An atopic dermatitis-like murine model by skin-brushed cockroach Per a 2 and oral tolerance induction by Lactococcus lactis-derived Per a 2.

Atopic dermatitis (AD) is a complex, chronic inflammatory skin disease. An estimated 57.5% of asthmatic patients and 50.7% of rhinitis patients are allergic to cockroaches in Taiwan. However, the role of cockroaches in the pathogenesis of AD is undetermined. Oral tolerance might be another strategy for protecting against AD and allergic inflammation by regulating T helper 2 (Th2) immune responses. Aim to examine the underlying immunologic mechanism, we developed an AD-like murine model by skin-brushing with cockroach Per a 2. We also investigated whether the systemic inflammation of AD in this murine model could be improved by specific tolerance to Lactococcus lactis-expressing Per a 2, which was administered orally. Repeated painting of Per a 2 without adjuvant to the skin of mice resulted in increased total IgE, Per a 2-specific IgE, and IgG1, but not IgG2a. In addition, epidermal thickening was significantly increased, there were more scratch episodes, and there were increases in total white blood cells (eosinophil, neutrophil, and lymphocyte) and Th2 cytokines (Interleukin (IL)-4, IL-5, IL-9, and IL-13) in a dose-dependent manner. The results revealed that oral administration of L. lactis-Per a 2 ameliorated Per a 2-induced scratch behavior and decreased the production of total IgE, Per a 2-specific IgE, and IgG1. Furthermore, L. lactis-Per a 2 treatment also suppressed inflammatory infiltration, expressions of thymic stromal lymphopoietin (TSLP) and IL-31 in skin lesions, and downregulated splenic IL-4 and IL-13 in Per a 2-induced AD mice. This study provides evidence supporting that repeated brushing of aeroallergens to the skin leads to atopic dermatitis phenotypes and oral allergen-specific immune tolerance can ameliorate AD-like symptoms and systemic inflammation and prevent progression of atopic march.

Indoor aeroallergens from American cockroaches and mites initiate atopic march via cutaneous contact in a murine model.

The progression of allergic diseases from atopic dermatitis in childhood to other allergic conditions such as asthma in later life is often referred to as the atopic march. In order to study the relationship between cutaneous sensitization by aeroallergen and atopic march, we established a mouse model to test the hypothesis using American cockroaches and house dust mites as the model allergens. Mice were sensitized via skin with native cockroach extract (CraA) or recombinant Per a 2 and Der p 2 proteins without adjuvant. Each mouse was subjected to a total of three 1-week patching sensitizations with a 2-week interval in between each application. The resulting immunological variables in sera, scratching behavior, airway hyperresponsiveness (AHR), and pathology of skin lesions and nasal mucosa were evaluated. In mice, application of CraA, rPer a 2, and rDer p 2 aeroallergens through skin patching induced significantly high levels of both total IgE and specific IgEs. The epicutaneous sensitization after a subsequent allergen challenge showed a significant increase in scratch bouts, AHR, epidermal thickness, and eosinophil counts in the skin compared with the control mice. In addition, stimulation of murine splenocytes with allergens increased higher levels of Th2 cytokines, anti-inflammatory cytokines, and chemokines excretion. Our study provides evidence supporting that epicutaneous sensitization to aeroallergens also led to nasal and airway symptoms comparable to atopic march as described in humans. We hope this new allergy model will be useful in the development of new preventive and therapeutic strategies aimed at stopping the atopic march.

Calreticulin Expression Controls Cellular Redox, Stemness, and Radiosensitivity to Function as a Novel Adjuvant for Radiotherapy in Neuroblastoma.

Radiotherapy (RT) is currently only used in children with high-risk neuroblastoma (NB) due to concerns of long-term side effects as well as lack of effective adjuvant. Calreticulin (CALR) has served distinct physiological roles in cancer malignancies; nonetheless, impact of radiation on chaperones and molecular roles they play remains largely unknown. In present study, we systemically analyzed correlation between CALR and NB cells of different malignancies to investigate potential role of CALR in mediating radioresistance of NB. Our data revealed that more malignant NB cells are correlated to lower CALR expression, greater radioresistance, and elevated stemness as indicated by colony- and neurospheroid-forming abilities and vice versa. Of note, manipulating CALR expression in NB cells of varying endogenous CALR expression manifested changes in not only stemness but also radioresistant properties of those NB cells. Further, CALR overexpression resulted in greatly enhanced ROS and led to increased secretion of proinflammatory cytokines. Importantly, growth of NB tumors was significantly hampered by CALR overexpression and was synergistically ablated when RT was also administered. Collectively, our current study unraveled a new notion of utilizing CALR expression in malignant NB to diminish cancer stemness and mitigate radioresistance to achieve favorable therapeutic outcome for NB.

