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.     

The tyrosine kinase inhibitor ZD1839 ("Iressa") inhibits HER2-driven signaling and suppresses the growth of HER2-overexpressing tumor cells

The epidermal growth factor receptor (EGFR) is commonly overexpressed in many human tumors and provides a new target for anticancer drug development. ZD1839 ("Iressa"), a quinazoline tyrosine kinase inhibitor selective for the EGFR, has shown good activity in preclinical studies and in the early phase of clinical trials. However, because it remains unclear which tumor types are the best targets for treatment with this agent, the molecular characteristics that correlate with tumor sensitivity to ZD1839 have been studied. In a panel of human breast cancer and other epithelial tumor cell lines, HER2-overexpressing tumors were particularly sensitive to ZD1839. Growth inhibition of these tumor cell lines was associated with the dephosphorylation of EGFR, HER2, and HER3, accompanied by the loss of association of HER3 with phosphatidylinositol 3-kinase, and down-regulation of Akt activity. These studies suggest that HER2-overexpressing tumors are particularly susceptible to the inhibition of HER family tyrosine kinase signaling and suggest novel strategies to treat these particularly aggressive tumors.     

Cooperative inhibitory effect of ZD1839 (Iressa) in combination with trastuzumab (Herceptin) on human breast cancer cell growth.

BACKGROUND: Co-expression of the epidermal growth factor receptor (EGFR) and of ErbB-2 is found in a subset of primary human breast cancer. MATERIALS AND METHODS: The antiproliferative effects of anti-EGFR and anti-ErbB-2 agents were evaluated using a monolayer assay. The effects of these agents on the activation of EGFR, ErbB-2, AKT and p42/p44 MAP kinases (MAPK) were investigated by western blot analysis. RESULTS: We found that both ZD1839 (Iressa), a specific EGFR tyrosine kinase inhibitor, and trastuzumab (Herceptin) (TRA), a humanized anti-ErbB-2 monoclonal antibody, were able to inhibit the growth of SK-Br-3 and BT-474 breast carcinoma cells, which express both EGFR and ErbB-2. Treatment of breast carcinoma cells with a combination of ZD1839 and TRA resulted in a synergistic inhibitory effect. Treatment of SK-Br-3 cells with ZD1839 produced a significant, dose-dependent reduction of the tyrosine phosphorylation of both EGFR and ErbB-2. Phosphorylation of MAPK and AKT were significantly reduced in SK-Br-3 cells following treatment with ZD1839, whereas treatment with TRA produced a reduction of AKT but not MAPK phosphorylation. Finally, treatment with ZD1839, but not with TRA, produced a significant increase in fragmented DNA in breast carcinoma cells. However, a more pronounced increase in the levels of fragmented DNA was observed following combined treatment with ZD1839 and TRA. CONCLUSIONS: These data suggest that combined treatment with drugs that target EGFR and ErbB-2 might result in an efficient inhibition of tumor growth in those breast carcinoma patients whose tumors co-express both receptors.     

ZD1839 (IRESSA), an EGFR-selective tyrosine kinase inhibitor, enhances taxane activity in bcl-2 overexpressing, multidrug-resistant MCF-7 ADR human breast cancer cells

Constitutive bcl-2 overexpression increases the tumorigenic and metastatic potential of doxorubicin-resistant, estrogen-independent, MCF-7 ADR human breast cancer cells. We evaluated the sensitivity to taxanes (paclitaxel, docetaxel and IDN 5109) of 2 bcl-2-overexpressing MCF-7 ADR clones and control neomycin-transfected MCF-7 ADR neo cells. The 2 bcl-2-overexpressing MCF-7 ADR clones were relatively resistant to all 3 taxanes, whereas the MCF-7 ADR neo cells were relatively resistant to paclitaxel and docetaxel, but sensitive to IDN 5109. We found that both MCF-7 ADR neo and bcl-2-overexpressing MCF-7 ADR clones express high levels of the epidermal growth factor receptor (EGFR) and its ligand, transforming growth factor-alpha (TGF-alpha). Therefore, we tested the growth inhibitory effect of ZD1839 (Iressa, AstraZeneca, Macclesfield, UK), an orally active, selective EGFR tyrosine kinase inhibitor (EGFR-TKI) that is in clinical development. ZD1839 inhibited the growth in soft agar of all 3 clones in a dose-dependent manner (IC(50) of approximately 0.1 microm). This effect was accompanied by a dose-dependent inhibition of EGFR tyrosine autophosphorylation and of the production of TGF-alpha, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). To determine whether the blockade of EGFR signaling might affect the sensitivity of bcl-2-overexpressing MCF-7 ADR cells to taxanes, cells were treated with ZD1839 in combination with paclitaxel, docetaxel or IDN 5109, and dose-dependent cooperative growth inhibition as well as apoptosis potentiation were observed. Combined treatment with IDN 5109 and ZD1839 also resulted in a significant inhibition of bcl-2 expression in bcl-2-overexpressing MCF-7 ADR cells. These results demonstrate the ability of ZD1839 to overcome taxane resistance in a model of hormone-independent, multidrug-resistant, human breast cancer.     

