Lukas Vrba | University of Arizona (original) (raw)

Papers by Lukas Vrba

BackgroundmiRNAs are small, endogenous non-coding RNAs approximately 22 nucleotides in length tha... more BackgroundmiRNAs are small, endogenous non-coding RNAs approximately 22 nucleotides in length that account for approximately 1% of the genome and play key regulatory roles in multiple signaling pathways. mir-218-1 is an intronic miRNA located within intron 15 of the SLIT2 gene. Public datasets showed enrichment of H3K4me3 within intron 4 of the SLIT2 gene. Therefore, we sought to determine the genomic location and transcriptional regulatory elements of the mir-218-1 candidate alternative promoter in pancreatic ductal adenocarcinoma.MethodsExpression of mir-218 was evaluated in a panel of pancreatic ductal adenocarcinoma cell lines. The mir-218-1 candidate alternative promoter was characterized by chromatin immunoprecipitation, Sequenom, and luciferase assays. Transcriptional regulation of the mir-218-1 candidate alternative promoter was assessed using chromatin immunoprecipitation and an inhibitor to NF-kB.ResultsWe found that expression of mir-218-1 does not correlate with SLIT2 ex...

Journal of Clinical Oncology, 2020

e15546 Background: Pancreatic cancer (PC) is a high mortality malignancy typically found when cur... more e15546 Background: Pancreatic cancer (PC) is a high mortality malignancy typically found when curative surgery is not an option. Liquid biopsies are a minimally invasive option that may allow earlier detection of PC. Using a bioinformatic analysis of TCGA and GEO we identified a novel set of blood-based biomarkers to detect PC with high sensitivity and specificity (AUROC = 0.999) (Vrba et al, Epigenetics 2019). Here we tested the ability of this DNA methylation signature (MS) to distinguish metastatic PC from pancreatic cysts (Cy) and healthy controls (HC) through quantitative DNA methylation analysis of cell free DNA (cfDNA). Methods: A 10 gene DNA methylation marker set that identifies PC was used to evaluate the cfDNA component of the plasma of PC patients, Cy patients and HC. The ROC analysis and AUC calculations were performed using the R library pROC (Robin et al, BMC Bioinformatics 2011). Plasma samples were collected in cfDNA collection tubes from patients with benign Cy, PC...

Cancer Research, 2015

Guidance molecules from the Slit gene family were originally described as cues for the directiona... more Guidance molecules from the Slit gene family were originally described as cues for the directional guidance of axons in the developing nervous system. More recently, members of these families have been found to play critical roles in epithelial development, angiogenesis and tumorigenesis. SLIT2 has properties of a potential tumor suppressor, is mutated at a low frequency in pancreatic tumors and is epigenetically silenced in many cancers. mir-218-1 is an intronic microRNA found within intron 15 of the SLIT2 gene. We propose that cell intrinsic mir-218-1 expression and function is independent of SLIT2 signaling in pancreatic ductal adenocarcinoma. This dynamic expression of mir-218-1 can inhibit tumor dissemination following the conversion of precursor lesions to invasive carcinoma. In normal pancreatic tissue, SLIT2 is highly expressed in the ductal compartment and expressed at a reduced level in the majority of primary pancreatic ductal adenocarcinomas (PDACs). We determined that K...

Neoplasia, 2008

Aberrations of p53 occur in most, if not all, human cancers. In breast cancer, p53 mutation is th... more Aberrations of p53 occur in most, if not all, human cancers. In breast cancer, p53 mutation is the most common genetic defect related to a single gene. Immortalized human mammary epithelial cells resemble the earliest forms of aberrant breast tissue growth but do not express many malignancy-associated phenotypes. We created a model of human mammary epithelial tumorigenesis by infecting hTERT-HME1 immortalized human mammary epithelial cells expressing wild-type p53 with four different mutant p53 constructs to determine the role of p53 mutation on the evolution of tumor phenotypes. We demonstrate that different mutant/wild-type p53 heterozygous models generate loss of function, dominant negative activity, and a spectrum of gain of function activities that induce varying degrees of invasive potential. We suggest that this model can be used to elucidate changes that occur in early stages of human mammary epithelial tumorigenesis. These changes may constitute novel biomarkers or reveal novel treatment modalities that could inhibit progression from primary to metastatic breast disease.

