Cell fate determination factor Dachshund reprograms breast cancer stem cell function

The cell fate determination factor Dachshund was cloned as a dominant inhibitor of the hyperactive epidermal growth factor receptor ellipse. The expression of Dachshund is lost in human breast cancer associated with poor prognosis. Breast tumor-initiating cells (TIC) may contribute to tumor progression and therapy resistance. Here, endogenous DACH1 was reduced in breast cancer cell lines with high expression of TIC markers and in patient samples of the basal breast cancer phenotype. Re-expression of DACH1 reduced new tumor formation in serial transplantations in vivo, reduced mammosphere formation, and reduced the proportion of CD44(high)/CD24(low) breast tumor cells. Conversely, lentiviral shRNA to DACH1 increased the breast (B)TIC population. Genome-wide expression studies of mammary tumors demonstrated DACH1 repressed a molecular signature associated with stem cells (SOX2, Nanog, and KLF4) and genome-wide ChIP-seq analysis identified DACH1 binding to the promoter of the Nanog, KLF4, and Lin28 genes. KLF4/c-Myc and Oct4/Sox2 antagonized DACH1 repression of BTIC. Mechanistic studies demonstrated DACH1 directly repressed the Nanog and Sox2 promoters via a conserved domain. Endogenous DACH1 regulates BTIC in vitro and in vivo.     

DACH1 regulates cell cycle progression of myeloid cells through the control of cyclin D, Cdk 4/6 and p21Cip1

The cell-fate determination factor Dachshund, a component of the Retinal Determination Gene Network (RDGN), has a role in breast tumor proliferation through the repression of cyclin D1 and several key regulators of embryonic stem cell function, such as Nanog and Sox2. However, little is known about the role of DACH1 in a myeloid lineage as a cell cycle regulator. Here, we identified the differential expression levels of extensive cell cycle regulators controlled by DACH1 in myeloid progenitor cells. The forced expression of DACH1 induced p27(Kip1) and repressed p21(Cip1), which is a pivotal characteristic of the myeloid progenitor. Furthermore, DACH1 significantly increased the expression of cyclin D1, D3, F, and Cdk 1, 4, and 6 in myeloid progenitor cells. The knockdown of DACH1 blocked the cell cycle progression of HL-60 promyeloblastic cells through the decrease of cyclin D1, D3, F, and Cdk 1, 4, and 6 and increase in p21(Cip1), which in turn decreased the phosphorylation of the Rb protein. The expression of Sox2, Oct4, and Klf4 was significantly up-regulated by the forced expression of DACH1 in mouse myeloid progenitor cells.     

A cyclin D1/microRNA 17/20 regulatory feedback loop in control of breast cancer cell proliferation

Decreased expression of specific microRNAs (miRNAs) occurs in human tumors, which suggests a function for miRNAs in tumor suppression. Herein, levels of the miR-17-5p/miR-20a miRNA cluster were inversely correlated to cyclin D1 abundance in human breast tumors and cell lines. MiR-17/20 suppressed breast cancer cell proliferation and tumor colony formation by negatively regulating cyclin D1 translation via a conserved 3' untranslated region miRNA-binding site, thereby inhibiting serum-induced S phase entry. The cell cycle effect of miR-17/20 was abrogated by cyclin D1 siRNA and in cyclin D1-deficient breast cancer cells. Mammary epithelial cell-targeted cyclin D1 expression induced miR-17-5p and miR-20a expression in vivo, and cyclin D1 bound the miR-17/20 cluster promoter regulatory region. In summary, these studies identify a novel cyclin D1/miR-17/20 regulatory feedback loop through which cyclin D1 induces miR-17-5p/miR-20a. In turn, miR-17/20 limits the proliferative function of cyclin D1, thus linking expression of a specific miRNA cluster to the regulation of oncogenesis.     

