Annexin A1 Preferentially Predicts Poor Prognosis of Basal-Like Breast Cancer Patients by Activating mTOR-S6 Signaling

<div><p>Introduction</p><p>Annexin A1 (ANXA1) is an anti-inflammatory protein reported to play a role in cell proliferation and apoptosis, and to be deregulated in breast cancer. The exact role of annexin A1 in the biology of breast cancer remains unclear. We hypothesized that the annexin A1 plays an oncogenic role in basal subtype of breast cancer by modulating key growth pathway(s).</p><p>Methods</p><p>By mining the Cancer Genome Atlas (TCGA)-Breast Cancer dataset and manipulating annexin A1 levels in breast cancer cell lines, we studied the role of annexin A1 in breast cancer and underlying signaling pathways.</p><p>Results</p><p>Our in-silico analysis of TCGA-breast cancer dataset demonstrated that annexin A1 mRNA expression is higher in basal subtype compared to luminal and HER2 subtypes. Within the basal subtype, patients show significantly poorer overall survival associated with higher expression of annexin A1. In both TCGA patient samples and cell lines, annexin A1 levels were significantly higher in basal-like breast cancer than luminal and Her2/neu-positive breast cancer. Stable annexin A1 knockdown in TNBC cell lines suppressed the mTOR-S6 pathway likely through activation of AMPK but had no impact on the MAPK, c-Met, and EGFR pathways. In a cell migration assay, annexin A1-depleted TNBC cells showed delayed migration as compared to wild-type cells, which could be responsible for poor patient prognosis in basal like breast cancers that are known to express higher annexin A1.</p><p>Conclusions</p><p>Our data suggest that annexin A1 is prognostic only in patients with basal like breast cancer. This appears to be in part due to the role of annexin A1 in activating mTOR-pS6 pathway.</p></div

Annexin A1 knockdown blocks mTOR-S6 signaling.

<p>(A and B) Western blot showing levels of pAKT, AKT, pmTOR, mTOR, S6, pS6, and annexin A1 relative to vinculin (loading control) in MDA-MB-436 and MDA-MB-468 parental cells and annexin A1 silenced clones. Representative images from multiple experiments are shown. Quantitation of these westerns is summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127678#pone.0127678.s003" target="_blank">S3</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127678#pone.0127678.s004" target="_blank">S4</a> Figs</p

High levels of annexin A1 are associated with low overall survival rates in basal-like breast cancer.

<p>The Kaplan-Meier curves indicate the overall survival rates for basal-like (A), luminal (B), and Her2/neu⁺ (C) cancers. The p values refer to comparisons between patients with annexin A1 levels above the median (top 50%) and patients with annexin A1 levels below the median (bottom 50%); in the basal group, p = 0.019.</p

Activation status of oncogenic pathways and knockdown of annexin A1 in TNBC cell lines.

<p>(A) Western blot showing expression of EGFR, c-Met, pAKT, annexin A1, and MAPK relative to vinculin (loading control) in TNBC and ER⁺ cell lines. (B) Western blot showing annexin A1 knockdown in the MDA-MB-436 and MDA-MB-468 cell lines.—ve, scramble control. Representative figures from multiple experiments are shown here.</p

Annexin A1 knockdown activates AMPK.

<p>(A and B) Western blot showing levels of pAMPK, AMPK and annexin A1 relative to vinculin (loading control) in MDA-MB-436 and MDA-MB-468 parental cells and annexin A1 silenced clones. Representative images from multiple experiments are shown. Quantitation of these westerns is summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127678#pone.0127678.s003" target="_blank">S3</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127678#pone.0127678.s004" target="_blank">S4</a> Figs</p

Annexin A1 depletion inhibits cell migration in TNBC cells.

<p>Migration was measured by scratch assays of annexin A1 shRNA-transfected MDA-MB-436 (A) and MDA-MB-468 (B) cells. The mean distances covered in 24hrs (in %, relative to the untreated cells, with standard errors) are shown. Scramble clone indicates non-silencing shRNA. Experiments were repeated three times.</p

TNBC cell lines and basal-like breast cancer patients express high annexin A1.

<p>(A–C) Western blots showing annexin A1 protein expression relative to vinculin loading control in TNBC cell lines (A), ER⁺ cell lines (B), and ER-,Her2/neu⁺ and ER+, Her2/neu⁺ cell lines (C). (D) Proportion of cell lines in which annexin A1 protein was expressed at a level that was at least twice as high as the level in the MCF7 reference (ER⁺) cells. For this analysis, all the Her2+ lines (whether ER+/Her2+ or ER-/Her2+) were combined). (E) Box plots showing annexin A1 mRNA levels that are normalized by quantile normalization within basal, luminal, and HER2/neu+ breast cancers subtype patients. * indicates that basal subtype is statistically different than others at p<0.001. Cell line based experiments were repeated three times.</p