Intracellular targeting of annexin A2 inhibits tumor cell adhesion, migration, and in vivo grafting

Abstract

Cytoskeletal-associated proteins play an active role in coordinating the adhesion and migration machinery in cancer progression. To identify functional protein networks and potential inhibitors, we screened an internalizing phage (iPhage) display library in tumor cells, and selected LGRFYAASG as a cytosol-targeting peptide. By affinity purification and mass spectrometry, intracellular annexin A2 was identified as the corresponding binding protein. Consistently, annexin A2 and a cell-internalizing, penetratin-fused version of the selected peptide (LGRFYAASG-pen) co-localized and specifically accumulated in the cytoplasm at the cell edges and cell-cell contacts. Functionally, tumor cells incubated with LGRFYAASG-pen showed disruption of filamentous actin, focal adhesions and caveolae-mediated membrane trafficking, resulting in impaired cell adhesion and migration in vitro. These effects were paralleled by a decrease in the phosphorylation of both focal adhesion kinase (Fak) and protein kinase B (Akt). Likewise, tumor cells pretreated with LGRFYAASG-pen exhibited an impaired capacity to colonize the lungs in vivo in several mouse models. Together, our findings demonstrate an unrecognized functional link between intracellular annexin A2 and tumor cell adhesion, migration and in vivo grafting. Moreover, this work uncovers a new peptide motif that binds to and inhibits intracellular annexin A2 as a candidate therapeutic lead for potential translation into clinical applications.

Figure 1

The LGRFYAASG peptide motif is a specific intracellular ligand for annexin A2. (a) iPhage binding assays on protein fractions obtained by affinity chromatography. BSA was used as a negative protein control. TU values were normalized on binding of the insertless iPhage to annexin A2. (b) In silico analysis of potential interactions among the mass spectrometry-identified proteins in the canonical actin cytoskeleton pathway, as determined by the IPA protein network prediction software. ANXA2, annexin A2. (c) Binding of iPhage to GST-fused recombinant annexin A2, LASP1, and CAPZA1. GST and BSA served as protein controls. (d) Binding of LGRFYAASG-iPhage to recombinant annexin A1 (ANXA1), A2 (ANXA2), A4 (ANXA4), and A5 (ANXA5). (e) Binding of LGRFYAASG-iPhage to annexin A2 in the presence of increasing concentrations of the synthetic peptide LGRFYAASG-pen. Unconjugated pen was used as a control, and binding in the absence of the inhibiting peptide was set at 100%. (f) Binding of alanine scanning variants of LGRFYAASG-iPhage. TU values were normalized on binding of an insertless iPhage, and bars represent mean values of triplicate experimental points ± standard error of the mean (SEM).

Figure 2

LGRFYAASG-pen co-localizes with annexin A2 and disrupts actin filaments, focal adhesions and caveolin-rich microdomains. (a) Co-localization analysis: KS1767 cells grown overnight onto circular coverslips were incubated for 2 h at 37 °C with biotinylated LGRFYAASG-pen, or unconjugated LGRFYAASG (10 µM). Untreated cells were used as controls. IF staining of biotinylated peptides (avidin-FITC, green) and annexin A2 (Cy3-conjugated secondary antibody, red) was analyzed with Fiji ImageJ. Co-localized pixels are visualized in white (arrows), and Manders’ overlap coefficients are reported in the graph (**P < 0.01; ***P < 0.001 versus LGRFYAASG-pen). (b,c) Disassembly of actin filaments: IF staining of paxillin (FITC-conjugated secondary antibody, green) and actin (rhodamine-phalloidin, red) in KS1767 cells grown onto (b) fibronectin/vitronectin- or (c) type I collagen-coated coverslips. (d) Localization of annexin A2 at the focal adhesions: KS1767 cells co-stained for paxillin (FITC-conjugated secondary antibody, green) and annexin A2 (Alexa 647-conjugated secondary antibody, red). An enlarged inset (white dotted line) is included for prompt visualization. (e) Western blot of Fak, Akt and Erk1/2 phosphorylation following KS1767 cell incubation with pen only or LGRFYAASG-pen for the indicated periods of time. Total Fak, Akt, Erk1/2 were used as a reference, and actin served as loading control. (f) Disruption of caveolin-rich microdomains: KS1767 cells incubated with the peptides (30 µM) followed by IF staining of caveolin-1 (FITC-conjugated secondary antibody, green) and actin (red). In (b), (c), (d), (f), nuclei are visualized with DAPI (blue); in all micrographs: scale bar, 10 µm.

Figure 3

LGRFYAASG-pen inhibits adhesion of tumor cells. Adhesion of KRIB, KS1767, B16F10 and LLC cells to fibronectin or vitronectin in the presence of LGRFYAASG-pen (30 µM). Unconjugated LGRFYAASG and pen served as negative controls (***P < 0.0001 versus control experimental points).

Figure 4

LGRFYAASG-pen inhibits tumor cell migration. (a) Transwell cell migration assays: 10⁵ cells were applied to the upper chamber of transwell inserts in the presence of each peptide (100 µM for KS1767, KRIB, B16F10, 10 µM for LCC). After 24 h, migrated cells were fixed, stained in crystal violet and counted (5 fields/well) under a light microscope (**P < 0.001; ***P < 0.0001 versus the control experimental points). (b) Wound-healing cell motility assays performed with KS1767, KRIB, LLC and B16F10 cells. A wound was produced with a pipetman tip in confluent cell layers, followed by incubation with either medium only or the indicated peptides (3–30 μM for KS1767, 10–100 μM for KRIB and B16F10, 1–10 μM for LLC). The width of the wounds was imaged with a phase-contrast microscope, and is represented as percent of wound closure. Micrographs refer to the highest peptide concentrations. Scale bar, 100 µm. (**P < 0.001; ***P < 0.0001 versus control experimental points).

Figure 5

LGRFYAASG-pen inhibits the growth of experimental tumors in several preclinical models. Representative gross morphology of lungs explanted after 8 weeks (KRIB, (a), 21 days (B16F10, (b), 13 days (LLC, (c) after fixation in Bouin’s fixative, and corresponding lung weights (n = 8 animals/group) shown as mean ± SEM (****P < 0.0001, **P < 0.001, *P < 0.01 versus vehicle experimental points).