Prognostic value of an APOBEC3 deletion polymorphism for glioma patients in Taiwan.

OBJECTIVE: The molecular pathogenesis of malignant gliomas, characterized by diverse tumor histology with differential prognosis, remains largely unelucidated. An APOBEC3 deletion polymorphism, with a deletion in APOBEC3B, has been correlated to risk and prognosis in several cancers, but its role in glioma is unclear. The authors aimed to examine the clinical relevance of the APOBEC3 deletion polymorphism to glioma risk and survival in a glioma patient cohort in Taiwan. METHODS: The authors detected deletion genotypes in 403 glioma patients and 1365 healthy individuals in Taiwan and correlated the genotypes with glioma risk, clinicopathological factors, patient survival, and patient sex. APOBEC3 gene family expression was measured and correlated to the germline deletion. A nomogram model was constructed to predict patient survival in glioma. RESULTS: The proportion of APOBEC3B-/- and APOBEC3B+/- genotypes was higher in glioblastoma (GBM) patients than healthy individuals and correlated with higher GBM risk in males. A higher percentage of cases with APOBEC3B- was observed in male than female glioma patients. The presence of APOBEC3B-/- was correlated with better overall survival (OS) in male astrocytic glioma patients. No significant correlation of the genotypes to glioma risk and survival was observed in the female patient cohort. Lower APOBEC3B expression was observed in astrocytic glioma patients with APOBEC3B-/- and was positively correlated with better OS. A 5-factor nomogram model was constructed based on male patients with astrocytic gliomas in the study cohort and worked efficiently for predicting patient OS. CONCLUSIONS: The germline APOBEC3 deletion was associated with increased GBM risk and better OS in astrocytic glioma patients in the Taiwan male population. The APOBEC3B deletion homozygote was a potential independent prognostic factor predicting better survival in male astrocytic glioma patients.

Effectiveness and safety of oral lactococci-based vaccine encoding triple common allergens to prevent airway allergy in mice.

Allergic airway disease is the most common chronic airway inflammatory disorder in developed countries. House dust mite, cockroach, and mold are the leading allergens in most tropical and subtropical countries, including Taiwan. As allergen avoidance is difficult for patients allergic to these perennial indoor allergens, allergen-specific immunotherapy (ASIT) is the only available allergen-specific and disease-modifying treatment. However, for patients sensitized to multiple allergens, ASIT using each corresponding allergen is cumbersome. In the present study, we developed a recombinant L. lactis vaccine against the three most common indoor aeroallergens and investigated its effectiveness for preventing respiratory allergy and safety in mice. Three recombinant clones of Der p 2 (mite), Per a 2 (roach), and Cla c 14 (mold) were constructed individually in pNZ8149 vector and then electroporated into host strain L.lactis NZ3900. BALB/c mice were fed with the triple vaccine 5 times per week for 4 weeks prior to sensitization. The effectiveness and safety profile were then determined. Oral administration of the triple vaccine significantly alleviated allergen-induced airway hyper-responsiveness in the vaccinated mice. The allergen-specific IgG2a was upregulated. IL-4 and IL-13 mRNA expressions as well as inflammatory cell infiltration in the lungs decreased significantly in the vaccinated groups. No body weight loss or abnormal findings in the liver and kidneys were found in any of the groups of mice. This is the first report to describe a triple-aeroallergen vaccine using a food-grade lactococcal expression system. We developed a convenient oral delivery system and intend to extend this research to develop a vaccination that can be self-administered at home by patients.

ASC modulates HIF-1α stability and induces cell mobility in OSCC.

