Analysis of tissue and salivary nicotinamide N-methyltransferase in oral squamous cell carcinoma: basis for the development of a noninvasive diagnostic test for early-stage disease

Oral squamous cell carcinoma (OSCC) is the most common form of head and neck cancer worldwide. In recent decades, the 5-year mortality rate is approximately 50% around the world. As reliable biomarkers of oral cancer are still lacking, it is necessary to identify new target molecules for early diagnosis, effective therapy, and monitoring of the disease. In the present work, we focused on the expression of the enzyme nicotinamide N-methyltransferase (NNMT). We analyzed enzyme activity in 37 paired tumor and non-tumor tissues and found that activity levels are significantly higher in tumor compared with adjacent normal oral mucosa. Interestingly, oral epithelium surrounding tumor of unfavorable cases (N+) seems to display higher activity levels compared with that of favorable ones (N0). Western blot analyses were performed to evaluate protein levels in saliva samples from patients with OSCC and healthy subjects. Preliminary results indicated an up-regulation of salivary NNMT in tumor. This study shows a marked increase in enzyme activity in oral cancer and suggests that adjacent normal tissue of unfavorable cases seems to change toward cancer. Moreover, it is conceivable to hypothesize that NNMT could represent a potential biomarker for early and non-invasive diagnosis of oral cancer.     

Accumulation of Nicotinamide N-Methyltransferase (NNMT) in Cancer-associated Fibroblasts: A Potential Prognostic and Predictive Biomarker for Gastric Carcinoma

Nicotinamide N-methyltransferase (NNMT), a major metabolic regulator, has been identified as a predictor of cancer prognosis in ovarian and colorectal cancers. The study aims to evaluate the significance of stromal NNMT in gastric cancer (GC). Expression of stromal NNMT in 612 GC and 92 non-malignant tissues specimens was investigated by immunohistochemistry (IHC). The association between NNMT expression and occurrence of cancer or patient outcome was further analyzed, and the factors contributing to disease prognosis were evaluated by multiple Cox models. Stromal NNMT expression was higher in the malignant tissue (p<0.001). NNMT expression was significantly associated with GC stage (p=0.006). Compared to stromal “NNMT-low” cases, “NNMT-high” cases has lower disease-specific survival (hazard ratio [HR], 2.356; 95% confidence interval [CI] = 1.591–3.488; p<0.001) and disease-free survival (HR = 2.265; 95% CI = 1.529–3.354; p<0.001), as observed by multivariate Cox analysis after adjusting for stromal NNMT expression with other factors such as tumor grade and size. Notably, patients with stage II NNMT-low GC might be negatively affected by adjuvant chemotherapy, but lower stromal NNMT expression predicted a more favorable prognosis for GC. Our study confirmed that stromal NNMT expression is significantly increased in GC, which predicts an unfavorable post-operative prognosis for GC:     

Nicotinamide N-methyltransferase in endothelium protects against oxidant stress-induced endothelial injury

Nicotinamide N-methyltransferase (NNMT, EC 2.1.1.1.) plays an important role in the growth of many different tumours and is also involved in various non-neoplastic disorders. However, the presence and role of NNMT in the endothelium has yet to be specifically explored. Here, we characterized the functional activity of NNMT in the endothelium and tested whether NNMT regulates endothelial cell viability. NNMT in endothelial cells (HAEC, HMEC-1 and EA.hy926) was inhibited using two approaches: pharmacological inhibition of the enzyme by NNMT inhibitors (5-amino-1-methylquinoline – 5MQ and 6-methoxynicotinamide – JBSF-88) or by shRNA-mediated silencing. Functional inhibition of NNMT was confirmed by LC/MS/MS-based analysis of impaired MNA production. The effects of NNMT inhibition on cellular viability were analyzed in both the absence and presence of menadione.Our results revealed that all studied endothelial lines express relatively high levels of functionally active NNMT compared with cancer cells (MDA-MB-231). Although the aldehyde oxidase 1 enzyme was also expressed in the endothelium, the further metabolites of N1-methylnicotinamide (N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-3-carboxamide) generated by this enzyme were not detected, suggesting that endothelial NNMT-derived MNA was not subsequently metabolized in the endothelium by aldehyde oxidase 1. Menadione induced a concentration-dependent decrease in endothelial viability as evidenced by a decrease in cell number that was associated with the upregulation of NNMT and SIRT1 expression in the nucleus in viable cells. The suppression of the NNMT activity either by NNMT inhibitors or shRNA-based silencing significantly decreased the endothelial cell viability in response to menadione. Furthermore, NNMT inhibition resulted in nuclear SIRT1 expression downregulation and upregulation of the phosphorylated form of SIRT1 on Ser47. In conclusion, our results suggest that the endothelial nuclear NNMT/SIRT1 pathway exerts a cytoprotective role that safeguards endothelial cell viability under oxidant stress insult.     

