IntroductionInsulin-like growth factor 1 (IGF-1) receptor (IGF-1R) is phosphorylated in all breast cancer subtypes. Past findings have shown that IGF-1R mediates antiestrogen resistance through cross-talk with estrogen receptor (ER) signaling and via its action upstream of the epidermal growth factor receptor and human epidermal growth factor receptor 2. Yet, the direct role of IGF-1R signaling itself in antiestrogen resistance remains obscure. In the present study, we sought to elucidate whether antiestrogen resistance is induced directly by IGF-1R signaling in response to its ligand IGF-1 stimulation.MethodsA breast cancer cell line ectopically expressing human wild-type IGF-1R, MCF7/IGF-1R, was established by retroviral transduction and colony selection. Cellular antiestrogen sensitivity was evaluated under estrogen-depleted two-dimensional (2D) and 3D culture conditions. Functional activities of the key IGF-1R signaling components in antiestrogen resistance were assessed by specific kinase inhibitor compounds and small interfering RNA.ResultsEctopic expression of IGF-1R in ER-positive MCF7 human breast cancer cells enhanced IGF-1R tyrosine kinase signaling in response to IGF-1 ligand stimulation. The elevated IGF-1R signaling rendered MCF7/IGF-1R cells highly resistant to the antiestrogens tamoxifen and fulvestrant. This antiestrogen-resistant phenotype involved mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol 3-kinase/protein kinase B pathways downstream of the IGF-1R signaling hub and was independent of ER signaling. Intriguingly, a MAPK/ERK-dependent agonistic behavior of tamoxifen at low doses was triggered in the presence of IGF-1, showing a mild promitogenic effect and increasing ER transcriptional activity.ConclusionsOur data provide evidence that the IGF-1/IGF-1R signaling axis may play a causal role in antiestrogen resistance of breast cancer cells, despite continuous suppression of ER transcriptional function by antiestrogens.

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

Drug-induced liver injury (DILI) is an important clinical problem. Here, we used a genomics approach to in detail investigate the hypothesis that critical drug-induced toxicity pathways act in synergy with the pro-inflammatory cytokine tumor necrosis factor α (TNFα) to cause cell death of liver HepG2 cells. Transcriptomics of the cell injury stress response pathways initiated by two hepatoxicants, diclofenac and carbamazepine, revealed the endoplasmic reticulum (ER) stress/translational initiation signaling and nuclear factor-erythroid 2 (NF-E2)-related factor 2 (Nrf2) antioxidant signaling as two major affected pathways, which was similar to that observed for the majority of ∼80 DILI compounds in primary human hepatocytes. Compounds displaying weak or no TNFα synergism, namely ketoconazole, nefazodone, and methotrexate, failed to synchronously induce both pathways. The ER stress induced was primarily related to protein kinase R-like ER kinase (PERK) and activating transcription factor 4 (ATF4) activation and subsequent expression of C/EBP homologous protein (CHOP), which was all independent of TNFα signaling. Identical ATF4 dependent transcriptional programs were observed in primary human hepatocytes as well as primary precision-cut human liver slices. Targeted RNA interference studies revealed that whereas ER stress signaling through inositol-requiring enzyme 1α (IRE1α) and activating transcription factor 6 (ATF6) acted cytoprotective, activation of the ER stress protein kinase PERK and subsequent expression of CHOP was pivotal for the onset of drug/TNFα-induced apoptosis. Whereas inhibition of the Nrf2-dependent adaptive oxidative stress response enhanced the drug/TNFα cytotoxicity, Nrf2 signaling did not affect CHOP expression. Both hepatotoxic drugs enhanced expression of the translational initiation factor EIF4A1, which was essential for CHOP expression and drug/TNFα-mediated cell killing. Our data support a model in which enhanced drug-induced translation initiates PERK-mediated CHOP signaling in an EIF4A1 dependent manner, thereby sensitizing toward caspase-8-dependent TNFα-induced apoptosis.