Molecular targeting of retinoic acid metabolism in neuroblastoma: the role of the CYP26 inhibitor R116010 in vitro and in vivo

Isomerisation to all-trans-retinoic acid (ATRA) is widely accepted as the key mechanism underlying the favourable clinical properties of 13-cis-retinoic acid (13cisRA). As intracellular metabolism of ATRA by CYP26 may result in clinical resistance to 13cisRA, an increase in efficacy may be achieved through modulation of this metabolic pathway. We have evaluated the effect of the CYP26 inhibitor R116010 on retinoid metabolism in neuroblastoma cell lines and a xenograft model. In neuroblastoma cells, which showed a high level of CYP26 induction in response to ATRA, R116010 selectively inhibited ATRA metabolism. In addition, siRNA-mediated knockdown of CYP26 selectively increased ATRA levels and the expression of retinoid-responsive marker genes was potentiated by R116010. Treatment of mice bearing SH-SY5Y xenografts with 13cisRA (100 mg kg(-1)) revealed substantial levels (16%) of intratumoral ATRA after 6 h, despite plasma ATRA levels representing only 1% total retinoids under these conditions. Co-administration of R116010 with 13cisRA in this mouse model resulted in significant increases in plasma ATRA and 13cisRA concentrations. Furthermore, R116010 induced significant decreases in levels of 4-oxo metabolites in hepatic tissue after co-administration with either ATRA or 13cisRA. These data suggest considerable potential for CYP26 inhibitors in the future treatment of neuroblastoma with 13cisRA.     

Differential induction of CYP1A1 and CYP1B1 by benzo[a]pyrene in oral squamous cell carcinoma cell lines and by tobacco smoking in oral mucosa

Polyaromatic hydrocarbons, including benzo[a]pyrene (BP), are major tobacco carcinogens. Their carcinogenic effects require metabolic activation by cytochrome p450 (CYP) enzymes. Relative CYP isoform expression is related to tissue-specific tobacco-related squamous cell carcinoma (SCC) susceptibility. There have been conflicting reports regarding relative CYP1A1 and CYP1B1 oral expression, and information regarding CYP1B1 expression in oral tissues is limited. To quantify BP- and tobacco-induced CYP1A1 and CYP1B1 expression in oral SCC cells and oral mucosa. Study Design: Real-time qPCR was performed to measure (1) BP-induced CYP1A1 and CYP1B1 mRNA expression in seven oral/other head and neck SCC cell lines (2) CYP1A1 and CYP1B1 mRNA expression in gingiva from 22 smokers and 24 nonsmokers. SCC lines exhibited either similar induction of both isoforms or preferential CYP1A1 induction (CYP1A1-to-CYP1B1 ratios 0.8–4.3). In contrast, gingival tissues from smokers exhibited preferential CYP1B1 induction. Marked interindividual variation in CYP1A1 and CYP1B1 expression was observed among smokers. In vitro conditions may not account for factors that modulate expression in vivo. Interindividual variation in inducible CYP1A1 and CYP1B1 expression may account in part for variation in tobacco-related oral SCC risk.     

ANO1蛋白在食管鳞状细胞癌中表达的瘤内异质性

目的:探讨ANO1蛋白在食管鳞状细胞癌组织中表达的瘤内异质性及与肿瘤临床病理学特征的关系.方法:采用组织微阵列免疫组化法检测122例食管鳞癌每例4个肿瘤取材区域及相应正常组织中ANO1蛋白的表达情况,比较每例肿瘤不同取材区域的表达差异,分析ANO1蛋白表达情况与患者临床病理学特征的相关性.结果:ANO1蛋白在所有正常组织中呈阴性表达,而在肿瘤组织的表达阳性率为53.3％(65/122).统计学分析显示,ANO1蛋白的表达情况在有、无淋巴结转移之间及不同肿瘤临床分期之间存在显著性差异(P＜0.05).在阳性表达的病例中,绝大多数(55/65)肿瘤内显示不同程度的ANO1蛋白表达异质性,其中32.3％(21/65)的病例存在不同取材区域之间较大的异质性.结论:ANO1蛋白在正常组织中呈阴性表达,在食管鳞状细胞癌组织中呈部分阳性表达,与临床病理学参数存在显著相关性(P＜0.05).而且,食管鳞状细胞癌ANO1蛋白表达存在明显的瘤内异质性,多位点取材有助于提高ANO1表达的检出率并可减少漏检.     

