(Expression Of Novel Markers In Head And Neck Carcinomas And Nasal Polyposis And Study Of Growth Dynamics Of HNESPCs)

Background:Head and neck squamous cell carcinoma (HNSCC) is the sixth most malignancy in the world. Approximately, one-fourth of all HNSCCs are laryngeal squamous cell carcinoma (LSCC). In contrast, hypopharyngeal squamous cell carcinoma (HSCC) is not so common, but it is usually diagnosed in the advanced stage with poor prognosis. Although several treatment strategies, including surgery, radiotherapy, gene therapy and immunotherapy, have been developed for LSCC and HSCC, no treatment could achieve a satisfactory therapeutic outcome for them and the survival rate has not been improved significantly. Therefore, identification of prognostic markers will be important for prevention and therapy of LSCC and HSCC.Sirtuin1(SIRT1), which is nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, belongs to the silent information regulator2family (Sir2) of sirtuin histone deacetylases (HDACs). It may play an important role in tumorigenesis. It is reported that SIRT1is increased in many human tumors, such as breast cancer, ovarian cancer, prostate cancer, gastric cancer, colon cancer, diffuse large B-cell lymphoma, acute myeloid leukemia and Bowen’s disease. However, there is also some evidence to show that the expression of SIRT1in breast cancer, ovarian carcinoma, bladder carcinoma, prostate carcinoma, and glioblastoma, is lower than that in normal tissues. SIRT1can be activated by SRT, AROS and SUMO-1and can be inhibited by tenovins, DBC1and Tat.DBC1(Deleted in Breast Cancer1) is well known as a negative regulator of SIRT1. Some studies reveal that DBC1promotes p53-mediated apoptosis through specific inhibition of SIRT1. Therefore, DBC1has been suggested as a tumor suppressor based on this evidence. However, some recent studies showed that DBC1is much more overexpressed in breast cancers and colorectal cancers than in normal tissues and it promotes the survival of breast cancer cells. Therefore, it is still difficult to determine whether DBC1is a tumor suppressor or a tumor promotor. HIC1is a tumor suppressor and SIRT1is one of its transcriptional targets. HIC1plays an important role in cell survival and apoptosis and it may also participate in tumorigenesis.Objective:In the present study, we aim to elucidate whether SIRT1, DBC1and HIC1are involved in the development of laryngeal squamous cell carcinomas (LSCCs) and hypopharyngeal squamous cell carcinomas (HSCCs) and to identify the potential role of those markers in the prevention and therapy of the tumors.Methods:The mRNA levels of SIRT1, DBC1and HIC1were measured in54paired LSCC or HSCC tumors and corresponding adjacent noncancerous mucosae using quantitative RT-PCR (qRT-PCR). The protein levels of SIRT1and DBC1were also evaluated in120cases of patients with LSCC or HSCC using immunohistochemical staining. SPSS18.0was used for statistical analysis. The correlation between the protein expression (SIRT1and DBC1) and clinical parameters was analyzed with Pearson chi-square test. Log Rank test was performed for the survival analysis.Results:qRT-PCR assay showed that, compared with the paired adjacent noncancerous mucosae, SIRT1mRNA and HIC1mRNA were significantly decreased in tumors. In contrast, DBC1mRNA was significantly increased in tumors compared with noncancerous mucosae. The immunohistochemical results indicated that the SIRT1protein was downregulated in tumors compared with noncancerous mucosae and the DBC1protein was downregulated in tumors, which is inconsistent with the results obtained by qRT-PCR. Moreover, decreased SIRT1was significantly correlated with the tumor clinical stage and lymph node metastasis. The decreased DBC1protein was significantly correlated with tumor differentiation, lymph node metastasis, and p53expression. SIRT1was positively correlated with DBC1and HIC1both in tumors and in controls. Besides, we performed the survival analysis using Log Rank test and found that SIRT1(Fig.5A) and DBC1(Fig.5B) were correlated with patient survival.Conclusion:SIRT1and DBC1play key roles in laryngeal and hypopharyngeal squamous cell