Cyclin E is a G 1 cyclin essential for S-phase entry and has a profound role in oncogenesis. Previously this laboratory found that cyclin E is overexpressed and present in lower-molecular-weight (LMW) isoforms in breast cancer cells and tumor tissues compared to normal cells and tissues. Such alteration of cyclin E is linked to poor patient outcome. Here we report that the LMW forms of cyclin E are hyperactive biochemically and they can more readily induce G 1 -to-S progression in transfected normal cells than the full-length form of the protein can. Through biochemical and mutational analyses we have identified two proteolytically sensitive sites in the amino terminus of human cyclin E that are cleaved to generate the LMW isoforms found in tumor cells. Not only are the LMW forms of cyclin E functional, as they phosphorylate substrates such as histone H1 and GSTRb, but also their activities are higher than the full-length cyclin E. These nuclear localized LMW forms of cyclin E are also biologically functional, as their overexpression in normal cells increases the ability of these cells to enter S and G 2 /M. Lastly, we show that cyclin E is selectively cleaved in vitro by the elastase class of serine proteases to generate LMW forms similar to those observed in tumor cells. These studies suggest that the defective entry into and exit from S phase by tumor cells is in part due to the proteolytic processing of cyclin E, which generates hyperactive LMW isoforms whose activities have been modified from that of the full-length protein.In the past decade, new findings in the fields of cell biology and molecular genetics of cancer have revealed a deregulation of the cell cycle as a critical event for the onset of tumorigenesis. Progression through the cell cycle, the sequence of events between two cell divisions, is governed by the actions of positive and negative regulators in the eukaryotic cell. The mammalian cell cycle is positively regulated by heterodimeric complexes of stable cyclin-dependent kinases (CDKs) and unstable regulatory cyclin subunits (1, 51). Mitogenic stimuli result in the phosphorylation and thereby activation of cyclin-CDK complexes by CDK-activating kinase (17,22,35). The activated cyclin-CDK complexes in turn phosphorylate substrates such as the retinoblastoma protein (pRb) throughout the cell cycle (16,36,51).The connection between cyclins and cancer has been substantiated with G 1 -type cyclins (25-27, 55). Cyclin E, a G 1 cyclin which forms complexes with CDK2, is essential for Sphase entry (41, 47) and has a profound role in oncogenesis (29,31). In dividing cells, the expression of cyclin E increases to a maximum at the G 1 /S transition, with a peak expression level near the restriction point (13, 34). When coupled to CDK2, the active kinase follows cyclin D-CDK4 in progressively phosphorylating pRb, releasing it from members of the E2F family (15). As E2F is released it activates a number of S-phase genes, including cyclin E and E2F-1 (18,40). This state of readiness to enter S phase requi...
The ubiquitously expressed 14-
Purpose: Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma. Here, we examined the biological mechanisms and therapeutic potential of targeting a critical platinum resistance gene, ATP7B, using both in vitro and in vivo models. Experimental Design: Expression of ATP7A and ATP7B was examined in ovarian cancer cell lines by real-time reverse transcription-PCR and Western blot analysis. ATP7A and ATP7B gene silencing was achieved with targeted small interfering RNA (siRNA) and its effects on cell viability and DNA adduct formation were examined. For in vivo therapy experiments, siRNA was incorporated into the neutral nanoliposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). Results: ATP7A and ATP7B genes were expressed at higher levels in platinum-resistant cells compared with sensitive cells; however, only differences in ATP7B reached statistical significance. ATP7A gene silencing had no significant effect on the sensitivity of resistant cells to cisplatin, but ATP7B silencing resulted in 2.5-fold reduction of cisplatin IC 50 levels and increased DNA adduct formation in cisplatin-resistant cells (A2780-CP20 and RMG2). Cisplatin was found to bind to the NH 2 -terminal copper-binding domain of ATP7B, which might be a contributing factor to cisplatin resistance. For in vivo therapy experiments, ATP7B siRNA was incorporated into DOPC and was highly effective in reducing tumor growth in combination with cisplatin (70-88% reduction in