MAGEB2 is activated by promoter demethylation in head and neck squamous cell carcinoma.

PURPOSE: Although promoter hypermethylation has been an accepted means of tumor suppressor gene inactivation, activation of otherwise normally repressed proto-oncogenes by promoter demethylation has been infrequently documented. EXPERIMENTAL DESIGN: In this study we performed an integrative, whole-genome analysis for discovery of epigenetically activated proto-oncogenes in head and neck cancer tumors. We used the 47K GeneChip U133 Plus 2.0 Affymetrix expression microarray platform to obtain re-expression data from 5-aza treated normal cell line and expression data from primary head and neck squamous cell carcinoma (HNSCC) tumor tissues and normal mucosa tissues. We then investigated candidate genes by screening promoter regions for CpG islands and bisulfite sequencing followed by QUMSP and RT PCR for the best candidate genes. Finally, functional studies were performed on the top candidate gene. RESULTS: From the top 178 screened candidates 96 had CpG islands in their promoter region. Seven candidate genes showed promoter region methylation in normal mucosa samples and promoter demethylation in a small cohort of primary HNSCC tissues. We then studied the demethylation of the top 3 candidate genes in an expanded cohort of 76 HNSCC tissue samples and 17 normal mucosa samples. We identified MAGEB2 as having significant promoter demethylation in primary head and neck squamous cell carcinoma tissues. We then found significantly higher expression of MAGEB2 in tumors in a separate cohort of 73 primary HNSCC tissues and 31 normal tissues. Finally, we found that MAGEB2 has growth promoting effects on minimally transformed oral keratinocyte cell lines but not a definite effect on HNSCC cell lines. CONCLUSION: In conclusion, we identified MAGEB2 as activated by promoter demethylation in HNSCCand demonstrates growth promoting effects in a minimally transformed oral keratinocyte cell line. More studies are needed to evaluate MAGBE2's exact role in HNSCC.

MAGEA4 induces growth in normal oral keratinocytes by inhibiting growth arrest and apoptosis.

Cancer testis antigens (CTAs) are proteins that are normally expressed only in male germ cells and are aberrantly upregulated in a variety of cancers such as melanomas and lung cancer. MAGEA proteins belong to Class I CTAs and are being utilized as targets for cancer immunotherapy. Despite the discovery of the first CTA (MAGEA1) 20 years ago, the functions of these proteins remain poorly understood and evidence suggests both oncogenic as well as tumor suppressive roles for these proteins. Herein, we investigated the role of MAGEA4 in promoting cell growth. When overexpressed, MAGEA4 promotes growth of spontaneously transformed normal oral keratinocytes (NOK-SI). To understand the mechanism of growth stimulation by MAGEA4, we explored the effect of overexpressing MAGEA4 on cell cycle and apoptosis. MAGEA4 inhibits growth arrest of cells in the G1 phase of the cell cycle. We also found that overexpression of MAGEA4 inhibits G418-induced apoptosis of NOK-SI cells. Interestingly, this inhibition was accompanied by repression of two p53 downstream genes, BAX and CDKN1A. Our results indicate that MAGEA4 promotes growth by preventing cell cycle arrest and by inhibiting apoptosis mediated by the p53 transcriptional targets.

Dose-dependent activation of putative oncogene SBSN by BORIS.

Testis-specific transcription factor BORIS (Brother of the Regulator of Imprinted Sites), a paralog and proposed functional antagonist of the widely expressed CTCF, is abnormally expressed in multiple tumor types and has been implicated in the epigenetic activation of cancer-testis antigens (CTAs). We have reported previously that suprabasin (SBSN), whose expression is restricted to the epidermis, is epigenetically derepressed in lung cancer. In this work, we establish that SBSN is a novel non-CTA target of BORIS epigenetic regulation. With the use of a doxycycline-inducible BORIS expressing vector, we demonstrate that relative BORIS dosage is critical for SBSN activation. At lower concentrations, BORIS induces demethylation of the SBSN CpG island and disruption and activation of chromatin around the SBSN transcription start site (TSS), resulting in a 35-fold increase in SBSN expression in the H358 human lung cancer cell line. Interestingly, increasing BORIS concentrations leads to a subsequent reduction in SBSN expression via chromatin repression. In a similar manner, increase in BORIS concentrations leads to eventual decrease of cell growth and colony formation. This is the first report demonstrating that different amount of BORIS defines its varied effects on the expression of a target gene via chromatin structure reorganization.

