Lomberk, Gwen

Abstract Purpose: The limited supply of oxygen and nutrients is thought to result in rigorous selection of cells that will eventually form the tumor. Experimental Design: Nupr1 expression pattern was analyzed in human tissue microarray (TMA) and correlated with survival time of the patient. Microarray analysis was conducted on MiaPaCa2 cells subjected to metabolic stress in Nupr1-silenced conditions. DNA repair and cell cycle–associated gene expression was confirmed by real-time quantitative PCR (qRT-PCR). Nupr1 and AURKA protective role were analyzed using RNA interference (RNAi) silencing or overexpression. DNA damage and autophagy were analyzed by Western blot analysis and immunofluorescence. Results: We showed that both Nupr1 and HIF1α are coexpressed in human pancreatic ductal adenocarcinoma (PDAC) samples and negatively correlate with survival time. PDAC-derived cells submitted to hypoxia and/or glucose starvation induce DNA damage–dependent cell death concomitantly to the overexpression of stress protein Nupr1. Affymetrix-based transcriptoma analysis reveals that Nupr1 knockdown enhances DNA damage and alters the expression of several genes involved in DNA repair and cell-cycle progression. Expression of some of these genes is common to hypoxia and glucose starvation, such as Aurka gene, suggesting that Nupr1 overexpression counteracts the transcriptional changes occurring under metabolic stress. The molecular mechanism by which hypoxia and glucose starvation induce cell death involves autophagy-associated, but not caspase-dependent, cell death. Finally, we have found that AURKA expression is partially regulated by Nupr1 and plays a major role in this response. Conclusions: Our data reveal that Nupr1 is involved in a defense mechanism that promotes pancreatic cancer cell survival when exposed to metabolic stress. Clin Cancer Res; 18(19); 5234–46. ©2012 AACR.

Abstract Purpose: The therapeutic benefit of temozolomide in glioblastoma multiforme (GBM) is limited by resistance. The goal of this study was to elucidate mechanisms of temozolomide resistance in GBM. Experimental Design: We developed an in vivo GBM model of temozolomide resistance and used paired parental and temozolomide-resistant tumors to define the mechanisms underlying the development of resistance and the influence of histone deacetylation (HDAC) inhibition. Results: Analysis of paired parental and resistant lines showed upregulation of O6-methylguanine-DNA methyltransferase (MGMT) expression in 3 of the 5 resistant xenografts. While no significant change was detected in MGMT promoter methylation between parental and derivative-resistant samples, chromatin immunoprecipitation showed an association between MGMT upregulation and elevated acetylation of lysine 9 of histone H3 (H3K9-ac) and decreased dimethylation (H3K9-me2) in GBM12 and GBM14. In contrast, temozolomide resistance development in GBM22 was not linked to MGMT expression, and both parental and resistant lines had low H3K9-ac and high H3K9-me2 within the MGMT promoter. In the GBM12TMZ-resistant line, MGMT reexpression was accompanied by increased recruitment of SP1, C-JUN, NF-κB, and p300 within the MGMT promoter. Interestingly, combined treatment of GBM12 flank xenografts with temozolomide and the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) favored the evolution of temozolomide resistance by MGMT overexpression as compared with treatment with temozolomide alone. Conclusion: This study shows, for the first time, a unique mechanism of temozolomide resistance development driven by chromatin-mediated MGMT upregulation and highlights the potential for epigenetically directed therapies to influence the mechanisms of resistance development in GBM. Clin Cancer Res; 18(15); 4070–9. ©2012 AACR.