carcinomas as tumor suppressors and are associated with lymph node metastasis in LSCCs and HSCCs. HIC1is also served as a tumor suppressor in LSCCs and HSCCs, which is closely correlated with SIRT1at mRNA level. Those novel markers (SIRT1, DBC1and HIC1) could be served as diagnostic indicators for LSCCs and HSCCs and they may become the new therapeutic targets of anticancer drugs. Background:Nasal polyposis (NP) is a common chronic inflammatory disease of the upper airways. The main characteristics of NP are the infiltration of various inflammatory cells and epithelial damage followed by aberrant tissue repair or remodeling. There are many studies about the molecular mechanisms of nasal polyposis. However, the pathogenesis of NP is still unknown. In our previous study, EMP1, EGF and NRG3were found to be downregulated in NP compared with controls by microarray analysis. Basal cells in nasal epithelium have sternness/progenitor characters and play essential roles in the epithelial remodeling in nasal polyposis (NP). We have successfully isolated and cultured human nasal epithelial stem/progenitor cells in vitro which will help us to study the mechanism of human airway inflammatory diseases including nasal polyposis.Epithelial membrane protein1(EMP1) is an integral membrane glycoprotein. It was first identified as a peripheral myelin protein22(PMP22) related transcript and was detected in epithelial tissues from gastrointestinal tract, skin, lung, and brain. EMP1was found to regulate epithelial cell proliferation and differentiation. EGF and NRG3are ligands of type I growth factor receptor (ERBB family). EGF is an important growth factor and there are many studies of it in various tissues. NRGs are a group of cell-cell signaling proteins, including NRG1, NRG2, NRG3and NRG4, which have the EGF-like domain structure. Glucocorticosteroids (GCs) are the most effective anti-inflammatory therapy for NP and they can inhibit the infiltration of eosinophils. An important aspect of GCs is to promote tissue repair and remodeling in NP and asthma through transforming growth factor (TGF)-P and epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) signaling pathways. GCs can also upregulate AP-1related genes which are connected with epithelial repair and downregulate p63which relates to tissue remodeling.Objective:We sought to determine the expression of EMP1and EGF-ERBB family in nasal polyposis and to find out if they play a role in the remodeling of chronically inflamed nasal epithelium in nasal polyposis.Methods:NP tissues were obtained from55NP patients,18of whom were treated with nasal glucocorticosteroid (GC)(i.e., these18patients had2sets of NP biopsies taken before and after treatment). Biopsies of inferior turbinate mucosa from30healthy subjects were used as controls. Quantitative real-time PCR was performed to determine the mRNA expression levels of EMP1and EGF-ERBB family. Immunohistochemistry was performed to evaluate the protein level of EMP1, p63and Ki67in nasal polyps and controls. And we also investigated p63, CK18and EMP1expressions in epithelial cells by immunocytochemistry. SPSS18.0was used for the statistical analysis.Results:The EMP1mRNA expression (2.77-fold) was significantly lower in tissues from NP patients before GC treatment than those from controls, and it was increased after GC treatment. EMP1was stained in nasal epithelium and was co-localized with both basal (p63+) and differentiated (CK18+) epithelial cells. Their immunoreactivity was significantly greater in controls than in NP patients, especially in those with severe hyperplastic (1.79-fold) or metaplastic (1.85-fold) epithelium. Positive correlations between EMP1and other epithelial cell related gene (e.g. JUN, PTGS2, and AREG, etc.) mRNAs were observed.The mRNA expression of EGF-ERBB family was significantly lower in tissues from NP patients before GC treatment than those from controls, especially for EGF (26.32-fold) and NRG3(37.91-fold). After GC treatment, most of the family members (EGF, NRG3, EGFR, ERBB2, ERBB3and ERBB4) were increased. EGF was positively correlated with NRG3at mRNA level (r=0.621). Positive correlations between EGF-ERBB