Suppression of KLF5 basal expression in oral carcinoma-derived cells through three intact CREB1-binding sites in the silencer region.

OBJECTIVES: Krüppel-like factor (KLF)5, which is overexpressed in carcinomas such as oral cancer, inhibits epidermal differentiation. KLF5 induces dedifferentiation of carcinoma cells, which effectuates carcinoma progression; nevertheless, the regulatory mechanism affecting the transcription of the KLF5 gene remains ambiguous. METHODS: Transcriptional activity of the KLF5 silencer, specifically the 425-bp region (425-region), was examined using reporter assays. An additional analysis was conducted to assess the impact of the minimal essential region (MER) of KLF5 on its basal expression. The affinity of cAMP responsive element binding protein 1 (CREB1) for three potential CREB1-binding sites in the 425-region was analyzed using DNA pull-down and quantitative chromatin immunoprecipitation assays. Reporter assays employing a human oral squamous carcinoma cell line, HSC2, transfected with small interfering RNA or complementary DNA for CREB1, were performed to investigate the effect of CREB1 binding sites on MER activity. RESULTS: The 425-region exhibited no transcriptional activity and suppressed MER transcriptional activity. This region encodes three putative CREB1-binding sites, and CREB1 demonstrated equal binding affinity for all three sites. The deletion of each of these binding sites reduced CREB1 precipitation and enhanced MER activity. Endogenous CREB1 knockdown and overexpression elevated and reduced MER activity, respectively, at the intact sites. Conversely, site deletion hampered and improved MER activity upon CREB1 knockdown and overexpression, respectively. CONCLUSIONS: Suppression of KLF5 basal expression via CREB1 binding to the 425-region requires all three CREB1-binding sites to remain intact in oral carcinoma cells. Consequently, deletion of the CREB1-binding site relieves suppression of KLF5 basal expression.

Suppression of Krüppel-like factor 5 basal expression by CREB1 binding to far distal element.

BACKGROUND: Krüppel-like factor 5 (KLF5) is a transcription factor regulating the proliferation and differentiation of epithelial cells, and its uncontrolled expression is closely associated with carcinoma progression. Sp3 binding to the minimal essential region (MER) of KLF5 gene is critical for KLF5 basal expression, but the expression control mechanism is unknown. OBJECTIVE: This study aimed to identify a regulatory region for KLF5 basal expression and the binding protein in carcinoma cells by analyzing the promoter upstream region. METHODS: Reporter assays determined the silencer region. The protein binding to the region was identified by database analysis and ChIP assay. The protein mediating the interaction between the region and the MER was confirmed through chromosome conformation capture (3 C) on ChIP assay. The effects of the protein on KLF5 expression were analyzed using qRT-PCR and western blot. RESULTS: Reporter assay localized the 425-region from upstream KLF5 gene as the silencer. Database analysis and ChIP assay found CREB1 binding to the 425-region. CREB1 siRNA or mutation of CREB1-binding site in the 425-region increased luciferase activities and decreased the binding to 425-region. 3 C on ChIP assay showed that CREB1 mediated interaction of the 425-region and the MER. CREB1 overexpression decreased endogenous KLF5 expression and luciferase activity. CONCLUSIONS: The 425-region is the silencer of KLF5 basal expression, and CREB1 binding suppresses the expression.

NF-κB subunit RELA suppression of mucosa-associated lymphoid tissue lymphoma translocation protein 1 expression in oral carcinoma cells.

Loss of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) expression closely associates with increased aggressive behaviors of oral carcinoma cells. It emphasizes that a mechanism to suppress the expression is an important subject for understanding carcinoma progression pathway. However, nothing is known at present. This study conducted on transcriptional regulation of the gene down-regulation. Reporter assays showed the presence of the silencer region between +402 and +501 region of MALT1 gene in oral carcinoma cells. It encoded a binding site of nuclear factor-κB subunit, RELA. RELA binding to the site was confirmed by the chromatin immunoprecipitation analyses, and deletion and mutations of the site significantly decreased the RELA binding. Short interfering RNAs for RELA up-regulated reporter gene and endogenous MALT1 protein expressions, and deletion and mutations of RELA binding site increased reporter gene expression. These results demonstrated RELA-binding to the site suppresses MALT1 expression that may facilitate oral carcinoma progression.

Domain structures of mucosa-associated lymphoid tissue lymphoma translocation 1 protein for nuclear localization in oral carcinoma cells and the proliferation inhibition.

