Synergistic inhibition with a dual epidermal growth factor receptor/HER-2/neu tyrosine kinase inhibitor and a disintegrin and metalloprotease inhibitor

The ErbB family of receptors is overexpressed in numerous human tumors. Overexpression correlates with poor prognosis and resistance to therapy. Use of ErbB-specific antibodies to the receptors (Herceptin or Erbitux) or ErbB-specific small-molecule inhibitors of the receptor tyrosine kinase activity (Iressa or Tarceva) has shown clinical efficacy in several solid tumors. An alternative method of affecting ErbB-initiated tumor growth and survival is to block sheddase activity. Sheddase activity is responsible for cleavage of multiple ErbB ligands and receptors, a necessary step in availability of the soluble, active form of the ligand and a constitutively activated ligand-independent receptor. This sheddase activity is attributed to the ADAM (a disintegrin and metalloprotease) family of proteins. ADAM 10 is the main sheddase of epidermal growth factor (EGF) and HER-2/neu cleavage, whereas ADAM17 is required for cleavage of additional EGF receptor (EGFR) ligands (transforming growth factor-alpha, amphiregulin, heregulin, heparin binding EGF-like ligand). This study has shown that addition of INCB3619, a potent inhibitor of ADAM10 and ADAM17, reduces in vitro HER-2/neu and amphiregulin shedding, confirming that it interferes with both HER-2/neu and EGFR ligand cleavage. Combining INCB3619 with a lapatinib-like dual inhibitor of EGFR and HER-2/neu kinases resulted in synergistic growth inhibition in MCF-7 and HER-2/neu-transfected MCF-7 human breast cancer cells. Combining the INCB7839 second-generation sheddase inhibitor with lapatinib prevented the growth of HER-2/neu-positive BT474-SC1 human breast cancer xenografts in vivo. These results suggest that there may be an additional clinical benefit of combining agents that target the ErbB pathways at multiple points.     

Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2-overexpressing and trastuzumab-treated breast cancer cells

Lapatinib (GW572016) is a selective inhibitor of both epidermal growth factor receptor (EGFR) and HER-2 tyrosine kinases. Here, we explore the therapeutic potential of lapatinib by testing its effect on tumor cell growth in a panel of 31 characterized human breast cancer cell lines, including trastuzumab-conditioned HER-2-positive cell lines. We further characterize its activity in combination with trastuzumab and analyze whether EGFR and HER-2 expression or changes induced in the activation of EGFR, HER-2, Raf, AKT, or extracellular signal-regulated kinase (ERK) are markers of drug activity. We report that concentration-dependent antiproliferative effects of lapatinib were seen in all breast cancer cell lines tested but varied significantly between individual cell lines with up to 1,000-fold difference in the IC(50)s (range, 0.010-18.6 micromol/L). Response to lapatinib was significantly correlated with HER-2 expression and its ability to inhibit HER-2, Raf, AKT, and ERK phosphorylation. Long-term in vivo lapatinib studies were conducted with human breast cancer xenografts in athymic mice. Treatment over 77 days resulted in a sustained and significant reduction in xenograft volume compared with untreated controls. For the combination of lapatinib plus trastuzumab, synergistic drug interactions were observed in four different HER-2-overexpressing cell lines. Moreover, lapatinib retained significant in vitro activity against cell lines selected for long-term outgrowth (>9 months) in trastuzumab-containing (100 microg/mL) culture medium. These observations provide a clear biological rationale to test lapatinib as a single agent or in combination with trastuzumab in HER-2-overexpressing breast cancer and in patients with clinical resistance to trastuzumab.     

