Inactivation of Lgi1 in murine neuronal precursor cells leads to dysregulation of axon guidance pathways.

LGI1 mutations predispose to a rare epilepsy syndrome and when inactivated in mice leads to early onset seizures and premature death. Histopathology of the mature brain soon after birth shows cortical dysplasia in Lgi1 null mice with hypercellularity in the outer cortical layers. Here we show extensive gene expression changes in neuronal precursor cells from Lgi1 null mice compared with wild type mice. The most significantly dysregulated pathway involves canonical axon guidance signaling with multiple networks involved in cell movement, adhesion and invasion related to actin cytoskeleton reorganization. The Lgi1 null NPCs show increased cell motility in vitro compared with normal counterparts. Dysregulation of genes critical to cell movement/migration and critical transcription factors involved in early neuronal development is a prominent feature. These studies provide a critical mechanistic link to the observation of increased cellularity in the outer layers of the developing cortex in Lgi1 null mice.

Genomic analysis of racial differences in triple negative breast cancer.

Triple negative breast cancer (TNBC) is more prevalent in African Americans (AAs), has a more aggressive clinical course including a higher mortality rate and an increased occurrence of metastases. This study was designed to determine if racial differences at the molecular level might explain the more aggressive phenotype in AAs. Mutation profiling, was performed on 51 AA and 77 CA tumor/ normal pairs. Transcript expression analysis was performed on 35AA and 37CA. Genes with high frequency mutation rates such as MUC4 and TP53 were common to both racial populations, however genes that were less frequently mutated differed between the races suggesting that those cause the more aggressive nature of TNBC in AA women. JAK-Stat and HER2 signaling were unique to the AA and PTEN and mTOR were unique to the CA profiles. Many pathways identified by the mutational profiles were predicted to be down-regulated by the transcript expression profiles.

Distinct signaling programs associated with progression of FGFR1 driven leukemia in a mouse model of stem cell leukemia lymphoma syndrome.

Constitutive activation of FGFR1 as a result of chromosome translocations is responsible for the development of a hematopoietic stem cell disorder that progresses to AML. We have developed a syngeneic mouse model of BCR-FGFR1 driven AML and used RNASeq to define gene expression signatures associated with disease progression. The development of the leukemic stem cells (LSC) is associated with a profound downregulation of specific transcription factors that normally maintain stem cell quiescence as well as cell adhesion and motility gene sets related to confinement to the stem cell niche. A prominent feature of the LSCs is the upregulation of genes involved in T-cell function, activation, migration and development. Despite this apparent T-cell priming in the LSCs, however, the majority of these genes are subsequently inactivated in the leukemic blast cells that derive from them. These studies provide insights into the molecular etiology of development and progression of FGFR1 driven AML.

A systematic evaluation of miRNA:mRNA interactions involved in the migration and invasion of breast cancer cells.

In this study we performed a systematic evaluation of functional miRNA-mRNA interactions associated with the invasiveness of breast cancer cells using a combination of integrated miRNA and mRNA expression profiling, bioinformatics prediction, and functional assays. Analysis of the miRNA expression identified 11 miRNAs that were differentially expressed, including 7 down-regulated (miR-200c, miR-205, miR-203, miR-141, miR-34a, miR-183, and miR-375) and 4 up-regulated miRNAs (miR-146a, miR-138, miR-125b1 and miR-100), in invasive cell lines when compared to normal and less invasive cell lines. Transfection of miR-200c, miR-205, and miR-375 mimics into MDA-MB-231 cells led to the inhibition of in vitro cell migration and invasion. The integrated analysis of miRNA and mRNA expression identified 35 known and novel target genes of miR-200c, miR-205, and mir-375, including CFL2, LAMC1, TIMP2, ZEB1, CDH11, PRKCA, PTPRJ, PTPRM, LDHB, and SEC23A. Surprisingly, the majority of these genes (27 genes) were target genes of miR-200c, suggesting that miR-200c plays a pivotal role in regulating the invasiveness of breast cancer cells. We characterized one of the target genes of miR-200c, CFL2, and demonstrated that CFL2 is overexpressed in aggressive breast cancer cell lines and can be significantly down-regulated by exogenous miR-200c. Tissue microarray analysis further revealed that CFL2 expression in primary breast cancer tissue correlated with tumor grade. The results obtained from this study may improve our understanding of the role of these candidate miRNAs and their target genes in relation to breast cancer invasiveness and ultimately lead to the identification of novel biomarkers associated with prognosis.

