ANO1 amplification and expression in HNSCC with a high propensity for future distant metastasis and its functions in HNSCC cell lines.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is associated with poor survival. To identify prognostic and diagnostic markers and therapeutic targets, we studied ANO1, a recently identified calcium-activated chloride channel (CaCC). METHODS: High-resolution genomic and transcriptomic microarray analysis and functional studies using HNSCC cell line and CaCC inhibitors. RESULTS: Amplification and overexpression of genes within the 11q13 amplicon are associated with the propensity for future distance metastasis of HPV-negative HNSCC. ANO1 was selected for functional studies based on high correlations, cell surface expression and CaCC activity. ANO1 overexpression in cells that express low endogenous levels stimulates cell movement, whereas downregulation in cells with high endogenous levels has the opposite effect. ANO1 overexpression also stimulates attachment, spreading, detachment and invasion, which could account for its effects on migration. CaCC inhibitors decrease movement, suggesting that channel activity is required for the effects of ANO1. In contrast, ANO1 overexpression does not affect cell proliferation. INTERPRETATION: ANO1 amplification and expression could be markers for distant metastasis in HNSCC. ANO1 overexpression affects cell properties linked to metastasis. Inhibitors of CaCCs could be used to inhibit the tumourigenic properties of ANO1, whereas activators developed to increase CaCC activity could have adverse effects.

Head and neck squamous cell carcinoma transcriptome analysis by comprehensive validated differential display.

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.

Defect in the p53-Mdm2 autoregulatory loop resulting from inactivation of TAF(II)250 in cell cycle mutant tsBN462 cells.

The cell cycle arrest and proapoptotic functions of p53 are under tight control by Mdm2. After stress activation of p53 by nontranscriptional mechanisms, transcription of the mdm2 gene results in increased synthesis of Mdm2 and down-regulation of p53. Disruption of this autoregulatory loop has profound effects on cell survival and tumorigenesis. We show that a defective p53-Mdm2 autoregulatory loop results from inactivation of a basal transcription factor, TAF(II)250, in tsBN462 cells. We found that Mdm2 expression rescues the temperature-sensitive phenotype of tsBN462 cells, as shown by activation of cell cycle-regulated gene promoters (B-myb, cyclin A, and cdc25C), increased cell growth and DNA synthesis, and inhibition of apoptosis. These effects of Mdm2 are mediated by p53. Exogenous Mdm2 expression apparently complements endogenous Mdm2 synthesis in tsBN462 cells, which is reduced compared to that in the equivalent parental cells with wild-type TAF(II)250, BHK21. Expression of wild-type TAF(II)250 in tsBN462 stimulates and prolongs the synthesis of Mdm2 and rescues the temperature-sensitive phenotype. The TAF(II)250 rescue is blocked by inhibition of Mdm2-p53 interactions. We also show that Mdm2 promoter activation, after transfer to the nonpermissive temperature, is attenuated in cells with mutant TAF(II)250. The temperature-sensitive phenotype apparently results from inefficient inhibition of heat-induced p53 by reduced Mdm2 synthesis due to low mdm2 promoter activity. These results raise the possibility that the p53-Mdm2 autoregulatory loop could guard against transcriptional defects in cells.

Cell-specific regulation of oncogene-responsive sequences of the c-fos promoter.

We have identified oncogene-responsive sequences in the human c-fos promoter that mediate induction of transcription by several nonnuclear oncoproteins and the tumor promoter TPA. These sequences are regulated in a cell-specific manner. (i) In NIH 3T3 cells, the CArG box of the c-fos promoter is sufficient to mediate activation by oncogenes. (ii) In contrast, in HeLa cells, additional flanking sequences are also required, including the outer arm of the serum response element and the FAP site. We also show that the serum response factor, which binds to the CArG box, activates transcription in vivo in NIH 3T3 cells but not in HeLa cells. Finally, we present evidence that the intracellular level of the c-Fos protein could be a major determinant of cell-specific regulation of these oncogene-responsive elements of the c-fos promoter.

Negative regulation contributes to tissue specificity of the immunoglobulin heavy-chain enhancer.

We have identified in and around the immunoglobulin heavy-chain enhancer two apparently distinct negative regulatory elements which repress immunoglobulin H enhancer, simian virus 40 enhancer, and heterologous promoter activity in fibroblasts but not in myeloma cells. We propose that in nonlymphoid cells, negative regulatory elements prevent activation of the immunoglobulin H enhancer by ubiquitous stimulatory trans-acting factors.

