Geoffrey Childs

Making and reading microarrays

There are a variety of options for making microarrays and obtaining microarray data. Here, we describe the building and use of two microarray facilities in academic settings. In addition to specifying technical detail, we comment on the advantages and disadvantages of components and approaches, and provide a protocol for hybridization. The fact that we are now making and using microarrays to answer biological questions demonstrates that the technology can be implemented in a university environment.

Low-level expression of microRNAs let-7d and miR-205 are prognostic markers of head and neck squamous cell carcinoma.

Small noncoding microRNAs (miRNAs) have been shown to be abnormally expressed in every tumor type examined. The importance of miRNAs as potential cancer prognostic indicators is underscored by their involvement in the regulation of basic cellular processes such as cell proliferation, differentiation, and apoptosis. In this study, miRNA expression profiles of head and neck squamous cell carcinoma (HNSCC) tumor and adjacent normal tissue were examined by microarray analysis and validated by quantitative TaqMan real-time polymerase chain reaction. Using TaqMan real-time polymerase chain reaction we measured the quantitative associations between a subset of miRNAs identified on microarrays in primary tumors at diagnosis and cancer survival in a cohort of 104 HNSCC patients undergoing treatment with curative intent. The majority of miRNAs exhibiting altered expression in primary human HNSCC tumors (including miR-1, miR-133a, miR-205, and let-7d) show lower expression levels relative to normal adjacent tissue. In contrast, hsa-miR-21 is frequently overexpressed in human HNSCC tumors. Using univariate and multivariable statistical models we show that low levels of hsa-miR205 are significantly associated with loco-regional recurrence independent of disease severity at diagnosis and treatment. In addition, combined low levels of hsa-miR-205 and hsa-let-7d expression in HNSCC tumors are significantly associated with poor head and neck cancer survival Our results show that miRNA expression levels can be used as prognostic markers of head and neck cancer.

Modulation of Cardiac Growth and Development by HOP, an Unusual Homeodomain Protein

We have discovered an unusual homeodomain protein, called HOP, which is comprised simply of a homeodomain. HOP is highly expressed in the developing heart where its expression is dependent on the cardiac-restricted homeodomain protein Nkx2.5. HOP does not bind DNA and acts as an antagonist of serum response factor (SRF), which regulates the opposing processes of proliferation and myogenesis. Mice homozygous for a HOP null allele segregate into two phenotypic classes characterized by an excess or deficiency of cardiac myocytes. We propose that HOP modulates SRF activity during heart development; its absence results in an imbalance between cardiomyocyte proliferation and differentiation with consequent abnormalities in cardiac morphogenesis.

Gene Discovery in Bladder Cancer Progression using cDNA Microarrays

To identify gene expression changes along progression of bladder cancer, we compared the expression profiles of early-stage and advanced bladder tumors using cDNA microarrays containing 17,842 known genes and expressed sequence tags. The application of bootstrapping techniques to hierarchical clustering segregated early-stage and invasive transitional carcinomas into two main clusters. Multidimensional analysis confirmed these clusters and more importantly, it separated carcinoma in situ from papillary superficial lesions and subgroups within early-stage and invasive tumors displaying different overall survival. Additionally, it recognized early-stage tumors showing gene profiles similar to invasive disease. Different techniques including standard t-test, single-gene logistic regression, and support vector machine algorithms were applied to identify relevant genes involved in bladder cancer progression. Cytokeratin 20, neuropilin-2, p21, and p33ING1 were selected among the top ranked molecular targets differentially expressed and validated by immunohistochemistry using tissue microarrays (n = 173). Their expression patterns were significantly associated with pathological stage, tumor grade, and altered retinoblastoma (RB) expression. Moreover, p33ING1 expression levels were significantly associated with overall survival. Analysis of the annotation of the most significant genes revealed the relevance of critical genes and pathways during bladder cancer progression, including the overexpression of oncogenic genes such as DEK in superficial tumors or immune response genes such as Cd86 antigen in invasive disease. Gene profiling successfully classified bladder tumors based on their progression and clinical outcome. The present study has identified molecular biomarkers of potential clinical significance and critical molecular targets associated with bladder cancer progression.

Genome-wide profiling of papillary thyroid cancer identifies MUC1 as an independent prognostic marker.