ZD1839 (Iressa), a novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, potently inhibits the growth of EGFR-positive cancer cell lines with or without erbB2 overexpression.

Overexpression of the growth factor receptors EGFR and erbB2 occurs frequently in several human cancers and is associated with aggressive tumour behaviour and poor patient prognosis. We have investigated the effects of ZD1839 (Iressa), a novel EGFR tyrosine kinase inhibitor, on the growth, in vitro and in vivo, of human cancer cell lines expressing various levels of EGFR and erbB2. Proliferation of EGFR-overexpressing A431 and MDA-MB-231 cells in vitro was potently inhibited (50%-70%) by ZD1839 with half-maximally effective doses in the low nanomolar range. In parallel, ZD1839 blocked autophosphorylation of EGFR and prevented activation of PLC-gamma 1, ERK MAP kinases and PKB/Akt by EGF. It also inhibited proliferation in EGFR(+) cancer cell lines overexpressing erbB2 (SKBr3, SKOV3, BT474) by between 20% and 80%, effects which correlated with inhibition of EGF-dependent erbB2 phosphorylation and activation of ERK MAP kinase and PKB/Akt in SKOV3 cells. Oral administration of ZD1839 inhibited the growth of MDA-MB-231 and SKOV3 tumours, established as xenografts in athymic mice, by 71% and 32%, respectively. Growth inhibition coincided with reduced proliferation but no change in apoptotic index. Collectively, these results show that ZD1839, at the doses studied, is a potent inhibitor of proliferation not only in cells overexpressing EGFR but also in EGFR(+) cells that overexpress erbB2.     

Effects of the EGFR/HER2 kinase inhibitor GW572016 on EGFR- and HER2-overexpressing breast cancer cell line proliferation, radiosensitization, and resistance

PURPOSE: Two members of the epidermal growth factor receptor family, EGFR and HER2, have been implicated in radioresistance in breast cancer and other malignancies. To gauge the potential clinical utility of targeting both EGFR and HER2 to control growth and radiosensitize human breast cancers, we examined the effect of a dual EGFR/HER2 inhibitor, GW572016, on the proliferation and radiation response of either EGFR- or HER2-overexpressing human breast cancer cell lines. METHODS AND MATERIALS: Primary human breast cancer cell lines that endogenously overexpress EGFR or HER2 and luminal mammary epithelial H16N2 cells stably transfected with HER2 were evaluated for the effect of GW572016 on inhibition of ligand-induced or constitutive receptor phosphorylation, proliferation, radiosensitization, and inhibition of downstream signaling. RESULTS: GW572016 inhibited constitutive and/or ligand-induced EGFR or HER2 tyrosine phosphorylation of all five cell lines, which correlated with the antiproliferative response in all but one cell line. GW572016 radiosensitized EGFR-overexpressing cell lines, but HER2-overexpressing cells were unable to form colonies after brief exposure to GW572016 even in the absence of radiation, and thus could not be evaluated for radiosensitization. One cell line was resistant to the antiproliferative and radiosensitizing effects of GW572016, despite receptor inhibition. Exploration of potential mechanisms of resistance in SUM185 cells revealed failure of GW572016 to inhibit downstream ERK and Akt activation, despite inhibition of HER2 phosphorylation. In contrast, sensitive HER2-overexpressing cell lines demonstrated inhibition of both ERK and Akt phosphorylation. CONCLUSION: GW572016 potently inhibits receptor phosphorylation in either EGFR- or HER2-overexpressing cell lines and has both antiproliferative and radiosensitizing effects. Resistance to GW572016 was not due to a lack of receptor inhibition, but rather with a lack of inhibition of ERK and Akt, suggesting that measurement of inhibition of crucial signaling pathways may better predict response than inhibition of receptor phosphorylation. The SUM185 cell line provides a valuable model for studying mechanisms of resistance of EGFR/HER2 inhibitor therapy.     

