Minimally invasive longitudinal intravital imaging of cellular dynamics in intact long bone.

Intravital two-photon microscopy enables deep-tissue imaging at high temporospatial resolution in live animals. However, the endosteal bone compartment and underlying bone marrow pose unique challenges to optical imaging as light is absorbed, scattered and dispersed by thick mineralized bone matrix and the adipose-rich bone marrow. Early bone intravital imaging methods exploited gaps in the cranial sutures to bypass the need to penetrate through cortical bone. More recently, investigators have developed invasive methods to thin the cortical bone or implant imaging windows to image cellular dynamics in weight-bearing long bones. Here, we provide a step-by-step procedure for the preparation of animals for minimally invasive, nondestructive, longitudinal intravital imaging of the murine tibia. This method involves the use of mixed bone marrow radiation chimeras to unambiguously double-label osteoclasts and osteomorphs. The tibia is exposed by a simple skin incision and an imaging chamber constructed using thermoconductive T-putty. Imaging sessions up to 12 h long can be repeated over multiple timepoints to provide a longitudinal time window into the endosteal and marrow niches. The approach can be used to investigate cellular dynamics in bone remodeling, cancer cell life cycle and hematopoiesis, as well as long-lived humoral and cellular immunity. The procedure requires an hour to complete and is suitable for users with minimal prior expertise in small animal surgery.

Synergistic targeting of BRCA1 mutated breast cancers with PARP and CDK2 inhibition.

Basal-like breast cancers (BLBC) are aggressive breast cancers that respond poorly to targeted therapies and chemotherapies. In order to define therapeutically targetable subsets of BLBC we examined two markers: cyclin E1 and BRCA1 loss. In high grade serous ovarian cancer (HGSOC) these markers are mutually exclusive, and define therapeutic subsets. We tested the same hypothesis for BLBC. Using a BLBC cohort enriched for BRCA1 loss, we identified convergence between BRCA1 loss and high cyclin E1 protein expression, in contrast to HGSOC in which CCNE1 amplification drives increased cyclin E1. In cell lines, BRCA1 loss was associated with stabilized cyclin E1 during the cell cycle, and BRCA1 siRNA led to increased cyclin E1 in association with reduced phospho-cyclin E1 T62. Mutation of cyclin E1 T62 to alanine increased cyclin E1 stability. We showed that tumors with high cyclin E1/BRCA1 mutation in the BLBC cohort also had decreased phospho-T62, supporting this hypothesis. Since cyclin E1/CDK2 protects cells from DNA damage and cyclin E1 is elevated in BRCA1 mutant cancers, we hypothesized that CDK2 inhibition would sensitize these cancers to PARP inhibition. CDK2 inhibition induced DNA damage and synergized with PARP inhibitors to reduce cell viability in cell lines with homologous recombination deficiency, including BRCA1 mutated cell lines. Treatment of BRCA1 mutant BLBC patient-derived xenograft models with combination PARP and CDK2 inhibition led to tumor regression and increased survival. We conclude that BRCA1 status and high cyclin E1 have potential as predictive biomarkers to dictate the therapeutic use of combination CDK inhibitors/PARP inhibitors in BLBC.

Cyclin E2 Promotes Whole Genome Doubling in Breast Cancer.

Genome doubling is an underlying cause of cancer cell aneuploidy and genomic instability, but few drivers have been identified for this process. Due to their physiological roles in the genome reduplication of normal cells, we hypothesised that the oncogenes cyclins E1 and E2 may be drivers of genome doubling in cancer. We show that both cyclin E1 (CCNE1) and cyclin E2 (CCNE2) mRNA are significantly associated with high genome ploidy in breast cancers. By live cell imaging and flow cytometry, we show that cyclin E2 overexpression promotes aberrant mitosis without causing mitotic slippage, and it increases ploidy with negative feedback on the replication licensing protein, Cdt1. We demonstrate that cyclin E2 localises with core preRC (pre-replication complex) proteins (MCM2, MCM7) on the chromatin of cancer cells. Low CCNE2 is associated with improved overall survival in breast cancers, and we demonstrate that low cyclin E2 protects from excess genome rereplication. This occurs regardless of p53 status, consistent with the association of high cyclin E2 with genome doubling in both p53 null/mutant and p53 wildtype cancers. In contrast, while cyclin E1 can localise to the preRC, its downregulation does not prevent rereplication, and overexpression promotes polyploidy via mitotic slippage. Thus, in breast cancer, cyclin E2 has a strong association with genome doubling, and likely contributes to highly proliferative and genomically unstable breast cancers.

