CGH, cDNA and tissue microarray analyses implicate FGFR2 amplification in a small subset of breast tumors.

Multiple regions of the genome are often amplified during breast cancer development and progression, as evidenced in a number of published studies by comparative genomic hybridization (CGH). However, only relatively few target genes for such amplifications have been identified. Here, we indicate how small-scale commercially available cDNA and CGH microarray formats combined with the tissue microarray technology enable rapid identification of putative amplification target genes as well as analysis of their clinical significance. According to CGH, the SUM-52 breast cancer cell line harbors several high-level DNA amplification sites, including the 10q26 chromosomal region where the fibroblast growth factor receptor 2 (FGFR2) gene has been localized. High level amplification of FGFR2 in SUM-52 was identified using CGH analysis on a microarray of BAC clones. A cDNA microarray survey of 588 genes showed >40-fold overexpression of FGFR2. Finally, a tissue microarray based FISH analysis of 750 uncultured primary breast cancers demonstrated in vivo amplification of the FGFR2 gene in about 1% of the tumors. In conclusion, three consecutive microarray (CGH, cDNA and tissue) experiments revealed high-level amplification and overexpression of the FGFR2 in a breast cancer cell line, but only a low frequency of involvement in primary breast tumors. Applied to a genomic scale with larger arrays, this strategy should facilitate identification of the most important target genes for cytogenetic rearrangements, such as DNA amplification sites detected by conventional CGH. Figures on http://www.esacp.org/acp/2001/22-4/heiskanen.htm     

Suicide HSVtk Gene Delivery by Neurotensin-Polyplex Nanoparticles via the Bloodstream and GCV Treatment Specifically Inhibit the Growth of Human MDA-MB-231 Triple Negative Breast Cancer Tumors Xenografted in Athymic Mice

The human breast adenocarcinoma cell line MDA-MB-231 has the triple-negative breast cancer (TNBC) phenotype, which is an aggressive subtype with no specific treatment. MDA-MB-231 cells express neurotensin receptor type 1 (NTSR1), which makes these cells an attractive target of therapeutic genes that are delivered by the neurotensin (NTS)-polyplex nanocarrier via the bloodstream. We addressed the relevance of this strategy for TNBC treatment using NTS-polyplex nanoparticles harboring the herpes simplex virus thymidine kinase (HSVtk) suicide gene and its complementary prodrug ganciclovir (GCV). The reporter gene encoding green fluorescent protein (GFP) was used as a control. NTS-polyplex successfully transfected both genes in cultured MDA-MB-231 cells. The transfection was demonstrated pharmacologically to be dependent on activation of NTSR1. The expression of HSVtk gene decreased cell viability by 49% (P<0.0001) and induced apoptosis in cultured MDA-MB-231 cells after complementary GCV treatment. In the MDA-MB-231 xenograft model, NTS-polyplex nanoparticles carrying either the HSVtk gene or GFP gene were injected into the tumors or via the bloodstream. Both routes of administration allowed the NTS-polyplex nanoparticles to reach and transfect tumorous cells. HSVtk expression and GCV led to apoptosis, as shown by the presence of cleaved caspase-3 and Apostain immunoreactivity, and significantly inhibited the tumor growth (55–60%) (P<0.001). At the end of the experiment, the weight of tumors transfected with the HSVtk gene was 55% less than that of control tumors (P<0.05). The intravenous transfection did not induce apoptosis in peripheral organs. Our results offer a promising gene therapy for TNBC using the NTS-polyplex nanocarrier.     

A fine balance between CCNL1 and TIMP1 contributes to the development of breast cancer cells

Cyclin L1 (CCNL1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP1) are candidate genes involved in several types of cancer. However, the expression of CCNL1 and the relationship between CCNL1 and TIMP1 in breast cancer cells is unknown. Using patients’ breast cancer tissues, the expression of CCNL1 and TIMP1 was measured by cDNA microarray and further confirmed by real-time RT-PCR and western blotting. Overexpression or repression of CCNL1 and TIMP1, individually or together, was performed in breast cancer MDA-MB-231 cells by transient transformation methods to investigate their role in breast cancer cell growth. Simultaneously, mRNA and protein expression levels of CCNL1 and TIMP1 were also measured. CCNL1 and TIMP1 expression was significantly elevated in breast cancer tissues compared with that in peri-breast cancer tissues of patients by cDNA microarray and these results were further confirmed by real-time RT-PCR and western blotting. Interestingly, in vitro experiments showed a stimulatory effect of TIMP1 and an inhibitory effect of CCNL1 on growth of MDA-MB-231 cells. Co-expression or co-repression of these two genes did not affect cell growth. Overexpression of CCNL1 and TIMP1 individually induced overexpression of each other. These data demonstrate that there is a fine balance between CCNL1 and TIMP1, which may contribute to breast cancer development.     

