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
ABSTRACT 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.
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- "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). "
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.Genes & Nutrition 05/2011; 7(1):63-74. DOI:10.1007/s12263-011-0225-y · 3.42 Impact Factor
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- "In the absence of ectopically expressed cyclin D1, silencing RSK2 decreased the S-phase population to ~50% of the control (Fig. 5F). As expected, over-expression of cyclin D1 increased the number of control cells in S-phase by ~ 1.7X (Grillo et al., 2006). Importantly, the forced expression of cyclin D1 after RSK2 knockdown not only prevented the decrease in S-phase but even increased the number of cells in S-phase. "
ABSTRACT: Stress granules aid cell survival in response to environmental stressors by acting as sites of translational repression. We report an unanticipated link between stress granules and the serine/threonine kinase RSK2. In stressed breast cells, endogenous RSK2 colocalizes in granules with TIA-1 and poly(A)-binding protein 1, and the sequestration of RSK2 and TIA-1 exhibits codependency. The RSK2 N-terminal kinase domain controls the direct interaction with the prion-related domain of TIA-1. Silencing RSK2 decreases cell survival in response to stress. Mitogen releases RSK2 from the stress granules and permits its nuclear import via a nucleocytoplasmic shuttling sequence in the C-terminal domain. Nuclear accumulation is dependent on TIA-1. Surprisingly, nuclear localization of RSK2 is sufficient to enhance proliferation through induction of cyclin D1, in the absence of other active signaling pathways. Hence, RSK2 is a pivotal factor linking the stress response to survival and proliferation.Molecular cell 10/2008; 31(5):722-36. DOI:10.1016/j.molcel.2008.06.025 · 14.46 Impact Factor
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- "Accordingly, another study showed that the major antiproliferative effect of the antiestrogen tamoxifen could be related to its inhibition of CCND1 expression (Hodges et al. 2003). CCND1 silencing experiments in BCC further confirmed the correlation between CCND1 expression levels and cellular proliferation, emphasizing CCND1 as a potential therapeutic target for breast cancer (Arnold and Papanikolaou 2005; Grillo et al. 2006). "
ABSTRACT: Estrogen stimulates the proliferation of the most common type of human breast cancer that expresses estrogen receptor alpha (ERalpha) through the activation of the cyclin D1 (CCND1) oncogene. However, our knowledge of ERalpha transcriptional mechanisms remains limited. Hence, it is still elusive why ERalpha ectopically expressed in ER-negative breast cancer cells (BCC) is functional on ectopic reporter constructs but lacks activity on many endogenous target genes, including CCND1. Here, we show that estradiol (E2) stimulation of CCND1 expression in BCC depends on a novel cell-type-specific enhancer downstream from the CCND1 coding region, which is the primary ERalpha recruitment site in estrogen-responsive cells. The pioneer factor FoxA1 is specifically required for the active chromatin state of this enhancer and as such is crucial for both CCND1 expression and subsequent cell cycle progression. Interestingly, even in BCC, CCND1 levels and proliferation are tightly controlled by E2 through the establishment of a negative feedforward loop involving the induction of NFIC, a putative tumor suppressor capable of directly repressing CCND1 transcription. Taken together, our results reveal an estrogen-regulated combinatorial network including cell-specific cis- and trans-regulators of CCND1 expression where ERalpha collaborates with other transcription factors associated with the ER-positive breast cancer phenotype, including FoxA1 and NFIC.Genes & Development 10/2006; 20(18):2513-26. DOI:10.1101/gad.1446006 · 12.64 Impact Factor