Epidermal growth factor receptor (HER1) tyrosine kinase inhibitor ZD1839 (Iressa) inhibits HER2/neu (erbB2)-overexpressing breast cancer cells in vitro and in vivo.

Aberrrant signaling by the epidermal growth factor receptor [EGFR (HER1, erbB1)] and/or HER2/neu tyrosine kinases is present in a cohort of breast carcinomas. Because HER2 is constitutively phosphorylated in some breast tumors, we speculated that, in these cancers, transmodulation of HER2 may occur via EGFR signaling. To test this possibility, we examined the effect of EGFR-specific kinase inhibitors against the HER2-overexpressing human breast tumor lines BT-474, SKBR-3, MDA-361, and MDA-453. ZD1839 (Iressa) is an ATP-mimetic that inhibits the purified EGFR and HER2 kinases in vitro with an IC(50) of 0.033 and >3.7 microM, respectively. The specificity of ZD1839 against EGFR was confirmed in Rat1 fibroblasts transfected with EGFR or HER2 chimeric receptors activated by synthetic ligands without the interference of endogenous receptors. Treatment of all breast cancer cell lines (except MDA-453) with 1 microM ZD1839 almost completely eliminated HER2 phosphorylation. In contrast, the incorporation of [gamma-(32)P]ATP in vitro onto HER2 receptors isolated from BT-474 cells was unaffected by 1 microM ZD1839. EGFR is expressed by BT-474, SKBR-3, and MDA-361 but not by MDA-453 cells, suggesting that ZD1839-mediated inhibition of the EGFR kinase explained the inhibition of HER2 phosphorylation in vivo. In SKBR-3 cells, ZD1839 exhibited a greater growth-inhibitory effect than Herceptin, a monoclonal antibody against the HER2 ectodomain. In both SKBR-3 and BT-474 cells, treatment with ZD1839 plus Herceptin induced a greater apoptotic effect than either inhibitor alone. Finally, ZD1839 completely prevented growth of BT-474 xenografts established in nude mice and enhanced the antitumor effect of Herceptin. These data imply that EGFR tyrosine kinase inhibitors will be effective against HER2-overexpressing breast tumor cells that also express EGFR and support their use in combination with HER2 antibodies, such as Herceptin, against mammary carcinomas with high levels of the HER2 proto-oncogene.

Identification of a novel membrane protein, HP59, with therapeutic potential as a target of tumor angiogenesis.

CM101, a polysaccharide isolated from the culture medium of Group B streptococcus, a neonatal pathogen, targets pathological angiogenesis and inhibits tumor growth in mice and humans. CM101 also targets neonatal lung and adult sheep lung endothelial cells. A gene encoding a transmembrane protein that interacts with CM101 was isolated from a sheep lung endothelial cell cDNA library. The gene, termed sp55, encodes a 495-amino acid polypeptide. COS-7 cells transfected with a vector containing sp55 express the SP55 protein-bound CM101 in a concentration-dependent manner. Stably transfected CHO cells also bound CM101. The corresponding human gene, hp59, was isolated from a human fetal lung cDNA library and had a predicted identity to SP55 of 86% over 495 amino acids. HP59 protein was shown by immunohistochemistry to be present in the pathological tumor vasculature of the lung, breast, colon, and ovary, but not in the normal vasculature, suggesting that the protein may be critical to pathological angiogenesis. The hp59 gene and/or the HP59 protein was not expressed in a variety of normal tissues, but was significantly expressed in human fetal lung, consistent with the pathophysiology of Group B streptococcus infections in neonates. Mice immunized with HP59 and SP55 peptides showed significant attenuation of tumor growth. Immunization effectively inhibited both the tumor angiogenesis and vasculogenesis processes, as evidenced by lack of both HP59- and CD34-positive vessels. These results and the immunohistochemistry data suggest a therapeutic potential for the CM101 target protein HP59 both as a drug target and as a vaccine against pathoangiogenesis.

