Anti-HER2 antibody trastuzumab inhibits CDK2-mediated NPAT and histone H4 expression via the PI3K pathway. Cell Cycle 5:1654-1661

Department of Experimental Therapeutics, University of Texas MD Anderson Cancer Center, Houston, Texas 77030-4009, USA.
Cell cycle (Georgetown, Tex.) (Impact Factor: 4.57). 09/2006; 5(15):1654-61. DOI: 10.4161/cc.5.15.3007
Source: PubMed


The anti-HER2 antibody trastuzumab (Herceptin) has been used to treat patients with breast cancers that overexpress HER2. We have demonstrated that p27(Kip1) upregulation is one of the key events that cause G(1) arrest upon trastuzumab treatment. Here, we have examined the effect of trastuzumab on expression of CDK2, Rb, E2F, NPAT and histone H4 in breast cancer cells that overexpress HER2. Trastuzumab treatment dramatically inhibited the kinase activity and expression of CDK2, whereas the kinase activity and expression of CDK4 were not affected. Unlike the p27(Kip1) upregulation that occurs primarily through post-translational mechanisms, CDK2 was downregulated primarily at a transcriptional level as shown by Northern blotting and real-time RT-PCR analyses. With a decrease in CDK2 activity, trastuzumab decreased the kinase activity of cyclin E but had little effect on cyclin E protein level. Overexpression of wild-type cyclin E or its lower molecular weight forms did not influence the response to trastuzumab. Levels and activities of CDK6, cyclin A, and cyclin D1 were all suppressed by trastuzumab. As a result, trastuzumab inhibited Rb phosphorylation that associates with CDK2, cyclin E, CDK6, cyclin A, or cyclin D1. As predicted from these changes, trastuzumab decreased the DNA-binding activity of E2F, decreased the level of NPAT protein, and decreased the level of histone H4 mRNA. Blockade of the PI3K pathway with LY294002 produced similar effects to trastuzumab treatment on expression of each of these genes. Taken together, treatment of breast cancer cells that overexpress HER2 with the anti-HER2 antibody trastuzumab inhibits CDK2, Rb phosphorylation, E2F activity, NPAT, and histone H4 via PI3K signaling that are needed for both DNA and histone synthesis during progression from G(1) phase to S phase of the cell cycle.

