RAR beta trans-repression of AP-1 transcription factor in HeLa cervical cancer cells : consequences on transcription of viral and cellular AP-1 controlled genes

In many types of cancer, including cervical cancer, the nuclear retinoic receptor ß2 (RARß2) gene is epigenetically modified and unable to be significantly induced. RARß2 can act as a tumor suppressor, since loss of its expression is associated with human tumor progression. One mechanism of RARß2-mediated growth inhibition is based on its ability to constitutively repress the AP-1 transcription factor, but this mechanism has never been demonstrated in cervical cancer cells. In this thesis the biological consequences of reconstitute RARß2 receptor expression in cervical cancer cells was investigated. For this purpose, human papillomavirus HPV-18 positive HeLa cells were stably transfected with RARß2 cDNA under the control of the ß-actin promoter. The characterization of the RARß2 tranfectants revealed a strongly reduced AP-1 binding to the corresponding specific oligonucleotides, even in the absence of atRA treatment. In HeLa cells, the AP-1 reduction correlates with diminished HPV-18 oncogene transcription and slower cellular growth. Western blot analysis demonstrated that the only member of the AP-1 family consistently reduced in HeLa RARß2 clones was c-Jun, despite ongoing gene expression. In order to understand by which mechanism c-Jun is reduced, and since the phosphorylation of c-Jun is important for protein stabilization, HeLa RARß2 clones were treated with different c-Jun-N-terminal kinase (JNK) stimulators. The treatments resulted in a c-Jun increase at RNA and protein levels, and led to a reconstitution of AP-1 binding similar to non-transfected HeLa controls. However, the reconstitution of AP-1 binding levels did not have an inductor effect on the HPV-18 oncogenes, the expression of which has been postulated to be AP-1 dependent. Other classical AP-1 regulated genes such as metalloproteinases were up regulated as expected. In conclusion, the data of this thesis indicate that RARß2 induced a destabilization and accelerated degradation of c-Jun at the protein level responsible for the AP-1 reduction. The mechanism of AP-1 trans-repression in HeLa cells is different from that postulated for other cancer cell models. All changes produced in HeLa cells after RARß2 constitutive expression lead to reduced cell proliferation, which can be associated with the RARß2 tumor suppressor function

PIK3CA-mediated PI3-kinase signalling is essential for HPV-induced transformation in vitro

<p>Abstract</p> <p>Background</p> <p>High-risk human papillomavirus (hrHPV) infections are causally related to cervical cancer development. The additional (epi)genetic alterations driving malignant transformation of hrHPV-infected cells however, are not yet fully elucidated. In this study we experimentally assessed the role of the PI3-kinase pathway and its regulator PIK3CA, which is frequently altered in cervical cancer, in HPV-induced transformation.</p> <p>Methods</p> <p>Cervical carcinomas and ectocervical controls were assessed for PIK3CA mRNA and protein expression by quantitative RT-PCR and immunohistochemical staining, respectively. A longitudinal <it>in vitro </it>model system of hrHPV-transfected keratinocytes, representing the immortal and anchorage independent phenotype, was assayed for PI3-kinase activation and function using chemical pathway inhibition i.e. LY294002 treatment, and PIK3CA RNA interference. Phenotypes examined included cellular viability, migration, anchorage independent growth and differentiation. mRNA expression of hTERT and HPV16 E6E7 were studied using quantitative RT-PCR and Northern blotting.</p> <p>Results</p> <p>Cervical carcinomas showed significant overexpression of PIK3CA compared to controls. During HPV-induced transformation <it>in vitro</it>, expression of the catalytic subunit PIK3CA as well as activation of downstream effector PKB/AKT progressively increased in parallel. Inhibition of PI3-kinase signalling in HPV16-transfected keratinocytes by chemical interference or siRNA-mediated silencing of PIK3CA resulted in a decreased phosphorylation of PKB/AKT. Moreover, blockage of PI3-kinase resulted in reduced cellular viability, migration, and anchorage independent growth. These properties were accompanied with a downregulation of HPV16E7 and hTERT mRNA expression. In organotypic raft cultures of HPV16- and HPV18-immortalized cells, phosphorylated PKB/AKT was primarily seen in differentiated cells staining positive for cytokeratin 10 (CK10). Upon PI3-kinase signalling inhibition, there was a severe impairment in epithelial tissue development as well as a dramatic reduction in p-PKB/AKT and CK10.</p> <p>Conclusion</p> <p>The present data indicate that activation of the PI3-kinase/PKB/AKT pathway through PIK3CA regulates various transformed phenotypes as well as growth and differentiation of HPV-immortalized cells and may therefore play a pivotal role in HPV-induced carcinogenesis.</p

Gene Expression of the Tumour Suppressor LKB1 Is Mediated by Sp1, NF-Y and FOXO Transcription Factors

The serine/threonine kinase LKB1 is a tumour suppressor that regulates multiple biological pathways, including cell cycle control, cell polarity and energy metabolism by direct phosphorylation of 14 different AMP-activated protein kinase (AMPK) family members. Although many downstream targets have been described, the regulation of LKB1 gene expression is still poorly understood. In this study, we performed a functional analysis of the human LKB1 upstream regulatory region. We used 200 base pair deletion constructs of the 5′-flanking region fused to a luciferase reporter to identify the core promoter. It encompasses nucleotides −345 to +52 relative to the transcription start site and coincides with a DNase I hypersensitive site. Based on extensive deletion and substitution mutant analysis of the LKB1 promoter, we identified four cis-acting elements which are critical for transcriptional activation. Using electrophoretic mobility shift assays as well as chromatin immunoprecipitations, we demonstrate that the transcription factors Sp1, NF-Y and two forkhead box O (FOXO) family members FOXO3 and FOXO4 bind to these elements. Overexpression of these factors significantly increased the LKB1 promoter activity. Conversely, small interfering RNAs directed against NF-Y alpha and the two FOXO proteins greatly reduced endogenous LKB1 expression and phosphorylation of LKB1's main substrate AMPK in three different cell lines. Taken together, these results demonstrate that Sp1, NF-Y and FOXO transcription factors are involved in the regulation of LKB1 transcription

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements