Inhibition of HDAC and Signal Transduction Pathways Induces Tight Junctions and Promotes Differentiation in p63-Positive Salivary Duct Adenocarcinoma.

BACKGROUND: The p53 family p63 is essential for the proliferation and differentiation of various epithelial basal cells. It is overexpressed in several cancers, including salivary gland neoplasia. Histone deacetylases (HDACs) are thought to play a crucial role in carcinogenesis, and HDAC inhibitors downregulate p63 expression in cancers. METHODS: In the present study, to investigate the roles and regulation of p63 in salivary duct adenocarcinoma (SDC), human SDC cell line A253 was transfected with siRNA-p63 or treated with the HDAC inhibitors trichostatin A (TSA) and quisinostat (JNJ-26481585). RESULTS: In a DNA array, the knockdown of p63 markedly induced mRNAs of the tight junction (TJ) proteins cingulin (CGN) and zonula occuludin-3 (ZO-3). The knockdown of p63 resulted in the recruitment of the TJ proteins, the angulin-1/lipolysis-stimulated lipoprotein receptor (LSR), occludin (OCLN), CGN, and ZO-3 at the membranes, preventing cell proliferation, and leading to increased cell metabolism. Treatment with HDAC inhibitors downregulated the expression of p63, induced TJ structures, recruited the TJ proteins, increased the epithelial barrier function, and prevented cell proliferation and migration. CONCLUSIONS: p63 is not only a diagnostic marker of salivary gland neoplasia, but it also promotes the malignancy. Inhibition of HDAC and signal transduction pathways is, therefore, useful in therapy for p63-positive SDC cells.

HDAC inhibitors suppress the proliferation, migration and invasiveness of human head and neck squamous cell carcinoma cells via p63­mediated tight junction molecules and p21­mediated growth arrest.

In human head and neck squamous cell carcinoma (HNSCC), the invasion and metastatic properties of cancer cells are promoted by junctional adhesion molecule­A (JAM­A) and claudin­1; these are epithelial tight junction molecules regulated by histone deacetylases (HDACs) and transcription factor p63. HDAC expression is reportedly upregulated in HNSCC, and HDAC inhibitors suppress cancer cell proliferation by initiating proliferative arrest or apoptosis. However, little is known of the anti­cancer mechanisms of HDAC inhibitors in HNSCC. Thus, in the present study, the HNSCC Detroit 562 cell line and primary cultured HNSCC cells were treated with HDAC inhibitors to investigate their effects in HNSCC. Higher expression of p63, HDAC1, JAM­A and claudin­1 was observed in HNSCC tissues compared with the adjacent dysplastic regions. In Detroit 562 cells, treatment with trichostatin A (TSA), an inhibitor of HDAC1 and 6, downregulated the expression of p63, JAM­A and claudin­1, and upregulated that of acetylated tubulin; conversely, p63 knockdown resulted in the downregulation of JAM­A and claudin­1. Collectively, inhibiting HDAC suppressed the migration and invasiveness of cancer cells. In addition, treatment with TSA suppressed cancer cell proliferation via G2/M arrest, as well as upregulating p21 and downregulating cyclin D1 expression. TSA also downregulated the expression of epidermal growth factor receptor (EGFR) and phospho­ERK1/2. p63 knockdown and treatment with an EGFR inhibitor induced G1 arrest and downregulated EGFR and phospho­ERK1/2 levels, respectively. HDAC inhibition also suppressed the migration and invasiveness of primary cultured HNSCC cells. Collectively, the results of the present study indicate that HDAC inhibitors suppress the proliferation, migration and invasiveness of HNSCC by downregulating the p63­mediated tight junction molecules JAM­A and claudin­1, and inducing p63 or p21­mediated growth arrest.

Dysregulation of junctional adhesion molecule-A via p63/GATA-3 in head and neck squamous cell carcinoma.

Junctional adhesion molecule-A (JAM-A), which belongs to the IgG superfamily, is a tight junction molecule associated with epithelial and endothelial barrier function. Overexpression of JAM-A is also closely associated with invasion and metastasis of cancers such as breast cancer, lung cancer and pancreatic cancer. However, little is known about the mechanism in overexpression of JAM-A in head and neck squamous cell carcinoma (HNSCC). In the present study, we found high expression of JAM-A at the protein and mRNA levels in HNSCC tissues, including those of the oropharynx, larynx, and hypopharynx, together with high protein expression of ß-catenin, p63, ΔNp63 and GATA-3. Furthermore, in ELISA, a significant increase of soluble JAM-A in the sera of HNSCC patients was observed compared to healthy subjects. Knockdown of JAM-A by siRNA inhibited cell proliferation, invasion and migration in the HNSCC cell line Detroit562 in vitro. JAM-A expression in Detroit562 was increased via a distinct signal transduction pathway including NF-κB. Expression of JAM-A, ß-catenin, p63 and ΔNp63 in Detroit562 was decreased under hypoxia. Knockdown of p63, ΔNp63 or GATA-3 by siRNAs reduced JAM-A expression in Detroit562. In primary cultured HNSCC cells in which CK7, p63, ΔNp63 and GATA-3 were detected, JAM-A expression was decreased by knockdown of p63 or ΔNp63. These results indicate that JAM-A is a biomarker of malignancy in HNSCC and that plasma soluble JAM-A may contribute to serum-based diagnosis of HNSCC. The mechanism of dysregulation of JAM-A via p63/GATA-3 is important in possible molecular targeted therapy for HNSCC.

