Johanna De-Castro Arce

Alternative splicing of human papillomavirus type-16 E6/E6* early mRNA is coupled to EGF signaling via Erk1/2 activation

Certain types of human papillomaviruses (HPVs) are etiologically linked to cervical cancer. Their transforming capacity is encoded by a polycistronic premRNA, where alternative splicing leads to the translation of functional distinct proteins such as E6, E6*, and E7. Here we show that splicing of HPV16 E6/E7 ORF cassette is regulated by the epidermal growth factor (EGF) pathway. The presence of EGF was coupled to preferential E6 expression, whereas depletion of EGF, or treatment with EGF receptor (EGFR) neutralizing antibodies or the EGFR inhibitor tyrphostin AG1478, resulted in E6 exon exclusion in favor of E6*. As a consequence, increased p53 levels and enhanced translation of E7 with a subsequent reduction of the retinoblastoma protein pRb could be discerned. E6 exon exclusion upon EGF depletion was independent from promoter usage, mRNA stability, or selective mRNA transport. Time-course experiments and incubation with cycloheximide demonstrated that E6 alternative splicing is a direct and reversible effect of EGF signal transduction, not depending on de novo protein synthesis. Within this process, Erk1/2-kinase activation was the critical event for E6 exon inclusion, mediated by the upstream MAP kinase MEK1/2. Moreover, siRNA knockdown experiments revealed an involvement of splicing factors hnRNPA1 and hnRNPA2 in E6 exon exclusion, whereas the splicing factors Brm and Sam68 were found to promote E6 exon inclusion. Because there is a natural gradient of EGF and EGF receptor expression in the stratified epithelium, it is reasonable to assume that EGF modulates E6/E7 splicing during the viral life cycle and transformation.

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 LKB1s 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.

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

BackgroundHigh-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.MethodsCervical carcinomas and ectocervical controls were assessed for PIK3CA mRNA and protein expression by quantitative RT-PCR and immunohistochemical staining, respectively. A longitudinal in vitro 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.ResultsCervical carcinomas showed significant overexpression of PIK3CA compared to controls. During HPV-induced transformation in vitro, 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.ConclusionThe 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.

Alterations in AP-1 and AP-1 regulatory genes during HPV-induced carcinogenesis

Background: Previous studies demonstrated a functional involvement of the AP-1 transcription factor in HPV-induced cervical carcinogenesis. Here, we aimed to obtain further insight in expression alterations of AP-1 family members during HPV-mediated transformation and their relationship to potential regulatory (Notch1, Net) and target (CADM1) genes. Methods: mRNA expression levels of c-Jun, JunB, junD, c-Fos, FosB, Fra-1, Fra-2, Notch1, Net and CADM1 were determined by quantitative RT-PCR in primary keratinocytes (n=5), early (n=4) and late (n=4) passages of non-tumorigenic HPV-immortalized keratinocytes and in tumorigenic cervical cancer cell lines (n=7). In a subset of cell lines protein expression and AP-1 complex composition was determined. Results: Starting in immortal stages c-Fos, Fra-2 and JunB expression became up regulated towards tumorigenicity, whereas Fra-1, c-Jun, Notch1, Net and CADM1 became down regulated. The onset of deregulated expression varied amongst the AP-1 members and was not directly related to altered Notch1, Net or CADM1 expression. Nevertheless, a shift in AP-1 complex composition from Fra-1/c-Jun to c-Fos/c-Jun heterodimers was only observed in tumorigenic cells. Conclusion: HPV-mediated transformation is associated with altered AP-1, Notch1, Net and CADM1 transcription. Whereas the onset of deregulated expression of various AP-1 family members became already manifest during the immortal state, a shift in AP-1 complex composition appeared a rather late event associated with tumorigenicity.

Interference with energy metabolism by 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside induces HPV suppression in cervical carcinoma cells and apoptosis in the absence of LKB1

Carcinogenesis is a dynamic and stepwise process, which is accompanied by a variety of somatic and epigenetic alterations in response to a changing microenvironment. Hypoxic conditions will select for cells that have adjusted their metabolic profile and can maintain proliferation by successfully competing for scarce nutritional and oxygen resources. In the present study we have investigated the effects of energy depletion in the context of HPV (human papillomavirus)-induced pathogenesis. We show that cervical carcinoma cell lines are susceptible to undergoing either growth arrest or cell death under conditions of metabolic stress induced by AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside), a known activator of the AMPK (AMP-activated protein kinase). Our results reveal that AICAR treatment leads to a reduced binding affinity of the transcription factor AP-1 (activator protein-1) and in turn to a selective suppression of HPV transcription. Moreover, the outcome of AICAR on proliferation and survival was dependent on p53 activation and the presence of LKB1, the major upstream kinase of AMPK. Using non-malignant LKB1-expressing somatic cell hybrids, which lose expression after tumorigenic segregation, as well as small interfering RNA LKB1 knockdown approaches, we could further demonstrate that expression of LKB1 protects cells from cytotoxicity induced by agents which modulate the ATP/AMP ratio. Since simulation of low energy status can selectively eradicate LKB1-negative cervical carcinoma cells, AICAR may represent a novel drug in the treatment of cervical cancer.

The functional role of Notch signaling in HPV-mediated transformation is dose-dependent and linked to AP-1 alterations

BackgroundThe role of Notch signaling in HPV-mediated transformation has been a long standing debate, as both tumor suppressive and oncogenic properties have been described. We examined whether the dual findings in literature may be explained by gene dosage effects and determined the relation with AP-1, a downstream target of Notch.MethodsSiHa cervical cancer cells were transfected with two doses of intracellular active Notch. Non-tumorigenic HPV16-immortalized keratinocytes (FK16A) were transfected with Fra1 specific siRNAs and non-targeting controls. Transfectants were analysed for Notch, Hes, cJun, cFos and Fra1 mRNA expression, Notch pathway activation using luciferase assays, cell viability using MTT assays, anchorage independent growth, AP-1 activity and/or AP-1 complex composition using EMSA.ResultsIn SiHa cells two activation states of Notch signaling pathway were obtained. Moderate Notch activation contributed to increased viability and anchorage independent growth, whereas high level Notch activation decreased anchorage independent growth. The shift in phenotypical outcome was correlated to altered AP-1 activity and complex composition. Moderate Notch expression led to an increased AP-1 transcriptional activity and DNA binding activity, but did not affect complex composition. High levels of Notch additionally led to a change in AP-1 complex composition, from cJun/cFos to cJun/Fra1 dimers, which is exemplary for non-tumorigenic HPV-immortalized cell lines. Conversely, silencing of Fra1 in non-tumorigenic HPV16-immortalized keratinocytes, leading to an enrichment of cJun/cFos dimers, was accompanied with increased colony formation.ConclusionThe functional role of Notch in HPV-mediated transformation is dosage dependent and correlated to a change in AP-1.