BORIS binding to the promoters of cancer testis antigens, MAGEA2, MAGEA3, and MAGEA4, is associated with their transcriptional activation in lung cancer.

PURPOSE: Aim of this study was to determine whether BORIS (Brother of the Regulator of Imprinted Sites) is a regulator of MAGEA2, MAGEA3, and MAGEA4 genes in lung cancer. EXPERIMENTAL DESIGN: Changes in expression of MAGEA genes upon BORIS induction/knockdown were studied. Recruitment of BORIS and changes in histone modifications at their promoters upon BORIS induction were analyzed. Luciferase assays were used to study their activation by BORIS. Changes in methylation at these promoters upon BORIS induction were evaluated. RESULTS: Alteration of BORIS expression by induction/knockdown directly correlated with expression of MAGEA genes. BORIS was enriched at their promoters in H1299 cells, which show high expression of these cancer testis antigens (CTA), compared with normal human bronchial epithelial (NHBE) cells which show low expression of the target CTAs. BORIS induction in A549 cells resulted in increased amounts of BORIS and activating histone modifications at their promoters along with a corresponding increase in their expression. Similarly, BORIS binding at these promoters in H1299 correlates with enrichment of activating modifications, whereas absence of BORIS binding in NHBE is associated with enrichment of repressive marks. BORIS induction of MAGEA3 was associated with promoter demethylation, but no methylation changes were noted with activation of MAGEA2 and MAGEA4. CONCLUSIONS: These data suggest that BORIS positively regulates these CTAs by binding and inducing a shift to a more open chromatin conformation with promoter demethylation for MAGEA3 or independent of promoter demethylation in case of MAGEA2 and MAGEA4 and may be a key effector involved in their derepression in lung cancer.

Integrated, genome-wide screening for hypomethylated oncogenes in salivary gland adenoid cystic carcinoma.

PURPOSE: Salivary gland adenoid cystic carcinoma (ACC) is a rare malignancy that is poorly understood. To look for relevant oncogene candidates under the control of promoter methylation, an integrated, genome-wide screen was conducted. EXPERIMENTAL DESIGN: Global demethylation of normal salivary gland cell strains using 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin A (TSA), followed by expression array analysis was conducted. ACC-specific expression profiling was generated using expression microarray analysis of primary ACC and normal samples. Next, the two profiles were integrated to identify a subset of genes for further validation of promoter demethylation in ACC versus normal. Finally, promising candidates were further validated for mRNA, protein, and promoter methylation levels in larger ACC cohorts. Functional validation was then conducted in cancer cell lines. RESULTS: We found 159 genes that were significantly re-expressed after 5-aza-dC/TSA treatment and overexpressed in ACC. After initial validation, eight candidates showed hypomethylation in ACC: AQP1, CECR1, C1QR1, CTAG2, P53AIP1, TDRD12, BEX1, and DYNLT3. Aquaporin 1 (AQP1) showed the most significant hypomethylation and was further validated. AQP1 hypomethylation in ACC was confirmed with two independent cohorts. Of note, there was significant overexpression of AQP1 in both mRNA and protein in the paraffin-embedded ACC cohort. Furthermore, AQP1 was upregulated in 5-aza-dC/TSA-treated SACC83. Finally, AQP1 promoted cell proliferation and colony formation in SACC83. CONCLUSIONS: Our integrated, genome-wide screening method proved to be an effective strategy for detecting novel oncogenes in ACC. AQP1 is a promising oncogene candidate for ACC and is transcriptionally regulated by promoter hypomethylation.

The role of MAGEA2 in head and neck cancer.

OBJECTIVE: To examine the role of MAGEA2 in the tumorigenesis of head and neck squamous cell carcinoma (HNSCC). DESIGN: Primary tissue microarray data and quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed that MAGEA2 is differentially overexpressed in HNSCC. Functional analyses were then performed using MAGEA2 transfections and small-interfering RNA knockdowns with subsequent anchorage-dependent growth studies and cell cycle analyses. Quantitative RT-PCR was used to evaluate expression changes in p53 downstream targets after transfection of MAGEA2 into normal upper aerodigestive cell lines. RESULTS: MAGEA2 is differentially overexpressed in HNSCC. In addition, MAGEA2 promotes growth in normal oral keratinocytes, whereas knockdown of MAGEA2 in HNSCC cells decreases growth. Using the HCT116 p53 wt and null cell line system, transfection of MAGEA2 induced growth in the p53 wt cell line while providing no growth advantage in the p53 mutant cells. Subsequently, transfection of MAGEA2 induced a decrease in messenger RNA expression of the p53 downstream targets CDKN1A and BAX and decreased G1 arrest in cells allowed to remain confluent for longer than 48 hours. CONCLUSIONS: These data suggest that MAGEA2 is differentially expressed in HNSCC and functions, in part, through the p53 pathway by increasing cellular proliferation and abrogating cell cycle arrest. This improved understanding of MAGEA2 function and expression patterns will potentially allow for the improved ability to use MAGEA2 for detection, surveillance, and targeted therapeutics.