Reexpression of LGI1 in glioma cells results in dysregulation of genes implicated in the canonical axon guidance pathway.

The LGI1 gene suppresses invasion in glioma cells and predisposes to epilepsy. In a gene expression array comparison between parental cells and T98G cell clones forced to express LGI1, we demonstrate that the canonical axon guidance pathway is the most significantly affected. In particular, aspects of axon guidance that involve reorganization of the actin cytoskeleton, which is also involved in cell movement and invasion, were affected. Analysis of actin fiber organization using fluorescence microscopy demonstrated that different T98G cell clones expressing the exogenous LGI1 gene show high levels of stress fibers compared with controls. Since stress fiber formation is associated with loss of cell mobility, we used scratch wound assays to demonstrate that LGI1-expressing clones show a significant reduction in cell mobility. LGI1 reexpression also resulted in loss of the PDGFRA and EGFR proteins, suggesting a rapid turnover of these receptors despite increased mRNA levels for PDGFRA. LGI1 suppression of invasion is associated with loss of ERK/MAPK1 activation. LGI1 is a secreted protein, and when the culture supernatant from cells expressing FLAG- and GFP-tagged proteins were applied to parental T98G cells, ERK/MAPK1 phosphorylation and cell mobility was suppressed, demonstrating that the LGI1 protein acts as a suppressive agent for cell movement in this assay. These observations support a previous suggestion that LGI1 can reduce cellular invasion in in vitro assays and, as a secreted agent, may be developed as a means of treating metastatic cancer. In addition, this observation provides a mechanistic link for LGI1's common role in metastasis and epilepsy development.

Normal colon epithelium: a dataset for the analysis of gene expression and alternative splicing events in colon disease.

BACKGROUND: Studies using microarray analysis of colorectal cancer have been generally beleaguered by the lack of a normal cell population of the same lineage as the tumor cell. One of the main objectives of this study was to generate a reference gene expression data set for normal colonic epithelium which can be used in comparisons with diseased tissues, as well as to provide a dataset that could be used as a baseline for studies in alternative splicing. RESULTS: We present a dependable expression reference data set for non-neoplastic colonic epithelial cells. An enriched population of fresh colon epithelial cells were obtained from non-neoplastic, colectomy specimens and analyzed using Affymetrix GeneChip EXON 1.0 ST arrays. For demonstration purposes, we have compared the data derived from these cells to a publically available set of tumor and matched normal colon data. This analysis allowed an assessment of global gene expression alterations and demonstrated that adjacent normal tissues, with a high degree of cellular heterogeneity, are not always representative of normal cells for comparison to tumors which arise from the colon epithelium. We also examined alternative splicing events in tumors compared to normal colon epithelial cells. CONCLUSIONS: The findings from this study represent the first comprehensive expression profile for non-neoplastic colonic epithelial cells reported. Our analysis of splice variants illustrate that this is a very labor intensive procedure, requiring vigilant examination of the data. It is projected that the contribution of this set of data derived from pure colonic epithelial cells will enhance studies in colon-related disease and offer a vital baseline for studies aimed at elucidating the mechanisms of alternative splicing.

Integration of transcript expression, copy number and LOH analysis of infiltrating ductal carcinoma of the breast.