Cytochrome P450 CYP1B1 activity in renal cell carcinoma

Renal cell carcinoma (RCC) is the most common malignancy of the kidney and has a poor prognosis due to its late presentation and resistance to current anticancer drugs. One mechanism of drug resistance, which is potentially amenable to therapeutic intervention, is based on studies in our laboratory. CYP1B1 is a cytochrome P450 enzyme overexpressed in a variety of malignant tumours. Our studies are now elucidating a functional role for CYP1B1 in drug resistance. Cytochrome P450 reductase (P450R) is required for optimal metabolic activity of CYP1B1. Both CYP1B1 and P450R can catalyse the biotransformation of anticancer drugs at the site of the tumour. In this investigation, we determined the expression of CYP1B1 and P450R in samples of normal kidney and RCC (11 paired normal and tumour and a further 15 tumour samples). The O-deethylation of ethoxyresorufin to resorufin was used to measure CYP1B1 activity in RCC. Cytochrome P450 reductase activity was determined by following the reduction of cytochrome c at 550 nm. The key finding of this study was the presence of active CYP1B1 in 70% of RCC. Coincubation with the CYP1B1 inhibitor alpha-naphthoflavone (10 nM) inhibited this activity. No corresponding CYP1B1 activity was detected in any of the normal tissue examined (n=11). Measurable levels of active P450R were determined in all normal (n=11) and tumour samples (n=26). The presence of detectable CYP1B1, which is capable of metabolising anticancer drugs in tumour cells, highlights a novel target for therapeutic intervention.     

SPHK1在人食管鳞癌组织中的表达及意义

目的：探讨SPHK1（sphingosine kinases 1）在人食管鳞癌组织中的表达及其与临床病理参数之间的关系.方法：采用免疫组化SP法检测40例人食管鳞癌组织、35例癌旁组织中SPHK1的表达.结果：40例人食管鳞癌组织中29例SPHK1高表达,主要定位于细胞质,占所有食管鳞癌组织的72.5％,明显高于SPHK1在癌旁组织中的表达（9/35,25.7％）.且SPHK1的表达与患者是否淋巴结转移（P =0.018）、TNM分期（P=0.035）相关,而与年龄、性别、组织学分化程度、肿瘤位置无关（P＜0.05）.结论：SPHK1在人食管鳞癌组织中高表达,可能作为食管鳞癌患者早期诊断与靶向治疗的分子标记物.     

Functional evaluation of novel single nucleotide polymorphisms and haplotypes in the promoter regions of CYP1B1 and CYP1A1 genes

Interindividual variation in the expression of the carcinogen- and estrogen-metabolizing enzymes cytochrome P4501B1 and 1A1 (CYP1B1 and CYP1A1) has been detected in human lung. To search for polymorphisms with functional consequences for CYP1B1 and CYP1A1 gene expression, we examined 1.5 kb of the promoter region of each gene. Genomic DNA from 21 Caucasian individuals was amplified by polymerase chain reaction (PCR) for direct cycle sequencing. Eight single nucleotide polymorphisms (SNPs) for CYP1B1 and 13 SNPs for CYP1A1 were found. The majority of polymorphisms occurred as multiSNP combinations for individual subjects. The wild-type sequences were cloned into a luciferase reporter construct. The most frequent polymorphisms were then recreated by iterative site-directed mutagenesis, replicating single polymorphisms and multiSNP combinations. These wild-type and variant constructs were functionally evaluated in transient transfection experiments employing exposures to either the index polycyclic aromatic hydrocarbon (PAH) inducer benzo[a]pyrene (B[a]P), a composite mixture of cigarette smoke extract (CSE), or the repressor chemopreventive agent trans-3,4,5-trihydroxystilbene (reseveratrol). Results indicated that all wild-type and variant constructs responded in qualitatively concordant fashion to the inducers and to the repressor. The CYP1B1 haplotypes and the majority of CYP1A1 haplotypes were shown to have no functional consequence, as compared to those of the wild-type promoter sequences. Two constructs of composite polymorphisms of CYP1A1 appeared to result in a statistically significant increase in basal promoter activity (1.38- and 1.50-fold, respectively), but the degree of functional impact was judged unlikely to be biologically important in vivo. We conclude that the observed promoter region polymorphisms in these genes are common, but are of unclear functional consequence.     

