Sp1 activates the transcription of many cellular and viral genes with the GC-box in either the proximal promoter or the enhancer. Sp1 is composed of several functional domains, such as the inhibitory domain (ID), two serine/threonine-rich domains, two glutamine-rich domains, three C 2 H 2 -type zinc finger DNA binding domains (ZFDBD), and a C-terminal D domain. The ZDDBD is the most highly conserved domain among the Sp-family transcription factors and plays a critical role in GCbox recognition. In this study, we investigated the protein-protein interactions occurring at the Sp1ZFDBD and the Sp1ID, and the molecular mechanisms controlling the interaction. Our results found that Sp1ZFDBD and Sp1ID repressed transcription once they were targeted to the proximal promoter of the pGal4 UAS reporter fusion gene system, suggesting molecular interaction with the repressor molecules. Indeed, mammalian two-hybrid assays, GST fusion protein pull-down assays, and co-immunoprecipitation assays showed that Sp1ZFDBD and Sp1ID are able to interact with corepressor proteins such as SMRT, NcoR, and BCoR. The molecular interactions appear to be regulated by MAP kinase/Erk kinase kinase (MEK). The molecular interactions between Sp1ID and the corepressor might explain the role of Sp1 as a repressor under certain circumstances. The siRNA-induced degradation of the corepressors resulted in an up-regulation of Sp1-dependent transcription. The cellular context of the corepressors and the regulation of molecular interaction between corepressors and Sp1ZFDBD or Sp1ID might be important in controlling Sp1 activity.Transcriptional regulation of the eukaryotic gene is a complicated process, involving a series of complex molecular interactions among regulatory and transcription factors. Specificity protein 1 (Sp1) 1 is probably one of the best characterized sequence-specific transcription factors, and has numerous functions in the transcription of many cellular and viral genes harboring GC boxes in their promoters (1, 2). Sp1 and its family of proteins have been implicated in a host of essential biological processes, and have been proven important in apoptosis, cell growth inhibition, differentiation, and carcinogenesis (Refs. 3-5 and references therein).Sp1 is a member of the Sp-multigene family, which also includes Sp2, Sp3, Sp4, Sp5, Sp6, Sp7, and Sp8 (3-5). The Sp-family proteins exhibit similar domain structures and are evolutionarily closely related. All of these proteins possess highly conserved C 2 H 2 -type zinc finger DNA binding domains at their C termini, and all belong to the Krü ppel-like zinc finger superfamily (3-5). In addition, the Sp-family proteins have been demonstrated to undergo post-translational modifications as the result of diverse mechanisms. For example, Sp1 is glycosylated and phosphorylated by Erk2, protein kinase C, casein kinase II, and cAMP-dependent protein kinase; Sp3 is acetylated and SUMOylated by the protein inhibitor of activated STAT (PIAS1, Ref. 3 and references therein, Refs. 6 -10).The Sp1 protein co...
Reliable biomarkers are required to predict the response to sorafenib. We investigated genomic variations associated with responsiveness to sorafenib for patients with unresectable hepatocellular carcinoma (HCC). Blood samples from 2 extreme, 2 strong and 3 poor responders to sorafenib were subjected to whole-genome analysis. Then, we validated candidate genomic variations with another 174 HCC patients, and performed in vitro functional analysis and in silico analyses. Genomic data of >96 gigabases/sample was generated at average of ~34X sequencing depth. In total, 1813 genomic variations were matched to sorafenib responses in clinical data; 708 were located within regions for sorafenib-target genes or drug absorption, distribution, metabolism, and excretion (ADME)-related genes. From them, 36 variants were within the coding regions and 6 identified as non-synonymous single-nucleotide variants from 4 ADME-related genes (ABCB1, FMO3, MUSK, and SLC15A2). Validation genotyping confirmed sequencing results and revealed patients genotype for rs2257212 in SLC15A2 showed longer progression-free survival (HR = 2.18). In vitro study displayed different response to sorafenib depending on the genotype of SLC15A2. Structural prediction analysis revealed changes of the phosphorylation levels in protein, potentially affecting sorafenib-associated enzymatic activity. Our finding using extreme responder seems to generate robust biomarker to predict the response of sorafenib treatment for HCC.
