Purpose: Recent evidence indicates that a tumor suppressor gene CEBPD (CCAAT/enhancer-binding protein delta) is downregulated in many cancers including cervical cancer, which provides a therapeutic potential associated with its reactivation. However, little is known for CEBPD activators and the effect of reactivation of CEBPD transcription upon anticancer drug treatment. In this study, we identified a novel CEBPD activator, 1-(2-hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione (HMDB). The purpose of this study is to characterize the mechanism of HMDB-induced CEBPD activation and its potential effect in cancer therapy.Experimental Design: Methylation-specific PCR assay, reporter assay, and chromatin immunoprecipitation (ChIP) assay were performed to dissect the signaling pathway of HMDB-induced CEBPD transcription. Furthermore, a consequence of HMDB-induced CEBPD expression was linked with E2F1 and retinoblastoma (RB), which discloses the scenario of CEBPD, E2F1, and RB bindings and transcriptional regulation on the promoters of proapoptotic genes, PPARG2 and GADD153. Finally, the anticancer effect of HMDB was examined in xenograft mice.Results: We demonstrate that CEBPD plays an essential role in HMDB-mediated apoptosis of cancer cells. HMDB up-regulates CEBPD transcription through the p38/CREB pathway, thus leading to transcriptional activation of PPARG2 and GADD153. Furthermore, increased level of CEBPD attenuates E2F1-induced cancer cell proliferation and partially rescues RB/E2F1-mediated repression of PPARG2 and GADD153 transcription. Moreover, HMDB treatment attenuates the growth of A431 xenografts in severe combined immunodeficient mice mice.Conclusions: These results clearly demonstrate that HMDB kills cancer cells through activation of CEBPD pathways and suggest that HMDB can serve as a superior chemotherapeutic agent with limited potential for adverse side effects. Clin Cancer Res; 16(23); 5770-80. Ó2010 AACR.

Early studies of the retinoblastoma gene (RB) have uncovered its critical role as a regulator of the G 1 /S cell cycle phase progression. Surprisingly, genetic approaches in mammals and nematodes have also shown RB controls cell lineage specification and aspects of differentiation. The RB gene product accomplishes this by diverse mechanisms such as by interacting with tissue-specific transcription factors, enhancing RNA interference, and modifying chromatin structure. We review recent studies uncovering novel mechanisms by which RB works in several cell lineages and we provide perspectives on how these new findings might relate to RB tumor suppression.

<p>Supplementary Figures S1-S4.</p>

<p>Supplementary Figures S1-S4.</p>

<p>Supplementary Methods</p>

<p>Supplementary Table 1. Expression of CEBPD and its correlation with clinicopathological parameters. Supplementary Table 2. Univariate log rank analysis for prognostic factors in 90 patients with follow-up. Supplementary Table 3. Multivariate survival analyses for prognostic factors in 90 patients with follow-up. Supplementary Table 4: List of primer sequences Supplementary Table 5: List of antibodies for Western blot</p>

<p>Supplemental Figure 1. Anti-cancer drugs induce CEBPD expression in liver cancer cells. Supplemental Figure 2. Scheme of depression of epigenetic effects to enhance HMDB-induced CEBPD expression and apoptosis. Supplemental Figure 3. (Related to Figure 5) The p38/CREB pathway mediates HMDB-induced CEBPD transcriptional activation and CEBPD participates in HMDB-induced apoptosis in liver cancer cells. Supplemental Figure 4. (Related to Figure 5) Combinatorial treatment of HMDB and 5-AzadC can additively affect cell death. Supplemental Figure 5. (Related to Figure 5) Enhanced cytotoxic effects of HMDB combined with 5-AzadC on Huh7 and HepG2 cells. Supplemental Figure 6. (Related to Figure 5) Loss of CEBPD attenuated dual treatment-induced tumor killing in liver cancer cells.</p>

<div>Abstract<p><b>Purpose:</b> Recent evidence indicates that a tumor suppressor gene <i>CEBPD</i> (CCAAT/enhancer-binding protein delta) is downregulated in many cancers including cervical cancer, which provides a therapeutic potential associated with its reactivation. However, little is known for CEBPD activators and the effect of reactivation of <i>CEBPD</i> transcription upon anticancer drug treatment. In this study, we identified a novel CEBPD activator, 1-(2-hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione (HMDB). The purpose of this study is to characterize the mechanism of HMDB-induced CEBPD activation and its potential effect in cancer therapy.</p><p><b>Experimental Design:</b> Methylation-specific PCR assay, reporter assay, and chromatin immunoprecipitation (ChIP) assay were performed to dissect the signaling pathway of HMDB-induced <i>CEBPD</i> transcription. Furthermore, a consequence of HMDB-induced CEBPD expression was linked with E2F1 and retinoblastoma (RB), which discloses the scenario of CEBPD, E2F1, and RB bindings and transcriptional regulation on the promoters of proapoptotic genes, <i>PPARG2</i> and <i>GADD153</i>. Finally, the anticancer effect of HMDB was examined in xenograft mice.</p><p><b>Results:</b> We demonstrate that CEBPD plays an essential role in HMDB-mediated apoptosis of cancer cells. HMDB up-regulates <i>CEBPD</i> transcription through the p38/CREB pathway, thus leading to transcriptional activation of <i>PPARG2</i> and <i>GADD153</i>. Furthermore, increased level of CEBPD attenuates E2F1-induced cancer cell proliferation and partially rescues RB/E2F1-mediated repression of PPARG2 and GADD153 transcription. Moreover, HMDB treatment attenuates the growth of A431 xenografts in severe combined immunodeficient mice mice.</p><p><b>Conclusions:</b> These results clearly demonstrate that HMDB kills cancer cells through activation of CEBPD pathways and suggest that HMDB can serve as a superior chemotherapeutic agent with limited potential for adverse side effects. <i>Clin Cancer Res; 16(23); 5770–80. ©2010 AACR.</i></p></div>