth-supporting matrix (see below). In this context, however, it should be noted that TGFBI knockout mice, despite a slower postnatal development, showed increased spontaneous and induced tumor development.
      • Zhang Y.
      • Wen G.
      • Shao G.
      • Wang C.
      • Lin C.
      • Fang H.
      • Balajee A.S.
      • Bhagat G.
      • Hei T.K.
      • Zhao Y.
      TGFBI deficiency predisposes mice to spontaneous tumor development.
      Of specific interest, similarly to our findings, TGFBI knockdown by small interfering RNAs (siRNAs)
      • Ahmed A.A.
      • Mills A.D.
      • Ibrahim A.E.
      • Temple J.
      • Blenkiron C.
      • Vias M.
      • Massie C.E.
      • Iyer N.G.
      • McGeoch A.
      • Crawford R.
      • Nicke B.
      • Downward J.
      • Swanton C.
      • Bell S.D.
      • Earl H.M.
      • Laskey R.A.
      • Caldas C.
      • Brenton J.D.
      The extracellular matrix protein TGFBI induces microtubule stabilization and sensitizes ovarian cancers to paclitaxel.
      and knockout
      • Zhang Y.
      • Wen G.
      • Shao G.
      • Wang C.
      • Lin C.
      • Fang H.
      • Balajee A.S.
      • Bhagat G.
      • Hei T.K.
      • Zhao Y.
      TGFBI deficiency predisposes mice to spontaneous tumor development.
      have been found to result in abnormal mitoses. Loss of chromosomal integrity might thus explain the increased tumor tendency in the TGFBI knockout mice.
      The results of our tumorigenicity assays suggest that TGFBI is important for the invasive growth of melanoma cells. In addition to this, studies with colon cancer cells have indicated that TGFBI may promote the extravasation step of metastasis.
      • Ma C.
      • Rong Y.
      • Radiloff D.R.
      • Datto M.B.
      • Centeno B.
      • Bao S.
      • Cheng A.W.
      • Lin F.
      • Jiang S.
      • Yeatman T.J.
      • Wang X.F.
      Extracellular matrix protein betaig-h3/TGFBI promotes metastasis of colon cancer by enhancing cell extravasation.
      Combined, these data suggest a key role for TGFBI in tumor progression. However, studies with some cell lines have shown the opposite: a decrease in tumor growth after TGFBI overexpression.
      • Skonier J.
      • Bennett K.
      • Rothwell V.
      • Kosowski S.
      • Plowman G.
      • Wallace P.
      • Edelhoff S.
      • Disteche C.
      • Neubauer M.
      • Marquardt H.
      Beta ig-h3: a transforming growth factor-beta-responsive gene encoding a secreted protein that inhibits cell attachment in vitro and suppresses the growth of CHO cells in nude mice.
      • Zhao Y.L.
      • Piao C.Q.
      • Hei T.K.
      Downregulation of Betaig-h3 gene is causally linked to tumorigenic phenotype in asbestos treated immortalized human bronchial epithelial cells.
      • Becker J.
      • Erdlenbruch B.
      • Noskova I.
      • Schramm A.
      • Aumailley M.
      • Schorderet D.F.
      • Schweigerer L.
      Keratoepithelin suppresses the progression of experimental human neuroblastomas.
      Thus, similarly to the cell adhesion studies, the results on tumor growth may depend on the characteristics of the cells and the microenvironment. Indeed, in a recent study, Ween et al
      • Ween M.P.
      • Lokman N.A.
      • Hoffmann P.
      • Rodgers R.J.
      • Ricciardelli C.
      • Oehler M.K.
      Transforming growth factor-beta-induced protein secreted by peritoneal cells increases the metastatic potential of ovarian cancer cells.
      found TGFBI to increase the motility and invasiveness of two ovarian cancer cell lines, but not that of a third, less metastatic one. Here, it should also be noted that the effects of the TGFBI inducer, TGF-β, on different cells (normal versus transformed or primary versus metastatic) may differ, with TGF-β acting as both a tumor suppressor and an oncogene.
      The facts that our control melanoma tumors showed intense TGFBI staining at the tumor edges, invasion fronts, and that the knockdown of TGFBI inhibited tumor growth and invasion, strongly support a role for TGFBI in advancing melanoma tumor growth. Of further interest, in the control tumors TGFBI was found to localize, together with FN, TN-C, and POSTN, in special fibrillar structures surrounding the invading melanoma cells. Previously, we have found similar organization of FN, TN-C, PCOL-I, and POSTN in clinical specimens of invasive melanomas,
      • Kääriäinen E.
      • Nummela P.