RNA-Mediated Gene Silencing of Nicotinamide N-Methyltransferase Is Associated with Decreased Tumorigenicity in Human Oral Carcinoma Cells

Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer. Despite progress in the treatment of OSCC, overall survival has not improved substantially in the last three decades. Therefore, identification of reliable biomarkers becomes essential to develop effective anti-cancer therapy. In this study, we focused on the enzyme Nicotinamide N-methyltransferase (NNMT), which plays a fundamental role in the biotransformation of many xenobiotics. Although several tumors have been associated with abnormal NNMT expression, its role in cancer cell metabolism remains largely unknown. In this report, 7 human oral cancer cell lines were examined for NNMT expression by Real-Time PCR, Western blot and HPLC-based catalytic assay. Subsequently, we evaluated the in vitro effect of shRNA-mediated silencing of NNMT on cell proliferation. In vivo tumorigenicity of oral cancer cells with stable knockdown of NNMT was assayed by using xenograft models. High expression levels of NNMT were found in PE/CA PJ-15 cells, in keeping with the results of Western blot and catalytic activity assay. PE/CA PJ-15 cell line was stably transfected with shRNA plasmids against NNMT and analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and soft agar Assays. Transfected and control cells were injected into athymic mice in order to evaluate the effect of NNMT silencing on tumor growth. NNMT downregulation resulted in decreased cell proliferation and colony formation ability on soft agar. In athymic mice, NNMT silencing induced a marked reduction in tumour volume. Our results show that the downregulation of NNMT expression in human oral carcinoma cells significantly inhibits cell growth in vitro and tumorigenicity in vivo. All these experimental data seem to suggest that NNMT plays a critical role in the proliferation and tumorigenic capacity of oral cancer cells, and its inhibition could represent a potential molecular approach to the treatment of oral carcinoma.     

let-7b and miR-126 Are Down-Regulated in Tumor Tissue and Correlate with Microvessel Density and Survival Outcomes in Non–Small–Cell Lung Cancer

Angiogenesis is a critical event in the development, progression, and spread of various human cancers, including lung cancer. Molecular mechanisms that underlie the complex regulation of angiogenic processes are poorly understood. However, an increasing body of evidence indicates miRNAs as important regulators of tumor angiogenesis. Forceps biopsies were collected from tumor tissue, surrounding tissue, and non-tumor tissue from 50 NSCLC patients. Lung tissue samples from individuals with no clinical evidence of a cancerous disease served as controls. Immunohistochemical staining for Factor VIII was used to evaluate microvessel density (MVD). TaqMan® primer-probe sets were used in quantitative real-time RT-PCR reactions to determine expression levels of let-7b, miR-126, miR-9, and miR-19a. We demonstrated significantly higher MVD and decreased expression levels of let-7b and miR-126 in tumor tissue and surrounding tissue in comparison to corresponding non-tumor tissue or lung tissue from the control group. In addition, no differences in MVD and expression levels of both miRNAs between tumor tissue and surrounding tissue from NSCLC patients were observed. Low expression of both miRNAs correlated with high MVD and worse progression-free survival and overall survival. These observations strongly suggest similar molecular alternations within tumor tissue and surrounding tissue that comprise a specific microenvironment. Low expression of let-7b and miR-126 seems to have a possible anti-angiogenic role in lung tumor tissue and significantly correlates with worse survival outcomes for lung cancer patients. Moreover, the regulation of let-7b and miR-126 expression could have therapeutic potential because it could reduce tumor angiogenesis and therefore suppress tumor growth in lung cancer patients.     