PLoS ONE, 2010

Background: The microRNA-200 family participates in the maintenance of an epithelial phenotype an... more Background: The microRNA-200 family participates in the maintenance of an epithelial phenotype and loss of its expression can result in epithelial to mesenchymal transition (EMT). Furthermore, the loss of expression of miR-200 family members is linked to an aggressive cancer phenotype. Regulation of the miR-200 family expression in normal and cancer cells is not fully understood. Methodology/Principal Findings: Epigenetic mechanisms participate in the control of miR-200c and miR-141 expression in both normal and cancer cells. A CpG island near the predicted mir-200c/mir-141 transcription start site shows a striking correlation between miR-200c and miR-141 expression and DNA methylation in both normal and cancer cells, as determined by MassARRAY technology. The CpG island is unmethylated in human miR-200/miR-141 expressing epithelial cells and in miR-200c/miR-141 positive tumor cells. The CpG island is heavily methylated in human miR-200c/miR-141 negative fibroblasts and miR-200c/miR-141 negative tumor cells. Mouse cells show a similar inverse correlation between DNA methylation and miR-200c expression. Enrichment of permissive histone modifications, H3 acetylation and H3K4 trimethylation, is seen in normal miR-200c/miR-141-positive epithelial cells, as determined by chromatin immunoprecipitation coupled to real-time PCR. In contrast, repressive H3K9 dimethylation marks are present in normal miR-200c/miR-141negative fibroblasts and miR-200c/miR-141 negative cancer cells and the permissive histone modifications are absent. The epigenetic modifier drug, 5-aza-2'-deoxycytidine, reactivates miR-200c/miR-141 expression showing that epigenetic mechanisms play a functional role in their transcriptional control. 3 Conclusions/Significance: We report that DNA methylation plays a role in the normal cell type-specific expression of miR-200c and miR-141 and this role appears evolutionarily conserved, since similar results were obtained in mouse. Aberrant DNA methylation of the miR-200c/141 CpG island is closely linked to their inappropriate silencing in cancer cells. Since the miR-200c cluster plays a significant role in EMT, our results suggest an important role for DNA methylation in the control of phenotypic conversions in normal cells.

Journal of Agricultural and Food Chemistry, 2005

The concentrations of prenylated chalcones and bitter acids were analyzed in Czech hop varieties.... more The concentrations of prenylated chalcones and bitter acids were analyzed in Czech hop varieties. The highest levels of (xanthohumol + desmethylxanthohumol) (0.97%, m/m) and of total bitter acids (17.19%, m/m) were observed for cv. Agnus. The concentration ratios of bitter acids to prenylated chalcones varied depending on the genotype, thereby suggesting genetic determination by different set(s) of structural and regulatory genes. Promoter elements of the chs_H1 gene encoding a "true" chalcone synthase, a candidate gene to co-determine the biosynthesis of prenylated chalcones, were analyzed, and several boxes for cis-regulatory elements including Myb transcription factors were discovered. A cDNA library was established from glandular tissue-enriched cones of cv. Osvald's clone 72 and used to screen for Myb regulatory elements. The cDNA of the first Myb regulatory factor from hop, called HlMyb1, was cloned and analyzed. The HlMyb1 open reading frame encodes 272 amino acids (29.8 kDa), and the protein showed highest homology to the light-regulated factor AtMyb68 from Arabidopsis thaliana within the Myb domain, whereas there was no significant homology with known MYB proteins outside this domain. Unlike AtMyb68, which is expressed in mature leaves, HlMyb1 is strongly expressed in hop inflorescences and could participate in the regulation of developmental processes involved in the production of hop cones and bioactive secondary metabolites.