Cooperative cell-growth inhibition by combination treatment with ZD1839 (Iressa) and trastuzumab (Herceptin) in non-small-cell lung cancer

An important recent advance in anticancer therapy was the development of molecular-targeting drugs, such as the epidermal growth-factor receptor (EGFR)-targeting drug ZD1839 (Iressa) and the HER2-trageting anti-HER2 monoclonal antibody trastuzumab (Herceptin). ZD1839 and trastuzumab are reported to improve the therapeutic efficacy of treatment for non-small-cell lung cancer (NSCLC) and breast cancer, respectively, although the effectiveness of either drug alone is not satisfactory. NSCLC cells often express both EGFR and HER2. We therefore investigated whether a combination of ZD1839 and trastuzumab had an additive or synergistic antitumor effect. In culture ZD1839 inhibited the growth of four NSCLC cell lines (A549, NCI-H23, NCI-H727, and NCI-H661) that expressed various levels of EGFR, HER2, HER3, and HER4. A significant cytotoxic effect was observed when ZD1839 was combined with trastuzumab in A549 cells. However, this combination had no apparent effect in NCI-H23 cells. Significant G(1)-phase arrest, increased p27 expression and decreased cyclin E or D1 levels were detected in A549 cells treated with ZD1839 and trastuzumab. No significant effects were detected in NCI-H23 cells examined. The combination treatment significantly inhibited the phosphorylation of EGFR, HER2, retinoblastoma, extracellular signal-regulated kinase-1/2, and protein kinase B/Akt in A549 cells, but not in NCI-H23 cells. Our results indicated that increased levels of constitutive EGFR/HER2 heterodimers were formed in A549 cells in the presence of ZD1839, whereas no heterodimer formation was detected in NCI-H23 cells. We therefore suggest that combination treatment with ZD1839 and trastuzumab might have improved therapeutic efficacy against NSCLC cells expressing both EGFR and HER2.     

EGFR over-expression and activation in high HER2, ER negative breast cancer cell line induces trastuzumab resistance

HER2 is gene amplified or over-expressed in 20–25% of breast cancers resulting in elevated HER2 activation. Trastuzumab (Herceptin), a humanized monoclonal antibody, targets activated HER2 and is clinically effective in HER2-over-expressing breast cancers. However, despite prolonged survival, treated breast cancer patients develop resistance. Resistance to trastuzumab occurs upon inactivation of HER2 regulatory proteins or upon up-regulation of alternative receptors. In particular, elevated levels of EGFR, present in estrogen receptor (ER) positive, trastuzumab-resistant BT-474 xenografts caused, a trastuzumab-resistant phenotype (Ritter et al. Clin Cancer Res 13:4909–4919, 2007). However, the role of EGFR in acquired trastuzumab resistance in ER negative cell models is not well defined. In this study, SKBR3 cell line clones expressing EGFR were generated to examine the role of EGFR over-expression on trastuzumab sensitivity in an, ER-negative breast carcinoma cell line. A stable clone, SKBR3/EGFR (clone 4) expressing moderate levels of EGFR remained sensitive to trastuzumab, whereas a stable clone, SKBR3/EGFR (clone 5) expressing high levels of EGFR, became resistant to trastuzumab. Depletion of EGFR by EGFR small-interfering RNAs in the SKBR3/EGFR (clone 5) reversed trastuzumab resistance. However, the SKBR3/EGFR (clone 5) cell line remained sensitive to lapatinib, an EGFR/HER2 inhibitor. Biochemical analysis using co-immunoprecipitation and proximity-based quantitative VeraTag assays demonstrated that high levels of EGFR phosphorylation, EGFR/EGFR homo-dimerization, and EGFR/HER2 hetero-dimerization were present in the trastuzumab-resistant cells. We conclude that EGFR over-expression can mediate trastuzumab resistance in both ER positive and ER negative cells and hypothesize that a threshold level of EGFR, in the absence of autocrine ligand production, is required to induce the resistant phenotype.     

