Spectral clustering of microarray data elucidates the roles of microenvironment remodeling and immune responses in survival of head and neck squamous cell carcinoma.

PURPOSE: To identify functionally related prognostic gene sets for head and neck squamous cell carcinoma (HNSCC) by unsupervised statistical analysis of microarray data. PATIENTS AND METHODS: Microarray analysis was performed on 14 normal oral epithelium and 71 HNSCCs from patients with outcome data. Spectral clustering (SC) analysis of the data set identified multiple vectors representing distinct aspects of gene expression heterogeneity between samples. Gene ontology (GO) analysis of vector gene lists identified gene sets significantly enriched within defined biologic pathways. The prognostic significance of these was established by Cox survival analysis. RESULTS: The most influential SC vectors were V2 and V3. V2 separated normal from tumor samples. GO analysis of V2 gene lists identified pathways with heterogeneous expression between HNSCCs, notably focal adhesion (FA)/extracellular matrix remodeling and cytokine-cytokine receptor (CR) interactions. Similar analysis of V3 gene lists identified further heterogeneity in CR pathways. V2CR genes represent an innate immune response, whereas high expression of V3CR genes represented an adaptive immune response that was not dependent on human papillomavirus status. Survival analysis demonstrated that the FA gene set was prognostic of poor outcome, whereas classification for adaptive immune response by the CR gene set was prognostic of good outcome. A combined FA&CR model dramatically exceeded the performance of current clinical classifiers (P < .001 in our cohort and, importantly, P = .007 in an independent cohort of 60 HNSCCs). CONCLUSION: The application of SC and GO algorithms to HNSCC microarray data identified gene sets highly significant for predicting patient outcome. Further large-scale studies will establish the usefulness of these gene sets in the clinical management of HNSCC.

Telomere dysfunction in human keratinocytes elicits senescence and a novel transcription profile.

The uncapping of telomeres has been shown to precipitate senescence in normal human fibroblasts and apoptosis in lymphocytes and p53-competent cancer cell lines. However, the effects of telomere uncapping on normal epithelial cells have not previously been examined. We have used the well characterised telomere repeat binding factor 2 (TRF2) dominant-negative mutant, TRF2(DeltaBDeltaM), to deplete Normal Human Epidermal Keratinocytes (NHEK) telomeres of TRF2. We observed only a two fold increase in both phosphorylation of p53 at serine 15 and 53BP1 DNA damage foci and no detectable increase in p21(WAF). Despite the weak DNA damage response, the keratinocytes growth arrest, demonstrate reduced colony formation and senescence. The small, abortive senescent colonies did not incorporate Brd-U within 48 h and expressed senescence-associated beta galactosidase (SA-beta-gal). Transcriptional profiling of TRF2-depleted keratinocytes showed a reproducible up-regulation of several genes. These included histones, genes associated with DNA damage and keratinocyte terminal differentiation. Several of the same genes were also shown to be up-regulated when keratinocytes undergo natural telomere-mediated senescence and down-regulated by ectopic telomerase expression. This study has thus revealed highly sensitive and specific candidate indicators of telomere dysfunction that may find use in identifying telomere-mediated keratinocyte senescence in ageing, cancer and other diseases.

Divergent routes to oral cancer.

Most head and neck squamous cell carcinoma (HNSCC) patients present with late-stage cancers, which are difficult to treat. Therefore, early diagnosis of high-risk premalignant lesions and incipient cancers is important. HNSCC is currently perceived as a single progression mechanism, resulting in immortal invasive cancers. However, we have found that approximately 40% of primary oral SCCs are mortal in culture, and these have a better prognosis. About 60% of oral premalignancies (dysplasias) are also mortal. The mortal and immortal tumors are generated in vivo as judged by p53 mutations and loss of p16(INK4A) expression being found only in the original tumors from which the immortal cultures were derived. To investigate the relationships of dysplasias to SCCs, we did microarray analysis of primary cultures of 4 normal oral mucosa biopsies, 19 dysplasias, and 16 SCCs. Spectral clustering using the singular value decomposition and other bioinformatic techniques showed that development of mortal and immortal SCCs involves distinct transcriptional changes. Both SCC classes share most of the transcriptional changes found in their respective dysplasias but have additional changes. Moreover, high-risk dysplasias that subsequently progress to SCCs more closely resemble SCCs than nonprogressing dysplasias. This indicates for the first time that there are divergent mortal and immortal pathways for oral SCC development via intermediate dysplasias. We believe that this new information may lead to new ways of classifying HNSCC in relation to prognosis.

DRAM, a p53-induced modulator of autophagy, is critical for apoptosis.

Inactivation of cell death is a major step in tumor development, and p53, a tumor suppressor frequently mutated in cancer, is a critical mediator of cell death. While a role for p53 in apoptosis is well established, direct links to other pathways controlling cell death are unknown. Here we describe DRAM (damage-regulated autophagy modulator), a p53 target gene encoding a lysosomal protein that induces macroautophagy, as an effector of p53-mediated death. We show that p53 induces autophagy in a DRAM-dependent manner and, while overexpression of DRAM alone causes minimal cell death, DRAM is essential for p53-mediated apoptosis. Moreover, analysis of DRAM in primary tumors revealed frequent decreased expression often accompanied by retention of wild-type p53. Collectively therefore, these studies not only report a stress-induced regulator of autophagy but also highlight the relationship of DRAM and autophagy to p53 function and damage-induced programmed cell death.

