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Despite their potential to regulate approximately one-third of the whole genome, relatively few microRNA (miRNA) targets have been experimentally validated, particularly in stratified squamous epithelia. Here we demonstrate not only that the lipid phosphatase SHIP2 is a target of miRNA-205 (miR-205) in epithelial cells, but, more importantly, that the corneal epithelial-specific miR-184 can interfere with the ability of miR-205 to suppress SHIP2 levels. This is the first example of a miRNA negatively regulating another to maintain levels of a target protein. Interfering with miR-205 function by using a synthetic antagomir, or by the ectopic expression of miR-184, leads to a coordinated damping of the Akt signaling pathway via SHIP2 induction. This was associated with a marked increase in keratinocyte apoptosis and cell death. Aggressive squamous cell carcinoma (SCC) cells exhibited elevated levels of miR-205. This was associated with a concomitant reduction in SHIP2 levels, Partial knockdown of endogenous miR-205 in SCCs markedly decreased phosphorylated Akt and phosphorylated BAD levels and increased apoptosis. We were able to increase SHIP2 levels in SCC cells after inhibition of miR-205. Therefore, miR-205 might have diagnostic value in determining the aggressivity of SCCs. Blockage of miR-205 activity with an antagomir or via ectopic expression of miR-184 could be novel therapeutic approaches for treating aggressive SCCs.

The transforming growth factor type [beta]-1 (TGF-[beta]) signaling pathway is a major tumor suppressor during early carcinogenesis, and its growth-suppressive activity is commonly lost during early tumor progression. I[kappa]B kinase [alpha] (IKK[alpha]) also acts as a tumor suppressor in stratified epithelia, and its expression and nuclear localization are progressively down-regulated during malignant progression of squamous cell carcinoma (SCC) and acquisition of an invasive phenotype. A critical role for IKK[alpha] in TGF-[beta] signaling in stratified epithelia was identified recently during normal keratinocyte differentiation, and both IKK[alpha] and components of the TGF-[beta] signaling pathway are required for induction of antiproliferative Myc antagonists in such cells. We now describe that the interaction between IKK[alpha] and the TGF-[beta] signaling pathway is also important in a subset of SCCs. In SCCs that are unable to shuttle IKK[alpha] to the nucleus, defective TGF-[beta]-induced growth arrest was rescued by introduction of a constitutively nuclear IKK[alpha] variant. These results suggest that the tumor-suppressive activity of IKK[alpha] in stratified epithelia may be exerted in part via the TGF-[beta] signaling pathway. squamous cell carcinoma | Myc | skin cancer

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 ancl 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)-1[alpha] 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. p53 | reactive oxygen species | MitoChip

There is a longstanding but poorly understood epidemiologic link between inflammation and cancer. Consistent with this, we previously showed that [alpha][beta] T cell deficiency can increase resistance to chemical carcinogenesis initiated by 7,12-dimethylbenz[a]anthracene and promoted by phorbol 12-myristate 13-acetate. This provoked the hypothesis that [alpha][beta] T cell deficiency removed T regulatory cells that limit the anti-tumor response or removed a specific tumor-promoting (T-pro) T cell population. Here we provide evidence for the latter, identifying a novel CD8+ subset that is a candidate for T-pro cells. We demonstrate that CD8 cell-deficient mice show substantially less tumor incidence and progression to carcinoma, whereas susceptibility is restored by CD8+ cell reconstitution. To characterize the putative T-pro cells, tumor-infiltrating lymphocytes were isolated from normal and CD4-/- mice, revealing an activated population of T cell receptor [[alpha][beta].sub.+]CD[8.sub.+]CD4[4.sub.+]CD62[L.sub.-] cells expressing the inflammatory mediators IFN[gamma] TNF[alpha], and cyclooxygenase-2, but deficient in perforin, relative to recirculating cells of equivalent phenotype. This novel population of CD[8.sub.+] T cells has intriguing similarities with other lymphocytes that have been associated with tissue growth and invasiveness and has implications for inflammation-associated carcinogenesis, models of cancer immunosurveillance, and immunotherapeutic strategies. inflammation | IFN[gamma] | squamous cell carcinoma | T cell receptor

Like many epithelial tumors, head and neck squamous cell carcinoma (HNSCC) contains a heterogeneous population of cancer cells. We developed an immunodeficient mouse model to test the tumorigenic potential of different populations of cancer cells derived from primary, unmanipulated human HNSCC samples. We show that a minority population of CD[44.sup.+] cancer cells, which typically comprise BMI1 | CD44

Immortalization and malignant transformation are important steps in tumor development. The ability to induce these processes from normal human epithelial cells with genetic alterations frequently found in the corresponding human cancer would significantly enhance our understanding of tumor development. Alterations in several key intracellular regulatory pathways (the pRB, p53, and mitogenic signaling pathways and the telomere maintenance system) appear to be sufficient for the neoplastic transformation of normal human cells. Nevertheless, in vitro transformation models to date depend on viral oncogenes, most prominently the simian virus 40 early region, to induce immortalization and malignant transformation of normal human epithelial cells. Here, we demonstrate a transformation model creating oral--esophageal cancer cells by using a limited set of genetic alterations frequently observed in the corresponding human cancer. In a stepwise model, cyclin D1 overexpression and p53 inactivation led to immortalization of oral keratinocytes. Additional ectopic epithelial growth factor receptor overexpression followed by c-myc overexpression as well as consecutive reactivation of telomerase induced by epithelial growth factor receptor sufficed to transform oral epithelial cells, truly recapitulating the development of the corresponding human disease. cyclin D1 | epithelial growth factor receptor | in vitro transformation | telomerase | c-myc