SWI/SNF Chromatin-Remodeling Factor Smarcd3/Baf60c Controls Epithelial-Mesenchymal Transition by Inducing Wnt5a Signaling

We previously identified a gene signature predicted to regulate the epithelial-mesenchymal transition (EMT) in both epithelial tissue stem cells and breast cancer cells. A phenotypic RNA interference (RNAi) screen identified the genes within this 140-gene signature that promoted the conversion of mesenchymal epithelial cell adhesion molecule-negative (EpCAM⁻) breast cancer cells to an epithelial EpCAM^+/high phenotype. The screen identified 10 of the 140 genes whose individual knockdown was sufficient to promote EpCAM and E-cadherin expression. Among these 10 genes, RNAi silencing of the SWI/SNF chromatin-remodeling factor Smarcd3/Baf60c in EpCAM⁻ breast cancer cells gave the most robust transition from the mesenchymal to epithelial phenotype. Conversely, expression of Smarcd3/Baf60c in immortalized human mammary epithelial cells induced an EMT. The mesenchymal-like phenotype promoted by Smarcd3/Baf60c expression resulted in gene expression changes in human mammary epithelial cells similar to that of claudin-low triple-negative breast cancer cells. These mammary epithelial cells expressing Smarcd3/Baf60c had upregulated Wnt5a expression. Inhibition of Wnt5a by either RNAi knockdown or blocking antibody reversed Smarcd3/Baf60c-induced EMT. Thus, Smarcd3/Baf60c epigenetically regulates EMT by activating WNT signaling pathways.     

Network Analysis of Breast Cancer Progression and Reversal Using a Tree-Evolving Network Algorithm

The HMT3522 progression series of human breast cells have been used to discover how tissue architecture, microenvironment and signaling molecules affect breast cell growth and behaviors. However, much remains to be elucidated about malignant and phenotypic reversion behaviors of the HMT3522-T4-2 cells of this series. We employed a “pan-cell-state” strategy, and analyzed jointly microarray profiles obtained from different state-specific cell populations from this progression and reversion model of the breast cells using a tree-lineage multi-network inference algorithm, Treegl. We found that different breast cell states contain distinct gene networks. The network specific to non-malignant HMT3522-S1 cells is dominated by genes involved in normal processes, whereas the T4-2-specific network is enriched with cancer-related genes. The networks specific to various conditions of the reverted T4-2 cells are enriched with pathways suggestive of compensatory effects, consistent with clinical data showing patient resistance to anticancer drugs. We validated the findings using an external dataset, and showed that aberrant expression values of certain hubs in the identified networks are associated with poor clinical outcomes. Thus, analysis of various reversion conditions (including non-reverted) of HMT3522 cells using Treegl can be a good model system to study drug effects on breast cancer.     

Long non-coding RNA UCA1 promotes breast tumor growth by suppression of p27 (Kip1)

Functional genomics studies have led to the discovery of a large amount of non-coding RNAs from the human genome; among them are long non-coding RNAs (lncRNAs). Emerging evidence indicates that lncRNAs could have a critical role in the regulation of cellular processes such as cell growth and apoptosis as well as cancer progression and metastasis. As master gene regulators, lncRNAs are capable of forming lncRNA–protein (ribonucleoprotein) complexes to regulate a large number of genes. For example, lincRNA-RoR suppresses p53 in response to DNA damage through interaction with heterogeneous nuclear ribonucleoprotein I (hnRNP I). The present study demonstrates that hnRNP I can also form a functional ribonucleoprotein complex with lncRNA urothelial carcinoma-associated 1 (UCA1) and increase the UCA1 stability. Of interest, the phosphorylated form of hnRNP I, predominantly in the cytoplasm, is responsible for the interaction with UCA1. Moreover, although hnRNP I enhances the translation of p27 (Kip1) through interaction with the 5′-untranslated region (5′-UTR) of p27 mRNAs, the interaction of UCA1 with hnRNP I suppresses the p27 protein level by competitive inhibition. In support of this finding, UCA1 has an oncogenic role in breast cancer both in vitro and in vivo. Finally, we show a negative correlation between p27 and UCA in the breast tumor cancer tissue microarray. Together, our results suggest an important role of UCA1 in breast cancer.     

