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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 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 ...

Insulin-like growth factor-binding protein-3 (IGFBP-3) belongs to a family of IGF-binding proteins. Humanin is a peptide known to bind residues 215-232 of mature IGFBP-3 in the C-terminal region of the protein. This region of IGFBP-3 was shown earlier to bind certain glycosaminoglycans including hyaluronan (HA). Here, we characterized the binding affinities of the IGFBP-3 protein and peptide (-KKGFYKKKQCRPSKGRKR-) to HA and to humanin and found that HA binds with a weaker affinity to this region than does humanin. Either HA or humanin could bind to this IGFBP-3 segment, but not simultaneously. The HA receptor, CD44, blocked HA binding to IGFBP-3 but had no effect on binding of humanin to either IGFBP-3 or its peptide. Upon incubation of HA with CD44 and either IGFBP-3 protein or peptide, humanin was effective at binding and sequestering IGFBP-3 or peptide, thereby enabling access of CD44 to HA. We show that IGFBP-3 and humanin in the medium of A549 lung cancer cells can immunoprecipitate in a complex. However, the fraction of IGFBP-3 in the medium that is able to bind HA was not complexed with humanin suggesting that HA binding to the 215-232 segment renders it inaccessible for binding to humanin. Moreover, while the cytotoxic effects of IGFBP-3 on cell viability were reversed by humanin, blocking HA-CD44 interaction with an anti-CD44 antibody in combination with IGFBP-3 did not have an additive negative effect on cell viability suggesting that IGFBP-3 exerts its cytotoxic effects on cell survival through a mechanism that depends on HA-CD44 interactions.

It is known that the humanin (HN) peptide binding to amyloid-β (Aβ) protects against its cytotoxic effects, while acetylcholinesterase (AChE) binding to Aβ increases its aggregation and cytotoxicity. HN is also known to bind the insulin-like growth factor binding protein-3 (IGFBP-3). Here, we examined the regulation of Aβ conformations by HN, AChE, and IGFBP-3 both in vitro and in the conditioned media from A549 and H1299 lung cancer cells. Our in vitro results showed the following: IGFBP-3 binds HN and blocks it from binding Aβ in the absence or presence of AChE; HN and AChE can simultaneously bind Aβ but not when in the presence of IGFBP-3; HN is unable to reduce the aggregation of Aβ in the presence of IGFBP-3; and HN abolishes the aggregation of Aβ induced by the addition of AChE in the absence of IGFBP-3. In the media, AChE and HN can simultaneously bind Aβ. While both AChE and HN are detected when using 6E10 Aβ antibodies, only AChE is detected when using the Aβ 17–24 antibody 4G8, the anti-oligomer A11, and the anti-amyloid fibril LOC antibodies. No signal was observed for IGFBP-3 with any of the anti-amyloid antibodies used. Exogenously added IGFBP-3 reduced the amount of HN found in a complex when using 6E10 antibodies and correlated with a concomitant increase in the amyloid oligomers. Immunodepletion of HN from the media of the A549 and H1299 cells increased the relative abundance of the oligomer vs the total amount of Aβ, the A11-positive prefibrillar oligomers, and to a lesser extent the LOC-positive fibrillar oligomers, and was also correlated with diminished cell viability and increased apoptosis.

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