BACKGROUND: A major challenge in the interpretation of genomic profiling data generated from breast cancer samples is the identification of driver genes as distinct from bystander genes which do not impact tumorigenesis. One way to assess the relative importance of alterations in the transcriptome profile is to combine parallel analyses that assess changes in the copy number alterations (CNAs). This integrated analysis permits the identification of genes with altered expression that map within specific chromosomal regions which demonstrate copy number alterations, providing a mechanistic approach to identify the 'driver genes'. METHODS: We have performed whole genome analysis of CNAs using the Affymetrix 250K Mapping array on 22 infiltrating ductal carcinoma samples (IDCs). Analysis of transcript expression alterations was performed using the Affymetrix U133 Plus2.0 array on 16 IDC samples. Fourteen IDC samples were analyzed using both platforms and the data integrated. We also incorporated data from loss of heterozygosity (LOH) analysis to identify genes showing altered expression in LOH regions. RESULTS: Common chromosome gains and amplifications were identified at 1q21.3, 6p21.3, 7p11.2-p12.1, 8q21.11 and 8q24.3. A novel amplicon was identified at 5p15.33. Frequent losses were found at 1p36.22, 8q23.3, 11p13, 11q23, and 22q13. Over 130 genes were identified with concurrent increases or decreases in expression that mapped to these regions of copy number alterations. LOH analysis revealed three tumors with whole chromosome or p arm allelic loss of chromosome 17. Genes were identified that mapped to copy neutral LOH regions. LOH with accompanying copy loss was detected on Xp24 and Xp25 and genes mapping to these regions with decreased expression were identified. Gene expression data highlighted the PPARα/RXRα Activation Pathway as down-regulated in the tumor samples. CONCLUSION: We have demonstrated the utility of the application of integrated analysis using high resolution CGH and whole genome transcript analysis for detecting driver genes in IDC. The high resolution platform allowed a refined demarcation of CNAs and gene expression profiling provided a mechanism to detect genes directly impacted by the CNA. This is the first report of LOH integrated with gene expression in IDC using a high resolution platform.

Interpreting aCGH-defined karyotypic changes in gliomas using copy number status, loss of heterozygosity and allelic ratios.

We have used SNP mapping arrays to simultaneously record copy number changes, loss of heterozygosity and allele ratios (ploidy) in a series of 13 gliomas. This combined analysis has defined novel amplification events in this tumor type involving chr1:241544532-243005121 and chr18:54716681-54917277 which contain the AKT3 and ZNF532 genes, respectively. The high resolution of this analysis has also identified homozygous deletions involving chr17:25600031-26490848 and Chr19:53883612-55061878. Throughout the karyotypes of these tumors, the combined analysis revealed counter intuitive relationships between copy number and LOH that requires reinterpretation of the significance of copy number gains and losses. It was not uncommon to observe copy number gains that were associated with loss of heterozygosity as well as copy number losses that were not. These events appeared to be related to ploidy status in the tumors as determined using allelic ratio calculations. Overall, this analysis of gliomas provides evidence for the need to perform more comprehensive interpretation of the CGH data beyond copy number analysis alone to evaluate the significance of individual events in the karyotypes.

Variant profiles of genes mapping to chromosome 16q loss in Wilms tumors reveals link to cilia-related genes and pathways.

BACKGROUND: Wilms tumor is the most common pediatric renal tumor and the fourth most common malignancy in children. Chromosome 16q deletion(del) or loss of heterozygosity (LOH) has been correlated with recurrence and overall poor prognosis, such that patients with 16qLOH and 1p allelic loss are treated with more aggressive chemotherapeutic regimens. METHODS: In the present study, we have compared the variant profiles of Wilms tumors with and without 16q del/LOH using both data available from the TARGET database (42 samples) and tumors procured from our legacy collection (8 samples). Exome-Seq data was analyzed for tumor specific variants mapping to 16q. Whole exome analysis was also performed. An unbiased approach for somatic variant analysis was used to detect tumor-specific, somatic variants. RESULTS: Of the 72 genes mapping to 16q, 42% were cilia-related genes and 28% of these were found to carry somatic variants specific to those tumors with 16qdel/LOH. Whole exome analyses further revealed that 30% of cilia-related genes across the genome carried alterations in tumors both with and without 16qdel/LOH. Additional pathway analyses revealed that many cilia-related pathway members also carried deleterious variant in these tumors including Sonic Hedgehog (SHh), Wnt, and Notch signaling pathways. CONCLUSIONS: The data suggest that cilia-related genes and pathways are compromised in Wilms tumors. The genes on chromosome 16q that carry deleterious variants in cilia-related genes may account for the more aggressive nature of tumors with 16q del/LOH.