Expression of raf oncogenes activates the PEA1 transcription factor motif.

PEA1 (AP1) motif transcription enhancer activity was stimulated by v-raf and more efficiently by activated c-raf-1 or A-raf than by their normal counterparts, in agreement with a role for PEA1 in transformation by raf. Mutations in the ATP-binding site of v-raf prevented activation, suggesting that phosphorylation is somehow required.

Functional interaction of c-Ets-1 and GHF-1/Pit-1 mediates Ras activation of pituitary-specific gene expression: mapping of the essential c-Ets-1 domain.

The mechanism by which activation of common signal transduction pathways can elicit cell-specific responses remains an important question in biology. To elucidate the molecular mechanism by which the Ras signaling pathway activates a cell-type-specific gene, we have used the pituitary-specific rat prolactin (rPRL) promoter as a target of oncogenic Ras and Raf in GH4 rat pituitary cells. Here we show that expression of either c-Ets-1 or the POU homeo-domain transcription factor GHF-1/Pit-1 enhance the Ras/Raf activation of the rPRL promoter and that coexpression of the two transcription factors results in an even greater synergistic Ras response. By contrast, the related GHF-1-dependent rat growth hormone promoter fails to respond to Ras or Raf, indicating that GHF-1 alone is insufficient to mediate the Ras/Raf effect. Using amino-terminal truncations of c-Ets-1, we have mapped the c-Ets-1 region required to mediate the optimal Ras response to a 40-amino-acid segment which contains a putative mitogen-activated protein kinase site. Finally, dominant-negative Ets and GHF constructs block Ras activation of the rPRL promoter, and each blocks the synergistic activation mediated by the other partner protein, further corroborating that a functional interaction between c-Ets-1 and GHF-1 is required for an optimal Ras response. Thus, the functional interaction of a pituitary-specific transcription factor, GHF-1, with a widely expressed nuclear proto-oncogene product, c-Ets-1, provides one important molecular mechanism by which the general Ras signaling cascade can be interpreted in a cell-type-specific manner.

Interaction of Ets-1 and the POU-homeodomain protein GHF-1/Pit-1 reconstitutes pituitary-specific gene expression.

The pituitary-specific, POU-homeodomain factor GHF-1/Pit-1 is necessary, but not sufficient, for cell-specific expression of prolactin (PRL), growth hormone (GH), and thyrotropin. Combinatorial interactions of GHF-1 with other factors are likely to be required; however, such factors and their mechanisms of action remain to be elucidated. Here we identify Ets-1 as a factor that functionally and physically interacts with GHF-1 to fully reconstitute proximal PRL promoter activity. In contrast, Ets-2 has no effect, and the alternatively spliced GHF-2/Pit-1beta variant fails to synergize with Ets-1. The Ets-1-GHF-1 synergy requires a composite Ets-1-GHF-1 cis element and is dependent on an Ets-1-specific protein domain. Furthermore, the ancestrally related and GHF-1-dependent GH promoter, which lacks this composite element, does not exhibit this response. Finally, Ets-1, but not Ets-2, binds directly to GHF-1 and GHF-2. These data show that a functional interaction of GHF-1 and Ets-1, acting via a composite DNA element, is required to establish lactotroph-specific PRL gene expression, thus providing a molecular mechanism by which GHF-1 can discriminate between the GH and PRL genes. These results underscore the importance of transcription factors that are distinct from, but interact with, homeobox proteins to establish lineage-specific gene expression.

Oncogene v-jun modulates DNA replication.

Cell transformation leads to alterations in both transcription and DNA replication. Activation of transcription by the expression of a number of transforming oncogenes is mediated by the transcription factor AP1 (Herrlich & Ponta, 1989; Imler & Wasylyk, 1989). AP1 is a composite transcription factor, consisting of members of the jun and fos gene-families. c-jun and c-fos are progenitors of oncogenes, suggestion that an important transcriptional event in cell transformation is altered activity of AP1, which may arise either indirectly by oncogene expression or directly by structural modification of AP1. We report here that the v-jun oncogene and its progenitor c-jun, as fusion proteins with the lex-A-repressor DNA binding domain, can activate DNA replication from the Polyoma virus (Py) origin of replication, linked to the lex-A operator. The transcription-activation region of v-jun is required for activation of replication. When excess v-jun is expressed in the cell, replication is inhibited or 'squelched'. These results suggest that one consequence of deregulated jun activity could be altered DNA replication and that there are similarities in the way v-jun activates replication and transcription.

