Joseph Abecassis

Identification of genes associated with tumorigenesis and metastatic potential of hypopharyngeal cancer by microarray analysis

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer among men in the developed world. There is a need, for both clinical and scientific reasons, to find markers to identify patients with aggressive disease as early as possible, and to understand the events leading to malignant transformation and susceptibility to metastasis. We report the first large-scale gene expression analysis of a unique HNSCC location, the hypopharynx. Four normal and 34 tumour samples were analysed with 12 600 gene microarrays. Clusters of differentially expressed genes were identified in the chromosomal regions 3q27.3, 17q21.2–q21.31, 7q11.22–q22.1 and 11q13.1–q13.3, which, interestingly, have already been identified by comparative genomic hybridization (CGH) as major regions of gene amplification. We showed that six overexpressed genes (EIF4G1, DVL3, EPHB4, MCM7, BRMS1 and SART1) located in these regions are indeed amplified. We report 119 genes that are highly differentially expressed between ‘early’ tumours and normal samples. Of these, we validated by quantitative PCR six novel poorly characterized genes. These genes are potential new markers of HNSCC. Comparing patients with relatively nonaggressive and aggressive tumours (without or with clinical evidence of metastasis 3 years after surgery), we identified 164 differentially expressed genes potentially involved in the acquisition of metastatic potential. This study contributes to the understanding of HNSCC, staging patients into prognostic groups and identifying high-risk patients who may benefit from more aggressive treatment.

Biological and clinical relevance of transcriptionally active human papillomavirus (HPV) infection in oropharynx squamous cell carcinoma.

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.

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.

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.

Frequent amplification of 11q13 DNA markers is associated with lymph node involvement in human head and neck squamous cell carcinomas

Amplification of 11q13 DNA markers, particularly hst-1/FGF4 oncogene and the bcl-1 locus, was evaluated in 178 head and neck squamous cell carcinomas (SCCs) by Southern blot and slot blot hybridisation. Coamplification of hst-1/FGF4 and bcl-1 genes was found in 57% of primary tumours and in 60% of the 89 metastatic lymph nodes tested. The pattern of amplification was significantly similar in matched sets of primary SCCs and metastatic lymph nodes. Levels of amplification, quantified by densitometric analysis of slot blots, ranged from 2 to 18-fold normal gene dosage. Also, c-myc oncogene (8q24) was found amplified less frequently, since 7% of 169 SCCs tested contained amplification of this gene, the level of which ranged from 2 to 8-fold. Hst-1/bcl-1 gene amplification was observed more frequently in the tumours arising from the hypopharynx. Coamplification of hst-1 and bcl-1 genes was significantly positively associated with tumours with nodal involvement (P = 0.001). Incidence of hst-1/bcl-1 gene amplification is higher in the tumours with a clinical stage III or IV. Hst-1/bcl-1 gene amplification was not related to tumour differentiation or local invasiveness. This prospective study shows that amplification of 11q13 DNA markers is a prominent event occurring in head and neck SCC and may contribute to the pathogenesis and evolution of a subset of patients bearing this type of cancer.

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.

MDM2 induces hyperplasia and premalignant lesions when expressed in the basal layer of the epidermis

The MDM2 oncogene is overexpressed in 5–10% of human tumours. Its major physiological role is to inhibit the tumour suppressor p53. However, MDM2 has p53‐independent effects on differentiation and does not predispose to tumorigenesis when it is expressed in the granular layer of the epidermis. These unexpected properties of MDM2 could be tissue specific or could depend on the differentiation state of the cells. Strikingly, we found that MDM2 has p53‐dependent effects on differentiation, proliferation and apoptosis when it is expressed in the less differentiated basal layer cells. MDM2 inhibits UV induction of p53, the cell cycle inhibitor p21WAF1/CIP1 and apoptosis (‘sunburn cells’). Importantly, MDM2 increases papilloma formation induced by chemical carcinogenesis and predisposes to the appearance of premalignant lesions and squamous cell carcinomas. p53 has a natural role in the protection against UV damage in the basal layer of the epidermis. Our results show that MDM2 predisposes to tumorigenesis when expressed at an early stage of differentiation, and provide a mouse model of MDM2 tumorigenesis relevant to p53s tumour suppressor functions.

Bcl-2/Bax protein ratio predicts 5-fluorouracil sensitivity independently of p53 status.

p53 tumour-suppressor gene is involved in cell growth control, arrest and apoptosis. Nevertheless cell cycle arrest and apoptosis induction can be observed in p53-defective cells after exposure to DNA-damaging agents such as 5-fluorouracil (5-FU) suggesting the importance of alternative pathways via p53-independent mechanisms. In order to establish relationship between p53 status, cell cycle arrest, Bcl-2/Bax regulation and 5-FU sensitivity, we examined p53 mRNA and protein expression and p53 protein functionality in wild-type (wt) and mutant (mt) p53 cell lines. p53 mRNA and p53 protein expression were determined before and after exposure to equitoxic 5-FU concentration in six human carcinoma cell lines differing in p53 status and displaying marked differences in 5-FU sensitivity, with IC50values ranging from 0.2–22.6 mM. 5-FU induced a rise in p53 mRNA expression in mt p53 cell lines and in human papilloma virus positive wt p53 cell line, whereas significant decrease in p53 mRNA expression was found in wt p53 cell line. Whatever p53 status, 5-FU altered p53 transcriptional and translational regulation leading to up-regulation of p53 protein. In relation with p53 functionality, but independently of p53 mutational status, after exposure to 5-FU equitoxic concentration, all cell lines were able to arrest in G1. No relationship was evidenced between G1 accumulation ability and 5-FU sensitivity. Moreover, after 5-FU exposure, Bax and Bcl-2 proteins regulation was under p53 protein control and a statistically significant relationship (r= 0.880,P= 0.0097) was observed between Bcl-2/Bax ratio and 5-FU sensitivity. In conclusion, whatever p53 status, Bcl-2 or Bax induction and Bcl-2/Bax protein ratio were correlated to 5-FU sensitivity.

G3BP is overexpressed in human tumors and promotes S phase entry.

The expression of the human Ras-GTPase activating protein (GAP)-binding protein (G3BP) was studied in human tumors and cell lines of different origins. Northern blot analysis and immunoblotting experiments showed enhanced expression of G3BP in all tumor samples as compared to healthy tissue. The enhanced expression does not seem to be related to the tumor site or to the stage of development of the cancer. In light of the proposed functions of G3BP, its increased expression in tumors suggest that it plays a role in dedifferentiation and proliferation processes. We also show that G3BP promotes S phase entry in cultured fibroblasts deprived of serum and that this function is dependent on the presence of the RNA binding domain of the protein.