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The management of locally advanced head and neck squamous cell carcinoma (HNSCC) with Cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor (EGFR), achieves only moderate response rates, and clinical trials that evaluated EGFR-blockade with tyrosine kinase inhibitors (TKI) yielded disappointing results. Inter-tumor heterogeneity may hinder the therapeutic efficiency of anti-EGFR treatments. HNSCC heterogeneity was addressed in several studies, which all converged towards the definition of molecular subgroups. They include the basal subgroup, defined by the deregulated expression of factors involved in the EGFR signaling pathway, including the epiregulin EGFR ligand encoded by the EREG gene. These observations indicate that basal tumors could be more sensitive to anti-EGFR treatments. To test this hypothesis, we performed a screen of a representative collection of basal versus non-basal HNSCC cell lines for their sensitivity to several anti-EGFR drugs (Cetuximab, Afatinib, and Gefitinib), tested as monotherapy or in combination with drugs that target closely-linked pathways [Mitogen-activated protein kinase kinase/ extracellular signal&ndash, regulated kinases (MEK), mammalian Target of Rapamycine (mTOR) or Human Epidermal growth factor Receptor 2 (HER2)]. Basal-like cell lines were found to be more sensitive to EGFR blockade alone or in combination with treatments that target MEK, mTOR, or HER2. Strikingly, the basal-like status was found to be a better predictor of cell response to EGFR blockade than clinically relevant mutations [e.g., cyclin-dependent kinase Inhibitor 2A (CDKN2A)]. Interestingly, we show that EGFR blockade inhibits EREG expression, and that EREG knock-down decreases basal cell clonogenic survival, suggesting that EREG expression could be a predictive functional marker of sensitivity to EGFR blockade in basal-like HNSCC.

Head and neck squamous cell carcinoma (HNSCC) is common worldwide and is associated with poor survival (Parkin et al, 1999, 2005; Dobrossy, 2005), mainly due to relapse, metastasis and second cancer (Forastiere et al, 2001; Le Tourneau et al, 2005). Owing to the increasing incidence of HNSCC, there is a growing need for new prognostic and diagnostic markers and therapeutic targets, as well as a better understanding of the biological functions that contribute to clinical outcome. We have studied changes in gene expression in HNSCC tumours compared with histologically normal matched tissues, using differential display and microarray analysis (Lemaire et al, 2003; Carles et al, 2006). This led to the identification of a novel uncharacterised gene located on chromosomal region 11q13 that is overexpressed in HNSCC, which is now called anoctamin 1 (ANO1, also called TMEM16A, ORAOV2, DOG1, TAOS2 and FLJ10261). We showed that ANO1 is overexpressed at the RNA and protein levels in HNSCC, predicted that ANO1 is a membrane protein, and also suggested that it might be a good candidate for targeted anticancer therapy (Carles et al, 2006). In later studies, we compared tumours from patients with different clinical outcomes (Rickman et al, 2008; Jung et al, 2010). The propensity for subsequent distant metastasis in HNSCC was analysed using primary tumours from patients initially treated by surgery that developed (M) or did not develop (NM) metastases as the first recurrent event. Transcriptome (Affymetrix HGU133_Plus2 (Paris, France), quantitative reverse transcriptase PCR) and array comparative genomic hybridisation analysis identified transcripts that were significantly associated with M/NM status, as well as genomic areas including 11q13. However, the genomic analysis was of low resolution, precluding the identification of changes at the single gene level that could be linked to individual transcripts (Rickman et al, 2008). We report here a reanalysis of the same samples with genomic microarrays (Illumina 370K SNP, Paris, France) that provided individual gene resolution, which rekindled our interest in ANO1. ANO1 consists of 26 exons and has been predicted to code for a variety of proteins. It belongs to a protein family with eight transmembrane helices and N- and C-termini that face the cytoplasm (Katoh and Katoh, 2004; Galindo and Vacquier, 2005). ANO1 has two conserved domains of unknown function, a domain that could interfere in meiotic segregation, and multiple potential glycosylation and phosphorylation sites (Katoh and Katoh, 2003). ANO1 has recently been reported to function as a calcium-activated chloride channel (CaCC) (Caputo et al, 2008; Schroeder et al, 2008; Yang et al, 2008). It is highly expressed in tissues with high CaCC activity, and is required for normal development of the trachea (Rock et al, 2008). Chloride channels are relatively underexplored for drug discovery. They have roles in diverse physiological processes, such as fluid secretion, olfactory perception, and neuronal and smooth muscle excitability. Mutations in these channels cause human diseases, including cystic fibrosis, and have applications in treating disorders, such as hypertension and others. There are many opportunities for chloride channels in drug discovery, including, for example, increasing ANO1 activity to treat cystic fibrosis (Verkman and Galietta, 2009). The ANO1 gene is located on 11q13 (Katoh and Katoh, 2003), a chromosomal region that is frequently amplified in HNSCC and is associated with poor outcome (for reviews, see Gollin, 2001; Katoh and Katoh, 2003; West et al, 2004; Perez-Ordonez et al, 2006; Espinosa et al, 2008; Haddad and Shin, 2008). Furthermore, ANO1 is one of a cassette of genes that have been suggested to drive 11q13 amplification by providing growth or metastatic advantage to cancer cells (Huang et al, 2006). In this study, we report that amplification and overexpression of ANO1 is highly correlated with the future development of metastasis in HPV-negative HNSCC. ANO1 is involved in cell motility, invasion and adhesion of HNSCC cells, which could account for this clinical association. Inhibitors of CaCC activity inhibit ANO1-induced migration, suggesting that CaCC activity is important for cell movement. These results raise the possibility that CaCC inhibitors could be explored for tumour therapy.

