Tamara Jones

Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF

The vertebrate transcription factors TCF (T cell factor) and LEF (lymphocyte enhancer binding factor) interact with beta-catenin and are hypothesized to mediate Wingless/Wnt signaling. We have cloned a maternally expressed Drosophila TCF family member, dTCF. dTCF binds a canonical TCF DNA motif and interacts with the beta-catenin homolog Armadillo. Previous studies have identified two regions in Armadillo required for Wingless signaling. One of these interacts with dTCF, while the other constitutes a transactivation domain. Mutations in dTCF and expression of a dominant-negative dTCF transgene cause a segment polarity phenotype and affect expression of the Wingless target genes engrailed and Ultrabithorax. Epistasis analysis positions dTCF downstream of armadillo. The Armadillo-dTCF complex mediates Wingless signaling as a bipartite transcription factor.

The identification of microRNAs in a genomically unstable region of human chromosome 8q24.

The PVT1 locus is identified as a cluster of T(2;8) and T(8;22) “variant” MYC-activating chromosomal translocation breakpoints extending 400 kb downstream of MYC in a subset (≈20%) of Burkitts lymphoma (vBL). Recent reports that microRNAs (miRNA) may be associated with fragile sites and cancer-associated genomic regions prompted us to investigate whether the PVT1 region on chromosome 8q24 may contain miRNAs. Computational analysis of the genomic sequence covering the PVT1 locus and experimental verification identified seven miRNAs. One miRNA, hsa-miR-1204, resides within a previously described PVT1 exon (1b) that is often fused to the immunoglobulin light chain constant region in vBLs and is present in high copy number in MYC/PVT1–amplified tumors. Like its human counterpart, mouse mmu-miR-1204 represents the closest miRNA to Myc (∼50 kb) and is found only 1 to 2 kb downstream of a cluster of retroviral integration sites. Another miRNA, mmu-miR-1206, is close to a cluster of variant translocation breakpoints associated with mouse plasmacytoma and exon 1 of mouse Pvt1. Virtually all the miRNA precursor transcripts are expressed at higher levels in late-stage B cells (including plasmacytoma and vBL cell lines) compared with immature B cells, suggesting possible roles in lymphoid development and/or lymphoma. In addition, lentiviral vector-mediated overexpression of the miR-1204 precursor (human and mouse) in a mouse pre–B-cell line increased expression of Myc. High levels of expression of the hsa-miR-1204 precursor is also seen in several epithelial cancer cell lines with MYC/PVT1 coamplification, suggesting a potentially broad role for these miRNAs in tumorigenesis. (Mol Cancer Res 2008;6(2):212–21)

Implication of Checkpoint Kinase-dependent Up-regulation of Ribonucleotide Reductase R2 in DNA Damage Response

To investigate drug mechanisms of action and identify molecular targets for the development of rational drug combinations, we conducted synthetic small interfering RNA (siRNA)-based RNAi screens to identify genes whose silencing affects anti-cancer drug responses. Silencing of RRM1 and RRM2, which encode the large and small subunits of the human ribonucleotide reductase complex, respectively, markedly enhanced the cytotoxicity of the topoisomerase I inhibitor camptothecin (CPT). Silencing of RRM2 was also found to enhance DNA damage as measured by histone γ-H2AX. Further studies showed that CPT up-regulates both RRM1 and RRM2 mRNA and protein levels and induces the nuclear translocation of RRM2. The checkpoint kinase 1 (Chk1) was up-regulated and activated in response to CPT, and CHEK1 down-regulation by siRNA and small molecule inhibitors of Chk1 blocked RRM2 induction by CPT. CHEK1 siRNA also suppressed E2F1 up-regulation by CPT, and silencing of E2F1 suppressed the up-regulation of RRM2. Silencing of ATR or ATM and inhibition of ATM activity by KU-55933 blocked Chk1 activation and RRM2 up-regulation. This study links the known components of CPT-induced DNA damage response with proteins required for the synthesis of dNTPs and DNA repair. Specifically, we propose that upon DNA damage, Chk1 activation, mediated by ATM and ATR, up-regulates RRM2 expression through the E2F1 transcription factor. Up-regulation in RRM2 expression levels coupled with its nuclear recruitment suggests an active role for ribonucleotide reductase in the cellular response to CPT-mediated DNA damage that could potentially be exploited as a strategy for enhancing the efficacy of topoisomerase I inhibitors.

