Accumulation of annexin A2 and S100A10 prevents apoptosis of apically delaminated, transformed epithelial cells

Abstract

In various epithelial tissues, the epithelial monolayer acts as a barrier. To fulfill its function, the structural integrity of the epithelium is tightly controlled. When normal epithelial cells detach from the basal substratum and delaminate into the apical lumen, the apically extruded cells undergo apoptosis, which is termed anoikis. In contrast, transformed cells often become resistant to anoikis and able to survive and grow in the apical luminal space, leading to the formation of multilayered structures, which can be observed at the early stage of carcinogenesis. However, the underlying molecular mechanisms still remain elusive. In this study, we first demonstrate that S100A10 and ANXA2 (Annexin A2) accumulate in apically extruded, transformed cells in both various cell culture systems and murine epithelial tissues in vivo. ANXA2 acts upstream of S100A10 accumulation. Knockdown of ANXA2 promotes apoptosis of apically extruded RasV12-transformed cells and suppresses the formation of multilayered epithelia. In addition, the intracellular reactive oxygen species (ROS) are elevated in apically extruded RasV12 cells. Treatment with ROS scavenger Trolox reduces the occurrence of apoptosis of apically extruded ANXA2-knockdown RasV12 cells and restores the formation of multilayered epithelia. Furthermore, ROS-mediated p38MAPK activation is observed in apically delaminated RasV12 cells, and ANXA2 knockdown further enhances the p38MAPK activity. Moreover, the p38MAPK inhibitor promotes the formation of multilayered epithelia of ANXA2-knockdown RasV12 cells. These results indicate that accumulated ANXA2 diminishes the ROS-mediated p38MAPK activation in apically extruded transformed cells, thereby blocking the induction of apoptosis. Hence, ANXA2 can be a potential therapeutic target to prevent multilayered, precancerous lesions.

Keywords: RasV12-transformed; S100A10; annexin A2; apical extrusion; apoptosis.

Fig. 1.

S100A10 is accumulated in apically extruded, transformed cells. (A) Schematics for phage antibody display screening to identify antibodies that bound to molecules of which expression or membrane localization is up-regulated under the coculture condition of normal and RasV12-transformed MDCK cells. (B and C) Immunofluorescence images of RasV12 cells with 1D2-single chain Fv (scFv) (B) or 1D2-IgG (C). The phage antibody isolated by the screening is 1D2-scFv. 1D2-IgG was generated by adding the constant regions of IgG to 1D2-scFv, which was used in the following immunofluorescence analyses. MDCK-pTR GFP-RasV12 cells were cocultured with normal MDCK cells at the indicated ratio. After 24 h of doxycycline treatment, cells were stained with1D2-scFv (red) (B) or 1D2-IgG (red) (C), Hoechst (blue), and/or Alexa Fluor 647-conjugated phalloidin (gray). (B) White and yellow arrowheads indicate RasV12 cells with and without intense 1D2-scFv immunofluorescence, respectively. (D) Quantification of the 1D2-IgG fluorescence intensity in (C). Values are expressed as a ratio relative to Not extruded. Data are mean ± SD. ****P < 0.0001 (Mann–Whitney test); n = 98 and 124 cells from three independent experiments. (E and F) Identification of S100A10 by immunoprecipitation. Lysates from normal MDCK cells were subjected to immunoprecipitation with 1D2-IgG, followed by western blotting with 1D2-IgG (E) or silver staining (F). The black arrowhead indicates the band analyzed by mass spectrometry. (G and H) Validation of S100A10 as an epitope protein of 1D2 antibody. (G) Lysates from MDCK-pTR GFP-RasV12 Luc-shRNA, S100A10-shRNA1, or S100A10-shRNA2 cells were analyzed by western blotting with 1D2-IgG, commercially obtained anti-S100A10 antibody, or anti-GAPDH antibody. (H) MDCK-pTR GFP-RasV12 Luc-shRNA or S100A10-shRNA1 cells were cocultured with normal cells, and cells were stained with 1D2-IgG (red), Alexa Fluor 647-conjugated phalloidin (gray), and Hoechst (blue). (I–L) Immunofluorescence analyses of S100A10. (I and K) Immunofluorescence images of S100A10 in apically delaminated RasV12 cells (I) and PANC-1 cells (K) under the single culture condition. MDCK-pTR GFP-RasV12 (I) or PANC-1 (K) cells were cultured alone and stained with 1D2-IgG (red), Hoechst (blue), and/or Alexa Fluor 488-conjugated phalloidin (green). (J and L) Quantification of the fluorescence intensity of S100A10 in (I) and (K). Values are expressed as a ratio relative to Not extruded. Data are mean ± SD. ****P < 0.0001 (Mann-Whitney test); (J) n = 201 and 206 cells and (L) n = 103 and 131 cells from three independent experiments. (M) Immunofluorescence images of S100A10 in apically extruded RasV12-expressing cells in the murine pancreatic epithelium. At 7 d after the induction of RasV12 expression by tamoxifen administration, the pancreatic tissue samples from CK19-RasV12-GFP mice were stained with anti-S100A10 (red), anti-GFP (green), and anti-E-cadherin (gray) antibodies, and Hoechst (blue). The asterisks indicate RasV12-expressing cells. [Scale bars, 50 μm (B) and 10 μm (C, H, I, K, and M).]