EIF2C is overexpressed and amplified in head and neck squamous cell carcinoma.

AIM: To discover putative oncogenes in head and neck squamous cell carcinoma (HNSCC) by integrating data from whole-genome comparison of array-based comparative genomic hybridization (CGH) and expression microarray analysis of HNSCC. METHODS: We integrated published data defining regions of loss/gain identified from the profiling of 21 HNSCC using high-resolution (<1 Mb) CGH arrays and data from an mRNA expression microarray (approx. 12,000 genes) comparing 6 normal tissues and 8 HNSCC tumor tissues. Eukaryotic translation initiation factor 2C subunit 2 (EIF2C2) was found to be the most significantly overexpressed gene by mRNA expression array, and corresponded to the most common region of amplification found by the CGH array described by Sparano et al. We validated EIF2C2 overexpression in primary tissue, overexpression and amplification in HNSCC lines (JHU-011, JHU-012, FADU) relative to a minimally transformed oral keratinocyte cell line (OKF6) and performed knockdown experiments. RESULTS: The tumor tissues had an average mRNA expression level of 123 (SD = 49) compared to the normal tissues (18.6, SD = 10) (p = 0.0005) by expression array. Quantitative RT-PCR validation of our expression arrays found that normal tissues had an average expression of 0.76 (SE = 0.08) and tumor tissues of 2.1 (SE = 0.35) (p = 0.0008). EIF2C2 was found to be amplified and overexpressed in 3 HNSCC cell lines. Knockdown of EIF2C2 in cell lines (JHU-012 and JHU-011) inhibited proliferation. CONCLUSION: EIF2C2 is amplified and overexpressed in HNSCC cell lines and primary tumors and functionally significant in cell lines.

Endothelin receptor type B gene promoter hypermethylation in salivary rinses is independently associated with risk of oral cavity cancer and premalignancy.

Endothelin receptor type B (EDNRB) and kinesin family member 1A (KIF1A) are candidate tumor suppressor genes that are inactivated in cancers. In this study, we evaluated the promoter hypermethylation of EDNRB and KIF1A and their potential use for risk classification in prospectively collected salivary rinses from patients with premalignant/malignant oral cavity lesions. Quantitative methylation-specific PCR was performed to analyze the methylation status of EDNRB and KIF1A in salivary rinses of 191 patients. We proceeded to determine the association of methylation status with histologic diagnosis and estimate classification accuracy. On univariate analysis, diagnosis of dysplasia/cancer was associated with age and KIF1A or EDNRB methylation. Methylation of EDNRB highly correlated with that of KIF1A (P < 0.0001). On multivariable modeling, histologic diagnosis was independently associated with EDNRB (P = 0.0003) or KIF1A (P = 0.027) methylation. A subset of patients analyzed (n = 161) without prior biopsy-proven malignancy received clinical risk classification based on examination. On univariate analysis, EDNRB and risk classification were associated with diagnosis of dysplasia/cancer and remained significant on multivariate analysis (EDNRB: P = 0.047, risk classification: P = 0.008). Clinical risk classification identified dysplasia/cancer with a sensitivity of 71% and a specificity of 58%. The sensitivity of clinical risk classification combined with EDNRB methylation improved to 75%. EDNRB methylation in salivary rinses was independently associated with histologic diagnosis of premalignancy and malignancy and may have potential in classifying patients at risk for oral premalignant and malignant lesions in settings without access to a skilled dental practitioner. This may also potentially identify patients with premalignant and malignant lesions that do not meet the criteria for high clinical risk based on skilled dental examination.

TKTL1 is activated by promoter hypomethylation and contributes to head and neck squamous cell carcinoma carcinogenesis through increased aerobic glycolysis and HIF1alpha stabilization.