Transformation of MCF-10A cells by random mutagenesis with frameshift mutagen ICR191: a model for identifying candidate breast-tumor suppressors.

BACKGROUND: Widely accepted somatic mutation theory of carcinogenesis states that mutations in oncogenes and tumor suppressor genes in genomes of somatic cells is the cause of neoplastic transformation. Identifying frequent mutations in cancer cells suggests the involvement of mutant genes in carcinogenesis. RESULTS: To develop an in vitro model for the analysis of genetic alterations associated with breast carcinogenesis, we used random mutagenesis and selection of human non-tumorigenic immortalized breast epithelial cells MCF-10A in tissue-culture conditions that mimic tumor environment. Random mutations were generated in MCF-10A cells by cultivating them in a tissue-culture medium containing the frameshift-inducing agent ICR191. The first selective condition we used to transform MCF1-10A cells was cultivation in a medium containing mutagen at a concentration that allowed cell replication despite p53 protein accumulation induced by mutagen treatment. The second step of selection was either cell cultivation in a medium with reduced growth-factor supply or in a medium that mimics a hypoxia condition or growing in soft agar. Using mutagenesis and selection, we have generated several independently derived cultures with various degrees of transformation. Gene Identification by Nonsense-mediated mRNA decay Inhibition (GINI) analysis has identified the ICR191-induced frameshift mutations in the TP53, smoothelin, Ras association (RalGDS/AF-6) domain family 6 (RASSF6) and other genes in the transformed MCF-10A cells. The TP53 gene mutations resulting in the loss of protein expression had been found in all independently transformed MCF-10A cultures, which form large progressively growing tumors with sustained angiogenesis in nude mice. CONCLUSION: Identifying genes containing bi-allelic ICR191-induced frameshift mutations in the transformed MCF-10A cells generated by random mutagenesis and selection indicates putative breast-tumor suppressors. This can provide a model for studying the role of mutant genes in breast carcinogenesis.

Identification of genes involved in squamous cell carcinoma of the lung using synchronized data from DNA copy number and transcript expression profiling analysis.

Lung cancer is the leading cause of cancer deaths in the world and squamous cell carcinoma (SqCC) is the second most common in this group. Genomic DNA copy number alterations are fundamental genetic events in the development and progression of SqCC as well as other epithelial-derived cancers. The ability to identify tumor suppressor genes (TSGs) and oncogenes that are affected during tumor initiation and progression could facilitate the identification of novel molecular targets for therapeutic intervention and provide diagnostic biomarkers. Despite the association of many genetic alterations in lung cancer the molecular mechanisms of tumor progression remain ambiguous since often too many candidates are revealed using conventional genetic microarray analysis. To overcome this limitation, we have identified genes in SqCC which show concordant gene expression changes defined using microarray analysis with DNA copy number alterations defined by BAC-array comparative genomic hybridization (aCGH) in the same tumors. An in-house overlay algorithm was used to synchronize the data resulting from the two analyses. Although the expression levels of many genes were altered when compared to normal controls, those which correlated with copy number changes were far fewer, providing a manageable number for biological studies. We identified over 2000 genes which displayed both gene expression alterations and mapped to BACs which demonstrated a corresponding loss or gain. A further stringent statistical analysis identified minimal regions of overlap for losses or gains which displayed a coincident decrease or increase in the expression of genes mapping to those regions. Consistent gains involved 3q23-q29, 5p15.1-q11.1 and chromosomes 18 and 20, while consistent losses involved 3p26.3-p12.3, 9p24.3-q34.3, and chromosomes 17 and 19. The concordance finding between these two approaches suggests that DNA copy number alterations can directly influence gene expression patterns that impact on tumorigenesis in SqCC of the lung.

