[Show abstract][Hide abstract] ABSTRACT: The molecular response to hypoxia is a critical cellular process implicated in cancer, and a target for drug development. The activity of the major player, HIF1α, is regulated at different levels by various factors, including the transcription factor ELK3. The molecular mechanisms of this intimate connection remain largely unknown. Whilst investigating global ELK3-chromatin interactions, we uncovered an unexpected connection that involves the microRNA hsa-miR-155-5p, a hypoxia-inducible oncomir that targets HIF1α. One of the ELK3 chromatin binding sites, detected by Chromatin Immuno-Precipitation Sequencing (ChIP-seq) of normal Human Umbilical Vein Endothelial Cells (HUVEC), is located at the transcription start site of the MIR155HG genes that expresses hsa-miR-155-5p. We confirmed that ELK3 binds to this promoter by ChIP and quantitative polymerase chain reaction (QPCR). We showed that ELK3 and hsa-miR-155-5p form a double-negative regulatory loop, in that ELK3 depletion induced hsa-miR-155-5p expression and hsa-miR-155-5p expression decreased ELK3 expression at the RNA level through a conserved target sequence in its 3'-UTR. We further showed that the activities of hsa-miR-155-5p and ELK3 are functionally linked. Pathway analysis indicates that both factors are implicated in related processes, including cancer and angiogenesis. Hsa-miR-155-5p expression and ELK3 depletion have similar effects on expression of known ELK3 target genes, and on in-vitro angiogenesis and wound closure. Bioinformatic analysis of cancer RNA-seq data shows that hsa-miR-155-5p and ELK3 expression are significantly anti-correlated, as would be expected from hsa-miR-155-5p targeting ELK3 RNA. Finally, hypoxia (0% oxygen) down-regulates ELK3 mRNA in a microRNA and hsa-miR-155-5p dependent manner. These results tie ELK3 into the hypoxia response pathway through an oncogenic microRNA and into a circuit implicated in the dynamics of the hypoxic response. This crosstalk could be important for the development of new treatments for a range of pathologies.
PLoS ONE 11/2014; 9(11):e113050. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The DNA repair protein DDB2 has been implicated in promoting cell cycle progression by regulating gene expression. DDB2 is selectively overexpressed in breast tumor cells that are non-invasive, but not in those that are invasive. We found that its overexpression in invasive human breast tumor cells limited their motility and invasiveness in vitro and blocked their ability to colonize lungs in vivo, defining a new function for DDB2 in malignant progression. DDB2 overexpression attenuated the activity of NF-kB and the expression of its target matrix metalloprotease MMP9. Mechanistic investigations indicated that DDB2 decreased NF-kB activity by upregulating expression of IkBa by binding the proximal promoter of this gene. This effect was causally linked to invasive capacity. Indeed, knockdown of DDB2-induced IkBa gene expression restored NF-kB activity and MMP9 expression, along with the invasive properties of breast tumor cells overexpressing DDB2. Taken together, our findings enlighten understanding of how breast cancer cells progress to an invasive phenotype and underscore potential clinical interest in DDB2 as a prognostic marker or therapeutic target in this setting.
[Show abstract][Hide abstract] ABSTRACT: Purpose Distant metastasis after treatment is observed in about 20% of Head and Neck Squamous Cell Carcinoma (HNSCC). In the absence of any validated robust biomarker, patients at higher risk for metastasis cannot be provided with tailored therapy. In order to identify prognostic HNSCC molecular subgroups and potential biomarkers, we have performed genome-wide integrated analysis of four omic sets of data. Experimental Design Using state-of-the art technologies, a core set of 45 metastasizing and 55 non-metastasizing HPV-unrelated HNSCC patient samples were analyzed at four different levels: gene expression (transcriptome), DNA methylation (methylome), DNA copy number (genome) and miRNA expression (miRNome). Molecular subgroups were identified by a model-based clustering analysis. Their clinical relevance was evaluated by survival analysis, and functional significance by pathway enrichment analysis. Results Patient subgroups selected by transcriptome, methylome or miRNome integrated analysis are associated with shorter metastasis-free survival. A common subgroup, R1, selected by all three omic approaches, is statistically more significantly associated with metastasis-free survival than any of the single omic selected subgroups. R1 and non-R1 samples display similar DNA copy number landscapes, but more frequent chromosomal aberrations are observed in the R1 cluster (especially loss at 13q14.2-3). R1 tumors are characterized by alterations of pathways involved in cell-cell adhesion, ECM, EMT, immune response and apoptosis. Conclusion Integration of data across several omic profiles leads to better selection of patients at higher risk, identification of relevant molecular pathways of metastasis, and potential to discover biomarkers and drug targets.