The inhibition of DDR2 by RNA interference suppressed in vivo and in vitro growth of human HCC cells. Our results may support that the use of DDR2 as a novel target of HCC treatment through control of tumor apoptosis, migration, and invasion.
Expression of cyclooxygenase-2 (COX-2) is involved in the chronic inflammation-related development of hepatocellular carcinoma (HCC), and the use of selective COX-2 inhibitors might provide new chemoprevention strategies for HCC. However, the role of the COX-2 in hepatocarcinogenesis remains obscure, particularly as it has been primarily studied with selective COX-2 inhibitors that may affect other cellular proteins involved in cell proliferation. Therefore, we investigated the effects of the inhibition of COX-2 by the selective COX-2 inhibitor NS-398 as well as by COX-2 specific small interfering RNA (siRNA) in the human HCC cell lines Hep3B and SNU-387. These cell lines expressed COX-2, and NS-398 induced apoptosis of these cells. NS-398 inhibited more than 60% of prostaglandin E(2) (PGE2) production and cell proliferation in a concentration-dependent manner in these cells. The inhibition of proliferation was almost restored with PGE2 supplement, suggesting that NS-398 may inhibit cell growth partially through inhibition of COX-2 and PGE2 production in human HCC cells. However, treatment with NS-398 led to increased expression of COX-2 in Hep3B and SNU-387 cells. To examine the effect of COX-2 depletion on these cells, we electroporated COX-2-specific siRNAs into SNU-387 cells. We observed significant, sequence-specific reductions in COX-2 expression, PGE2 production, and cell proliferation, though the reduction in cell proliferation was less than that induced by NS-398. In conclusion, these data suggest that COX-2 itself is directly involved, though not decisively, in proliferation of human HCC cells. RNA interference may provide a useful tool for manipulating COX-2-related hepatocarcinogenesis in research and therapeutic settings.
308 Background: Reliable biomarkers are required to predict patient response to sorafenib. We attempted to investigate genomic variations associated with responsiveness to sorafenib treatment in patients with unresectable hepatocellular carcinoma (HCC) and their functional relevance. Methods: We obtained blood samples from 4 strong and 3 poor responders to sorafenib treatment and subjected these samples to whole-genome analysis. Next, we performed validation tests for candidate single-nucleotide polymorphisms (SNPs) in the samples of 174 HCC patients who were treated with sorafenib, followed by in vitro functional analysis and in silico analyses of candidate SNPs. Results: On average, 90 gigabases/sample was generated at ~34X sequencing depth. In total, 1813 genomic variations were perfectly matched to sorafenib responses in the clinical data; 708 were located within regions for sorafenib-target genes or drug absorption, distribution, metabolism, and excretion (ADME)-related genes—36 within the coding regions and 6 identified as non-synonymous single-nucleotide variants from 4 ADME-related genes (ABCB1, FMO3, MUSK, and SLC15A2), which potentially cause functional alterations. Validation tests of 174 patients confirmed sequencing results and revealed that patients with the C/C genotype for rs2257212 in SCL15A2 displayed higher risk for cancer progression than did patients with C/T or T/T genotypes (HR: 2.18; 95% CI, 1.15–4.15; P = 0.018). In vitro functional analysis revealed that cells harboring C/C genotype for this SNP displayed lower response to sorafenib treatment than did cells harboring the T/T genotype. Structural prediction analysis revealed change in protein phosphorylation levels, potentially affecting sorafenib-associated enzymatic activity. Conclusions: SLC15A2 could be a robust biomarker of response to sorafenib treatment in HCC patients.
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