Author Correction: An atlas of genetic influences on osteoporosis in humans and mice.

In the version of this article initially published, in Fig. 5a, the data in the right column of 'DAAM2 gRNA1' were incorrectly plotted as circles indicating 'untreated' rather than as squares indicating 'treated'. The error has been corrected in the HTML and PDF versions of the article.

An atlas of genetic influences on osteoporosis in humans and mice.

Osteoporosis is a common aging-related disease diagnosed primarily using bone mineral density (BMD). We assessed genetic determinants of BMD as estimated by heel quantitative ultrasound in 426,824 individuals, identifying 518 genome-wide significant loci (301 novel), explaining 20% of its variance. We identified 13 bone fracture loci, all associated with estimated BMD (eBMD), in ~1.2 million individuals. We then identified target genes enriched for genes known to influence bone density and strength (maximum odds ratio (OR) = 58, P = 1 × 10-75) from cell-specific features, including chromatin conformation and accessible chromatin sites. We next performed rapid-throughput skeletal phenotyping of 126 knockout mice with disruptions in predicted target genes and found an increased abnormal skeletal phenotype frequency compared to 526 unselected lines (P < 0.0001). In-depth analysis of one gene, DAAM2, showed a disproportionate decrease in bone strength relative to mineralization. This genetic atlas provides evidence linking associated SNPs to causal genes, offers new insight into osteoporosis pathophysiology, and highlights opportunities for drug development.

ELF5 Drives Lung Metastasis in Luminal Breast Cancer through Recruitment of Gr1+ CD11b+ Myeloid-Derived Suppressor Cells.

During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasis-free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer.

Targeting cyclin-dependent kinase 1 (CDK1) but not CDK4/6 or CDK2 is selectively lethal to MYC-dependent human breast cancer cells.

BACKGROUND: Although MYC is an attractive therapeutic target for breast cancer treatment, it has proven challenging to inhibit MYC directly, and clinically effective pharmaceutical agents targeting MYC are not yet available. An alternative approach is to identify genes that are synthetically lethal in MYC-dependent cancer. Recent studies have identified several cell cycle kinases as MYC synthetic-lethal genes. We therefore investigated the therapeutic potential of specific cyclin-dependent kinase (CDK) inhibition in MYC-driven breast cancer. METHODS: Using small interfering RNA (siRNA), MYC expression was depleted in 26 human breast cancer cell lines and cell proliferation evaluated by BrdU incorporation. MYC-dependent and MYC-independent cell lines were classified based on their sensitivity to siRNA-mediated MYC knockdown. We then inhibited CDKs including CDK4/6, CDK2 and CDK1 individually using either RNAi or small molecule inhibitors, and compared sensitivity to CDK inhibition with MYC dependence in breast cancer cells. RESULTS: Breast cancer cells displayed a wide range of sensitivity to siRNA-mediated MYC knockdown. The sensitivity was correlated with MYC protein expression and MYC phosphorylation level. Sensitivity to siRNA-mediated MYC knockdown did not parallel sensitivity to the CDK4/6 inhibitor PD0332991; instead MYC-independent cell lines were generally sensitive to PD0332991. Cell cycle arrest induced by MYC knockdown was accompanied by a decrease in CDK2 activity, but inactivation of CDK2 did not selectively affect the viability of MYC-dependent breast cancer cells. In contrast, CDK1 inactivation significantly induced apoptosis and reduced viability of MYC-dependent cells but not MYC- independent cells. This selective induction of apoptosis by CDK1 inhibitors was associated with up-regulation of the pro-apoptotic molecule BIM and was p53-independent. CONCLUSIONS: Overall, these results suggest that further investigation of CDK1 inhibition as a potential therapy for MYC-dependent breast cancer is warranted.

BCL-2 hypermethylation is a potential biomarker of sensitivity to antimitotic chemotherapy in endocrine-resistant breast cancer.