Leptin and high glucose stimulate cell proliferation in MCF-7 human breast cancer cells: reciprocal involvement of PKC-alpha and PPAR expression

Glucose concentration may be an important factor in breast cancer cell proliferation, and the prevalence of breast cancer is high in diabetic patients. Leptin may also be an important factor since plasma levels of leptin correlated with TNM staging for breast cancer patients. The effects of glucose and leptin on breast cancer cell proliferation were evaluated by examining cell doubling time, DNA synthesis, levels of cell cycle related proteins, protein kinase C (PKC) isozyme expression, and peroxisome proliferator-activated receptor (PPAR) subtypes were determined following glucose exposure at normal (5.5 mM) and high (25 mM) concentrations with/without leptin in MCF-7 human breast cancer cells. In MCF-7 cells, leptin and high glucose stimulated cell proliferation as demonstrated by the increases in DNA synthesis and expression of cdk2 and cyclin D1. PKC-alpha, PPARgamma, and PPARalpha protein levels were up-regulated following leptin and high glucose treatment in drug-sensitive MCF-7 cells. However, there was no significant effect of leptin and high glucose on cell proliferation, DNA synthesis, levels of cell cycle proteins, PKC isozymes, or PPAR subtypes in multidrug-resistant human breast cancer NCI/ADR-RES cells. These results suggested that hyperglycemia and hyperleptinemia increase breast cancer cell proliferation through accelerated cell cycle progression with up-regulation of cdk2 and cyclin D1 levels. This suggests the involvement of PKC-alpha, PPARalpha, and PPARgamma.     

Cyclin D1 determines mitochondrial function in vivo

The cyclin D1 gene encodes a regulatory subunit of the holoenzyme that phosphorylates and inactivates the pRb tumor suppressor to promote nuclear DNA synthesis. cyclin D1 is overexpressed in human breast cancers and is sufficient for the development of murine mammary tumors. Herein, cyclin D1 is shown to perform a novel function, inhibiting mitochondrial function and size. Mitochondrial activity was enhanced by genetic deletion or antisense or small interfering RNA to cyclin D1. Global gene expression profiling and functional analysis of mammary epithelial cell-targeted cyclin D1 antisense transgenics demonstrated that cyclin D1 inhibits mitochondrial activity and aerobic glycolysis in vivo. Reciprocal regulation of these genes was observed in cyclin D1-induced mammary tumors. Cyclin D1 thus integrates nuclear DNA synthesis and mitochondrial function.     

Bone morphogenetic protein-2 blocks MDA MB 231 human breast cancer cell proliferation by inhibiting cyclin-dependent kinase-mediated retinoblastoma protein phosphorylation

Bone morphogenetic protein-2 (BMP-2) has been shown to act as an antiproliferative agent for a number of different cell types. We show that BMP-2 dose-dependently inhibits growth of MDA MB 231 human breast cancer cells. Epidermal growth factor (EGF) stimulates DNA synthesis and entry of these cells into the S-phase. BMP-2 inhibits EGF-induced DNA synthesis by arresting them in G1 phase of the cell cycle. BMP-2 increases the level of cyclin kinase inhibitor p21. Furthermore, we show that exposure of MDA MB 231 cells to BMP-2 stimulates association of p21 with cyclin D1 and with cyclin E resulting in the inhibition of their associated kinase activities. Finally, BMP-2 treatment is found to cause hypophosphorylation of the retinoblastoma protein (pRb), a key regulator of cell cycle progression. Our data provide a mechanism for the antiproliferative effect of BMP-2 in the breast cancer cells.     

p14ARF Post-Transcriptional Regulation of Nuclear Cyclin D1 in MCF-7 Breast Cancer Cells: Discrimination between a Good and Bad Prognosis?

