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Chromatin Remodeling Factors and BRM/BRG1 Expression as Prognostic Indicators in Non-Small Cell Lung Cancer

We immunohistochemically examined 12 core proteins involved in the chromatin remodeling machinery using a tissue microarray composed of 150 lung adenocarcinoma (AD) and 150 squamous cell carcinoma (SCC) cases. Most of the proteins showed nuclear staining, whereas some also showed cytoplasmic or membranous staining. When the expression patterns of all tested antigens were considered, proteins with nuclear staining clustered into two major groups. Nuclear signals of BRM, Ini-1, retinoblastoma, mSin3A, HDAC1, and HAT1 clustered together, whereas nuclear signals of BRG1, BAF155, HDAC2, BAF170, and RbAP48 formed a second cluster. Additionally, two thirds of the cases on the lung tissue array had follow-up information, and survival analysis was performed for each of the tested proteins. Positive nuclear BRM (N-BRM) staining correlated with a favorable prognosis in SCC and AD patients with a 5 year-survival of 53.5% compared with 32.3% for those whose tumors were negative for N-BRM (P = 0.015). Furthermore, patients whose tumors stained positive for both N-BRM and nuclear BRG1 had a 5 year-survival of 72% compared with 33.6% (P = 0.013) for those whose tumors were positive for either or negative for both markers. In contrast, membranous BRM (M-BRM) staining correlated with a poorer prognosis in AD patients with a 5 year-survival of 16.7% compared with those without M-BRM staining (38.1%; P = 0.016). These results support the notion that BRM and BRG1 participate in two distinct chromosome remodeling complexes that are functionally complementary and that the nuclear presence of BRM, its coexpression with nuclear BRG1, and the altered cellular localization of BRM (M-BRM) are useful markers for non-small cell lung cancer prognosis.

Nonclassic Functions of Human Topoisomerase I: Genome-Wide and Pharmacologic Analyses

The biological functions of nuclear topoisomerase I (Top1) have been difficult to study because knocking out TOP1 is lethal in metazoans. To reveal the functions of human Top1, we have generated stable Top1 small interfering RNA (siRNA) cell lines from colon and breast carcinomas (HCT116-siTop1 and MCF-7-siTop1, respectively). In those clones, Top1 is reduced approximately 5-fold and Top2alpha compensates for Top1 deficiency. A prominent feature of the siTop1 cells is genomic instability, with chromosomal aberrations and histone gamma-H2AX foci associated with replication defects. siTop1 cells also show rDNA and nucleolar alterations and increased nuclear volume. Genome-wide transcription profiling revealed 55 genes with consistent changes in siTop1 cells. Among them, asparagine synthetase (ASNS) expression was reduced in siTop1 cells and in cells with transient Top1 down-regulation. Conversely, Top1 complementation increased ASNS, indicating a causal link between Top1 and ASNS expression. Correspondingly, pharmacologic profiling showed L-asparaginase hypersensitivity in the siTop1 cells. Resistance to camptothecin, indenoisoquinoline, aphidicolin, hydroxyurea, and staurosporine and hypersensitivity to etoposide and actinomycin D show that Top1, in addition to being the target of camptothecins, also regulates DNA replication, rDNA stability, and apoptosis. Overall, our studies show the pleiotropic nature of human Top1 activities. In addition to its classic DNA nicking-closing functions, Top1 plays critical nonclassic roles in genomic stability, gene-specific transcription, and response to various anticancer agents. The reported cell lines and approaches described in this article provide new tools to perform detailed functional analyses related to Top1 function.

Global Expression Analysis of Prostate Cancer-associated Stroma and Epithelia

Characterization of gene expression profiles in tumor cells and the tumor microenvironment is an important step in understanding neoplastic progression. To date, there are limited data available on expression changes that occur in the tumor-associated stroma as either a cause or consequence of cancer. In the present study, we employed a 54,000 target oligonucleotide microarray to compare expression profiles in the 4 major components of the microenvironment: tumor epithelium, tumor-associated stroma, normal epithelium, and normal stroma. Cells from 5 human, whole-mount prostatectomy specimens were microdissected and the extracted and amplified mRNA was hybridized to an Affymetrix Human Genome U133 Plus 2.0 GeneChip. Using the intersection of 2 analysis methods, we identified sets of differentially expressed genes among the 4 components. Forty-four genes were found to be consistently differentially expressed in the tumor-associated stroma; 35 were found in the tumor epithelium. Interestingly, the tumor-associated stroma showed a predominant up-regulation of transcripts compared with normal stroma, in sharp contrast to the overall down-regulation seen in the tumor epithelium relative to normal epithelium. These data provide insight into the molecular changes occurring in tumor-associated stromal cells and suggest new potential targets for future diagnostic, imaging, or therapeutic intervention.

