Hiroyuki Hamakawa

Human FAT1 cadherin controls cell migration and invasion of oral squamous cell carcinoma through the localization of β-catenin

FAT1 [Homo sapiens FAT tumor suppressor homologxa01 (Drosophila)] is an intrinsic membrane protein classified as a member of the cadherin superfamily. The FAT1 gene is a tumor suppressor in humans as well as being the pivotal gene for cell morphogenesis and migration. Deletion of this gene could play a role in the characteristics of oral squamous cell carcinomas (OSCCs), involving cell adhesion, migration and/or invasion. This study investigated the mechanisms by which FAT1 is involved in the biological behavior of OSCCs. First, a rat monoclonal antibody was developed against a FAT1 intra-cellular domain epitope, and used for an immunohistochemical study of FAT1 in clinically obtained OSCC samples. FAT1 was localized at lamellipodial edges or cell-cell boundaries in normal cells and well differentiated OSCCs, but showed a diffuse cytoplasmic and nuclear distribution in moderately-poorly differentiated OSCCs. FAT1-siRNA was transfected into OSCCs resulting in a drastic inhibition of cell migration and invasion based on the suppression of FAT1 expression and disorganized localization of β-catenin which is associated with cell polarity and migration. These results suggested that FAT1 may be involved in the migration and invasion mechanisms of OSCCs and, therefore, it could be an important target for the development of new therapeutic strategies.

Knockdown of Akt isoforms by RNA silencing suppresses the growth of human prostate cancer cells in vitro and in vivo

The serine/threonine kinase Akt has three highly homologous isoforms in mammals: Akt1, Akt2, and Akt3. Recent studies indicate that Akt is often constitutively active in many types of human malignancy. Here we investigated the expression and function of Akt isoforms in human prostatic carcinoma cells. Initially, we used Western blotting to examine Akt expression in four human prostate cancer cell lines. Next, small-interfering RNAs (siRNAs) specific for Akt isoforms were used to elucidate their role on the in vitro and in vivo growth of prostate cancer cells. Expression of Akt1 and Akt2 was detected in all cells tested, but Akt3 was expressed only in cancer cells that did not express androgen receptors. All synthetic siRNAs against Akt isoforms suppressed their expression and inhibited the growth of cancer cells in vitro. Furthermore, atelocollagen-mediated systemic administration of siRNAs significantly reduced the growth of tumors that had been subcutaneously xenografted. These results suggest that targeting Akt isoforms could be an effective treatment for prostate cancers.

Silencing Id-1 inhibits lymphangiogenesis through down-regulation of VEGF-C in oral squamous cell carcinoma

Our previous study demonstrated that overexpression of Id-1 (inhibitor of differentiation/DNA binding) was associated with lymphatic metastasis in human oral squamous cell carcinoma (OSCC). In this study, we further unveiled the association of Id-1 with vascular endothelial growth factor-C (VEGF-C) and peritumoral lymphatic vessel density (PLVD), and the effect of silencing Id-1 on inhibiting lymphangiogenesis in OSCC. We found that Id-1 was associated with VEGF-C (r=0.569, p<0.001) and PLVD (r=0.240, p<0.001) in OSCC. Lentivirus-mediated RNA interference targeting Id-1 in an OSCC cell line Tca8113 resulted in down-regulation of VEGF-C (p=0.003, 0.007). Moreover, when Id-1 was suppressed by injecting Id-1-siRNA-lentivirus into the transplanted tumors in nude mice, VEGF-C was down-regulated (p=0.018) and the PLVD decreased (p=0.001). Our results suggest that Id-1 was correlated with lymphangiogenesis in OSCC. Silencing Id-1 could inhibit lymphangiogenesis through down-regulation of VEGF-C and it might be a promising treatment modality for the lymphatic metastasis of OSCC.

One-step nucleic acid amplification for detecting lymph node metastasis of head and neck squamous cell carcinoma.

OBJECTIVESnLymph node stage is an important prognostic factor in head and neck squamous cell carcinoma (HNSCC). We previously reported the clinical usefulness of sentinel lymph node biopsy diagnosed by genetic analysis using quantitative RT-PCR. However, this method takes about 3h. In this study, we attempted to develop a more efficient method for the intraoperative genetic detection of lymph node metastasis in HNSCC.nnnMATERIALS AND METHODSnA total of 312 lymph nodes (65 patients) were diagnosed by the one-step nucleic acid amplification (OSNA) method using GD-100. OSNA consists of a short homogenization step followed by amplification of cytokeratin 19 (CK19) mRNA directly from the lysate. Each lymph node was divided into two to diagnose metastasis. One half was used for the OSNA assay, and the other was subjected to semi-serial sectioning, sliced at 200-μm intervals and examined by H&E and cytokeratin AE1/AE3 immunohistochemical staining. The accuracy of OSNA assay was evaluated based on histopathological diagnosis.nnnRESULTSnSixty-one of 312 lymph nodes were pathologically metastasis-positive. The overall concordance rate between the OSNA assay using breast cancer criteria and histopathology was 94.2%. The optimal cut-off for the copy number of CK19 mRNA in assessing lymph node metastasis of HNSCC was 300 copies/μl, which had the highest diagnostic accuracy (95.2%). The OSNA assay can be completed within 30 min.nnnCONCLUSIONnThe OSNA assay, which shows high sensitivity and specificity, suggests the possibility to be used as a novel tool for the genetic detection of lymph node metastasis in HNSCC patients.