      • Soikkeli J.
      • Yin M.
      • Lukk M.
      • Jahkola T.
      • Virolainen S.
      • Ora A.
      • Ukkonen E.
      • Saksela O.
      • Hölttä E.
      Switch to an invasive growth phase in melanoma is associated with tenascin-C, fibronectin, and procollagen-I forming specific channel structures for invasion.
      • Soikkeli J.
      • Podlasz P.
      • Yin M.
      • Nummela P.
      • Jahkola T.
      • Virolainen S.
      • Krogerus L.
      • Heikkilä P.
      • von Smitten K.
      • Saksela O.
      • Hölttä E.
      Metastatic outgrowth encompasses COL-I, FN1, and POSTN up-regulation and assembly to fibrillar networks regulating cell adhesion, migration, and growth.
      suggesting that our murine melanoma tumor model can faithfully reflect the changes in real melanoma tumors. Indeed, we found TGFBI to also form fibrillar structures in clinical melanoma specimens and to show co-localization with the fibrillar structures formed by FN. It is also notable that tight co-expression of TGFBI and FN has previously been reported in ovarian and breast cancers.
      • Ahmed A.A.
      • Mills A.D.
      • Ibrahim A.E.
      • Temple J.
      • Blenkiron C.
      • Vias M.
      • Massie C.E.
      • Iyer N.G.
      • McGeoch A.
      • Crawford R.
      • Nicke B.
      • Downward J.
      • Swanton C.
      • Bell S.D.
      • Earl H.M.
      • Laskey R.A.
      • Caldas C.
      • Brenton J.D.
      The extracellular matrix protein TGFBI induces microtubule stabilization and sensitizes ovarian cancers to paclitaxel.
      Furthermore, by microarray analysis of human melanoma lymph node metastases (n = 13) versus normal lymph nodes (n = 11), we have found TGFBI to show up-regulation in the metastases (1.65-fold change; data not shown), albeit less than that observed with FN.
      • Soikkeli J.
      • Podlasz P.
      • Yin M.
      • Nummela P.
      • Jahkola T.
      • Virolainen S.
      • Krogerus L.
      • Heikkilä P.
      • von Smitten K.
      • Saksela O.
      • Hölttä E.
      Metastatic outgrowth encompasses COL-I, FN1, and POSTN up-regulation and assembly to fibrillar networks regulating cell adhesion, migration, and growth.
      In striking contrast to murine control tumors, in TGFBI-KD–induced tumors, FN, TN-C, and POSTN did not show the organized fibrillar staining pattern, but showed instead an amorphous mass-like staining at the tumor edges. As TGFBI has been previously reported to bind to FN (and other ECM proteins) and integrins (see above), it is possible that TGFBI is somehow modulating the integrin-dependent assembly of FN into the fibrillar ECM structures needed for cell growth and migration.
      • Soikkeli J.
      • Podlasz P.
      • Yin M.
      • Nummela P.
      • Jahkola T.
      • Virolainen S.
      • Krogerus L.
      • Heikkilä P.
      • von Smitten K.
      • Saksela O.
      • Hölttä E.
      Metastatic outgrowth encompasses COL-I, FN1, and POSTN up-regulation and assembly to fibrillar networks regulating cell adhesion, migration, and growth.
      • Sottile J.
      • Hocking D.C.
      • Langenbach K.J.
      Fibronectin polymerization stimulates cell growth by RGD-dependent and -independent mechanisms.
      In any case, our data demonstrating that TGFBI down-regulation may lead to a complete tumor regression highlight how critically important a single ECM protein may be for the invasive growth of melanoma cells. In further studies, it will be interesting to see the exact mechanism(s) of TGFBI action. Altogether, TGFBI offers an attractive, ideal new target for therapy, as knockdown of its expression not only caused inhibition of melanoma tumor growth, but also resulted in myxoid degeneration and complete regression of the tumors. In further support of this proposition, Turtoi et al
      • Turtoi A.
      • Musmeci D.
      • Wang Y.
      • Dumont B.
      • Somja J.
      • Bevilacqua G.
      • De Pauw E.
      • Delvenne P.
      • Castronovo V.
      Identification of novel accessible proteins bearing diagnostic and therapeutic potential in human pancreatic ductal adenocarcinoma.
      recently identified TGFBI as a novel overexpressed protein bearing diagnostic and therapeutic potential in human pancreatic ductal adenocarcinomas. As an extracellular and thus potentially accessible/targetable protein, TGFBI could indeed be of high clinical value in the diagnostics and therapy of several malignancies.