Clinicopathological variables used at present for prognostication and treatment selection for papillary thyroid carcinomas (PTCs) do not uniformly predict tumor behavior, necessitating identification of novel prognostic markers. Complicating the assessment is the long natural history of PTC and our rudimentary knowledge of its genetic composition. In this study we took advantage of differences in clinical behavior of two distinct variants of PTC, the aggressive tall-cell variant (TCV) and indolent conventional PTC (cPTC), to identify molecular prognosticators of outcome using complementary genome wide analyses. Comparative genome hybridization (CGH) and cDNA microarray (17,840 genes) analyses were used to detect changes in DNA copy number and gene expression in pathological cPTC and TCV. The findings from CGH and cDNA microarray analyses were correlated and validated by real-time PCR and immunohistochemical analyses on a series of 100 cases of cPTC and TCV. Genes identified by this approach were evaluated as prognostic markers in cPTC by immunohistochemistry on tissue arrays. CGH identified significant differences in the presence (76 versus 27%; P = 0.001) and type of DNA copy number aberrations in TCV compared with cPTC. Recurrent gains of 1p34–36, 1q21, 6p21–22, 9q34, 11q13, 17q25, 19, and 22 and losses of 2q21–31, 4, 5p14-q21, 6q11–22, 8q11–22, 9q11–32, and 13q21–31 were unique to TCV. Hierarchical clustering of gene expression profiles revealed significant overlap between TCV and cPTC, but further analysis identified 82 dysregulated genes differentially expressed among the PTC variants. Of these, MUC1 was of particular interest because amplification of 1q by CGH correlated with MUC1 amplification by real-time PCR analysis and protein overexpression by immunohistochemistry in TCV (P = 0.005). Multivariate analysis revealed a significant association between MUC1 overexpression and treatment outcome, independent of histopathological categorization (P = 0.03). Analysis of a validation series containing a matched group of aggressive and indolent cPTCs confirmed the association between MUC1 overexpression and survival (relative risk, 2.3; 95% confidence interval, 1.1–5.5; P = 0.03). Our data suggest that MUC1 dysregulation is associated with aggressive behavior of PTC and may serve as a prognostic marker and potential therapeutic target in this disease.

Gene expression profiles in HPV-infected head and neck cancer.

Epidemiological and laboratory evidence indicate that, in addition to tobacco and alcohol, human papillomaviruses (HPV) play an important aetiological role in a subset of head and neck squamous cell carcinoma (HNSCC). To evaluate the molecular pathogenesis of HPV‐infected HNSCC, we compared gene expression patterns between HPV‐positive and ‐negative HNSCC tumours using cDNA microarrays. Tumour tissue was collected from 42 histologically confirmed HNSCC patients from an inner‐city area of New York. Total DNA and RNA were extracted and purified from frozen tumour samples and gene expression levels were compared to a universal human reference RNA standard using a 27 323 cDNA microarray chip. HPV detection and genotyping were performed using an MY09/11‐PCR system and RT‐PCR. HPV was detected in 29% of HNSCC tumours. Most harboured only HPV16 and expressed the HPV16‐E6 oncogene. HPV prevalence was highest in pharyngeal tumours (45%). Gene expression patterns that differentiated HPV‐positive from negative tumours were compared by supervised classification analysis, and a multiple‐gene signature was found to predict HPV16 prevalence in primary HNSCC with a false discovery rate < 0.2. Focusing on never‐smokers, we further identified a distinct subset of 123 genes that were specifically dysregulated in HPV16‐positive HNSCC. Overexpressed genes in HPV‐positive HNSCC tumours included the retinoblastoma‐binding protein (p18), replication factor‐C gene, and an E2F‐dimerization partner transcription factor (TFDP2) that have also been found to be overexpressed in cervical cancer. An additional subset of genes involved in viral defence and immune response, including interleukins and interferon‐induced proteins, was found to be down‐regulated in HPV‐positive tumours, supporting a characteristic and unique transcriptional profile in HPV‐induced HNSCC. Copyright

The transcriptional repressor ZFM1 interacts with and modulates the ability of EWS to activate transcription.

The ZFM1 protein is both a transcriptional repressor and identical to the splicing factor SF1. ZFM1 was shown to interact with and repress transcription from the glycine, glutamine, serine, and threonine-rich transcription activation domain of the sea urchin transcription factor, stage-specific activator protein (SSAP). EWS, a human protein involved in cellular transformation in Ewing’s sarcoma tumors, contains an NH2-terminal transcriptional activation domain (NTD) which resembles that of SSAP in both amino acid composition and the ability to drive transcription to levels higher than VP16 in most cell types. Here we report that ZFM1 also interacts with EWS in both two-hybrid assays and glutathioneS-transferase pull-down experiments. The region on EWS which interacts with ZFM1 maps to 37 amino acids within its NTD. Overexpression of ZFM1 in HepG2 cells represses the transactivation of reporter gene expression driven by Gal4-EWS-NTD fusion protein and this repression correlates with ZFM1 binding to EWS. Furthermore, two proteins, TLS and hTAFII68, which have extensive homology to EWS, also interact with ZFM1. Recently, it was discovered that EWS/TLS/hTAFII68 are each present in distinct TFIID populations and EWS and hTAFII68 were also found to be associated with the RNA polymerase II holoenzyme. The association of ZFM1 with these proteins implies that one normal cellular function for ZFM1 may be to negatively modulate transcription of target genes coordinated by these cofactors.

