C/EBP-b Regulates Cell Death Figure 7. Cleaved caspase-3 staining of tumor sections. Sections of control F10.9-4 tumors and LIP-expressing F10.9-4 tumors of similar sizes, isolated at day 8 (top panels), were immunostained with an antibody against cleaved caspase 3, and nuclei were counter-stained with Hoechst 33258. The fraction of cleaved-caspase3-positive cells was determined by counting sections from 3 control and 3 LIP-expressing tumors (57–94 cells/field, middle top panel). Sections of control F10.9-3 tumors and LAP-expressing F10.9-3 tumors of similar sizes, isolated at day 8 (bottom panels), were prepared and stained as above. The fraction of cleaved-caspase3-positive cells was determined by counting sections from 3 control and 3 LAP- expressing tumors (67–109 cells/field, middle bottom panel). Representative images of sections from 3 tumors are shown. Bar = 0.1 mm. doi:10.1371/journal.pone.0009516.g007 LAP Attenuates Tumor ER Stress Hypoxia, nutrient deprivation and accumulation of toxic metab- olites lead to tumor cell death by triggering ER stress. Therefore, we examined the impact of LAP inhibition or over-expression on ER stress in the tumors. To this end, we immunostained sections obtained from tumors of similar size at day 8 for two ER stress markers, TRIB3 and HERPUD [38,39]. Inhibition of endogenous LAP by over- expression of LIP significantly increased the expression of TRIB3 (P,0.0003, N = 3) and HERPUD (P,0.0005, N = 5), whereas over- expression of LAP reduced the expression of these markers (P,0.01, N = 3 for TRIB3; P,0.01, N = 4 for HERPUD) (Figure 10). The reduced expression of the CHOP-induced death executor TRIB3 indicated that the tumor progression and the inhibition of ER stress– triggered cell death in vitro share a common mechanism, mediated by C/EBP-b. Discussion Tumor progression depends on the ability of its cells to adapt to ER stress due to lagging development of the neovasculature, which lead to nutrient limitation, impaired clearance of toxic metabolites and hypoxia. The combination of these constraints with the inherent hypermutability of cancer cells selects for cells resistant to ER stress–triggered cell death. Such resistance may be achieved by constitutive activation of the protective phase of the UPR [40,41]. Despite significant advances in recent years [22], many features of tumor cell resistance to ER stress are unknown. The present study demonstrates the critical role of resistance to ER stress in tumor progression and shows that C/EBP-b modulates the ER stress- triggered cell death. We suggest that selection of LAP over- expressing cells, as reported in many types of cancers, enables the growing tumor to thrive despite continuous exposure to an ER stress–inducing environment. Although further studies are re- quired to confirm the mode of action of C/EBP-b in the tumor, it is tempting to speculate that LAP or certain LAP-induced genes may serve as potential therapeutic targets for cancer and for other ER stress–mediated diseases. The reported induction of C/EBP-b by ER stress [3] provided a general physiological framework for these studies on top of the tumor-related pathophysiology. Earlier studies with C/EBP-b- deficient cells and mice yielded conflicting results on the role of C/ EBP-b in cell survival and death. Over-expression of LAP has been described in several cancer cell lines and was implicated in oncogenic transformation [25,42]. Other studies found that C/ EBP-b was pro-apoptotic [43,44]. Such conflicting observations are not unexpected, as the single C/EBP-b mRNA is alternatively translated to both transcriptional activators (LAP* and LAP) and to their natural, dominant-negative inhibitor (LIP). Interactions of LAP and LIP with other transcription factors may also explain these conflicting results, as seen by us upon over-expression of LIP in HeLa cells. However, by using B16 melanoma clones that individually express either inducible LAP or LIP (Figure S1), we were able to determine the specific impact of LAP and LIP on the cell fate under ER stress in vitro and in tumors in vivo. Induction of LIP results in suppression of endogenous LAP activity. Induction of LAP, on the other hand, mimics the over-expression seen in many tumors. Our finding that LAP attenuated whereas LIP augmented ER stress–triggered cell death in vitro (Figures 1, 2 &S2) correlated with the observed fourfold increase in the size of LAP-expressing tumors during days 8-13 and the mirror image fourfold decrease in size of LIP-expressing tumors (Figure 5). The protective effect exerted by LAP in cells exposed to nutrient deprivation or hypoxia further supports this correlation (Figure 9). The inhibition of the expression of death executor CHOP by LAP under ER stress (Figure 4) provides one mechanism by which LAP attenuated ER PLoS ONE | www.plosone.org 8 March 2010 | Volume 5 | Issue 3 | e9516