A6 Peptide Activates CD44 Adhesive Activity, Induces FAK and MEK Phosphorylation, and Inhibits the Migration and Metastasis of CD44-Expressing Cells

Abstract

The A6 peptide (acetyl-KPSSPPEE-amino) has antitumor activity in the absence of significant adverse events in murine tumor models and clinical trials. A6 shares sequence homology with CD44, an adhesion receptor involved in metastasis that is also a marker of cancer stem cells and drug-resistant phenotypes. We investigated the mechanism of action of A6 by examining its effects on CD44 activity, cell migration, and metastasis. A6 inhibited the migration of a subset of ovarian and breast cancer cell lines, exhibiting IC 50 values of 5 to 110 nmol/L. The ability of A6 to inhibit migration in vitro correlated with CD44 expression. Immunopreciptation studies showed that CD44 binds A6 and that biotin-tagged A6 can be cross-linked to CD44. The binding of A6 altered the structure of CD44 such that it was no longer recognized by a monoclonal antibody to a specific epitope. Importantly, A6 potentiated the CD44-dependent adhesion of cancer cells to hyaluronic acid and activated CD44-mediated signaling, as evidenced by focal adhesion kinase and MAP/ERK kinase phosphorylation. In vivo, A6 (100 mg/kg delivered s.c. twice daily) reduced the number of lung foci generated by the i.v. injection of B16-F10 melanoma cells by 50% (P ¼ 0.029 in an unpaired t test). We conclude that A6 potently blocks the migration of CD44-positive cells in vitro through an interaction with CD44 that alters its structure and activates CD44 to enhance ligand binding and downstream signaling. The concurrent ability of A6 to agonize the CD44 receptor suggests that CD44 activation may represent a novel strategy for inhibiting metastatic disease.

The Cell Surface Receptor CD44: NMR‐Based Characterization of Putative Ligands

“…These results are in agreement with what was found very recently by Liu et al [24] using a sensitive Surface Plasmon Resonance (SPR) binding assay. Because the activity of A6 against CD44 was probed in cellular studies using DF1485 [25] , we also investigated the possibility of a direct interaction between the antibody and A6. For these studies we synthesized a 13 C labeled A6 peptide by introducing a 13 C methyl in its N-terminal acetyl group ( 13 C-A6 peptide).…”

“…Next, we focused our studies on the binding properties of the A6 peptide given that it is in phase 2 clinical trials for CLL and SLL patients [9] (ClinicalTrials.gov Identifier: NCT02046928). The detailed mechanism of action of A6 has not been defined, but studies on metastatic disease suggested that it functions through a CD44-mediated pathway [25] . While cellular studies indicated a possible direct interaction between CD44 and A6, we could not observe any significant binding of A6 to 15 N-labeled sample of hCD44(21–178) in 2D [ 1 H, 15 N]-sofast-HMQC (Figure 2B) and ITC (supplementary Figure S1) experiments.…”

Hyaluronan as a therapeutic target in human diseases

“…Some new peptide inhibitors such as A-6 peptide [305] and P6 peptide [306] have recently been reported. A6 is a capped eight amino acid peptide (Acetyl-KPSSPPEE-NH2) derived from human urokinase plasminogen activator (uPA).…”

Perspectives of CD44 targeting therapies

“…This peptide turned out to bind specifically to CD44 (Piotrowicz et al 2011) and is now examined in a trial phase II study for its efficacy in the treatment of human ovarian cancer (Ghamande et al 2008;Gold et al 2012). Another CD44 binding peptide, was identified in an indirect way, namely in a screen for overlapping synthetic peptides from the laminin α5 globular domain.…”