Polymorphisms in the CYP1B1 gene are associated with increased risk of prostate cancer

CYP1B1 has been evaluated as a candidate gene for various cancers because of its function in activating environmental procarcinogens and catalysing the conversion of oestrogens to genotoxic catechol oestrogens. To test the hypothesis that genetic polymorphisms in the CYP1B1 gene may associate with the risk for prostate cancer (CaP), we compared the allele, genotype, and haplotype frequencies of 13 single nucleotide polymorphisms (SNPs) of CYP1B1 among 159 hereditary prostate cancer (HPC) probands, 245 sporadic CaP cases, and 222 unaffected men. When each of the SNPs was analysed separately, marginally significant differences were observed for allele frequencies between sporadic cases and controls for three consecutive SNPs (-1001C/T, -263G/A, and -13C/T, P=0.04-0.07). Similarly, marginally significant differences between sporadic cases and controls in the frequency of variant allele carriers were observed for five consecutive SNPs (-1001C/T, -263G/A, -13C/T, +142C/G, and +355G/T, P=0.02-0.08). Interestingly, when the combination of these five SNPs was analysed using a haplotype approach, a larger difference was found (P=0.009). One frequent haplotype (C-G-C-C-G of -1001C/T, -263G/A, -13C/T, +142C/G, and +355G/T) was associated with an increased risk for CaP, while the other frequent haplotype (T-A-T-G-T) was associated with a decreased risk for CaP. These findings suggest that genetic polymorphisms in CYP1B1 may modify the risk for CaP.     

TSLC1在食管鳞癌中的表达及临床意义

目的：研究食管鳞状细胞癌组织中肺癌肿瘤抑制因子1（TSLC1）基因的表达及临床病理意义,探讨其在食管鳞状细胞癌发生、发展过程中的作用。方法：应用免疫组化（SP法）检测50例食管鳞状细胞癌标本及癌旁正常食管组织中TSLC1基因的表达情况,分析其与食管鳞状细胞癌患者临床病理学参数之间的关系。结果：50例食管鳞状细胞癌组织中TSLC1基因表达阳性率为48.0%（24/50）,其表达与癌组织浸润深度、区域淋巴结转移及TNM分期有关（P〈0.05）。与性别、年龄、肿瘤大体分型、肿瘤大小、位置、分化程度无关（P〉0.05）。50例癌旁正常食管组织中TSLC1基因高表达,阳性率为94.0%（47/50）。食管鳞状细胞癌组织中的阳性表达率显著低于癌旁正常食管组织,两者相比差异有统计学意义（P〈0.01）。结论：食管鳞状细胞癌组织中有明显的TSLC1基因的表达缺失,TSLC1蛋白表达缺失与食管鳞状细胞癌的发生、发展有关。     

Genetic variants at 9p21.3 are associated with risk of esophageal squamous cell carcinoma in a Chinese population

Genome‐wide association studies have linked genetic variants at 9p21.3 to the risk of multiple cancers. However, the roles of genetic variants at 9p21.3 in esophageal squamous cell carcinoma (ESCC) development are largely unknown. We evaluated the genetic variants at 9p21.3 reported in cancer genome‐wide association studies with a case–control study including 2139 ESCC cases and 2273 controls in a Chinese population, and measured the mRNA expression levels of MTAP, CDKN2A, CDKN2B, and CDKN2B‐AS1 in paired ESCC tumor and adjacent normal tissues. We found that the G allele of rs7023329 was significantly associated with a decreased risk of ESCC with a per‐allele odds ratio of 0.84 (95% confidence interval, 0.77–0.91; P = 2.95 × 10^?5). The rs7023329‐G allele was related to a high expression of MTAP (P = 0.020). The rs1679013‐C allele was independently associated with an increased risk of ESCC with a per‐allele odds ratio of 1.12 (95% confidence interval, 1.01–1.24; P = 0.039). We also found that the carriers of the risk allele rs1679013‐C had lower expression of CDKN2B than non‐carriers (P = 0.035). CDKN2B was also significantly downregulated in ESCC tumor tissues compared with adjacent normal tissues (P = 3.50×10^?5). Therefore, our findings indicate that genetic variants at 9p21.3 may modulate the expression of MTAP and CDKN2B and contribute to ESCC susceptibility. This may further advance our understanding of the 9p21.3 locus in cancer development.     