Bmi-1 在非小细胞肺癌中的表达及相关性探讨

目的:探讨Bmi-1在非小细胞肺癌(NSCLC)组织中的表达及其与临床病理特征的关系。方法:采用RT-PCR检测30例非小细胞肺癌及20例癌旁组织中Bmi-1mRNA的表达情况,同时应用免疫组织化学SP法检测52例非小细胞肺癌及30例癌旁组织中Bmi-1蛋白表达情况。结果:非小细胞肺癌组织中Bmi-1mRNA表达量明显高于癌旁组织(t=5.188,P<0.01)。肺癌组中Bmi-1蛋白的阳性表达率为67.3%(35/52),明显高于癌旁组13.3%(4/30),且表达量差别有高度统计学意义(Z=-4.837,P<0.01);肺癌组中Bmi-1蛋白阳性表达与癌组织的TNM分期及有无淋巴结转移有关(Z=-2.567,-2.366,P<0.05),而与患者的性别、年龄、组织类型、分化程度等无关(P均>0.05);结论:Bmi-1在非小细胞肺癌中的高表达与肿瘤发生发展及转移相关,可成为早期诊断肺癌及判断转移的重要参考指标。     

Nicotinamide N-methyltransferase upregulation inversely correlates with lymph node metastasis in oral squamous cell carcinoma

We investigated expression levels of Nicotinamide N-Methyltransferase (NNMT), an enzyme involved in the biotransformation of many drugs and xenobiotic compounds, in oral squamous cell carcinoma (OSCC). Measurements were performed by semi-quantitative RT-PCR and quantitative real-time PCR in tumor and matched adjacent healthy tissue. Interestingly, NNMT was up-regulated in most of the favorable OSCCs, while no marked NNMT expression alterations between tumor and normal mucosa were detected in most of the unfavorable OSCCs. Western blot and immunohistochemical analyses also were performed and the relationship between tumor characteristics and NNMT levels in OSCC were studied to evaluate the effectiveness of NNMT as a prognostic marker in the squamous cell carcinoma of the oral cavity. In summary, the present study suggests that NNMT may have potential as a biomarker and a therapeutic target for OSCC.     

Prostate Tissue Metal Levels and Prostate Cancer Recurrence in Smokers

Although smoking is not associated with prostate cancer risk overall, smoking is associated with prostate cancer recurrence and mortality. Increased cadmium (Cd) exposure from smoking may play a role in progression of the disease. In this study, inductively coupled plasma mass spectrometry was used to determine Cd, arsenic (As), lead (Pb), and zinc (Zn) levels in formalin-fixed paraffin embedded tumor and tumor-adjacent non-neoplastic tissue of never- and ever-smokers with prostate cancer. In smokers, metal levels were also evaluated with regard to biochemical and distant recurrence of disease. Smokers (N?=?25) had significantly higher Cd (median ppb, p?=?0.03) and lower Zn (p?=?0.002) in non-neoplastic tissue than never-smokers (N?=?21). Metal levels were not significantly different in tumor tissue of smokers and non-smokers. Among smokers, Cd level did not differ by recurrence status. However, the ratio of Cd ppb to Pb ppb was significantly higher in both tumor and adjacent tissue of cases with distant recurrence when compared with cases without distant recurrence (tumor tissue Cd/Pb, 6.36 vs. 1.19, p?=?0.009, adjacent non-neoplastic tissue Cd/Pb, 6.36 vs. 1.02, p?=?0.038). Tissue Zn levels were also higher in smokers with distant recurrence (tumor, p?=?0.039 and adjacent non-neoplastic, p?=?0.028). These initial findings suggest that prostate tissue metal levels may differ in smokers with and without recurrence. If these findings are confirmed in larger studies, additional work will be needed to determine whether variations in metal levels are drivers of disease progression or are simply passengers of the disease process.     

Bmi-1在非小细胞肺癌中的表达及相关性探讨

目的：探讨Bmi-1在非小细胞肺癌（NSCLC）组织中的表达及其与临床病理特征的关系。方法：采用RT-PCR检测30例非小细胞肺癌及20例癌旁组织中Bmi-1mRNA的表达情况,同时应用免疫组织化学SP法检测52例非小细胞肺癌及30例癌旁组织中Bmi-1蛋白表达情况。结果：非小细胞肺癌组织中Bmi-1mRNA表达量明显高于癌旁组织（t=5.188,P〈0.01）。肺癌组中Bmi-1蛋白的阳性表达率为67.3%（35/52）,明显高于癌旁组13.3%（4/30）,且表达量差别有高度统计学意义（Z=-4.837,P〈0.01）;肺癌组中Bmi-1蛋白阳性表达与癌组织的TNM分期及有无淋巴结转移有关（Z=-2.567,-2.366,P〈0.05）,而与患者的性别、年龄、组织类型、分化程度等无关（P均〉0.05）;结论：Bmi-1在非小细胞肺癌中的高表达与肿瘤发生发展及转移相关,可成为早期诊断肺癌及判断转移的重要参考指标。     

