Gene expression phenotypic models that predict the activity of oncogenic pathways.

High-density DNA microarrays measure expression of large numbers of genes in one assay. The ability to find underlying structure in complex gene expression data sets and rigorously test association of that structure with biological conditions is essential to developing multi-faceted views of the gene activity that defines cellular phenotype. We sought to connect features of gene expression data with biological hypotheses by integrating 'metagene' patterns from DNA microarray experiments in the characterization and prediction of oncogenic phenotypes. We applied these techniques to the analysis of regulatory pathways controlled by the genes HRAS (Harvey rat sarcoma viral oncogene homolog), MYC (myelocytomatosis viral oncogene homolog) and E2F1, E2F2 and E2F3 (encoding E2F transcription factors 1, 2 and 3, respectively). The phenotypic models accurately predict the activity of these pathways in the context of normal cell proliferation. Moreover, the metagene models trained with gene expression patterns evoked by ectopic production of Myc or Ras proteins in primary tissue culture cells properly predict the activity of in vivo tumor models that result from deregulation of the MYC or HRAS pathways. We conclude that these gene expression phenotypes have the potential to characterize the complex genetic alterations that typify the neoplastic state, whether in vitro or in vivo, in a way that truly reflects the complexity of the regulatory pathways that are affected.

IKKalpha regulates mitogenic signaling through transcriptional induction of cyclin D1 via Tcf.

The Wnt/beta-catenin/Tcf and IkappaB/NF-kappaB cascades are independent pathways involved in cell cycle control, cellular differentiation, and inflammation. Constitutive Wnt/beta-catenin signaling occurs in certain cancers from mutation of components of the pathway and from activating growth factor receptors, including RON and MET. The resulting accumulation of cytoplasmic and nuclear beta-catenin interacts with the Tcf/LEF transcription factors to induce target genes. The IkappaB kinase complex (IKK) that phosphorylates IkappaB contains IKKalpha, IKKbeta, and IKKgamma. Here we show that the cyclin D1 gene functions as a point of convergence between the Wnt/beta-catenin and IkappaB pathways in mitogenic signaling. Mitogenic induction of G(1)-S phase progression and cyclin D1 expression was PI3K dependent, and cyclin D1(-/-) cells showed reduced PI3K-dependent S-phase entry. PI3K-dependent induction of cyclin D1 was blocked by inhibitors of PI3K/Akt/IkappaB/IKKalpha or beta-catenin signaling. A single Tcf site in the cyclin D1 promoter was required for induction by PI3K or IKKalpha. In IKKalpha(-/-) cells, mitogen-induced DNA synthesis, and expression of Tcf-responsive genes was reduced. Reintroduction of IKKalpha restored normal mitogen induction of cyclin D1 through a Tcf site. In IKKalpha(-/-) cells, beta-catenin phosphorylation was decreased and purified IKKalpha was sufficient for phosphorylation of beta-catenin through its N-terminus in vitro. Because IKKalpha but not IKKbeta induced cyclin D1 expression through Tcf activity, these studies indicate that the relative levels of IKKalpha and IKKbeta may alter their substrate and signaling specificities to regulate mitogen-induced DNA synthesis through distinct mechanisms.

The inhibitor of cyclin-dependent kinase 4a/alternative reading frame (INK4a/ARF) locus encoded proteins p16INK4a and p19ARF repress cyclin D1 transcription through distinct cis elements.

The Ink4a/Arf locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF) (murine p19(ARF)). Invariant inactivation of either the p16(INK4a)-cyclin D/CDK-pRb pathway and/or p53-p14(ARF) pathway occurs in most human tumors. Cyclin D1 is frequently overexpressed in breast cancer cells contributing an alternate mechanism inactivating the p16(INK4a)/pRb pathway. Targeted overexpression of cyclin D1 to the mammary gland is sufficient for tumorigenesis, and cyclin D1-/- mice are resistant to Ras-induced mammary tumors. Recent studies suggest cyclin D1 and p16(INK4a) expression are reciprocal in human breast cancers. Herein, reciprocal regulation of cyclin D1 and p16(INK4a) was observed in tissues of mice mutant for the Ink4a/Arf locus. p16(INK4a) and p19(ARF) inhibited DNA synthesis in MCF7 cells. p16(INK4a) repressed cyclin D1 expression and transcription. Repression of cyclin D1 by p16(INK4a) occurred independently of the p16(INK4a)-cdk4-binding function and required a cAMP-response element/activating transcription factor-2-binding site. p19(ARF) repressed cyclin D1 through a novel distal cis-element at -1137, which bound p53 in chromatin-immunoprecipitation assays. Transcriptional repression of the cyclin D1 gene through distinct DNA sequences may contribute to the tumor suppressor function of the Ink4a/Arf locus.

The role of Ink4a/Arf in ErbB2 mammary gland tumorigenesis.