High-level expression of ASC (Apoptosis-associated speck-like protein containing a CARD) leads to lymph node metastasis in OSCC, but the underlying mechanism remains unclear. Here, we show that HIF-1α participates in ASC-induced metastasis. We identified 195 cell-motion-associated genes that were highly activated in ASC-overexpressed SAS_ASC cells; of them, 14 representative genes were found to be overexpressed in OSCC tissues in our previously reported RNA-seq dataset, OSCC-Taiwan. Nine of the 14 genes were also upregulated in OSCC-TCGA samples. Among the nine genes, RRAS2, PDGFA, and VEGFA, were correlated with poor overall survival of patients in OSCC-TCGA dataset. We further demonstrated that the promoters of these 14 ASC-induced genes contained binding motifs for the transcription-regulating factor, HIF-1α. We observed that ASC interacted with and stabilized HIF-1α in both the cytoplasm and the nucleus under normoxia. Molecules involved in the HIF-1α pathway, such as VHL and PHD2, showed no difference in their gene and protein levels in the presence or absence of ASC, but the expression of HIF-1α-OH, and the ubiquitination of HIF-1α were both decreased in SAS_ASC cells versus SAS_con cells. The migration and invasion activities of SAS_ASC cells were reduced when cells were treated with the HIF-1α synthesis inhibitor, digoxin. Taken together, our results demonstrate that the novel ASC-HIF-1α regulatory pathway contributes to lymph node metastasis in OSCC, potentially suggesting a new treatment strategy for OSCC.

Characterization of Copy Number Variations in Oral Cavity Squamous Cell Carcinoma Reveals a Novel Role for MLLT3 in Cell Invasiveness.

BACKGROUND: DNA copy number variations (CNVs) are a hallmark of cancer, and the current study aimed to demonstrate the profile of the CNVs for oral cavity squamous cell carcinoma (OSCC) and elucidate the clinicopathological associations and molecular mechanisms of a potential marker derived from CNVs, mixed-lineage leukemia translocated to chromosome 3 protein (MLLT3), in OSCC carcinogenesis. MATERIALS AND METHODS: CNVs in 37 OSCC tissue specimens were analyzed using a high-resolution microarray, the OncoScan array. Gene expression was analyzed by real-time polymerase chain reaction in 127 OSCC and normal tissue samples. Cell function assays included cell cycle, migration, invasion and chromatin immunoprecipitation assays. RESULTS: We found a novel copy number amplified region, chromosome 9p, encompassing MLLT3 via the comparison of our data set with six other OSCC genome-wide CNV data sets. MLLT3 overexpression was associated with poorer overall survival in patients with OSCC (p = .048). MLLT3 knockdown reduced cell migration and invasion. The reduced invasion ability in MLLT3-knockdown cells was rescued with double knockdown of MLLT3 and CBP/p300-interacting transactivator with ED rich carboxy-terminal domain 4 (CITED4; 21.0% vs. 61.5%). Knockdown of MLLT3 impaired disruptor of telomeric silencing-1-like (Dot1L)-associated hypermethylation in the promoter of the tumor suppressor, CITED4 (p < .001), and hence dysregulated HIF-1α-mediated genes (TWIST, MMP1, MMP2, VIM, and CDH1) in OSCC cells. CONCLUSION: We identified unique CNVs in tumors of Taiwanese patients with OSCC. Notably, MLLT3 overexpression is related to the poorer prognosis of patients with OSCC and is required for Dot1L-mediated transcriptional repression of CITED4, leading to dysregulation of HIF-1α-mediated genes. IMPLICATIONS FOR PRACTICE: This article reports unique copy number variations in oral cavity squamous cell carcinoma (OSCC) tumors of Taiwanese patients. Notably, MLLT3 overexpression is related to the poorer prognosis of patients with OSCC and is required for Dot1L-mediated transcriptional repression of CITED4, leading to dysregulation of HIF-1α-mediated genes.

Participation of NADPH Oxidase-Related Reactive Oxygen Species in Leptin-Promoted Pulmonary Inflammation: Regulation of cPLA2α and COX-2 Expression.