VEGF、P-ACC、LKB1在非小细胞肺癌组织中的表达及意义

目的:探究非小细胞肺癌组织中血管内皮生长因子(VEGF)、磷酸化乙酰辅酶A羟化酶(P-ACC)、肝激酶B1(LKB1)表达及其与肿瘤血管生成的关系。方法:将我院收治的83例非小细胞肺癌(NSCLC)患者作为研究对象,取其NSCLC病理组织样本进行研究,同时取其远离肿瘤的外周正常肺组织作为对照。采用免疫组化法测定其NSCLC病理组织样本和正常组织样本VEGF、P-ACC、LKB1的表达情况,分析比较NSCLC病理组织的VEGF、P-ACC、LKB1表达情况与其病理特征及肿瘤血管生成的关系。结果:NSCLC组织样本的VEGF阳性表达率为72.29%,明显高于癌旁正常组织样本(22.89%)(P0.05);同时,其P-ACC、LKB1阳性表达率分别为31.33%、61.45%,明显低于癌旁正常组织样本(分别为75.90%、90.36%)(P0.05)。NSCLC组织VEGF阳性表达与N分期、临床分期以及肿瘤微血管密度(MVD)有关,P-ACC阳性表达与T分期、临床分期以及MVD有关,LKB1阳性表达与N分期、临床分期、分化程度以及MVD有关(P0.05)。在样本中,VEGF阳性NSCLC组织的MVD水平明显高于VEGF阴性样本,而P-ACC、LKB1阳性NSCLC组织的MVD水平明显低于阴性样本(P0.05)。结论:非小细胞肺癌组织中VEGF在呈高表达,P-ACC、LKB1呈现低表达。VEGF、P-ACC、LKB1的表达与NSCLC临床病理特征及肿瘤血管生成均存在密切联系,对于预测NSCLC癌细胞的生长、浸润和转移具有重要意义。     

Low expression of ITIH5 in adenocarcinoma of the lung is associated with unfavorable patients' outcome

Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) is supposed to be involved in extracellular matrix stability and thus may play a key role in the inhibition of tumor progression. The current study is the first to analyze in depth ITIH5 expression and DNA methylation, as well as its potential clinical impact in non-small-cell lung carcinoma (NSCLC). We examined ITIH5 mRNA expression in tumor and adjacent normal lung tissue specimens of NSCLC patients. In addition, methylation frequency of the ITIH5 promoter was investigated using methylation-specific PCR and pyrosequencing. Significance of our data was validated by independent data sets from The Cancer Genome Atlas and the Kaplan-Meier Plotter platform. Furthermore, ITIH5 protein expression was evaluated by immunohistochemistry utilizing a tissue microarray with 385 distinct lung tissue samples. Based on our tissue collections, ITIH5 mRNA expression was significantly decreased in NSCLC compared to normal lung tissue in line with an increased methylation frequency in lung cancer tissue. Independent TCGA data confirmed significant expression loss of ITIH5 in lung cancer concordant with ITIH5 promoter hypermethylation in NSCLC. Of interest, low ITIH5 mRNA expression was particularly found in the magnoid and squamoid ADC expression subtype, concordant with an unfavorable patients'' outcome in squamoid as well as tobacco smoking ADC patients. In conclusion, ITIH5 may be a novel putative tumor suppressor gene in NSCLC with a potential molecular significance in the squamoid ADC subtype and further clinical impact for risk stratification of adenocarcinoma patients. In addition, ITIH5 may serve as a novel biomarker for prognosis of tobacco smoking ADC patients.     

Complex roles of nicotinamide N-methyltransferase in cancer progression

Nicotinamide N-methyltransferase (NNMT) is an intracellular methyltransferase, catalyzing the N-methylation of nicotinamide (NAM) to form 1-methylnicotinamide (1-MNAM), in which S-adenosyl-l-methionine (SAM) is the methyl donor. High expression of NNMT can alter cellular NAM and SAM levels, which in turn, affects nicotinamide adenine dinucleotide (NAD⁺)-dependent redox reactions and signaling pathways, and remodels cellular epigenetic states. Studies have revealed that NNMT plays critical roles in the occurrence and development of various cancers, and analysis of NNMT expression levels in different cancers from The Cancer Genome Atlas (TCGA) dataset indicated that NNMT might be a potential biomarker and therapeutic target for tumor diagnosis and treatment. This review provides a comprehensive understanding of recent advances on NNMT functions in different tumors and deciphers the complex roles of NNMT in cancer progression.Subject terms: Cancer, Mechanisms of disease     

