Suspension of cultured human foreskin keratinocytes (HKs) with trypsin phosphorylates tyrosine residues on an 80-kDa membrane glycoprotein, p80 (Xia, Y., Gil, S. G., and Carter, W. G. (1996) J. Cell Biol. 132, 727-740). Readhesion dephosphorylates p80. Sequencing of a p80 cDNA established identity to CUB domain-containing protein 1 (CDCP1), a gene elevated in carcinomas. CDCP1/p80 cDNA encodes three extracellular CUB domains, a transmembrane domain, and two putative cytoplasmic Tyr phosphorylation sites. Treatment of adherent HKs with suramin, a heparin analogue, or inhibitors of phosphotyrosine phosphatases (PTPs; vanadate or calpeptin) increases phosphorylation of p80 and a novel 140-kDa membrane glycoprotein, gp140. Phosphorylated gp140 was identified as a trypsin-sensitive precursor to p80. Identity was confirmed by digestion and phosphorylation studies with recombinant gp140-GFP. Plasmin, a serum protease, also converts gp140 to p80, providing biological significance to the cleavage in wounds. Phosphorylation of gp140 and p80 are mediated by Src family kinases at multiple Tyr residues including Tyr 734 . Dephosphorylation is mediated by PTP(s). Conversion of gp140 to p80 prolongs phosphorylation of p80 in response to suramin and changes in adhesion. This distinguishes gp140 and p80 and explains the relative abundance of phosphorylated p80 in trypsinized HKs. We conclude that phosphorylation of gp140 is dynamic and balanced by Src family kinase and PTPs yielding low equilibrium phosphorylation. We suggest that the balance is altered by conversion of gp140 to p80 and by adhesion, providing a novel transmembrane phosphorylation signal in epithelial wounds.Wounding of quiescent epidermis activates changes in adhesion and cell signaling resulting in keratinocyte migration, basement membrane (BM) 1 repair, and wound closure (1-3).Based on an in vitro model for wound activation, we reported that trypsin detachment of cultured human foreskin keratinocytes (HKs) promotes phosphorylation of tyrosine residues on an 80-kDa membrane glycoprotein (p80) (4). Phosphorylated p80 (P-p80) in suspended HKs is dephosphorylated upon readhesion to laminin 5 via integrins ␣ 6 ␤ 4 and ␣ 3 ␤ 1 . In work here, we purified and characterized p80. We wished to understand whether phosphorylated p80 identified in an in vitro deadhesion/readhesion screen (4) might be involved in physiologically significant wound activation. Quiescent epidermis adheres to laminin 5 in the BM via integrin ␣ 6 ␤ 4 in hemidesmosome (HD) cell junctions. Wounding epidermis generates leading and following subpopulations of keratinocytes at the wound margin (5). Leading keratinocytes migrate over exposed dermal collagen via integrin ␣ 2 ␤ 1 and fibronectin via integrin ␣ 5 ␤ 1 . However, leading cells also deposit laminin 5 as a provisional BM and interact with these deposits via integrin ␣ 3 ␤ 1 (5-7). The interaction of leading cells with deposited laminin 5 generates distinct transmembrane signals when compared with interaction with dermal ligands. For example,...

Introduction: During development of primary prostate carcinomas alterations to the extracellular matrix occur, including a loss of laminin 332 (LM332) and one of its cell surface receptors, β4 integrin. There are few published studies on what the laminin composition of metastases may be or how alterations in laminin expression affect prostate cancer cell behavior. Expression of the insulin-like growth factor receptor (IGF-IR) also is altered in prostate cancer progression and some studies suggest cross-talk between integrins and IGF-IR. Purpose: To determine if laminin and integrin expression is altered in prostate cancer metastases and how these alterations affect prostate cancer cell behavior in light of the interactions between integrins and IGF-IR. Methods: We performed immunofluorescent (IF) staining on tissue samples of primary PCa and metastases. We also used various prostate cancer cell lines, including M12 cells transfected with the LMα4, β2, or α4β2 chains, to perform adhesion, migration, and proliferation assays in the presence of inhibitory integrin and IGF-IR antibodies. Results: Our IF staining demonstrated that in addition to a loss in LM332 expression, the LMα4 and β2 chains were absent from the prostate basal membrane in primary prostate tumors. Expression of the LMα5 and β1γ1 chains was retained in primary tumors as well as in lymph node metastases. We saw a loss of β4 integrin in primary tumors as well as in metastatic cancer, but α6 and β1 integrins were retained; in lymph node mets, α2 and α3 integrins also were expressed. For M12 laminin cells, enhanced adhesion to both collagen and fibronectin was further increased by treatment with the inhibitory IGF-IR antibody A12. An inhibitory β1 integrin antibody, but not individual alpha integrin antibodies, blocked this enhancement. Inhibitory β1 as well as alpha integrin antibodies blocked adhesion of the empty-vector control cells. Similar results were obtained for cell migration studies using a scratch-assay. Staining of cells following exposure to A12 showed there was an increase in LM332 and LMβ1γ1 expression compared to untreated cells, which may account for the increased adhesion and migration following A12 treatment. We then examined laminin expression of various prostate cancer cell lines: LNCaP, LNCaP C4-2, PC3, and DU145. We found no expression of LM332 in any of these lines. Cytoplasmic expression of the LMβ1γ1 and LMβ2 (LNCaP lines) chains was detected, but deposition of these laminins was not present. These lines demonstrated increased adhesion to laminin-rich ECM compared with adhesion to fibronectin or collagen. And these lines showed enhanced proliferation when grown on laminin-rich ECM from the M12 laminin cells. Summary: Alterations in laminins affect the adhesion, migration, and proliferation of prostate cancer cells, in part, through integrin and IGF-IR actions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2329.