Obesity is a worldwide epidemic problem and correlates to varieties of acute or chronic lung diseases such as acute respiratory distress syndrome, chronic obstructive pulmonary disease, and pulmonary fibrosis. An increase of leptin, a kind of adipokine, in lean mice plasma has been found to impair immune responses and facilitate the infection of Klebsiella pneumoniae, resulting in increased pneumonia severity. Also, a higher leptin level is found in exhaled breath condensates of obese or asthmatic subjects, compared to healthy ones, suggesting that leptin is involved in the occurrence or exacerbation of lung injury. In previous studies, we showed that leptin stimulated cytosolic phospholipase A2-α (cPLA2α) gene expression in lung alveolar type II cells via mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB)-activated coactivator p300. Herein, we show that the in vivo application of leptin in the respiratory system upregulated the expression of inflammatory proteins cPLA2α and cyclooxygenase-2 (COX-2) together with leukocyte infiltration. Treatment with an ROS scavenger (N-acetylcysteine, NAC), an NADPH oxidase inhibitor (apocynin), or an activating protein (AP)-1 inhibitor (tanshinone IIA) attenuated leptin-mediated cPLA2α/COX-2 expression and leukocyte recruitment in the lung. Leptin increased intracellular oxidative stress in a leptin receptor (OB-R) and NADPH oxidase-dependent manner, leading to the phosphorylation of the AP-1 subunit c-Jun. In summation, leptin increased lung cPLA2α/COX-2 expression and leukocyte recruitment via the NADPH oxidase/ROS/AP-1 pathway. Understanding the inflammatory effects of leptin on the pulmonary system provides opportunities to develop strategies against lung injury related to metabolic syndrome or obesity.

APOBEC3A is an oral cancer prognostic biomarker in Taiwanese carriers of an APOBEC deletion polymorphism.

Oral squamous cell carcinoma is a prominent cancer worldwide, particularly in Taiwan. By integrating omics analyses in 50 matched samples, we uncover in Taiwanese patients a predominant mutation signature associated with cytidine deaminase APOBEC, which correlates with the upregulation of APOBEC3A expression in the APOBEC3 gene cluster at 22q13. APOBEC3A expression is significantly higher in tumors carrying APOBEC3B-deletion allele(s). High-level APOBEC3A expression is associated with better overall survival, especially among patients carrying APOBEC3B-deletion alleles, as examined in a second cohort (n = 188; p = 0.004). The frequency of APOBEC3B-deletion alleles is ~50% in 143 genotyped oral squamous cell carcinoma -Taiwan samples (27A3B -/-:89A3B +/-:27A3B +/+), compared to the 5.8% found in 314 OSCC-TCGA samples. We thus report a frequent APOBEC mutational profile, which relates to a APOBEC3B-deletion germline polymorphism in Taiwanese oral squamous cell carcinoma that impacts expression of APOBEC3A, and is shown to be of clinical prognostic relevance. Our finding might be recapitulated by genomic studies in other cancer types.Oral squamous cell carcinoma is a prevalent malignancy in Taiwan. Here, the authors show that OSCC in Taiwanese show a frequent deletion polymorphism in the cytidine deaminases gene cluster APOBEC3 resulting in increased expression of A3A, which is shown to be of clinical prognostic relevance.

Optimized Collection Protocol for Plasma MicroRNA Measurement in Patients with Cardiovascular Disease.

Background. Various microRNAs (miRNAs) are used as markers of acute coronary syndrome, in which heparinization is considered mandatory therapy. Nevertheless, a standard method of handling plasma samples has not been proposed, and the effects of heparin treatment on miRNA detection are rarely discussed. Materials and Method. This study used quantitative polymerase chain reaction (qPCR) analysis to investigate how storage temperature, standby time, hemolysis, and heparin treatment affect miRNA measurement in plasma samples from 25 patients undergoing cardiac catheterization. Results. For most miRNAs, the qPCR results remained consistent during the first 2 hours. The miRNA signals did not significantly differ between samples stored at 4°C before processing and samples stored at room temperature (RT) before processing. miR-451a/miR-23a ratio < 60 indicated < 0.12% hemolysis with 100% sensitivity and 100% specificity. Pretreatment with 0.25 U heparinase I recovered qPCR signals that were reduced by in vivo heparinization. Conclusions. For miRNA measurement, blood samples stored at RT should be processed into plasma within 2 hours after withdrawal and should be pretreated with 0.25 U heparinase I to overcome heparin-attenuated miRNA signals. The miR-451a/miR-23a ratio is a reliable indicator of significant hemolysis.

Saliva protein biomarkers to detect oral squamous cell carcinoma in a high-risk population in Taiwan.