Universal Reference RNA is not a representative normal sample for oligonucleotide microarray studies.

Translational research has been defined as the scientific study using human material that will ultimately generate patient specific data. A major caveat in human directed study is the availability of high quality and quantities of patient derived homogeneous cells for analysis. Whereas there exist sources for which tumor tissue and blood samples can be made available, the same cannot be said for normal tissue. The absence of normal control tissue has led to the creation of pooled cell lines and tissues for purchase known as "reference RNA". Although initially created for purposes of standardization, the difficulty associated with acquiring normal tissue has led some investigators to use sources of universal pooled RNA for comparative analysis with clinical tissue specimens. In order to study the effects of using Universal Reference RNA on expression profiling experiments we have evaluated the performance of universal RNA compared to RNA obtained from a purified population of colon epithelial cells in defining a set of altered transcripts in colon cancer.

The application of microarray technology to the analysis of the cancer genome.

The identification of genetic events that are involved in the development of human cancer has been facilitated through the development and application of a diverse series of high resolution, high throughput microarray platforms. Essentially there are two types of array; those that carry PCR products from cloned nucleic acids (e.g. cDNA, BACs, cosmids) and those that use oligonucleotides. Each has advantages and disadvantages but it is now possible to survey genome wide DNA copy number abnormalities and expression levels to allow correlations between losses, gains and amplifications in tumor cells with genes that are over- and under-expressed in the same samples. The gene expression arrays that provide estimates of mRNA levels in tumors have given rise to exon-specific arrays that can identify both gene expression levels, alternative splicing events and mRNA processing alterations. Oligonucleotide arrays are also being used to interrogate single nucleotide polymorphisms (SNPs) throughout the genome for linkage and association studies and these have been adapted to quantify copy number abnormalities and loss of heterozygosity events. To identify as yet unknown transcripts tiling arrays across the genome have been developed which can also identify DNA methylation changes and be used to identify DNA-protein interactions using ChIP on Chip protocols. Ultimately DNA sequencing arrays will allow resequencing of chromosome regions and whole genomes. With all of these capabilities becoming routine in genomics laboratories, the idea of a systematic characterization of the sum genetic events that give rise to a cancer cell is rapidly becoming a reality.

miR-339 Promotes Development of Stem Cell Leukemia/Lymphoma Syndrome via Downregulation of the BCL2L11 and BAX Proapoptotic Genes.

The development of myeloid and lymphoid neoplasms related to overexpression of FGFR1 kinases as a result of chromosome translocations depends on the promotion of a stem cell phenotype, suppression of terminal differentiation, and resistance to apoptosis. These phenotypes are related to the stem cell leukemia/lymphoma syndrome (SCLL), which arises through the effects of the activated FGFR1 kinase on gene transcription, which includes miRNA dysregulation. In a screen for miRNAs that are directly regulated by FGFR1, and which stimulate cell proliferation and survival, we identified miR-339-5p, which is highly upregulated in cells carrying various different chimeric kinases. Overexpression of miR-339-5p in SCLL cell types enhances cell survival and inhibition of its function leads to reduced cell viability. miR-339-5p overexpression protects cells from the consequences of FGFR1 inactivation, promoting cell-cycle progression and reduced apoptosis. Transient luciferase reporter assays and qRT-PCR detection of endogenous miR-339-5p expression in stably transduced cell lines demonstrated that BCR-FGFR1 can directly regulate miR-339-5p expression. This correlation between miR-339-5p and FGFR1 expression is also seen in primary human B-cell precursor acute lymphoblastic leukemia. In a screen to identify targets of miR-339-5p, we identified and verified the BCL2L11 and BAX genes, which can promote apoptosis. In vivo, SCLL cells forced to overexpress miR-339-5p show a more rapid onset of disease and poorer survival compared with parental cells expressing endogenous levels of miR-339-5p. Analysis of human primary B-cell precursor ALL shows a significant higher expression of miR339-5p compared with the two cohorts of CLL patient samples, suggesting direct roles in disease progression and supporting the evidence generated in mouse models of SCLL.Significance: Proapoptiotic genes that are direct targets of miR-339-5p significantly influence promotion and aggressive development of leukemia/lymphomas associated with FGFR1 overexpression. Cancer Res; 78(13); 3522-31. ©2018 AACR.