Identification of WEE1 as a potential molecular target in cancer cells by RNAi screening of the human tyrosine kinome

Breast cancers can be classified into those that express the estrogen (ER) and progesterone (PR) receptors, those with ERBB2 (HER-2/Neu) amplification, and those without expression of ER, PR, or amplification of ERBB2 (referred to as triple-negative or basal-like breast cancer). In order to identify potential molecular targets in breast cancer, we performed a synthetic siRNA-mediated RNAi screen of the human tyrosine kinome. A primary RNAi screen conducted in the triple-negative/basal-like breast cancer cell line MDA-MB231 followed by secondary RNAi screens and further studies in this cell line and two additional triple-negative/basal-like breast cancer cell lines, BT20 and HCC1937, identified the G2/M checkpoint protein, WEE1, as a potential therapeutic target. Similar sensitivity to WEE1 inhibition was observed in cell lines from all subtypes of breast cancer. RNAi-mediated silencing or small compound inhibition of WEE1 in breast cancer cell lines resulted in an increase in γH2AX levels, arrest in the S-phase of the cell cycle, and a significant decrease in cell proliferation. WEE1-inhibited cells underwent apoptosis as demonstrated by positive Annexin V staining, increased sub-G1 DNA content, apoptotic morphology, caspase activation, and rescue by the pan-caspase inhibitor, Z-VAD-FMK. In contrast, the non-transformed mammary epithelial cell line, MCF10A, did not exhibit any of these downstream effects following WEE1 silencing or inhibition. These results identify WEE1 as a potential molecular target in breast cancer.

Systems-wide RNAi analysis of CASP8AP2 / FLASH shows transcriptional deregulation of the replication-dependent histone genes and extensive effects on the transcriptome of colorectal cancer cells

BackgroundColorectal carcinomas (CRC) carry massive genetic and transcriptional alterations that influence multiple cellular pathways. The study of proteins whose loss-of-function (LOF) alters the growth of CRC cells can be used to further understand the cellular processes cancer cells depend upon for survival.ResultsA small-scale RNAi screen of ~400 genes conducted in SW480 CRC cells identified several candidate genes as required for the viability of CRC cells, most prominently CASP8AP2/FLASH. To understand the function of this gene in maintaining the viability of CRC cells in an unbiased manner, we generated gene specific expression profiles following RNAi. Silencing of CASP8AP2/FLASH resulted in altered expression of over 2500 genes enriched for genes associated with cellular growth and proliferation. Loss of CASP8AP2/FLASH function was significantly associated with altered transcription of the genes encoding the replication-dependent histone proteins as a result of the expression of the non-canonical polyA variants of these transcripts. Silencing of CASP8AP2/FLASH also mediated enrichment of changes in the expression of targets of the NFκB and MYC transcription factors. These findings were confirmed by whole transcriptome analysis of CASP8AP2/FLASH silenced cells at multiple time points. Finally, we identified and validated that CASP8AP2/FLASH LOF increases the expression of neurofilament heavy polypeptide (NEFH), a protein recently linked to regulation of the AKT1/ß-catenin pathway.ConclusionsWe have used unbiased RNAi based approaches to identify and characterize the function of CASP8AP2/FLASH, a protein not previously reported as required for cell survival. This study further defines the role CASP8AP2/FLASH plays in the regulating expression of the replication-dependent histones and shows that its LOF results in broad and reproducible effects on the transcriptome of colorectal cancer cells including the induction of expression of the recently described tumor suppressor gene NEFH.

Genetic Amplification of the NOTCH Modulator LNX2 Upregulates the WNT/β-Catenin Pathway in Colorectal Cancer

Chromosomal copy number alterations (aneuploidy) define the genomic landscape of most cancer cells, but identification of the oncogenic drivers behind these imbalances remains an unfinished task. In this study, we conducted a systematic analysis of colorectal carcinomas that integrated genomic copy number changes and gene expression profiles. This analysis revealed 44 highly overexpressed genes mapping to localized amplicons on chromosome 13, gains of which occur often in colorectal cancers (CRC). RNA interference (RNAi)-mediated silencing identified eight candidates whose loss-of-function reduced cell viability 20% or more in CRC cell lines. The functional space of the genes NUPL1, LNX2, POLR1D, POMP, SLC7A1, DIS3, KLF5, and GPR180 was established by global expression profiling after RNAi exposure. One candidate, LNX2, not previously known as an oncogene, was involved in regulating NOTCH signaling. Silencing LNX2 reduced NOTCH levels but also downregulated the transcription factor TCF7L2 and markedly reduced WNT signaling. LNX2 overexpression and chromosome 13 amplification therefore constitutively activates the WNT pathway, offering evidence of an aberrant NOTCH-WNT axis in CRC.

RNAi screening identifies TAK1 as a potential target for the enhanced efficacy of topoisomerase inhibitors.

