Tadashi Yoshii

Nuclear Export of Phosphorylated Galectin-3 Regulates Its Antiapoptotic Activity in Response to Chemotherapeutic Drugs

ABSTRACT Galectin-3 (Gal-3), a member of the β-galactoside binding protein family containing the NWGR antideath motif of the Bcl-2 protein family, is involved in various aspects of cancer progression. Previously, it has been shown that the antiapoptotic activity of Gal-3 is regulated by the phosphorylation at Ser6 by casein kinase 1 (CK1). Here we questioned how phosphorylation at Ser6 regulates Gal-3 function. We have generated serine-to-alanine (S6A) and serine-to-glutamic acid (S6E) Gal-3 mutants and transfected them into the BT-549 human breast carcinoma cell line, which does not express Gal-3. BT-549 cell clones expressing wild-type (wt) and mutant Gal-3 were exposed to chemotherapeutic anticancer drugs. In response to the apoptotic insults, phosphorylated wt Gal-3 was exported from the nucleus to the cytoplasm and protected the BT-549 cells from drug-induced apoptosis while nonphosphorylated mutant Gal-3 neither was exported from the nucleus nor protected BT-549 cells from drug-induced apoptosis. Furthermore, leptomycin B, a nuclear export inhibitor, increased the cisplatin-induced apoptosis of Gal-3 expressing BT-549 cells. These results suggest that Ser6 phosphoryaltion acts as a molecular switch for its cellular translocation from the nucleus to the cytoplasm and, as a result, regulates the antiapoptotic activity of Gal-3.

Endogenous Galectin-3 Determines the Routing of CD95 Apoptotic Signaling Pathways

Studies of CD95 (APO-1/Fas), a member of the death receptor family, have revealed that it is involved in two primary CD95 apoptotic signaling pathways, one regulated by the large amount of active caspase-8 (type I) formed at the death-inducing signaling complex and the other by the apoptogenic activity of mitochondria (type II). To date, it is still unclear which pathway will be activated in response to an apoptotic insult. Here, we demonstrate that the antiapoptotic molecule galectin-3, which contains the four amino acid-anti-death-motif (NWGR) conserved in the BH1 domain of the Bcl-2 member proteins, is expressed only in type I cells. Transfection of galectin-3 cDNA into galectin-3 null cells (type II) resulted converting them to type I apoptotic phenotype. In addition, we show that galectin-3 is complexed with CD95 in vivo identifying galectin-3 as a novel CD95-binding partner that determines which of the CD95 apoptotic signaling pathways the cell will select.

Malignant transformation of thyroid follicular cells by galectin-3

Galectin-3, a beta-galactoside binding lectin, is highly expressed in thyroid carcinomas of follicular cell origin, whereas neither benign thyroid adenomas nor normal thyroid tissues express galectin-3. We previously showed that antisense inhibition of galectin-3 expression markedly reduced the malignant phenotype of thyroid papillary carcinoma cells. In the present study we transfected galectin-3 cDNA into TAD-2 normal thyroid follicular cells. Stable transfectants expressing galectin-3 acquired the phenotype of serum-independent growth, clonogenicity in soft agar, as well as loss of contact inhibition. We also compared the gene expression profile of the galectin-3 transfectants to that of the vehicle control, which revealed that a series of genes were differentially expressed between the two. They include proliferating cell nuclear antigen, replication factor C, and retinoblastoma genes that participate in G1-S transition. These results indicate the transformation of thyroid follicular cells by galectin-3 and possible involvement of galectin-3 in cell cycle.

Human papillomavirus and cystic node metastasis in oropharyngeal cancer and cancer of unknown primary origin.

