Hongyu Zhang

A Novel Set of DNA Methylation Markers in Urine Sediments for Sensitive/Specific Detection of Bladder Cancer

Purpose: This study aims to provide a better set of DNA methylation markers in urine sediments for sensitive and specific detection of bladder cancer. Experimental Design: Fifty-nine tumor-associated genes were profiled in three bladder cancer cell lines, a small cohort of cancer biopsies and urine sediments by methylation-specific PCR. Twenty-one candidate genes were then profiled in urine sediments from 132 bladder cancer patients (8 cases for stage 0a; 68 cases for stage I; 50 cases for stage II; 4 cases for stages III; and 2 cases for stage IV), 23 age-matched patients with noncancerous urinary lesions, 6 neurologic diseases, and 7 healthy volunteers. Results: Despite six incidences of four genes reported in 3 of 23 noncancerous urinary lesion patients analyzed, cancer-specific hypermethylation in urine sediments were reported for 15 genes (P < 0.05). Methylation assessment of an 11-gene set (SALL3, CFTR, ABCC6, HPR1, RASSF1A, MT1A, RUNX3, ITGA4, BCL2, ALX4, MYOD1, DRM, CDH13, BMP3B, CCNA1, RPRM, MINT1, and BRCA1) confirmed the existing diagnosis of 121 among 132 bladder cancer cases (sensitivity, 91.7%) with 87% accuracy. Significantly, more than 75% of stage 0a and 88% of stage I disease were detected, indicating its value in the early diagnosis of bladder cancer. Interestingly, the cluster of reported methylation markers used in the U.S. bladder cancers is distinctly different from that identified in this study, suggesting a possible epigenetic disparity between the American and Chinese cases. Conclusions: Methylation profiling of an 11-gene set in urine sediments provides a sensitive and specific detection of bladder cancer.

Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis

BackgroundHepatocellular carcinoma (HCC) presents one of the major health threats in China today. A better understanding of the molecular genetics underlying malignant transformation of hepatocytes is critical to success in the battle against this disease. The methylation state of C5 of the cytosine in the CpG di-nucleotide that is enriched within or near the promoter region of over 50 % of the polymerase II genes has a drastic effect on transcription of these genes. Changes in the methylation profile of the promoters represent an alternative to genetic lesions as causative factors for the tumor-specific aberrant expression of the genes.MethodsWe have used the methylation specific PCR method in conjunction with DNA sequencing to assess the methylation state of the promoter CpG islands of twenty genes. Aberrant expression of these genes have been attributed to the abnormal methylation profile of the corresponding promoter CpG islands in human tumors.ResultsWhile the following sixteen genes remained the unmethylated in all tumor and normal tissues: CDH1, APAF1, hMLH1, BRCA1, hTERC, VHL, RARβ, TIMP3, DAPK1, SURVIVIN, p14ARF, RB1, p15INK4b, APC, RASSF1c and PTEN, varying degrees of tumor specific hypermethylation were associated with the p16INK4a , RASSF1a, CASP8 and CDH13 genes. For instance, the p16INK4a was highly methylated in HCC (17/29, 58.6%) and less significantly methylated in non-cancerous tissue (4/29. 13.79%). The RASSF1a was fully methylated in all tumor tissues (29/29, 100%), and less frequently methylated in corresponding non-cancerous tissue (24/29, 82.75%).ConclusionsFurthermore, co-existence of methylated with unmethylated DNA in some cases suggested that both genetic and epigenetic (CpG methylation) mechanisms may act in concert to inactivate the p16INK4a and RASSF1a in HCC. Finally, we found a significant association of cirrhosis with hypermethylation of the p16INK4a and hypomethylation of the CDH13 genes. For the first time, the survey was carried out on such an extent that it would not only provide new insights into the molecular mechanisms underscoring the aberrant expression of the genes in this study in HCC, but also offer essential information required for a good methylation-based diagnosis of HCC.

