Adult cancers may derive from stem or early progenitor cells 1,2 . Epigenetic modulation of gene expression is essential for normal function of these early cells, but is highly abnormal in cancers, which often exhibit aberrant promoter CpG island hypermethylation and transcriptional silencing of tumor suppressor genes and pro-differentiation factors [3][4][5] . We find that, for such genes, both normal and malignant embryonic cells generally lack the gene DNA hypermethylation found in adult cancers. In embryonic stem (ES) cells, these genes are held in a "transcription ready" state mediated by a "bivalent" promoter chromatin pattern consisting of the repressive polycomb group (PcG) H3K27me mark plus the active mark, H3K4me. However, embryonic carcinoma (EC) cells add two key repressive marks, H3K9me2 and H3K9me3, both associated with DNA hypermethylated genes in adult cancers [6][7][8] . We hypothesize that cell chromatin patterns and transient silencing of these important growth regulatory genes in stem or progenitor cells of origin for cancer may leave these genes vulnerable to aberrant DNA hypermethylation and heritable gene silencing in adult tumors.Correspondence may be addressed to S.B.B. at sbaylin@jhmi.edu. Competing Interests Statement. The commercial rights to the MSP technique belong to Oncomethylome. S.B.B and J.G.H. serve as consultants to Oncomethylome and is entitled to royalties from any commercial use of this procedure. Epigenetic gene silencing and associated promoter CpG island DNA hypermethylation are prevalent in all cancer types, and provide an alternative mechanism to mutations by which tumor suppressor genes may be inactivated within a cancer cell [3][4][5] . These epigenetic changes may precede genetic changes in pre-malignant cells and foster the accumulation of additional genetic and epigenetic hits 9 . Adult cancers may derive from stem or early progenitor cells 1, 2 , and epigenetic modulation of gene expression is essential for normal function of these early cells. We now explore whether DNA hypermethylation and heritable silencing of groups of genes in adult tumor initiation and progression might reflect chromatin properties for these genes associated with a stem or precursor cell of origin. NIH Public AccessWe compared the epigenetic status of a group of genes frequently hypermethylated and silenced in adult cancers ( Fig. 1-all (Fig. 1). Among the genes studied, 13 of 29 (45%) are hypermethylated in a single line, HCT-116, of adult colon cancer, but none are hypermethylated in ES cells, and only 3% and 7% were completely methylated in the Tera-1 and Tera-2 EC lines, respectively. Thus, the key epigenetic parameter of promoter CpG island hypermethylation which is common in a large group of genes in adult cancer cells does not seem to be a common feature of EC cells.In murine ES cells, many developmental genes are maintained in a state of low transcriptional activity and are available for transcription increases or decreases when differentiation cues are received 11 . Our s...
Microarray technology is a powerful tool for measuring RNA expression for thousands of genes at once. Various studies have been published comparing competing platforms with mixed results: some find agreement, others do not. As the number of researchers starting to use microarrays and the number of cross-platform meta-analysis studies rapidly increases, appropriate platform assessments become more important. Here we present results from a comparison study that offers important improvements over those previously described in the literature. In particular, we noticed that none of the previously published papers consider differences between labs. For this study, a consortium of ten laboratories from the Washington, DC-Baltimore, USA, area was formed to compare data obtained from three widely used platforms using identical RNA samples. We used appropriate statistical analysis to demonstrate that there are relatively large differences in data obtained in labs using the same platform, but that the results from the best-performing labs agree rather well.
We report here that butyrate, a naturally occurring fatty acid commonly used as a nutritional supplement and differentiation agent, greatly enhances the efficiency of induced pluripotent stem (iPS) cell derivation from human adult or fetal fibroblasts. After transient butyrate treatment, the iPS cell derivation efficiency is enhanced by 15-to 51-fold using either retroviral or piggyBac transposon vectors expressing 4 to 5 reprogramming genes. Butyrate stimulation is more remarkable (>100-to 200-fold) on reprogramming in the absence of either KLF4 or MYC transgene. Butyrate treatment did not negatively affect properties of iPS cell lines established by either 3 or 4 retroviral vectors or a single piggyBac DNA transposon vector. These characterized iPS cell lines, including those derived from an adult patient with sickle cell disease by either the piggyBac or retroviral vectors, show normal karyotypes and pluripotency. To gain insights into the underlying mechanisms of butyrate stimulation, we conducted genome-wide gene expression and promoter DNA methylation microarrays and other epigenetic analyses on established iPS cells and cells from intermediate stages of the reprogramming process. By days 6 to 12 during reprogramming, butyrate treatment enhanced histone H3 acetylation, promoter DNA demethylation, and the expression of endogenous pluripotency-associated genes, including DPPA2, whose overexpression partially substitutes for butyrate stimulation. Thus, butyrate as a cell permeable small molecule provides a simple tool to further investigate molecular mechanisms of cellular reprogramming. Moreover, butyrate stimulation provides an efficient method for reprogramming various human adult somatic cells, including cells from patients that are more refractory to reprogramming. STEM CELLS 2010;28:713-720 Disclosure of potential conflicts of interest is found at the end of this article.