CTHRC1在食管鳞癌中的表达及其临床意义

目的 检测CTHRC1在食管鳞癌组织中的表达并分析其临床意义。方法 通过组织芯片和免疫组化技术检测215例食管鳞癌组织中CTHRC1蛋白表达,分析其异常表达与临床病理特征以及预后之间的关系。结果 CTHRC1在癌旁鳞状上皮组织中基本不表达,而在食管鳞癌组织中阳性表达率为61.9％（133／215）。CTHRC1表达与食管鳞癌TNM分期（P＜0.001）、浸润深度（P=0.006）和淋巴结转移（P=0.002）有关。单因素生存分析显示食管癌CTHRC1阳性表达者预后较CTHRC1阴性表达者差（中位OS：57个月 vs. 73个月,P=0.016）;多因素回归分析显示CTHRC1表达不是食管鳞癌独立的预后因素。结论 CTHRC1在食管鳞癌的发生和发展过程中异常活化和表达,可能是食管癌治疗的潜在靶点之一。     

miRNA-26b-3p通过靶向STAT3调控食管鳞状细胞癌细胞的增殖和迁移

目的:观察miR-26b-3p在食管鳞状细胞癌(ESCC)组织中的表达水平及其对ESCC细胞增殖、侵袭和迁移能力的影响,并探讨其分子调控机制.方法:选取河北医科大学第四医院2018年4月1日至2018年12月25日手术切除的ESCC组织及相应癌旁组织各60例,利用qPCR法检测ESCC组织、癌旁组织和ESCC细胞中mi...     

Polymorphisms in the CYP1A1 gene are associated with prostate cancer risk

CYP1A1 is likely to play an important role in the etiology of CaP through its function in activating environmental procarcinogens and catalyzing the oxidative metabolites of estrogens. To test the hypothesis that genetic polymorphisms in the CYP1A1 gene may be associated with the risk for CaP, we compared the allele, genotype and haplotype frequencies of 3 SNPs (3801T>C, 2455A>G and 2453C>A) of CYP1A1 among 159 HPC probands, 245 sporadic CaP cases and 222 unaffected men. Two SNPs (3801T>C and 2455A>G) were each individually associated with CaP risk when the allele and genotype frequencies were compared between CaP patients and unaffected controls. Furthermore, a combined SNP analysis using a haplotype approach revealed an even stronger association in Caucasians. Specifically, 4 major haplotypes (T-A-C, C-A-C, C-G-C and T-A-A) accounted for 99.8% of all observed haplotypes. These 4 haplotypes correspond to the previously described nomenclature (CYP1A1*1A, CYP1A1*2A, CYP1A1*2B and CYP1A1*4). The frequencies of these 4 haplotypes were significantly different among CaP patients and controls. The haplotype T-A-C (CYP1A1*1A) was significantly associated with increased risk for CaP, and the haplotype C-A-C (CYP1A1*2A) was significantly associated with decreased risk for CaP. These findings suggest that genetic polymorphisms in CYP1A1 may modify the risk for CaP.     

BRCA2 loss-of-function germline mutations are associated with esophageal squamous cell carcinoma risk in Chinese

Esophageal squamous cell carcinoma (ESCC) occurs with highest frequency in China with over 90% mortality, highlighting the need for early detection and improved treatment strategies. We aimed to identify ESCC cancer predisposition gene(s). Our study included 4,517 individuals. The discovery phase using whole-exome sequencing (WES) included 186 familial ESCC patients from high-risk China. Targeted gene sequencing validation of 598 genes included 3,289 Henan and 1,228 moderate-risk Hong Kong Chinese. A WES approach identified BRCA2 loss-of-function (LOF) mutations in 3.23% (6/186) familial ESCC patients compared to 0.21% (9/4300) in the ExAC East Asians (odds ratio [OR] = 15.89, p = 2.48 × 10⁻¹⁰). BRCA2 LOF mutation frequency in the combined Henan cohort has significantly higher prevalence (OR = 10.55, p = 0.0035). Results were independently validated in an ESCC Hong Kong cohort (OR = 10.64, p = 0.022). One Hong Kong pedigree was identified to carry a BRCA2 LOF mutation. BRCA2 inactivation in ESCC was via germline LOF mutations and wild-type somatic allelic loss via loss of heterozygosity. Gene-based association analysis, including LOF mutations and rare deleterious missense variants defined with combined annotation dependent depletion score ≥30, confirmed the genetic predisposition role of BRCA2 (OR = 9.50, p = 3.44 × 10⁻⁵), and provided new evidence for potential association of ESCC risk with DNA repair genes (POLQ and MSH2), inflammation (TTC39B) and angiogenesis (KDR). Our findings are the first to provide compelling evidence of the role of BRCA2 in ESCC genetic susceptibility in Chinese, suggesting defective homologous recombination is an underlying cause in ESCC pathogenesis, which is amenable to therapeutic options based on synthetic lethality approaches such as targeting BRCA2 with PARP1 inhibitors in ESCC.     