The clinical significance of human papillomavirus (HPV) in neck node metastasis from cancer of unknown primary (CUP) is not well established. We aimed to address the relationship of HPV status between node metastasis and the primary tumor, and also the relevance of HPV status regarding radiographically detected cystic node metastasis in head and neck squamous cell carcinoma (HNSCC) and CUP. HPV DNA was examined in 68 matched pairs of node metastasis and primary tumor, and in node metastasis from 27 CUPs. In surgically treated CUPs, p16 was examined immunohistochemically. When tonsillectomy proved occult tonsillar cancer in CUP, HPV DNA and p16 were also examined in the occult primary. Cystic node metastasis on contrast-enhanced computed tomography scans was correlated with the primary site and HPV status in another series of 255 HNSCCs and CUPs with known HPV status. Node metastasis was HPV-positive in 19/37 (51%) oropharyngeal SCCs (OPSCCs) and 10/27 (37%) CUPs, but not in non-OPSCCs. Fluid was collected from cystic node metastasis using fine needle aspiration in two OPSCCs and one CUP, and all fluid collections were HPV-positive. HPV status, including the presence of HPV DNA, genotype, and physical status, as well as the expression pattern of p16 were consistent between node metastasis and primary or occult primary tumor. Occult tonsillar cancer was found more frequently in p16-positive CUP than in p16-negative CUP (odds ratio (OR), 39.0; 95% confidence interval (CI), 1.4–377.8; P = 0.02). Radiographically, cystic node metastasis was specific to OPSCC and CUP, and was associated with HPV positivity relative to necrotic or solid node metastasis (OR, 6.2; 95% CI, 1.2–45.7; P = 0.03). In conclusion, HPV status remains unchanged after metastasis. The occult primary of HPV-positive CUP is most probably localized in the oropharynx. HPV status determined from fine needle aspirates facilitates the diagnosis of cystic node metastasis.

Human papillomavirus and p53 mutations in head and neck squamous cell carcinoma among Japanese population

We aimed to reveal the prevalence and pattern of human papillomavirus (HPV) infection and p53 mutations among Japanese head and neck squamous cell carcinoma (HNSCC) patients in relation to clinicopathological parameters. Human papillomavirus DNA and p53 mutations were examined in 493 HNSCCs and its subset of 283 HNSCCs. Oropharyngeal carcinoma was more frequently HPV‐positive than non‐oropharyngeal carcinoma (34.4% vs 3.6%, P < 0.001), and HPV16 accounted for 91.1% of HPV‐positive tumors. In oropharyngeal carcinoma, which showed an increasing trend of HPV prevalence over time (P < 0.001), HPV infection was inversely correlated with tobacco smoking, alcohol drinking, p53 mutations, and a disruptive mutation (P = 0.003, <0.001, <0.001, and <0.001, respectively). The prevalence of p53 mutations differed significantly between virus‐unrelated HNSCC and virus‐related HNSCC consisting of nasopharyngeal and HPV‐positive oropharyngeal carcinomas (48.3% vs 7.1%, P < 0.001). Although p53 mutations were associated with tobacco smoking and alcohol drinking, this association disappeared in virus‐unrelated HNSCC. A disruptive mutation was never found in virus‐related HNSCC, whereas it was independently associated with primary site, such as the oropharynx and hypopharynx (P = 0.01 and 0.03, respectively), in virus‐unrelated HNSCC. Moreover, in virus‐unrelated HNSCC, G:C to T:A transversions were more frequent in ever‐smokers than in never‐smokers (P = 0.04), whereas G:C to A:T transitions at CpG sites were less frequent in ever‐smokers than in never‐smokers (P = 0.04). In conclusion, HNSCC is etiologically classified into virus‐related and virus‐unrelated subgroups. In virus‐related HNSCC, p53 mutations are uncommon with the absence of a disruptive mutation, whereas in virus‐unrelated HNSCC, p53 mutations are common, and disruptive mutagenesis of p53 is related with oropharyngeal and hypopharyngeal carcinoma.

Cytoplasmic and serum galectin-3 in diagnosis of thyroid malignancies

In order to address whether galectin-3 in the sera and fine needle aspirates serve as a diagnostic marker distinguishing between benign and malignant thyroid nodules, we developed an enzyme-linked immunosorbent assay. We quantified galectin-3 in fine needle aspirates from a series of 118 patients with thyroid nodules and serum galectin-3 from another series of 46 patients, which were compared with final histology after thyroidectomy. Relative galectin-3 value (ng/mg), defined as galectin-3 concentration (ng/ml) divided by total protein concentration (mg/ml) in fine needle aspirates, was significantly higher in papillary carcinoma than in the other thyroid entities. There was no significant difference in serum galectin-3 level among patients with thyroid nodules and healthy individuals. Accordingly, relative galectin-3 value allows a definitive diagnosis of papillary carcinoma independent of cellular morphology, whereas serum galectin-3 does not serve as a marker for papillary carcinoma.