Journal of Agricultural and Food Chemistry, 2006

Screening of a cDNA library of the hop cv. Osvald's 72 and genomic cloning were used to isolate m... more Screening of a cDNA library of the hop cv. Osvald's 72 and genomic cloning were used to isolate members of an oligofamily of chs_H1 genes that codetermine the biosynthesis of prenylated chalcones known to be valuable medicinal compounds present in hop (Humulus lupulus L.). chs_H1 oligofamily members showed more than 99% and 98% identity on nucleotide and amino acid levels, respectively, and retained all conserved amino acids that form the catalytic center characteristic for "true" chalcone synthases. The chs_H1 promoter exhibited low sequence variability in addition to conservation of all predicted cis-regulatory elements. Possible transactivation of the chs_H1 gene with the transcription factor PAP1 from Arabidopsis thaliana was assayed using Agrobacterium tumefaciens infiltrations of Nicotiana benthamiana and Petunia hybrida plants. Infiltration of N. benthamiana leaves with chs_H1 promoter/GUS chimeras led to a 24.8-fold increase of the GUS activity when coinfiltrated with the pap1 gene. Coinfiltration of the "native" chs_H1 gene with pap1 led to an increased accumulation of chs_H1 mRNA as observed by semiquantitative reverse transcription-polymerase chain reaction. Transgenic lines of P. hybrida expressing the pap1 gene showed unusual patterns of UV-A-inducible pigmentation and anthocyanin accumulation in parenchymatic and medulla cells. Infiltration of transgenic leaves of P. hybrida with chs_H1 and pap1 genes arranged as a tandem led to quick pigmentation within 12 h after UV-A irradiation. It is indicated that the chs_H1 promoter contains functional element(s) mediating an efficient response to PAP1 expression and UV-A irradiation. UV-A also induced chs_H1 mRNA and accumulation of flavonol glycosides in hop leaves. It can be expected that the PAP1 factor could significantly influence the expression of the chs_H1 oligofamily in transgenic hop and modify the hop metabolome.

Clinical Epigenetics

We tested the ability of a novel DNA methylation biomarker set to distinguish metastatic pancreat... more We tested the ability of a novel DNA methylation biomarker set to distinguish metastatic pancreatic cancer cases from benign pancreatic cyst patients and to monitor tumor dynamics using quantitative DNA methylation analysis of cell-free DNA (cfDNA) from blood samples. The biomarkers were able to distinguish malignant cases from benign disease with high sensitivity and specificity (AUC = 0.999). Furthermore, the biomarkers detected a consistent decline in tumor-derived cfDNA in samples from patients undergoing chemotherapy. The study indicates that our liquid biopsy assay could be useful for management of pancreatic cancer patients.

Epigenetics

Cancer-specific DNA methylation from the tumor derived fraction of cell free DNA found in blood s... more Cancer-specific DNA methylation from the tumor derived fraction of cell free DNA found in blood samples could be used for minimally invasive detection and monitoring of cancer. The knowledge of marker regions with cancer-specific DNA methylation is necessary to the success of such a process. We analyzed the largest cancer DNA methylation dataset available-TCGA Illumina HumanMethylation450 data with over 8,500 tumors-in order to find cancer-specific DNA methylation markers for most common human cancers. First, we identified differentially methylated regions for individual cancer types and those were further filtered against data from normal tissues to obtain marker regions with cancer-specific methylation, resulting in a total of 1,250 hypermethylated and 584 hypomethylated marker CpGs. From hypermethylated markers, optimal sets of six markers for each TCGA cancer type were chosen that could identify most tumors with high specificity and sensitivity [area under the curve (AUC): 0.969-1.000] and a universal 12 marker set that can detect tumors of all 33 TCGA cancer types (AUC >0.84). In addition to hundreds of new DNA methylation markers, our approach also identified markers that are in current clinical use, SEPT9 and GSTP1, indicating the validity of our approach and a significant potential utility for the newly discovered markers. The hypermethylated markers are linked to polycomb associated loci and a significant fraction of the discovered markers is within noncoding RNA genes; one of the best markers is MIR129-2. Future clinical testing of herein discovered markers will confirm new markers that will improve minimally invasive diagnosis and monitoring for multiple cancers.

Epigenetics

Identification of cancer-specific methylation of DNA released by tumours can be used for noninvas... more Identification of cancer-specific methylation of DNA released by tumours can be used for noninvasive diagnostics and monitoring. We previously reported in silico identification of DNA methylation loci specifically hypermethylated in common human cancers that could be used as epigenetic biomarkers. Using DNA methylation specific qPCR we now clinically tested a group of these cancer-specific loci on cell-free DNA (cfDNA) extracted from the plasma fraction of blood samples from healthy controls and non-small cell lung cancer (NSCLC) patients. These DNA methylation biomarkers distinguish lung cancer cases from controls with high sensitivity and specificity (AUC = 0.956), and furthermore, the signal from the markers correlates with tumour size and decreases after surgical resection of lung tumours. Presented observations suggest the clinical value of these DNA methylation biomarkers for NSCLC diagnostics and monitoring. Since we successfully validated the biomarkers using independent DNA methylation data from multiple additional common carcinoma cohorts (bladder, breast, colorectal, oesophageal, head and neck, pancreatic or prostate cancer) we predict that these DNA methylation biomarkers will detect additional carcinoma types from plasma samples as well.

