Hannah L.F. Swa1, Walter P. Blackstock1, Lina H.K Lim2, and Jayantha Gunaratne1
1 - Institute of Molecular and Cell Biology, Singapore
2 - Department of Physiology & NUS Immunology Program, National University of Singapore
Published in Mol Cell Proteomics, April 16, 2012 (Epub ahead of print)
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 physio-pathological 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 (SILAC) 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 ANXA1's possible roles 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 to 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 suggest 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.
(A) A schematic workflow of the SILAC forward (Fwd) and reverse (Rev) experiments (B) A scatter plot showing up-regulated (red), down-regulated (blue) and unchanged protein clusters (black) in the merged Fwd and Rev experiments. (C)/(D) Biological pathway analysis of down-and up-regulated proteins clusters (E) Comet assay showing slower recovery for ANXA1-/- cells after DNA damage (heat stress). (F) Wound healing assay showing slower wound closure for ANXA1-/- cells. (G)Proliferative response of ANXA1-/- cells upon oxidative damage is rescued by over-expressing the most down-regulated protein in the DNA-damage response pathway, Yap1. (H) Migration assay showing better migratory capability in ANXA1-/- cells upon silencing β-parvin, the most up-regulated protein in cell adhesion/motility sub-cluster.
For more information on Quantitative Proteomics Group, please click here.