Mechanisms of tumor regression and resistance to estrogen deprivation and fulvestrant in a model of estrogen receptor-positive, HER-2/neu-positive breast cancer

HER-2/neu in breast cancer is associated with tamoxifen resistance, but little data exist on its interaction with estrogen deprivation or fulvestrant. Here, we used an in vivo xenograft model of estrogen receptor (ER)-positive breast cancer with HER-2/neu overexpression (MCF7/HER-2/neu-18) to investigate mechanisms of growth inhibition and treatment resistance. MCF7/HER-2/neu-18 tumors were growth inhibited by estrogen deprivation and with fulvestrant, but resistance developed in 2 to 3 months. Inhibited tumors had reductions in ER, insulin-like growth factor-I receptor (IGF-IR), phosphorylated HER-2/neu (p-HER-2/neu), and phosphorylated p42/44 mitogen-activated protein kinase (p-MAPK). p27 was increased especially in tumors sensitive to estrogen deprivation. Tumors with acquired resistance to these therapies had complete loss of ER, increased p-HER-2/neu, increased p-MAPK, and reduced p27. In contrast, IGF-IR and phosphorylated AKT (p-AKT) levels were markedly reduced in these resistant tumors. The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib, which can block EGFR/HER-2/neu signaling, significantly delayed the emergence of resistance to both estrogen deprivation and fulvestrant. Levels of p-MAPK and p-AKT decreased with gefitinib, whereas high ER levels were restored. Eventually, however, tumors progressed in mice treated with gefitinib combined with estrogen deprivation or fulvestrant accompanied again by loss of ER and IGF-IR, increased p-HER-2/neu, high p-MAPK, and now increased p-AKT. Thus, estrogen deprivation and fulvestrant can effectively inhibit HER-2/neu-overexpressing tumors but resistance develops quickly. EGFR/HER-2/neu inhibitors can delay resistance, but reactivation of HER-2/neu and signaling through AKT leads to tumor regrowth. Combining endocrine therapy with EGFR/HER-2/neu inhibitors should be tested in clinical breast cancer, but a more complete blockade of EGFR/HER-2/neu may be optimal.     

Chemoprevention of 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamster cheek pouch by topical application of a dual inhibitor of epidermal growth factor receptor (EGFR) and ErbB2 tyrosine kinases

Oral cancer is a common neoplasm worldwide with tobacco and alcohol being the major etiological factors contributing to its pathogenesis. Epidermal growth factor receptor (EGFR) and ErbB2 are known to be involved in the development of oral cancer with the former up-regulated in up to 90% human cases. The goal of this study was to evaluate the chemopreventive effects of a dual inhibitor of EGFR and ErbB2 tyrosine kinases, GW2974, in the 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster cheek pouch model. A short-term experiment (3-week topical DMBA followed by 1-week topical GW2974) was conducted to examine the effects of GW2974 on aberrant arachidonic acid (AA) metabolism and cell proliferation in the hamster oral epithelium. Topical application of 0.1 ml GW2974 (160 microM, three times a week) significantly reduced the levels of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), 5-, 12-, 15-hydroxyeicosatetraenoic acid (HETE), and cell proliferation (BrdU-labeling index). In a long-term post-initiation experiment (6-week topical DMBA followed by 18-week topical GW2974), GW2974 (4 mM and 8 mM) significantly inhibited the incidence, number and size of visible tumors. Under microscope, the numbers of oral lesions (hyperplasia, dysplasia, carcinoma) and the incidence of squamous cell carcinoma (SCC) were also significantly suppressed by GW2974. In summary, our study indicated that dual inhibition of EGFR and ErbB2 tyrosine kinases by GW2974 was effective in preventing oral carcinogenesis in DMBA-induced hamster cheek pouch model. GW2974 exerted its chemopreventive effects in part by suppressing aberrant AA metabolism.     