Reversion of Ras transformed cells by Ets transdominant mutants.

Considerable progress has been made in elucidating the components of the Ras signalling pathway, from both biochemical and genetic investigations. However little is known about the nuclear targets of the pathway, and in particular those that mediate the long-term changes in gene expression resulting from Ras transformation. Ets family members may be involved in these processes since Ras stimulates transcription through ets-DNA binding sites. We show that a mutated Ets protein, delta PU.1, inhibits Ras activation of transcription. Stable expression of delta PU.1 in Ras transformed NIH3T3 fibroblasts reverts the transformed phenotype by many characteristics, including morphology, anchorage independent growth, saturation density, growth in low serum, tumour formation in nude mice and to some extent sensitivity to apoptotic cell death. Similar trans-dominant mutants of c-Ets-1 and c-Ets-2, the most divergent members of the Ets-family to PU.1, also revert Ras transformed cells, as indicated by morphology, anchorage-independent growth, saturation density and doubling time in low serum. Reversion may result from a shared property of the mutants, such as binding to ets motifs in promoters. These results provide evidence for an important role for Ets proteins in Ras transformation.

Conserved mechanisms of Ras regulation of evolutionary related transcription factors, Ets1 and Pointed P2.

Cell transformation by the Ras oncogene is mediated by members of the ets gene family. To analyse the mechanisms of regulation, we have studied activation of several ets factors by Ras expression. We show that expression of Ha-Ras strongly activates the Ets1 p68 and p54 isoforms and Ets2 in F9 EC cells. We have mapped the Ras responsive elements of Ets1 p68 to two domains, RI+II and RIII. Mutation of threonine 82 to alanine in RI+II abolishes both Ras activation and phosphorylation by MAP kinase. Threonine 82 is part of a sequence that is conserved in Drosophila Pointed P2, an ets protein that has been shown both genetically and biochemically to mediate Ras signalling in Drosophila cells. We extend the comparison of these evolutionary related proteins by showing that Pointed P2 is activated by Ras in mammalian cells and mutation of the homologous threonine abolishes activation. Furthermore, we show that Pointed P2 resembles Ets1, in that it has conserved sequences in a similar position adjacent to the ets DNA binding domain that negatively auto-regulates DNA binding. These results go towards showing that the Drosophila Pointed and vertebrate Ets1 are evolutionary related proteins that have remarkably conserved Ras regulatory mechanisms downstream from MAP kinase.

p53 mediated death of cells overexpressing MDM2 by an inhibitor of MDM2 interaction with p53.

The p53 tumour suppressor is frequently inactivated in human tumours. One form of inactivation results from overexpression of MDM2, that normally forms a negative auto-regulatory loop with p53 and inhibits its activity through complex formation. We have investigated whether disrupting the MDM2-p53 complex in cells that overexpress MDM2 is sufficient to trigger p53 mediated cell death. We find that expression of a peptide homologue of p53 that binds to MDM2 leads to increased p53 levels and transcriptional activity. The consequences are increased expression of the downstream effectors MDM2 and p21WAF1/CIP1, inhibition of colony formation, cell cycle arrest and cell death. There is also a decrease in E2F activity, that might have been due to the known physical and functional interactions of MDM2 with E2F1/DP1. However, this decrease is p53 dependent, as are also colony formation, cell cycle arrest and cell death. These results show that a peptide homologue of p53 is sufficient to induce p53 dependent cell death in cells overexpressing MDM2, and support the notion that disruption of the p53-MDM2 complex is a target for the development of therapeutic agents.

Prediction of future metastasis and molecular characterization of head and neck squamous-cell carcinoma based on transcriptome and genome analysis by microarrays.

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.

Oncogenic conversion alters the transcriptional properties of ets.

The vEts oncoprotein and its progenitor cEts1(p68) belong to a growing family of transcription factors that are related by the conserved ets domain. We show here that the ets domain and adjacent COOH-terminal amino acids are required for DNA binding by cEts1(p68). vEts differs from cEts1(p68) in both the COOH-terminal sequence and an amino acid substitution in the ets domain. The change in the COOH-terminal sequence markedly decreases its affinity for specific DNA, and the ets domain mutation further diminishes binding. vEts does not trans-activate through the ets (PEA3) motif in vivo. Surprisingly, vEts still efficiently trans-activates the promoters of two genes, stromelysin and collagenase, that are found to be overexpressed in transformed cells. The AP1 motifs of both promoters are required for efficient activation. vEts does not bind to the AP1 motif, even in the presence of cJun and cFos. The DNA-binding domain of Ets1 is required for activation through the AP1 element. Activation is inhibited by the expression of the glucocorticoid and retinoic acid receptors, suggesting that activation by Ets does not involve reversal of negative regulators of AP1. We suggest that activation is by an indirect mechanism involving activation of endogenous genes. Our results show that vEts differs from its progenitor cEts1(p68) in its trans-activating properties. The findings suggest that activation of the Jun and Fos oncoprotein pathway is important for transformation by Ets.

