Mark D'Amico

Cyclin D1 is required for transformation by activated Neu and is induced through an E2F-dependent signaling pathway

ABSTRACT The neu (c-erbB-2) proto-oncogene encodes a tyrosine kinase receptor that is overexpressed in 20 to 30% of human breast tumors. Herein, cyclin D1 protein levels were increased in mammary tumors induced by overexpression of wild-type Neu or activating mutants of Neu in transgenic mice and in MCF7 cells overexpressing transforming Neu. Analyses of 12 Neu mutants in MCF7 cells indicated important roles for specific C-terminal autophosphorylation sites and the extracellular domain in cyclin D1 promoter activation. Induction of cyclin D1 by NeuT involved Ras, Rac, Rho, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38, but not phosphatidylinositol 3-kinase. NeuT induction of the cyclin D1 promoter required the E2F and Sp1 DNA binding sites and was inhibited by dominant negative E2F-1 or DP-1. Neu-induced transformation was inhibited by a cyclin D1 antisense or dominant negative E2F-1 construct in Rat-1 cells. Growth of NeuT-transformed mammary adenocarcinoma cells in nude mice was blocked by the cyclin D1 antisense construct. These results demonstrate that E2F-1 mediates a Neu-signaling cascade tocyclin D1 and identify cyclin D1 as a critical downstream target of neu-induced transformation.

The integrin-linked kinase regulates the cyclin D1 gene through glycogen synthase kinase 3beta and cAMP-responsive element-binding protein-dependent pathways.

The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the pRB tumor suppressor protein. Cyclin D1 is overexpressed in 20–30% of human breast tumors and is induced both by oncogenes including those for Ras, Neu, and Src, and by the β-catenin/lymphoid enhancer factor (LEF)/T cell factor (TCF) pathway. The ankyrin repeat containing serine-threonine protein kinase, integrin-linked kinase (ILK), binds to the cytoplasmic domain of β1 and β3integrin subunits and promotes anchorage-independent growth. We show here that ILK overexpression elevates cyclin D1 protein levels and directly induces the cyclin D1 gene in mammary epithelial cells. ILK activation of the cyclin D1 promoter was abolished by point mutation of a cAMP-responsive element-binding protein (CREB)/ATF-2 binding site at nucleotide −54 in the cyclin D1 promoter, and by overexpression of either glycogen synthase kinase-3β (GSK-3β) or dominant negative mutants of CREB or ATF-2. Inhibition of the PI 3-kinase and AKT/protein kinase B, but not of the p38, ERK, or JNK signaling pathways, reduced ILK induction of cyclin D1 expression. ILK induced CREB transactivation and CREB binding to the cyclin D1 promoter CRE. Wnt-1 overexpression in mammary epithelial cells induced cyclin D1 mRNA and targeted overexpression of Wnt-1 in the mammary gland of transgenic mice increased both ILK activity and cyclin D1 levels. We conclude that the cyclin D1 gene is regulated by the Wnt-1 and ILK signaling pathways and that ILK induction of cyclin D1 involves the CREB signaling pathway in mammary epithelial cells.

Activation of the cyclin D1 gene by the E1A-associated protein p300 through AP-1 inhibits cellular apoptosis

