Expression of HOXB2, a retinoic acid signaling target in pancreatic cancer and pancreatic intraepithelial neoplasia

Purpose: Despite significant progress in understanding the molecular pathology of pancreatic cancer and its precursor lesion: pancreatic intraepithelial neoplasia (PanIN), there remain no molecules with proven clinical utility as prognostic or therapeutic markers. Here, we used oligonucleotide microarrays to interrogate mRNA expression of pancreatic cancer tissue and normal pancreas to identify novel molecular pathways dysregulated in the development and progression of pancreatic cancer. Experimental Design: RNA was hybridized to Affymetrix Genechip HG-U133 oligonucleotide microarrays. A relational database integrating data from publicly available resources was created to identify candidate genes potentially relevant to pancreatic cancer. The protein expression of one candidate, homeobox B2 (HOXB2), in PanIN and pancreatic cancer was assessed using immunohistochemistry. Results: We identified aberrant expression of several components of the retinoic acid (RA) signaling pathway (RARa, MUC4, Id-1, MMP9, uPAR, HB-EGF, HOXB6, and HOXB2), many of which are known to be aberrantly expressed in pancreatic cancer and Pan IN. HOXB2, a downstream target of RA, was up-regulated 6.7-fold in pancreatic cancer compared with normal pancreas. Immunohistochemistry revealed ectopic expression of HOXB2 in 15% of early Pan IN lesions and 48 of 128 (38%) pancreatic cancer specimens. Expression of HOXB2 was associated with nonresectable tumors and was an independent predictor of poor survival in resected tumors. Conclusions: We identified aberrant expression of RA signaling components in pancreatic cancer, including HOXB2, which was expressed in a proportion of PanIN lesions. Ectopic expression of HOXB2 was associated with a poor prognosis for all patients with pancreatic cancer and was an independent predictor of survival in patients who underwent resection.

Ultraconserved elements in insect genomes: A highly conserved intronic sequence implicated in the control of homothorax mRNA splicing

Recently, we identified a large number of ultraconserved (uc) sequences in noncoding regions of human, mouse, and rat genomes that appear to be essential for vertebrate and amniote ontogeny. Here, we used similar methods to identify ultraconserved genomic regions between the insect species Drosophila melanogaster and Drosophila pseudoobscura, as well as the more distantly related Anopheles gambiae. As with vertebrates, ultraconserved sequences in insects appear to Occur primarily in intergenic and intronic sequences, and at intron-exon junctions. The sequences are significantly associated with genes encoding developmental regulators and transcription factors, but are less frequent and are smaller in size than in vertebrates. The longest identical, nongapped orthologous match between the three genomes was found within the homothorax (hth) gene. This sequence spans an internal exon-intron junction, with the majority located within the intron, and is predicted to form a highly stable stem-loop RNA structure. Real-time quantitative PCR analysis of different hth splice isoforms and Northern blotting showed that the conserved element is associated with a high incidence of intron retention in hth pre-mRNA, suggesting that the conserved intronic element is critically important in the post-transcriptional regulation of hth expression in Diptera.

HOXA1 is required for E-cadherin-dependent anchorage-independent survival of human mammary carcinoma cells

Forced expression of HOXA1 is sufficient to stimulate oncogenic transformation of immortalized human mammary epithelial cells and subsequent tumor formation. We report here that the expression and transcriptional activity of HOXA1 are increased in mammary carcinoma cells at full confluence. This confluence-dependent expression of HOXA1 was abrogated by incubation of cells with EGTA to produce loss of intercellular contact and rescued by extracellular addition of Ca2+. Increased HOXA1 expression at full confluence was prevented by an E-cadherin function-blocking antibody and attachment of non-confluent cells to a substrate by homophilic ligation of E-cadherin increased HOXA1 expression. E-cadherin-directed signaling increased HOXA1 expression through Rac1. Increased HOXA1 expression consequent to E-cadherin-activated signaling decreased apoptotic cell death and was required for E-cadherin-dependent anchorage-independent proliferation of human mammary carcinoma cells. HOXA1 is therefore a downstream effector of E-cadherin-directed signaling required for anchorage-independent proliferation of mammary carcinoma cells.