Platinum drug effects on the expression of genes in the polyamine pathway: time-course and concentration-effect analysis based on Affymetrix gene expression profiling of A2780 ovarian carcinoma cells.

PURPOSE: As a follow-up to our previous findings that platinum drugs induce a key enzyme in polyamine catabolism, gene expression profiling and mathematical modeling were used to define the effects of cisplatin and oxaliplatin on the expression of polyamine metabolic pathway genes in A2780 human ovarian carcinoma cells. METHODS: Time-course and concentration-effect experiments were each carried out with cisplatin or oxaliplatin in two separate experiments and cells subjected to gene expression profiling using Affymetrix array technology. Time-course data were modeled using exponential increase and decrease models. Concentration-effect data were modeled using a four parameter Hill model. RESULTS: Gene expression profiling of human ovarian carcinoma A2780 cells after exposure to either cisplatin or oxaliplatin indicates that the expression of several genes involved in polyamine pathway is affected by the platinum drugs. Mathematical/Statistical modeling of the data from time-course and concentration-effect experiments of gene expression from nine polyamine pathway genes represented on the HGU95Av2 chip, indicates that three biosynthetic pathway genes (SAMDC, ODC1 and SRM) are down-regulated and one catabolic pathway gene (SSAT) is up-regulated. Expression changes were similar for different probesets for a given gene on the array. Studies on the induction of SSAT by platinum drugs suggested by the Affymetrix data have been previously validated from this laboratory (Hector et al. in Mol Cancer Ther 3:813-822, 2004). Here, the effects of oxaliplatin exposure on SAMDC and ODC observed by Affymetix are validated with real time QRT-PCR. CONCLUSION: The data indicate a concerted effect of platinum drugs on the polyamine metabolic pathway with down-regulation in the expression of several enzyme genes involved in biosynthesis and many-fold up-regulation in expression of SSAT, an acetylating enzyme gene that is critically involved in polyamine catabolism and export.

An exfoliation and enrichment strategy results in improved transcriptional profiles when compared to matched formalin fixed samples.

BACKGROUND: Identifying the influence formalin fixation has on RNA integrity and recovery from clinical tissue specimens is integral to determining the utility of using archival tissue blocks in future molecular studies. For clinical material, the current gold standard is unfixed tissue that has been snap frozen. Fixed and frozen tissue however, both require laser capture microdissection to select for a specific cell population to study. The recent development of a sampling method capable of obtaining a viable, enriched cell population represents an alternative option in procuring cells from clinical material for molecular research purposes. The expression profiles of cells obtained by using this procurement approach, in conjunction with the profiles from cells laser capture microdissected from frozen tissue sections, were compared to the expression profiles from formalin fixed cells to determine the influence fixation has on expression profiles in clinical material. METHODS: Triplicate samples of non-neoplastic colonic epithelial cells were recovered from a hemicolectomy specimen using three different procurement methods from the same originating site: 1) an exfoliation and enrichment strategy 2) laser capture microdissection from formalin fixed tissue and 3) laser capture microdissection from frozen tissue. Parameters currently in use to assess RNA integrity were utilized to assess the quality of recovered RNA. Additionally, an expression microarray was performed on each sample to assess the influence each procurement technique had on RNA recovery and degradation. RESULTS: The exfoliation/enrichment strategy was quantitatively and qualitatively superior to tissue that was formalin fixed. Fixation negatively influenced the expression profile of the formalin fixed group compared to both the frozen and exfoliated/enrichment groups. CONCLUSION: The exfoliation/enrichment technique represents a superior alternative in tissue procurement and RNA recovery relative to formalin fixed tissue. None of the deleterious effects associated with formalin fixation are encountered in the exfoliated/enriched samples because of the absence of its use in this protocol. The exfoliation/enrichment technique also represents an economical alternative that will yield comparable results to cells enriched by laser capture microdissection from frozen tissue sections.