DJ-1 may contribute to metastasis of non-small cell lung cancer

Lung cancer is a leading cause of cancer-related death, about 40% human non-small cell lung cancer (NSCLC) patients showed lymph node involvements. However, the precise mechanism for the metastasis is still not fully understood. This study was to analyze the potential molecular mechanism for lung cancer metastasis. In the current study, proteomics analysis by two-dimensional electrophoresis (2-DE) was performed first to identify the differentially expressed protein between the higher metastasis lung adenocarcinoma cell line Anip973 and the lower metastasis lung adenocarcinoma cell line AGZY83-a. We confirmed the result by RT-PCR, immunoblotting and immunocytochemistry analyses in these two cell lines. Then we examined the expression of the differentially expressed protein in tumor tissues of NSCLC patients by immunoblotting and immunohistochemistry analyses. Using 2-DE analysis, we have identified DJ-1 was expressed higher in the higher metastasis Anip973 compared to the parental cell line AGZY83-a, that was confirmed by RT-PCR, immunoblotting and immunocytochemistry analyses. In NSCLC patients?? tumor tissues study, immunoblotting data showed that, DJ-1 expression level was significantly higher in 72.2% (13/18) of NSCLC tissue samples compared to that in paired normal lung tissues (P?=?0.044). Immunohistochemistry analysis demonstrated increased DJ-1 expression in 85 NSCLC tumor tissue samples compared with 7 normal lung tissue samples (P?=?0.044). DJ-1 expression was also found to be significantly correlated with cancer lymphatic metastasis (P?=?0.039). DJ-1 might contribute to the metastasis of NSCLC.     

Noninvasive diagnostics of bladder cancer based on analysis of telomerase activity and expression hTERT and hTR coding its subunits

In order to develop noninvasive diagnostics of bladder cancer (BC), telomerase activity has been examined by means the TRAP method (telomerase repeat amplification protocol) in tumor tissue and urine pellet samples taken from patients with bladder cancer. The levels of relative expression of genes encoding telomerase catalytic subunit (hTERT) and its RNA subunit (hTR) were evaluated by RT-PCR. Telomerase activity and expression of genes encoding its subunits were detected in both tumor tissues and in the urine cell pellet from each BC patient. Results of our study demonstrate possibility of noninvasive BC diagnostics using combination of these methods with sensitivity of 96% and specificity of 100% in the case of telomerase detection and with sensitivity of 80% and specificity of 100% in the case of hTERT detection in urine pellet samples.     

Decrease of PDSS2 expression,a novel tumor suppressor,in non-small cell lung cancer

Background: Prenyl diphosphate synthase subunit 2 (PDSS2) gene has recently been reported as a potential tumor suppressor. The association of PDSS2 and non-small cell lung cancer (NSCLC) has not been known. Methods: To investigate its association with NSCLC, we examined the expression level of PDSS2 in 28 paired clinical samples of non-small cell lung cancer tissues and surrounding normal tissues. Results: PDSS2 was constitutionally expressed in normal lung tissues regardless of sex, race and smoking history. An overall decreased PDSS2 expression was found in the tumor tissues compared to surrounding normal tissues. Decrease in PDSS2 expression was more severe in poorly and poor-to-moderately differentiated lung cancers, while the decrease was not significant in moderately to well-differentiated tumors. Moreover, the expression of PDSS2 decreased more in higher pathological stage, and in patients with lymph node metastasis. The decrease in PDSS2 expression in tumor tissues was not related to sex or histological type of NSCLC, but was related to smoking history. No correlation has been found between PDSS2 and the clinical factors of EGRF, Ki-67 and p53. Conclusion: Taken together, decreased expression of PDSS2 in NSCLC was evident. This is an initial report for the expression of PDSS2 in relation to different factors in lung cancer. Loss of PDSS2 could serve as a potential biomarker in NSCLC development. The role of PDSS2 as a tumor suppressor, and the mechanism of its potential anti-tumor action in NSCLC warrant further investigation.     