An in vitro system to characterize prostate cancer progression identified signaling required for self-renewal.

Mutations in RB and PTEN are linked to castration resistance and poor prognosis in prostate cancer. Identification of genes that are regulated by these tumor suppressors in a context that recapitulates cancer progression may be beneficial for discovering novel therapeutic targets. Although various genetically engineered mice thus far provided tumor models with various pathological stages, they are not ideal for detecting dynamic changes in gene transcription. Additionally, it is difficult to achieve an effect specific to tumor progression via gain of functions of these genes. In this study, we developed an in vitro model to help identify RB- and PTEN-loss signatures during the malignant progression of prostate cancers. Trp53-/- ; Rbf/f , Trp53-/- ; Ptenf/f , and Trp53-/- ; Rbf/f ; Ptenf/f prostate epithelial cells were infected with AD-LacZ or AD-Cre. We found that deletion of Rb, Pten or both stimulated prostasphere formation and tumor development in immune-compromised mice. The GO analysis of genes affected by the deletion of Rb or Pten in Trp53-/- prostate epithelial cells identified a number of genes encoding cytokines, chemokines and extracellular matrix remodeling factors, but only few genes related to cell cycle progression. Two genes (Il-6 and Lox) were further analyzed. Blockade of Il-6 signaling and depletion of Lox significantly attenuated prostasphere formation in 3D culture, and in the case of IL-6, strongly suppressed tumor growth in vivo. These findings suggest that our in vitro model may be instrumental in identifying novel therapeutic targets of prostate cancer progression, and further underscore IL-6 and LOX as promising therapeutic targets. © 2015 Wiley Periodicals, Inc.

Undifferentiated State Induced by Rb-p53 Double Inactivation in Mouse Thyroid Neuroendocrine Cells and Embryonic Fibroblasts.

Retinoblastoma tumor suppressor protein (RB) is inactivated more frequently during tumor progression than during tumor initiation. However, its exact role in controlling the malignant features associated with tumor progression is poorly understood. We established in vivo and in vitro models to investigate the undifferentiated state induced by Rb inactivation. Rb heterozygous mice develop well-differentiated thyroid medullary carcinoma. We found that additional deletion of Trp53, without change in lineage, converted these Rb-deficient tumors to a poorly differentiated type associated with higher self-renewal activity. Freshly prepared mouse embryonic fibroblasts (MEFs) of Rb(-/-) ; Trp53(-/-) background formed stem cell-like spheres that expressed significant levels of embryonic genes despite of lacking the ability to form colonies on soft agar or tumors in immune-deficient mice. This suggested that Rb-p53 double inactivation resulted in an undifferentiated status but without carcinogenic conversion. We next established Rb(-/-) ; N-ras(-/-) MEFs that harbored a spontaneous carcinogenic mutation in Trp53. These cells (RN6), in an Rb-dependent manner, efficiently generated spheres that expressed very high levels of embryonic genes, and appeared to be carcinogenic. We then screened an FDA-approved drug library to search for agents that suppressed the spherogenic activity of RN6 cells. Data revealed that RN6 cells were sensitive to specific agents including ones those are effective against cancer stem cells. Taken together, all these findings suggest that the genetic interaction between Rb and p53 is a critical determinant of the undifferentiated state in normal and tumor cells.

Hop water extract inhibits double-stranded RNA-induced thymic stromal lymphopoietin release from human nasal epithelial cells.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) acts as a master switch for allergic inflammation and plays a key role in allergic diseases, including allergic rhinitis. Double-stranded RNA (dsRNA) recognized by Toll-like receptor 3 (TLR3) strongly activates TSLP release from human nasal epithelial cells (HNECs). Hop (Humulus lupulus L.) extracts have been shown to have potent pharmacologic effects on inflammation. METHODS: To investigate whether a hop water extract (HWE) prevents TSLP release from HNECs, human telomerase reverse transcriptase (hTERT)-transfected HNECs, used as a model of normal HNECs, were pretreated with HWE before treatment with the TLR3 ligand Polyinosine-polycytidylic acid (poly[I:C]). RESULTS: In the hTERT-transfected HNECs, treatment with HWE significantly reduced poly(I:C)-induced production and release of TSLP in a dose-dependent manner, as well as dexamethasone. Treatment with the protein kinase C (PKC) inhibitor GF109203X and NF-κB inhibitor IMD-0354 also reduced poly(I:C)-induced TSLP release from hTERT-transfected HNECs. Treatment with HWE efficiently prevented up-regulation of PKC activity by 12-O-tetradecanoyl phorbol-13-acetate but not NF-κB activity induced by IL-1ß in hTERT-transfected HNECs. CONCLUSION: Our results clearly indicated that HWE inhibited dsRNA-induced production and release of TSLP via a PKC signal pathway in HNECs and it may have potent preventive effects against allergic rhinitis.