Laser capture microdissection, microarrays and the precise definition of a cancer cell.

Most expression profiling studies of solid tumors have used biopsy samples containing large numbers of contaminating stromal and other cell types, thereby complicating any precise delineation of gene expression in nontumor versus tumor cell types. Combining laser capture microdissection, RNA amplification protocols, microarray technologies and our knowledge of the human genome sequence, it is possible to isolate pure populations of cells or even a single cell and interrogate the expression of thousands of sequences for the purpose of more precisely defining the biology of the tumor cell. Although many of the studies that currently allow for characterization of small sample preparations and single cells were performed utilizing noncancer cell types, and in some cases isolation protocols other than laser capture microdissection, a list of protocols are described that could be used for the expression analysis of individual tumor cells. Application of these experimental approaches to cancer studies may permit a more accurate definition of the biology of the cancer cell, so that ultimately, more specific targeted therapies can be developed.

Identification of EpCAM as a Molecular Target of Prostate Cancer Stroma

To delineate the molecular changes that occur in the tumor microenvironment, we previously performed global transcript analysis of human prostate cancer specimens using tissue microdissection and expression microarrays. Epithelial and stromal compartments were individually studied in both tumor and normal fields. Tumor-associated stroma showed a distinctly different expression pattern compared with normal stroma, having 44 differentially expressed transcripts, the majority of which were up-regulated. In the present study, one of the up-regulated transcripts, epithelial cell adhesion activating molecule, was further evaluated at the protein level in 20 prostate cancer cases using immunohistochemistry and a histomathematical analysis strategy. The epithelial cell adhesion activating molecule showed a 76-fold expression increase in the tumor-associated stroma, as compared with matched normal stroma. Moreover, Gleason 4 or 5 tumor stroma was increased 170-fold relative to matched normal stroma, whereas the Gleason 3 tumor area showed only a 36-fold increase, indicating a positive correlation with Gleason tumor grade. Since the stromal compartment may be particularly accessible to vascular-delivered agents, epithelial cell adhesion activating molecule could become a valuable molecular target for imaging or treatment of prostate cancer.

Identification of TDE2 gene and its expression in non-small cell lung cancer

TDE2, a gene with sequence similarity to the mouse testicular tumor‐differentially‐expressed (Tde1/MUSTETU) gene, was identified by serial analysis of gene expression (SAGE) in nonsmall cell lung cancers (NSCLC). Here we characterized the TDE2 gene and determined its transcript levels in a panel of lung tumors, adjacent nonmalignant lung tissues and a variety of normal human tissues. In addition, we show that TDE2 is a potential transmembrane protein with 11 putative transmembrane helices. Using real‐time quantitative PCR, we showed that TDE2 transcript levels were higher in NSCLC compared to nonmalignant samples. In nonpulmonary normal tissues, the level of TDE2 was the highest in bladder, kidney and muscle; moderate to low in stomach, liver, skin, placenta and ovary tissues; and undetectable in brain, spleen and heart. By in situ hybridization, we showed that 10 of 18 lung tumors and only 1 of 14 adjacent nonmalignant regions had high levels of TDE2 transcripts. Alternatively, only 2 of 18 tumors and 8 of 14 adjacent nonmalignant bronchiole epithelium regions demonstrated negative to low levels of TDE2 signals.