Fig. 2.

ANXA2 is accumulated in apically extruded, transformed cells. (A) Immunofluorescence images of ANXA2 in apically extruded RasV12 cells. MDCK-pTR GFP-RasV12 Luc-shRNA or ANXA2-shRNA2 cells were cocultured with normal MDCK cells. After 24 h of doxycycline treatment, cells were stained with anti-ANXA2 antibody (red), Alexa Fluor 647-conjugated phalloidin (gray), and Hoechst (blue). (B) Quantification of the fluorescence intensity of ANXA2 in MDCK-pTR GFP-RasV12 Luc-shRNA cells in (A). Values are expressed as a ratio relative to Not extruded. Data are mean ± SD. ****P < 0.0001 (Mann–Whitney test); n = 103 and 86 cells from three independent experiments. (C–F) Immunofluorescence analyses of ANXA2. (C, E, and F) Immunofluorescence images of ANXA2 in apically delaminated RasV12 cells (C and E) and PANC-1 cells (F) under the single culture condition. MDCK-pTR GFP-RasV12 (C and E) or PANC-1 cells (F) were cultured alone and stained with anti-ANXA2 antibody (red), Hoechst (blue), and/or Alexa Fluor 488-conjugated phalloidin (green). (E) Cells were also stained with anti-S100A10 antibody (cyan) to examine the colocalization of ANXA2 and S100A10. (D) Quantification of the fluorescence intensity of ANXA2 in MDCK-pTR GFP-RasV12 Luc-shRNA cells in (C). Values are expressed as a ratio relative to Not extruded. Data are mean ± SD. ****P < 0.0001 (Mann–Whitney test); n = 171 and 130 cells from three independent experiments. (G) Quantification of the fluorescence intensity of ANXA2 in HPDE-pTRE3G GFP-KRasV12, PANC-1, and AsPC-1 cells. Values are expressed as a ratio relative to Not extruded. Data are mean ± SD. ****P < 0.0001 (Mann–Whitney test); n = 100 and 104 cells for HPDEC-RasV12, n = 121 and 167 cells for PANC-1, n = 133 and 152 cells for AsPC-1 from three independent experiments. Representative images of HPDE-pTRE3G GFP-KRasV12 and AsPC-1 cells are shown in SI Appendix, Fig. S2 B and C. (H) Immunofluorescence images of ANXA2 in apically extruded RasV12-expressing cells in the murine pancreatic epithelium. At 7 d after tamoxifen administration, the pancreatic tissue samples from CK19-RasV12-GFP mice were stained with anti-ANXA2 (red), anti-GFP (green), and anti-E-cadherin (gray) antibodies, and Hoechst (blue). [Scale bars, 10 μm (A, C, E, F, and H).]

Fig. 3.

ANXA2 acts upstream of S100A10. (A–D) Immunoblotting analyses for the effect of knockdown of ANXA2 (A and C) or S100A10 (B and D) on the expression of ANXA2 and S100A10. MDCK-pTR GFP-RasV12 Luc-shRNA, ANXA2-shRNA1 or 2 (A), or S100A10-shRNA1 or 2 (B) cells, or PANC-1 cells transfected with control-siRNA, ANXA2-siRNA1 or 2 (C), or S100A10-siRNA1 or 2 (D) were cultured alone. Cell lysates were analyzed by western blotting with anti-ANXA2, anti-S100A10, or anti-GAPDH antibody. (E–J) Immunofluorescence analyses for the effect of knockdown of ANXA2 or S100A10 on the accumulation of S100A10 or ANXA2 in apically extruded transformed cells. (E–H) MDCK-pTR GFP-RasV12 Luc-shRNA, ANXA2-shRNA2 (E and F), or S100A10-shRNA1 (G and H) cells were cocultured with normal MDCK cells (E and G) or cultured alone (F and H). (I and J) PANC-1 cells transfected with control-siRNA, ANXA2-siRNA2 (I), or S100A10-siRNA2 (J) were cultured alone. (E–J) Cells were stained with anti-S100A10 antibody (red), anti-ANXA2 antibody (magenta), Hoechst (blue), and/or Alexa Fluor 647- or 488-conjugated phalloidin (gray or green). [Scale bars, 10 μm (E–J).]

Fig. 4.