family and AP-1related genes (JUN, PTGS2, IL6and EGR1) mRNAs were observed. EGF-ERBB family was positively correlated with T help cell related markers (T-bet, RORC and GATA3) and other markers (CXCL12, CXCR4and CDKN1A). Besides, EGF-ERBB family was negatively correlated with eosinophil related markers (CCL13and CCL18).Conclusion:EMP1could be a specific marker for aberrant epithelial remodeling and metaplasia in chronic inflammatory upper airway mucosa (e.g. NP). EGF-ERBB family may participate in the pathogenesis of nasal polyps. EMP1and EGF-ERBB family may serve as the drug treatment targets. Background:There are four major cell types in healthy nasal epithelium, including basal cells, ciliated cells, non-ciliated columnar cells and goblet cells. Basal cells are considered to have stemness and progenitor properties, which can self-renew and differentiate into other epithelial cell types. In our recent study, we have successfully isolated and cultured human nasal epithelial stem/progenitor cells (hNESPCs) from human inferior turbinate tissues in a serum-free culture method. In this study, we investigated growth and proliferation properties of hNESPCs in an in vitro cell culture system and further confirmation was performed in nasal mucosal tissue obtained from healthy subjects and NP patients.Objective:We investigate whether the human nasal epithelial stem/progenitor cells (hNESPCs) from patients with NP are inherently distinct from those obtained from healthy controls.Methods:Epithelial basal cells were isolated and cultured for four passages from NP tissues and control nasal mucosa. The immunocytochemistry and qRT-PCR was performed to analyze the expression of markers.Results:More than90%of the hNESPCs in the colonies were p63positive, and among these cells, approximately90%were co-localized with KRT5; while they did not express any differentiated nasal epithelial cell markers (e.g., betaⅣ-tubulin and MUC5AC). Another common stem cell marker KRT14was also stained in the colonies, but only a subset of p63or Ki67positive cells expressed KRT14. hNESPCs from both NP and control tissues showed a similar growth pattern throughout the4passages. More "fried-egg" shape phenotype cells were seen from the cell cultures of NP tissues in both P2and P3than those from controls. These cells were almost all β-galactosidase positive, showing a light blue staining mostly in perinuclear region, indicating more senescent cells in the colonies. Cells from control tissues proliferated more rapidly than those from NP tissues over subsequent passages, showing higher CFE values (difference from11.3%to8.5%through PI to P3) and shorter doubling times (difference from6.5hr to22hr through P1to P3). In addition, the variations (based on standard deviation) of CFE and doubling time values were larger in cell cultures derived from NP tissues.The immunocytochemistry results showed that, in hNESPCs from NP samples, the ratio of Ki67+/p63+cells was significantly lower in P2and P3as compared to those cells from controls and the percentage of Ki67+over p63+cells decreased from P1to P3(median value,84%,61%, and36%respectively). The ki67expression level decreased from P1to P2in cells from NP and control samples. In addition, ki67mRNA level was lower in hNESPCs from NP as compared to controls in P1(1.5-fold, a borderline significant trend, p=0.05) and P2(2.1-fold, approaching borderline of significance, p=0.07).We further analyzed expression levels of p63and Ki67in nasal mucosa from the same NP and control subjects. Ki67immunostaining decreased in the hyperplasia or metaplasia areas of NP epithelium as compared to control epithelium. Similarly, the percentage (median,25th-75th percentile) of Ki67+cells among p63+cells was significantly lower (p＜0.001) in the hyperplasic NP epithelium (7.3%,4.3%-9.9%) than in healthy controls (13.1%,11.4%-17.3%) by immuno fluorescence staining.Conclusion:In conclusion, our study demonstrated significantly reduced growth and proliferation dynamics in hNESPCs from NP epithelium. These intrinsic differences in growth and proliferation properties could be the main cause of the persistence and aberrant remodeling in NPs.