Targeting Id1 reduces proliferation and invasion in aggressive human salivary gland cancer cells

BackgroundSalivary gland cancer (SGC) is one of the common malignancies of the head and neck area. It develops in the minor and major salivary glands and sometimes metastasizes to other organs, particularly to the lungs. Inhibitors of differentiation (Id) proteins are negative regulators of basic helix-loop-helix transcription factors that control malignant cell behavior and tumor aggressiveness in many tissues. In this study, our goal was to determine the potential role of Id proteins, particularly Id1, during human SGC cell progression.MethodsWe first determined the expression levels of Id1 and Id2 in four SGC cell lines: two adenocarcinoma of the salivary gland (HSG and HSY) and two adenoid cystic carcinoma (ACC2 and ACCM) cell lines. We then used constructs that expressed antisense cDNAs to Id1 or Id2 to knockdown the expression of these proteins in cell lines where they were highly expressed, and determined the effects of the knockdown on cell proliferation, migration and invasion.ResultsId1 mRNA and protein were detectable in all cell lines, and expression of Id2 was variable, from absent to high. The ACC2 and ACCM cell lines expressed both Id1 and Id2, but Id1 was expressed at a higher level in the more aggressive ACCM cell line in comparison toACC2 cells as confirmed by Id1 promoter-reporter assays. We therefore focused on the ACCM cells for the remainder of the study. We found that proliferation and invasiveness of ACCM cells were strongly reduced after Id1 knockdown whereas Id2 suppression had only a slight effect. Results of scratch and colony formation assays also confirmed that ACCM cell aggressiveness was significantly reduced upon Id1 knockdown. Finally, this knockdown resulted in reduced c-myc and enhanced cyclin-dependent kinase inhibitor p21 expression.ConclusionsThese results demonstrate that Id1 plays an important role in the control of human SGC cell aggressiveness and suggest a potential role as a marker of diagnosis, prognosis and progression of SGCs. Id1 suppression could represent a novel and effective approach for the treatment of salivary gland cancer.

Targeting Aurora kinase A suppresses the growth of human oral squamous cell carcinoma cells in vitro and in vivo

OBJECTIVESnOncogene addiction has provided therapeutic opportunities in many human malignancies, but molecular targeted therapy for oral squamous cell carcinoma (OSCC) is not yet available. In this study, we attempted to identify an appropriate target molecule for treatment of patients with OSCC.nnnMATERIALS AND METHODSnMicroarray analysis was performed to determine the gene expression profiles in nine human OSCC cell lines and a non-neoplastic keratinocyte cell line. The expression levels of Aurora kinase A (AURKA) mRNA and protein in human OSCC cells and tissues were examined. We investigated the effect of small interfering RNAs specific for AURKA (siAURKAs) and MLN8237, an AURKA selective inhibitor on the growth of OSCC cells in vitro and in vivo. We also analyzed clinical significance in AURKA mRNA expression levels in OSCC.nnnRESULTSnAURKA was overexpressed in human OSCC cell lines and tissues. All siAURKAs almost completely suppressed the expression of AURKA protein, and significantly inhibited the growth of OSCC cells by 31-89%. MLN8237 also reduced the cellular growth rate by 38-74%. Both siAURKA and MLN8237 significantly reduced the size of subcutaneously xenografted OSCC tumors by 66% and 40%. Knockdown of AURKA expression and MLN8237 induced the growth inhibition of primary cultured cells established from patients OSCC tumors. Furthermore, we found a significant association between AURKA mRNA expression levels and histological differentiation and lymph node metastasis.nnnCONCLUSIONSnAURKA plays a critical role in the growth of human OSCC cells and targeting AURKA may be a useful therapeutic strategy for OSCC.

