Jingde Zhu

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.

Stabilization of PML nuclear localization by conjugation and oligomerization of SUMO-3

The PML gene of acute promyelocytic leukemia (APL) encodes a cell-growth and tumor suppressor. PML localizes to discrete nuclear bodies (NBs) that are disrupted in APL cells, resulting from a reciprocal chromosome translocation t (15;17). Here we show that the nuclear localization of PML is also regulated by SUMO-3, one of the three recently identified SUMO isoforms in human cells. SUMO-3 bears similar subcellular distribution to those of SUMO-1 and -2 in the interphase nuclear body, which is colocalized with PML protein. However, both SUMO-2 and -3 are also localized to nucleoli, a region lacking SUMO-1. Immunoprecipitated PML protein bears SUMO-3 moiety in a covalently modified form, supporting the notion that PML is conjugated by SUMO-3. To determine the functional relevance of SUMO-3 conjugation on PML molecular dynamics, we suppressed SUMO-3 protein expression using a siRNA-mediated approach. Depletion of SUMO-3 markedly reduced the number of PML-containing NBa and their integrity, which is rescued by exogenous expression of SUMO-3 but not SUMO-1 or SUMO-2. The specific requirement of SUMO-3 for PML nuclear localization is validated by expression of SUMO-3 conjugation defective mutant. Moreover, we demonstrate that oligomerization of SUMO-3 is required for PML retention in the nucleus. Taken together, our studies provide first line of evidence showing that SUMO-3 is essential for PML localization and offer novel insight into the pathobiochemistry of APL.

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.

The mitotic checkpoint kinase NEK2A regulates kinetochore microtubule attachment stability

Loss or gain of whole chromosome, the form of chromosome instability commonly associated with cancers is thought to arise from aberrant chromosome segregation during cell division. Chromosome segregation in mitosis is orchestrated by the interaction of kinetochores with spindle microtubules. Our studies show that NEK2A is a kinetochore-associated protein kinase essential for faithful chromosome segregation. However, it was unclear how NEK2A ensures accurate chromosome segregation in mitosis. Here we show that NEK2A-mediated Hec1 (highly expressed in cancer) phosphorylation is essential for faithful kinetochore microtubule attachments in mitosis. Using phospho-specific antibody, our studies show that NEK2A phosphorylates Hec1 at Ser165 during mitosis. Although such phosphorylation is not required for assembly of Hec1 to the kinetochore, expression of non-phosphorylatable mutant Hec1S165 perturbed chromosome congression and resulted in a dramatic increase in microtubule attachment errors, including syntelic and monotelic attachments. Our in vitro reconstitution experiment demonstrated that Hec1 binds to microtubule in low affinity and phosphorylation by NEK2A, which prevents aberrant kinetochore-microtubule connections in vivo, increases the affinity of the Ndc80 complex for microtubules in vitro. Thus, our studies illustrate a novel regulatory mechanism in which NEK2A kinase operates a faithful chromosome attachment to spindle microtubule, which prevents chromosome instability during cell division.

Phosphorylation of HsMis13 by Aurora B kinase is essential for assembly of functional kinetochore.

Chromosome movements in mitosis are orchestrated by dynamic interactions between spindle microtubules and the kinetochore, a multiprotein complex assembled onto centromeric DNA of the chromosome. Here we show that phosphorylation of human HsMis13 by Aurora B kinase is required for functional kinetochore assembly in HeLa cells. Aurora B interacts with HsMis13 in vitro and in vivo. HsMis13 is a cognate substrate of Aurora B, and the phosphorylation sites were mapped to Ser-100 and Ser-109. Suppression of Aurora B kinase by either small interfering RNA or chemical inhibitors abrogates the localization of HsMis13 but not HsMis12 to the kinetochore. In addition, non-phosphorylatable but not wild type and phospho-mimicking HsMis13 failed to localize to the kinetochore, demonstrating the requirement of phosphorylation by Aurora B for the assembly of HsMis13 to kinetochore. In fact, localization of HsMis13 to the kinetochore is spatiotemporally regulated by Aurora B kinase, which is essential for recruiting outer kinetochore components such as Ndc80 components and CENP-E for functional kinetochore assembly. Importantly, phospho-mimicking mutant HsMis13 restores the assembly of CENP-E to the kinetochore, and tension developed across the sister kinetochores in Aurora B-inhibited cells. Thus, we reason that HsMis13 phosphorylation by Aurora B is required for organizing a stable bi-oriented microtubule kinetochore attachment that is essential for faithful chromosome segregation in mitosis.

Rho Kinase Phosphorylation Promotes Ezrin-Mediated Metastasis in Hepatocellular Carcinoma

During progression of hepatocellular carcinoma, multiple genetic and epigenetic alterations act to posttranslationally modulate the function of proteins that promote cancer invasion and metastasis. To define such abnormalities that contribute to liver cancer metastasis, we carried out a proteomic comparison of primary hepatocellular carcinoma and samples of intravascular thrombi from the same patient. Mass spectrometric analyses of the liver cancer samples revealed a series of acidic phospho-isotypes associated with the intravascular thrombi samples. In particular, we found that Thr567 hyperphosphorylation of the cytoskeletal protein ezrin was tightly correlated to an invasive phenotype of clinical hepatocellular carcinomas and to poor outcomes in tumor xenograft assays. Using phospho-mimicking mutants, we showed that ezrin phosphorylation at Thr567 promoted in vitro invasion by hepatocarcinoma cells. Phospho-mimicking mutant ezrinT567D, but not the nonphosphorylatable mutant ezrinT567A, stimulated formation of membrane ruffles, suggesting that Thr567 phosphorylation promotes cytoskeletal-membrane remodeling. Importantly, inhibition of Rho kinase, either by Y27632 or RNA interference, resulted in inhibition of Thr567 phosphorylation and a blockade to cell invasion, implicating Rho kinase-ezrin signaling in hepatocellular carcinoma cell invasion. Our findings suggest a strategy to reduce liver tumor metastasis by blocking Rho kinase-mediated phosphorylation of ezrin.

Use of DNA methylation for cancer detection: Promises and challenges

DNA methylation is an important and reversible epigenetic modification, which regulates genomic stability and cellular plasticity. Faithful DNA methylation is essential for mammalian development and health, and perturbation of methylation dynamics contributes to the development of disease, including cancer. The discovery and validation of the biological indicators (biomarkers) for human cancers are essential steps in the development of methods for accurate subtype classification and outcome prediction in clinic oncology. While genetics (SNP, LOH and mutation) and expression profiling (mRNA and protein) of biomarkers have been extensively assessed for cancer diagnosis and prognosis, the potential for using epigenetic fingerprints for early diagnosis and outcome prediction in clinic oncology propels the exploration of using DNA methylation as a biomarker for cancer prognosis. Both the promises and challenges to realizing the clinical utility of the DNA methylation in cancer management are discussed in this review.