Mucosa-associated lymphoid tissue lymphoma translocation 1 protein (MALT1) consisting of death domain, Ig-like domains and caspase-like domain is expressed in nucleus of oral carcinoma cells, and loss of the expression closely associates with disease progression and stimulates proliferation of the cells. However, nothing is known about the molecular backgrounds. In this study, eight constructs with different domain constitution of human MALT1 and six constructs were transiently and stably transfected into oral carcinoma cell lines, respectively. The immunoblot analysis showed that constructs containing caspase-like domain was expressed in nucleus and the domain-deleted constructs in cytoplasm. Immunocytochemistry of stably transfected HSC2 oral carcinoma cells confirmed the caspase-like domain-dependent nuclear localization. Involvement of domains in proliferation of stably transfected HSC2 cells was quantified by the real-time and conventional colorimetric assays. In contrast to suppression of the proliferation by full-length wild-type MALT1, any domain-deleted constructs enhanced the proliferation. Death domain construct without caspase-like domain suppressed the proliferation when it was localized in nucleus by ligating with the nuclear localization signal. These results demonstrate that nuclear localization of MALT1 in oral carcinoma cells depends on the presence of caspase-like domain and that death domain nuclear entity is responsible for MALT1 inhibition of oral carcinoma cell proliferation. Nuclear localization of death domain led by caspase-like domain may suppress oral carcinoma progression.

Vascular endothelial growth factor signaling in VE-cadherin expression and tube-like formation by rheumatoid arthritic synovial fibroblast-like cells.

An increase in the vasculature is one of most representative changes in the synovial tissue of joints in rheumatoid arthritis (RA) and is closely associated with disease progression. Although the vasculatures are believed to be a result of VE-cadherin-dependent angiogenesis and a possible therapeutic target of the disease, synovial fibroblastic cells express VE-cadherin and form tube-like structures, suggesting that vasculatures in RA synovium may not simply result from angiogenesis. This paper analyzes a mechanism of VE-cadherin expression by rheumatoid arthritic synovial fibroblast-like cells (RSFLs) and their involvement in the tube-like formation. A representative angiogenic factor, vascular endothelial growth factor (VEGF), and its binding to a predominant receptor (VEGFR2) activated VE-cadherin expression and the signaling pathways of ERK/MAPK and PI3K/AKT/mTOR. Treatment of RSFLs with signaling pathway inhibitors, VEGFR2 siRNA and a VEGF-antagonizing mimicking peptide inhibited VE-cadherin expression dose-dependently. VEGF-stimulated tube-like formation by RSFLs on Matrigel was hindered by the mimicking peptide and inhibitor treatment. This data demonstrates that RSFLs activated by VEGF binding of VEGFR2 express VE-cadherin and formed tube-like structure under the control of ERK/MAPK and PI3K/AKT/mTOR pathways suggesting that the inhibition suppresses vascular development in RA synovium.

Minimal essential region for krüppel-like factor 5 expression and the regulation by specificity protein 3-GC box binding.

Krüppel-like factor 5 (KLF5) transcriptionally controls the proliferation-differentiation balance of epithelium and is overexpressed in carcinomas. Although genomic region modifying KLF5 expression is widespread in different types of cells, the region that commonly regulates basal expression of the genes across cell-types is uncertain. In this study we determined the minimal essential region for the expression and its regulatory transcription factors using oral carcinoma cells. A reporter assay defined a 186bp region downstream of the transcription start site and a cluster of six GC boxes (GC1-GC6) as the minimal essential region. Mutation in the GC1 or GC6 regions but not other GC boxes significantly decreased the reporter expression. The decrease by the GC1 mutation was reproduced in the 2kbp full-length promoter, but not by the GC6 mutation. Additionally, specificity proteins (Sp) that can be expressed in epithelial cells and bind GC box, Sp3 co-localized with KLF5 in oral epithelium and carcinomas and chromatin immunoprecipitation analyses showed Sp3 as the prime GC1-binding protein. Inhibition of Sp-GC box binding by mithramycin A and knockdown of Sp3 by the short interfering RNA decreased expression of the reporter gene and endogenous KLF5. These data demonstrate that a 186bp region is the minimal essential region and that Sp3-GC1 binding is essential to the basal expression of KLF5.

Krüppel-like factor 4 expression in oral carcinoma cells and hypermethylation at the gene promoter.