PLoS ONE, 2010

Background: The microRNA-200 family participates in the maintenance of an epithelial phenotype an... more Background: The microRNA-200 family participates in the maintenance of an epithelial phenotype and loss of its expression can result in epithelial to mesenchymal transition (EMT). Furthermore, the loss of expression of miR-200 family members is linked to an aggressive cancer phenotype. Regulation of the miR-200 family expression in normal and cancer cells is not fully understood. Methodology/Principal Findings: Epigenetic mechanisms participate in the control of miR-200c and miR-141 expression in both normal and cancer cells. A CpG island near the predicted mir-200c/mir-141 transcription start site shows a striking correlation between miR-200c and miR-141 expression and DNA methylation in both normal and cancer cells, as determined by MassARRAY technology. The CpG island is unmethylated in human miR-200/miR-141 expressing epithelial cells and in miR-200c/miR-141 positive tumor cells. The CpG island is heavily methylated in human miR-200c/miR-141 negative fibroblasts and miR-200c/miR-141 negative tumor cells. Mouse cells show a similar inverse correlation between DNA methylation and miR-200c expression. Enrichment of permissive histone modifications, H3 acetylation and H3K4 trimethylation, is seen in normal miR-200c/miR-141-positive epithelial cells, as determined by chromatin immunoprecipitation coupled to real-time PCR. In contrast, repressive H3K9 dimethylation marks are present in normal miR-200c/miR-141negative fibroblasts and miR-200c/miR-141 negative cancer cells and the permissive histone modifications are absent. The epigenetic modifier drug, 5-aza-29-deoxycytidine, reactivates miR-200c/miR-141 expression showing that epigenetic mechanisms play a functional role in their transcriptional control.

Clinical Epigenetics, 2022

We tested the ability of a novel DNA methylation biomarker set to distinguish metastatic pancreat... more We tested the ability of a novel DNA methylation biomarker set to distinguish metastatic pancreatic cancer cases from benign pancreatic cyst patients and to monitor tumor dynamics using quantitative DNA methylation analysis of cellfree DNA (cfDNA) from blood samples. The biomarkers were able to distinguish malignant cases from benign disease with high sensitivity and specificity (AUC = 0.999). Furthermore, the biomarkers detected a consistent decline in tumor-derived cfDNA in samples from patients undergoing chemotherapy. The study indicates that our liquid biopsy assay could be useful for management of pancreatic cancer patients.

F1000Research

Tumor-specific DNA methylation can be used for cancer diagnostics and monitoring. We have recent... more Tumor-specific DNA methylation can be used for cancer diagnostics and monitoring. We have recently reported a set of DNA methylation biomarkers that can distinguish plasma samples from lung cancer patients versus healthy controls with high sensitivity and specificity. Furthermore, the DNA methylation signal from the biomarker loci detected in plasma samples correlated with tumor size and decreased after surgical resection of lung tumors. In order to determine the timing of DNA methylation of these loci during carcinogenesis and thus the potential of the biomarkers to detect early stages of the disease we analyzed the DNA methylation of the biomarker loci in five precancerous conditions using available data from the GEO database. We found that the DNA methylation of the biomarker loci is gained early in carcinogenesis since most of the precancerous conditions already have biomarker loci hypermethylated. Moreover, these DNA methylation biomarkers are able to distinguish between p...