Characterization of cell-type specific profiles in tissues and isolated cells from squamous cell carcinomas of the lung.

Lung cancer accounts for 28% of all cancer deaths, a higher percentage than any other human cancer. Squamous Cell Carcinoma (SqCC) is the most common lung neoplasm and is a tumor that is extensively associated with tobacco use. Despite the association of many genetic alterations with lung cancer, the precise molecular mechanisms of tumorigenesis, for the most part, remain ambiguous. Although many studies of lung cancer have used global transcript profiling approaches designed to uncover genes or pathways that are important in lung tumorigenesis, no strong candidates have emerged. A lack of concurrence amongst these various studies can be attributed, in a large part, to the cellular heterogeneity within lung tissue. We have attempted to reduce this complication by designing a profiling strategy that will minimize the confounding involvement of tissue heterogeneity in gene expression of lung tumors. Specifically, we have profiled transcript expression levels in both isolated cells and tissues from SqCC and normal samples. Our strategy consists of combining and subtracting the input of these various cell types which has produced a unique transcript profile of the squamous carcinoma cell. We then analyzed the data using Pathways Assist analysis software to determine which processes may be involved in SqCC tumorigenesis. The MAP/ERK pathway involved in growth and differentiation was the pathway that was most frequently identified across all comparisons. In addition, biological interaction networks of the SqCC profile identified IL-8 as playing a potentially important role SqCC development.

Aberrant expression of novel and previously described cell membrane markers in human breast cancer cell lines and tumors.

PURPOSE: In a previous gene expression array study, we identified some 300 genes that were differentially expressed in human epidermal growth factor receptor tyrosine kinase 2 (HER2)-positive versus HER2-negative breast cancer cells. We have now done validation experiments on a group of three cell membrane components that had previously not been implicated in breast cancer. We also studied the expression of three other cell membrane proteins known to play a role in mammary neoplasia. EXPERIMENTAL DESIGN: By immunohistochemistry, we examined up to 130 archival breast carcinomas for Celsr2, E-cadherin, Kai1, and CD9 expression. The expression levels of NET-6 and TROP-2 were determined by quantitative reverse transcription-PCR in a subset of frozen tumors. We also studied fresh pellets and paraffin-embedded cell buttons of nine human breast cell lines. The relationship between the expression of all six membrane proteins and a variety of pathologic and biological variables, including estrogen receptor, HER2, and epidermal growth factor receptor status, was also examined. The NET-6 gene was transfected into a low-expressing cell line, and the effect on cellular morphology, growth, and invasion in vitro was recorded. RESULTS: Celsr2 was down-regulated in one cell line and in 7% of breast cancers. E-cadherin, Kai1, and CD9 were down-regulated in 35%, 76%, and 79% of tumors, respectively, confirming the important role of these markers in human mammary neoplasia. In breast cancer cell lines and tissues, TROP-2 was generally expressed at low levels, although a few specimens showed relative overexpression. NET-6 levels were lower in HER2-negative breast carcinoma cells. In addition, NET-6 was markedly down-regulated in estrogen receptor-negative breast cancers, and expression was lowest in "basal-like" tumors. Ectopic expression of NET-6 in low-expressing MDA-MB-231 cells altered cellular morphology, inhibited growth in vitro, and decreased invasion in a Boyden chamber assay. CONCLUSIONS: We have confirmed the expression of three new membrane markers that had previously not been implicated in human breast cancer, and one of them (NET-6) was correlated with HER2 and estrogen receptor status. NET-6 levels were decreased in estrogen receptor-negative and high-grade tumors, and ectopic expression of this gene had an inhibitory effect on proliferation and invasion. Thus, NET-6 may represent a novel breast cancer suppressor gene.