Most human tumors display inactivation of the p53 and the p16(INK4)/pRb pathway. The Ink4a/alternative reading frame (ARF) locus encodes the p16(INK4a) and p14(ARF) (murine p19(ARF)) proteins. p16(INK4a) is deleted in 40-60% of breast cancer cell lines, and p16(INK4a) inactivation by DNA methylation occurs in < or =30% of human breast cancers. In mice genetically heterozygous for p16(INK4a) or Ink4a/Arf, predisposition to specific tumor types is enhanced. Ink4a/Arf(+/-) mice have increased E micro -Myc-induced lymphomagenesis and epidermal growth factor receptor-induced gliomagenesis. ErbB2 (epidermal growth factor receptor-related protein B2) is frequently overexpressed in human breast cancer and is sufficient for mammary tumorigenesis in vivo. We determined the role of heterozygosity at the Ink4a/Arf locus in ErbB2-induced mammary tumorigenesis. Compared with mouse mammary tumor virus-ErbB2 Ink4a/Arf(+/-) mice, mouse mammary tumor virus-ErbB2 Ink4a/Arf(wt) mammary tumors showed increased p16(INK4a), reduced Ki-67 expression, and reduced cyclin D1 protein but increased mammary tumor apoptosis with no significant change in the risk of developing mammary tumors. These studies demonstrate the contribution of Ink4a/Arf heterozygosity to tumor progression is tissue specific in vivo. In view of the important role of Ink4a/Arf in response to chemotherapy, these transgenic mice may provide a useful model for testing breast tumor therapies.

Flavopiridol and trastuzumab synergistically inhibit proliferation of breast cancer cells: association with selective cooperative inhibition of cyclin D1-dependent kinase and Akt signaling pathways.

Cyclin D1 is essential for Neu-induced cell growth and is induced by growth factors through Ras-dependent and Ras-independent signaling pathways (1). Because flavopiridol, a novel flavanoid cyclin-cyclin-dependent kinase inhibitor, may function through Ras-dependent and/or -independent pathways, we hypothesized that treatment of breast cancer cells with inhibitors of Neu signaling and flavopiridol might synergize to inhibit proliferation. Human breast cancer cell lines, which express high levels of endogenous Neu receptor, were treated with the anti-Neu antibody, trastuzumab, together with flavopiridol and subject to MTT assay. Cell lines were assayed for alterations in cell cycle by fluorescence-activated cell sorter and signaling proteins by Western blot. Flavopiridol and trastuzumab synergistically inhibited DNA synthesis, cellular proliferation, and contact-dependent growth. Cytotoxic synergy was observed independent of the sequence of addition of the two drugs to cultured cells. In SKBR3 cells, a combination of trastuzumab and flavopiridol inhibited the Ras-MAPK-Akt pathway, decreased cyclin D1 abundance, and kinase activity to a greater extent than either drug alone. Compared with single-agent treatment, combination treatment selectively inhibited Akt and pRB phosphorylation. Cytotoxic synergy was observed with flavopiridol plus LY294002 (selective phosphatidylinositol 3-kinase inhibitor) but not with PD98059 (selective mitogen-activated protein kinase kinase 1 inhibitor) suggesting that Akt inhibition may be important in synergy. Zinc-induced overexpression of cyclin D1 in T-47D deltaMTcycD1 cells were more resistant to drug-induced cell death when compared with vector-transfected T-47D deltaMT cells. Cyclin D1 overexpression reverses drug treatment induced cell cycle arrest in SKBR3 cells. Flavopiridol and trastuzumab yield cytotoxic synergy in human breast cancer cells overexpressing Neu. Cyclin D1 overexpression results in combination drug resistance. In addition, inhibition of Akt may prove to be a useful therapeutic strategy in combination with flavopiridol.

Inhibition of cyclin D1 gene transcription by Brg-1.

The evolutionarily conserved SWI-SNF chromatin remodeling complex regulates cellular proliferation. A catalytic subunit, BRG-1, is frequently down regulated, silenced or mutated in malignant cells, however, the mechanism by which BRG-1 may function as a tumor suppressor or block breast cancer cellular proliferation is not understood. The cyclin D1 gene is a collaborative oncogene overexpressed in greater than 50% of human breast cancers. Herein, BRG-1 inhibited DNA synthesis and cyclin D1 expression in human MCF-7 breast cancer epithelial cells. The cyclin D1 promoter AP-1 and CRE sites were required for repression by BRG-1 in promoter assays. BRG-1 deficient cells abolished and siRNA to BRG-1 reduced, formation of the BRG-1 chromatin complex. The endogenous cyclin D1 promoter AP-1 site bound BRG-1. Estradiol treatment of MCF-7 cells induced recruitment of BRG-1 to the endogenous hpS2 gene promoter. Estradiol, which induced cyclin D1 abundance, was associated with a reduction in recruitment of the co-repressors HP1alpha/HDAC1 to the endogenous cyclin D1 promoter AP-1/BRG-1 binding sites. These studies suggest the endogenous cyclin D1 promoter BRG-1 binding site functions as a molecular scaffold in the context of local chromatin upon which coactivators and corepressors are recruited to regulate cyclin D1.