Genistein sensitizes inhibitory effect of tamoxifen on the growth of estrogen receptor-positive and HER2-overexpressing human breast cancer cells

Although tamoxifen (TAM) is used for the front-line treatment and prevention of estrogen receptor-positive (ER+) breast tumors, nearly 40% of estrogen-dependent breast tumors do not respond to TAM treatment. Moreover, the positive response is usually of short duration, and most tumors eventually develop TAM-resistance. Overexpression of HER2 gene is associated with TAM-resistance of breast tumor, and suppression of HER2 expression enhances the TAM activity. Soy isoflavone genistein has been shown to have anti-cancer activities and suppress expression of HER2 and ERalpha. The objective of this study was to test the hypothesis that genistein may sensitize the response of ER+ and HER2-overexpressing breast cancer cells to TAM treatment. The combination treatment of TAM and genistein inhibited the growth of ER+/HER2-overexpressing BT-474 human breast cancer cells in a synergistic manner in vitro. Determination of cellular markers indicated that this synergistic inhibitory effect might be contributed in part from combined effects on cell-cycle arrest at G(1) phase and on induction of apoptosis. Further determination of the molecular markers showed that TAM and genistein combination synergistically induced BT-474 cell apoptosis in part by synergistic downregulation of the expression of survivin, one of the apoptotic effectors, and downregulation of EGFR, HER2, and ERalpha expression. Our research may provide a novel approach for the prevention and/or treatment of TAM insensitive/resistant human breast cancer, and warrants further in vivo studies to verify the efficacy of genistein and TAM combination on the growth of ER+/HER2-overexpressing breast tumors and to elucidate the in vivo mechanisms of synergistic actions.     

Trastuzumab has preferential activity against breast cancers driven by HER2 homodimers

In breast cancer cells with HER2 gene amplification, HER2 receptors exist on the cell surface as monomers, homodimers, and heterodimers with EGFR/HER3. The therapeutic antibody trastuzumab, an approved therapy for HER2(+) breast cancer, cannot block ligand-induced HER2 heterodimers, suggesting it cannot effectively inhibit HER2 signaling. Hence, HER2 oligomeric states may predict the odds of a clinical response to trastuzumab in HER2-driven tumors. To test this hypothesis, we generated nontransformed human MCF10A mammary epithelial cells stably expressing a chimeric HER2-FKBP molecule that could be conditionally induced to homodimerize by adding the FKBP ligand AP1510, or instead induced to heterodimerize with EGFR or HER3 by adding the heterodimer ligands EGF/TGFα or heregulin. AP1510, EGF, and heregulin each induced growth of MCF10A cells expressing HER2-FKBP. Trastuzumab inhibited homodimer-mediated but not heterodimer-mediated cell growth. In contrast, the HER2 antibody pertuzumab, which blocks HER2 heterodimerization, inhibited growth induced by heregulin but not AP1510. Lastly, the HER2/EGFR tyrosine kinase inhibitor lapatinib blocked both homodimer- and heterodimer-induced growth. AP1510 triggered phosphorylation of Erk1/2 but not AKT, whereas trastuzumab inhibited AP1510-induced Erk1/2 phosphorylation and Shc-HER2 homodimer binding, but not TGFα-induced AKT phosphorylation. Consistent with these observations, high levels of HER2 homodimers correlated with longer time to progression following trastuzumab therapy in a cohort of patients with HER2-overexpressing breast cancer. Together, our findings confirm the notion that HER2 oligomeric states regulate HER2 signaling, also arguing that trastuzumab sensitivity of homodimers may reflect their inability to activate the PI3K (phosphoinositide 3-kinase)/AKT pathway. A clinical implication of our results is that high levels of HER2 homodimers may predict a positive response to trastuzumab.     

Human breast cancer cells selected for resistance to trastuzumab in vivo overexpress epidermal growth factor receptor and ErbB ligands and remain dependent on the ErbB receptor network.