      Acknowledgments

      We thank Leena Saikko, Merja Haukka, and Ulla Kiiski for technical assistance and Leif C. Andersson and Susanna Virolainen for expert histopathological evaluations.

      Supplementary Data

      • Supplemental Figure S1

        Percentage of bound and spread human skin fibroblasts in 96 wells coated with BSA (control), pFN, cFN, COL-I, LAM, and recombinant human TGFBI. Values are mean ± SD.

      • Supplemental Figure S3

        Migration of control and TGFBI-KD cells (30,000) through COL-I-coated transwell chambers. A: Photographs of cells passed through the membrane during the 16-hour incubation and stained with Crystal Violet. Cells were transfected with no vector (WM239), non-target shRNA vector (nt3A), or TGFBI shRNA vectors G5 (G5B) and G6 (G6C). Original magnification, ×100. B: Graph showing the average number of migrated cells per area (calculated from five areas photographed from both replicas). Error bars represent SD.

      • Supplemental Figure S4

        Staining of control cells (non-target shRNA clone 3A, A–D) and TGFBI-KD cells (clone G6C, E–H) with phalloidin (red staining in A, C, E, G), vinculin antibody (green staining in B, F), and phospho-FAK antibody (green staining in D, H). Original magnification, ×630.

      • Supplemental Figure S5

        Histological/immunohistochemical analyses of the inner parts of control (non-target shRNA) tumor (A, D, G) and TGFBI-KD-induced tumor (B, E, H), and the edge of TGFBI-KD–induced tumor (C, F, I) with S100 immunostaining (brown in A–C), Herovici's stain (D–F), and Alcian blue–PAS stain (G–I). Original magnification, ×200.

      • Supplemental Figure S6

        Immunostaining of control (non-target shRNA) tumors (A, C) and TGFBI-KD–induced tumors (B, D) using antibody specific to minichromosome maintenance complex component 7 (MCM7; brown staining). Original magnification, ×100.

      • Supplemental Figure S7

        Growth curves of tumors induced by the non-target shRNA expressing control cells and TGFBI-KD cells injected into nude mice without Matrigel. Cells (3 × 106) in Opti-MEM were subcutaneously injected into the lower abdominal regions of the mice, and tumor growth was monitored by measuring the tumor volume with a caliper two to three times per week. A: Growth of the tumors for the first 25 days. B: Growth until the first control mouse had to be sacrificed for ethical reasons. Note the y axis scale differences in A and B. Values are mean ± SD (six animals per group).

