Levent Bekir Beder

Expression and mutation analysis of epidermal growth factor receptor in head and neck squamous cell carcinoma.

The epidermal growth factor receptor (EGFR)–RAS–RAF–mitogen‐activated protein kinase signaling cascade is an important pathway in cancer development and recent reports show that EGFR and its downstream signaling molecules are mutated in a number of cancers. We have analyzed 91 Japanese head and neck squamous cell carcinomas (HNSCC) and 12 HNSCC cell lines for mutations in EGFR, ErbB2, and K‐ras. Exons encoding the hot‐spot regions in the tyrosine kinase domain of both EGFR (exons 18, 19, and 21) and ErbB2 (exons 18–23), as well as exons 1 and 2 of K‐ras were amplified by polymerase chain reaction and sequenced directly. EGFR expression was also analyzed in 65 HNSCC patients using immunohistochemistry. Only one silent mutation, C836T, was found in exon 21 of EGFR in the UT‐SCC‐16A cell line and its corresponding metastasic cell line UT‐SCC‐16B. No other mutation was found in EGFR, ErbB2, or K‐ras. All tumors showed EGFR expression. In 21 (32%) tumors, EGFR was expressed weakly (+1). In 27 (42%) tumors it was expressed (+2) moderately, and in 17 (26%) tumors high expression (+3) was detected. Overexpression (+2, +3) was found in 44 tumors (68%). A worse tumor differentiation and a positive nodal stage were significantly associated with EGFR overexpression (P = 0.02, P = 0.032, respectively). Similar to patients from western ethnicity, mutations are absent or rare in Japanese HNSCC. Protein overexpression rather than mutation might be responsible for activation of the EGFR pathway in HNSCC. (Cancer Sci 2008; 99: 1589–1594)

Downregulation of ING3 mRNA expression predicts poor prognosis in head and neck cancer

Although many clinical and pathological prognostic factors such as tumor stage and lymph‐node involvement have been described, to date no reliable or clinically applicable marker or tumor aggressiveness has been identified for head and neck cancer. In an attempt to identify such a molecular prognostic marker, we analyzed the mRNA expression status of ING3 by quantitative reverse transcription–polymerase chain reaction. We also examined p53 mutation status and investigated its relationship with ING3, as well its clinicopathological characteristics. About half of the 71 tumor samples demonstrated downregulation of ING3 compared to their matched normal counterparts. Although most clinicopathological variables were not significantly related to ING3 downregulation or p53 mutation status, a significant relationship was detected in terms of overall survival between the cases with low and normal to high ING3 expression. At 5 years follow up, approximately 60% of the patients with normal to high ING3 expression survived, whereas this was 35% in the patients with low ING3 expression. Multivariate analysis also showed downregulation of ING3 as an independent prognostic factor for poor overall survival. These results reveal that ING3 would function as a potential tumor suppressor molecule and that low levels of ING3 may indicate an aggressive nature of head and neck cancer. (Cancer Sci 2008; 99: 531–538)

Role of miR-200c/miR-141 in the regulation of epithelial-mesenchymal transition and migration in head and neck squamous cell carcinoma

Epithelial-mesenchymal-transition (EMT) is a critical step in tumor invasion and metastasis, while its fate is mainly defined by the balanced expression between the miR-200 family and ZEB transcription factors. In this study, we observed a reciprocal correlation between miR-200c/mir-141 and ZEB1, as well as between ZEB2 and E-cadherin expression in a panel of 13 head and neck squamous cell carcinoma (HNSCC) cell lines. We also confirmed that the enforced expression of miR-200c and miR-141 significantly reduced the migration capacity of HNSCC cells. Accordingly, the enforced expression of miR-200c and mir-141 resulted in a significant upregulation in E-cadherin expression, contrary to the significant downregulation in ZEB1 expression in 3 cell lines (UTSCC-24A, UTSCC-24B and UTSCC-6A cells). Another pair of cell lines, UTSCC-60A and UTSCC‑ 60B failed to show a significant change in the expression of E-cadherin or ZEB1/ZEB2 during the enforced expression of miR-200c/miR-141. To address the issue, we focused on the hypermethylation status of the ZEB1/2 promoters, which have both been shown to include wide CpG islands. We observed a marked upregulation in both ZEB1 and ZEB2 mRNA expression following treatment with a demethylating agent in both pairs of UTSCC cell lines. In conclusion, our findings confirm the existence of a reciprocal correlation between the mir-200 family and the ZEB family, and demonstrate the role of the miR-200 family in EMT, as well as in the migration and invasion ability of HNSCC cells. Furthermore, our data suggest that the promoter hypermethylation of ZEB1 and ZEB2 may play an essential role and may overshadow the effects of the miR-200 family in the regulation of EMT during carcinogenesis.