Lung cancer is the leading cause of cancer mortality worldwide, yet there exists a limited view of the genetic lesions driving this disease. In this study, an integrated high-resolution survey of regional amplifications and deletions, coupled with gene-expression profiling of non-small-cell lung cancer subtypes, adenocarcinoma and squamous-cell carcinoma (SCC), identified 93 focal copynumber alterations, of which 21 span array comparative genomic hybridization | expression profiling | lung adenocarcinoma | squamous-cell lung carcinoma | TP73L

In solid tumors, the relationship between DNA copy number and global expression over large chromosomal regions has not been systematically explored. We used a 12,626-gene expression array analysis of head and neck squamous cell carcinoma and normal oral mucosa and annotated gene expression levels to specific chromosomal loci. Expression alterations correlated with reported data using comparative genomic hybridization. When genes with significant differences in expression between normal and malignant lesions, as defined by significance analysis of microarrays (SAM), were compared to nonsignificant genes, similar chromosomal patterns of alteration in expression were noted. Individual tumors underwent microsatellite analysis and [X.sup.2] analysis of expression at 3p and 22q. Significant 3p underexpression and 22q over-expression were found in all primary tumors with 3p and 22q allelic imbalance, respectively, whereas no tumor without allelic imbalance on these chromosomal arms demonstrated expression differences. Loss and gain of chromosomal material in solid cancers can alter gene expression over large chromosomal regions, including multiple genes unrelated to malignant progression. chromosomal mapping I microarrays I comparative genomic hybridization I allelic imbalance I head and neck squamous cell carcinoma

To determine whether the FHIT gene at 3p14.2 is altered in head and neck squamous cell carcinomas (HNSCC), we examined 26 HNSCC cell lines for deletions within the FHIT locus by Southern analysis, for allelic losses of specific exons FHIT by fluorescence in situ hybridization (FISH) and for integrity of FHIT transcripts. Three cell lines exhibited homozygous deletions within the FHIT gene, 55% (15/25) showed the presence of aberrant transcripts, and 65% (13/20) showed the presence of multiple cell populations with losses of different portions of FHIT alleles by FISH of FHIT genomic clones to interphase nuclei. When the data obtained by FISH and by reverse transcriptase-PCR analyses are combined, 22 of 26 cell lines showed alterations of at least one allele of the FHIT gene. Our data indicate that the FHIT gene is disrupted in HNSCCs and hence, loss of FHIT function may be important in the development and/or progression of head and neck cancers.

We and others recently isolated a human p53 homologue (p40/p51/p63/p73L) and localized the gene to the distal long arm of chromosome 3. Here we sought to examine the role of p40/p73L, two variants lacking the N-terminal transactivation domain, in cancer. Fluorescent in situ hybridization (FISH) analysis revealed frequent amplification of this gene locus in primary squamous cell carcinoma of the lung and head and neck cancer cell lines. (We named this locus AIS for amplified in squamous cell carcinoma.) Furthermore, amplification of the AIS locus was accompanied by RNA and protein overexpression of a variant [p68.sup.AIS] lacking the terminal transactivation domain. Protein overexpression in primary lung tumors was limited to squamous cell carcinoma and tumors known to harbor a high frequency of p53 mutations. Overexpression of [p40.sup.AIS] in Rat 1a cells led to an increase in soft agar growth and tumor size in mice. Our results support the idea that AIS plays an oncogenic role in human cancer. p63 tumor-suppressor gene

Field cancerization predisposes the upper aerodigestive tract mucosa to the formation of multiple primary tumors, when exposed to environmental carcinogens. Up-regulation of epidermal growth factor receptor occurs early in squamous cell carcinogenesis and is critical for the loss of growth control in a variety of human cancers, including head and neck squamous cell carcinomas. In these tumor cells in culture, epidermal growth factor receptor stimulation initiates signaling via persistent activation of selective STAT proteins. To determine the timing of Stat3 activation in head and neck carcinogenesis, we studied the expression and constitutive activation of Stat3 in tumors and normal mucosa from patients with head and neck cancer compared with mucosa from controls without cancer. Stat3 was up-regulated and constitutively activated in both primary human head and neck tumors as well as in normal mucosa from these cancer patients compared with control normal mucosa from patients without cancer. In vivo liposome-mediated gene therapy with a Stat3 antisense plasmid efficiently inhibited Stat3 activation, increased tumor cell apoptosis, and decreased Bcl-[X.sub.L] expression in a head and neck xenograft model. These findings provide evidence that constitutively activated Stat3 is an early event in head and neck carcinogenesis that contributes to the loss of growth control by an anti-apoptotic mechanism.