      • Supplemental Figure S8

        Immunostaining of control (non-target shRNA) tumors using antibodies specific to TGFBI (A), FN (B), tenascin-C (TN-C; C), and periostin (POSTN; D). m, Muscle. Images A–C are from the same tumor. Original magnification, ×200.

      References

        • Steeg P.S.
        Tumor metastasis: mechanistic insights and clinical challenges.
        Nat Med. 2006; 12: 895-904
        • Gupta G.P.
        • Massague J.
        Cancer metastasis: building a framework.
        Cell. 2006; 127: 679-695
        • Skonier J.
        • Bennett K.
        • Rothwell V.
        • Kosowski S.
        • Plowman G.
        • Wallace P.
        • Edelhoff S.
        • Disteche C.
        • Neubauer M.
        • Marquardt H.
        Beta ig-h3: a transforming growth factor-beta-responsive gene encoding a secreted protein that inhibits cell attachment in vitro and suppresses the growth of CHO cells in nude mice.
        DNA Cell Biol. 1994; 13: 571-584
        • Zhao Y.L.
        • Piao C.Q.
        • Hei T.K.
        Downregulation of Betaig-h3 gene is causally linked to tumorigenic phenotype in asbestos treated immortalized human bronchial epithelial cells.
        Oncogene. 2002; 21: 7471-7477
        • Ivanov S.V.
        • Ivanova A.V.
        • Salnikow K.
        • Timofeeva O.
        • Subramaniam M.
        • Lerman M.I.
        Two novel VHL targets, TGFBI (BIGH3) and its transactivator KLF10, are up-regulated in renal clear cell carcinoma and other tumors.
        Biochem Biophys Res Commun. 2008; 370: 536-540
        • Skonier J.
        • Neubauer M.
        • Madisen L.
        • Bennett K.
        • Plowman G.D.
        • Purchio A.F.
        cDNA cloning and sequence analysis of beta ig-h3, a novel gene induced in a human adenocarcinoma cell line after treatment with transforming growth factor-beta.
        DNA Cell Biol. 1992; 11: 511-522
        • Thapa N.
        • Lee B.H.
        • Kim I.S.
        TGFBIp/betaig-h3 protein: a versatile matrix molecule induced by TGF-beta.
        Int J Biochem Cell Biol. 2007; 39: 2183-2194
        • Runager K.
        • Enghild J.J.
        • Klintworth G.K.
        Focus on molecules: transforming growth factor beta induced protein (TGFBIp).
        Exp Eye Res. 2008; 87: 298-299
        • Klintworth G.K.
        Corneal dystrophies.
        Orphanet J Rare Dis. 2009; 4: 7
        • Zhang L.
        • Zhou W.
        • Velculescu V.E.
        • Kern S.E.
        • Hruban R.H.
        • Hamilton S.R.
        • Vogelstein B.
        • Kinzler K.W.
        Gene expression profiles in normal and cancer cells.
        Science. 1997; 276: 1268-1272
        • Kitahara O.
        • Furukawa Y.
        • Tanaka T.
        • Kihara C.
        • Ono K.
        • Yanagawa R.
        • Nita M.E.
        • Takagi T.
        • Nakamura Y.
        • Tsunoda T.
        Alterations of gene expression during colorectal carcinogenesis revealed by cDNA microarrays after laser-capture microdissection of tumor tissues and normal epithelia.
        Cancer Res. 2001; 61: 3544-3549
        • Buckhaults P.
        • Rago C.
        • St Croix B.
        • Romans K.E.
        • Saha S.
        • Zhang L.
        • Vogelstein B.
        • Kinzler K.W.
        Secreted and cell surface genes expressed in benign and malignant colorectal tumors.
        Cancer Res. 2001; 61: 6996-7001
        • Ma C.
        • Rong Y.
        • Radiloff D.R.