Tyrosine kinase inhibitors: rationale, mechanisms of action, and implications for drug resistance.

Tyrosine kinases play a role in normal cellular regulatory processes. However, aberrant tyrosine kinase activity can lead to cellular transformation and can be causally associated with tumor maintenance and progression. In the last few years, high-throughput screening and the use of combinatorial, computational, and medicinal chemistry have led to the identification of small molecules that compete with the adenosine triphosphate binding site of the catalytic domain of several oncogenic tyrosine kinases. Some of these compounds are highly specific to a single tyrosine kinase, while others can inhibit several homologous kinase pockets simultaneously. At a practical level, the relative promiscuity of these inhibitors against more than one oncogenic tyrosine kinase may have clinical merit as well as implications for host tissue toxicity. Many of these small molecules are in different stages of preclinical and clinical development against several solid tumors and will be discussed.

ErbB2/neu kinase modulates cellular p27(Kip1) and cyclin D1 through multiple signaling pathways.

It is well established that ErbB1 and ErbB2 can cooperate in mammary epithelial cell transformation. Therefore, to understand how ErbB1/ErbB2 signaling contributes to this process, we used the ErbB kinase inhibitor AG1478in ErbB2-dependent BT-474 and SKBR-3 human breast cancer cells. These cells overexpress ErbB2 and also display moderate levels of ErbB1. Treatment with AG1478 resulted in rapid ErbB2 dephosphorylation, reversible G(1) arrest, and interruption of constitutive mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Consequently, both MAPK-dependent transcription of cyclin D1 and phosphorylation of the cyclin-dependent kinase (Cdk) inhibitor p27 were inhibited. The inhibition of PI3K/Akt resulted in increased activity of glycogen synthase kinase-3beta, which phosphorylated cyclin D1, potentially reducing its steady-state levels. The loss of cyclin D1 reduced the amount of cyclin D1/Cdk4 complexes that can sequester p27 in the cytosol. This plus the reduced phosphorylation of p27 by MAPK enhanced the stability of p27 that associated with nuclear Cdk2 at high stoichiometry and inhibited its kinase activity. Antisense p27 oligonucleotides decreased p27 levels and abrogated the G(1) arrest induced by AG1478. Similarly, infection with an adenovirus encoding inducible cyclin D1 also counteracted the antiproliferative effect of AG1478. These data imply that: (a) modulation of both p27 and cyclin D1 are required for the growth arrest that results from blockade of the ErbB2 kinase; and (b) ErbB2 overexpressing cells use both MAPK and PI3K/Akt to modulate p27 and cyclin D1 and, hence, subvert the G(1)-to-S transition.

Cyclin-dependent kinase inhibitor p27(Kip1) is required for mouse mammary gland morphogenesis and function.

We have studied the role of the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) in postnatal mammary gland morphogenesis. Based on its ability to negatively regulate cyclin/Cdk function, loss of p27 may result in unrestrained cellular proliferation. However, recent evidence about the stabilizing effect of p27 on cyclin D1-Cdk4 complexes suggests that p27 deficiency might recapitulate the hypoplastic mammary phenotype of cyclin D1-deficient animals. These hypotheses were investigated in postnatal p27-deficient (p27(-/-)), hemizygous (p27(+/)-), or wild-type (p27(+/+)) mammary glands. Mammary glands from p27(+/)- mice displayed increased ductal branching and proliferation with delayed postlactational involution. In contrast, p27(-/-) mammary glands or wild-type mammary fat pads reconstituted with p27(-/-) epithelium produced the opposite phenotype: hypoplasia, low proliferation, decreased ductal branching, impaired lobuloalveolar differentiation, and inability to lactate. The association of cyclin D1 with Cdk4, the kinase activity of Cdk4 against pRb in vitro, the nuclear localization of cyclin D1, and the stability of cyclin D1 were all severely impaired in p27(-/-) mammary epithelial cells compared with p27(+/+) and p27(+/-) mammary epithelial cells. Therefore, p27 is required for mammary gland development in a dose-dependent fashion and positively regulates cyclin D-Cdk4 function in the mammary gland.