PURPOSE: This study aims to investigate the role of the aberrant expression of Transkelolase-like 1 (TKTL1) in head and neck squamous cell carcinoma (HNSCC) tumorigenesis and to characterize TKTL1 contribution to HNSCC tumorigenesis through aerobic glycolysis and HIF1alpha stabilization. EXPERIMENTAL DESIGN: TKTL1 promoter hypomethylation and mRNA/protein aberrant expression were studied in human HNSCC tumor samples and normal mucosas. Oncogenic functions of TKTL1 were examined in HNSCC cell line panels and tumor xenograft models with TKTL1 expression construct. The metabolite levels of fructose-6-phosphate, glyceraldehydes-3-phosphate, pyruvate, lactate, and the levels of HIF1alpha protein and its downsteam glycolytic targets were compared between the TKTL1-expressing and vehicle-expressing HNSCC cells. Meanwhile, the effects of HIF1alpha/glycolytic inhibitors were evaluated on the TKTL1 transfectants. RESULTS: TKTL1 exhibits high frequency of promoter hypomethylation in HNSCC tumors compared with the normal mucosas, correlating with its overexpression in HNSCC. Overexpression of TKTL1 in HNSCC cells promoted cellular proliferation and enhanced tumor growth in vitro and in vivo. Overexpression of TKTL1 increased the production of fructose-6-phosphate and glyceraldehyde-3-phosphate, in turn elevating the production of pyruvate and lactate, resulting in the normoxic stabilization of the malignancy-promoting transcription factor HIF1alpha and the upregulation of downstream glycolytic enzymes. Notably, the reduction of TKTL1 expression decreased HIF1alpha accumulation and inhibition with HIF1alpha and/or the glycolysis inhibitor could abrogate the growth effects mediated by TKTL1 overexpression. CONCLUSION: TKTL1 is a novel candidate oncogene that is epigenetically activated by aberrant hypomethlation and contributes to a malignant phenotype through altered glycolytic metabolism and HIF1alpha accumulation.

Chronic cigarette smoke extract treatment selects for apoptotic dysfunction and mitochondrial mutations in minimally transformed oral keratinocytes.

Cigarette smoke demonstrates a carcinogenic effect through chronic exposure, not acute exposures. However, current cell line models study only the acute effects of cigarette smoke. Using a cell line model, we compared the effects of acute versus chronic cigarette smoke extract (CSE) on mitochondria in minimally transformed oral keratinocytes (OKF6). OKF6 cells were treated with varying concentrations of CSE for 6 months. Cells were analyzed monthly by flow cytometry for mitochondrial membrane potential (MMP), cytochrome c release, caspase 3 activation and viability after CSE exposure. At each time point, the same assays were performed after 24 hr of valinomycin (MMP-depolarizing agent) treatment. The mitochondrial DNA of chronically CSE-treated cells was sequenced. After 6 months of CSE treatment, the cells were increasingly resistant to CSE-mediated and valinomycin-induced cell death. In addition, chronic CSE treatment caused chronic depolarization of MMP, cytochrome c release and caspase activation. Cells grown in the presence of only CSE vapor also exhibited the same resistance and chronic baseline apoptotic activation. Mitochondrial DNA sequencing found that chronic CSE-treated cells had more amino acid-changing mitochondrial mutations than acutely treated cells. CSE treatment of normal cells select for apoptotic dysfunction as well as mitochondrial mutations. These findings suggest that chronic tobacco exposure induces carcinogenesis via selection of apoptosis resistance and mitochondrial mutation in addition to previously known genotoxic effects that were found by acute treatments. Chronic models of tobacco exposure on upper aerodigestive epithelia may be more insightful than models of acute exposure in studying head and neck carcinogenesis.

Integrative discovery of epigenetically derepressed cancer testis antigens in NSCLC.