As part of a cell's inherent protection against carcinogenesis, p14ARF is upregulated in response to hyperproliferative signalling to induce cell cycle arrest. This property makes p14ARF a leading candidate for cancer therapy. This study explores the consequences of reactivating p14ARF in breast cancer and the potential of targeting p14ARF in breast cancer treatment. Our results show that activation of the p14ARF-p53-p21-Rb pathway in the estrogen sensitive MCF-7 breast cancer cells induces many hallmarks of senescence including a large flat cell morphology, multinucleation, senescence-associated-β-gal staining, and rapid G1 and G2/M phase cell cycle arrest. P14ARF also induces the expression of the proto-oncogene cyclin D1, which is most often associated with a transition from G1-S phase and is highly expressed in breast cancers with poor clinical prognosis. In this study, siRNA knockdown of cyclin D1, p21 and p53 show p21 plays a pivotal role in the maintenance of high cyclin D1 expression, cell cycle and growth arrest post-p14ARF induction. High p53 and p14ARF expression and low p21/cyclin D1 did not cause cell-cycle arrest. Knockdown of cyclin D1 stops proliferation but does not reverse senescence-associated cell growth. Furthermore, cyclin D1 accumulation in the nucleus post-p14ARF activation correlated with a rapid loss of nucleolar Ki-67 protein and inhibition of DNA synthesis. Latent effects of the p14ARF-induced cellular processes resulting from high nuclear cyclin D1 accumulation included a redistribution of Ki-67 into the nucleoli, aberrant nuclear growth (multinucleation), and cell proliferation. Lastly, downregulation of cyclin D1 through inhibition of ER abrogated latent recurrence. The mediation of these latent effects by continuous expression of p14ARF further suggests a novel mechanism whereby dysregulation of cyclin D1 could have a double-edged effect. Our results suggest that p14ARF induced-senescence is related to late-onset breast cancer in estrogen responsive breast cancers and/or the recurrence of more aggressive breast cancer post-therapy.     

Cell cycle regulators cyclin D1 and CDK4/6 have estrogen receptor-dependent divergent functions in breast cancer migration and stem cell-like activity

Cyclin D1 and its binding partners CDK4/6 are essential regulators of cell cycle progression and are implicated in cancer progression. Our aim was to investigate a potential regulatory role of these proteins in other essential tumor biological characteristics. Using a panel of breast cancer cell lines and primary human breast cancer samples, we have demonstrated the importance of these cell cycle regulators in both migration and stem-like cell activity. siRNA was used to target cyclin D1 and CDK4/6 expression, having opposing effects on both migration and stem-like cell activity dependent upon estrogen receptor (ER) expression. Inhibition of cyclin D1 or CDK4/6 increases or decreases migration and stem-like cell activity in ER−ve (ER-negative) and ER+ve (ER-positive) breast cancer, respectively. Furthermore, overexpressed cyclin D1 caused decreased migration and stem-like cell activity in ER−ve cells while increasing activity in ER+ve breast cancer cells. Treatment of breast cancer cells with inhibitors of cyclin D1 and CDK4/6 (Flavopiridol/PD0332991), currently in clinical trials, mimicked the effects observed with siRNA treatment. Re-expression of ER in two ER−ve cell lines was sufficient to overcome the effects of either siRNA or clinical inhibitors of cyclin D1 and CDK4/6.   In conclusion, cyclin D1 and CDK4/6 have alternate roles in regulation of migration and stem-like cell activity. Furthermore, these effects are highly dependent upon expression of ER. The significance of these results adds to our general understanding of cancer biology but, most importantly, could be used diagnostically to predict treatment response to cell cycle inhibition in breast cancer.     

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.     

Hexane/ethanol extract of Glycyrrhiza uralensis and its active compound isoangustone A induce G1 cycle arrest in DU145 human prostate and 4T1 murine mammary cancer cells

Although licorice is known to exert anticarcinogenic effects, it contains large quantities of glycyrrhizin (GL), which causes severe hypertension. We have previously demonstrated that the hexane/ethanol extract of Glycyrrhiza uralensis (HEGU) contains no detectable GL and suppresses doxorubicin-induced apoptosis in H9c2 rat cardiac myoblasts. The principal objective of this study was to determine whether and by what mechanism HEGU and its active component, isoangustone A, inhibit cell-cycle progression in DU145 human prostate and 4T1 mouse breast cancer cells. HEGU and isoangustone A dose-dependently decreased DNA synthesis and induced G1 phase arrest in both DU145 and 4T1 cells. HEGU and isoangustone A reduced the levels of CDK2 and CDK4 as well as cyclin A and cyclin D1 proteins, and also induced a decrease in CDK2 activity. The addition of HEGU to drinking water significantly suppressed the orthotopic growth of 4T1 allografts and the expression of the proliferating nuclear cell antigen, CDK2 and CDK4 proteins in the tumor tissues. These results demonstrate the potential of HEGU containing isoangustone A as an antitumor agent.     