Knockdown of ANXA2 promotes apoptosis of apically extruded RasV12-transformed cells. (A–D) NucView analyses for apically extruded RasV12 cells. MDCK-pTR GFP-RasV12 Luc-shRNA or ANXA2-shRNA2 cells were cocultured with normal MDCK cells (A) or cultured alone (C) and stained with NucView 530 (red). Yellow and white arrowheads indicate NucView-positive and -negative, extruded RasV12 cells, respectively. (B and D) Quantification of the ratio of NucView-positive cells in apically shifted/extruded RasV12 cells in (A) and (C). (B) Note that the number of NucView-positive surrounding normal cells is very few. Only NucView-positive GFP-RasV12-expressing cells were analyzed. Data are mean ± SD from three independent experiments. *P < 0.05 (paired two-tailed Student’s test). The total number of analyzed extruded cells is 1,043 and 766 (B) or 491 and 257 (D) from three independent experiments. (E) XZ-images of MDCK-pTR GFP-RasV12 Luc-shRNA, ANXA2-shRNA2, or S100A10-shRNA1 cells. Yellow lines indicate the bilayered regions. (F) Effect of knockdown of ANXA2 or S100A10 on the formation of bilayered epithelia. Normal MDCK, MDCK-pTR GFP-RasV12 Luc-shRNA, ANXA2-shRNA1 or 2, or S100A10-shRNA1 or 2 cells were cultured alone, and the bilayered regions were quantified from XZ-images. Data are mean ± SD from three independent experiments. *P < 0.05 and **P < 0.01 (one-way ANOVA with Dunnett’s test). (G) Effect of cell death inhibitors on the formation of bilayered epithelia of ANXA2-knockdown RasV12 cells. MDCK-pTR GFP-RasV12 ANXA2-shRNA1 or 2 cells were cultured alone and treated with Z-VAD-FMK or Ferrostatin-1. Data are mean ± SD. *P < 0.05; ns, not significant (one-way ANOVA with Dunnett’s test). [Scale bars, 50 μm (A and C) and 10 μm (E).]

Fig. 5.

The ROS-p38MAPK pathway positively regulates apoptosis in extruded ANXA2-knockdown RasV12-transformed cells. (A) Fluorescence images of CellROX in apically extruded RasV12 cells. MDCK-pTR GFP-RasV12 Luc-shRNA or ANXA2-shRNA2 cells were cultured alone in the presence of Z-VAD-FMK and stained with CellROX (red) and Hoechst (blue). (B) Quantification of the fluorescence intensity of CellROX in (A). Values are expressed as a ratio relative to Not extruded Luc-sh. Data are mean ± SD. *P < 0.05, ****P < 0.0001 (one-way ANOVA with Dunnett’s test); n = 227, 282, 176, and 203 cells from four independent experiments. (C) The effect of Trolox on CellROX fluorescence in apically extruded ANXA2-knockdown RasV12 cells. (D) Quantification of the ratio of NucView-positive cells in apically extruded RasV12 cells. MDCK-pTR GFP-RasV12 Luc-shRNA or ANXA2-shRNA2 cells were cultured with or without Trolox and analyzed with NucView. Data are mean ± SD from three independent experiments. *P < 0.05, **P < 0.01; ns, not significant (one-way ANOVA with Dunnett’s test). The total number of analyzed extruded cells is 322, 374, 215, and 341 from three independent experiments. (E) Effect of Trolox on the formation of bilayered epithelia of ANXA2-knockdown RasV12 cells. MDCK-pTR GFP-RasV12 Luc-shRNA, ANXA2-shRNA1 or 2 cells were cultured with Trolox. Data are mean ± SD from three independent experiments. *P < 0.05; ns, not significant. Paired two-tailed Student’s test. (F) Immunofluorescence images of phosphorylated p38MAPK (p-p38MAPK) in apically extruded RasV12 cells. (G) Quantification of p-p38MAPK immunofluorescence intensity. (F and G) MDCK-pTR GFP-RasV12 Luc-shRNA or ANXA2-shRNA2 cells were cultured with or without Trolox in the presence of Z-VAD-FMK and stained with anti-p-p38MAPK antibody (magenta) and Hoechst (blue). (G) Values are expressed as a ratio relative to control Luc-sh_Not extruded. Data are mean ± SD. ****P < 0.0001 (one-way ANOVA with Dunnett’s test); n = 110, 99, 159, 114, 91, 81, 74, and 56 cells from three independent experiments. (H) Effect of p38MAPK inhibitor or JNK inhibitor on the formation of bilayered epithelia of ANXA2-knockdown RasV12 cells. MDCK-pTR GFP-RasV12 ANXA2-shRNA1 or 2 cells were cultured with p38MAPK inhibitor (SB203580) or JNK inhibitor (SP600125). Data are mean ± SD from three independent experiments. *P < 0.05; ns, not significant. One-way ANOVA with Dunnett’s test. [Scale bars, 10 μm (A, C, and F).]

Fig. 6.

Schematics for the functional role of ANXA2 in apically extruded transformed cells. Accumulated ANXA2 suppresses the ROS-mediated activation of p38MAPK and induction of apoptosis, leading to the formation of multilayered epithelia.