Analysis and results of Ku and XRCC4 expression in hypopharyngeal cancer tissues treated with chemoradiotherapy.

DNA double-strand break (DSB) is one of the most serious forms of damage induced by ionizing irradiation. Non-homologous end-joining (NHEJ) is a key mechanism of DNA DSB repair. The immunohistochemical analysis of proteins involved in NHEJ may have potential as a predictive assay for tumor radiosensitivity. We examined the correlation between the expression of proteins involved in DNA DSB in biopsy specimens and the results of chemoradiotherapy in hypopharyngeal cancers. Fifty-seven patients with previously untreated squamous cell carcinoma of the hypopharynx were treated between March 2002 and December 2009. Most patients (75%) had stage III or IV disease. The chemotherapy consisted of cisplatin plus 5FU or S-1. A tumor dose of 50 Gy was usually administered to the primary tumor and regional lymph nodes. Doses of 10-20 Gy were usually added to the primary tumor with reduced fields after 50 Gy. The 5-year disease-free survival rate was 100% for patients in stage I, 90% in stage II, 64% in stage III and 50% in stage IV. In stages I-III, patients with a lower expression of Ku70 or XRCC4 tended to have better locoregional control. These results indicated that a lower expression of Ku70 or XRCC4 may be correlated with higher radiosensitivity. Two patients had distant metastasis alone, of which one had 0% expression of Ku70 and the other had 0% expression of Ku86. The absence of Ku70 or Ku86 expression indicates low DNA-PK activity. Low DNA-PK activity due to a low expression of Ku may result in the genetic alteration of cancer cells, leading to a higher tendency of distant metastasis. This finding suggests that proteins involved in NHEJ may have an impact on the treatment results of chemoradiotherapy in hypopharyngeal cancer.

Altered expression of claudin-1, claudin-7, and tricellulin regardless of human papilloma virus infection in human tonsillar squamous cell carcinoma.

CONCLUSIONS: Altered expression of claudin-1, claudin-7, and tricellulin in early tonsillar squamous cell carcinoma (SCC) independent of human papilloma virus (HPV) infection may lead to tumor progression. OBJECTIVES: Integral tight junction proteins, the claudins and tricellulin, play a crucial role in all tissues. HPV is significantly associated with tonsillar SCC. We sought to determine the expression of claudin-1, claudin-7, and tricellulin in HPV-infected and HPV-free tonsillar SCC. METHODS: Twenty-eight tonsillar SCCs were studied by immunohistochemical analysis and real-time reverse transcription polymerase chain reaction with in situ hybridization analysis. RESULTS: Compared with normal tissues, claudin-1 was strongly expressed, whereas claudin-7 and tricellulin were weakly expressed or were absent in primary SCC and metastatic lymph nodes. Claudin-7 and tricellulin were markedly reduced at all stages of tumor development. In situ hybridization analysis showed no correlation between HPV infection and altered expression of the tight junction proteins.

Administration route-dependent induction of antitumor immunity by interferon-alpha gene transfer.

Type I interferon (IFN) protein is a cytokine with pleiotropic biological functions that include induction of apoptosis, inhibition of angiogenesis, and immunomodulation. We have demonstrated that intratumoral injection of an IFN-alpha-expressing adenovirus effectively induces cell death of cancer cells and elicits a systemic tumor-specific immunity in several animal models. On the other hand, reports demonstrated that an elevation of IFN in the serum following an intramuscular delivery of a vector is able to activate antitumor immunity. In this study, we compared the intratumoral and systemic routes of IFN gene transfer with regard to the effect and safety of the treatment. Intratumoral injection of an IFN-alpha adenovirus effectively activated tumor-responsive lymphocytes and caused tumor suppression not only in the gene-transduced tumors but also in distant tumors, which was more effective than the intravenous administration of the same vector. The expression of co-stimulatory molecules on CD11c(+) cells isolated from regional lymph nodes was enhanced by IFN gene transfer into the tumors. Systemic toxicity such as an elevation of hepatic enzymes was much lower in mice treated by intratumoral gene transfer than in those treated by systemic gene transfer. Our data suggest that the intratumoral route of the IFN vector is superior to intravenous administration, due to the effective induction of antitumor immunity and the lower toxicity.