        • Datto M.B.
        • Centeno B.
        • Bao S.
        • Cheng A.W.
        • Lin F.
        • Jiang S.
        • Yeatman T.J.
        • Wang X.F.
        Extracellular matrix protein betaig-h3/TGFBI promotes metastasis of colon cancer by enhancing cell extravasation.
        Genes Dev. 2008; 22: 308-321
        • Sasaki H.
        • Kobayashi Y.
        • Nakashima Y.
        • Moriyama S.
        • Yukiue H.
        • Kaji M.
        • Kiriyama M.
        • Fukai I.
        • Yamakawa Y.
        • Fujii Y.
        Beta IGH3, a TGF-beta inducible gene, is overexpressed in lung cancer.
        Jpn J Clin Oncol. 2002; 32: 85-89
        • Schneider D.
        • Kleeff J.
        • Berberat P.O.
        • Zhu Z.
        • Korc M.
        • Friess H.
        • Buchler M.W.
        Induction and expression of betaig-h3 in pancreatic cancer cells.
        Biochim Biophys Acta. 2002; 1588: 1-6
        • Becker J.
        • Erdlenbruch B.
        • Noskova I.
        • Schramm A.
        • Aumailley M.
        • Schorderet D.F.
        • Schweigerer L.
        Keratoepithelin suppresses the progression of experimental human neuroblastomas.
        Cancer Res. 2006; 66: 5314-5321
        • Tso C.L.
        • Shintaku P.
        • Chen J.
        • Liu Q.
        • Liu J.
        • Chen Z.
        • Yoshimoto K.
        • Mischel P.S.
        • Cloughesy T.F.
        • Liau L.M.
        • Nelson S.F.
        Primary glioblastomas express mesenchymal stem-like properties.
        Mol Cancer Res. 2006; 4: 607-619
        • Yoo H.J.
        • Yun B.R.
        • Kwon J.H.
        • Ahn H.S.
        • Seol M.A.
        • Lee M.J.
        • Yu G.R.
        • Yu H.C.
        • Hong B.
        • Choi K.
        • Kim D.G.
        Genetic and expression alterations in association with the sarcomatous change of cholangiocarcinoma cells.
        Exp Mol Med. 2009; 41: 102-115
        • Zajchowski D.A.
        • Bartholdi M.F.
        • Gong Y.
        • Webster L.
        • Liu H.L.
        • Munishkin A.
        • Beauheim C.
        • Harvey S.
        • Ethier S.P.
        • Johnson P.H.
        Identification of gene expression profiles that predict the aggressive behavior of breast cancer cells.
        Cancer Res. 2001; 61: 5168-5178
        • Genini M.
        • Schwalbe P.
        • Scholl F.A.
        • Schafer B.W.
        Isolation of genes differentially expressed in human primary myoblasts and embryonal rhabdomyosarcoma.
        Int J Cancer. 1996; 66: 571-577
        • Tang J.
        • Zhou H.W.
        • Jiang J.L.
        • Yang X.M.
        • Li Y.
        • Zhang H.X.
        • Chen Z.N.
        • Guo W.P.
        BetaIg-h3 is involved in the HAb18G/CD147-mediated metastasis process in human hepatoma cells.
        Exp Biol Med (Maywood). 2007; 232: 344-352
        • Kääriäinen E.
        • Nummela P.
        • Soikkeli J.
        • Yin M.
        • Lukk M.
        • Jahkola T.
        • Virolainen S.
        • Ora A.
        • Ukkonen E.
        • Saksela O.
        • Hölttä E.
        Switch to an invasive growth phase in melanoma is associated with tenascin-C, fibronectin, and procollagen-I forming specific channel structures for invasion.
        J Pathol. 2006; 210: 181-191
        • Soikkeli J.
        • Podlasz P.
        • Yin M.
        • Nummela P.
        • Jahkola T.
        • Virolainen S.
        • Krogerus L.
        • Heikkilä P.
        • von Smitten K.