Profiling early head and neck cancer.

Head and neck squamous-cell carcinoma (HNSCC) is the sixth most common cancer worldwide and, disappointingly, survival rates are not improving. Moreover, HNSCC has a severe impact on the quality of life of patients and survivors, and the significant morbidity subsequent to treatment often mandates long-term multidisciplinary care, which places significant financial pressures on the treating institution. Therefore, prevention and early diagnosis of high-risk pre-malignant lesions are high priorities for reducing deaths due to head and neck cancer. Recent advances have begun to elucidate the different aetiologies of HNSCCs in relation to previous pre-malignancies and to identify which pre-malignant lesions are likely to progress to malignancy.

Effects of dietary flavonoids on major signal transduction pathways in human epithelial cells.

Flavonoids (FVs) are an important class of plant compounds postulated to be one of the constituents responsible for the beneficial effects of fruits and vegetables on health, including heart disease and cancer. At pharmacological levels, various naturally-occurring flavonoids have been shown to be cancer-protective in a variety of animal models and flavonoid derivatives, such as flavopyridol, are being assessed as chemotherapy drugs in clinical trials. This report has investigated the effects of the most common dietary FVs on several major signalling pathways in biopsies of human epithelial cells using primary cultures freshly isolated from biopsies and has obtained evidence for the previously unrecognised importance of stress kinase responses induced by kaempferol (KF), apigenin (AP) and luteolin (LU). KF, AP and LU all activated ATM/ATR (mutated in ataxia-telangiectasia and related) kinases and the p38 stress kinase and this was associated with induction of GADD45 and cell cycle arrest in G2, but not induction of apoptosis. These effects were not due to general toxicity since they were reversible on removal of FV. The inductions of ATM/ATR and p38 were functionally important since caffeine, an inhibitor of ATM/ATR, and the p38-specific inhibitor, SB203580, prevented induction of GADD45 and growth arrest by these three flavonoids. In contrast, although quercetin (QU) activated ATM (but not ATR), it did not activate p38 kinase, GADD45 or p53. QU may interfere with one of the lipoxygenase (LOX) pathways since the growth inhibitory effects of QU (but not the other three flavonoids) could be reversed by addition of LOX metabolites, particularly 12- and 15-hydroxyeicostetraenic acids.

Senescing oral dysplasias are not immortalized by ectopic expression of hTERT alone without other molecular changes, such as loss of INK4A and/or retinoic acid receptor-beta: but p53 mutations are not necessarily required.

Our previous work showed that acquisition of immortality at the dysplasia stage of oral cancer progression was consistently associated with four changes: loss of retinoic acid receptor (RAR)-beta and p16INK4A expression, p53 mutations and activation of telomerase. One atypical dysplasia (D17) that underwent delayed senescence after an extended lifespan showed loss of RAR-beta and p16INK4A/p14ARF expression, but retained functional wild-type p53 and telomerase was not activated. We now demonstrate that retroviral delivery of hTERT results in telomere lengthening and immortalization of D17 without loss of functional wild-type p53 activity. In contrast, the expression of hTERT in two other typical mortal dyplasia cultures (that retain RAR-beta and p16INK4A expression) does not extend their lifespan, even though telomeres are lengthened.

Molecular changes associated with oral dysplasia progression and acquisition of immortality: potential for its reversal by 5-azacytidine.

This study has identified molecular changes characteristic of early oral cancer progression. We reported previously that acquisition of the immortal phenotype is an early event in oral cancer development (F. McGregor et al., Cancer Res., 57: 3886-3889, 1997); our current data indicate that about half of oral dysplasia cultures are immortal, and this is associated with loss of expression of retinoic acid receptor (RAR)-beta and the cell cycle inhibitor p16(ink4a) (p16), p53 mutations, and increased levels of telomerase/human telomerase reverse transcriptase mRNA. In contrast, increased expression of the epidermal growth factor receptor, known to be a characteristic of oral cancer, does not occur until after the dysplasia stage in squamous cell carcinomas. Acquisition of invasive properties as judged by an in vitro Matrigel invasion assay also does not occur until the carcinoma stage and is further increased in metastases. Interestingly, one atypical mortal dysplasia with a considerably extended life span has lost expression of RAR-beta and p16, but it still expresses only wild-type p53 (albeit at a higher level than normal) and has not activated telomerase. RAR-beta and/or p16 re-expression can be induced by treatment with 5-aza-2-deoxycytidine (Aza-C) in some immortal dysplasias, and this has been shown to be due to silencing of gene expression by promoter methylation. Aza-C treatment also down-regulated telomerase activity and human telomerase reverse transcriptase mRNA. Interestingly, with one dysplasia, Aza-C was able to reverse its immortal phenotype, as judged by morphological criteria and expression of the senescence-associated acid beta-galactosidase activity during terminal growth arrest; this immortal dysplasia was the only one in which Aza-C treatment not only down-regulated telomerase activity but also induced re-expression of both RAR-beta and p16. The possibility of reversing the immortal phenotype of some dysplasias by Aza-C may be of clinical usefulness.