Tumor‐specific mutation and downregulation of ING5 detected in oral squamous cell carcinoma

Our previous study showed high frequency of allelic loss at chromosome 2q37 region in oral cancer. This location contains several candidate tumor suppressor genes such as PPP1R7, ILKAP, DTYMK and ING5. We previously showed 3 members of inhibitor of growth (ING) family, ING1, ING3 and ING4 as tumor suppressor gene in head and neck cancer. As ING5 shows high homology with other members of ING genes including highly conserved carboxy‐terminal plant homeodomain and nuclear localization signal, we first picked up ING5 and examined it as a possible tumor suppressor in oral cancer. For this aim, mutation and mRNA expression status of ING5 in paired normal and oral squamous cell carcinoma samples were examined by reverse transcription polymerase chain reaction (RT‐PCR) and sequencing. Three missense mutations located within leucine zipper like (LZL) finger and novel conserved region (NCR) domains in ING5 protein were detected, probably abrogating its normal function. We also found 5 different alternative splicing variants of ING5. Then, we examined mRNA level of ING5 by quantitative real time reverse transcription polymerase chain reaction (qRT‐PCR) analysis, which demonstrated decreased expression of ING5 mRNA in 61% of the primary tumors as compared to the matched normal samples. In conclusion, tumor‐specific mutation and downregulation of ING5 mRNA suggested it as a tumor suppressor gene in oral squamous cell carcinoma.

Nontypeable Haemophilus influenzae isolated from intractable acute otitis media internalized into cultured human epithelial cells.

OBJECTIVES The aim of this study is to examine the internalization of nontypeable Haemophilus influenzae (NTHi) into human epithelial cells. METHODS Bactericidal assay was applied to examine the effects of antibiotics against cell-adherent NTHi using HEp-2 cells. A trans-well chamber assay was applied to examine the internalization and penetration of NTHi using Detroit562 cells. RESULTS The adherence of NTHi to HEp-2 cells was noted after 2h of incubation. Azithromycin had a strong bactericidal effect against both cell-associated and non-adherent NTHi, while ceftriaxone did not show bactericidal effects on NTHi adhered to the HEp-2 cells. Three (60.0%) out of five NTHi isolates from the nasopharynx of children with intractable acute otitis media (AOM) internalized into and subsequently penetrated through the epithelial cells at various degrees. Azithromycin had a strong bactericidal effect against the cell-internalized NTHi, while ceftriaxone was bactericidal only against extracellular NTHi. CONCLUSION The potential of NTHi as the intracellular pathogen may contribute to the persistent existence of this pathogen that result in the prolonged and intractable clinical course of AOM. Azithromycin may be a therapeutically significant antibiotic for patients with prolonged respiratory tract infections due to NTHi.

Increased mRNA expression of ADAMTS metalloproteinases in metastatic foci of head and neck cancer.

Although contribution of matrix metalloproteinases in cancer progression and dissemination is now well known, roles of recently discovered metalloproteinases, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), in cancer development and progression remain mostly unknown.

Radical mastoidectomy cavity myiasis caused by Wohlfahrtia magnifica

In this article, a Wohlfahrtia magnifica otomyiasis case, a 31-year-old, non-mentally retarded patient who had undergone radical mastoidectomy previously is presented. Maggots in the radical mastoidectomy cavity were removed then topical treatment was applied. The maggots were identified as W. magnifica. In cases of myiasis, identification of larvae following direct extraction and application of preventative methods is essential.