        • Saksela O.
        • Hölttä E.
        Metastatic outgrowth encompasses COL-I, FN1, and POSTN up-regulation and assembly to fibrillar networks regulating cell adhesion, migration, and growth.
        Am J Pathol. 2010; 177: 387-403
        • Nummela P.
        • Yin M.
        • Kielosto M.
        • Leaner V.
        • Birrer M.J.
        • Hölttä E.
        Thymosin beta4 is a determinant of the transformed phenotype and invasiveness of S-adenosylmethionine decarboxylase-transfected fibroblasts.
        Cancer Res. 2006; 66: 701-712
        • Soikkeli J.
        • Lukk M.
        • Nummela P.
        • Virolainen S.
        • Jahkola T.
        • Katainen R.
        • Harju L.
        • Ukkonen E.
        • Saksela O.
        • Hölttä E.
        Systematic search for the best gene expression markers for melanoma micrometastasis detection.
        J Pathol. 2007; 213: 180-189
        • Ravanko K.
        • Järvinen K.
        • Helin J.
        • Kalkkinen N.
        • Hölttä E.
        Cysteine cathepsins are central contributors of invasion by cultured adenosylmethionine decarboxylase-transformed rodent fibroblasts.
        Cancer Res. 2004; 64: 8831-8838
        • Kielosto M.
        • Nummela P.
        • Katainen R.
        • Leaner V.
        • Birrer M.J.
        • Hölttä E.
        Reversible regulation of the transformed phenotype of ornithine decarboxylase- and ras-overexpressing cells by dominant-negative mutants of c-Jun.
        Cancer Res. 2004; 64: 3772-3779
        • Kielosto M.
        • Nummela P.
        • Järvinen K.
        • Yin M.
        • Hölttä E.
        Identification of integrins alpha6 and beta7 as c-Jun- and transformation-relevant genes in highly invasive fibrosarcoma cells.
        Int J Cancer. 2009; 125: 1065-1073
        • Hsu M.Y.
        • Elder D.E.
        • Herlyn M.
        Melanoma: the Wistar melanoma (WM) cell lines.
        in: Masters J.R.W. Palsson B. Human Cell Culture. Volume 1. UK, Kluwer Academic Publishers, Dordrecht, The Netherlands1998: 259-274
        • Cruz-Munoz W.
        • Man S.
        • Xu P.
        • Kerbel R.S.
        Development of a preclinical model of spontaneous human melanoma central nervous system metastasis.
        Cancer Res. 2008; 68: 4500-4505
        • Blackburn J.S.
        • Liu I.
        • Coon C.I.
        • Brinckerhoff C.E.
        A matrix metalloproteinase-1/protease activated receptor-1 signaling axis promotes melanoma invasion and metastasis.
        Oncogene. 2009; 28: 4237-4248
        • Joensuu H.
        • Roberts P.J.
        • Sarlomo-Rikala M.
        • Andersson L.C.
        • Tervahartiala P.
        • Tuveson D.
        • Silberman S.
        • Capdeville R.
        • Dimitrijevic S.
        • Druker B.
        • Demetri G.D.
        Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor.
        N Engl J Med. 2001; 344: 1052-1056
        • Hoek K.S.
        DNA microarray analyses of melanoma gene expression: a decade in the mines.
        Pigment Cell Res. 2007; 20: 466-484
        • Javelaud D.
        • Alexaki V.I.
        • Mauviel A.
        Transforming growth factor-beta in cutaneous melanoma.
        Pigment Cell Melanoma Res. 2008; 21: 123-132
        • Shelton L.
        • Troilo D.
        • Lerner M.R.
        • Gusev Y.
        • Brackett D.J.
        • Rada J.S.
        Microarray analysis of choroid/RPE gene expression in marmoset eyes undergoing changes in ocular growth and refraction.
