Annexin A1 (AnxA1) is a candidate regulator of the epithelial-to mesenchymal (EMT)-like phenotypic switch, a pivotal event in breast cancer progression. We show here that AnxA1 expression is associated with a highly invasive basal-like breast cancer subtype both in a panel of human breast cancer cell lines as in breast cancer patients and that AnxA1 is functionally related to breast cancer progression. AnxA1 knockdown in invasive basal-like breast cancer cells reduced the number of spontaneous lung metastasis, whereas additional expression of AnxA1 enhanced metastatic spread. AnxA1 promotes metastasis formation by enhancing TGFβ/Smad signaling and actin reorganization, which facilitates an EMT-like switch, thereby allowing efficient cell migration and invasion of metastatic breast cancer cells.

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Dynamic remodeling of the actin cytoskeleton is required for cell spreading, motility, and migration and can be regulated by tyrosine kinase activity. Phosphotyrosine proteomic screening revealed phosphorylation of the lipid-, calcium-, and actin-binding protein annexin A2 (AnxA2) at Tyr23 as a major event preceding ts-v-Src kinase-induced cell scattering. Expression of the phospho-mimicking mutant Y23E-AnxA2 itself was sufficient to induce actin reorganization and cell scattering in MDCK cells. While Y23E-AnxA2, but not Y23A-AnxA2, enhanced Src- or hepatocyte growth factor (HGF)-induced cell scattering, short hairpin RNA-mediated knockdown of AnxA2 inhibited both v-Src- and HGF-induced cell scattering. Three-dimensional branching morphogenesis was induced in wild-type-AnxA2-expressing cells only in the presence of HGF, while Y23E-AnxA2 induced HGF-independent branching morphogenesis. Knockdown of AnxA2 prevented lumen formation during cystogenesis. The Y23E-AnxA2-induced scattering was associated with dephosphorylation/activation of the actin-severing protein cofilin. Likewise, inactive S3E-cofilin and constitutively active LIM kinase, a direct upstream kinase of cofilin, inhibited Y23E-AnxA2-induced scattering. Together, our studies indicate an essential role for AnxA2 phosphorylation in regulating cofilin-dependent actin cytoskeletal dynamics in the context of cell scattering and branching morphogenesis.

Drug-induced liver injury (DILI) is an important clinical problem. It involves crosstalk between drug toxicity and the immune system, but the exact mechanism at the cellular hepatocyte level is not well understood. Here we studied the mechanism of crosstalk in hepatocyte apoptosis caused by diclofenac and the proinflammatory cytokine tumor necrosis factor a (TNF-a). HepG2 cells were treated with diclofenac followed by TNF-a challenge and subsequent evaluation of necrosis and apoptosis. Diclofenac caused a mild apoptosis of HepG2 cells, which was strongly potentiated by TNF-a. A focused apoptosis machinery short interference RNA (siRNA) library screen identified that this TNF-a-mediated enhancement involved activation of caspase-3 through a caspase-8/Bid/APAF1 pathway. Diclofenac itself induced sustained activation of c-Jun N-terminal kinase (JNK) and inhibition of JNK decreased both diclofenac and diclofenac/TNF-a-induced apoptosis. Live cell imaging of GFPp65/RelA showed that diclofenac dampened the TNF-a-mediated nuclear factor kappaB (NF-jB) translocation oscillation in association with reduced NF-jB transcriptional activity. This was associated with inhibition by diclofenac of the TNF-ainduced phosphorylation of the inhibitor of NF-jB alpha (IjBa). Finally, inhibition of IjB kinase b (IKKb) with BMS-345541 as well as stable lentiviral short hairpin RNA (shRNA)-based knockdown of p65/RelA sensitized hepatocytes towards diclofenac/TNFa-induced cytotoxicity. Conclusion: Together, our data suggest a model whereby diclofenac-mediated stress signaling suppresses TNF-a-induced survival signaling routes and sensitizes cells to apoptosis. (HEPATOLOGY 2011;53:2027-2041 A dverse drug reactions are an important cause of morbidity and mortality in humans and druginduced liver injuries (DILIs) are the leading cause of acute liver failure. 1 In addition, DILI accounts for most of the drug attritions 2 and more than 10% of the occurring liver failures happen due to idiosyncratic DILIs. 1 We propose that the crosstalk between drug reactive metabolite-mediated stress responses and cytokine-mediated pro-and antiapoptotic signaling is an important component in the pathophysiology of DILI.Abbreviations: AIF, apoptosis inducing factor; AnxV, annexin V; APAF1, apoptotic protease activating factor 1; c-FLIP, cellular FLICE-like inhibitory protein; DILI, drug-induced liver injury; GFP, green fluorescent protein; IjBa, inhibitor of NF-jB a; IKKb, IjB kinase b; JNK, c-Jun N-terminal kinase; LDH, lactate dehydrogenase; MAPK, mitogen activated protein kinase; NF-jB, nuclear factor-jB; NSAID, nonsteroidal antiinflammatory drug; PUMA, p53 up-regulated modulator of apoptosis; ROS, reactive oxygen species; RAIDD, RIP-associated protein with a death domain, siRNA, short interfering RNA; TNFR-1, TNF receptor-1; TNF-a, tumor necrosis factor a.From the