The development of acquired resistance to ErbB2 tyrosine kinase inhibitors limits the clinical efficacy of this class of cancer therapeutics. Little is known about the mechanism(s) of acquired resistance to these agents. Here we establish a model of acquired resistance to N-{3-chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2 (methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (lapatinib), an inhibitor of ErbB2 and ErbB1 tyrosine kinases by chronically exposing lapatinib-sensitive ErbB2-overexpressing breast cancer cells to lapatinib, simulating the clinic where lapatinib is administered on a daily chronic basis. Analysis of baseline gene expression in acquired lapatinib-resistant and parental cells indicates estrogen receptor (ER) signaling involvement in the development of resistance. Using gene interference, we confirm that acquired resistance to lapatinib is mediated by a switch in cell survival dependence and regulation of a key antiapoptotic mediator from ErbB2 alone to codependence upon ER and ErbB2 rather than loss of ErbB2 expression or insensitivity of ErbB2 signaling to lapatinib. Increased ER signaling in response to lapatinib is enhanced by the activation of factors facilitating the transcriptional activity of ER, notably FOXO3a and caveolin-1. Importantly, we confirm that lapatinib induces ER signaling in tumor biopsies from patients with ErbB2-overexpressing breast cancers receiving lapatinib therapy. These findings provided the rationale for preventing the development of acquired resistance by simultaneously inhibiting both ER and ErbB2 signaling pathways. Establishing clinically relevant models of acquired resistance to ErbB2 kinase inhibitors will enhance therapeutic strategies to improve clinical outcomes for patients with ErbB2-overexpressing breast cancers.estrogen receptor ͉ lapatinib ͉ resistance A berrant activation of oncogenic tyrosine kinases and steroid receptors plays a key role in breast carcinogenesis. Overexpression or gene amplification of ErbB2, a member of the ErbB receptor tyrosine kinase family, occurs in 25-30% of breast cancers where it predicts for a poor clinical outcome (1). Consequently, therapies targeting ErbB2 represent an attractive strategy in breast cancer (2, 3). Trastuzumab, a humanized anti-ErbB2 monoclonal antibody, is an approved treatment for patients with ErbB2-overexpressing breast cancers (4). ErbB2 signaling can also be blocked using small-molecule tyrosine kinase inhibitors that compete with ATP for binding at the ErbB2 catalytic kinase domain. N- {3-chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2 (methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (lapatinib; GW572016), a potent reversible inhibitor of ErbB2 and ErbB1 tyrosine kinases is currently in Phase III clinical trials in breast and other carcinomas (5). Inhibition of ErbB2 tyrosine autophosphorylation by lapatinib abrogates downstream mitogenactivated protein kinase (MAPK)-Erk1͞2 and PI3K-Akt growth͞ survival signaling in ErbB2-overexpressing breast cancer cell lines, xen...

Here we show that ectopic expression of a kinase-active Plk3 (Plk3-A) induced apoptosis. In response to DNA damage, the kinase activity of Plk3 was rapidly increased in an ATMdependent manner, whereas that of Plk1 was markedly inhibited. Recombinant Plk3 phosphorylated in vitro a glutathione S-transferase fusion protein containing p53, but not glutathione S-transferase alone. Recombinant Plk1 also phosphorylated p53 but on residues that differed from those targeted by Plk3. Co-immunoprecipitation and pull-down assays demonstrated that Plk3 physically interacted with p53 and that this interaction was enhanced upon DNA damage. In vitro kinase assays followed by immunoblotting showed that serine 20 of p53 was a target of Plk3. Furthermore, expression of a kinase-defective Plk3 mutant (Plk3 K52R ) resulted in significant reduction of p53 phosphorylation on serine 20, which was correlated with a decrease in the expression of p21 and with a concomitant increase in cell proliferation. These results strongly suggest that Plk3 functionally links DNA damage to cell cycle arrest and apoptosis via the p53 pathway.

UvrABC excision nuclease (UvrA, UvrB, and In this communication using proteins purified to near homogeneity we demonstrate that helicase II alone has no effect on the initial rate of excision nuclease activity and only a small effect on its turnover. Similarly we find that pol I alone does not stimulate the ABC excision nuclease. However, the combination of the two causes the enzyme to turn over such that in a complete excision repair system (UvrABC excision nuclease, helicase II, pol I, DNA ligase) the rate of the removal of nucleotide adducts approaches 0.08 adduct per UvrABC excision nuclease complex per min. This value is in reasonable agreement with the 0.12-0.50 min-1 that can be calculated from the in vivo data published by several workers (5, 13-15). MATERIALS AND METHODSEnzymes. The UvrA, UvrB, and UvrC subunits of UvrABC excision nuclease were purified separately as described previously (1,16 Substrates. Radiolabeled or unlabeled pBR322 DNA was prepared by standard methods and superhelical DNA was purified through two successive ethidium bromide/CsCl density gradient centrifugations (19). The tritiated DNA used in the incision and filter binding assays had a specific activity of 1.5 x 105 cpm/,ug. The substrate for these assays was prepared by irradiating the DNA with 254-nm UV light that produced 10 lethal hits per molecule as measured by the transformation assay (19). The DNA used for the incision and filter binding assays contained 70-75% superhelical molecules. The substrate for the excision assay was prepared as described (20).Assays. The activity and turnover rate of UvrABC excision nuclease were measured by three separate assays: incision, excision, and filter binding. All the assays were conducted in a nucleotide-excision-repawr buffer which contained 50 mM Tris-HCI, pH 7.4/50 mM KCl/10 mM MgCl2/2 mM ATP/33 ,uM each of dATP, dGTP, dTTP, and dCTP/10 mM dithiothreitol/10% glycerol (vol/vol)/bovine serum albumin at 50 ,g/ml plus DNA and repair proteins at the amounts indicated. The incision assay measures the conversion of superhelical DNA to open circles and has been described elsewhere (19 6774The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.