3′-End Sequencing for Expression Quantification (3SEQ) from Archival Tumor Samples

Gene expression microarrays are the most widely used technique for genome-wide expression profiling. However, microarrays do not perform well on formalin fixed paraffin embedded tissue (FFPET). Consequently, microarrays cannot be effectively utilized to perform gene expression profiling on the vast majority of archival tumor samples. To address this limitation of gene expression microarrays, we designed a novel procedure (3′-end sequencing for expression quantification (3SEQ)) for gene expression profiling from FFPET using next-generation sequencing. We performed gene expression profiling by 3SEQ and microarray on both frozen tissue and FFPET from two soft tissue tumors (desmoid type fibromatosis (DTF) and solitary fibrous tumor (SFT)) (total n = 23 samples, which were each profiled by at least one of the four platform-tissue preparation combinations). Analysis of 3SEQ data revealed many genes differentially expressed between the tumor types (FDR<0.01) on both the frozen tissue (∼9.6K genes) and FFPET (∼8.1K genes). Analysis of microarray data from frozen tissue revealed fewer differentially expressed genes (∼4.64K), and analysis of microarray data on FFPET revealed very few (69) differentially expressed genes. Functional gene set analysis of 3SEQ data from both frozen tissue and FFPET identified biological pathways known to be important in DTF and SFT pathogenesis and suggested several additional candidate oncogenic pathways in these tumors. These findings demonstrate that 3SEQ is an effective technique for gene expression profiling from archival tumor samples and may facilitate significant advances in translational cancer research.     

Anchoring Ethinylestradiol Induced Gene Expression Changes with Testicular Morphology and Reproductive Function in the Medaka

Environmental estrogens are ubiquitous in the environment and can cause detrimental effects on male reproduction. In fish, a multitude of effects from environmental estrogens have been observed including altered courting behavior and fertility, sex reversal, and gonadal histopathology. However, few studies in fish assess the impacts of estrogenic exposure on a physiological endpoint, such as reproduction, as well as the associated morphologic response and underlying global gene expression changes. This study assessed the implications of a 14 day sub-chronic exposure of ethinylestradiol (EE2; 1.0 or 10.0 µg/L EE2) on male medaka fertility, testicular histology and testicular gene expression. The findings demonstrate that a 14 day exposure to EE2 induced impaired male reproductive capacity and time- and dose-dependent alterations in testicular morphology and gene expression. The average fertilization rate/day following the exposure for control, 1.0 and 10.0 µg/L EE2 was 91.3% (±4.4), 62.8% (±8.3) and 28.8% (±5.8), respectively. The testicular morphologic alterations included increased germ cell apoptosis, decreased germinal epithelium and thickening of the interstitium. These changes were highly associated with testicular gene expression changes using a medaka-specific microarray. A pathway analysis of the differentially expressed genes emphasized genes and pathways associated with apoptosis, cell cycle and proliferation, collagen production/extracellular matrix organization, hormone signaling, male reproduction and protein ubiquitination among others. These findings highlight the importance of anchoring global gonadal gene expression changes with morphology and ultimately with tissue/organ function.     

WDR5 Expression Is Prognostic of Breast Cancer Outcome

WDR5 is a core component of the human mixed lineage leukemia-2 complex, which plays central roles in ER positive tumour cells and is a major driver of androgen-dependent prostate cancer cell proliferation. Given the similarities between breast and prostate cancers, we explore the potential prognostic value of WDR5 gene expression on breast cancer survival. Our findings reveal that WDR5 over-expression is associated with poor breast cancer clinical outcome in three gene expression data sets and BreastMark. The eQTL analysis reveals 130 trans-eQTL SNPs whose genes mapped with statistical significance are significantly associated with patient survival. These genes together with WDR5 are enriched with “cellular development, gene expression, cell cycle” signallings. Knocking down WDR5 in MCF7 dramatically decreases cell viability, but does not alter tumour cell response to doxorubicin. Our study reveals the prognostic value of WDR5 expression in breast cancer which is under long-range regulation of genes involved in cell cycle, and anthracycline could be coupled with treatments targeting WDR5 once such a regimen is available.     

Retinoic acid and ascorbic acid act synergistically in inhibiting human breast cancer cell proliferation