Oncogenic conversion of Ets affects redox regulation in-vivo and in-vitro.

The avian acute leukemia virus E26 encodes a fusion protein between viral Gag and the cellular transcription factors cMyb and cEts1(p68). vEts on its own transforms more mature erythroid cells. We have compared the properties of vEts and cEts1(p68). vEts interacts preferentially with an antibody that recognizes the active conformation of the DNA-binding domain. The DNA-binding activity of vEts is particularly sensitive to incubation conditions for band-shift assays, phosphorylation and modification by sulphydryl-specific reagents. Increased sensitivity is due to loss of a protective function of cEts1 C-terminal sequences. cEts2 has a related C-terminal sequence with a similar role. These results suggest that the vEts DNA-binding domain is more accessible to protein-protein interactions and to regulatory mechanisms. Indeed, vEts DNA binding is preferentially inactivated by oxidizing conditions in-vivo. We suggest that the 'open' conformation of the vEts DNA-binding domain favours interactions with other proteins or DNA and facilitates transformation.

The immunoglobulin heavy-chain B-lymphocyte enhancer efficiently stimulates transcription in non-lymphoid cells.

The mouse immunoglobulin heavy-chain (IgH) B-lymphocyte enhancer stimulates transcription from heterologous promoters 20- to 40-fold when transfected into several non-lymphoid cell lines. Stimulation in B-lymphocyte melanoma cell-lines is only about 5--10 times better. A central sequence is equally active in both cell types, whilst flanking sequences, on either side of the common enhancer sequences, specifically stimulate transcription in myeloma cells. These results suggest that there are factors in non-lymphoid cells that can interact with the IgH enhancer to stimulate transcription.

A novel eukaryotic promoter element: the simian virus 40 72 base pair repeat.

Using both clones of mouse LMTK- cells cotransformed with various chimeric conalbumin promoter simian virus 40 (SV40) early gene recombinants and the herpes thymidine kinase gene, and HeLa cells transfected with the same chimeric recombinants, we show that the SV40 72 base pair (bp) repeat sequence is a bidirectional potentiator of initiation of transcription from adjacent T-A-T-A box-dependent and -independent start sites. These results are consistent with our previous model based mainly on the results of T antigen gene expression assays that the 72-bp repeat acts as a bidirectional entry site for RNA polymerase B. We also show that the conalbumin T-A-T-A box is an important element for efficient and accurate in vivo initiation of transcription.

Short and long range activation by the SV40 enhancer.

Activation of transcription by the SV40 enhancer decreases in an apparently biphasic manner when DNA sequences are interposed between the SV40 enhancer and either the homologous SV40 early or the heterologous conalbumin promoter elements. With increasing lengths of short DNA fragments (up to about 150 bp) activation of transcription decreases to less than 10% of the maximum. This short range effect is observed for both the SV40 early and conalbumin promoter elements and for either orientation of the SV40 enhancer. With the conalbumin promoter, increasing the length of the interposing DNA to 275 bp decreases activation to approximately 4%. Larger inserts, of 650 or 3737 bp, lead to an activation of 0.5%. However, in these recombinants, transcription is still activated at least 10 fold compared to an enhancerless recombinant. The implication of these results is discussed.

The SV40 72 bp repeat preferentially potentiates transcription starting from proximal natural or substitute promoter elements.

Activation of gene expression by the SV40 72 bp repeat was studied at the transcriptional level by quantitative S1 nuclease mapping of total RNA isolated from Hela cells transfected with chimeric conalbumin promoter-SV40 early gene recombinants. Our results demonstrate that, irrespective of its orientation, the 72 bp repeat is a potentiator of initiation of transcription from "TATA"-box-dependent and -independent "natural" or "substitute" promoter elements. In addition, we show that potential proximal promoter sequences are activated in preference to more distal ones. These results are consistent with the bidirectional entry site model for transcription activation by the 72 bp repeat.

The c-Ets oncoprotein activates the stromelysin promoter through the same elements as several non-nuclear oncoproteins.

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.