BACKGROUND: Krüppel-like factor 4 (KLF4) is a transcription factor regulating proliferation-differentiation balance of epithelium, and down-regulated in less-differentiated and advanced oral carcinomas. Although the expression is inactivated by the promoter hypermethylation in malignant tumor cells, it remains unknown in oral carcinoma cells. METHODS: Genomic DNA isolated from nine different oral carcinoma cell lines and a normal keratinocyte line was treated with sodium bisulfite, and methylation at KLF4 gene promoter was determined by PCR direct-sequence analysis. KLF4 expression in cells cultured with or without demethylation reagent was monitored by quantitative real-time PCR and immunoblot. RESULTS: A 237-bp promoter region spanning - 718 and - 482 of KLF4 gene was hypermethylated in oral carcinoma cells that express KLF4 at a low level, but the methylation was infrequent in cells expressing KLF4 high amount. The downstream region from - 481 to +192 was not methylated in any cell lines. Demethylation treatment of cells up-regulated the expression at mRNA and protein levels. CONCLUSION: This study demonstrated that hypermethylation at a narrow range of the promoter region down-regulates KLF4 expression, and suggests that the loss of expression by the hypermethylation contributes to oral carcinoma progression.

Krüppel-like factors 4 and 5 expression and their involvement in differentiation of oral carcinomas.

Proliferation-differentiation balance of epithelial cells is regulated by Krüppel-like factors (KLF) 4 and 5, and the unbalanced expression relates to carcinoma progression. However, little is known about the expression and role in oral carcinomas. This study examined expression of KLF4 and KLF 5 in the carcinomas by immunohistochemistry (n = 67) and the involvement in proliferation and differentiation of carcinoma cells. KLF4 was detected in keratinizing carcinoma cells and KLF5 in non-keratinizing cells. KLF4 staining declined in the patient with lymph node metastasis (P < 0.05) and in parallel with the histological dedifferentiation (P = 0.09). Exogenous overexpression of KLF4 arranged cells in a cobble-like structure with desmosomes and KLF5 elongated cells like fibroblasts without desmosomes. KLF4 suppressed fibronectin expression, and KLF5 down-regulated and degraded E-cadherin. The proliferation was not affected by KLFs. Thus, down-regulation of KLF4 and up-regulation of KLF5 may stimulate oral carcinoma progression through the dedifferentiation of carcinoma cells.

Heterogeneous tumor stromal microenvironments of oral squamous cell carcinoma cells in tongue and nodal metastatic lesions in a xenograft mouse model.

BACKGROUND: Oral squamous cell carcinoma exhibits a poor prognosis, caused by aggressive progression and early-stage metastasis to cervical lymph nodes. Here, we developed a xenograft mouse model to explore the heterogeneity of the tumor microenvironment that may govern local invasion and nodal metastasis of tumor cells. METHODS: We transplanted five oral carcinoma cell lines into the tongues of nude mice and determined tongue tumor growth and micrometastatic dissemination by serially sectioning the tongue and lymph node lesions in combination with immunohistochemistry and computer-assisted image analysis. Our morphometric analysis enabled a quantitative assessment of blood and lymphatic endothelial densities in the intratumoral and host stromal regions. RESULTS: All cell lines tested were tumorigenic in mouse tongue. The metastatic lesion-derived carcinoma cell lines (OSC19, OSC20, and HSC2) yielded a 100% nodal metastasis rate, whereas the primary tumor-derived cell lines (KOSC2 and HO-1-u-1) showed <40% metastatic potential. Immunohistochemistry showed that the individual cell lines gave rise to heterogeneous tumor architecture and phenotypes and that their micrometastatic lesions assimilated the immunophenotypic properties of the corresponding tongue tumors. Notably, OSC19 and OSC20 cells shared similar aggressive tumorigenicity in both the tongue and lymph node environments but displayed markedly diverse immunophenotypes and gene expression profiles. CONCLUSIONS: Our model facilitated comparing the tumor microenvironments in tongue and lymph node lesions. The results support that tumorigenicity and tumor architecture in the host tongue environment depend on the origin and properties of the carcinoma cell lines and that metastatic progression may take place through heterogeneous tumor-host interactions.

Differential expression of fatty acid-binding proteins and pathological implications in the progression of tongue carcinoma.

