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ABSTRACT: Connexin43 (Cx43) expression is lost in cancer cells and many studies have reported that Cx43 is a tumor suppressor gene. Paradoxically, in a cellular NIH3T3 model, we have previously shown that Ha-Ras-mediated oncogenic transformation results in increased Cx43 expression. Although the examination of transcriptional regulation revealed essential regulatory elements, it could not solve this paradox. Here we studied post-transcriptional regulation of Cx43 expression in cancer using the same model in search of novel gene regulatory elements. Upon Ras transformation, both Cx43 mRNA stability and translation efficiency were increased. We investigated the role of Cx43 mRNA 3' and 5'Untranslated regions (UTRs) and found an opposing effect; a 5'UTR-driven positive regulation is observed in Ras-transformed cells (NIH-3T3(Ras)), while the 3'UTR is active only in normal NIH-3T3(Neo) cells and completely silenced in NIH-3T3(Ras) cells. Most importantly, we identified a previously unknown regulatory element within the 3'UTR, named S1516, which accounts for this 3'UTR-mediated regulation. We also examined the effect of other oncogenes and found that Ras- and Src-transformed cells show a different Cx43 UTRs post-transcriptional regulation than ErbB2-transformed cells, suggesting distinct regulatory pathways. Next, we detected different patterns of S1516 RNA-protein complexes in NIH-3T3(Neo) compared to NIH-3T3(Ras) cells. A proteomic approach identified most of the S1516-binding proteins as factors involved in post-transcriptional regulation. Building on our new findings, we propose a model to explain the discrepancy between the Cx43 expression in Ras-transformed NIH3T3 cells and the data in clinical specimens.
PLoS ONE 01/2013; 8(3):e58500. · 4.09 Impact Factor
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Martin Loignon,
Weimin Miao,
Lianggao Hu, Andrew Bier,
Tarek A Bismar,
P James Scrivens,
Koren Mann,
Mark Basik,
Amélie Bouchard,
Pierre O Fiset,
Zachary Batist,
Gerald Batist
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ABSTRACT: Nrf2 is the key transcription factor for cytoprotective gene programs. Nrf2 is normally maintained at very low concentrations by proteasomal degradation, through its interaction with the adapter protein Keap1 and the Cul3 E3 ligase. Increased Nrf2 concentration resulting from loss of function Keap1 mutations has been described in chemoresistant non-small cell lung cancer. Previous studies in breast cancer showed low levels of some Nrf2-regulated detoxification genes, but the mechanism has not been systematically examined. We found that half of the breast cancer cell lines examined have decreased concentration of Nrf2 compared with normal mammary epithelial cell lines, associated with variable but detectable levels in Keap1 levels, and consistently increased Cul3 mRNA and protein. Immunochemistry showed that 7 of 10 breast cancer specimens examined also have low Nrf2 levels and increased Cul3. Keap1 protein levels are variable. We found no C23Y mutation in Keap1 of any of the cell lines. Using siRNA, we silenced Cul3 in MCF-7 breast cancer cells, and microarray analysis reveals the induction of GCL, NQO1, AKR1C1, UGDH, and TXN by at least 2-fold. The Nrf2-regulated ABCC1 drug transporter was also found to be increased. These Cul3-silenced MCF7 cells are highly resistant to oxidative stress induced by H(2)O(2,) to the carcinogen benzo(a)pyrene, and to both Doxorubicin and Paclitaxel. This high Cul3/low Nrf2 signature may be key to cellular sensitivity to both chemical carcinogeneic stimuli as well as to cytotoxicity of commonly used chemotherapeutic drugs in established breast cancers.
Molecular Cancer Therapeutics 08/2009; 8(8):2432-40. · 5.23 Impact Factor
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ABSTRACT: Connexin43 (Cx43) is often deregulated in breast cancer tissue compared with normal adjacent tissue. Stable reexpression of Cx43 in cancer slows growth and renders the cells more sensitive to cytotoxic chemotherapeutics. Pseudogenes are often considered nonfunctional copies of DNA. The Cx43 pseudogene (PsiCx43) possesses all the features of an expressed gene and is exclusively transcribed in breast cancer cell lines and not in normal cells. PsiCx43 can be translated in vivo, and its protein exhibits growth-suppressive behavior similar to Cx43. We showed that PsiCx43 binds to the polyribosomes in breast cancer cells and that exogenous expression of PsiCx43 induces translational inhibition of Cx43. Furthermore, PsiCx43 is translated and binds more efficiently to the translational machinery than does Cx43 in an in vitro system. Following knockdown of PsiCx43 in breast cancer cells, we observed an increase in Cx43 RNA and protein. This results in increased cellular sensitivity to cytotoxic chemotherapy. Our results show that PsiCx43 acts as a posttranscriptional regulator of Cx43 in breast cancer cells, and that this represents an example of the regulation of genes by pseudogenes with potential therapeutic implications in cancer.
Molecular Cancer Therapeutics 05/2009; 8(4):786-93. · 5.23 Impact Factor
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ABSTRACT: Pseudogenes are classically thought of as nonfunctional DNA sequences due to their inability to be translated, or to produce a functional protein. Gap junctions, a multiprotein complex made of proteins called connexins, are involved in intercellular communication and are deregulated in many cancers. Connexin43 (Cx43) is the only connexin for which a pseudogene has been reported so far. The Cx43 pseudogene (PsiCx43) has all of the features of an expressed gene. We identified the presence of a PsiCx43 mRNA transcript in several cancer cell lines and in none of the normal mammary epithelial cells studied. Using an in vitro translation assay, we found that the PsiCx43 coding plasmid could be translated into a 43 kDa protein. This was further confirmed by expressing a PsiCx43-green fluorescence protein fusion protein in breast cancer MCF-7 cells. We then examined the functional significance of the PsiCx43. In both MTT growth and colony formation assays, significant growth inhibition was observed, a feature common to cells overexpressing the Cx43 gene. However, using a scrape-loading assay, we could not detect any effect on gap junctional intercellular communication. Based on our findings, PsiCx43 joins and enlarges the thus far restricted group of functionally transcribed and translated pseudogenes.
Oncogene 07/2004; 23(27):4763-70. · 6.37 Impact Factor
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ABSTRACT: Gap junctional intercellular communication (GJIC) is a form of cell–cell communication mediating the exchange of small molecules between neighboring cells. Gap junctions (GJs) are formed by connexins (Cxs), and are subject to tight and dynamic regulation. They are involved in the cell cycle, differentiation, and cell signaling. The loss of Cxs and GJs is a hallmark of carcinogenesis, while their induction in cancer cells leads to a reversal of the cancer phenotype, induction of differentiation, and regulation of cell growth. On the basis of the observations about Cx loss in breast cancer, this review examines Cxs' involvement in breast cancer metastasis. Previous work indicates that Cx expression is inversely correlated to metastatic potential. This is probably because of the loss of cooperation between neighboring cells, leading to cell heterogeneity and cell dissociation in the tumor. The possible involvement of Cx activity during metastasis will be discussed.
Journal of Mammary Gland Biology and Neoplasia 09/2001; 6(4):431-440. · 6.74 Impact Factor
