HER2-overexpressing Breast Cancer Cells Research Articles

Overcoming Trastuzumab Resistance in HER2-Overexpressing Breast Cancer Cells by Using a Novel Celecoxib-Derived Phosphoinositide-Dependent Kinase-1 Inhibitor

Although trastuzumab has been successfully used in patients with HER2-overexpressing metastatic breast cancer, resistance is a common problem that ultimately culminates in treatment failure. In light of the importance of Akt signaling in trastuzumab's antitumor action, we hypothesized that concurrent inhibition of Akt could enhance trastuzumab sensitivity and moreover reverse the resistant phenotype in HER2-positive breast cancer cells. Based on our finding that celecoxib mediates antitumor effects through the inhibition of phosphoinositide-dependent kinase-1 (PDK-1)/Akt signaling independently of cyclooxygenase-2 (COX-2), we used celecoxib as a scaffold to develop a COX-2-inactive PDK-1 inhibitor, 2-amino-N-[4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-acetamide (OSU-03012). Here, we investigated the effect of OSU-03012 on trastuzumab-mediated apoptosis in four breast cancer cell lines with different HER2 expression and trastuzumab-resistance status, including MDA-MB-231, BT474, SKBR3, and insulin-like growth factor-I receptor-overexpressing SKBR3 (SKBR3/IGF-IR). Effects of trastuzumab and OSU-03012, individually or in combination, on cell viability and changes in pertinent biomarkers including HER2 expression, phosphorylation of Akt, p27(kip1), and the PDK-1 substrate p70(S6K) were assessed. OSU-03012 alone was able to trigger apoptosis in all cell lines with equal potency (IC(50) = 3-4 microM), suggesting no cross-resistance with trastuzumab. Medium dose-effect analysis indicates that OSU-03012 potentiated trastuzumab's antiproliferative effect in HER2-positive cells, especially in SKBR3/IGF-IR cells, through the down-regulation of PDK-1/Akt signaling. This synergy, however, was not observed in HER2-negative MDA-MB-231 cells. This combination treatment represents a novel strategy to increase the efficacy of trastuzumab and to overcome trastuzumab resistance in the treatment of HER2-positive breast cancer.

Adding paclitaxel to trastuzumab enhances antibody-dependent cell-mediated cytotoxicity via increment of natural killer cells in patients with Her-2 overexpressing breast cancer

10548 Background: One of the mechanisms by which trastuzumab (TZ) inhibits the growth of Her-2 overexpressing breast cancer cells is the activation of a host tumor response via antibody-dependent cell-mediated cytotoxicity (ADCC). We questioned whether adding PTX to TZ enhances ADCC and also investigated kinetics of effector cells in ADCC. Methods: In ADCC, 5 cases with metastatic breast cancer receiving TZ (4mg/kg as a loading and 2 mg/kg weekly) with PTX (2qw, 100 mg/m2) were investigated. ADCC was analyzed by 51Cr releasing assay using SK-BR-3 cell line and white blood cells taken at the time of pretreatment, after 4 mg/kg of TZ, before/after 2 mg/kg of TZ, and before/after 2 mg/kg of TZ plus PTX. In of effector cell studies, we investigated fractions of natural killer (NK) cell, monocyte, and neutrophil taken at pre-administration and 10 minutes post-administration in 16 patients with Her-2 overexpressing breast cancer receiving weekly TZ and PTX (80 mg/m2). We defined NK cells as being both CD16+ (FcγRIII) and CD56+ and neutrophils as CD64+ (FcγRI) by flow cytometry. Results: Compared with pretreatment ADCC level, cytotoxicity were enhanced to 220% (median, ranging 30–259) after 4 mg/kg of TZ, 129% (78–210) after 2 mg/kg of TZ, and 148% (42–557) after the combination of TZ and PTX, whereas suppressed to 94% (48–163) after PTX alone. 2 weeks after the combination therapy, ADCC was significantly enhanced to 169% (113–257, p<0.05), compared with pre-treatment. In effector cells, NK cells increased in 131% (74–175, p<0.05) by TZ and in 224% (169–286, p<0.05) by the combination therapy. No significant changes were found in monocytes (98%, 50–160) and in neutrophils (100%, 50–160) by TZ alone, however monocytes decreased to 44% (34–50, p<0.05) and also neutrophils did to 49% (9–133) by the combination. Conclusions: Higher doses of TZ induced NK cell recruitment and the combination of TZ and PTX has a significant increase in recruitment and activation of NK cells. Adding PTX to TZ significantly enhanced ADCC via the rapid kinetics of NK cells. This may reflect that the combination of TZ and PTX has a strong synergistic effect than we expected in patients with Her-2 overexpressing breast cancer. [Table: see text]

Evaluation of a recombinant HER2 vaccine: Induction of specific antibodies, T-cells and preliminary activity in metastatic breast cancer patients