BACKGROUND: Cancer/testis antigens (CTAs) were first discovered as immunogenic targets normally expressed in germline cells, but differentially expressed in a variety of human cancers. In this study, we used an integrative epigenetic screening approach to identify coordinately expressed genes in human non-small cell lung cancer (NSCLC) whose transcription is driven by promoter demethylation. METHODOLOGY/PRINCIPAL FINDINGS: Our screening approach found 290 significant genes from the over 47,000 transcripts incorporated in the Affymetrix Human Genome U133 Plus 2.0 expression array. Of the top 55 candidates, 10 showed both differential overexpression and promoter region hypomethylation in NSCLC. Surprisingly, 6 of the 10 genes discovered by this approach were CTAs. Using a separate cohort of primary tumor and normal tissue, we validated NSCLC promoter hypomethylation and increased expression by quantitative RT-PCR for all 10 genes. We noted significant, coordinated coexpression of multiple target genes, as well as coordinated promoter demethylation, in a large set of individual tumors that was associated with the SCC subtype of NSCLC. In addition, we identified 2 novel target genes that exhibited growth-promoting effects in multiple cell lines. CONCLUSIONS/SIGNIFICANCE: Coordinated promoter demethylation in NSCLC is associated with aberrant expression of CTAs and potential, novel candidate protooncogenes that can be identified using integrative discovery techniques. These findings have significant implications for discovery of novel CTAs and CT antigen directed immunotherapy.

Coordinated activation of candidate proto-oncogenes and cancer testes antigens via promoter demethylation in head and neck cancer and lung cancer.

BACKGROUND: Epigenetic alterations have been implicated in the pathogenesis of solid tumors, however, proto-oncogenes activated by promoter demethylation have been sporadically reported. We used an integrative method to analyze expression in primary head and neck squamous cell carcinoma (HNSCC) and pharmacologically demethylated cell lines to identify aberrantly demethylated and expressed candidate proto-oncogenes and cancer testes antigens in HNSCC. METHODOLOGY/PRINCIPAL FINDINGS: We noted coordinated promoter demethylation and simultaneous transcriptional upregulation of proto-oncogene candidates with promoter homology, and phylogenetic footprinting of these promoters demonstrated potential recognition sites for the transcription factor BORIS. Aberrant BORIS expression correlated with upregulation of candidate proto-oncogenes in multiple human malignancies including primary non-small cell lung cancers and HNSCC, induced coordinated proto-oncogene specific promoter demethylation and expression in non-tumorigenic cells, and transformed NIH3T3 cells. CONCLUSIONS/SIGNIFICANCE: Coordinated, epigenetic unmasking of multiple genes with growth promoting activity occurs in aerodigestive cancers, and BORIS is implicated in the coordinated promoter demethylation and reactivation of epigenetically silenced genes in human cancers.

Applying the molecular biology and epigenetics of head and neck cancer in everyday clinical practice.

During the past decade, there has been a significant increase in knowledge regarding the molecular biology and epigenetics of head and neck squamous cell carcinoma (HNSCC). This has been aided by the steady development of new technology and novel techniques aimed at elucidating additional aberrant molecular alterations characteristic of HNSCC, including the advent of high throughput assays and the development of more sophisticated bioinformatics tools. In addition, advancements in the field of cancer epigenetics and microRNA have increased the complexity of understanding HNSCC tumorigenesis. These advances have lead to an increasing number of translational studies in the diagnosis, prognosis, and treatment of head and neck cancer. The end result is that molecular biomarkers, gene detection panels and targeted therapeutics are becoming a reality for the care of patients with HNSCC. In this article, we will focus on the many implications of this research as it pertains to clinical practice and the treatment of HNSCC patients.

Molecular techniques and genetic alterations in head and neck cancer.

It is well known that cellular DNA alterations can lead to the formation of cancer, and there has been much discovery in the pathways involved in the development of head and neck squamous cell carcinoma (HNSCC). With novel genome-wide molecular assays, our ability to detect these abnormalities has increased. We now have a better understanding of the molecular complexity of HNSCC, but there is still much research to be done. In this review, we discuss the well described genetic alterations and touch on the newer findings, as well as some of the future directions of head and neck cancer research.

MicroRNA alterations in head and neck squamous cell carcinoma.