Overexpression of the antiapoptotic factor BCL-2 is a frequent feature of malignant disease and is commonly associated with poor prognosis and resistance to conventional chemotherapy. In breast cancer, however, high BCL-2 expression is associated with favorable prognosis, estrogen receptor (ER) positivity, and low tumor grade, whereas low expression is included in several molecular signatures associated with resistance to endocrine therapy. In the present study, we correlate BCL-2 expression and DNA methylation profiles in human breast cancer and in multiple cell models of acquired endocrine resistance to determine whether BCL-2 hypermethylation could provide a useful biomarker of response to cytotoxic therapy. In human disease, diminished expression of BCL-2 was associated with hypermethylation of the second exon, in a region that overlapped a CpG island and an ER-binding site. Hypermethylation of this region, which occurred in 10% of primary tumors, provided a stronger predictor of patient survival (P = 0.019) when compared with gene expression (n = 522). In multiple cell models of acquired endocrine resistance, BCL-2 expression was significantly reduced in parallel with increased DNA methylation of the exon 2 region. The reduction of BCL-2 expression in endocrine-resistant cells lowered their apoptotic threshold to antimitotic agents: nocodazole, paclitaxel, and the PLK1 inhibitor BI2536. This phenomenon could be reversed with ectopic expression of BCL-2, and rescued with the BCL-2 inhibitor ABT-737. Collectively, these data imply that BCL-2 hypermethylation provides a robust biomarker of response to current and next-generation cytotoxic agents in endocrine-resistant breast cancer, which may prove beneficial in directing therapeutic strategy for patients with nonresectable, metastatic disease.

Cyclin E2 induces genomic instability by mechanisms distinct from cyclin E1.

Cyclins E1 drives the initiation of DNA replication, and deregulation of its periodic expression leads to mitotic delay associated with genomic instability. Since it is not known whether the closely related protein cyclin E2 shares these properties, we overexpressed cyclin E2 in breast cancer cells. This did not affect the duration of mitosis, nor did it cause an increase in p107 association with CDK2. In contrast, cyclin E1 overexpression led to inhibition of the APC complex, prolonged metaphase and increased p107 association with CDK2. Despite these different effects on the cell cycle, elevated levels of either cyclin E1 or E2 led to hallmarks of genomic instability, i.e., an increased proportion of abnormal mitoses, micronuclei and chromosomal aberrations. Cyclin E2 induction of genomic instability by a mechanism distinct from cyclin E1 indicates that these two proteins have unique functions in a cancer setting.

Differences in degradation lead to asynchronous expression of cyclin E1 and cyclin E2 in cancer cells.

Cyclin E1 is expressed at the G 1/S phase transition of the cell cycle to drive the initiation of DNA replication and is degraded during S/G2M. Deregulation of its periodic degradation is observed in cancer and is associated with increased proliferation and genomic instability. We identify that in cancer cells, unlike normal cells, the closely related protein cyclin E2 is expressed predominantly in S phase, concurrent with DNA replication. This occurs at least in part because the ubiquitin ligase component that is responsible for cyclin E1 downregulation in S phase, Fbw7, fails to effectively target cyclin E2 for proteosomal degradation. The distinct cell cycle expression of the two E-type cyclins in cancer cells has implications for their roles in genomic instability and proliferation and may explain their associations with different signatures of disease.

Cyclin E2 overexpression is associated with endocrine resistance but not insensitivity to CDK2 inhibition in human breast cancer cells.

Cyclin E2, but not cyclin E1, is included in several gene signatures that predict disease progression in either tamoxifen-resistant or metastatic breast cancer. We therefore examined the role of cyclin E2 in antiestrogen resistance in vitro and its potential for therapeutic targeting through cyclin-dependent kinase (CDK) inhibition. High expression of CCNE2, but not CCNE1, was characteristic of the luminal B and HER2 subtypes of breast cancer and was strongly predictive of shorter distant metastasis-free survival following endocrine therapy. After antiestrogen treatment of MCF-7 breast cancer cells, cyclin E2 mRNA and protein were downregulated and cyclin E2-CDK2 activity decreased. However, this regulation was lost in tamoxifen-resistant (MCF-7 TAMR) cells, which overexpressed cyclin E2. Expression of either cyclin E1 or E2 in T-47D breast cancer cells conferred acute antiestrogen resistance, suggesting that cyclin E overexpression contributes to the antiestrogen resistance of tamoxifen-resistant cells. Ectopic expression of cyclin E1 or E2 also reduced sensitivity to CDK4, but not CDK2, inhibition. Proliferation of tamoxifen-resistant cells was inhibited by RNAi-mediated knockdown of cyclin E1, cyclin E2, or CDK2. Furthermore, CDK2 inhibition of E-cyclin overexpressing cells and tamoxifen-resistant cells restored sensitivity to tamoxifen or CDK4 inhibition. Cyclin E2 overexpression is therefore a potential mechanism of resistance to both endocrine therapy and CDK4 inhibition. CDK2 inhibitors hold promise as a component of combination therapies in endocrine-resistant disease as they effectively inhibit cyclin E1 and E2 overexpressing cells and enhance the efficacy of other therapeutics.