both models compared with controls). This reduction in tumor growth was accompanied by reduced proliferation, increased tumor cell apoptosis, and reduced angiogenesis. Conclusion: These data provide a new understanding of cisplatin resistance in cancer cells and may have implications for therapeutic reversal of drug resistance.Ovarian cancer has the highest mortality rate among all gynecologic malignancies (1). Following cytoreductive surgery, treatment with paclitaxel and platinum has become a recommended approach for initial chemotherapy (2). Current combination chemotherapy regimens produce complete remission in up to 80% of patients with advanced ovarian cancer. However, despite these initial high response rates, most patients suffer relapse and require treatment with multiple subsequent chemotherapy regimens (3). Successful management of advanced or recurrent gynecologic malignancies is often difficult due to both
Vascular endothelial growth factor receptor (VEGFR) has recently been discovered on ovarian cancer cells, but its functional significance is unknown and is the focus of this study. By protein analysis, A2780‐par and HeyA8 ovarian cancer cell lines expressed VEGFR‐1 and HeyA8 A2774, and SKOV3ip1 expressed VEGFR‐2. By in situ hybridization (ISH), 85% of human ovarian cancer specimens showed moderate to high VEGFR‐2 expression, whereas only 15% showed moderate to high VEGFR‐1 expression. By immunofluorescence, little or no VEGFR‐2 was detected in normal ovarian surface epithelial cells, whereas expression was detected in 75% of invasive ovarian cancer specimens. To differentiate between the effects of tumor versus host expression of VEGFR, nude mice were injected with SKOV3ip1 cells and treated with either human VEGFR‐2 specific antibody (1121B), murine VEGFR‐2 specific antibody (DC101) or the combination. Treatment with 1121B reduced SKOV3ip1 cell migration by 68% (p < 0.01) and invasion by 72% (p < 0.01), but exposure to VEGFR‐1 antibody had no effect. Treatment with 1121B effectively blocked VEGF‐induced phosphorylation of p130Cas. In vivo treatment with either DC101 or 1121B significantly reduced tumor growth alone and in combination in the SKOV3ip1 and A2774 models. Decreased tumor burden after treatment with DC101 or 1121B correlated with increased tumor cell apoptosis, decreased proliferative index, and decreased microvessel density. These effects were significantly greater in the combination group (p < 0.001). We show functionally active VEGFR‐2 is present on most ovarian cancer cells. The observed anti‐tumor activity of VEGF‐targeted therapies may be mediated by both anti‐angiogenic and direct anti‐tumor effects. © 2008 Wiley‐Liss, Inc.
Recent progress in diagnostic tools allows many breast cancers to be detected at an early preinvasive stage. Thus, a better understanding of the molecular basis of early breast cancer progression is essential. Previously, we discovered that 14-3-3Z is overexpressed in >40% of advanced breast cancers, and this overexpression predicts poor patient survival. Here, we examined at what stage of breast disease 14-3-3Z overexpression occurs, and we found that increased expression of 14-3-3Z begins at atypical ductal hyperplasia, an early stage of breast disease. To determine whether 14-3-3Z overexpression is a decisive early event in breast cancer, we overexpressed 14-3-3Z in MCF10A cells and examined its effect in a three-dimensional culture model. We discovered that 14-3-3Z overexpression severely disrupted the acini architecture resulting in luminal filling. Proper lumen formation is a result of anoikis, apoptosis due to detachment from the basement membrane. We found that 14-3-3Z overexpression conferred resistance to anoikis. Additionally, 14-3-3Z overexpression in MCF10A cells and in mammary epithelial cells (MEC) from 14-3-3Z transgenic mice reduced expression of p53, which is known to mediate anoikis. Mechanistically, 14-3-3Z induced hyperactivation of the phosphoinositide 3-kinase/Akt pathway which led to phosphorylation and translocation of the MDM2 E3 ligase resulting in increased p53 degradation. Ectopic expression of p53 restored luminal apoptosis in 14-3-3Z-overexpressing MCF10A acini in threedimensional cultures. These data suggest that 14-3-3Z overexpression is a critical event in early breast disease, and down-regulation of p53 is one of the mechanisms by which 14-3-3Z alters MEC acini structure and increases the risk of breast cancer. [Cancer Res 2008;68(6):1760-7]
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