Intratumoral interferon-alpha gene transfer enhances tumor immunity after allogeneic hematopoietic stem cell transplantation.

One of the major challenges in the treatment of solid cancers by allogenic hematopoietic stem cell transfer (alloHSCT) is the specific enhancement of antitumor immunity. Interferon (IFN) is a cytokine with pleiotropic biological functions including an immunomoduration, and our preclinical studies have shown that an intratumoral IFN-alpha gene transfer induced strong local tumor control and systemic tumor-specific immunity. In the present study, we examined whether the IFN-alpha gene transfer could enhance recognition of tumor-associated antigens by donor T cells and augment the antitumor activity of alloHSCT. First, when a mouse IFN-alpha adenovirus vector (Ad-mIFN) was injected into subcutaneous xenografts of syngeneic renal and colon cancer cells, tumor growth was significantly suppressed in a dose-dependent manner. A significant tumor cell death and infiltration of immune cells was recognized in the Ad-mIFN-injected tumors, and the dendritic cells isolated from the tumors showed a strong Th1-oriented response. The antitumor effect of Ad-mIFN was then examined in a murine model of minor histocompatibility antigen-mismatched alloHSCT. The intratumoral IFN-alpha gene transfer caused significant tumor suppression in the alloHSCT recipients, and this suppression was evident not only in the gene-transduced tumors but also in simultaneously inoculated distant tumors which did not receive the vector injection. A cytotoxicity assay showed specific tumor cell lysis by donor T cells responding to IFN-alpha. Graft-versus-host disease was not exacerbated serologically or clinically in the mice treated with IFN-alpha. This combination strategy deserves evaluation in future clinical trials for human solid cancers.

Extracellular matrix interacts with interferon alpha protein: retention and display of cytotoxicity.

We have been investigating the efficacy of an intratumoral interferon (IFN)-alpha gene transfer against solid cancers, and found that when the gene is transduced into the subcutaneous tumors, IFN-alpha concentration is markedly increased in the injected tumor but not in the serum. To explain this effective confinement of IFN-alpha to target tissues, we hypothesized that the extracellular matrix in the tumors interacts with IFN-alpha. In this study, a solid-phase-binding assay and immunoprecipitation demonstrated that the IFN-alpha binds directly to matrix proteins. Immunohistochemical staining showed a co-localization of IFN-alpha with pericellular fibronectin. In addition, matrix-bound IFN-alpha protein transduced intracellular signaling and potentiated its cytotoxic activity, suggesting that the retention of IFN-alpha protein on extracellular matrix is likely to play a role in its in vivo biological activity. The data suggest a therapeutic advantage of the intratumoral IFN-alpha gene transfer over the conventional parenteral therapy both in the safety and efficacy.

Endoscopic resection with hypertonic saline-solution-epinephrine injection plus band ligation for large pedunculated or semipedunculated gastric polyp.

BACKGROUND: Endoscopic resection of pedunculated polyps with heads 1 cm or greater in diameter presents a risk of bleeding. To minimize this complication, we performed endoscopic resection with hypertonic saline-solution-epinephrine injection plus band ligation and evaluated its safety and effectiveness. METHODS: Seventeen patients with 20 pedunculated or semipedunculated polyps with heads 1 cm or greater in diameter were treated with this technique. Conventional upper-GI endoscope, hypertonic saline-solution and epinephrine, sclerotherapy needle, and endoscopic band ligator device are needed for the procedure. OBSERVATIONS: All lesions were easily and safely resected. During this procedure, a band ligation chamber proved to be satisfactory for accurate recognition of a postpolypectomy ulcer under good visual control. No hemorrhage, perforation, or other complication occurred as a result of the use of this technique. The histologic resection margin was affected by nonneoplastic components in 6 of 20 lesions. Follow-up endoscopy 1 week later revealed a small, shallow ulcer without residual polyp in all lesions. CONCLUSIONS: This preliminary study suggests that endoscopic resection with hypertonic saline-solution-epinephrine injection plus band ligation is a simple and effective method for the prevention of polypectomy-associated bleeding. Prospective trials, including randomized controlled studies, are required to evaluate the suitability of this modality for wide clinical use.

Clathrate-formation mediated adsorption of methane on Cu-complex crystals.

We measured adsorption and desorption isotherms of methane on [Cu(4, 4'-bipyridine)2(BF4)2] (LPC) at 258, 273, and 303 K. Adsorption proceeds almost vertically at a definite pressure, which is named gate pressure. The lower the measurement temperature, the smaller the gate pressure. The temperature dependence of the gate pressure is expressed by the Clapeyron-Clausius equation, giving a thermodynamic evidence on the clathrate formation between the Cu complex and methane.