Developmental expression of transcription factor genes in a demosponge: insights into the origin of metazoan multicellularity

Demosponges are considered part of the most basal evolutionary lineage in the animal kingdom. Although the sponge body plan fundamentally differs from that of other metazoans, their development includes many of the hallmarks of bilaterian and eumetazoan embryogenesis, namely fertilization followed by a period of cell division yielding distinct cell populations, which through a gastrulation-like process become allocated into different cell layers and patterned within these layers. These observations suggest that the last common ancestor (LCA) to all living animals was developmentally more sophisticated than is widely appreciated and used asymmetric cell division and morphogen gradients to establish localized populations of specified cells within the embryo. Here we demonstrate that members of a range of transcription factor gene classes, many of which appear to be metazoan-specific, are expressed during the development of the demosponge Reniera, including ANTP, Pax, POU, LIM-HD, Sox, nuclear receptor, Fox (forkhead), T-box, Mef2, and Ets genes. Phylogenetic analysis of these genes suggests that not only the origin but the diversification of some of the major developmental metazoan transcription factor classes took place before sponges diverged from the rest of the Metazoa. Their expression during demosponge development suggests that, as in today's sophisticated metazoans, these genes may have functioned in the regulatory network of the metazoan LCA to control cell specification and regionalized gene expression during embryogenesis.

Epidermal Growth Factor Impairs Palatal Shelf Adhesion and Fusion in the T gf-β3 Null Mutant

The cleft palate presented by transforming growth factor-β3 (Tgf-β3 ) null mutant mice is caused by altered palatal shelf adhesion, cell proliferation, epithelial-to-mesenchymal transformation and cell death. The expression of epidermal growth factor (EGF), transforming growth factor-β1 ( Tgf-β1 ) and muscle segment homeobox-1 (Msx-1) is modified in the palates of these knockout mice, and the cell proliferation defect is caused by the change in EGF expression. In this study, we aimed to determine whether this change in EGF expression has any effect on the other mechanisms altered in Tgf-β 3 knockout mouse palates. We tested the effect of inhibiting EGF activity in vitro in the knockout palates via the addition of Tyrphostin AG 1478. We also investigated possible interactions between EGF, Tgf-β 1 and Msx-1 in Tgf-β 3 null mouse palate cultures. The results show that the inhibition of EGF activity in Tgf-β 3 null mouse palate cultures improves palatal shelf adhesion and fusion, with a particular effect on cell death, and restores the normal distribution pattern of Msx-1 in the palatal esenchyme. Inhibition of TGF-β 1 does not affect either EGF or Msx-1 expression.

"Mutações no gene OCT4 em células eritroleucêmicas humanas: implicações sobre o fenótipo de resistência a múltiplas drogas (MDR)”

O fator de transcrição OCT4 é um importante marcador de células tronco e tem sido relacionado com o conceito de células tronco tumorais (CTTs). Recentemente, ele tem sido também relacionado ao fenótipo de resistência a múltiplas drogas (MDR). O objetivo deste trabalho foi testar se o OCT4 está ligado ao fenótipo MDR em células eritroleucêmicas derivadas da linhagem LMC-K562. Para isso, foi realizada a análise de expressão de genes associados à superfamília de transportadores ABC (ATP Binding Cassette) e, também, de fatores de transcrição relacionados a células tronco. Os primeiros resultados apontaram uma relação direta entre OCT4 e transportadores ABC na linhagem MDR derivada de K562 (Lucena). O sequenciamento de promotores ABC não revelou qualquer mutação que pudesse explicar a expressão diferenciada do OCT4 na linhagem MDR. Posteriormente, o sequenciamento da região contendo o domínio homeobox do gene OCT4 de ambas as linhagens celulares evidenciou, pela primeira vez, que este fator de transcrição é alvo de mutações que podem estar relacionados com o fenótipo MDR. As mutações encontradas implicam substituições de vários aminoácidos em ambas as linhagens celulares. K562 teve sete substituições (três exclusivas), enquanto Lucena teve 13 (nove exclusivas). Além disso, um busca in silico por motivos de fosforilação dentro da sequência de aminoácidos comparada, revelou que o OCT4 humano normal possui sete motivos potenciais de fosforilação. Entretanto, K562 perdeu um motivo e Lucena dois deles. Moléculas com diferentes padrões de fosforilação podem ter sua função modificada. Estes resultados indicam o OCT4 com uma alvo potencial para o tratamento do câncer, especialmente aqueles resistentes à quimioterapia.