A small molecule pan-Bcl-2 family inhibitor, GX15-070, induces apoptosis and enhances cisplatin-induced apoptosis in non-small cell lung cancer cells

Purpose Overexpression of Bcl-2 family members as well as deregulated apoptosis pathways are known hallmarks of lung cancer. Non-small cell lung cancer (NSCLC) cells are typically resistant to cytotoxic chemotherapy and approaches that alter the balance between pro-survival and pro-death Bcl-2 family members have shown promise in preclinical models of NSCLC. Methods Here we evaluated the effects of a novel pan-Bcl-2 inhibitor GX15-070 on NSCLC survival and when combined with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors as well as traditional cytotoxic agents. GX15-070 is a small molecule agent that binds anti-apoptotic Bcl-2 proteins and interferes with their ability to interact with pro-apoptotic proteins. We evaluated the effect of GX15-070 and correlated the effect on EGFR status as well as Bcl-2 family protein expression. Results We show that GX15-070 can disrupt Mcl-1:Bak interactions in lung cancer cells. We identified differential sensitivity of a panel of lung cancer cells to GX15-070 and no clear relationship existed between EGFR status or Bcl-2 family protein expression and sensitivity to GX15-070. GX15-070 could induce apoptosis in a subset of lung cancer cell lines and this correlated with the effects on cell viability. GX15-070 combined with gefitinib was synergistic in a cell line dependent on EGFR for survival but GX15-070 could not reverse resistance to gefitinib in cell lines not dependent on EGFR for survival. Finally, we observed synergy between GX15-070 and cisplatin in NSCLC cells. Conclusions Based on these results, GX15-070 can trigger apoptosis in NSCLC cells and can enhance chemotherapy-induced death. These data suggest that clinical trials with GX15-070 in combination with cytotoxic chemotherapy are indicated.     

Decreased response to paclitaxel versus docetaxel in HER-2/neu transfected human breast cancer cells

Taxanes are effective in the treatment of metastatic breast cancer. Docetaxel has been shown to be more potent than paclitaxel in inducing bcl-2 phosphorylation and apoptosis and is clinically active in some paclitaxel-resistant breast tumors. HER-2/neu overexpression has been shown to correlate with resistance to hormonal therapy as well as chemotherapy. Using a HER-2/neu transfected MCF-7 human breast cancer cell line, we investigated the role of HER-2/neu overexpression on resistance to paclitaxel and docetaxel treatment. A control vector transfected MCF-7 human breast cancer cell line (MCF/neo) and a HER-2/neu transfected MCF-7 line (MCF/18) were treated with various concentrations of docetaxel or paclitaxel. Cell number was assessed using the MTT tetrazolium dye assay. In the control vector transfected MCF/neo cell line, paclitaxel and docetaxel gave similar dose-dependent growth inhibition ( p = 0.175). In HER-2/neu transfected MCF/18 cells, docetaxel treatment resulted in a dose-dependent inhibition similar to that seen in MCF/neo cells. Paclitaxel, however, gave significantly less growth inhibition than docetaxel in the HER-2/neu overexpressing MCF/18 cells (p = 0.0003). These data suggest that HER-2/neu overexpression may contribute to paclitaxel resistance. In contrast, the cytotoxic effects of docetaxel in these breast carcinoma cells are not affected by HER-2/neu expression. Therefore, docetaxel may be the preferred taxane therapy in HER-2/neu overexpressing breast tumors.     

Overexpression of ErbB2 induces invasion of MCF10A human breast epithelial cells via MMP-9

Metastasis is the principal cause of death from breast cancer. ErbB2 (HER-2/neu) has been identified as an important regulator of metastatic potential of breast cancer. The present study investigated the molecular mechanism underlying the role of ErbB2 in malignant phenotypic conversion of MCF10A human breast epithelial cells which originally have ‘normal’ cell character. Here we report that ErbB2 induces invasion and migration of MCF10A cells though up-regulation of matrix metalloproteinase (MMP)-9. We also observed a marked reduction of an epithelial cell marker, E-cadherin, and an induction of vimentin in ErbB2-MCF10A cells, suggesting that epithelial-mesenchymal transition may play a role in the ErbB2-induced invasion and migration of MCF10A cells. Overexpression of ErbB2 significantly activated p38 MAPK and Akt, while Raf-1/MEK/ERK pathway was not activated by ErbB2. Using pharmacological inhibitors, we further show that p38 MAPK and Akt signaling pathways are crucial for the ErbB2-induced MMP-9 up-regulation, invasion and migration of MCF10A cells. Given that ErbB2 is one of the most important oncogenes in human breast cancer and thus is an attractive therapeutic target, our findings may provide a molecular basis for the promoting role of ErbB2 in breast cancer progression.     

