Satoshi Toh

Genomic Structure of the Canalicular Multispecific Organic Anion–Transporter Gene (MRP2/cMOAT) and Mutations in the ATP-Binding–Cassette Region in Dubin-Johnson Syndrome

Summary Dubin-Johnson syndrome (DJS) is an autosomal recessive disease characterized by conjugated hyperbilirubinemia. Previous studies of the defects in the human canalicular multispecific organic anion transporter gene ( MRP2/cMOAT ) in patients with DJS have suggested that the gene defects are responsible for DJS. In this study, we determined the exon/intron structure of the human MRP2/cMOAT gene and further characterized mutations in patients with DJS. The human MRP2/cMOAT gene contains 32 exons, and it has a structure that is highly conserved with that of another ATP–binding–cassette gene, that for a multidrug resistance–associated protein. We then identified three mutations, including two novel ones. All mutations identified to date are in the cytoplasmic domain, which includes the two ATP-binding cassettes and the linker region, or adjacent putative transmembrane domain. Our results confirm that MRP2/cMOAT is the gene responsible for DJS. The finding that mutations are concentrated in the first ATP-binding–cassette domain strongly suggests that a disruption of this region is a critical route to loss of function.

Nuclear translocation of the Y‐box binding protein by ultraviolet irradiation

The Y‐box binding protein, YB‐1, is a member of a DNA binding protein family with a structurally and functionally conserved cold shock domain. Using Western blotting and immunohistochemical methods, larger amounts of YB‐1 were detected in the cytosol, particularly at the perinuclear region, than in the nucleus of human cancer cells. UV irradiation increased accumulation of YB‐1 in the nucleus at 20 min and thereafter. This translocation of YB‐1 into the nucleus by UV irradiation was blocked by the protein kinase inhibitor H‐7, but not HA‐1004. Both green fluorescent protein (GFP)‐YB‐1 and GFP‐YB‐1C with the C‐terminus (248–317) of YB‐1 were located mainly in the cytosol, but GFP‐YB‐1ΔC with a deletion at the C‐terminus of YB‐1 was located in the nucleus. YB‐1 is translocated into the nucleus by UV irradiation, possibly through a protein kinase C‐mediated signal transduction pathway, and the C‐terminal region of YB‐1 might be important for cytoplasmic retention of YB‐1.

Chemoprevention of head and neck cancer by green tea extract: EGCG-the role of EGFR signaling and "lipid raft"

Over the past decade dose-intensified chemo-radiotherapy or molecular targeted therapy has been introduced into the treatments of head and neck squamous cell carcinoma (HNSCC) to improve the outcomes of this dismal disease. However, these strategies have revealed only limited efficacy so far. Moreover, the frequent occurrences of second primary tumor further worsen the prognosis of patients. In this context, early detection and chemoprevention appear to be a realistic and effective method to improve the prognosis as well as quality of life in patients with HNSCC. In this short paper, we discuss the potential of green tea extract, (-)-epigallocatechin-3-galate (EGCG) in HNSCC chemoprevention, focusing on two aspects that are provided recently: (1) evidence of clinical efficacy and (2) unique biological effects on “lipid raft” that emerged as an important platform of numerous biophysical functions, for example, receptor tyrosin kinases (RTKs) signalings including epidermal growth factor receptor (EGFR), which play critical roles in HNSCC carcinogenesis.

The Roles of JNK1 and Stat3 in the Response of Head and Neck Cancer Cell Lines to Combined Treatment with All trans‐retinoic Acid and 5‐Fluorouracil

We have used a combination of vitamin A (all‐trans‐retinyl palmitate), 5‐fluorouracil (5‐FU) and radiation to treat human head and neck squamous cell carcinoma (HNSCC). This chemoradiother‐ apy is called “FAR therapy” In this study we examined the effects of all‐trans‐retinoic acid (ATRA), the active metabolite of vitamin A, and ATRA plus 5‐FU on two HNSCC cell lines (YCU‐ N861 and YCU‐H891) to gain insight into the molecular mechanisms of FAR therapy. ATRA at 1 μM (the order of concentration found in HNSCC tumors treated with FAR therapy) inhibited cell proliferation and caused Gl cell cycle arrest in both cell lines. This was associated with a decrease in cyclin D1, an increase in p27Kipl and a reduction in the hyperphosphorylated form of retinoblastoma protein (pRB). With YCU‐N861 cells, ATRA also caused a decrease in Bcl‐2 and Bcl‐XL and an increase in Bax. Both ATRA and 5‐FU activated c‐Jun N‐terminal kinase (JNK) 1 and the combination of both agents resulted in additive or synergistic activation of JNK1, and also enhanced the induction of apoptosis. The YCU‐H891 cells, in which the epidermal growth factor receptor (EGFR)‐signal transducer and activator of transcription 3 (Stat3) pathway is constitutively activated, were more resistant to treatments with ATRA, 5‐FU and the combination of both agents than YCU‐N861 cells. A dominant negative Stat3 construct strongly enhanced the cellular sensitivity of this cell line to 5‐FU but not to ATRA. In addition there is evidence that activation of Stat3 is associated with cellular resistance to radiation in HNSCC. Therefore, the addition to FAR therapy of agents that inhibit activation of the Stat3 pathway may enhance the clinical response of patients with HNSCC to FAR therapy.

