Quantitative proteomics profiling of murine mammary gland cells unravels impact of annexin-1 on DNA damage response, cell adhesion, and migration

Abstract

Annexin 1 (ANXA1), the first characterized member of the annexin superfamily, is known to bind or annex to cellular membranes in a calcium-dependent manner. Besides mediating inflammation, ANXA1 has also been reported to be involved in important physiopathological implications including cell proliferation, differentiation, apoptosis, cancer, and metastasis. However, with controversies in ANXA1 expression in breast carcinomas, its role in breast cancer initiation and progression remains unclear. To elucidate how ANXA1 plays a role in breast cancer initiation, we performed stable isotope labeling of amino acids in cell culture analysis on normal mammary gland epithelial cells from ANXA1-heterozygous (ANXA1(+/-)) and ANXA1-null (ANXA1(-/-)) mice. Among over 4000 quantified proteins, we observed 214 up-regulated and 169 down-regulated with ANXA1(-/-). Bioinformatics analysis of the down-regulated proteins revealed that ANXA1 is potentially implicated in DNA damage response, whereas the analysis of up-regulated proteins showed the possible roles of ANXA1 in cell adhesion and migration pathways. These observations were supported by relevant functional assays. The assays for DNA damage response demonstrated an accumulation of more DNA damage with slower recovery on heat stress and an impaired oxidative damage response in ANXA1(-/-) cells in comparison with ANXA1(+/-) cells. Overexpressing Yes-associated protein 1 or Yap1, the most down-regulated protein in DNA damage response pathway cluster, rescued the proliferative response in ANXA1(-/-) cells exposed to oxidative damage. Both migration and wound healing assays showed that ANXA1(+/-) cells possess higher motility with better wound closure capability than ANXA1(-/-) cells. Knocking down of β-parvin, the protein with the highest fold change in the cell adhesion protein cluster, indicated an increased cell migration in ANXA1(-/-) cells. Altogether our quantitative proteomics study on ANXA1 suggests that ANXA1 plays a protective role in DNA damage and modulates cell adhesion and motility, indicating its potential role in cancer initiation as well as progression in breast carcinoma.

Fig. 1.

SILAC workflow and protein quantification analysis. A, a schematic workflow of the SILAC forward (Fwd) and reverse (Rev) experiments where ANXA1^+/− and ANXA1^−/− mammary gland cells were adapted in K0R0 and K8R10 (and vice versa). B, distribution of unchanged, up- and down-regulated proteins from merged Fwd and Rev experiments with high confidence ratios. Statistically significant changes in protein levels (unit by log₂ ratio) between the two mammary gland cell lines are marked by the cutoffs shown in blue and red. C, a scatter plot showing the up-regulated (red), down-regulated (blue), and unchanged (black) protein clusters.

Fig. 2.

Heat stress responses of ANXA1^−/−versus ANXA1^+/− mammary gland cells and the effect of Yap1 on intrinsic ROS accumulation/damage in ANXA1^−/−. A, comet assay was performed with the mammary gland cells with and without DNA damage (heat treatment). After 30 min of 45 °C (heat stress) or 37 °C (control), the cells were allowed to recover at the various time points for comet assay. DNA damage was measured as mean tail moment in the graph plotted. B, representatives of SYBER Green-stained comets 24 h after heat stress for both mammary gland cells. Comet Imager Software measures the tail moments of the comets as indicated by the sky blue trail. C, representative pictures from comet assays of the two mammary gland cells at the 0-h time point without any treatment (control) indicated a much higher mean tail moment (1.068) for the knockout than the ANXA1^+/− (0.308). D–F, FACS analyses from the ROS assays. The intrinsic increase in ROS in the ANXA1-deficient is evidenced by the slight shift of the peak to the right relative to ANXA1^+/− (D). No significant difference was observed between control and Yap-1-transfected cells (E and F). The data are representative of three repeats of the experiments.

Fig. 3.

Oxidative stress responses of ANXA1^−/− mammary gland cells with Yap1 overexpression. A, graph showing the cell proliferation of ANXA1^+/− and ANXA1^−/− cells with and without H₂O₂. The cells were treated with 1 mm H₂O₂ overnight, and the absorbance was measured over 2 h using WST reagent. B, Western blot showing the overexpression of mouse Yap1 in ANXA1^−/− cells (left) and graph showing proliferation profile between ANXA1^−/− and Yap1-transfected ANXA1^−/− cells (right). The cells were transfected with pCDNA-Yap1 (mouse) for 24 h before seeding into 96-well for cell proliferation/viability assay by WST reagent. C, graph showing the rescued cellular proliferation response made by Yap1-transfected ANXA1^−/− mammary gland cells. Transfected cells were treated overnight with 1 mm H₂O₂ before measurement. All of the data are representatives of triplicates of two repeats of the experiments.

Fig. 4.

Migration and wound healing capabilities of ANXA1^−/−versus ANXA1^+/− mammary gland cells. A, graph showing an overall representation of four separate sets of migration assays. ANXA1^+/− and ANXA1^−/− mammary gland cells without any treatment were trypsinized for Transwell migration assays using 10% FBS in lower chamber for 48 h. B, representatives of real time snapshots of the directive migration or wound healing assay at different time points (from supplemental Movie 1). C, nearly complete closure of wound by ANXA1^+/− mammary gland cells was observed at 24 h (left with 1.2% scratch area), whereas ANXA1^−/− lagged with 15.8% scratched area as depicted.

Fig. 5.

Effect of β-parvin on migration and wound healing capabilities in ANXA1^−/− mammary gland cells. A, RT-PCR showing the expression levels of β-parvin after knocking down in ANXA1^−/− cells as compared with mock control. β-Actin was used as a normalizing control. B, graph showing an overall representation of two separate sets of migration assays. ANXA1^−/− cells were mock-treated (control) or knocked down with β-parvin siRNA before trypsinized for Transwell migration assays using 10% FBS in lower chamber for 24 h. C, representatives of real time snapshots of the directive or wound healing assay at different time points for the mock control and si-β-parvin ANXA1^−/− cells (from supplemental Movie 2). D, graph depicting nearly complete wound closure by si-β-parvin ANXA1^−/− cells (left with 1% scratch area), whereas the mock control cells lagged with an 11% scratch area at 12 h.

Fig. 6.

Comparison of the cell adhesive/migratory abilities of mock-treated or antisense β-parvin-transfected ANXA^−/− cells. A, hematoxylin and eosin staining of ANXA1^−/− cells transfected with antisense β-parvin that have passed through the porous membrane and adhered to the collagen on the underside of the cell culture insert over 24 h as compared with mock and ANXA1^+/− cells. Pictures (representatives of triplicates from two separate sets of the experiments) were taken under 2.5× and 10× magnification using a light microscope, showing an observable abolition of collagen adhesion by si-β-parvin-transfected ANXA1^−/− cells as compared with the mock control. B, graph showing the total number of cells left in the upper chamber and those that have migrated through the collagen into the lower chamber after 24 h. No statistical significance was observed across the number of cells left in the upper chambers, whereas a statistical significance of p < 0.05 was observed between the mock and si-β-parvin and between the mock and ANXA1^+/− cells but not between si-β-parvin and ANXA1^+/− cells.