Growth inhibition and apoptosis by an active component of OK-432, a streptococcal agent, via Toll-like receptor 4 in human head and neck cancer cell lines

Toll-like receptor 4 (TLR4) plays a significant role in cancer therapy as receptors of bacteria-derived immunotherapeutic agents such as OK-432, a streptococcal immunotherapeutic agent. In addition, recent reports demonstrated that TLRs, including TLR4, are also expressed in cancer cells as well as in immunocompetent cells. It is a problem in cancer therapy that the immunoadjuvant may activate survival signals such as nuclear factor (NF)-κB or mitogen-activated protein kinases (MAPKs) in cancer cells via TLRs. In the current study, we investigated responsiveness of human head and neck cancer cell lines against TLR4 ligands, OK-PSA, an active component of OK-432, and a lipopolysaccharide (LPS). Stimulation with LPS or OK-PSA resulted in the activation of NF-κB in these cell lines expressing TLR4 and MD-2 that is a significant coreceptor for TLR4 signaling. Interestingly, OK-PSA induced cell-growth inhibition, while LPS enhanced the proliferation of the cancer cells. OK-PSA induced NF-κB activation more slowly than that induced by LPS. In addition, phosphorylation of p38 MAPK by OK-PSA was only slight compared with that by LPS. OK-PSA also induced apoptosis of the cancer cells mediated by the activation of caspase 1, 3 and 8 in a p53-independent manner. These findings strongly suggest that active components of OK-432 may elicit anti-cancer effects via enhancing host immunity as well as via directly inducing the growth inhibition and apoptosis of head and neck cancer cells through TLR4 signal.

Novel Therapeutic Strategies for Malignant Salivary Gland Tumors: Lessons Learned from Breast Cancer

Malignant salivary gland tumors (MSGTs) account for 2–6% of all head and neck cancers. Despite the rarity, MSGTs have been of great interest due to a wide variety of pathological features and high metastasis rates resulting in poor prognosis. Surgical resection followed by radiation therapy represents the main treatment of this malignancy. Adjuvant therapy is reserved for the management of local recurrence, no longer amenable to additional local therapy, and for metastasis. Based on the studies from other types of tumors, particularly breast cancer, the expression and function of sex steroid hormone receptors in cancer have been extensively studied and applied to diagnosis and treatment. Although a number of studies in MSGTs have been published, the rationale for hormone therapy is still controversial due to the disparate results and insufficient number of cases. However, some recent reports have demonstrated that certain salivary gland neoplasms are similar to breast cancer, not only in terms of the pathological features, but also at the molecular level. Here, we shed light on the biological similarity between MSGTs and certain types of breast cancer, and describe the potential use of hormone and additional therapies for MSGTs.

Carrier cell-mediated cell lysis of squamous cell carcinoma cells by squamous cell carcinoma antigen 1 promoter-driven oncolytic adenovirus

The squamous cell carcinoma antigen (SCCA) serves as a serological marker for squamous cell carcinomas. Molecular cloning of the SCCA genomic region has revealed the presence of two tandemly arrayed genes: SCCA1 and SCCA2. SCCA1 gene is up‐regulated in squamous cell carcinoma cells. We analyzed the proximal region of the SCCA1 promoter and the antitumor effect of oncolytic adenovirus driven by the SCCA1 promoter in squamous cell carcinoma cells.

Premetastatic vasculogenesis in oral squamous cell carcinoma xenograft-draining lymph nodes

OBJECTIVEnTo study vascular anatomy on oral cancer-draining lymph nodes before metastasis in mice.nnnMATERIAL AND METHODSnCell lines: highly lymph metastatic oral squamous cell carcinoma SASL1m and non-metastatic human adenoid cystic carcinoma ACC2. Bone marrow transplants and xenografts: Nude mice were lethally irradiated and transplanted with bone marrow cells from EGFP(+) mice. SASL1m or ACC2 cells were implanted in the tongue. Non-xenografted mice were used as controls. In addition, we injected conditioned medium from SASL1m or ACC2 in transplanted mice. Immunohistochemistry: Primary tumors and neck lymph nodes were resected and stained with anti-mouse Podoplanin and CD31. Images were visualized in a confocal microscope. Image analysis: Areas covered by EGFP, CD31 and Podoplanin were measured and compared statistically. Expression microarrays: Transcriptomic microarray analysis compared SASL1 to ACC2 cells. Interactomes were generated to reveal altered pathways.nnnRESULTSnSASL1m cells induced the assemblage of blood vessels in cancer-free, tumor-draining lymph nodes. These blood vessels incorporated bone marrow-derived EGFP(+)CD31(+) cells. Notably, SASL1m-conditioned medium induced a similar reaction. Non-metastatic cells failed to produce any change. Microarray and pathway analyses revealed the upregulation of Transforming Growth Factor-β, Lysyl Oxidase-like 2, Slit homolog 3 and Protease Serine 22. The upregulation of these genes was confirmed in xenografts.nnnCONCLUSIONSnThis study suggests that a blood supply for new tumors is established in lymph nodes before metastasis. It also suggests that premetastatic vasculogenesis and primary tumor angiogenesis may be mediated by different mechanisms.