PRISM/PRDM6, a Transcriptional Repressor That Promotes the Proliferative Gene Program in Smooth Muscle Cells

ABSTRACT Smooth muscle cells (SMCs) display remarkable phenotypic diversity and plasticity and can readily switch between proliferative and differentiated states in response to extracellular cues. In an effort to identify novel transcriptional regulators of smooth muscle phenotypes, we compared the gene expression profiles of arterial and venous SMCs by microarray-based transcriptional profiling. Among numerous genes displaying distinct expression patterns in these two SMC types, we discovered an expressed sequence tag encoding a previously uncharacterized zinc finger protein belonging to the PRDM (PRDI-BF1 and RIZ homology domain) family of chromatin-remodeling proteins and named it PRISM (PR domain in smooth muscle). PRISM is expressed in a variety of smooth muscle-containing tissues and displays especially robust expression in the cardiac outflow tract and descending aorta during embryogenesis. PRISM is localized to the nucleus and contains an amino-terminal PR domain and four Krüppel-like zinc fingers at the carboxy terminus. We show that PRISM acts as a transcriptional repressor by interacting with class I histone deacetylases and the G9a histone methyltransferase, thereby identifying PRISM as a novel SMC-restricted epigenetic regulator. Overexpression of PRISM in cultured primary SMCs induces genes associated with the proliferative smooth muscle phenotype while repressing regulators of differentiation, including myocardin and GATA-6. Conversely, small interfering RNA-mediated knockdown of PRISM slows cell growth and induces myocardin, GATA-6, and markers of SMC differentiation. We conclude that PRISM acts as a novel epigenetic regulator of SMC phenotypic plasticity by suppressing differentiation and maintaining the proliferative potential of vascular SMCs.

Use of Laser-Capture Microdissection for the Identification of Marker Genes for the Ventromedial Hypothalamic Nucleus

The ventromedial hypothalamic nucleus (VMH) plays an important role in the control of feeding and energy homeostasis. In contrast to other hypothalamic nuclei that are also known to regulate energy balance, there is a paucity of nucleus-specific marker genes for the VMH, limiting the application of molecular approaches for analyzing VMH information processing, function, and circuitry. Here, we report the use of laser-capture microdissection to isolate a set of cDNAs that are enriched in the VMH relative to two adjacent hypothalamic nuclei, the arcuate and dorsomedial hypothalamus. The relative expression levels of nine of the 12 most robustly expressed VMH-enriched genes were confirmed by real-time PCR analysis using separate RNAs from these three nuclei. Three of these VMH-enriched genes were further characterized by in situ hybridization histochemistry, including pituitary adenylate cyclase activating polypeptide, cerebellin 1, and an expressed sequence tag named LBH2. Finally, to test whether some of these genes were coordinately regulated, we monitored their expression in steroidogenic factor 1 (SF-1) knock-out mice. SF-1 is a transcription factor that controls the development of the VMH. The RNA levels for four of these genes were reduced in these knock-out animals, further suggesting that they are direct or indirect targets of this orphan nuclear receptor. The VMH-enriched genes identified here provide a basis for a functional analysis of VMH neuronal subpopulations via the use of bacterial artificial chromosome transgenics and related technologies. These results also demonstrate the utility of laser-capture microdissection coupled with microarray technology to identify nucleus-specific transcriptional networks.

Low-Level Expression of miR-375 Correlates with Poor Outcome and Metastasis While Altering the Invasive Properties of Head and Neck Squamous Cell Carcinomas

Small, noncoding microRNAs (miRNAs) have been shown to be abnormally expressed in every tumor type examined. We used comparisons of global miRNA expression profiles of head and neck squamous cell carcinoma (HNSCC) samples and adjacent normal tissue to rank those miRNAs that were most significantly altered in our patient population. Rank Consistency Score analysis revealed miR-375 to have the most significantly lowered miRNA levels in tumors relative to matched adjacent nonmalignant tissue from the same patient among 736 miRNAs that were evaluated. This result has been previously observed by other groups; however, we extend this finding with the unique observation that low miR-375 expression levels correlate significantly with cancer survival and distant metastasis. In a study of 123 primary HNSCC patients using multivariable Cox proportional hazard ratios (HR) and 95% confidence intervals (CI), both death from disease (HR: 12.8, 95% CI: 3 to 49) and incidence of distant metastasis (HR: 8.7, 95% CI: 2 to 31) correlated with lower expression levels of miR-375 regardless of the site or stage of the tumor. In addition, we found that oral cavity tumor cell lines (eg, UMSCC1 and UMSCC47) overexpressing miR-375 were significantly less invasive in vitro than their matched empty vector controls. We conclude that miR-375 represents a potential prognostic marker of poor outcome and metastasis in HNSCC and that it may function by suppressing the tumors invasive properties.