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Available from: Mollianne McGahren Murray
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    • "While significant progress has been achieved, molecular mechanisms underlying the action of trastuzumab are still not fully elucidated. Treatment with trastuzumab inhibits proliferation of breast cancer cells by blocking the phosphatidylinositol-3-kinase (PI3K) signaling pathway, upregulating the cyclin dependent kinase inhibitor p27Kip1 and inducing G1 arrest of the cell cycle [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]. We and others have also illustrated the ability of trastuzumab to interfere with DNA repair [14], [21] and tumor angiogenesis [22], [23], [24], [25], [26], [27]. "
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    ABSTRACT: Trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of the HER2 oncoprotein, can effectively target HER2-positive breast cancer through several mechanisms. Although the effects of trastuzumab on cancer cell proliferation, angiogenesis and apoptosis have been investigated in depth, the effect of trastuzumab on microRNA (miRNA) has not been extensively studied. We have performed miRNA microarray profiling before and after trastuzumab treatment in SKBr3 and BT474 human breast cancer cells that overexpress HER2. We found that trastuzumab treatment of SKBr3 cells significantly decreased five miRNAs and increased three others, whereas treatment of BT474 cells significantly decreased two miRNAs and increased nine. The only change in miRNA expression observed in both cell lines following trastuzumab treatment was upregulation of miRNA-194 (miR-194) that was further validated in vitro and in vivo. Forced expression of miR-194 in breast cancer cells that overexpress HER2 produced no effect on apoptosis, modest inhibition of proliferation, significant inhibition of cell migration/invasion in vitro and significant inhibition of xenograft growth in vivo. Conversely, knockdown of miR-194 promoted cell migration. Increased miR-194 expression markedly reduced levels of the cytoskeletal protein talin2 and specifically inhibited luciferase reporter activity of a talin2 wild-type 3'-untranslated region, but not that of a mutant reporter, indicating that talin2 is a direct downstream target of miR-194. Trastuzumab treatment inhibited breast cancer cell migration and reduced talin2 expression in vitro and in vivo. Knockdown of talin2 inhibited cell migration/invasion. Knockdown of trastuzumab-induced miR-194 expression with a miR-194 inhibitor compromised trastuzumab-inhibited cell migration in HER2-overexpressing breast cancer cells. Consequently, trastuzumab treatment upregulates miR-194 expression and may exert its cell migration-inhibitory effect through miR-194-mediated downregulation of cytoskeleton protein talin2 in HER2-overexpressing human breast cancer cells.
    Full-text · Article · Jul 2012 · PLoS ONE
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    • "Along with others, we have shown a linkage between tumorigenesis and cyclin E by correlating the altered expression of cyclin E to the loss of growth control in breast cancer (Buckley et al., 1993; Keyomarsi and Pardee, 1993; Keyomarsi and Herliczek, 1997). Le et al. have shown that treatment of SKBr3 cells with the anti-HER2 antibody 4D5 caused decreased cyclin E-associated kinase activity (Le et al., 2006). One question this study raised is whether the decrease in cyclin E-associated kinase activity was due to changes in full-length or LMW cyclin E expression. "
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    ABSTRACT: HER2/neu (HER2) and cyclin E are important prognostic indicators in breast cancer. Since both are involved in cell cycle regulation we investigated whether there was a direct interaction between the two. HER2 and cyclin E expression levels were determined in 395 breast cancer patients. Patients with HER2-overexpression and high levels of cyclin E had decreased 5-year disease-specific survival compared with low levels of cyclin E (14% versus 89%, P < .0001) In vitro studies were performed in which HER2-mediated activity in HER2-overexpressing breast cancer cell lines was downregulated by transfection with HER2 siRNA or treatment with trastuzumab. Cyclin E expression levels were determined, and functional effects investigated using kinase assays, MTT assays to assess cell viability as a marker of proliferation, and FACS analysis to determine cell cycle profiles. Decreased HER2-mediated signaling resulted in decreased expression of cyclin E, particularly the low molecular weight (LMW) isoforms. Decreased HER2 and LMW cyclin E expression had functional effects, including decreased cyclin E-associated kinase activity and decreased proliferation, due to increased apoptosis and an increased accumulation of cells in the G1 phase. In vivo studies performed in a HER2-overexpressing breast cancer xenograft model confirmed the effects of trastuzumab on cyclin E expression. Given the relationship between HER2 and cyclin E, in vitro clonogenic assays were performed to assess combination therapy targeting both proteins. Isobologram analysis showed a synergistic interaction between the two agents (trastuzumab targeting HER2 and roscovitine targeting cyclin E). Taken together, these studies demonstrate that HER2-mediated signaling effects LMW cyclin E expression, which in turn effects cell cycle regulation. LMW cyclin E has prognostic and predictive roles in HER2-overexpressing breast cancer, warranting further study of its potential as a therapeutic target.
    Full-text · Article · May 2010 · Oncogene
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    • "Quantitative models can separate the strengths of drug action on individual phases of the cell cycle. Previous molecular biological studies have shown that HER2 is associated with increases of both G1-S-specific cyclins (cyclins D and E) and G2-M-specific cyclin (cyclin A) [17,18], which are crucial for G1-S and G2-M progression, respectively. Our objective in this study is to use quantitative models to determine if HER2 inhibitors abolish the function on both phase transitions and how this contributes to cell cycle blockage. "
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    ABSTRACT: Oncogene signaling is known to deregulate cell proliferation resulting in uncontrolled growth and cellular transformation. Gene amplification and/or somatic mutations of the HER2/Neu (ErbB2) proto-oncogene occur in approximately 20% of breast cancers. A therapeutic strategy that has been used to block HER2 function is the small molecule tyrosine kinase inhibitor lapatinib. Using human mammary epithelial cells that overexpress HER2, we determined the anti-proliferative effect of lapatinib through measuring the total cell number and analyzing the cell cycle distribution. A mathematical model was used to interpret the experimental data. The model suggests that lapatinib acts as expected by slowing the transition through G1 phase. However, the experimental data indicated a previously unreported late cytotoxic effect, which was incorporated into the model. Both effects depend on the dosage of the drug, which shows saturation kinetics. The model separates quantitatively the cytostatic and cytotoxic effects of lapatinib and may have implications for preclinical studies with other anti-oncogene therapies.
    Full-text · Article · Feb 2007 · Theoretical Biology and Medical Modelling
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