CM101 treatment overrides tumor-induced immunoprivilege leading to apoptosis.

CM101, a bacterial polysaccharide exotoxin produced by group B Streptococcus (GBS), also referred to as GBS toxin, has been shown to target pathological neovasculature and activate complement (C3), thereby inducing neovascularitis, infiltration of inflammatory cells, inhibition of tumor growth, and apoptosis in murine tumor models. Data from refractory cancer patients in a Phase I clinical trial with CM101 indicated a similar mechanism of tumor-targeted inflammation. To further our understanding of the mechanism of action of CM101 as an antitumor agent, we examined the role of the inflammatory response in inducing tumor apoptosis in a normal mouse and tumor-bearing mouse model. The i.v. infusion of CM101 into B16BL-6 melanoma tumor-bearing mice elevated p53 mRNA in circulating leukocytes as measured by reverse transcription-PCR, and immunohistochemistry demonstrated infiltration and sequestration of leukocytes. Whole tumor lysates from excised tumors exhibited an increase in binding to the murine p21(Waf1/Cip1) derived p53 DNA binding sequence compared with control whole tumor lysates, in which minimal or no DNA binding was observed. CM101 infusion led to elevated levels of Fas protein within the tumors as well as a decrease in the expression of fas ligand (fasL). Furthermore, tumors were apoptotic as determined by terminal deoxynucleotidyl transferase-mediated nick end labeling and DNA fragmentation assays. Collectively, these data suggest that CM101 up-regulates p53 in tumor-infiltrating leukocytes, initiating a loss of tumor immunoprivilege and consequently rendering the tumor sensitive to Fas/fasL-mediated apoptosis. CM101 induced loss of tumor immunoprivilege through changes in the expression of leukocyte p53, tumor Fas and fasL coupled with neovascularitis and leukocyte infiltration, constitutes a plausible molecular pathway for tumor reduction observed in cancer patients.

Hydrogen peroxide- and peroxynitrite-induced mitochondrial DNA damage and dysfunction in vascular endothelial and smooth muscle cells.

The mechanisms by which reactive species (RS) participate in the development of atherosclerosis remain incompletely understood. The present study was designed to test the hypothesis that RS produced in the vascular environment cause mitochondrial damage and dysfunction in vitro and, thus, may contribute to the initiating events of atherogenesis. DNA damage was assessed in vascular cells exposed to superoxide, hydrogen peroxide, nitric oxide, and peroxynitrite. In both vascular endothelial and smooth muscle cells, the mitochondrial DNA (mtDNA) was preferentially damaged relative to the transcriptionally inactive nuclear beta-globin gene. Similarly, a dose-dependent decrease in mtDNA-encoded mRNA transcripts was associated with RS treatment. Mitochondrial protein synthesis was also inhibited in a dose-dependent manner by ONOO(-), resulting in decreased cellular ATP levels and mitochondrial redox function. Overall, endothelial cells were more sensitive to RS-mediated damage than were smooth muscle cells. Together, these data link RS-mediated mtDNA damage, altered gene expression, and mitochondrial dysfunction in cell culture and reveal how RS may mediate vascular cell dysfunction in the setting of atherogenesis.

Up-regulation of base excision repair correlates with enhanced protection against a DNA damaging agent in mouse cell lines.