The adenovirus E1A protein interferes with regulators of apoptosis and growth by physically interacting with cell cycle regulatory proteins including the retinoblastoma tumor suppressor protein and the coactivator proteins p300/CBP (where CBP is the CREB-binding protein). The p300/CBP proteins occupy a pivotal role in regulating mitogenic signaling and apoptosis. The mechanisms by which cell cycle control genes are directly regulated by p300 remain to be determined. The cyclin D1 gene, which is overexpressed in many different tumor types, encodes a regulatory subunit of a holoenzyme that phosphorylates and inactivates PRB. In the present study E1A12S inhibited the cyclin D1 promoter via the amino-terminal p300/CBP binding domain in human choriocarcinoma JEG-3 cells. p300 induced cyclin D1 protein abundance, and p300, but not CBP, induced the cyclin D1 promoter. cyclin D1 or p300 overexpression inhibited apoptosis in JEG-3 cells. The CH3 region of p300, which was required for induction of cyclin D1, was also required for the inhibition of apoptosis. p300 activated the cyclin D1 promoter through an activator protein-1 (AP-1) site at −954 and was identified within a DNA-bound complex with c-Jun at the AP-1 site. Apoptosis rates of embryonic fibroblasts derived from mice homozygously deleted of the cyclin D1 gene (cyclin D1 −/− ) were increased compared with wild type control on several distinct matrices. p300 inhibited apoptosis in cyclin D1+/+ fibroblasts but increased apoptosis in cyclin D1 −/− cells. The anti-apoptotic function of cyclin D1, demonstrated by sub-G1 analysis and annexin V staining, may contribute to its cellular transforming and cooperative oncogenic properties.

Inhibition of Cellular Proliferation through IκB Kinase-Independent and Peroxisome Proliferator-Activated Receptor γ-Dependent Repression of Cyclin D1

ABSTRACT The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-regulated nuclear receptor superfamily member. Liganded PPARγ exerts diverse biological effects, promoting adipocyte differentiation, inhibiting tumor cellular proliferation, and regulating monocyte/macrophage and anti-inflammatory activities in vitro. In vivo studies with PPARγ ligands showed enhancement of tumor growth, raising the possibility that reduced immune function and tumor surveillance may outweigh the direct inhibitory effects of PPARγ ligands on cellular proliferation. Recent findings that PPARγ ligands convey PPARγ-independent activities through IκB kinase (IKK) raises important questions about the specific mechanisms through which PPARγ ligands inhibit cellular proliferation. We investigated the mechanisms regulating the antiproliferative effect of PPARγ. Herein PPARγ, liganded by either natural (15d-PGJ2 and PGD2) or synthetic ligands (BRL49653 and troglitazone), selectively inhibited expression of the cyclin D1 gene. The inhibition of S-phase entry and activity of the cyclin D1-dependent serine-threonine kinase (Cdk) by 15d-PGJ2 was not observed in PPARγ-deficient cells. Cyclin D1 overexpression reversed the S-phase inhibition by 15d-PGJ2. Cyclin D1 repression was independent of IKK, as prostaglandins (PGs) which bound PPARγ but lacked the IKK interactive cyclopentone ring carbonyl group repressed cyclin D1. Cyclin D1 repression by PPARγ involved competition for limiting abundance of p300, directed through a c-Fos binding site of the cyclin D1 promoter. 15d-PGJ2 enhanced recruitment of p300 to PPARγ but reduced binding to c-Fos. The identification of distinct pathways through which eicosanoids regulate anti-inflammatory and antiproliferative effects may improve the utility of COX2 inhibitors.

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, p16INK4a and p14ARF (murine p19ARF). Invariant inactivation of either the p16INK4a-cyclin D/CDK-pRb pathway and/or p53-p14ARF pathway occurs in most human tumors. Cyclin D1 is frequently overexpressed in breast cancer cells contributing an alternate mechanism inactivating the p16INK4a/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 p16INK4a expression are reciprocal in human breast cancers. Herein, reciprocal regulation of cyclin D1 and p16INK4a was observed in tissues of mice mutant for the Ink4a/Arf locus. p16INK4a and p19ARF inhibited DNA synthesis in MCF7 cells. p16INK4a repressed cyclin D1 expression and transcription. Repression of cyclin D1 by p16INK4a occurred independently of the p16INK4a-cdk4-binding function and required a cAMP-response element/activating transcription factor-2-binding site. p19ARF 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.

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 MCF7 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 HP1α/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.

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 ∼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.

Erratum: Gene expression phenotypic models that predict the activity of oncogenic pathways (Nature Genetics (2003) 34 (226-230))

Nat. Genet. 34, 226–230 (2003). Figure 3 was reproduced poorly in print. A corrected version appears below.