The role of CT and 18F-FDG PET in managing the neck in node-positive head and neck cancer after chemoradiotherapy

Conclusion. Patients showing a complete response on computed tomography (CT) can be spared from neck dissection. Objective. To determine whether CT or fluorine-18-fluorodeoxyglucose positron emission tomography (18F-FDG PET) is superior in the evaluation of persistent nodal disease after chemoradiotherapy in patients with node-positive head and neck squamous cell carcinoma (HNSCC). Patients and methods. Study entry criteria included node-positive HNSCC treated with definitive chemoradiotherapy, a local complete response, and post-treatment CT and 18F-FDG PET studies 7 weeks after chemoradiotherapy. Forty-eight patients with 60 node-positive necks were eligible. Nodes larger than 1 cm, or with central necrosis on CT, or any visually hypermetabolic nodes on 18F-FDG PET were considered positive. Regardless of PET findings, necks with positive CT were subjected to neck dissection, whereas those with negative CT were observed without neck dissection. Results. Twenty-two necks showed positive CT, 20 and 2 of which underwent neck dissection and fine needle aspiration cytology, respectively, resulting in pathologic evidence of persistent nodal disease in 13 necks. Five of 38 necks with negative CT developed regional recurrence. Diagnostic accuracy was equivalent between CT and 18F-FDG PET. There was no difference in 3-year cause-specific survival between patients with positive and negative CT (79% and 81%, respectively).

Fatal sepsis caused by an unusual Klebsiella species that was misidentified by an automated identification system.

This is a description of fatal sepsis caused by infection with Klebsiella variicola, which is an isolate genetically related to Klebsiella pneumoniae. The patients condition was incorrectly diagnosed as common sepsis caused by K. pneumoniae, which was identified using an automated identification system, but next-generation sequencing and the non-fermentation of adonitol finally identified the cause of sepsis as K. variicola.

Immediate effectiveness of humming on the supraglottic compression in subjects with muscle tension dysphonia.

Aims: To verify whether humming corrects supraglottic compression in muscle tension dysphonia (MTD) patients. Methods: We enrolled 23 MTD participants (13 male, 10 female) showing supraglottic compression. Each individual was instructed to perform 3 types of phonation under transnasal laryngofiberscopy: natural phonation, humming phonation without pitch change and subsequent um-hum phonation, i.e. humming with pitch glide up as if agreeing with someone. The degree of supraglottic compression was estimated with 2 parameters. The false vocal fold and anterior-posterior indices (the FVF and AP indices) were calculated by normalizing the lateral width and AP length of the visible vocal cords at phonation normalized to the mean vocal cord length at inspiration, respectively. These indices were compared among the tasks. Results: All the MTD participants but 5 females accomplished decreases in the vocal roughness scores upon the phonatory tasks. The whole MTD group showed significant increases in the FVF and AP indices even after humming without pitch change with a dominance of the AP index. The humming-responsive MTD subgroup showed greater increases in both indices than the humming-resistant subgroup. Conclusion: These data demonstrate that humming corrects both the lateral and AP components of supraglottic compression in most MTD patients.

Synchronous bilateral tonsillar carcinomas associated with human papillomavirus.

Although the incidence of human papillomavirus (HPV)-positive oropharyngeal carcinoma is increasing, only a limited number of synchronous bilateral HPV-positive tonsillar carcinomas have been reported to date. Here, we describe an additional case of 61-year-old female. Pathological analysis proved squamous cell carcinoma in biopsy specimens from bilateral tonsillar lesions and a fine needle aspirate from an enlarged cervical node. Polymerase chain reaction (PCR) and direct sequencing showed HPV-16 DNA in all of the biopsy specimens and fine needle aspirate with completely concordant sequences. Bilateral tonsillar lesions were immunohistochemically positive for p16. Taken together with radiological findings, she was diagnosed to have bilateral tonsillar carcinomas (cT1N2bM0 on the right side and cT2N0M0 on the left side). We administered concurrent chemoradiotherapy to treat these synchronous lesions, and the restaging workup resulted in overall complete response. No recurrent and/or metastatic disease has been evident 20 months after the restaging. It seems reasonable to include bilateral tonsils as a therapeutic target in the treatment of HPV-positive unknown primary carcinoma.