Tongue carcinomas are common malignancies of the oral cavity. Understanding the molecular mechanisms behind the disease progression is a prerequisite for improving patient prognosis. Fatty acid-binding proteins (FABPs) are cytoplasmic lipid chaperones that affect cellular organization and energy production. Although their aberrant expression is involved in carcinoma progression, its role in the pathology of tongue carcinomas remains unclear. In the present study, the immunohistochemical expression of FABP4 and FABP5 in tongue carcinomas (n=58) and its involvement in the clinicopathological parameters were examined. Normal tongue epithelial cells expressed FABP5, an epidermal-type FABP, but not FABP4, an adipocyte-type FABP. The cytoplasmic staining of FABP5 was increased in carcinomas with advanced T-stage (P<0.05) and clinical stage (P<0.05). Ectopic expression of FABP4 was detected in almost all carcinomas, although its role in disease progression remains undetermined. Upregulation of FABP5 in the wounded skin of genetically normal mice indicated that microenvironmental tissue factors induce FABP5 expression. The results of the present study demonstrated the aberrant expression of FABP4 and FABP5 in tongue carcinomas and suggested the involvement of FABP5 in disease progression.

Concomitant loss of p120-catenin and ß-catenin membrane expression and oral carcinoma progression with E-cadherin reduction.

The binding of p120-catenin and ß-catenin to the cytoplasmic domain of E-cadherin establishes epithelial cell-cell adhesion. Reduction and loss of catenin expression degrades E-cadherin-mediated carcinoma cell-cell adhesion and causes carcinomas to progress into aggressive states. Since both catenins are differentially regulated and play distinct roles when they dissociate from E-cadherin, evaluation of their expression, subcellular localization and the correlation with E-cadherin expression are important subjects. However, the same analyses are not readily performed on squamous cell carcinomas in which E-cadherin expression determines the disease progression. In the present study, we examined expression and subcellular localization of p120-catenin and ß-catenin in oral carcinomas (n = 67) and its implications in the carcinoma progression and E-cadherin expression using immunohitochemistry. At the invasive front, catenin-membrane-positive carcinoma cells were decreased in the dedifferentiated (p120-catenin, P < 0.05; ß-catenin, P < 0.05) and invasive carcinomas (p120-catenin, P < 0.01; ß-catenin, P < 0.05) and with the E-cadherin staining (p120-catenin, P < 0.01; ß-catenin, P < 0.01). Carcinoma cells with ß-catenin cytoplasmic and/or nuclear staining were increased at the invasive front compared to the center of tumors (P < 0.01). Although the p120-catenin isoform shift from three to one associates with carcinoma progression, it was not observed after TGF-ß, EGF or TNF-α treatments. The total amount of p120-catenin expression was decreased upon co-treatment of TGF-ß with EGF or TNF-α. The above data indicate that catenin membrane staining is a primary determinant for E-cadherin-mediated cell-cell adhesion and progression of oral carcinomas. Furthermore, it suggests that loss of p120-catenin expression and cytoplasmic localization of ß-catenin fine-tune the carcinoma progression.

Proteomic identification of keratin alterations with enhanced proliferation of oral carcinoma cells by loss of mucosa-associated lymphoid tissue 1 expression.

Progression of oral carcinomas associates with aberrant activation and inactivation of molecules that work in established or unknown pathways. Although mucosa­associated lymphoid tissue 1 (MALT1) expressed in normal oral epithelium is inactivated in the aggressive subset of carcinomas with worse prognosis, phenotypic changes of carcinoma cells upon the loss of expression is unknown. We performed a proteomic analysis to identify MALT1­regulated proteins in oral carcinoma cells. Four different keratins were included in the ten most abundantly changed proteins. K8/18 were upregulated in MALT1 stably­expressing carcinoma cells and K5/14 in MALT1­marginal control cells. K8/18 upregulation and K5/14 downregulation were MALT1 dose­dependent and observed in a series of oral carcinoma cells. MALT1 suppressed cell proliferation (0.52-fold, P<0.01) and its dominant-negative form stimulated it (1.33-fold, P<0.01). The decreased proliferation associated with reduction of cyclin D1, which was recovered by the short interfering RNA against MALT1. Taken together, loss of MALT1 expression alters keratin expression and enhances proliferation of carcinoma cells, and may progress oral carcinomas into the advanced state.

Single nucleotide polymorphisms of mucosa-associated lymphoid tissue 1 in oral carcinoma cells and gingival fibroblasts.