We have developed a transcriptome-wide approach to identify genes affected by promoter CpG island DNA hypermethylation and transcriptional silencing in colorectal cancer. By screening cell lines and validating tumor-specific hypermethylation in a panel of primary human colorectal cancer samples, we estimate that nearly 5% or more of all known genes may be promoter methylated in an individual tumor. When directly compared to gene mutations, we find larger numbers of genes hypermethylated in individual tumors, and a higher frequency of hypermethylation within individual genes harboring either genetic or epigenetic changes. Thus, to enumerate the full spectrum of alterations in the human cancer genome, and to facilitate the most efficacious grouping of tumors to identify cancer biomarkers and tailor therapeutic approaches, both genetic and epigenetic screens should be undertaken.
We report that angiotensin-converting enzyme (ACE), a critical physiologic regulator of blood pressure, angiogenesis, and inflammation, is a novel marker for identifying hemangioblasts differentiating from human embryonic stem cells (hESC). We demonstrate that ACE ؉ CD45 ؊ CD34 ؉/؊ hemangioblasts are common yolk sac (YS)-like progenitors for not only endothelium but also both primitive and definitive human lymphohematopoietic cells. Thrombopoietin and basic fibroblast growth factor are identified as critical factors for the proliferation of human hemangioblasts.The developmental sequence of human embryoid body hematopoiesis is remarkably congruent to the timeline of normal human YS development, which occurs during weeks 2 to 6 of human gestation. Furthermore, ACE and the reninangiotensin system (RAS) directly regulate hemangioblast expansion and differentiation via signaling through the angiotensin II receptors AGTR1 and AGTR2. ACE enzymatic activity is required for hemangioblast expansion, and differentiation toward either endothelium or multipotent hematopoietic progenitors is dramatically augmented after manipulation of angiotensin II signaling with either AGTR1-or AGTR2-specific inhibitors. The RAS can therefore be exploited to direct the hematopoietic or endothelial fate of hESC-derived hemangioblasts, thus providing novel opportunities for human tissue engineering. Moreover, the initial events of human hematoendotheliogenesis can be delineated in a manner previously impossible because of inaccessibility to early human embryonic tissues. IntroductionHuman hematopoiesis initiates during the third week of development with formation of yolk sac (YS) blood islands derived from extraembryonic mesoderm. 1,2 The YS generates primarily nucleated primitive erythroblasts that express predominantly embryonic hemoglobins (eg, ε 2 2 , Gower I; ␣ 2 ε 2 , Gower II; and 2 ␥ 2 , Portland), and primitive macrophages. 3 After the onset of circulation at approximately 21 days of embryonic development, YS blood cells continue to circulate in embryonic blood, but the fetal liver (FL) eventually replaces the YS as the main hematopoietic organ beginning at 5 to 6 weeks. FL hematopoiesis is dominated by adult-type definitive erythrocytes (enucleated, macrocytic) expressing abundant fetal (HbF; ␣ 2 ␥ 2 ), adult (HbA; ␣ 2  2 ) hemoglobins, but limited embryonic hemoglobins. [4][5][6] The FL also produces rare megakaryocytes, granulocytes, macrophages, lymphocytes, and blast cells. 7 Definitive erythroid, myeloid, megakaryocytic, natural killer-B (NK-B) lymphoid, and multipotent progenitors, with limited long-term engraftment potential, are briefly detected during the late YS (eg, 4-5 weeks) stage, before fetal liver hematopoiesis, in both mice and humans. 2,[8][9] The ephemeral nature of definitivetype YS hematopoiesis suggests a gradual yolk sac-fetal liver transition. 1 Indeed, the classic embryonic 3 fetal 3 adult globin switch in early erythrocytes occurs sequentially over time and probably in the same clonal lineage. For examp...