Estrogen regulation of cyclin E2 requires cyclin D1 but not c-Myc.

During estrogen-induced proliferation, c-Myc and cyclin D1 initiate independent pathways that activate cyclin E1-Cdk2 by sequestration and/or downregulation of the CDK inhibitor p21(Waf1/Cip1), without significant increases in cyclin E1 protein levels. In contrast, cyclin E2 undergoes a marked increase in expression, which occurs within 9 to 12 h of estrogen treatment of antiestrogen-pretreated MCF-7 breast cancer cells. Both E cyclins are important to estrogen action, as small interfering RNA (siRNA)-mediated knockdown of either cyclin E1 or cyclin E2 attenuated estrogen-mediated proliferation. Inducible expression of cyclin D1 upregulated cyclin E2, while siRNA-mediated knockdown of cyclin D1 attenuated estrogen effects on cyclin E2. However, manipulation of c-Myc levels did not profoundly affect cyclin E2. Cyclin E2 induction by estrogen was accompanied by recruitment of E2F1 to the cyclin E1 and E2 promoters, and cyclin D1 induction was sufficient for E2F1 recruitment. siRNA-mediated knockdown of the chromatin remodelling factor CHD8 prevented cyclin E2 upregulation. Together, these data indicate that cyclin E2-Cdk2 activation by estrogen occurs via E2F- and CHD8-mediated transcription of cyclin E2 downstream of cyclin D1. This contrasts with the predominant regulation of cyclin E1-Cdk2 activity via CDK inhibitor association downstream of both c-Myc and cyclin D1 and indicates that cyclins E1 and E2 are not always coordinately regulated.

Identification of functional networks of estrogen- and c-Myc-responsive genes and their relationship to response to tamoxifen therapy in breast cancer.

BACKGROUND: Estrogen is a pivotal regulator of cell proliferation in the normal breast and breast cancer. Endocrine therapies targeting the estrogen receptor are effective in breast cancer, but their success is limited by intrinsic and acquired resistance. METHODOLOGY/PRINCIPAL FINDINGS: With the goal of gaining mechanistic insights into estrogen action and endocrine resistance, we classified estrogen-regulated genes by function, and determined the relationship between functionally-related genesets and the response to tamoxifen in breast cancer patients. Estrogen-responsive genes were identified by transcript profiling of MCF-7 breast cancer cells. Pathway analysis based on functional annotation of these estrogen-regulated genes identified gene signatures with known or predicted roles in cell cycle control, cell growth (i.e. ribosome biogenesis and protein synthesis), cell death/survival signaling and transcriptional regulation. Since inducible expression of c-Myc in antiestrogen-arrested cells can recapitulate many of the effects of estrogen on molecular endpoints related to cell cycle progression, the estrogen-regulated genes that were also targets of c-Myc were identified using cells inducibly expressing c-Myc. Selected genes classified as estrogen and c-Myc targets displayed similar levels of regulation by estrogen and c-Myc and were not estrogen-regulated in the presence of siMyc. Genes regulated by c-Myc accounted for 50% of all acutely estrogen-regulated genes but comprised 85% (110/129 genes) in the cell growth signature. siRNA-mediated inhibition of c-Myc induction impaired estrogen regulation of ribosome biogenesis and protein synthesis, consistent with the prediction that estrogen regulates cell growth principally via c-Myc. The 'cell cycle', 'cell growth' and 'cell death' gene signatures each identified patients with an attenuated response in a cohort of 246 tamoxifen-treated patients. In multivariate analysis the cell death signature was predictive independent of the cell cycle and cell growth signatures. CONCLUSIONS/SIGNIFICANCE: These functionally-based gene signatures can stratify patients treated with tamoxifen into groups with differing outcome, and potentially identify distinct mechanisms of tamoxifen resistance.