Genome Research, 2011

Epigenetic mechanisms are important regulators of cell type-specific genes, including miRNAs. In ... more Epigenetic mechanisms are important regulators of cell type-specific genes, including miRNAs. In order to identify cell type-specific miRNAs regulated by epigenetic mechanisms, we undertook a global analysis of miRNA expression and epigenetic states in three isogenic pairs of human mammary epithelial cells (HMEC) and human mammary fibroblasts (HMF), which represent two differentiated cell types typically present within a given organ, each with a distinct phenotype and a distinct epigenotype. While miRNA expression and epigenetic states showed strong interindividual concordance within a given cell type, almost 10% of the expressed miRNA showed a cell type-specific pattern of expression that was linked to the epigenetic state of their promoter. The tissue-specific miRNA genes were epigenetically repressed in nonexpressing cells by DNA methylation (38%) and H3K27me3 (58%), with only a small set of miRNAs (21%) showing a dual epigenetic repression where both DNA methylation and H3K27me3 were present at their promoters, such as MIR10A and MIR10B. Individual miRNA clusters of closely related miRNA gene families can each display cell type-specific repression by the same or complementary epigenetic mechanisms, such as the MIR200 family, and MIR205, where fibroblasts repress MIR200C/141 by DNA methylation, MIR200A/200B/429 by H3K27me3, and MIR205 by both DNA methylation and H3K27me3. Since deregulation of many of the epigenetically regulated miRNAs that we identified have been linked to disease processes such as cancer, it is predicted that compromise of the epigenetic control mechanisms is important for this process. Overall, these results highlight the importance of epigenetic regulation in the control of normal cell type-specific miRNA expression.

Journal of Breast Cancer

MORT expression. Thus, taken in toto, these results are consistent with MORT silencing occurring ... more MORT expression. Thus, taken in toto, these results are consistent with MORT silencing occurring early in breast carcinogenesis, during cellular immortalization, and before the development of luminal IBC. Delineating the molecular functions of the MORT lncRNA could provide new strategies for therapeutic or chemopreventive interventions.

Epigenetics, Jan 2, 2015

PLoS ONE, 2013

miRNAs are important regulators of gene expression that are frequently deregulated in cancer, wit... more miRNAs are important regulators of gene expression that are frequently deregulated in cancer, with aberrant DNA methylation being an epigenetic mechanism involved in this process. We previously identified miRNA promoter regions active in normal mammary cell types and here we analyzed which of these promoters are targets of aberrant DNA methylation in human breast cancer cell lines and breast tumor specimens. Using 5-methylcytosine immunoprecipitation coupled to miRNA tiling microarray hybridization, we performed comprehensive evaluation of DNA methylation of miRNA gene promoters in breast cancer. We found almost one third (55/167) of miRNA promoters were targets for aberrant methylation in breast cancer cell lines. Breast tumor specimens displayed DNA methylation of majority of these miRNA promoters, indicating that these changes in DNA methylation might be clinically relevant. Aberrantly methylated miRNA promoters were, similar to protein coding genes, enriched for promoters targeted by polycomb in normal cells. Detailed analysis of selected miRNA promoters revealed decreased expression of miRNA linked to increased promoter methylation for mir-31, mir-130a, let-7a-3/let-7b, mir-155, mir-137 and mir-34b/mir-34c genes. The proportion of miRNA promoters we found aberrantly methylated in breast cancer is several fold larger than that observed for protein coding genes, indicating an important role of DNA methylation in miRNA deregulation in cancer.

F1000Research

We have previously described a hominid-specific long non-coding RNA, MORT (also known as ZNF667-A... more We have previously described a hominid-specific long non-coding RNA, MORT (also known as ZNF667-AS1, Gene ID: 100128252), which is expressed in all normal cell types, but epigenetically silenced during cancer-associated immortalization of human mammary epithelial cells. Initial analysis of The Cancer Genome Atlas (TCGA) showed that 15 of 17 cancer types, which represent the 10 most common cancers in women and men, display DNA methylation associated MORT silencing in a large fraction of their tumors. In this study we analyzed MORT expression and DNA methylation state in the remaining 16 TCGA cancer types not previously reported. Seven of the 16 cancer types showed DNA methylation linked MORT silencing in a large fraction of their tumors. These are carcinomas (cervical cancer, and cancers of esophagus, stomach, and bile duct), and the non-epithelial tumors mesothelioma, sarcoma, and uterine carcinosarcoma. Together with the findings from our previous report, MORT expression is si...