Tyrosine kinase inhibitor emodin suppresses growth of HER-2/neu-overexpressing breast cancer cells in athymic mice and sensitizes these cells to the inhibitory effect of paclitaxel.

Overexpression of the HER-2/neu proto-oncogene, which encodes the tyrosine kinase receptor p185neu, has been observed in tumors from breast cancer patients. We demonstrated previously that emodin, a tyrosine kinase inhibitor, suppresses tyrosine kinase activity in HER-2/neu-overexpressing breast cancer cells and preferentially represses transformation phenotypes of these cells in vitro. In the present study, we examined whether emodin can inhibit the growth of HER-2/neu-overexpressing tumors in mice and whether emodin can sensitize these tumors to paclitaxel, a commonly used chemotherapeutic agent for breast cancer patients. We found that emodin significantly inhibited tumor growth and prolonged survival in mice bearing HER-2/neu-overexpressing human breast cancer cells. Furthermore, the combination of emodin and paclitaxel synergistically inhibited the anchorage-dependent and -independent growth of HER-2/neu-overexpressing breast cancer cells in vitro and synergistically inhibited tumor growth and prolonged survival in athymic mice bearing s.c. xenografts of human tumor cells expressing high levels of p185neu. Both immunohistochemical staining and Western blot analysis showed that emodin decreases tyrosine phosphorylation of HER-2/neu in tumor tissue. Taken together, our results suggest that the tyrosine kinase activity of HER-2/neu is required for tumor growth and chemoresistance and that tyrosine kinase inhibitors such as emodin can inhibit the growth of HER-2/neu-overexpressing tumors in mice and also sensitize these tumors to paclitaxel. The results may have important implications in chemotherapy for HER-2/neu-overexpressing breast tumors.     

Acquired antiestrogen resistance in MCF-7 human breast cancer sublines is not accomplished by altered expression of receptors in the ErbB-family

Development of acquired resistance against antiestrogen treatment is a serious problem in human breast cancer, and knowledge of alterations resulting in resistance is important for selection of further treatment. To mimic the clinical situation we have established a series of MCF-7 human breast cancer cell lines by long term treatment with the antiestrogens tamoxifen, ICI 164,384, and ICI 182,780. Common for these cell lines is a decreased expression of the estrogen receptor (ER). In human breast cancer, lack of response to endocrine therapy is often associated with decreased expression of the estrogen receptor and increased expression of epidermal growth factor receptor (EGFR) and/or HER-2/neu (ErbB-2). Our antiestrogen resistant cell lines did not express altered levels of EGFR, HER-2/neu, ErbB-3, or ErbB-4. Estrogen and antiestrogen regulation of HER-2/neu expression was essentially similar in parent and resistant MCF-7 cells. Treatment with antibodies to HER-2/neu (Herceptin^TM) did not affect growth of MCF-7 cells or resistant cells, indicating that in this in vitro model system, acquired antiestrogen resistance does not emerge from activation of the HER-2/neu signaling pathway. In MCF-7 cells transfected with HER-2/neu and/or ErbB-3, overexpression alone did not result in resistance. However, addition of heregulinl-1 abolished the inhibitory activity of ICI 182,780 on both vector and HER-2/neu/ErbB-3 transfected MCF-7 cells, demonstrating that activation of the HER-2/neu receptor signaling pathway can override the growth inhibitory effect of ICI 182,780.     