SP/KLF (Specificity protein/Krüppel-like factor) transcription factors comprise an emerging group of proteins that may behave as tumour suppressors. Incidentally, many cancers that display alterations in certain KLF proteins are also associated with a high incidence of KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue) mutations. Therefore in the present paper we investigate whether SP/KLF proteins suppress KRAS-mediated cell growth, and more importantly, the potential mechanisms underlying these effects. Using a comprehensive family-wide screening of the 24 SP/KLF members, we discovered that SP5, SP8, KLF2, KLF3, KLF4, KLF11, KLF13, KLF14, KLF15 and KLF16 inhibit cellular growth and suppress transformation mediated by oncogenic KRAS. Each protein in this subset of SP/KLF members individually inhibits BrdU (5-bromo-2-deoxyuridine) incorporation in KRAS oncogenic-mutant cancer cells. SP5, KLF3, KLF11, KLF13, KLF14 and KLF16 also increase apoptosis in these cells. Using KLF11 as a representative model for mechanistic studies, we demonstrate that this protein inhibits the ability of cancer cells to form both colonies in soft agar and tumour growth in vivo. Molecular studies demonstrate that these effects of KLF11 are mediated, at least in part, through silencing cyclin A via binding to its promoter and leading to cell-cycle arrest in S-phase. Interestingly, similar to KLF11, KLF14 and KLF16 mechanistically share the ability to modulate the expression of cyclin A. Collectively, the present study stringently defines a distinct subset of SP/KLF proteins that impairs KRAS-mediated cell growth, and that mechanistically some members of this subset accomplish this, at least in part, through regulation of the cyclin A promoter.

Abstract Reflux-induced injury promotes esophageal adenocarcinoma, one of the most rapidly increasing, highly lethal cancers in Western countries. Here, we investigate the efficacy of a combinatorial chemoprevention strategy for esophageal adenocarcinoma and characterize the underlying molecular mechanisms. Specifically, our approach involves the use of ursodeoxycholic acid (Urso) due to its ability to decrease injury-inducing bile salts in combination with Aspirin to mitigate the consequences of injury. We find that Urso-Aspirin combination reduces the risk of adenocarcinoma in vivo in animals with reflux, decreases the proliferation of esophageal adenocarcinoma cells, and downregulates a key cell cycle regulator, CDK2. Mechanistically, using cell growth, luciferase reporter, expression, and chromatin immunoprecipitation assays, we identify GLI1, a Hedgehog-regulated transcription factor, as a novel target of Urso-Aspirin combination. We show that GLI1 is upregulated during esophageal carcinogenesis, and GLI1 can bind to the CDK2 promoter and activate its expression. Although the Urso-Aspirin combination downregulates GLI1, the GLI1 overexpression not only abrogates the effect of this combination on proliferation but it also restores CDK-2 expression. These findings support that the chemopreventive effect of the Urso-Aspirin combination occurs, at least in part, through a novel GLI1-CDK2–dependent mechanism. To further understand the regulation of CDK2 by GLI1, both pharmacologic and RNAi-mediated approaches show that GLI1 is a transcriptional activator of CDK2, and this regulation occurs independent of Smoothened, the central transducer of the Hedgehog canonical pathway. Collectively, these results identify a novel GLI1-to-CDK2 pathway in esophageal carcinogenesis, which is a bona fide target for effective combinatorial chemoprevention with Urso and Aspirin. Cancer Res; 70(17); 6787–96. ©2010 AACR.

Sp1 is one of the best characterized transcriptional activators. The biological importance of Sp1 is underscored by the fact that several hundreds of genes are thought to be regulated by this protein. However, during the last 5 years, a more extended family of Sp1-like transcription factors has been identified and characterized by the presence of a conserved DNA-binding domain comprising three Krüppel-like zinc fingers. Each distinct family member differs in its ability to regulate transcription, and, as a consequence, to influence cellular processes. Specific activation and repression domains located within the N-terminal regions of these proteins are responsible for these differences by facilitating interactions with various co-activators and co-repressors. The present review primarily focuses on discussing the structural, biochemical and biological functions of the repressor members of this family of transcription factors. The existence of these transcriptional repressors provides a tightly regulated mechanism for silencing a large number of genes that are already known to be activated by Sp1.