MicroRNA 34c Gene Down-regulation via DNA Methylation Promotes Self-renewal and Epithelial-Mesenchymal Transition in Breast Tumor-initiating Cells

Background: The mechanisms for miRNA dysregulation in BT-ICs remain obscure. Results: Single hypermethylated CpG site in the promoter region of miR-34c gene repressed miR-34c expression by reducing DNA binding activities of Sp1 and promoted self-renewal and EMT of BT-ICs. Conclusion: Single hypermethylated CpG site in the promoter region contributes to the reduction of microRNA in BT-ICs. Significance: Methylation regulates the expression of microRNA in BT-ICs. Tumor-initiating cells (T-ICs), a subpopulation of cancer cells with stem cell-like properties, are related to tumor relapse and metastasis. Our previous studies identified a distinct profile of microRNA (miRNA) expression in breast T-ICs (BT-ICs), and the dysregulated miRNAs contribute to the self-renewal and tumorigenesis of these cells. However, the underlying mechanisms for miRNA dysregulation in BT-ICs remain obscure. In the present study, we demonstrated that the expression and function of miR-34c were reduced in the BT-ICs of MCF-7 and SK-3rd cells, a breast cancer cell line enriched for BT-ICs. Ectopic expression of miR-34c reduced the self-renewal of BT-ICs, inhibited epithelial-mesenchymal transition, and suppressed migration of the tumor cells via silencing target gene Notch4. Furthermore, we identified a single hypermethylated CpG site in the promoter region of miR-34c gene that contributed to transcriptional repression of miR-34c in BT-ICs by reducing DNA binding activities of Sp1. Therefore, miR-34c reduction in BT-ICs induced by a single hypermethylated CpG site in the promoter region promotes self-renewal and epithelial-mesenchymal transition of BT-ICs.

DNA Methylation-regulated miR-193a-3p Dictates Resistance of Hepatocellular Carcinoma to 5-Fluorouracil via Repression of SRSF2 Expression

Background: Chemoresistance prevents effective therapy of hepatocellular carcinoma (HCC). Results: Genomic and mechanistic studies suggested the role of miR-193a-3p via SRSF2 mediates up-regulation of the proapoptotic splicing form of caspase 2 in HCC 5-FU resistance. Conclusion: We identify a novel molecular mechanism underlying 5-FU resistance in HCC. Significance: These molecular events identified provide a set of prognostic markers for future rational 5-FU therapy in HCC. Chemoresistance prevents effective cancer therapy and is rarely predictable prior to treatment, particularly for hepatocellular carcinoma (HCC). Following the chemoresistance profiling of eight HCC cell lines to each of nine chemotherapeutics, two cell lines (QGY-7703 as a sensitive and SMMC-7721 as a resistant cell line to 5-fluorouracil (5-FU) treatment) were systematically studied for mechanistic insights underpinning HCC 5-FU chemoresistance. Genomic profiling at both DNA methylation and microRNA (miR) levels and subsequent mechanistic studies illustrate a new mechanism for how DNA methylation-regulated miR-193a-3p dictates the 5-FU resistance of HCC cells via repression of serine/arginine-rich splicing factor 2 (SRSF2) expression. In turn, SRSF2 preferentially up-regulates the proapoptotic splicing form of caspase 2 (CASP2L) and sensitizes HCC cells to 5-FU. Forced changes of miR-193a-3p level reverse all of the phenotypic features examined, including cell proliferation, cell cycle progression, and 5-FU sensitivity, in cell culture and in nude mice. Importantly, the siRNA-mediated repression of SRSF2 phenocopies all of the miR-193a-3p mimic-triggered changes in QGY-7703. This newly identified miR-193a-3p-SRSF2 axis highlights a new set of companion diagnostics required for optimal 5-FU therapy of HCC, which involve assaying both the DNA methylation state of the miR-193a gene and the expression of miR-193a-3p and SRSF2 and the relative level of the proapoptotic versus antiapoptotic splicing forms of caspase 2 in clinical samples.