Identification of downstream targets of estrogen and c-myc in breast cancer cells.

Estrogen (E) plays a pivotal regulatory role in the control of cell proliferation in the normal breast and breast cancer (BC). To identify genes with likely roles in proliferation control that are regulated by E and its downstream target c-myc, we compared transcript profiles of antiestrogens-arrested cells stimulated to reinitiate cell cycle progression by E treatment or c-myc induction. Approximately 2/3 of the probe sets significantly regulated by E (adjusted p < 0.01) increased in expression. Half of the E-regulated probe sets were also regulated by c-myc. Genes involved in cell growth, cell proliferation, and cell survival were over-represented in the E-regulated geneset. Analysis of selected candidates has identified a nucleolar protein whose expression is correlated with c-myc expression in BC cell lines. These data indicate that a significant component of E-induced mitogenesis is mediated by c-myc and that selected c-myc target genes may be surrogate markers of c-myc expression in BC.

The estrogen and c-Myc target gene HSPC111 is over-expressed in breast cancer and associated with poor patient outcome.

INTRODUCTION: Estrogens play a pivotal role in the initiation and progression of breast cancer. The genes that mediate these processes are not fully defined, but potentially include the known mammary oncogene MYC. Characterization of estrogen-target genes may help to elucidate further the mechanisms of estrogen-induced mitogenesis and endocrine resistance. METHODS: We used a transcript profiling approach to identify targets of estrogen and c-Myc in breast cancer cells. One previously uncharacterized gene, namely HBV pre-S2 trans-regulated protein 3 (HSPC111), was acutely upregulated after estrogen treatment or inducible expression of c-Myc, and was selected for further functional analysis using over-expression and knock-down strategies. HSPC111 expression was also analyzed in relation to MYC expression and outcome in primary breast carcinomas and published gene expression datasets. RESULTS: Pretreatment of cells with c-Myc small interfering RNA abrogated estrogen induction of HSPC111, identifying HSPC111 as a potential c-Myc target gene. This was confirmed by the demonstration of two functional E-box motifs upstream of the transcription start site. HSPC111 mRNA and protein were over-expressed in breast cancer cell lines and primary breast carcinomas, and this was positively correlated with MYC mRNA levels. HSPC111 is present in a large, RNA-dependent nucleolar complex, suggesting a possible role in ribosomal biosynthesis. Neither over-expression or small interfering RNA knock-down of HSPC111 affected cell proliferation rates or sensitivity to estrogen/antiestrogen treatment. However, high expression of HSPC111 mRNA was associated with adverse patient outcome in published gene expression datasets. CONCLUSION: These data identify HSPC111 as an estrogen and c-Myc target gene that is over-expressed in breast cancer and is associated with an adverse patient outcome.

Progestins reinitiate cell cycle progression in antiestrogen-arrested breast cancer cells through the B-isoform of progesterone receptor.

Estrogen treatment of MCF-7 human breast cancer cells allows the reinitiation of synchronous cell cycle progression in antiestrogen-arrested cells. Here, we report that progestins also reinitiate cell cycle progression in this model. Using clonal cell lines derived from progesterone receptor (PR)-negative MCF-7M13 cells expressing wild-type or mutant forms of PRA and PRB, we show that this effect is mediated via PRB, not PRA. Cell cycle progression did not occur with a DNA-binding domain mutant of PRB but was unaffected by mutation in the NH(2)-terminal, SH3 domain interaction motif, which mediates rapid progestin activation of c-Src. Thus, the progestin-induced proliferative response in antiestrogen-inhibited cells is mediated primarily by the transcriptional activity of PRB. Analysis of selected cell cycle targets showed that progestin treatment induced levels of cyclin D1 expression and retinoblastoma protein (Rb) phosphorylation similar to those induced by estradiol. In contrast, progestin treatment resulted in only a 1.2-fold induction of c-Myc compared with a 10-fold induction by estradiol. These results support the conclusion that progestin, in a PRB-dependent manner, can overcome the growth-inhibitory effects of antiestrogens in estrogen receptor/PR-positive breast cancer cells by the induction of cyclin D1 expression. The mediation of this effect by PRB, but not PRA, further suggests a mechanism whereby abnormal regulation of the normal expression ratios of PR isoforms in breast cancer could lead to the attenuation of antiestrogen-mediated growth arrest.

c-Myc overexpression and endocrine resistance in breast cancer.