International audience; Propensity for subsequent distant metastasis in head and neck squamous-cell carcinoma (HNSCC) was analysed using 186 primary tumours from patients initially treated by surgery that developed (M) or did not develop (NM) metastases as the first recurrent event. Transcriptome (Affymetrix HGU133_Plus2, QRT-PCR) and array-comparative genomic hybridization data were collected. Non-supervised hierarchical clustering based on Affymetrix data distinguished tumours differing in pathological differentiation, and identified associated functional changes. Propensity for metastasis was not associated with these subgroups. Using QRT-PCR data we identified a four-gene model (PSMD10, HSD17B12, FLOT2 and KRT17) that predicts M/NM status with 77% success in a separate 79-sample validation group of HNSCC samples. This prediction is independent of clinical criteria (age, lymph node status, stage, differentiation and localization). The most significantly altered transcripts in M versus NM were significantly associated to metastasis-related functions, including adhesion, mobility and cell survival. Several genomic modifications were significantly associated with M/NM status (most notably gains at 4q11-22 and Xq12-28; losses at 11q14-24 and 17q11 losses) and partly linked to transcription modifications. This work yields a basis for the development of prognostic molecular signatures, markers and therapeutic targets for HNSCC metastasis.

International audience; Head and neck squamous cell carcinoma (HNSCC) is common worldwide and is associated with a poor rate of survival. Identification of new markers and therapeutic targets, and understanding the complex transformation process, will require a comprehensive description of genome expression, that can only be achieved by combining different methodologies. We report here the HNSCC transcriptome that was determined by exhaustive differential display (DD) analysis coupled with validation by different methods on the same patient samples. The resulting 820 nonredundant sequences were analysed by high throughput bioinformatics analysis. Human proteins were identified for 73% (596) of the DD sequences. A large proportion (>50%) of the remaining unassigned sequences match ESTs (expressed sequence tags) from human tumours. For the functionally annotated proteins, there is significant enrichment for relevant biological processes, including cell motility, protein biosynthesis, stress and immune responses, cell death, cell cycle, cell proliferation and/or maintenance and transport. Three of the novel proteins (TMEM16A, PHLDB2 and ARHGAP21) were analysed further to show that they have the potential to be developed as therapeutic targets.

International audience; Net (Elk-3, Sap-2, Erp) and the related ternary complex factors Elk-1 and Sap-1 are effectors of multiple signalling pathways at the transcriptional level and play a key role in the dynamic regulation of gene expression. Net is distinct from Elk-1 and Sap-1, in that it is a strong repressor of transcription that is converted to an activator by the Ras/Erk signalling pathway. Two autonomous repression domains of Net, the NID and the CID, mediate repression. We have previously shown that the co-repressor CtBP is implicated in repression by the CID. In this report we show that repression by the NID involves a different pathway, sumoylation by Ubc9 and PIAS1. PIAS1 interacts with the NID in the two-hybrid assay and in vitro. Ubc9 and PIAS1 stimulate sumoylation in vivo of lysine 162 in the NID. Sumoylation of lysine 162 increases repression by Net and decreases the positive activity of Net. These results increase our understanding of how one of the ternary complex factors regulates transcription, and contribute to the understanding of how different domains of a transcription factor participate in the complexity of regulation of gene expression.

The c-ets protooncogenes have recently been shown to code for transcription factors that activate the oncogene responsive unit of the polyoma virus enhancer. We show that transcription of the stromelysin gene, which is highly expressed in transformed cells and tumours, is efficiently activated by c-Ets-1 and -2 through two DNA elements. The distal element is a highly conserved palindrome composed of two strong binding sites for c-Ets-1. The proximal element does not bind c-Ets-1, but may be activated indirectly by increased synthesis of c-Jun and c-Fos. Both ets responsive elements mediate activation by the oncoproteins Ha-Ras, v-Src and v-Mos. These results suggest that c-Ets participates in the mechanisms by which stromelysin gene expression is deregulated in transformed cells and tumours.

In contrast with our previous results (Hen, R., Borrelli, E. & Chambon, P. (1985) Science 230, 1391-1394), which demonstrated that the mouse immunoglobulin heavy chain transcriptional enhancer is repressed in lymphoid cells by the products of the adenovirus type 2 E1A transcription unit, we show here that these products activate the same enhancer in mouse fibroblast L cell lines that contain stably integrated copies of a recombinant in which the enhancer is inserted upstream from the chicken conalbumin promoter. In addition, competition experiments suggest that the activity of the heavy chain enhancer may be repressed by a trans-acting factor in mouse L cells. We speculate that the E1A products may prevent the action of this cellular repressor in these cells.