PURPOSE: We have investigated mechanisms of acquired resistance to the HER2 antibody trastuzumab in BT-474 human breast cancer cells. EXPERIMENTAL DESIGN: BT-474 xenografts established in athymic nude mice were eliminated by trastuzumab. Continuous cell lines (HR for Herceptin resistant) were generated from tumors that recurred in the presence of continuous antibody therapy. RESULTS: The isolated cells behaved resistant to trastuzumab in culture as well as when reinjected into nude mice. They retained HER2 gene amplification and trastuzumab binding and were exquisitely sensitive to peripheral blood mononuclear cells ex vivo in the presence of the antibody. The HR cells exhibited higher levels of phosphorylated epidermal growth factor receptor (EGFR) and EGFR/HER2 heterodimers. Phosphorylation of HER2 in HR cells was inhibited by the EGFR tyrosine kinase inhibitors erlotinib and gefitinib. Gefitinib also inhibited the basal association of p85 with phosphorylated HER3 in HR cells. Both inhibitors as well as the dual EGFR/HER2 inhibitor, lapatinib, induced apoptosis of the HR cells in culture. Growth of established HR5 xenografts was inhibited by erlotinib in vivo. In addition, the HR cells overexpressed EGFR, transforming growth factor alpha, heparin-binding EGF, and heregulin RNAs compared with the parental trastuzumab-sensitive cells. CONCLUSIONS: These results are consistent with the inability of trastuzumab to block the heterodimerization of HER2 and suggest that amplification of ligand-induced activation of ErbB receptors is a plausible mechanism of acquired resistance to trastuzumab that should be investigated in primary mammary cancers.     

Furanodienone induces cell cycle arrest and apoptosis by suppressing EGFR/HER2 signaling in HER2-overexpressing human breast cancer cells

Purpose  

Overexpression of EGFR and HER2 is seen in breast cancers and results in poor prognosis and decreased patient survival. Clinically, EGFR and HER2 are effective therapeutic targets. The objective of this study is to investigate the in vitro effects of furanodienone, an active chemical component isolated from Rhizoma Curcumae, on the activation of EGFR/HER2 signaling, cell cycle, and apoptosis in HER2-overexpressing BT474 and SKBR3 cells.     

Inhibitory Effect of Polyclonal Antibodies Against HER3 Extracellular Subdomains on Breast Cancer Cell Lines

Objective: Human epidermal growth factor receptor 3 (HER3) is a unique member of the tyrosine kinase receptors with an inactive kinase domain and is the preferable dimerization partner for HER2 which lead to potent tumorigenic signaling. Methods: In this study, the expression plasmids coding for the human HER3 subdomains were transfected into CHO-K1 cells. Produced proteins were characterized by ELISA and SDS-PAGE. Rabbits were immunized and produced polyclonal antibodies (pAbs) that were characterized by ELISA, Immunoblotting and flowcytometry and their inhibitory effects were assessed by XTT on BT-474 and JIMT-1 breast cancer cell lines. Result: The recombinant subdomains were highly immunogenic in rabbits. The pAbs reacted with the recombinant subdomains as well as commercial HER3 and the native receptor on tumor cell membranes and could significantly inhibit growth of Trastuzumab sensitive (BT-474) and resistant (JIMT-1) breast cancer cell lines in vitro. Conclusion: It seems that HER3 extra cellular domains (ECD) induce a strong anti-tumor antibody response and may prove to be potentially useful for immunotherapeutic applications.     

A central role for HER3 in HER2-amplified breast cancer: implications for targeted therapy