Suitability of Surgical Tumor Tissues,Biopsy, or Cytology Samples for Epidermal Growth Factor Receptor Mutation Testing in Non–Small Cell Lung Carcinoma Based on Chinese Population

BACKGROUND: Epidermal growth factor receptor (EGFR) mutation status is crucial in treatment selection for non–small cell lung cancer (NSCLC) patients; however, the detection materials’ availability remains challenging in clinical practice. In this study, we collected surgical resection tissues, lymph node biopsy, and cytological samples for EGFR mutation testing and investigated the associations between gene mutation and clinical characteristics. METHODS: Two hundred and seventy-six NSCLC adenocarcinoma specimens were collected, and highly sensitive amplification refractory mutation system method was implemented for EGFR mutation detection, with clinicopathologic characteristics involved in the final analysis. RESULTS: In the total of 276 samples, 96% (265/276) of tumors obtained evaluable EGFR mutation status, the frequency of mutation was 55.8% (148/265) in all specimens, and three different type samples shared a comparable successful testing rate: 97.4% (38/39) in surgical tumor tissues, 100% (108/108) in lymph node biopsy samples, and 92.2% (119/129) in cytological samples. EGFR mutation was significantly associated with sex, smoking history, lymph node metastasis status (N stage), primary tumor size, testing tissues origin, and sample type (P < .05). Multivariate analysis reconfirmed that smoking history and primary tumor size shared significant correlation with EGFR mutation after adjustment. CONCLUSIONS: Both lymph node biopsy and cytological samples were suitable surrogates for EGFR mutation detection in NSCLC compared with tumor tissues, gene status should be detected widely considering the high EGFR mutation rate, and nonsmoking history together with smaller primary tumor size was an independent indicator of EGFR mutation status.     

Cytosine 5-Hydroxymethylation of the LZTS1 Gene Is Reduced in Breast Cancer

Change of DNA cytosine methylation (5mC) is an early event in the development of cancer, and the recent discovery of a 5-hydroxymethylated form (5hmC) of cytosine suggests a regulatory epigenetic role that might be different from 5-methylcytosine. Here, we aimed at elucidating the role of 5hmC in breast cancer. To interrogate the 5hmC levels of the leucine zipper, putative tumor suppressor 1 (LZTS1) gene in detail, we analyzed 75 primary breast cancer tissue samples from initial diagnosis and 12 normal breast tissue samples derived from healthy persons. Samples were subjected to 5hmC glucosyltransferase treatment followed by restriction digestion and segment-specific amplification of 11 polymerase chain reaction products. Nine of the 11 5′LZTS1 fragments showed significantly lower (fold change of 1.61–6.01, P < .05) 5hmC content in primary breast cancer tissue compared to normal breast tissue samples. No significant differences were observed for 5mC DNA methylation. Furthermore, both LZTS1 and TET1 mRNA expressions were significantly reduced in tumor samples (n = 75, P < .001, Student''s t test), which correlated significantly with 5hmC levels in samples. 5hmC levels in breast cancer tissues were associated with unfavorable histopathologic parameters such as lymph node involvement (P < .05, Student''s t test). A decrease of 5hmC levels of LZTS1, a classic tumor suppressor gene known to influence metastasis in breast cancer progression, is correlated to down-regulation of LZTS1 mRNA expression in breast cancer and might epigenetically enhance carcinogenesis. The study provides support for the novel hypothesis that suggests a strong influence of 5hmC on mRNA expression. Finally, one may also consider 5hmC as a new biomarker.     

Aberrant methylation of p16, HIC1, N33, and GSTP1 in tumor epithelium and tumor-associated cells in prostate cancer

The methylation status of p16, HIC1, N33, and GSTP1, which are involved in prostate carcinogenesis, was studied in prostate tissue samples containing neoplasms. Malignant acini, prostatic intraepithelial neoplasia (PIN) and benign prostatic hyperplasia (BPH) foci, and stroma surrounding glandular structures of each type were detected in histological sections, using laser capture microdissection of prostate tissue. High levels of methylation were found in tumor epithelium and adjacent tumor-associated stromal cells. Epigenetic changes in the stroma are indicative of a major role of tumor microenvironment in cancer development and progression. The methylation status of p16, HIC1, N33, and GSTP1 was also assessed in prostate biopsy material and operative tumor samples without laser capture microdissection. The methylation frequencies of all genes in tumor samples were considerably lower than those in microdissected tumor samples (HIC1, 71% vs. 89%; p16, 22% vs. 78%; GSTP1, 32% vs. 100%; and N33, 20% vs. 33%, respectively). It was concluded that laser capture micro-dissection is required in molecular analysis of tumors of this type.