Delineation of the molecular basis for selenium-induced growth arrest in human prostate cancer cells by oligonucleotide array.

Despite the growing interest in selenium intervention of prostate cancer in humans, scanty information is currently available on the molecular mechanism of selenium action. Our past research indicated that methylseleninic acid (MSA) is an excellent reagent for investigating the anticancer effect of selenium in vitro. The present study was designed to examine the cellular and molecular effects of MSA in PC-3 human prostate cancer cells. After exposure to physiological concentrations of MSA, these cells exhibited a dose- and time-dependent inhibition of growth. MSA retarded cell cycle progression at multiple transition points without changing the proportion of cells in different phases of the cell cycle. Flow cytometric analysis of annexin V- and propidium iodide-labeled cells showed a marked induction of apoptosis by MSA. Array analysis with the Affymetrix human genome U95A chip was then applied to profile the gene expression changes that might mediate the effects of selenium. Gene profiling was done in a time course experiment (at 12, 24, 36, and 48 h) using synchronized cells. A large number of potential selenium-responsive genes with diverse biological functions were identified. These genes fell into 12 clusters of distinct kinetics pattern of modulation by MSA. The expression changes of 10 genes known to be critically involved in cell cycle regulation were selected for verification by Western analysis to determine the reliability of the array data. An agreement rate of 70% was obtained based on these confirmation experiments. The array data enabled us to focus on the role of potential key genes (e.g., GADD153, CHK2, p21(WAF1), cyclin A, CDK1, and DHFR) that might be targets of MSA in impeding cell cycle progression. The data also provide valuable insights into novel biological effects of selenium, such as inhibition of cell invasion, DNA repair, and stimulation of transforming growth factor beta signaling. The present study demonstrates the utility of a genome-wide analysis to elucidate the mechanism of selenium chemoprevention.

Up-regulation of c-Jun-NH2-kinase pathway contributes to the induction of mitochondria-mediated apoptosis by alpha-tocopheryl succinate in human prostate cancer cells.

Previously, alpha-tocopheryl succinate (alpha-TOS) has been reported to induce caspase-mediated apoptosis in PC-3 human prostate cancer cells. Caspase-9 was among several initiator caspases activated by alpha-TOS, suggesting a potential contribution of the intrinsic apoptotic pathway in mediating the response to alpha-TOS. Gene expression microarray was carried out as a screen to identify novel signaling molecules modulated by alpha-TOS, with a special focus on those known to play a role in mitochondria-mediated apoptosis. We discovered that Ask1, GADD45beta, and Sek1, three key components of the stress-activated mitogen-activated protein kinase pathway, are novel targets of alpha-TOS. Western blot analysis showed increased levels of phospho-Sek1 and phospho-c-Jun-NH2-kinase (JNK) in addition to total Ask1, GADD45beta, and Sek1. alpha-TOS also altered JNK-specific phosphorylation of Bcl-2 and Bim in a manner consistent with enhanced mitochondrial translocation of Bax and Bim. Because the expression level of most Bcl-2 family members remained unchanged, the posttranslational modification of Bcl-2 and Bim by JNK is likely to be a driving force in alpha-TOS activation of the intrinsic apoptotic pathway. Based on our findings, we propose a working model to capture the salient features of the apoptotic signaling circuitry of alpha-TOS.