Validation of cyclin D1/CDK4 as an anticancer drug target in MCF-7 breast cancer cells: Effect of regulated overexpression of cyclin D1 and siRNA-mediated inhibition of endogenous cyclin D1 and CDK4 expression
We have examined the role of cyclin D1 and cyclin-dependent kinase-4 (CDK4) in the cell cycle progression and proliferation of MCF-7 breast cancer cells. Forced expression of cyclin D1 using a tetracycline-regulated expression system, and suppression of endogenous cyclin D1 and CDK4 using small interfering RNA (siRNA) were used to validate this protein complex as a drug target in cancer drug discovery. Overexpression of cyclin D1 increased both phosphorylation of the retinoblastoma gene product (RB) and passage through the G1-S phase transition, resulting in increased proliferation of cells. When cyclin D1 expression was shut off, growth rates fell below those seen in control cell lines transfected with the vector, indicating an increased dependence on this protein for proliferation. Inhibition of endogenous cyclin D1 or CDK4 expression by RNA interference resulted in hypophosphorylation of RB and accumulation of cells in G1. These results support the prevailing view that pharmacological inhibition of cyclin D1/CDK4 complexes is a useful strategy to inhibit the growth of tumors. Furthermore, since MCF-7 cells appear to be dependent on this pathway for their continued proliferation, it is a suitable cell line to test novel cyclin D1/CDK4 inhibitors.
"The work of Yu et al.
 and Landis et al.
 suggests that inhibition of CDK4 might benefit patients with ErbB-2 initiated breast cancers . The CDK4/CyclinD1 complex as an anti-cancer drug target has been further validated in MCF-7 breast cancer cells . "
[Show abstract][Hide abstract] ABSTRACT: Cyclin-dependent kinases (CDKs) play a key role in the cell cycle and are important anti-cancer drug targets. The natural product fascaplysin inhibits CDK4 with surprising selectivity (IC(50) = 0.4 µM) compared to the close homolog CDK2 (IC(50) = 500 µM). Free energy calculations of the positively charged fascaplysin and an uncharged iso-electronic derivative in the CDK2 and CDK4 inhibitor complexes indicate that the positive charge of fascaplysin is crucial for selectivity. This finding will guide further improvements in the design of fascaplysin-based selective inhibitors for CDK4.
PLoS ONE 08/2012; 7(8):e42612. DOI:10.1371/journal.pone.0042612 · 3.23 Impact Factor
"Mitogenic stimuli increase the intracellular levels of cyclin D1, which then form an assembly with CDK4 or CDK6, and ultimately leading to the hyperphosphorylation of Rb protein (Sherr 1993; Matsushime et al. 1994). Elevations in cyclin D1 result in a corresponding increase in cyclin D1/CDK4 activity and hyperphosphorylation of Rb protein, whereas blockade of cyclin D1 activation blocks the hyperphosphorylation of Rb protein and results in cell cycle arrest in G1 (Grillo et al. 2006). Overexpression of cyclin D1 has been observed in approximately 50% of all breast cancers (Hall and Peters 1996; Arnold and Papanikolaou 2005; Caldon et al. 2006), whereas suppression of cyclin D1 activity significantly inhibits mammary tumorigenesis (Bartkova et al. 1994; Arber et al. 1997). "
[Show abstract][Hide abstract] ABSTRACT: Systemic chemotherapy is the only current method of treatment that provides some chance for long-term survival in patients with advanced or metastatic cancer. γ-Tocotrienol is a natural form of vitamin E found in high concentrations in palm oil and displays potent anticancer effects, but limited absorption and transport of by the body has made it difficult to obtain and sustain therapeutic levels in the blood and target tissues. Statins are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase and are an example of a promising cancer chemotherapeutic agent whose clinical usefulness has been limited due to high-dose toxicity. Similarly, erlotinib and gefitinib are anticancer agents that inhibit the activation of individual HER/ErbB receptor subtypes, but have shown limited clinical success because of heterodimerization between different EGF receptor family members that can rescue cancer cells from agents directed against a single receptor subtype. Recent studies have investigated the anticancer effectiveness of low-dose treatment of various statins or EGF receptor inhibitors alone and in combination with γ-tocotrienol on highly malignant +SA mouse mammary epithelial cells in vitro. Combined treatment with subeffective doses of γ-tocotrienol with these other chemotherapeutic agents resulted in a synergistic inhibition of +SA cell growth and viability. These findings strongly suggest that combined treatment of γ-tocotrienol with other anticancer agents may not only provide an enhanced therapeutic response but also provide a means to avoid the toxicity, low bioavailability, or limited therapeutic action associated with high-dose monotherapy.
"The biologically relevant role of cyclin D1 in breast tumourigenesis has been evidenced by several findings: mammary gland-targeted cyclin D1 over-expression resulted in mammary hyperplasia and adenocarcinoma in transgenic mice (9); cyclin D1 antisense blocked ErbB2-induced mammary tumour growth in vivo (10), and cyclin D1-deficient mice were resistant to ErbB2- or Ras-induced mammary tumourigenesis (11). In addition, the correlation between cyclin D1 expression levels and cellular proliferation in breast cancer cells has been further confirmed by silencing experiments (1,12). "
[Show abstract][Hide abstract] ABSTRACT: Cyclin D1 gene (CCND1) is a critical mitogen-regulated cell-cycle control element whose transcriptional modulation plays a crucial role in breast cancer growth and progression. Here we demonstrate that the non-aromatizable androgen 5-α-dihydrotestosterone (DHT) inhibits endogenous cyclin D1 expression, as evidenced by reduction of cyclin D1 mRNA and protein levels, and decrease of CCND1-promoter activity, in MCF-7 cells. The DHT-dependent inhibition of CCND1 gene activity requires the involvement and the integrity of the androgen receptor (AR) DNA-binding domain. Site directed mutagenesis, DNA affinity precipitation assay, electrophoretic mobility shift assay and chromatin immunoprecipitation analyses indicate that this inhibitory effect is ligand dependent and it is mediated by direct binding of AR to an androgen response element (CCND1-ARE) located at -570 to -556-bp upstream of the transcription start site, in the cyclin D1 proximal promoter. Moreover, AR-mediated repression of the CCND1 involves the recruitment of the atypical orphan nuclear receptor DAX1 as a component of a multiprotein repressor complex also embracing the participation of Histone Deacetylase 1. In conclusion, identification of the CCND1-ARE allows defining cyclin D1 as a specific androgen target gene in breast and might contribute to explain the molecular basis of the inhibitory role of androgens on breast cancer cells proliferation.
Nucleic Acids Research 09/2010; 38(16):5351-65. DOI:10.1093/nar/gkq278 · 9.11 Impact Factor
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