Epidermal growth factor receptor (EGFR) and HER3 each form heterodimers with HER2 and have independently been implicated as key coreceptors that drive HER2-amplified breast cancer. Some studies suggest a dominant role for EGFR, a notion of renewed interest given the development of dual HER2/EGFR small-molecule inhibitors. Other studies point to HER3 as the primary coreceptor. To clarify the relative contributions of EGFR and HER3 to HER2 signaling, we studied receptor knockdown via small interfering RNA technology across a panel of six HER2-overexpressing cell lines. Interestingly, HER3 was as critical as HER2 for maintaining cell proliferation in most cell lines, whereas EGFR was dispensable. Induction of HER3 knockdown in the HER2-overexpressing BT474M1 cell line was found to inhibit growth in three-dimensional culture and induce rapid tumor regression of in vivo xenografts. Furthermore, preferential phosphorylation of HER3, but not EGFR, was observed in HER2-amplified breast cancer tissues. Given these data suggesting HER3 as an important therapeutic target, we examined the activity of pertuzumab, a HER2 antibody that inhibits HER3 signaling by blocking ligand-induced HER2/HER3 heterodimerization. Pertuzumab inhibited ligand-dependent morphogenesis in three-dimensional culture and induced tumor regression in the heregulin-dependent MDA-MB-175 xenograft model. Importantly, these activities of pertuzumab were distinct from those of trastuzumab, a monoclonal antibody currently used for treatment of HER2-amplified breast cancer patients. Our data suggest that inhibition of HER3 may be more clinically relevant than inhibition of EGFR in HER2-amplified breast cancer and also suggest that adding pertuzumab to trastuzumab may augment therapeutic benefit by blocking HER2/HER3 signaling.     

HER2 kinase domain mutation results in constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine kinase inhibitors

HER2/Neu gene mutations have been identified in lung cancer. Expression of a HER2 mutant containing a G776(YVMA) insertion in exon 20 was more potent than wild-type HER2 in associating with and activating signal transducers, phosphorylating EGFR, and inducing survival, invasiveness, and tumorigenicity. HER2(YVMA) transphosphorylated kinase-dead EGFR(K721R) and EGFR(WT) in the presence of EGFR tyrosine kinase inhibitors (TKIs). Knockdown of mutant HER2 in H1781 lung cancer cells increased apoptosis and restored sensitivity to EGFR TKIs. The HER2 inhibitors lapatinib, trastuzumab, and CI-1033 inhibited growth of H1781 cells and cells expressing exogenous HER2(YVMA). These data suggest that (1) HER2(YVMA) activates cellular substrates more potently than HER2(WT); and (2) cancer cells expressing this mutation remain sensitive to HER2-targeted therapies but insensitive to EGFR TKIs.     

Silencing of the HER2/neu gene by siRNA inhibits proliferation and induces apoptosis in HER2/neu-overexpressing breast cancer cells

In eukaryotes, double-stranded (ds) RNA induces sequence-specific inhibition of gene expression referred to as RNA interference (RNAi). We exploited RNAi to define the role of HER2/neu in the neoplastic proliferation of human breast cancer cells. We transfected SK-BR-3, BT-474, MCF-7, and MDA-MB-468 breast cancer cells with short interfering RNA (siRNA) targeted against human HER2/neu and analyzed the specific inhibition of HER2/neu expression by Northern and Western blots. Transfection with HER2/neu-specific siRNA resulted in a sequence-specific decrease in HER2/neu mRNA and protein levels. Moreover, transfection with HER2/neu siRNA caused cell cycle arrest at G0/G1 in the breast cancer cell lines SK-BR-3 and BT-474, consistent with a powerful RNA silencing effect. siRNA treatment resulted in an antiproliferative and apoptotic response in cells overexpressing HER2/neu, but had no influence in cells with almost no expression of HER2/neu proteins like MDA-MB-468 cells. These data indicate that HER2/neu function is essential for the proliferation of HER2/neu-overexpressing breast cancer cells. Our observations suggest that siRNA targeted against human HER2/neu may be valuable tools as antiproliferative agents that display activity against neoplastic cells at very low doses.     

MicroSPECT/CT imaging of co-expressed HER2 and EGFR on subcutaneous human tumor xenografts in athymic mice using 111In-labeled bispecific radioimmunoconjugates