Most cases of oral squamous cell carcinoma (OSCC) develop from visible oral potentially malignant disorders (OPMDs). The latter exhibit heterogeneous subtypes with different transformation potentials, complicating the early detection of OSCC during routine visual oral cancer screenings. To develop clinically applicable biomarkers, we collected saliva samples from 96 healthy controls, 103 low-risk OPMDs, 130 high-risk OPMDs, and 131 OSCC subjects. These individuals were enrolled in Taiwan's Oral Cancer Screening Program. We identified 302 protein biomarkers reported in the literature and/or through in-house studies and prioritized 49 proteins for quantification in the saliva samples using multiple reaction monitoring-MS. Twenty-eight proteins were successfully quantified with high confidence. The quantification data from non-OSCC subjects (healthy controls + low-risk OPMDs) and OSCC subjects in the training set were subjected to classification and regression tree analyses, through which we generated a four-protein panel consisting of MMP1, KNG1, ANXA2, and HSPA5. A risk-score scheme was established, and the panel showed high sensitivity (87.5%) and specificity (80.5%) in the test set to distinguish OSCC samples from non-OSCC samples. The risk score >0.4 detected 84% (42/50) of the stage I OSCCs and a significant portion (42%) of the high-risk OPMDs. Moreover, among 88 high-risk OPMD patients with available follow-up results, 18 developed OSCC within 5 y; of them, 77.8% (14/18) had risk scores >0.4. Our four-protein panel may therefore offer a clinically effective tool for detecting OSCC and monitoring high-risk OPMDs through a readily available biofluid.

ASC contributes to metastasis of oral cavity squamous cell carcinoma.

ASC (Apoptosis-associated Speck-like protein containing a CARD) acts as a platform protein in the inflammasome cascade of some cancer types. However, its potential involvement in OSCC (oral cavity squamous cell carcinoma) has not yet been determined. Here, we investigated the potential role of ASC in OSCC. RT-qPCR analysis of 20 paired tumor and adjacent normal tissue samples revealed that the mRNA levels of ASC, along with IL-1ß, CASP1, and NLRP3 in ASC-associated NLRP3 inflammasome were significantly elevated in OSCC tissues. Immunohistochemical staining of these four proteins in 111 clinical specimens revealed that high-level expression of ASC was significantly associated with tumor stage, node stage (p=0.001), overall stage (p<0.001), extracapsular spread (p<0.001), perineural invasion (p=0.004) and tumor depth (p<0.001). Kaplan-Meier survival analysis further revealed that high-level ASC expression was correlated with poorer overall survival (p=0.001), disease-specific survival (p<0.001) and disease-free survival (p<0.001). Studies using OSCC cell lines indicated that high-level ASC expression enhanced cell migration and invasion, and experiments using an orthotropic nude mouse model confirmed that ASC overexpression induced metastasis of OSCC cells. This is the first report to show that ASC contributes to OSCC metastasis, and that high-level ASC expression is a marker for poor prognosis in OSCC patients.

AdipoR-increased intracellular ROS promotes cPLA2 and COX-2 expressions via activation of PKC and p300 in adiponectin-stimulated human alveolar type II cells.

Adiponectin, an adipokine, accumulated in lung system via T-cadherin after allergens/ozone challenge. However, the roles of adiponectin on lung pathologies were controversial. Here we reported that adiponectin stimulated expression of inflammatory proteins, cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), and production of reactive oxygen species (ROS) in human alveolar type II A549 cells. AdipoR1/2 involved in adiponectin-activated NADPH oxidase and mitochondria, which further promoted intracellular ROS accumulation. Protein kinase C (PKC) may involve an adiponectin-activated NADPH oxidase. Similarly, p300 phosphorylation and histone H4 acetylation occurred in adiponectin-challenged A549 cells. Moreover, adiponectin-upregulated cPLA2 and COX-2 expression was significantly abrogated by ROS scavenger (N-acetylcysteine) or the inhibitors of NADPH oxidase (apocynin), mitochondrial complex I (rotenone), PKC (Ro31-8220, Gö-6976, and rottlerin), and p300 (garcinol). Briefly, we reported that adiponectin stimulated cPLA2 and COX-2 expression via AdipoR1/2-dependent activation of PKC/NADPH oxidase/mitochondria resulting in ROS accumulation, p300 phosphorylation, and histone H4 acetylation. These results suggested that adiponectin promoted lung inflammation, resulting in exacerbation of pulmonary diseases via upregulating cPLA2 and COX-2 expression together with intracellular ROS production. Understanding the adiponectin signaling pathways on regulating cPLA2 and COX-2 may help develop therapeutic strategies on pulmonary diseases.