Loss of miR-200c up-regulates CYP1B1 and confers docetaxel resistance in renal cell carcinoma

Despite high protein expression and enzymatic activity of cytochrome P450 1B1 (CYP1B1) in renal cell cancer (RCC), its functional significance has not been elucidated. Here we explored the functional role and regulatory mechanism of CYP1B1 in RCC. Reduction of CYP1B1 levels fail to prevent in vitro tumorigenicity such as proliferation, apoptosis, and cell cycle progression of RCC cells. Moreover, the expression levels are not associated with tumor type, stage, Fuhrman grade and 5-year survival probability after surgery. Instead, alteration of CYP1B1 expression regulates the chemosensitivity of RCC cells to docetaxel suggesting its critical contribution to the chemoresistance. Additionally, miR-200c, which is significantly down-regulated in RCC regulates CYP1B1 expression and activity. An inverse association was also observed between the expression levels of miR-200c and CYP1B1 protein in RCC tissues. Finally, alteration of miR-200c levels affects the chemosensitivity of RCC cells. Restoration of docetaxel resistance by exogenous expression of CYP1B1 in miR-200c-over-expressing cells indicates that CYP1B1 is a functional target of miR-200c. These results suggest that CYP1B1 up-regulation mediated by low miR-200c is one of the mechanisms underlying resistance of RCC cells to docetaxel. Therefore, expression of CYP1B1 and miR-200c in RCC may be useful as a prediction for docetaxel response.     

miR-503 靶向ERCC1 抑制食管鳞状细胞癌放疗抵抗作用的机制

[摘要] 目的:探讨miR-503 通过调控人切除修复交叉互补基因1（ERCC1）介导食管鳞状细胞癌（ESCC）放疗抵抗作用的分子机制。方法：采用qPCR法检测在放疗抵抗的ESCC肿瘤组织及KYSE140、KYSE140R细胞中miR-503 的表达水平。将miR-503模拟物、miR-503 抑制物或si-ERCC1 转染至KYSE140 和KYSE140R细胞中,经射线照射后,克隆形成实验和CCK-8 实验检测KYSE140R细胞的增殖活力,流式细胞仪检测KYSE140R细胞的凋亡情况,WB实验检测ERCC1 蛋白表达水平的变化。双荧光素酶报告基因验证miR-503 与ERCC1 的靶向关系。结果：miR-503 在ESCC放疗抵抗组织和细胞中均低表达（均P<0.01）。过表达miR-503 可显著抑制KYSE140R细胞增殖并诱导细胞凋亡（均P<0.01）。双荧光素酶报告基因实验证实ERCC1 是miR-503 的靶基因,且miR-503 负调控ERCC1 的表达。过表达miR-503 显著下调KYSE140、KYSE140R 细胞中ERCC1 表达水平（均P<0.01）,并显著抑制细胞增殖活力（均P<0.01）、显著提高细胞凋亡率（均P<0.01）;敲降ERCC1 有类似作用,而同时敲降ERCC1 和miR-503 则逆转以上影响。结论：过表达miR-503 通过靶向ERCC1 调控KYSE140R细胞对放疗的敏感性。     

The tumor suppressive role of NUMB isoform 1 in esophageal squamous cell carcinoma

Esophageal quamous cell carcinoma (ESCC) is the predominant histological type of esophageal carcinoma in Asian populations. To date, few biomarkers have been identified for ESCC. In present study, we found a tumor suppressor, NUMB isoform 1 (NUMB-1), as a promising prognostic biomarker for patients with ESCC. NUMB-1 mRNA was downregulated in 66.7% of primary ESCC tissues when compared with matched adjacent non-tumor tissues. The low expression of NUMB-1 was significantly associated with high tumor recurrence (p=0.029) and poor post-operative overall survival (p=0.016). To further explore the underlying mechanisms by which NUMB-1 regulates ESCC, we demonstrated that ectopic expression of NUMB-1 inhibited cell proliferation through inducing G2/M phase arrest, which was accompanied by an increase in p21 and cyclin B1-cdc2 levels. However, it had no impact on apoptosis of ESCC cells. In addition, overexpression of NUMB-1 prevented epithelial-mesenchymal transition, inhibited invasion of ESCC cells and NOTCH pathway, suppressed Aurora-A activity by preventing phosphorylation of Aurora-A at T288 which resulted in cell cycle arrest. Taken together, our findings suggested NUMB-1 functions as a tumor-suppressor and serves as a prognositc biomarker for ESCC patients; thus, NUMB-1 may be a potential novel therapeutic target for treatment of ESCC.