Identification of unique expression signatures and therapeutic targets in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC), the predominant histological subtype of esophageal cancer, is characterized by high mortality. Previous work identified important mRNA expression differences between normal and tumor cells; however, to date there are limited ex vivo studies examining expression changes occurring during normal esophageal squamous cell differentiation versus those associated with tumorigenesis. In this study, we used a unique tissue microdissection strategy and microarrays to measure gene expression profiles associated with cell differentiation versus tumorigenesis in twelve cases of patient-matched normal basal squamous epithelial cells (NB), normal differentiated squamous epithelium (ND), and squamous cell cancer. Class comparison and pathway analysis were used to compare NB versus tumor in a search for unique therapeutic targets.ResultsAs a first step towards this goal, gene expression profiles and pathways were evaluated. Overall, ND expression patterns were markedly different from NB and tumor; whereas, tumor and NB were more closely related. Tumor showed a general decrease in differentially expressed genes relative to NB as opposed to ND that exhibited the opposite trend. FSH and IgG networks were most highly dysregulated in normal differentiation and tumorigenesis, respectively. DNA repair pathways were generally elevated in NB and tumor relative to ND indicating involvement in both normal and pathological growth. PDGF signaling pathway and 12 individual genes unique to the tumor/NB comparison were identified as therapeutic targets, and 10 associated ESCC gene-drug pairs were identified. We further examined the protein expression level and the distribution patterns of four genes: ODC1, POSTN, ASPA and IGF2BP3. Ultimately, three genes (ODC1, POSTN, ASPA) were verified to be dysregulated in the same pattern at both the mRNA and protein levels.ConclusionsThese data reveal insight into genes and molecular pathways mediating ESCC development and provide information potentially useful in designing novel therapeutic interventions for this tumor type.

Inactivation of LLC1 gene in nonsmall cell lung cancer

Serial analysis of gene expression studies led us to identify a previously unknown gene, c20orf85, that is present in the normal lung epithelium but absent or downregulated in most primary nonsmall cell lung cancers and lung cancer cell lines. We named this gene LLC1 for Low in Lung Cancer 1. LLC1 is located on chromosome 20q13.3 and has a 70% GC content in the promoter region. It has 4 exons and encodes a protein containing 137 amino acids. By in situ hybridization, we observed that LLC1 message is localized in normal lung bronchial epithelial cells but absent in 13 of 14 lung adenocarcinoma and 9 out of 10 lung squamous carcinoma samples. Methylation at CpG sites of the LLC1 promoter was frequently observed in lung cancer cell lines and in a fraction of primary lung cancer tissues. Treatment with 5‐aza deoxycytidine resulted in a reduced methylation of the LLC1 promoter concomitant with the increase of LLC1 expression. These results suggest that inactivation of LLC1 by means of promoter methylation is a frequent event in nonsmall cell lung cancer and may play a role in lung tumorigenesis.

Gene expression analysis of RNA purified from embryonic stem cells and embryoid body-derived cells using a high-throughput microarray platform.

In this unit, starting with purified RNA, experimental protocols for performing microarray expression analysis of embryonic stem cell lines compared to their corresponding differentiated embryocidal bodies are described. Methods for data analysis are suggested, with the goal of determining which genes are differentially expressed between the preparations. As an example, the use of the Affymetrix microarray expression platform is described, but alternative experimental options for analysis of RNA transcript levels are also summarized. This unit suggests quality control metrics, summarizes the critical parameters necessary for obtaining reproducible experimental results, and outlines quantitative PCR methods for validating microarray results.

Abstract 2168: Molecular pathway analysis of esophageal squamous cell differentiation versus carcinoma formation

Esophageal squamous cell carcinoma (ESCC) is the predominant histological subtype of esophageal cancer and is characterized by high mortality. Our previous work analyzing candidate tumor associated genes in ESCC highlighted the important differences in mRNA expression between the normal esophageal and tumor epithelial compartments. However, to date there are limited studies examining expression changes that occur during esophageal squamous cell differentiation versus those associated with ESCC tumorigenesis. To address this issue, we microdissected the basal cell layer and differentiated cell layer from normal epithelium and compared these expression profiles with matched tumor samples in twelve patients using gene expression microarrays (Hu133A 2.0, Affymetrix, Inc.) and qRT-PCR for selected miRNAs. Genes and pathways of interest were determined using class comparison and pathway analyses. Pathway expression of BRCA1 in the DNA damage response pathway presented the most significant difference (P Overall, the preliminary results indicate that tumor profiles were closer to basal cells than differentiated cells, consistent with their rapid growth rate and less well differentiated morphology. Investigation of gene expression patterns in normal cell types and ESCC provides novel insight into the transcript profiles associated with normal development versus carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2168.