DNA methylation mediated repression of miR-886-3p predicts poor outcome of human small cell lung cancer

Small cell lung cancer (SCLC) is one of the most aggressive types of cancer, yet the pathologic mechanisms underlying its devastating clinical outcome remain elusive. In this report, we surveyed 924 miRNA (miR) for their expressions in the formalin-fixed paraffin-embedded specimens from 42 patients with SCLC, and found that the downregulated miR-886-3p is closely correlated with the shorter survival of SCLC. This correlation was validated with another 40 cases. It was further discovered that loss of miR-886-3p expression was mediated by DNA hypermethylation of its promoter in both cultured SCLC cells and tumor samples. Moreover, miR-886-3p potently repressed cell proliferation, migration, and invasion of NCI-H446 cell in cell culture via suppression of the expression of its target genes: PLK1 and TGF-β1 at posttranscription levels. Forced upregulation of miR-886-3p greatly inhibited in vivo tumor growth, bone/muscle invasion, and lung metastasis of NCI-H446 cells. This newly identified miR-886-3p-PLK1/TGF-β1 nexus that modulates SCLC aggression suggests that both loss of miR-886-3p expression and hypermethylation of the miR-886 promoter are the promising indicators for poor outcome of as well as new therapeutic targets for SCLC.

miR-193a-3p regulates the multi-drug resistance of bladder cancer by targeting the LOXL4 gene and the Oxidative Stress pathway

BackgroundChemoresistance is a major obstacle to the curative cancer chemotherapy and presents one of the most formidable challenges in both research and management of cancer.ResultsFrom the detailed studies of a multi-chemosensitive (5637) versus a chemoresistant (H-bc) bladder cancer cell lines, we showed that miR-193a-3p [GenBank: NR_029710.1] promotes the multi-chemoresistance of bladder cancer cells. We further demonstrated that lysyl oxidase-like 4 (LOXL4) gene [GenBank: NM_032211.6] is a direct target of miR-193a-3p and executes the former’s impact on bladder cancer chemoresistance. The Oxidative Stress pathway activity is drastically affected by a forced reversal of miR-193a-3p or LOXL4 levels in cell and may act at the downstream of LOXL4 gene to relay the miR-193a-3p’s impact on the multi-chemoresistance in both cultured cells and the tumor xenografts in nude mice.ConclusionsIn addition to a new mechanistic insight, our results provide a set of the essential genes in this newly identified miR-193a-3p/LOXL4/Oxidative Stress axis as the diagnostic targets for a guided anti-bladder cancer chemotherapy.

MiR-193a-3p promotes the multi-chemoresistance of bladder cancer by targeting the HOXC9 gene

Chemoresistance prevents the curative cancer chemotherapy and presents a formidable challenge for both cancer researchers and clinicians. We have previously shown that miR-193a-3p promotes the multi-chemoresistance of bladder cancer cells via repressing its three target genes: SRSF2, PLAU and HIC2. Here, we showed that as a new direct target, the homeobox C9 (HOXC9) gene also executes the promoting effect of miR-193a-3p on the bladder cancer chemoresistance from a systematic study of multi-chemosensitive (5637) and resistant (H-bc) bladder cancer cell lines in both cell culture and tumor-xenograft/nude mice system. Paralleled with the changes in the drug-triggered cell death, the activities of both DNA damage response and oxidative stress pathways were drastically altered by a forced reversal of miR-193a-3p or HOXC9 levels in bladder cancer cells. In addition to a new mechanistic insight, our results provide a set of the essential genes in the miR-193a-3p/HOXC9/DNA damage response/oxidative stress pathway axis as the diagnostic targets for the guided anti-bladder cancer chemotherapy.