Epidermal growth factor receptors (EGFR) form heterodimers with HER2 in breast cancer, and increased EGFR expression has been found in HER2-positive tumors resistant to trastuzumab (Herceptin). Our objective was to synthesize bispecific radioimmunoconjugates (bsRICs) that recognize HER2 and EGFR and evaluate their ability to image tumors in athymic mice that express one or both receptors by microSPECT/CT. Bispecific radioimmunoconjugates were constructed by conjugating maleimide-derivatized trastuzumab Fab fragments that bind HER2 to a thiolated form of EGF with an intervening 24 mer polyethylene glycol (PEG₂₄) spacer. Bispecific radioimmunoconjugates were derivatized with diethylenetriaminepentaacetic acid for labeling with ¹¹¹In. The ability of ¹¹¹In-bsRICs to bind HER2 or EGFR was determined in competition assays using cells expressing one or both receptors. Tumor and normal tissue uptake were examined in CD1 athymic mice bearing subcutaneous tumor xenografts that expressed HER2, EGFR, or both receptors, with or without pre-administration of Fab or EGF to determine specificity. HER2 and EGFR binding and displacement of binding by competitors were found for ¹¹¹In-bsICs. The highest uptake of ¹¹¹In-bsRICs [7.3 ± 3.5 %ID/g] in 231-H2N human breast cancer xenografts (HER2+/EGFR+) occurred at 48 h post-injection. Pre-administration of trastuzumab Fab decreased uptake in SK-OV-3 (HER2+/EGFR?) human ovarian cancer xenografts from 7.1 ± 1.2 to 2.4 ± 1.5 %ID/g. Pre-administration of excess EGF decreased uptake in MDA-MB-231 (HER2?/EGFR+) human breast cancer xenografts from 5.9 ± 0.5 to 2.0 ± 0.1 %ID/g. All tumors were imaged by microSPECT/CT. We conclude that ¹¹¹In-bsRICs composed of trastuzumab Fab and EGF exhibited specific binding in vitro to tumor cells displaying HER2 or EGFR, and were taken up specifically in vivo in tumors expressing one or both receptors, permitting tumor visualization by microSPECT/CT. These agents may ultimately be useful for imaging heterodimerized HER2-EGFR complexes since their bivalent properties permit more avid binding to these complexes.     

Enhancement of radiosensitivity by dual inhibition of the HER family with ZD1839 ("Iressa") and trastuzumab ("Herceptin")

PURPOSE: The aims of this study were twofold: (1) to examine the effects of dual inhibition of 2 members of the HER family, the epidermoid growth factor receptor (EGFR) and HER2/neu, by gefitinib (ZD1839) and trastuzumab on radiosensitivity; and (2) to explore the molecular mechanism of radiosensitization especially focusing on the survival signal transduction pathways by using A431 human vulvar squamous carcinoma cells expressing EGFR and HER2/neu. METHODS AND MATERIALS: The effects of inhibitors on the radiation-induced activation of EGFR and/or HER2/neu, and the intracellular proteins that are involved in their downstream signaling, were quantified by the Western blot. Radiosensitizing effects by the blockage of EGFR and/or HER2/neu were determined by a clonogenic assay. RESULTS: Radiation-induced activation of the EGFR and HER2/neu was inhibited with ZD1839 and/or trastuzumab. ZD1839 also inhibited the radiation-induced phosphorylation of HER2/neu. Radiation in combination with the HER family inhibitors inhibited the activation of Akt and MEK1/2, the downstream survival signaling of the HER family. ZD1839 enhanced radiosensitivity with a dose-modifying factor (DMF) (SF3) of 1.45 and trastuzumab did so with a DMF (SF3) of 1.11. Simultaneous blockade of EGFR and HER2/neu induced a synergistic radiosensitizing effect with a DMF (SF3) of 2.29. CONCLUSIONS: The present data suggest that a dual EGFR and HER2/neu targeting may have potential for radiosensitization in tumors in which both of these pathways are active.     

Dual inhibition of ErbB1 (EGFR/HER1) and ErbB2 (HER2/neu)

Targeting of epidermal growth factor receptor (EGFR) and HER2 is a proven anti-cancer strategy. However, heterodimerisation, compensatory 'crosstalk' and redundancy exist in the ErbB network, and there is therefore a sound scientific rationale for dual inhibition of EGFR and HER2. Trials of approved agents in combination, for example trastuzumab and cetuximab, are underway. There is also a new generation of small molecule tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mABs) that target two or more ErbB receptors. Lapatinib, a TKI of EGFR and HER2, has shown clinical benefit in trastuzumab refractory breast cancer and is poised for FDA approval. Other agents include BIBW-2992 and HKI-272, irreversible TKIs of EGFR and HER2, and pertuzumab, a heterodimerisation inhibitor of EGFR and HER2.