Insulin-like growth factor binding protein-3 (IGFBP-3) belongs to a family of six IGF binding proteins. We previously found that IGFBP-3 exerts its cytotoxic effects on A549 (p53 wild-type) cell survival through a mechanism that depends on hyaluronan-CD44 interactions. To shed light on the mechanism employed, we used CD44-negative normal human lung cells (HFL1), A549, and H1299 (p53-null) lung cancer cells. A synthetic IGFBP-3 peptide ( 215 -KKGfYKKKQcRpSKGRKR-232 ) but not the mutant (K228AR230A), was able to bind hyaluronan more efficiently than the analogous sequences from the other IGFBPs. In a manner comparable to that of the IGFBP-3 protein, the peptide blocked hyaluronan-CD44 signaling, and more effectively inhibited viability of A549 cells than viability of either H1299 or HFL1 cell lines. Treatment with the IGFBP-3 protein or its peptide resulted in increased acetylcholinesterase concentration and activity in the A549 cell media but not in the media of either HFL1 or H1299, an effect that correlated with increased apoptosis and decreased cell viability. These effects were diminished upon the same treatment of A549 cells transfected with either p53 siRNA or acetylcholinesterase siRNA. Taken together, our results show that IGFBP-3 or its peptide blocks hyaluronan-CD44 signaling via a mechanism that depends on both p53 and acetylcholinesterase.Lung cancer is a devastating human disease and among the most common causes of cancer deaths worldwide 1,2 . Of all cases of the disease, non-small cell lung cancer (NSCLC) accounts for approximately 85% 3 .CD44 is a type 1 transmembrane cell-surface glycoprotein with tumor promoting functions in many types of cancer cells 4-7 . It is the main cell surface receptor for hyaluronan (HA) 5-9 . Found on the extracellular side of the cell membrane is the CD44 globular HA-binding domain (HABD) 9,10 shown previously to bind HA as a globular water-soluble protein 11 . CD44 is encoded by a single gene 5,6,12 and many different variant isoforms (CD44v) are generated by alternative splicing that yield different patterns of amino acid insertion into the stalk domain of CD44 with the smallest being the standard CD44 (CD44s) 5,13-15 . Residues 32-123 in the N-terminal domain of CD44, common to both CD44s and CD44v isoforms, contain the HA-binding motif 16 . Assessment of CD44 expression in human lung cancer cell lines 17 , including A549 and H1299 used in this study, showed that the predominant isoform expressed is CD44s 18 . Being a common marker for tumor-initiating cells/cancer stem cells in human carcinomas, CD44 has gained much attention in the cancer literature 14 . HA-CD44 binding is known to modulate numerous downstream signaling cascades, such as the ERK1/2/MAPK and PI3K/Akt pathways, leading to tumor cell proliferation, survival, chemoresistance, and invasiveness 5,7,12,19 .HA is a non-sulfated, anionic glycosaminoglycan 5,16,20,21 polymer composed of the disaccharide sequence (D-glucuronic acid and D-N-acetylglucosamine) without known post-synthetic modification 6,22-24 ...

IGFBP‐3, the most abundant IGFBP and the main carrier of insulin‐like growth factor I (IGF‐I) in the circulation, can bind IGF‐1 with high affinity, which attenuates IGF/IGF‐IR interactions, thereby resulting in antiproliferative effects. The C‐terminal domain of insulin‐like growth factor‐binding protein‐3 (IGFBP‐3) is known to contain an 18‐basic amino acid motif capable of interacting with either humanin (HN) or hyaluronan (HA). We previously showed that the 18‐amino acid IGFBP‐3 peptide is capable of binding either HA or HN with comparable affinities to the full‐length IGFBP‐3 protein and that IGFBP‐3 can compete with the HA receptor, CD44, for binding HA. Blocking the interaction between HA and CD44 reduced viability of A549 human lung cancer cells. In this study, we set out to better characterize IGFBP‐3‐HA interactions. We show that both stereochemistry and amino acid identity are important determinants of the interaction between the IGFBP‐3 peptide and HA and for the peptide's ability to exert its cytotoxic effects. Binding of IGFBP‐3 to either HA or HN was unaffected by glycosylation or reduction of IGFBP‐3, suggesting that the basic 18‐amino acid residue sequence of IGFBP‐3 remains accessible for interaction with either HN or HA upon glycosylation or reduction of the full‐length protein. Removing N‐linked oligosaccharides from CD44 increased its ability to compete with IGFBP‐3 for binding HA, while reduction of CD44 rendered the protein relatively ineffective at blocking IGFBP‐3‐HA interactions. We conclude that both deglycosylation and disulfide bond formation are important for CD44 to compete with IGFBP‐3 for binding HA.

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Insulin‐like growth factor binding protein‐3 (IGFBP‐3) belongs to a family of six IGF binding proteins. We previously found that IGFBP‐3 exerts its cytotoxic effects on A549 (p53 wild‐type) cell survival through a mechanism that depends on hyaluronan‐CD44 interactions. To shed light on the mechanism employed, we used CD44‐negative normal human lung cells (HFL1), A549, and H1299 (p53‐null) lung cancer cells. A synthetic IGFBP‐3 peptide (215‐KKGFYKKKQCRPSKGRKR‐232) but not the mutant (K228AR230A), was able to bind hyaluronan more efficiently than the analogous sequences from the other IGFBPs. In a manner comparable to that of the IGFBP‐3 protein, the peptide blocked HA‐CD44 signaling, and more effectively inhibited viability of A549 cells than viability of either H1299 or HFL1 cell lines. Treatment with the IGFBP‐3 protein or its peptide, affected acetylcholinesterase activity in the A549 cell media but not in the media of either HFL1 or H1299, an effect that correlated with changes in cell viability. These effects were diminished upon the same treatment of A549 cells transfected with either p53 siRNA or acetylcholinesterase siRNA. Taken together, our results show that IGFBP‐3 or its peptide blocks hyaluronan‐CD44 signaling via a mechanism involving p53 and acetylcholinesterase. Support or Funding Information Research reported in this work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM131222 to Hedeel Guy Evans.