Clinical Cancer Research 06/2013; · 8.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Patients with human papillomavirus (HPV)-related oropharyngeal tumors display improved prognosis. The biological basis of this tumor phenotype is poorly understood. We investigated whether increased lymphocyte infiltrate in HPV-positive oropharyngeal squamous cell carcinomas could account for better prognosis. We previously identified, in an Affymetrix GeneChip analysis of 83 HPV-unrelated and 11 HPV-related squamous cell carcinoma of the oropharynx, several candidate genes, including CD8α and CD3ζ. Their expression was validated in this study by qRT-PCR on an independent clinical series of 144 oropharyngeal tumors. Immunohistochemical staining of tumor specimens was performed to evaluate infiltration of tumor stroma by CD8+ and CD4+ lymphocytes. The prognostic value of CD8α and CD3ζ expression levels was measured by Kaplan-Meier and Cox regression model analyses. Immune response-related signaling pathways were found to be deregulated in HPV-positive oropharyngeal tumors. Expression of CD8α, CD3ζ, granzyme K, CD28 and integrin αL RNAs was upregulated in HPV-positive lesions when compared with HPV-unrelated tumors (p < 0.05). Stroma of HPV-positive tumors was frequently and strongly infiltrated by CD8α- and CD3ζ-positive T cells. CD8α RNA expression correlated with both improved global (Kaplan-Meier; p = 0.005; Cox regression: p = 0.003) and disease-free (Cox regression: p = 0.04) survival. CD3ζ RNA expression correlated with improved overall survival (Cox regression: p = 0.024). These results suggest that an increased cytotoxic T-cell-based antitumor immune response is involved in improved prognosis of patients with HPV-positive tumors.
International Journal of Cancer 08/2012; 132(2). · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human papillomavirus (HPV)-related oropharyngeal cancer represents a distinct head and neck squamous cell carcinoma (HNSCC) subpopulation, with improved disease-free and overall survival. In general, HPV-positive HNSCCs express wild-type TP53, which could explain its increased radiosensitivity. However, the molecular mechanisms underlying this higher sensitivity remain elusive. We have previously shown that HPV-related oropharyngeal carcinomas express decreased levels of the NEDD8-activating enzyme 1/amyloid β precursor protein-binding protein 1 (NAE1/APP-BP1) gene. NAE1/APP-BP1 function is required for the NEDDylation of target proteins, and has been shown to be a negative regulator of p53 transcriptional activity. In this study, we addressed the hypothesis that NAE1/APP-BP1 expression levels regulate p53 activity and cell survival upon ionizing irradiation. We used the radiosensitive and naturally HPV16-infected UPCI:SCC90 cell line and the radioresistant and HPV-negative SQ20B cell line as the control. NAE1/APP-BP1 expression levels were modulated with expression constructs and siRNAs. Radiosensitivity was evaluated with clonogenic survival assays. p53 transcriptional activity was measured with a luciferase assay. The overexpression of NAE1/APP-BP1 in UPCI:SCC90 cells resulted in the increased NEDDylation of p53, inhibition of p53 activity and increased cell resistance to ionizing radiation. Conversely, the inhibition of NAE1/APP-BP1 expression in SQ20B cells induced p53-dependent cell death after treatment with X-rays. Taken together, these results indicate that NAE1/APP-BP1 and NEDDylation are invovled in modulating p53 activity and regulating its role in the response of cells to ionizing radiation. Our findings bring new insights in the molecular mechanisms underlying the increased radiosensitivity of HPV-related oropharyngeal tumors. This is of importance, as no reliable and robust predictive biomarkers for tumor response to radiotherapy are currently available. These results also have potential clinical significance, as drugs targeting NAE1/APP-BP1 have recently emerged as a novel therapeutic modality in cancer treatment.