        Mol Vis. 2008; 14: 1465-1479
        • Shelton L.
        • Summers-Rada J.
        TGFBIp inhibits the attachment of human scleral fibroblasts to collagen type I.
        Invest Ophthalmol Vis Sci. 2009; 50: 3542-3552
        • LeBaron R.G.
        • Bezverkov K.I.
        • Zimber M.P.
        • Pavelec R.
        • Skonier J.
        • Purchio A.F.
        Beta IG-H3, a novel secretory protein inducible by transforming growth factor-beta, is present in normal skin and promotes the adhesion and spreading of dermal fibroblasts in vitro.
        J Invest Dermatol. 1995; 104: 844-849
        • Hashimoto K.
        • Noshiro M.
        • Ohno S.
        • Kawamoto T.
        • Satakeda H.
        • Akagawa Y.
        • Nakashima K.
        • Okimura A.
        • Ishida H.
        • Okamoto T.
        • Pan H.
        • Shen M.
        • Yan W.
        • Kato Y.
        Characterization of a cartilage-derived 66-kDa protein (RGD-CAP/beta ig-h3) that binds to collagen.
        Biochim Biophys Acta. 1997; 1355: 303-314
        • Ohno S.
        • Noshiro M.
        • Makihira S.
        • Kawamoto T.
        • Shen M.
        • Yan W.
        • Kawashima-Ohya Y.
        • Fujimoto K.
        • Tanne K.
        • Kato Y.
        RGD-CAP ((beta)ig-h3) enhances the spreading of chondrocytes and fibroblasts via integrin alpha(1)beta(1).
        Biochim Biophys Acta. 1999; 1451: 196-205
        • Kim J.E.
        • Kim S.J.
        • Lee B.H.
        • Park R.W.
        • Kim K.S.
        • Kim I.S.
        Identification of motifs for cell adhesion within the repeated domains of transforming growth factor-beta-induced gene, betaig-h3.
        J Biol Chem. 2000; 275: 30907-30915
        • Billings P.C.
        • Whitbeck J.C.
        • Adams C.S.
        • Abrams W.R.
        • Cohen A.J.
        • Engelsberg B.N.
        • Howard P.S.
        • Rosenbloom J.
        The transforming growth factor-beta-inducible matrix protein (beta)ig-h3 interacts with fibronectin.
        J Biol Chem. 2002; 277: 28003-28009
        • Nam J.O.
        • Kim J.E.
        • Jeong H.W.
        • Lee S.J.
        • Lee B.H.
        • Choi J.Y.
        • Park R.W.
        • Park J.Y.
        • Kim I.S.
        Identification of the alphavbeta3 integrin-interacting motif of betaig-h3 and its anti-angiogenic effect.
        J Biol Chem. 2003; 278: 25902-25909
        • Reinboth B.
        • Thomas J.
        • Hanssen E.
        • Gibson M.A.
        Beta ig-h3 interacts directly with biglycan and decorin, promotes collagen VI aggregation, and participates in ternary complexing with these macromolecules.
        J Biol Chem. 2006; 281: 7816-7824
        • Ferguson J.W.
        • Mikesh M.F.
        • Wheeler E.F.
        • LeBaron R.G.
        Developmental expression patterns of Beta-ig (betaIG-H3) and its function as a cell adhesion protein.
        Mech Dev. 2003; 120: 851-864
        • Kim J.E.
        • Park R.W.
        • Choi J.Y.
        • Bae Y.C.
        • Kim K.S.
        • Joo C.K.
        • Kim I.S.
        Molecular properties of wild-type and mutant betaIG-H3 proteins.
        Invest Ophthalmol Vis Sci. 2002; 43: 656-661
        • Kim B.Y.
        • Olzmann J.A.
        • Choi S.I.
        • Ahn S.Y.
        • Kim T.I.
        • Cho H.S.
        • Suh H.
        • Kim E.K.
        Corneal dystrophy-associated R124H mutation disrupts TGFBI interaction with Periostin and causes mislocalization to the lysosome.
