Background:Human papillomavirus (HPV) testing in oropharyngeal squamous cell carcinoma (OPSCC) is now advocated. Demonstration of transcriptionally active high-risk HPV (HR-HPV) in fresh tumour tissue is considered to be the analytical ‘gold standard'. Clinical testing has focused on formalin-fixed paraffin-embedded (FFPE) tissue at the expense of sensitivity and specificity. Recently, a novel RNA in situ hybridisation test (RNAscope) has been developed for the detection of HR-HPV in FFPE tissue; however, validation against the ‘gold standard' has not been reported.Methods:A tissue microarray comprising FFPE cores from 79 OPSCC was tested using HR-HPV RNAscope. Analytical accuracy and prognostic capacity were established by comparison with the reference test; qRT–PCR for HR-HPV on matched fresh-frozen samples.Results:High-risk HPV RNAscope had a sensitivity and specificity of 97 and 93%, respectively, against the reference test. Kaplan–Meier estimates of disease-specific survival (DSS, P=0.001) and overall survival (OS, P<0.001) by RNAscope were similar to the reference test (DSS, P=0.003, OS, P<0.001) and at least, not inferior to p16 immunohistochemistry +/− HR-HPV DNA-based tests.Conclusion:HR-HPV RNAscope demonstrates excellent analytical and prognostic performance against the ‘gold standard'. These data suggest that the test could be developed to provide the ‘clinical standard' for assigning a diagnosis of HPV-related OPSCC.

Bladder cancer displays an aggressive phenotype in the muscle-invasive phase, and is associated with a high mortality rate. Therefore, novel molecular therapeutic targets are needed to improve patient survival. A monoclonal antibody against the extracellular domain of the claudin-4 (CLDN4) tight junction protein was established by immunizing rats with a plasmid vector encoding human CLDN4. A hybridoma clone, producing a rat monoclonal antibody recognizing CLDN4 (clone 4D3), was obtained. Immunohistochemistry by using the 4D3 antibody showed that CLDN4 expression was associated with local invasion, nodal metastasis, distant metastasis, and advanced stage in 86 cases of bladder cancer. The 4D3 antibody inhibited growth, invasion, and survival, associated with abrogation of the intratumoral microenvironment; lowered concentrations of epidermal growth factor and vascular endothelial growth factor were found in three-dimensional cultures of T24 and RT4 cells. In combination with cisplatin therapy, 4D3 enhanced cisplatin cytotoxicity by increasing cellular permeability, leading to increased intracellular cisplatin concentrations. In mouse models of subcutaneous tumors and lung metastasis, 4D3 enhanced tumor growth inhibition, alone and with concurrent cisplatin treatment. The anti-tumor activity of the newly established 4D3 antibody suggests that it may be a powerful tool in CLDN4-targeting therapy, and in combination with chemotherapy.