Oral carcinoma patients with inactivation of mucosa-associated lymphoid tissue 1 (MALT1) expression worsen their prognoses. Although the genetic mutation could be responsible for the inactivation, no information is available at present. In the present study, genomic DNA of oral carcinoma cells (HOC313, TSU, HSC2, HSC3, KOSC2, KOSC3, SCCKN, OSC19, Ca9.22, and Ho1u1 cells) and normal gingival fibroblasts (GF12 cells) derived from a Japanese population were amplified by polymerase chain reaction using primer sets spanning MALT1 exons, and nucleotide substitutions were analyzed by the single strand conformation polymorphism analysis. The substitutions were commonly observed in all cells, which express MALT1 at various levels. The substitutions at exons 1 and 9 were located at the 5' untranslated region and replaced (336)Asp to Asn, respectively, and others were positioned at the introns. Among the intronic substitutions, four were matched with the single nucleotide polymorphisms (SNPs) registered at the database. Since all cells were derived from a Japanese population, all substitutions detected are the SNPs. Absence of the carcinoma cell-specific mutation suggests that the inactivation of MALT1 expression but not the mutation promotes oral carcinoma progression.

Progression of oral squamous cell carcinoma accompanied with reduced E-cadherin expression but not cadherin switch.

The cadherin switch from E-cadherin to N-cadherin is considered as a hallmark of the epithelial-mesenchymal transition and progression of carcinomas. Although it enhances aggressive behaviors of adenocarcinoma cells, the significance and role of cadherin switch in squamous cell carcinomas (SCCs) are largely controversial. In the present study, we immunohistochemically examined expression of E-cadherin and N-cadherin in oral SCCs (n = 63) and its implications for the disease progression. The E-cadherin-positive carcinoma cells were rapidly decreased at the invasive front. The percentage of carcinoma cells stained E-cadherin at the cell membrane was reduced in parallel with tumor dedifferentiation (P<0.01) and enhanced invasion (P<0.01). In contrast, N-cadherin-positive cells were very limited and did not correlate with the clinicopathological parameters. Mouse tongue tumors xenotransplantated oral SCC cell lines expressing both cadherins in vitro reproduced the reduction of E-cadherin-positive carcinoma cells at the invasive front and the negligible expression of N-cadherin. These results demonstrate that the reduction of E-cadherin-mediated carcinoma cell-cell adhesion at the invasive front, but not the cadherin switch, is an important determinant for oral SCC progression, and suggest that the environments surrounding carcinoma cells largely affect the cadherin expression.

Mutational analysis of HRAS and KRAS genes in oral carcinoma cell lines.

RAS overexpression and its active mutations are involved in malignant tumorigenesis. However, the mutation rates in oral carcinoma cells differ between populations. In the present study, genomic DNA of oral carcinoma cells (HOC313, TSU, HSC2, HSC3, KOSC2, KOSC3, SCCKN, OSC19, Ca9.22, and Ho1u1 cells) or normal gingival fibroblasts (GF12 cells) derived from a Japanese population were amplified by polymerase chain reaction using primer sets, spanning HRAS and KRAS exons. Nucleotide substitutions were analyzed by single strand conformation polymorphism. In contrast to no substitutions in KRAS, nine different substitutions were detected in HRAS. Of the nine, six substitutions were located at intron 1 (HSC2 and HSC3 cells) or intron 2 (HSC3, SCCKN and Ca9.22 cells), and one each of exon 1 (all cells), exon 2 (HOC313, TSU, HSC2 and HSC3 cells) and the 5' upstream region (all cells). Substitutions at exons 1 and 2 did not affect the amino acid sequence; the exon 1 substitution was positioned at the 5' untranslated region, which may be a single nucleotide polymorphism (SNP) sequence because all the cells were isolated from a Japanese population, and the mutations at exon 2 was a silent mutation. A substitution at the 5' upstream region was an SNP. These data demonstrate that SNPs and point mutations observed in HRAS do not change the amino acid sequence, and suggest that the mutations affecting the amino acid sequence may be a rare event in oral carcinomas of the Japanese population.

The divergent roles of secreted frizzled related protein-1 (SFRP1) in lung morphogenesis and emphysema.

Developmentally expressed genes are believed to play a central role in tissue repair after injury; however, in lung disease their role has not been established. This study demonstrates that SFRP1, an inhibitor of Wnt signaling normally expressed during lung embryogenesis, is induced in the lungs of emphysema patients and in two murine models of the disease. SFRP1 was found to be essential for alveolar formation as Sfrp1(-/-) mice exhibited aberrant Wnt signaling, mesenchymal proliferation, and impaired alveoli formation. In contrast, SFRP1 activated ERK and up-regulated MMP1 and MMP9 without altering TIMP1 production when expressed in human lung epithelial cells. These findings demonstrate that SFRP1 promotes normal alveolar formation in lung development, although its expression in the adult up-regulates proteins that can cause tissue destruction. Thus, SFRP1 induction during tissue injury is unlikely to contribute to the repair response but rather is a participatory factor in the pathogenesis of emphysema and tissue destruction.