BACKGROUND: Breast cancer is an increasingly common malignancy. Several vitamins such as retinoic acid (RA), ascorbic acid (AA), vitamin D and vitamin E are known to prevent the development and progression of breast cancer. OBJECTIVE: We sought to determine whether RA and AA together (RA+AA) acted synergistically in blocking the proliferation of human breast cancer cells. To elucidate the mechanism by which RA+AA inhibited breast carcinoma proliferation, we then evaluated the gene expression profiles of the treated and untreated cells by radioactive cDNA microarray analysis. METHODS: We cultured the human breast cancer cell line MCF-7 for 3 days with 100 nM RA and/or 1 mM AA, counted the cell numbers and harvested the total RNAs for cDNA microarray analysis. RESULTS: RA, AA and RA+AA reduced MCF-7 cell proliferation by 20.7%, 23.3% and 75.7% relative to the untreated cell proliferation, respectively. The synergistic ratio of RA and AA was 1.72. The MCF-7 gene expression profiles showed that 29 genes were up-regulated and 38 genes were down-regulated after RA+AA treatment. The nature of these genes suggests that the mechanism by which RA and AA act synergistically in inhibiting human breast cancer cell proliferation may involve the expression of genes that induce differentiation and block proliferation, and the up-regulation of antioxidant enzymes and proteins involved in apoptosis, cell cycle regulation and DNA repair. CONCLUSION: Combined treatment with RA and AA inhibits the proliferation of human breast cancer cells by altering their gene expression related to antioxidation processes as well as the proliferation inhibitory pathway.     

Ubiquitin-Conjugating Enzyme UBE2C Is Highly Expressed in Breast Microcalcification Lesions

Ubiquitin-conjugating enzyme 2C (UBE2C) contributes to ubiquitin-mediated proteasome degradation of cell cycle progression in breast cancer. Microcalcification (MC) is the most common mammographic feature of early breast cancer. In this study, we evaluated whether UBE2C could be a tumor marker of early breast cancer with MC found on screening mammography. UBE2C protein and mRNA expression were measured in breast core biopsy pairs of MC and adjacent non-MC breast tissue from each subject. Immunohistochemistry revealed UBE2C positivity in 69.4% of MC samples and 77.6% negativity in non-MC samples (p<0.0001). On RT-qPCR, 56.1% of malignant MC lesion samples showed high mRNA level of UBE2C and 80% of benign MC lesion samples showed a low level of UBE2C (p = 0.1766). We investigated the carcinogenic role of UBE2C in MCF-7 breast cancer cells with UBE2C knockdown; UBE2C knockdown downregulated cell proliferation and activated the cellular apoptosis pathway to inhibit cell colony formation. Furthermore, UBE2C expression was associated with that of carcinogenic genes human epidermal growth factor receptor type 2 (HER2), cellular c-Ki-ras2 proto-oncogene (KRAS), vascular endothelial growth factor (VEGF), CXC chemokine receptor 4 (CXCR4), C-C motif chemokine 5 (CCL5), neural precursor cell expressed, developmentally downregulated 9 (NEDD9) and Ras homolog family member C (RhoC). UBE2C may be a marker for diagnosis of nonpalpable breast lesions but not benign or malignant tumors in mammography core biopsies. Suppression of UBE2C may be a potential therapy target in breast cancer.     

Analysis of gene expression profiles associated with cisplatin resistance in human ovarian cancer cell lines and tissues using cDNA microarray

Gene expression profiles were analyzed by using cDNA microarray for a cisplatin-sensitive cell line (KF), and three- and thirty-fold cisplatin-resistant ovarian cancer cell lines (KFr and KFrP200) both showing no p53 mutation within exon 5, 6, 7, 8 and no pglycoprotein overexpression. Expression of GST-pi mRNA increased as the level of resistance to cisplatin became high. Microarray analysis revealed that DNA repair associated genes, i.e., XRCC5, XRCC6, ERCC5, hMLH1 were over-expressed in three-fold cisplatin-resistant cell line, KFr as compared to cisplatin-sensitive parental cell line, KF. Apoptosis inhibitors, i.e., IGFR type I and II were over-expressed, and apoptosis inducer, i.e., caspase 3 and BAK were underexpressed in highly cisplatin-resistant cell line, KFrP200 as compared to KFr. As for clinical cases, cDNA microarray was used to compare gene expression profiles directly between two groups, i.e., the chemotherapy (CAP) sensitive group (n = 2) and the resistant group (n = 2). Six genes such as beta tubulin, high-mobility group (nonhistone chromosomal) protein 1, connective tissue growth factor, insulin-like growth factor binding protein 2, alpha tubulin, and RAS-related gene were overexpressed in CAP therapy resistance group, whereas seven genes such as CD9 antigen, alpha-2-macroglobulin, caveolin 2, interleukin 1 receptor antagonist, Rho GTPase activating protein 1, reticulon 3, cyclin-dependent kinase 10, keratin 7 were underexpressed in CAP therapy resistance group. By increasing clinical case number and gene number of microarray to be used in the analysis of expression profile of gene cluster affecting anticancer drug resistance and sensitivity of the ovarian cancer, it would be possible to apply microarray analysis to personalization of chemotherapy such as selection of effective chemotherapy protocol and prediction of therapeutic effect in the near future.