MicroRNAs (mirs) are small noncoding RNA molecules (~22 nucleotides) that regulate posttranscriptional gene expression. Currently, there has not been a comprehensive study of their role in primary head and neck squamous cell carcinoma (HNSCC). To determine the role of mirs in HNSCC, we screened for altered microRNA expression in HNSCC primary tissue and cell lines. We then further tested the functional impact of alterations of specific mirs. An initial screening of 4 primary HNSCC, 4 normal mucosal controls and 4 HNSCC cell lines was analyzed for mature microRNA expression by microarray. Significance was determined using significance analysis of microarrays (SAM). Nine microRNAs were found by SAM to be upregulated or downregulated in tumor tissue including mir-21, let-7, 18, 29c, 142-3p, 155, 146b (overexpressed) and 494 (underexpressed). Mir-21 was validated by qRT-PCR. Functional validation by growth assays was performed, further validating mir-21. Transfection of mir-21 into JHU-011 and JHU-012 cell lines showed a 39% increase in cell growth at 72 hr relative to controls (p < 0.05). Transfection of the inhibitor into JHU-O12 cell lines showed a 92% decrease in cell growth relative to controls at 72 hr (p < 0.05). In addition, flow cytometry analysis of JHU-012 cells 48 hr after mir-21 inhibitor transfection showed a statistically significant increase in cytochrome c release and increased apoptosis. These differentially expressed microRNAs may be of interest as potential novel oncogenes and tumor suppressor genes in HNSCC. Mir-21 is a putative oncogenic microRNA in head and neck cancer.

Frequency and phenotypic implications of mitochondrial DNA mutations in human squamous cell cancers of the head and neck.

Mitochondrial genomic mutations are found in a variety of human cancers; however, the frequency of mitochondrial DNA (mtDNA) mutations in coding regions remains poorly defined, and the functional effects of mitochondrial mutations found in primary human cancers are not well described. Using MitoChip, we sequenced the whole mitochondrial genome in 83 head and neck squamous cell carcinomas. Forty-one of 83 (49%) tumors contained mtDNA mutations. Mutations occurred within noncoding (D-loop) and coding regions. A nonrandom distribution of mutations was found throughout the mitochondrial enzyme complex components. Sequencing of margins with dysplasia demonstrated an identical nonconservative mitochondrial mutation (A76T in ND4L) as the tumor, suggesting a role of mtDNA mutation in tumor progression. Analysis of p53 status showed that mtDNA mutations correlated positively with p53 mutations (P < 0.002). To characterize biological function of the mtDNA mutations, we cloned NADH dehydrogenase subunit 2 (ND2) mutants based on primary tumor mutations. Expression of the nuclear-transcribed, mitochondrial-targeted ND2 mutants resulted in increased anchorage-dependent and -independent growth, which was accompanied by increased reactive oxygen species production and an aerobic glycolytic metabolic phenotype with hypoxia-inducible factor (HIF)-1alpha induction that is reversible by ascorbate. Cancer-specific mitochondrial mutations may contribute to development of a malignant phenotype by direct genotoxic effects from increased reactive oxygen species production as well as induction of aerobic glycolysis and growth promotion.

Head and neck cancer cell lines exhibit differential mitochondrial repair deficiency in response to 4NQO.

Constituents of tobacco can cause DNA adduct formation and are implicated in head and neck squamous cell cancer (HNSC) development. We investigated the capacity of HNSC cell lines to repair mitochondrial DNA (mtDNA) damage induced by a DNA adduct-forming agent. HNSC cell lines underwent 4-nitroquinoline 1-oxide (4NQO) exposure with subsequent rescue with normal media. Real-time quantitative PCR for nuclear DNA (nDNA) and mtDNA was performed. mtDNA to nDNA ratios were calculated and standardized to mock-treated cells to assess mtDNA repair ability. Two of three tested cancer cell lines exposed to 4NQO exhibited consistent decreases in mtDNA/nDNA ratios throughout the different repair timepoints. At 24 h mtDNA/nDNA ratios of JHU-O19 and JHU-O22 decreased to 63% and 60% of controls, respectively. Conversely, a control keratinocyte cell line exhibited overall increases in mtDNA/nDNA ratios compared to baseline suggesting intact DNA repair mechanisms. By using a DNA adduct formation and repair model featuring 4NQO and HNSC cell lines, we have implicated faulty mtDNA repair as having a potential role in HNSC.

Mutant epidermal growth factor receptor up-regulates molecular effectors of tumor invasion.

The gene most commonly altered in human glioblastomas is the epidermalgrowth factor receptor (EGFR). We profiled transcripts induced by mutantEGFR to better understand its role in tumor progression. The pattern found suggested enhanced tumor invasion. The highly induced genes included extracellular matrix components, metalloproteases, and a serine protease. We confirmed that mutant EGFR did make glioblastoma cells both more motile and invasive using in vitro assays. Furthermore, inhibitors of EGFR (OSI-774 and Tyrphostin AG1478) selectively down-regulated these molecular effectors in glioblastoma cells, eliminating enhanced invasion.