The miR-193a-3p regulated PSEN1 gene suppresses the multi-chemoresistance of bladder cancer

Chemoresistance prevents the curative cancer therapy, our understanding of which remains inadequate. Among the differentially expressed genes between the chemosensitive (5637) and chemoresistant (H-bc) bladder cancer cell lines, the expression level of the PSEN1 gene (presenilin 1), a key component of the γ-secretase, is negatively correlated with chemoresistance. A small interfering RNA mediated repression of the PSEN1 gene suppresses cell apoptosis and de-sensitizes 5637 cells, while overexpression of the presenilin 1 sensitizes H-bc cells to the drug-triggered cell death. As a direct target of microRNA-193a-3p that promotes the multi-chemoresistance of the bladder cancer cell, PSEN1 acts as an important executor for the microRNA-193a-3ps positive impact on the multi-chemoresistance of bladder cancer, probably via its activating effect on DNA damage response pathway. In addition to the mechanistic insights, the key players in this microRNA-193a-3p/PSEN1 axis are likely the diagnostic and/or therapeutic targets for an effective chemotherapy of bladder cancer.

Hypermethylated SFRP1, but none of other nine genes “informative” for western countries, is valuable for bladder cancer detection in mainland China

PurposeA 11-gene set by methylation-specific PCR in urine sediments for sensitive/specific detection of bladder cancer has been identified previously. In this study, we have evaluated 10 DNA methylation biomarkers that have been reported informative in western countries for bladder cancer diagnosis for a better set.Materials and methodsThe promoter CpG Islands of the following 10 genes: CDH1, FANCF, LOXL1, LOXL4, p16INK4, SFRP1, SOX9, TIG1, TIMP3, and XAF1 have been subjected to methylation-specific PCR analysis in the DNA of 2 bladder cancer cell lines, 2 normal bladder tissues and urine sediments of 82 bladder cancer patients, 15 non-cancerous urogenital patients and 5 healthy volunteers.ResultsBoth XAF1 and LOXL1 genes were heterozygously methylated in the normal bladder tissues, showing no cancer state specificity. While the hypermethylated states were detected in urine sediments of bladder cancer at a frequency not less than 2.4% (2/82 cases), nine genes were also methylated in the patients of the non-cancerous urogenital diseases. The methylated SFRP1 was detected in 36.6% (30/82 cases) of bladder cancer and 6.7% (1/15 cases) of non-cancerous urogenital diseases, showing the bladder cancer specificity.ConclusionsInclusion of the SFRP1 gene into a set of 11 genes has improved the bladder cancer detection. The insufficiency of predicting disease onset in this study with the previously recommended targets in western countries suggests a possible disease disparity between these two populations. Alternatively, the tissue-specific methylation might be mistaken as the cancer specific in the studies where no non-cancerous lesion controls were involved.

Preclinical optimization of a broad-spectrum anti-bladder cancer tri-drug regimen via the Feedback System Control (FSC) platform

Therapeutic outcomes of combination chemotherapy have not significantly advanced during the past decades. This has been attributed to the formidable challenges of optimizing drug combinations. Testing a matrix of all possible combinations of doses and agents in a single cell line is unfeasible due to the virtually infinite number of possibilities. We utilized the Feedback System Control (FSC) platform, a phenotype oriented approach to test 100 options among 15,625 possible combinations in four rounds of assaying to identify an optimal tri-drug combination in eight distinct chemoresistant bladder cancer cell lines. This combination killed between 82.86% and 99.52% of BCa cells, but only 47.47% of the immortalized benign bladder epithelial cells. Preclinical in vivo verification revealed its markedly enhanced anti-tumor efficacy as compared to its bi- or mono-drug components in cell line-derived tumor xenografts. The collective response of these pathways to component drugs was both cell type- and drug type specific. However, the entire spectrum of pathways triggered by the tri-drug regimen was similar in all four cancer cell lines, explaining its broad spectrum killing of BCa lines, which did not occur with its component drugs. Our findings here suggest that the FSC platform holdspromise for optimization of anti-cancer combination chemotherapy.