Truncated ErbB2 receptor (p95ErbB2) is regulated by heregulin through heterodimer formation with ErbB3 yet remains sensitive to the dual EGFR/ErbB2 kinase inhibitor GW572016

The expression of the NH2 terminally truncated ErbB2 receptor (p95ErbB2) in breast cancer correlates with metastatic disease progression compared with the expression of full-length p185ErbB2. We now show that heregulin (HRG), but not EGF, stimulates p95ErbB2 phosphorylation in BT474 breast cancer cells. Furthermore, phospho-p95ErbB2 forms heterodimers with ErbB3, but not EGFR, while p185ErbB2 heterodimerizes with both EGFR and ErbB3. The predilection of p95ErbB2 to heterodimerize with ErbB3 provides an explanation for its regulation by HRG, an ErbB3 ligand. GW572016, a reversible small molecule inhibitor of EGFR and ErbB2 tyrosine kinases, inhibits baseline p95ErbB2 phosphorylation in BT474 cells and tumor xenografts. Inhibition of p95ErbB2, p185ErbB2, and EGFR phosphorylation by GW572016 resulted in the inhibition of downstream phospho-Erk1/2, phospho-AKT, and cyclin D steady-state protein levels. Increased phosphorylation of p95ErbB2 and AKT in response to HRG was abrogated to varying degrees by GW572016. In contrast, trastuzumab did not inhibit p95ErbB2 phosphorylation or the expression of downstream phospho-Erk1/2, phospho-AKT, or cyclin D. It is tempting to speculate that trastuzumab resistance may be mediated in part by the selection of p95ErbB2-expressing breast cancer cells capable of exerting potent growth and prosurvival signals through p95ErbB2-ErbB3 heterodimers. Thus, p95ErbB2 represents a target for therapeutic intervention, and one that is sensitive to GW572016 therapy.     

Combining lapatinib (GW572016), a small molecule inhibitor of ErbB1 and ErbB2 tyrosine kinases, with therapeutic anti-ErbB2 antibodies enhances apoptosis of ErbB2-overexpressing breast cancer cells

Antibodies and small molecule tyrosine kinase inhibitors targeting ErbB2 exhibit distinct, noncross resistant mechanisms of action. Here, apoptosis of ErbB2-overexpressing breast cancer cells was enhanced by combining lapatinib, an inhibitor of ErbB1 and ErbB2 tyrosine kinases, with anti-ErbB2 antibodies, including (i) trastuzumab, a humanized monoclonal antibody, and (ii) pAb, rabbit polyclonal antisera generated by vaccination with a human ErbB2 fusion protein. Treating ErbB2-overexpressing breast cancer cell lines with a relatively low concentration of lapatinib alone resulted in a minimal increase in tumor cell apoptosis with an associated decrease in steady-state protein levels of p-ErbB2, p-Akt, p-Erk1/2, and notably survivin, compared to baseline. Exposure to pAb alone reduced total ErbB2 protein, disrupting ErbB3 transactivation, leading to a marked inhibition of p-Akt; however, survivin protein levels remained unchanged and apoptosis only increased slightly. Treatment with trastuzumab alone had relatively little effect on survivin and apoptosis was unaffected. Combining lapatinib with either pAb or trastuzumab markedly downregulated survivin protein and enhanced tumor cell apoptosis. The association between the inhibition of survivin and enhanced apoptosis following the combination of ErbB2-targeted therapies provides a biological effect in order to identify therapeutic strategies that promote tumor cell apoptosis and might improve clinical response.     