Loss of Heterozygosity at the 9p21-24 Region and Identification of BRM as a Candidate Tumor Suppressor Gene in Head and Neck Squamous Cell Carcinoma

We have analyzed allelic loss of the short arm of chromosome 9 in 39 head and neck cancers using 13 polymorphic markers and found two deletions of hot spots at 9p21 and 9p24. Loss of heterozygosity was detected at least at one locus in 28 of 39 cases (71.8%). P16, a well-known tumor suppressor gene, is considered to be a target for deletion of the 9p21 region. However, novel frequent chromosomal deletion and a candidate tumor suppressor gene, BRM at the 9p24 region, were detected. Moreover, comparison of clinicopathological variables demonstrated that loss of heterozygosity at the BRM locus was associated with a worse prognosis.

Deletion at Dickkopf (dkk)-3 locus (11p15.2) is related with lower lymph node metastasis and better prognosis in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) is a frequently occurring cancer, and despite improvement of its treatment methods, including chemotherapy, radiotherapy, and surgery, the improvement of survival remains poor. Recent advances in molecular biology of human cancer indicated various molecular abnormalities in HNSCC, including activation of oncogenes and inactivation of tumor suppressor genes (TSGs). Dickkopf (Dkk)-3 gene is known as a negative regulator of Wnt signaling and is suggested to function as TSG in several kinds of malignancies. We hypothesized that Dkk-3 might play an important role in HNSCC, too. Thus, in the current study, we analyzed allelic alteration of Dkk-3 locus (chromosome 11p15.2) by means of loss of heterozygosity (LOH) analysis. The study population consisted of 50 patients with HNSCC (mean age of 65 years old). Furthermore, we also examined the correlation between LOH findings of Dkk-3 locus with clinicopathological parameters to investigate its use as a biomarker in HNSCC. A remarkable LOH ratio (57%) was detected in the cases studied, implying that Dkk-3 is likely to be involved in HNSCC carcinogenesis. However, interestingly and in contrast to the expectations, we found that the group with LOH of Dkk-3 locus had less lymph node metastasis, and showed a favorable overall survival compared to the patients with retention of Dkk-3 area in survival analysis. These results indicate that Dkk-3 can play a role in HNSCC carcinogenesis with unknown mechanism. Moreover, allelic loss at Dkk-3 locus may also be used as a novel prognostic biomarker in HNSCC.

Allelic loss of the ING gene family loci is a frequent event in ameloblastoma

Ameloblastoma is the most frequently encountered odontogenic tumor, characterized by a locally invasive behavior, frequent recurrences, and, although rare, metastatic capacity. Loss or inactivation of tumor suppressor genes (TSGs) allows cells to acquire neoplastic growth. The ING family proteins are tumor suppressors that physically and functionally interact with p53 to perform important roles in apoptosis, DNA repair, cell cycle regulation, and senescence. TP53 genetic alterations were reported to infrequently occur in ameloblastoma. Considering that other TSGs related to TP53 could be altered in this tumor, we focused our study on the ING family genes. We analyzed the loss of heterozygosity (LOH) status of the ING family (ING1-ING5) chromosomal loci in a group of ameloblastomas by microsatellite analysis, and correlated the ING LOH status with clinicopathological characteristics. By using specific microsatellite markers, high frequency of LOH was found at the loci of each ING gene family member (33.3-72.2%). A significant relationship was shown between LOH of D2S 140 (ING5 locus) and solid tumor type (p = 0.02). LOH of ING3MS (ING3 locus) was also high in solid type tumors, showing a near significant association. In addition, a notable tendency toward higher LOH for half of the markers was observed in recurrent cases. LOH of ING family genes appears as a common genetic alteration in solid ameloblastoma. The current study provides interesting novel information regarding the potential prognostic significance of the allelic loss of the ING gene family loci in ameloblastoma tumorigenesis.