Leptin Promotes cPLA2 Gene Expression through Activation of the MAPK/NF-κB/p300 Cascade.

Hyperplasia or hypertrophy of adipose tissues plays a crucial role in obesity, which is accompanied by the release of leptin. Recently, obesity was determined to be associated with various pulmonary diseases including asthma, acute lung injury, and chronic obstructive pulmonary disease. However, how obesity contributes to pulmonary diseases and whether leptin directly regulates lung inflammation remains unclear. We used cell and animal models to study the mechanisms of leptin mediation of pulmonary inflammation. We found that leptin activated de novo synthesis of cytosolic phospholipase A2-α (cPLA2-α) in vitro in the lung alveolar type II cells, A549, and in vivo in ICR mice. Upregulated cPLA2-α protein was attenuated by pretreatment with an OB-R blocking antibody, U0126, SB202190, SP600125, Bay11-7086, garcinol, and p300 siRNA, suggesting roles of p42/p44 MAPK, p38 MAPK, JNK1/2, NF-κB, and p300 in leptin effects. Leptin enhanced the activities of p42/p44 MAPK, p38 MAPK, JNK1/2, and p65 NF-κB in a time-dependent manner. Additional studies have suggested the participation of OB-R, p42/p44 MAPK, and JNK1/2 in leptin-increased p65 phosphorylation. Furthermore, p300 phosphorylation and histone H4 acetylation were reduced by blockage of OB-R, p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-κB in leptin-stimulated cells. Similarly, blockage of the MAPKs/NF-κB/p300 cascade significantly inhibited leptin-mediated cPLA2-α mRNA expression. Our data as a whole showed that leptin contributed to lung cPLA2-α expression through OB-R-dependent activation of the MAPKs/NF-κB/p300 cascade.

The relationship between secretory leukocyte protease inhibitor expression and Epstein-Barr virus status among patients with nasopharyngeal carcinoma.

AIM: The aim was to study the expression of Secretory Leukocyte Protease Inhibitor (SLPI) and to explore its correlation with the presence of Epstein-Barr Virus (EBV) among patients with nasopharyngeal carcinoma. MATERIALS AND METHODS: The expression levels of SLPI mRNA in NPC cell lines and in ten matched-pairs of NPC and adjacent normal tissue were examined by quantitative real-time Polymerase Chain Reaction (PCR). Furthermore, protein expression of SLPI in 71 paraffin-embedded NPC biopsies was assessed by immunohistochemistry. Finally, the serum level of SLPI in 177 NPC patients and 103 healthy controls was evaluated by enzyme-linked immunosorbent assay (ELISA). RESULTS: The expression of SLPI mRNA in NPC cells was significantly lower than in the adjacent normal epithelium (p<0.001). When the expression of SLPI in EBV-positive and -negative NPC cell lines was compared, we found that both mRNA and protein expressions of SLPI were significantly higher in EBV-negative cells. Furthermore, the results of immunohistochemical analysis demonstrated that the frequency of reduced SLPI expression in EBV-positive biopsies was significantly higher than that in EBV-negative biopsies. CONCLUSION: In this study, we have confirmed that SLPI is significantly down-regulated in NPC tissues. In addition, based on our preliminary results, we propose that the reduction of SLPI in NPC cells is associated with the presence of the EBV genome and/or the expression of EBV-encoded genes. SLPI may play an important role in EBV-mediated NPC tumorigenesis.