Immortality is an essential characteristic of human carcinoma cells. We recently developed an eff... more Immortality is an essential characteristic of human carcinoma cells. We recently developed an efficient, reproducible method that immortalizes human mammary epithelial cells (HMEC) in the absence of gross genomic changes by targeting 2 critical senescence barriers. Consistent transcriptomic changes associated with immortality were identified using microarray analysis of isogenic normal finite pre-stasis, abnormal finite post-stasis, and immortal HMECs from 4 individuals. A total of 277 genes consistently changed in cells that transitioned from post-stasis to immortal. Gene ontology analysis of affected genes revealed biological processes significantly altered in the immortalization process. These immortalization-associated changes showed striking similarity to the gene expression changes seen in The Cancer Genome Atlas (TCGA) clinical breast cancer data. The most dramatic change in gene expression seen during the immortalization step was the downregulation of an unnamed, incompletely annotated transcript that we called MORT, for mortality, since its expression was closely associated with the mortal, finite lifespan phenotype. We show here that MORT (ZNF667-AS1) is expressed in all normal finite lifespan human cells examined to date and is lost in immortalized HMEC. MORT gene silencing at the mortal/immortal boundary was due to DNA hypermethylation of its CpG island promoter. This epigenetic silencing is also seen in human breast cancer cell lines and in a majority of human breast tumor tissues. The functional importance of DNA hypermethylation in MORT gene silencing is supported by the ability of 5-aza-2 0 -deoxycytidine to reactivate MORT expression. Analysis of TCGA data revealed deregulation of MORT expression due to DNA hypermethylation in 15 out of the 17 most common human cancers. The epigenetic silencing of MORT in a large majority of the common human cancers suggests a potential fundamental role in cellular immortalization during human carcinogenesis.

Journal of Clinical Oncology, 2020

Cancer Research, 2015

Neoplasia, 2008

PLoS ONE, 2010

Journal of Agricultural and Food Chemistry, 2005

Journal of Agricultural and Food Chemistry, 2006

Clinical Epigenetics

Epigenetics

PLoS ONE, 2010

Background: The microRNA-200 family participates in the maintenance of an epithelial phenotype an... more Background: The microRNA-200 family participates in the maintenance of an epithelial phenotype and loss of its expression can result in epithelial to mesenchymal transition (EMT). Furthermore, the loss of expression of miR-200 family members is linked to an aggressive cancer phenotype. Regulation of the miR-200 family expression in normal and cancer cells is not fully understood. Methodology/Principal Findings: Epigenetic mechanisms participate in the control of miR-200c and miR-141 expression in both normal and cancer cells. A CpG island near the predicted mir-200c/mir-141 transcription start site shows a striking correlation between miR-200c and miR-141 expression and DNA methylation in both normal and cancer cells, as determined by MassARRAY technology. The CpG island is unmethylated in human miR-200/miR-141 expressing epithelial cells and in miR-200c/miR-141 positive tumor cells. The CpG island is heavily methylated in human miR-200c/miR-141 negative fibroblasts and miR-200c/miR-141 negative tumor cells. Mouse cells show a similar inverse correlation between DNA methylation and miR-200c expression. Enrichment of permissive histone modifications, H3 acetylation and H3K4 trimethylation, is seen in normal miR-200c/miR-141-positive epithelial cells, as determined by chromatin immunoprecipitation coupled to real-time PCR. In contrast, repressive H3K9 dimethylation marks are present in normal miR-200c/miR-141negative fibroblasts and miR-200c/miR-141 negative cancer cells and the permissive histone modifications are absent. The epigenetic modifier drug, 5-aza-29-deoxycytidine, reactivates miR-200c/miR-141 expression showing that epigenetic mechanisms play a functional role in their transcriptional control.

Clinical Epigenetics, 2022

We tested the ability of a novel DNA methylation biomarker set to distinguish metastatic pancreat... more We tested the ability of a novel DNA methylation biomarker set to distinguish metastatic pancreatic cancer cases from benign pancreatic cyst patients and to monitor tumor dynamics using quantitative DNA methylation analysis of cellfree DNA (cfDNA) from blood samples. The biomarkers were able to distinguish malignant cases from benign disease with high sensitivity and specificity (AUC = 0.999). Furthermore, the biomarkers detected a consistent decline in tumor-derived cfDNA in samples from patients undergoing chemotherapy. The study indicates that our liquid biopsy assay could be useful for management of pancreatic cancer patients.

F1000Research

Genome Research, 2011

Journal of Breast Cancer

Epigenetics, Jan 2, 2015

PLoS ONE, 2013

F1000Research