Cyclin D1 Inhibits Cell Proliferation through Binding to PCNA and Cdk2

Cyclin D1 is known as a promoting factor for cell growth. We previously showed, however, that the expression of cyclin D1 increases markedly in senescent human fibroblastsin vitro.Here we investigate whether the overexpression of cyclin D1 inhibits cell proliferation. Colony formation after transfection with the cyclin D1 expression vector was repressed in NIH-3T3, TIG-1, CHO-K1, and HeLa cells, compared with those with mock and cyclin E expression vectors. A transient transfection assay demonstrated that the overexpression of cyclin D1 inhibited DNA synthesis of TIG-1 cells. The complexes of cyclin D1 with PCNA and cdk2 increased remarkably in senescent cells, compared with young counterparts. Excessive glutathioneS-transferase (GST)–cyclin D1 inhibited DNA replication and repressed cdk2-dependent kinase activityin vitro.DNA synthesis of NIH-3T3 transfectants with PCNA or cdk2 expression vectors was not inhibited by the overexpression of cyclin D1. These results indicate that an excessive level of cyclin D1 represses cell proliferation by inhibiting DNA replication and cdk2 activity through the binding of cyclin D1 to PCNA and cdk2, as it does in senescent cells.     

The role of collagen structure in mitogen stimulation of ERK,cyclin D1 expression,and G1-S progression in rat hepatocytes

Adhesion to type 1 collagen can elicit different cellular responses dependent upon whether the collagen is in a fibrillar form (gel) or monomeric form (film). Hepatocytes adherent to collagen film spread extensively, express cyclin D1, and increase DNA synthesis in response to epidermal growth factor, whereas hepatocytes adherent to collagen gel have increased differentiated function, but lower DNA synthesis. The signaling mechanisms by which different forms of type I collagen modulate cell cycle progression are unknown. When ERK MAP kinase activation was analyzed in hepatocytes attached to collagen film, two peaks of ERK activity were demonstrated. Only the second peak, which correlated with an increase of cyclin D1, was required for G1-S progression. Notably, this second peak of ERK activity was absent in cells adherent to collagen gel, but not required in the presence of exogenous cyclin D1. Expression of activated mutants of the Ras/Raf/MEK signaling pathway in cells adherent to collagen gel restored ERK phosphorylation and DNA synthesis, but differentially affected cell shape. Although Ras, Raf, and MEK all increased expression of cyclin D1 on collagen film, only Ras and Raf significantly up-regulated cyclin D1 levels on collagen gel. These results demonstrate that adhesion to polymerized collagen induces growth arrest by inhibiting the Ras/ERK-signaling pathway to cyclin D1 required in late G1.     

Cell cycle control in breast cancer cells

In breast cancer, cyclins D1 and E and the cyclin-dependent kinase inhibitors p21 (Waf1/Cip1)and p27 (Kip1) are important in cell-cycle control and as potential oncogenes or tumor suppressor genes. They are regulated in breast cancer cells following mitogenic stimuli including activation of receptor tyrosine kinases and steroid hormone receptors, and their deregulation frequently impacts on breast cancer outcome, including response to therapy. The cyclin-dependent kinase inhibitor p16 (INK4A) also has a critical role in transformation of mammary epithelial cells. In addition to their roles in cell cycle control, some of these molecules, particularly cyclin D1, have actions that are not mediated through regulation of cyclin-dependent kinase activity but may be important for loss of proliferative control during mammary oncogenesis.     

Visfatin stimulates proliferation of MCF-7 human breast cancer cells

Obesity, a condition characterized by increased fat content and altered secretion of adipokines, is a risk factor for postmenopausal breast cancer. Visfatin has recently been established as a novel adipokine that is highly enriched in visceral fat. Here we report that visfatin regulated proliferation of MCF-7 human breast cancer cells. Exogenous administration of recombinant visfatin increased cell proliferation and DNA synthesis rate in MCF-7 cells. Furthermore, visfatin activated G1-S phase cell cycle progression by upregulation of cyclin D1 and cdk2 expression. Visfatin also increased the expression of matrix metalloproteinases 2, matrix metalloproteinases 9, and vascular endothelial growth factor genes, suggesting that it may function in metastasis and angiogenesis of breast cancer. Taken together, these findings suggest that visfatin plays an important role in breast cancer progression.     