Melanoma inhibitory activity (MIA) is an 11-kDa secretory protein isolated from malignant melanoma cells that is correlated with invasion and metastasis in various human malignancies. We examined MIA expression in 62 oral squamous cell carcinomas (OSCC) by immunohistochemistry. MIA expression was significantly associated with nodal metastasis (P = 0.00018). MIA expression was also associated with expression of high mobility group box-1 (HMGB1) (P < 0.0001) and lymph vessel density (P < 0.0001). Expression levels of MIA, HMGB1, nuclear factor kB (NFkB) p65 and HMGB1-NFkB p65 binding were significantly higher in a metastatic human OSCC cell line (HSC3) than those in a non-metastatic OSCC cell line (HSC4). H ead and neck cancer is the sixth most common malignancy worldwide and the first leading cause of cancer death in South Asia.(1) About 300 000 patients develop OSCC every year in the world.(2,3) OSCC has a high potential for nodal metastasis and locoregional invasion, (4) from which over 50% of patients die.(5,6) To control lymph-node metastasis of OSCC, we need to study the molecular aspects of the mechanism of metastasis.MIA is an 11-kDa secretory protein isolated from supernatants of HTZ-19 malignant melanoma cells, (7,8) the gene locus of which is mapped to chromosome 19q13.32-13.33.(9) Although previous reports indicated that MIA is correlated with invasion and metastasis in malignant melanoma, (10)(11)(12) breast cancer,chondrosarcoma, (13) glioma, (14) and pancreatic cancer,the definite functions of MIA for cancer cells are still unclear.HMGB1 has a dual role as an extracellular secretory protein and a chromosomal structural protein.(16) HMGB1 works as a cytokine or a growth factor in neural ontogenesis, septic inflammation and neoplasm. HMGB1 is also considered an amphoterin, which is isolated as a motility factor in neurite outgrowth.(17) We previously reported coexpression of HMGB1 and receptor for advanced glycation end products (RAGE), which is a major membrane receptor for HMGB1 and is significantly associated with tumor progression and metastasis (18)(19)(20)(21)(22)(23)(24) and suppression of tumor-associated macrophages. (25,26) As a chromatin structural protein, HMGB1 participates in gene expression, DNA repair and functions of the p53 family.(27) Recently, HMGB1 was revealed to interact with NFkB p65 to accelerate MIA expression. (28,29) HMGB1 and NFkB p65 concurrently bind to a 30-bp region in the promoter region of the MIA gene designated as the highly conserved region (HCR).MIA is suspected to play an important role as a pro-metastatic factor in HMGB1-overexpressing cancers. In the present study, we analyzed the relationship between MIA expression and nodal metastasis and HMGB1 expression in human OSCC. Materials and MethodsTumor specimens. Sixty-two formalin-fixed, paraffin-embedded specimens of primary OSCC were randomly selected at Nara Medical University Hospital, Kashihara, Japan. None of the samples was treated using neo-adjuvant therapy. Medical records and prognostic follow-up data w...

SIRT1 could protect degenerative human NP cells against apoptosis, and there were extensive and intimate connection between apoptosis and autophagy. Up to now, the role of autophagy in the process of human IVD degeneration is unclear. We sought to explore the relationship between autophagy and human IVD degeneration and to understand whether autophagy is involved in the protective effect of SIRT1 against apoptosis in NP cells. Our results showed that the autophagosomes number, the mRNA level of LC3 and Beclin-1, the protein expression of LC3-II/I and Beclin-1, decreased in NP from DDD. Resveratrol could increase the protein expression of LC3-II/I and Beclin-1, and reduce apoptosis in degenerative NP cells. In contrast, the protein levels of LC3-II/I and Beclin-1 were down-regulated and apoptosis level was significantly up-regulated in treatment with nicotinamide or SIRT1-siRNA transfection. Further analysis identified that the expression of cleaved Caspase3 and apoptosis incidence significantly increased with the pretreatment of bafilomycin A, whether resveratrol was added or not. These suggested that autophagy may play an important role in IVD degeneration, and SIRT1 protected degenerative human NP cells against apoptosis via promoting autophagy. These findings would aid in the development of novel therapeutic approaches for degenerative disc disease treatment.

Cancer cells produce energy through aerobic glycolysis, but contributions of host tissues to cancer energy metabolism are unclear. In this study, we aimed to elucidate the cancer-host energy production relationship, in particular, between cancer energy production and host muscle. During the development and progression of colorectal cancer, expression of the secreted autophagy-inducing stress protein HMGB1 increased in the muscle of tumor-bearing animals. This effect was associated with decreased expression of pyruvate kinase PKM1 and pyruvate kinase activity in muscle via the HMGB1 receptor for advanced glycation endproducts (RAGE). However, muscle mitochondrial energy production was maintained. In contrast, HMGB1 addition to colorectal cancer cells increased lactate fermentation. In the muscle, HMGB1 addition induced autophagy by decreasing levels of active mTOR and increasing autophagy-associated proteins, plasma glutamate, and 13 C-glutamine incorporation into acetyl-CoA. In a mouse model of colon carcinogenesis, a temporal increase in HMGB1 occurred in serum and colonic mucosa with an increase in autophagy associated with altered plasma free amino acid levels, increased glutamine, and decreased PKM1 levels. These differences were abolished by administration of an HMGB1 neutralizing antibody. Similar results were obtained in a mouse xenograft model of human colorectal cancer. Taken together, our findings suggest that HMGB1 released during tumorigenesis recruits muscle to supply glutamine to cancer cells as an energy source.