双重酪氨酸激酶抑制剂Lapatinib治疗乳腺癌研究进展

Lapatinib是一种新型的双重酪氨酸激酶抑制剂(TKI),能够同时作用于表皮生长因子受体(EGFR)和HER-2两个靶点。Lapatinib的临床前期及临床研究结果表明,其对乳腺癌患者有效,尤其对曲妥珠单抗耐药的局部晚期和转移性乳腺癌患者显示出可喜的疗效。以Lapatinib为代表的TKI有望为乳腺癌的治疗开辟一条崭新的途径。     

Combining flavopiridol with various signal transduction inhibitors

Treatment of human tumors with a combination of chemotherapeutic agents results in improved response as well as the ability to use less toxic concentrations of the drugs. Recent phase I clinical trials with the cyclin-dependent kinase inhibitor, flavopiridol, have shown some promise in the treatment of a variety of human tumors. Because of the severe toxicity, however, the use of less toxic doses in combination with other antiproliferative agents would be desirable. The purpose of this study was to examine the effects of combining flavopiridol with several signal transduction inhibitors: the SC236 COX-2 inhibitor, a PKC kinase inhibitor and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor in a control vector transfected MCF-7 human breast cancer cell line (MCF/neo) and a HER-2/neu transfected MCF-7 cell line (MCF/18). Enhanced (better than that seen with either agent alone but not additive) growth inhibition was observed in both cell lines with the combination of flavopiridol and the PKC kinase inhibitor. The combination of flavopiridol and the SC236 COX-2 inhibitor resulted in an enhanced effect in the MCF/18 cell line and a synergistic effect in the MCF/neo cells. The combination of flavopiridol and LY294002 resulted in a synergistic effect in the MCF/18 cell line and an additive effect in the MCF/neo cells. These data suggest that combinations of flavopiridol and signal transduction inhibitors warrant further studies as treatments for breast tumors, and that HER-2/neu expression may influence the choice of inhibitor to combine with flavopiridol.     

Effect of the combination of docetaxel,zoledronic acid,and a COX-2 inhibitor on the growth of human breast cancer cell lines

Current trends in the treatment of human tumors are with drug combinations that result in improved responses as well as the ability to use less toxic concentrations of the drugs. Recent reports have shown that COX-2 inhibitors and taxanes are effective in the suppression of human tumor growth. The bisphosphonate, zoledronic acid, primarily used in the treatment of bone metastases, also inhibits proliferation and induces apoptosis in human breast and prostate carcinoma and multiple myeloma. HER-2/neu overexpression has been suggested as a mechanism for resistance to both hormonal therapy as well as chemotherapy. This study examines the effects of combining a cyclooxygenase-2 inhibitor with zoledronic acid and/or docetaxel in a HER-2/neu transfected and control human breast cancer cell line. All three compounds produced dose-dependent growth inhibition in both cell lines. The HER-2/neu transfected MCF/18 cells, however, were less sensitive to zoledronic acid than the control MCF/neo cells, 9% to 53% inhibition and 18% to 67%, respectively. Enhanced growth inhibition was observed in both cell lines with the combination of docetaxel and SC236 and the combination of docetaxel and zoledronic acid. The combination of SC236 with zoledronic acid also gave an enhanced inhibitory effect in the MCF/neo line. This combination, however, was additive in the HER-2/neu transfected MCF/18 cell line. The triple combination of SC236, zoledronic acid and/or docetaxel resulted in a small increase in growth inhibition compared to that seen with the double combinations.     

Does lapatinib,a small-molecule tyrosine kinase inhibitor,constitute a breakthrough in the treatment of breast cancer?

ErbB/HER receptor or its signal transduction pathway is an attractive therapeutic target for breast cancer. Lapatinib, an orally administered dual inhibitor of ErbB1 (EGFR) and ErbB2 (HER2) receptor tyrosine kinases has shown promising results for metastatic breast cancer (MBC). Lapatinib exhibited activity against trastuzumab-refractory MBC and showed an acceptable adverse event profile such as transient mild rash, diarrhea and nausea. The addition of lapatinib to capecitabine resulted in significantly prolonged time to progression. Large randomized trials using lapatinib following chemotherapy and surgery are ongoing for early stage HER2-overexpressing breast cancer. Various combinations with agents such as paclitaxel, aromatase inhibitors, or other molecular targeted agents are currently being investigated in clinical trials. If these approaches overcome the limitations of trastuzumab, lapatinib will become an effective treatment option for breast cancer in the near future.