In an effort to develop strategies that improve the efficacy of existing anticancer agents, we have conducted a siRNA-based RNAi screen to identify genes that, when targeted by siRNA, improve the activity of the topoisomerase I (Top1) poison camptothecin (CPT). Screening was conducted using a set of siRNAs corresponding to over 400 apoptosisrelated genes in MDA-MB-231 breast cancer cells. During the course of these studies, we identified the silencing of MAP3K7 as a significant enhancer of CPT activity. Follow-up analysis of caspase activity and caspase-dependent phosphorylation of histone H2AX demonstrated that the silencing of MAP3K7 enhanced CPT-associated apoptosis. Silencing MAP3K7 also sensitized cells to additional compounds, including CPT clinical analogs. This activity was not restricted to MDA-MB-231 cells, as the silencing of MAP3K7 also sensitized the breast cancer cell line MDA-MB-468 and HCT-116 colon cancer cells. However, MAP3K7 silencing did not affect compound activity in the comparatively normal mammary epithelial cell line MCF10A, as well as some additional tumorigenic lines. MAP3K7 encodes the TAK1 kinase, an enzyme that is central to the regulation of many processes associated with the growth of cancer cells (e.g. NF- κB, JNK, and p38 signaling). An analysis of TAK1 signaling pathway members revealed that the silencing of TAB2 also sensitizes MDA-MB-231 and HCT-116 cells towards CPT. These findings may offer avenues towards lowering the effective doses of Top1 inhibitors in cancer cells and, in doing so, broaden their application.

Multiplexing siRNAs to compress RNAi-based screen size in human cells

Here we describe a novel strategy using multiplexes of synthetic small interfering RNAs (siRNAs) corresponding to multiple gene targets in order to compress RNA interference (RNAi) screen size. Before investigating the practical use of this strategy, we first characterized the gene-specific RNAi induced by a large subset (258 siRNAs, 129 genes) of the entire siRNA library used in this study (∼800 siRNAs, ∼400 genes). We next demonstrated that multiplexed siRNAs could silence at least six genes to the same degree as when the genes were targeted individually. The entire library was then used in a screen in which randomly multiplexed siRNAs were assayed for their affect on cell viability. Using this strategy, several gene targets that influenced the viability of a breast cancer cell line were identified. This study suggests that the screening of randomly multiplexed siRNAs may provide an important avenue towards the identification of candidate gene targets for downstream functional analyses and may also be useful for the rapid identification of positive controls for use in novel assay systems. This approach is likely to be especially applicable where assay costs or platform limitations are prohibitive.

Identification of the Drosophila melanogaster homologue of the mammalian signal transducer protein, Vav.

Mammalian Vav signal transducer protein couples tyrosine kinase signals with the activation of the Rho/Rac GTPases, thus leading to cell differentiation and/or proliferation. We have isolated and characterized the DroVav gene, the homologue of hVav in Drosophila melanogaster. DroVav encodes a protein (793 residues) whose similarity with hVav is 47% and with hVav2 and hVav3 is 45%. DroVav preserves the unique, complex structure of hVav proteins, including the ‘calponin homology’, dbl homology, pleckstrin homology; SH2 and SH3 domains in addition to regions that are acidic rich, proline rich and cysteine rich. DroVav is located on the X chromosome in polytene interval 18A5;18B and is expressed in all stages of development and in all tissues. In mammalian cells, DroVav is tyrosine‐phosphorylated in response to epidermal growth factor receptor (EGFR) induction; in vitro, the DroVav SH2 region is associated with tyrosine‐phosphorylated EGFR. Thus, DroVav probably plays a pivotal role as a signal transducer protein during fruit fly development.

Drosophila caliban, a nuclear export mediator, can function as a tumor suppressor in human lung cancer cells

We previously showed that the Drosophila DNA binding homeodomain of Prospero included a 28 amino-acid sequence (HDA) that functions as a nuclear export signal. We describe here the identification of a protein we named Caliban, which can directly interact with the HDA. Caliban is homologous to human Sdccag1, which has been implicated in colon and lung cancer. Here we show that Caliban and Sdccag1 are mediators of nuclear export in fly and human cells, as interference RNA abrogates export of EYFP-HDA in normal fly and human lung cells. Caliban functions as a bipartite mediator nuclear export as the carboxy terminus binds HDA and the amino terminus itself functions as an NES, which directly binds the NES receptor Exportin. Finally, while non-cancerous lung cells have functional Sdccag1, five human lung carcinoma cell lines do not, even though Exportin still functions in these cells. Expression of fly Caliban in these human lung cancer cells restores EYFP-HDA nuclear export, reduces a cells ability to form colonies on soft agar and reduces cell invasiveness. We suggest that Sdccag1 inactivation contributes to the transformed state of human lung cancer cells and that Caliban should be considered a candidate for use in lung cancer gene therapy.