Expression of EIF3-p48/INT6, TID1 and Patched in cancer, a profiling of multiple tumor types and correlation of expression.

Alterations in eIF3-p48/INT6 gene expression have been implicated in murine and human mammary carcinogenesis. We examined levels of INT6 protein in human tumors and determined that breast and colon tumors clustered into distinct groups based on levels of INT6 expression and clinicopathological variables. We performed multiplex tissue immunoblotting of breast, colon, lung, and ovarian tumor tissues and found that INT6 protein levels positively correlated with those of TID1, Patched, p53, c-Jun, and phosphorylated-c-Jun proteins in a tissue-specific manner. INT6 and TID1 showed significant positive correlation in all tissue types tested. These findings were confirmed by immunohistochemical staining of INT6 and TID1. Further evidence supporting a cooperative role for INT6 and TID1 is the presence of endogenous INT6 and TID1 proteins as complexes. We detected co-immunoprecipitation between INT6 and TID1, as well as between INT6 and Patched. These findings suggest potential integrated roles for INT6, TID1, and Patched proteins in cell growth, development, and tumorigenesis. Additionally, these data suggest that the combination of INT6, TID1, and Patched protein levels may be useful biomarkers for the development of diagnostic assays.

A study of cytotoxic synergy of UCN-01 and flavopiridol in syngeneic pair of cell lines.

Flavopiridol and UCN-01 are two novel protein kinase inhibitors with diverse cellular effects that may complement each other with regards to induction of apoptosis. HeLa cells engineered to overexpress human survivin (HeLa-S) were at least approximately 4.8-fold resistant to UCN-01 relative to proliferation observed in control HeLa cells (HeLa-V). Flavopiridol cytotoxicity as measured using the MTT assay was unaffected in HeLa-S cells when compared with HeLa-V cells. Similarly, simultaneous treatment of HeLa-V cells with flavopiridol and UCN-01 for 72 hours did not result in synergistic inhibition of proliferation; however, in HeLa-S cells, this combination resulted in synergistic inhibition of cell proliferation. Flavopiridol and UCN-01 augmented apoptosis in HeLa-S cells (as compared with HeLa-V cells) as measured by caspase-3 cellular activity assay, DNA fragmentation and PARP cleavage by western blot. In HeLa-V and -S cells, combination treatment resulted in caspase-8 cleavage. Caspase-9 was expressed in HeLa-V cells; however, there was a marked reduction of caspase-9 content in HeLa-S cells only. Combination treatment resulted in a significant reduction in survivin abundance in HeLa-S and SKBR3-UR cells, but not in their respective parental lines. The synergy of Flavopiridol and UCN-01 are selectively toxic to survivin-overexpressing cell lines and the mechanism of toxicity involves caspase-dependent cell death.

Indomethacin induces differential expression of beta-catenin, gamma-catenin and T-cell factor target genes in human colorectal cancer cells.

Indomethacin-induced G(1) arrest and apoptosis of human colorectal cancer (CRC) cells is associated with a dose-dependent decrease in beta-catenin protein levels. Beta-catenin plays a pivotal role in the WNT signalling pathway and its expression is frequently dysregulated at early stages of colorectal carcinogenesis. The objective of this study was to investigate the effect of indomethacin on catenin expression and downstream WNT signalling events in human CRC cells. Beta-catenin, gamma-catenin and T-cell factor (TCF) target gene (cyclin D1, c-MYC and PPARdelta) expression was studied following indomethacin treatment of SW480 and HCT116 cells. Cyclin D1 was used as a model TCF target gene for analysis of beta-catenin-TCF-4 DNA binding and trans-activation. Indomethacin treatment was associated with a specific decrease in beta-catenin (but not gamma-catenin) expression. Resulting TCF target gene expression was gene specific (cyclin D1, decreased; c-MYC, increased; PPARdelta, no significant change). Cyclin D1 promoter analysis revealed that indomethacin disrupted formation of a beta-catenin-TCF-4-DNA complex. Indomethacin-induced G(1) arrest and apoptosis is associated with specific beta-catenin down-regulation in human CRC cells in vitro. Differential expression of TCF target genes following indomethacin treatment implies complex effects on multiple genes which play an important role in colorectal carcinogenesis.

DACH1 inhibits transforming growth factor-beta signaling through binding Smad4.

The vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, we hypothesized that DACH1 might play a similar cellular function. Herein, DACH1 was found to be expressed in breast cancer cell lines and to inhibit transforming growth factor-beta (TGF-beta)-induced apoptosis. DACH1 repressed TGF-beta induction of AP-1 and Smad signaling in gene reporter assays and repressed endogenous TGF-beta-responsive genes by microarray analyses. DACH1 bound to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localized with NCoR in nuclear dotlike structures. NCoR enhanced DACH1 repression, and the repression of TGF-beta-induced AP-1 or Smad signaling by DACH1 required the DACH1 DS domain. The DS domain of DACH was sufficient for NCoR binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibited the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-beta signaling by interacting with NCoR and Smad4.