Cholangiocarcinomas (CCA) are aggressive cancers, with a high mortality and poor survival rate. Only radical surgery offers patients some hope of cure; however, most patients are not surgical candidates because of the late diagnosis secondary to relatively poor accuracy diagnostic means. MicroRNAs (miRs) are involved in every cancer examined, but they have not been evaluated in primary CCA. In this study, miR arrays were performed on 5 primary CCAs and 5 normal bile duct specimens (NBD). Several miRs were dysregulated, and miR-21 was overexpressed, in CCAs. miR-21 differential expression in these 10 specimens was verified with quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). To validate these findings, qRT-PCR for miR-21 was then performed on 18 additional primary CCAs and 12 normal liver specimens. MiR-21 was 95% sensitive and 100% specific in distinguishing between CCA and normal tissues, with an area under the Receiver Operating Characteristic (ROC) curve of 0.995. Inhibitors of miR-21 increased protein levels of programmed cell death 4 (PDCD4) and tissue inhibitor of metalloproteinases 3 (TIMP3). Notably, messenger RNA (mRNA) levels of TIMP3 were significantly lower in CCAs than in normals. Conclusions MiR-21 is overexpressed in human CCAs. Furthermore, miR-21 may be oncogenic, at least in part, by inhibiting PDCD4 and TIMP3. Finally, these data suggest that TIMP3 is a candidate tumor suppressor gene in the biliary tree.
Cancer cells differentiate along specific lineages that largely determine their clinical and biologic behavior. Distinct cancer phenotypes from different cells and organs likely result from unique gene expression repertoires established in the embryo and maintained after malignant transformation. We used comprehensive gene expression analysis to examine this concept in the prostate, an organ with a tractable developmental program and a high propensity for cancer. We focused on gene expression in the murine prostate rudiment at three time points during the first 48 h of exposure to androgen, which initiates proliferation and invasion of prostate epithelial buds into surrounding urogenital sinus mesenchyme. Here, we show that androgen exposure regulates genes previously implicated in prostate carcinogenesis comprising pathways for the phosphatase and tensin homolog (PTEN), fibroblast growth factor (FGF)/mitogen-activated protein kinase (MAPK), and Wnt signaling along with cellular programs regulating such 'hallmarks' of cancer as angiogenesis, apoptosis, migration and proliferation. We found statistically significant evidence for novel androgeninduced gene regulation events that establish and/or maintain prostate cell fate. These include modulation of gene expression through microRNAs, expression of specific transcription factors, and regulation of their predicted targets. By querying public gene expression databases from other tissues, we found that rather than generally characterizing androgen exposure or epithelial budding, the early prostate development program more closely resembles the program for human prostate cancer. Most importantly, early androgen-regulated genes and functional themes associated with prostate development were highly enriched in contrasts between increasingly lethal forms of prostate cancer, confirming a 'reactivation' of embryonic pathways for proliferation and invasion in prostate cancer progression. Among the genes with the most significant links to the development and cancer, we highlight coordinate induction of the transcription factor Sox9 and suppression of the proapoptotic phospholipidbinding protein Annexin A1 that link early prostate development to early prostate carcinogenesis. These results credential early prostate development as a reliable and valid model system for the investigation of genes and pathways that drive prostate cancer.
Highly tumorigenic cancer cell (HTC) populations have been identified for a variety of solid tumors and assigned stem cell properties. Strategies for identifying HTCs in solid tumors have been primarily empirical rather than rational, particularly in epithelial tumors, which are responsible for 80% of cancer deaths. We report evidence for a spatially restricted bladder epithelial (urothelial) differentiation program in primary urothelial cancers (UCs) and in UC xenografts. We identified a highly tumorigenic UC cell compartment that resembles benign urothelial stem cells (basal cells), co-expresses the 67-kDa laminin receptor and the basal cell-specific cytokeratin CK17, and lacks the carcinoembryonic antigen family member CEA-CAM6 (CD66c). This multipotent compartment resides at the tumor-stroma interface, is easily identified on histologic sections, and possesses most, if not all, of the engraftable tumor-forming ability in the parental xenograft. We analyzed differential expression of genes and pathways in basal-like cells versus more differentiated cells. Among these, we found significant enrichment of pathways comprising ''hallmarks'' of cancer, and pharmacologically targetable signaling pathways, including Janus kinase-signal transducer and activator of transcription, Notch, focal adhesion, mammalian target of rapamycin, epidermal growth factor receptor (erythroblastic leukemia viral oncogene homolog [ErbB]), and wingless-type MMTV integration site family (Wnt). The basal/HTC gene expression signature was essentially invisible within the context of nontumorigenic cell gene expression and overlapped significantly with genes driving progression and death in primary human UC. The spatially restricted epithelial differentiation program described here represents a conceptual advance in understanding cellular heterogeneity of carcinomas and identifies basal-like HTCs as attractive targets for cancer therapy.
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