Heterogeneous ribonucleoprotein K and thymidine phosphorylase are independent prognostic and therapeutic markers for oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the major cancer of head and neck with increasing incidence and mortality in Taiwan. We investigate hnRNP K, TP and FLIP expression and assess the prognostic and therapeutic potential of these markers in oral squamous cell carcinoma (OSCC). We analyzed hnRNP K, TP, and FLIP expression in 110 OSCC patients by immunohistochemistry. Statistical analyses were applied to correlate nuclear and cytoplasmic hnRNP K with elevated TP and FLIP, and to determine the associations of these three markers with clinicopathological manifestations, and assess their prognostic and therapeutic significance. The therapeutic implication of elevated TP was determined by measuring the sensitivity of OSCC cells to the TP-targeting drug, 5-fluoro-5'-deoxyuridine (5'-DFUR). We found that each of these proteins was overexpressed in OSCC tumors. Nuclear hnRNP K and cytoplasmic hnRNP K were strongly associated with TP (r(2)=0.344, P=0.0004) and FLIP (r(2)=0.201, P=0.035), respectively. High hnRNP K and TP levels were associated with clinicopathological parameters predictive of poorer treatment outcome. Multivariate analyses indicated that cytoplasmic hnRNP K and TP are independent predictors of overall survival (P=0.022 and 0.009, respectively) and disease-free survival (P=0.012 and 0.005, respectively). OSCC cells expressing high levels of TP were more sensitive to treatment with 5'-DFUR. Elevated cytoplasmic hnRNP K and TP overexpression are associated with poorer survival in OSCC patients. In vitro experiments suggest that OSCC tumors with high levels of TP are more sensitive to 5'-DFUR treatment. Thus, cytoplasmic hnRNP K and TP may be potential prognostic and therapeutic markers for OSCC.

Targeted methylation of two tumor suppressor genes is sufficient to transform mesenchymal stem cells into cancer stem/initiating cells.

Although DNA hypermethylation within promoter CpG islands is highly correlated with tumorigenesis, it has not been established whether DNA hypermethylation within a specific tumor suppressor gene (TSG) is sufficient to fully transform a somatic stem cell. In this study, we addressed this question using a novel targeted DNA methylation technique to methylate the promoters of HIC1 and RassF1A, two well-established TSGs, along with a two-component reporter system to visualize successful targeting of human bone marrow-derived mesenchymal stem cells (MSC) as a model cell system. MSCs harboring targeted promoter methylations of HIC1/RassF1A displayed several features of cancer stem/initiating cells including loss of anchorage dependence, increased colony formation capability, drug resistance, and pluripotency. Notably, inoculation of immunodeficient mice with low numbers of targeted MSC resulted in tumor formation, and subsequent serial xenotransplantation and immunohistochemistry confirmed the presence of stem cell markers and MSC lineage in tumor xenografts. Consistent with the expected mechanism of TSG hypermethylation, treatment of the targeted MSC with a DNA methyltransferase inhibitor reversed their tumorigenic phenotype. To our knowledge, this is the first direct demonstration that aberrant TSG hypermethylation is sufficient to transform a somatic stem cell into a fully malignant cell with cancer stem/initiating properties.

Targeted methylation of CMV and E1A viral promoters.

DNA methylation is a gene-silencing and host defense system that can down-regulate viral gene expression in mammalian cells. An established targeted DNA methylation method was used to demonstrate that genome-integrated CMV and adenovirus type 5 E1A promoters were hypermethylated after MCF7 and HEK293 cells were transfected with in vitro methylated viral promoter fragments. In both cases, the targeted methylation-induced gene silencing could be reversed by addition of 5-aza-2'-deoxycytidine, confirming that the CMV and E1A promoters are regulated by DNA methylation. The kinetics of the targeted DNA methylation was determined using a reporter system in live cells. In conclusion, targeted DNA methylation is able to efficiently silence susceptible viral promoters and provides an alternative strategy to study the impact of loci-specific DNA methylation in viral gene expression.

Aberrant methylation impairs low density lipoprotein receptor-related protein 1B tumor suppressor function in gastric cancer.

DNA methylation plays a significant role in tumor progression. In this study, we used CpG microarray and differential methylation hybridization approaches to identify low density lipoprotein receptor-related protein 1B (LRP1B) as a novel epigenetic target in gastric cancer. LRP1B was hypermethylated in four gastric cancer cell lines, and low LRP1B mRNA expression was associated with high methylation levels in gastric cancer cell lines. Addition of a DNA methylation inhibitor (5-Aza-dC) restored the mRNA expression of LRP1B in these cell lines, indicating that DNA methylation is involved in regulating LRP1B expression. In 45 out of 74 (61%) clinical samples, LRP1B was highly methylated; LRP1B mRNA expression was significantly lower in 15 out of 19 (79%, P < 0.001) gastric tumor tissues than in corresponding adjacent normal tissues. In addition, ectopic expression of mLRP1B4 in gastric cancer cell lines suppressed cell growth, colony formation and tumor formation in nude mice. These results collectively indicate that LRP1B is a functional tumor suppressor gene in gastric cancer and that is regulated by DNA methylation.