“FAR” chemoradiotherapy improves laryngeal preservation rates in patients with T2N0 glottic carcinoma†

The appropriate treatment approach for patients with T2N0 laryngeal cancer remains highly controversial. Because radiotherapy alone is associated with a high risk of local recurrence, we have developed a triple combination treatment approach consisting of 5‐fluorouracil (250 mg/day, i.v.), vitamin A (50,000 unit/day, i.m.) and external radiation (2.0 Gy/day), which we have termed “FAR therapy.”

Genomic organization of the human Y-box protein (YB-1) gene

The human Y-box protein (YB-1) is a member of a family of DNA-binding proteins containing a highly conserved cold shock domain. The genomic organization of the human YB-1 gene was determined from five overlapping genomic clones that encompassed all exons of the gene. Sequence analysis of these clones revealed that human YB-1 spans approximately 19 kb of genomic DNA and contains eight exons. The cold shock domain is encoded by exons 1-5. Both exon-splitting and codon-splitting in the region of the gene encoding the cold shock domain are similar to those in the corresponding region of another Y-box binding protein, dbpA. Exon-intron structures and nucleotide sequences of the regions encoding the N-terminal and C-terminal domains of the two proteins differ markedly between YB-1 and dbpA. These observations suggest that YB-1 and dbpA arose by duplication of a common ancestral gene encoding all these domains.

β-catenin nuclear accumulation in head and neck mucoepidermoid carcinoma: Its role in cyclin D1 overexpression and tumor progression

Nuclear/cytoplasmic accumulation of β‐catenin is mainly regulated by its degradation, which is initiated by interaction with adenomatous polyposis coli (APC) protein. Accumulation of β‐catenin activates the transcription of 1 of the target oncogenic genes, cyclin D1, in the Wnt/Wingless pathway. The role of β‐catenin and cyclin D1 in this pathway has not been previously studied in head and neck mucoepidermoid carcinoma (MEC). This study investigates abnormalities of β‐catenin and the APC gene in MEC and correlates the patterns of cyclin D1 overexpression and nuclear/cytoplasmic accumulation of β‐catenin with the clinical outcome.

Role of squamous cell carcinoma antigen 1 expression in the invasive potential of head and neck squamous cell carcinoma

Serine proteases have important roles in tumor invasion and metastasis, and their inhibitors, serine protease inhibitors (serpins), are attractive targets for therapeutic strategies. On chromosome 18q21, there is a cluster of serpins: maspin, headpin, and squamous cell carcinoma antigen 1 (SCCA1)/SCCA2. Others and we have reported that the expression of these serpins is downregulated in head and neck squamous cell carcinoma (HNSCC) cells compared with normal squamous epithelial cells. In this study, we hypothesized that expression of SCCA1 is biologically disadvantageous to HNSCC cells.

Somatic evolution of head and neck cancer – Biological robustness and latent vulnerability

Despite recent advancements in multidisciplinary treatments, the overall survival and quality of life of patients with advanced head and neck squamous cell carcinoma (HNSCC) have not improved significantly over the past decade. Molecular targeted therapies, which have been addressed and advanced by the concept of “oncogene addiction”, have demonstrated only limited successes so far. To explore a novel clue for clinically effective targeted therapies, we analyzed the molecular circuitry of HNSCC through the lens that HNSCC is an evolving system. In the trajectory of this somatic evolution, HNSCC acquires biological robustness under a variety of selective pressures including genetic, epigenetic, micro‐environmental and metabolic stressors, which well explains the major mechanism of “escaping from oncogene addiction”. On the other hand, this systemic view appears to instruct us approaches to target latent vulnerability of HNSCC that is masked behind the plasticity and evolvability of this complex adaptive system.

Molecular subclassification determined by human papillomavirus and epidermal growth factor receptor status is associated with the prognosis of oropharyngeal squamous cell carcinoma

Human papillomavirus (HPV) infection is an indicator of good response to chemoradiotherapy in oropharyngeal squamous cell carcinoma (OPSCC), and epidermal growth factor receptor (EGFR) is a molecular-therapeutic target in head and neck squamous cell carcinoma. Here we investigated the prevalence and prognostic significance of HPV infection and EGFR alteration in OPSCC. We analyzed the presence of high-risk HPV using in situ hybridization, protein expressions of p16 and EGFR using immunohistochemistry, and the EGFR gene copy number gain using chromogenic in situ hybridization (CISH) in 105 cases of OPSCC. The biopsy specimens before chemoradiotherapy were used for these analyses. HPV infection and p16 protein overexpression were detected in 53.3% and 52.4% of the OPSCCs, and each factor was associated with better overall survival (P = .0026 and P = .0026) and nonkeratinizing histology (P = .0002 and P = .0004), respectively. EGFR gene copy number gain (high polysomy or amplification) was detected in 12.4% of the OPSCCs and was correlated with EGFR protein overexpression (P = .0667) and worse overall survival (P < .0001). HPV infection and EGFR gene copy number gain (EGFR CISH positive) were mutually exclusive. The HPV-negative/EGFR CISH-positive OPSCCs had significantly worse overall survival than did the HPV-positive/EGFR CISH-negative OPSCCs and HPV-negative/EGFR CISH-negative OPSCCs (P < .0001 and P < .0001, respectively). The EGFR CISH-negative OPSCCs had favorable prognosis irrespective of HPV infection. Our results suggest that EGFR gene copy number gain-positive tumors represent an HPV-negative, aggressive subgroup of OPSCCs. The molecular subclassification of OPSCCs based on HPV infection and EGFR status may serve as important information for appropriate therapeutic strategy.