International Journal of Oncology 08/2012; · 2.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The infection of the head and neck epithelium by high-risk human papillomaviruses (HPV) is a risk factor for cancer onset and development. The incidence of HPV-related head and neck squamous cell carcinoma is currently increasing. These lesions display distinct clinical features. HPV positive patients are often younger and have a smaller history of tobacco smoking and alcohol drinking, but have a history of virus-transmitting sex practices. HPV-related tumours are mainly found in the oropharynx, are more associated to a local lymph node invasion and display a poorly differentiated morphology. Despite these more aggressive features, HPV-positive head and neck squamous cell carcinomas correlate with an improved local control, disease-free and global survival. It is thought that HPV-driven specific biologic abnormalities underlie higher tumour sensitivity to chemotherapeutic drugs and ionizing radiations. The expression of the HPV E6 and E7 oncoproteins induce cell transformation by interfering with cell signalling pathways involved in apoptosis, cell cycle, angiogenesis and induce the overexpression of the CDKN2A gene. Therefore, alternative treatments based on therapies targeting these pathways in combination with radiation dose de-escalation could be proposed to HPV-positive patients, if they are properly and reliably identified.
[Show abstract][Hide abstract] ABSTRACT: The aim of the present study was to determine the cytotoxic consequences of high-linear energy transfer (LET) irradiation in the presence of oxaliplatin on hepatocellular carcinoma (HCC) cells in vitro. We attempted to correlate the induction of apoptosis and autophagy with the formation of DNA double-strand breaks (DSBs). SK-Hep1 cells were irradiated by 65 MeV neutrons in the presence of oxaliplatin and/or the poly(ADP-ribose) polymerase (PARP) inhibitor PJ34. DSBs were measured by the formation of gammaH2AX foci. Results show that in SK-Hep1 cells exposed to fast neutrons in the presence of oxaliplatin, DSBs occurred and persisted with time after irradiation. While apoptosis remained low in co-treated cells, autophagy was considerably increased after irradiation and augmented by the addition of oxaliplatin. Thus, autophagic cell death appears to play a prominent role in the cytotoxicity of the combined treatment and may be linked to the generation of heavy damage to DNA.
Anticancer research 02/2010; 30(2):303-10. · 1.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human papillomaviruses (HPV) are associated with a subset of head and neck squamous cell carcinoma (HNSCC), particularly HPV16. This study analyzed the presence and genotype of high risk HPVs, viral DNA load and transcription of the E6/E7 mRNAs, in 231 consecutive HNSCC. Twelve out of 30 HPV16 DNA-positive tumors displayed high E6/E7 mRNAs levels and were localized in the oropharyngeal region. While HPV-free and non-transcriptionally active HPV-related patients showed similar 5-years survival rates, E6/E7 expression was associated with a better prognosis. This emphasizes the importance of considering the transcriptional status of HPV-positive tumors for patient stratification. A gene expression profiling analysis of these different types of tumors was carried out. The most significant differentially expressed gene was CDKN2A, a known biomarker for HPV-related cancer. Assessing both the expression level of the E6/E7 mRNAs and of CDKN2A in HNSCC is required to detect active HPV infection. Chromosomic alterations were investigated by Comparative Genomic Hybridation (CGH) analysis of tumors with transcriptionally active HPV and HPV-negative tumors. The loss of the chromosomal region 16q was found to be a major genetic event in HPV-positive lesions. A cluster of genes located in 16q21-24 displayed decreased expression levels, notably APP-BP1 that is involved in the modulation of the transcriptional activity of p53. In conclusion, this study highlights important criteria required to predict clinically active HPV infection, identifies new biological pathways implicated in HPV tumorigenesis and increases the understanding of HPV-HNSCC physiopathology that is required to develop new targets for therapy.
International Journal of Cancer 09/2009; 126(8):1882-94. · 6.20 Impact Factor