The oncoprotein c-Myc is frequently overexpressed in breast cancer and ectopic expression in breast cancer cell lines attenuates responses to antiestrogen treatment. Here, we review preliminary data aimed at further elucidating a potential role for c-Myc in clinical endocrine resistance in breast cancer. Immunohistochemical and semi-quantitative PCR revealed that c-Myc protein and c-myc mRNA were frequently overexpressed in both ER-positive and ER-negative breast carcinoma. Furthermore, both constitutive and inducible c-Myc overexpression in MCF-7 breast cancer cell lines markedly reduced their sensitivity to the growth inhibitory effects of the pure antiestrogen ICI 182,780. In order to identify potential downstream targets of c-Myc that mediate this effect, Affymetrix microarrays were employed to examine the patterns of gene expression shared by MCF-7 cells stimulated by estrogen, or by induction of c-Myc. Approximately 50% of estrogen target genes identified 6h after treatment were also regulated by c-Myc. One novel target, EMU4, was transcriptionally regulated by c-Myc. In addition, there was a strong correlation between c-myc and EMU4 mRNA expression in a battery of breast cancer cell lines. These data confirm that c-Myc overexpression is a common event in breast cancer, and that this is associated with resistance to antiestrogens in vitro. Furthermore, the development of an experimental paradigm for the discovery of c-Myc and estrogen target genes associated with endocrine resistance provides a framework for the discovery and validation of genes involved in estrogen signalling, and c-Myc-mediated-antiestrogen resistance.

Regulation of cyclin expression and cell cycle progression in breast epithelial cells by the helix-loop-helix protein Id1.

The helix-loop-helix protein Id1 has been implicated in regulating mammary epithelial cell proliferation and differentiation but the underlying molecular mechanisms are not well characterized. Under low serum conditions, ectopic expression of Id1, but not Id2, allowed continued proliferation of immortalized mammary epithelial cells and breast cancer cells. Conversely, downregulation of Id1 impaired proliferation. The effects of short interfering RNA (siRNA)-mediated downregulation of Id1 were the same as those following downregulation of c-Myc: decreased expression of cyclins D1 and E, reduced phosphorylation of pRb at Ser780 (a site targeted by cyclin D1-Cdk4) and reduced cyclin E-Cdk2 activity. Decreased cyclin D1 expression was an early response to Id1 antisense oligonucleotide treatment. Inhibition of c-Myc function by siRNA, antisense oligonucleotides or a dominant repressor resulted in downregulation of Id1, while ectopic expression of c-Myc resulted in rapid induction of Id1, suggesting that Id1 may be downstream of c-Myc. These data indicate that in mammary epithelial cells, Id1 has cell cycle regulatory functions that are similar to those of c-Myc, and suggest that cyclin D1 may be involved in Id1 regulation of cell cycle progression.

Estrogen and insulin/IGF-1 cooperatively stimulate cell cycle progression in MCF-7 breast cancer cells through differential regulation of c-Myc and cyclin D1.

Estrogen and insulin/insulin-like growth factor-I (IGF-I) are major mitogens for breast epithelial cells and when co-administered, synergistically induce G(1)-S phase cell cycle progression. We investigated this cooperativity by evaluating if the key cell cycle regulators, c-Myc and cyclin D1, represent points of convergence in the action of these mitogens in MCF-7 breast cancer cells. These studies demonstrated that estrogen significantly increased both c-Myc and cyclin D1 protein, while insulin predominantly increased cyclin D1 levels. This cumulative increase in c-Myc and cyclin D1 contributes to the cooperativity of these mitogens, since ectopic expression of c-Myc or cyclin D1 cooperates with either the estrogen or insulin signaling pathways to increase cell cycle progression. Inhibition of the MAPK or PI3-kinase pathways significantly reduced c-Myc and cyclin D1 protein levels and cell cycle progression. Ectopic expression of cyclin D1 partially overcame this inhibition, while ectopic expression of c-Myc partially overcame MAPK but not PI3-kinase inhibition. Therefore, estrogen and insulin/IGF-1 differentially regulate c-Myc and cyclin D1 to cooperatively stimulate breast cancer cell proliferation.