Epigenetic loss of mucosa-associated lymphoid tissue 1 expression in patients with oral carcinomas.

Mucosa-associated lymphoid tissue 1 (MALT1), which is located in a genomic region that encodes unknown tumor suppressor gene(s), activates nuclear factor-kappaB in lymphocyte lineages. However, its expression and role in the pathology of malignant tumors of epithelial origin is not known. In the present study, we examined MALT1 expression and its implications for the pathology of oral carcinomas. Immunostaining localized MALT1 in the nucleus of normal oral epithelial cells, but the expression was absent in 45.0% of carcinomas (49 of 109 cases) especially at the invasive front. The loss of expression was correlated with tumor recurrence (P = 0.007) and poor patient survival (P < 0.001), and it was an independent prognostic determinant (P < 0.001). MALT1-negative carcinomas exhibited microsatellite instability at the MALT1 locus and a specific cytosine methylation positioned at -256 from the gene, and the expression was recovered by demethylation treatment. In contrast to lymphocyte lineages, carcinoma cells showed MALT1 located at the nucleus independent of its domain structures, and its loss of expression induced the epithelial-mesenchymal transition. These results show that MALT1 is expressed in the nucleus of oral epithelial cells and that its expression is epigenetically inactivated during tumor progression, suggesting that the detection of MALT1 expression is a useful predictive and prognostic determinant in the clinical management of oral carcinomas.

The role of WNT in rheumatoid arthritis and its therapeutic implication.

Rheumatoid arthritis (RA) is a systematic inflammatory and intractable disease, which progressively affects multiple joints. Recent findings strongly suggest a key role of WNT signaling in the disease initiation and progression. In this review, we discuss the role and possibility of treatment by targeting WNT signaling.

Purification of matrix metalloproteinases by column chromatography.

Matrix metalloproteinases (MMPs) are zinc endopeptidases composed of 23 members in humans, which belong to a subfamily of the metzincin superfamily. They play important roles in many pathophysiological events including development, organogenesis, angiogenesis, tissue remodeling and destruction, and cancer cell proliferation and progression by degradation of extracellular matrix (ECM) and non-ECM proteins and interaction with various molecules. Here, we present standard protocols for purification of native proMMPs (proMMP-1, -2, -3, -7, -9 and -10) and recombinant MT1-MMP (MMP-14) using conventional column chromatography. Purification steps comprise the initial common step [diethylaminoethyl (DEAE)-cellulose, Green A Dyematrex gel and gelatin-Sepharose columns], the second step for removal of nontarget proMMPs by immunoaffinity columns (anti-MMP-1 and/or anti-MMP-3 IgG-Sepharose columns) and the final step for further purification (IgG-Sepharose, DEAE-cellulose, Zn2+-chelate-Sepharose and/or gel filtration columns). Purified proMMPs and MMP are functionally active and suitable for biochemical analyses. The basic protocol for the purification from culture media takes approximately 7-10 d.

Epigenetic inactivation of IkappaB Kinase-alpha in oral carcinomas and tumor progression.

PURPOSE: The loss of epithelial phenotypes in the process of carcinoma progression correlates with clinical outcome, and genetic/epigenetic changes accumulate aggressive clones toward uncurable disease. IkappaB kinase-alpha (IKKalpha) has a decisive role in the development of the skin and establishes keratinocyte phenotypes. We assessed clinical implications of IKKalpha expression in oral carcinomas and epigenetic aberrations for the loss of expression. EXPERIMENTAL DESIGN: We examined IKKalpha expression in oral carcinomas by immunostaining (n = 64) and genetic instability by microsatellite PCR (n = 46). Promoter methylation status was analyzed by bisulfite-modified sequence (n = 11). RESULTS: IKKalpha was expressed in the nucleus of basal cells of normal oral epithelium, but not or marginally detected in 32.8% of carcinomas. The immunoreactivity was significantly decreased in less differentiated carcinomas (P < 0.05) and correlated to long-term survival of patients (P < 0.01) with an independent prognostic value (P < 0.05). Although allelic/biallelic loss of the gene was limited to four cases, we detected microsatellite instability in 63.0% cases in which the immunoreactivities were decreased and the promoter was hypermethylated. CONCLUSION: These results showed that oral carcinomas exhibiting genetic instability and promoter hypermethylation down-regulate expression of IKK and suggest that the epigenetic loss of the expression closely associates with disease progression toward unfavorable prognosis.