        J Biol Chem. 2009; 284: 19580-19591
        • Kim M.O.
        • Yun S.J.
        • Kim I.S.
        • Sohn S.
        • Lee E.H.
        Transforming growth factor-beta-inducible gene-h3 (beta(ig)-h3) promotes cell adhesion of human astrocytoma cells in vitro: implication of alpha6beta4 integrin.
        Neurosci Lett. 2003; 336: 93-96
        • Thapa N.
        • Kang K.B.
        • Kim I.S.
        Beta ig-h3 mediates osteoblast adhesion and inhibits differentiation.
        Bone. 2005; 36: 232-242
        • Nam E.J.
        • Sa K.H.
        • You D.W.
        • Cho J.H.
        • Seo J.S.
        • Han S.W.
        • Park J.Y.
        • Kim S.I.
        • Kyung H.S.
        • Kim I.S.
        • Kang Y.M.
        Up-regulated transforming growth factor beta-inducible gene h3 in rheumatoid arthritis mediates adhesion and migration of synoviocytes through alpha v beta3 integrin: regulation by cytokines.
        Arthritis Rheum. 2006; 54: 2734-2744
        • Ahmed A.A.
        • Mills A.D.
        • Ibrahim A.E.
        • Temple J.
        • Blenkiron C.
        • Vias M.
        • Massie C.E.
        • Iyer N.G.
        • McGeoch A.
        • Crawford R.
        • Nicke B.
        • Downward J.
        • Swanton C.
        • Bell S.D.
        • Earl H.M.
        • Laskey R.A.
        • Caldas C.
        • Brenton J.D.
        The extracellular matrix protein TGFBI induces microtubule stabilization and sensitizes ovarian cancers to paclitaxel.
        Cancer Cell. 2007; 12: 514-527
        • Kim H.J.
        • Kim I.S.
        Transforming growth factor-beta-induced gene product, as a novel ligand of integrin alphaMbeta2, promotes monocytes adhesion, migration and chemotaxis.
        Int J Biochem Cell Biol. 2008; 40: 991-1004
        • Ziegler W.H.
        • Gingras A.R.
        • Critchley D.R.
        • Emsley J.
        Integrin connections to the cytoskeleton through talin and vinculin.
        Biochem Soc Trans. 2008; 36: 235-239
        • Mannherz H.G.
        • Hannappel E.
        The beta-thymosins: intracellular and extracellular activities of a versatile actin binding protein family.
        Cell Motil Cytoskeleton. 2009; 66: 839-851
        • Zhang Y.
        • Wen G.
        • Shao G.
        • Wang C.
        • Lin C.
        • Fang H.
        • Balajee A.S.
        • Bhagat G.
        • Hei T.K.
        • Zhao Y.
        TGFBI deficiency predisposes mice to spontaneous tumor development.
        Cancer Res. 2009; 69: 37-44
        • Ween M.P.
        • Lokman N.A.
        • Hoffmann P.
        • Rodgers R.J.
        • Ricciardelli C.
        • Oehler M.K.
        Transforming growth factor-beta-induced protein secreted by peritoneal cells increases the metastatic potential of ovarian cancer cells.
        Int J Cancer. 2011; 128: 1570-1584
        • Sottile J.
        • Hocking D.C.
        • Langenbach K.J.
        Fibronectin polymerization stimulates cell growth by RGD-dependent and -independent mechanisms.
        J Cell Sci. 2000; 23: 4287-4299
        • Turtoi A.
        • Musmeci D.
        • Wang Y.
        • Dumont B.
        • Somja J.
        • Bevilacqua G.
        • De Pauw E.
        • Delvenne P.
        • Castronovo V.
        Identification of novel accessible proteins bearing diagnostic and therapeutic potential in human pancreatic ductal adenocarcinoma.
        J Proteome Res. 2011; 10: 4302-4313