Integrin-binding Protein Nischarin Interacts with Tumor Suppressor Liver Kinase B1 (LKB1) to Regulate Cell Migration of Breast Epithelial Cells

Biallelic inactivation of LKB1, a serine/threonine kinase, has been detected in 30% of lung adenocarcinomas, and inhibition of breast tumor growth has been demonstrated. We have identified the tumor suppressor, Nischarin, as a novel binding partner of LKB1. Our mapping analysis shows that the N terminus of Nischarin interacts with amino acids 44–436 of LKB1. Time lapse microscopy and Transwell migration data show that the absence of both Nischarin and LKB1 from an invasive breast cancer cell line (MDA-MB-231) enhances migration as measured by increased distance and speed of migrating cells. Our data suggest that this is a result of elevated PAK1 and LIMK1 phosphorylation. Moreover, the absence of Nischarin and LKB1 increased tumor growth in vivo. Consistent with this, the percentage of S phase cells was increased, as demonstrated by flow cytometry and enhanced cyclin D1. The absence of Nischarin and LKB1 also led to a dramatic increase in the formation of lung metastases. Our studies, for the first time, demonstrate functional interaction between LKB1 and Nischarin to inhibit cell migration and breast tumor progression. Mechanistically, we show that these two proteins together regulate PAK-LIMK-Cofilin and cyclin D1/CDK4 pathways.     

The cyclin D1-CDK4 oncogenic interactome enables identification of potential novel oncogenes and clinical prognosis

Overexpression of cyclin D1 and its catalytic partner, CDK4, is frequently seen in human cancers. We constructed cyclin D1 and CDK4 protein interaction network in a human breast cancer cell line MCF7, and identified novel CDK4 protein partners. Among CDK4 interactors we observed several proteins functioning in protein folding and in complex assembly. One of the novel partners of CDK4 is FKBP5, which we found to be required to maintain CDK4 levels in cancer cells. An integrative analysis of the extended cyclin D1 cancer interactome and somatic copy number alterations in human cancers identified BAIAPL21 as a potential novel human oncogene. We observed that in several human tumor types BAIAPL21 is expressed at higher levels as compared to normal tissue. Forced overexpression of BAIAPL21 augmented anchorage independent growth, increased colony formation by cancer cells and strongly enhanced the ability of cells to form tumors in vivo. Lastly, we derived an Aggregate Expression Score (AES), which quantifies the expression of all cyclin D1 interactors in a given tumor. We observed that AES has a prognostic value among patients with ER-positive breast cancers. These studies illustrate the utility of analyzing the interactomes of proteins involved in cancer to uncover potential oncogenes, or to allow better cancer prognosis.     

Bcl-2 induces cyclin D1 promoter activity in human breast epithelial cells independent of cell anchorage

Cyclin D1 expression is co-regulated by growth factor and cell adhesion signaling. Cell adhesion to the extracellular matrix activates focal adhesion kinase (FAK), which is essential for cyclin D1 expression. Upon the loss of cell adhesion, cyclin D1 expression is downregulated, followed by apoptosis in normal epithelial cells. Since bcl-2 prevents apoptosis induced by the loss of cell adhesion, we hypothesized that bcl-2 induces survival signaling complementary to cell adhesion-mediated gene regulation. In the present study, we investigated the role of bcl-2 on FAK activity and cyclin D1 expression. We found that bcl-2 overexpression induces cyclin D1 expression in human breast epithelial cell line MCF10A independent of cell anchorage. Increased cyclin D1 expression in stable bcl-2 transfectants is not related to bcl-2-increased G1 duration, but results from cyclin D1 promoter activation. Transient transfection studies confirmed anchorage-independent bcl-2 induction of cyclin D1 promoter activity in human breast epithelial cell lines (MCF10A, BT549, and MCF-7). We provide evidence that bcl-2 induction of cyclin D1 expression involves constitutive activation of focal adhesion kinase, regardless of cell adhesion. The present study suggests a potential oncogenic activity for bcl-2 through cyclin D1 induction, and provides an insight into the distinct proliferation-independent pathway leading to increased cyclin D1 expression in breast cancer.