DNA polymerase beta is required in mammalian cells for the predominant pathway of base excision repair involving single nucleotide gap filling DNA synthesis. Here we examine the relationship between oxidative stress, cellular levels of DNA polymerase beta and base excision repair capacity in vitro , using mouse monocytes and either wild-type mouse fibroblasts or those deleted of the DNA polymerase beta gene. Treatment with an oxidative stress-inducing agent such as hydrogen peroxide, 3-morpholinosydnonimine, xanthine/xanthine oxidase or lipopolysaccharide was found to increase the level of DNA polymerase beta in both monocytes and fibroblasts. Base excision repair capacity in vitro , as measured in crude cell extracts, was also increased by lipopolysaccharide treatment in both cell types. In monocytes lipopolysaccharide-mediated up-regulation of the base excision repair system correlated with increased resistance to the monofunctional DNA alkylating agent methyl methanesulfonate. By making use of a quantitative PCR assay to detect lesions in genomic DNA we show that lipopolysaccharide treatment of fibroblast cells reduces the incidence of spontaneous DNA lesions. This effect may be due to the enhanced DNA polymerase beta-dependent base excision repair capacity of the cells, because a similar decrease in DNA lesions was not observed in cells deficient in base excision repair by virtue of DNA polymerase beta gene deletion. Similarly, fibroblasts treated with lipopolysaccharide were more resistant to methyl methanesulfonate than untreated cells. This effect was not observed in cells deleted of the DNA polymerase beta gene. These results suggest that the DNA polymerase beta-dependent base excision repair pathway can be up-regulated by oxidative stress-inducing agents in mouse cell lines.

Functional studies on the anti-pathoangiogenic properties of CM101.

Group B streptococcus (GBS) isolated from human neonates diagnosed with sepsis and respiratory distress produces a polysaccharide exotoxin (CM101) which has been previously described as GBS toxin. CM101 infused i.v. into tumor-bearing mice causes rapid tumor neovascularitis, infiltration of inflammatory cells, inhibition of tumor growth and tumor apoptosis. CM101 has successfully completed phase I studies in refractory cancer patients with very encouraging results. We have now demonstrated a mechanism of action for CM101. Using a normal mouse tumor model, we have examined tumor and normal tissues which were harvested at 0, 5, 15, 30 and 60min post-infusion of either CM101 or dextran. We present evidence that CM101 is rapidly (within the first 5min) bound to the tumor neovasculature. Complement is activated by the alternative pathway (C3) and leukocytes start to infiltrate the tumor within the first 5min. Through RT-PCR and immunohistochemical techniques, we demonstrate that proinflammatory cytokines, interleukin-6 and tumor necrosis factor (TNF)-alpha, are up-regulated in infiltrating leukocytes and TNF receptor 2 is up- regulated in the targeted tumor neovasculature. Combined, these events constitute possible explanations for the observed pathophysiology of tumor ablation.

Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress.

A significant amount of reactive oxygen species (ROS) is generated during mitochondrial oxidative phosphorylation. Several studies have suggested that mtDNA may accumulate more oxidative DNA damage relative to nuclear DNA. This study used quantitative PCR to examine the formation and repair of hydrogen peroxide-induced DNA damage in a 16.2-kb mitochondrial fragment and a 17.7-kb fragment flanking the beta-globin gene. Simian virus 40-transformed fibroblasts treated with 200 microM hydrogen peroxide for 15 or 60 min exhibited 3-fold more damage to the mitochondrial genome compared with the nuclear fragment. Following a 60-min treatment, damage to the nuclear fragment was completely repaired within 1.5 hr, whereas no DNA repair in the mitochondrion was observed. Mitochondrial function, as assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction, also showed a sharp decline. These cells displayed arrested-cell growth, large increases in p21 protein levels, and morphological changes consistent with apoptosis. In contrast, when hydrogen peroxide treatments were limited to 15 min, mtDNA damage was repaired with similar kinetics as the nuclear fragment, mitochondrial function was restored, and cells resumed division within 12 hr. These results indicate that mtDNA is a critical cellular target for ROS. A model is presented in which chronic ROS exposure, found in several degenerative diseases associated with aging, leads to decreased mitochondrial function, increased mitochondrial-generated ROS, and persistent mitochondrial DNA damage. Thus persistent mitochondrial DNA damage may serve as a useful biomarker for ROS-associated diseases.