631 Background: We designed a vaccine to induce T cells able to recognize epitopes from HER2 and to engender a polyclonal antibody reponse. Methods: The vaccine is a recombinant HER2 protein, including its extra and part of its intra-cellular domains (ECD/ICD), combined with a potent immunologic adjuvant. Cohorts of patients with Stage II/III breast cancer (BC) received 20, 100 or 500 μg in the adjuvant setting. Treatment comprised of six vaccinations over 14 weeks, for the 500-μg dose, recall injections were given on weeks 34 and 38. The trial was extended to include an alternative vaccination schedule: 500 μg on days 0, 28 and 98. In an on-going trial, patients with metastatic BC treated in the first line setting are receiving the 500-μg treatment and being assessed for clinical response. Results: The vaccine was well tolerated, with no symptomatic cardiotoxicity. Antibody (Ab) response against ECD was dose-dependent, with 2/12, 9/14 and 14/15 immune responders in the respective cohorts after four vaccinations. Response was dose-related. Ab isotypes were analyzed in the 500 μg cohort: in 50% of patients, high levels of IgM (30–60%) against ECD were found after four vaccinations. The switch towards IgG was complete in all patients after six vaccinations. The efficacy of booster vaccinations was observed mainly in patients with low IgM after eight weeks. After two vaccinations, Ab titers on the alternative 500 μg vaccination schedule were as high as after four vaccinations utilizing the initial schedule. The anti-ECD antibodies in 11/15 patients (500 μg level) bound HER2-overexpressing breast-cancer cell lines. In sera from 2 patients tested thus far, the gene-expression resembled that of trastuzumab. Assays show that specific T cells were obtained; detailed analysis is continuing. Among metastatic patients, two showed evidence of tumor regression after vaccination. Conclusions: The HER2 vaccine was well tolerated and induced (dose-dependently) anti-ECD Ab that bound the HER2 receptor. Data suggest that the vaccine also induced specific T-cell immunity. The alternative vaccination schedule may increase the Ab titers. This data justifies further evaluation of this vaccine in the phase II/III setting. [Table: see text]

Identification of ADAM10 as a major source of HER2 ectodomain sheddase activity in HER2 overexpressing breast cancer cells

ALL AUTHORS: Phillip C.C. Liu, Xiangdong Liu, Yanlong Li, Maryanne Covington, Richard Wynn, Reid Huber, Milton Hillman, Gengjie Yang, Dawn Ellis, Cindy Marando, Kamna Katiyar, Jodi Bradley, Kenneth Abremski, Mark Stow, Mark Rupar, Jincong Zhuo, Yun-Long Li, Qiyan Lin, David Burns, Meizhong Xu, Colin Zhang, Ding-Quan Qian, Chunhong He, Vaqar Sharief, Lingkai Weng, Costas Agrios, Eric Shi, Brian Metcalf, Robert Newton, Steven Friedman, Wenqing Yaol, Peggy Scherlel, Gregory Hollis, Timothy C. Burn Overexpression and activating mutations of ErbB family members have been implicated in the development and progression of a variety of tumor types. Cleavage of the HER2 receptor by an as yet unidentified ectodomain sheddase has been shown to liberate the HER2 extracellular domain (ECD) leaving a fragment with constitutive kinase activity that can provide ligand-independent growth and survival signals to the cell. This process is clinically relevant since HER2 ECD serum levels in metastatic breast cancer patients are associated with a poorer prognosis. Thus, inhibition of the HER2 sheddase may provide a novel therapeutic approach for breast cancer. We describe the use of transcriptional profiling, pharmacological and in vitro approaches to identify the major source of HER2 sheddase activity. Real-time PCR was used to identify those ADAM family members which were expressed in HER2 shedding cell lines. siRNAs that selectively inhibited ADAM10 expression reduced HER2 shedding. In addition, we profiled over 1000 small molecules for in vitro inhibition of a panel of ADAM and MMP proteins; a positive correlation was observed only between ADAM10 inhibition and reduction of HER2 ECD shedding in a cell based assay. Finally, in vitro studies demonstrate that in combination with low doses of Herceptin, selective ADAM10 inhibitors decrease proliferation in HER2 overexpressing cell lines while inhibitors, that do not inhibit ADAM10, have no impact. These results are consistent with ADAM10 being a major determinant of HER2 shedding, the inhibition of which, may provide a novel therapeutic approach for treating a variety of cancers with active HER2 signaling.

Derepression of the Her-2 uORF is mediated by a novel post-transcriptional control mechanism in cancer cells

Transcripts harboring 5' upstream open reading frames (uORFs) are often found in genes controlling cell growth including receptors, oncogenes, or growth factors. uORFs can modulate translation or RNA stability and mediate inefficient translation of these potent proteins under normal conditions. In dysregulated cancer cells, where the gene product, for example Her-2 receptor, is overexpressed, post-transcriptional processes must exist that serve to override the inhibitory effects of the uORFs. The 5' untranslated region (UTR) of Her-2 mRNA contains a short uORF that represses translation of the downstream coding region. We demonstrate that in Her-2 overexpressing breast cancer cells, the 3' UTR of the Her-2 mRNA can override translational inhibition mediated by the Her-2 uORF. Within this 3' UTR, a translational derepression element (TDE) that binds to a 38-kDa protein was identified. These results define a novel biological mechanism in which translational control of genes harboring a 5' uORF can be modulated by elements in their 3' UTRs.

Synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid induces growth arrest in HER2-overexpressing breast cancer cells

HER2 overexpression is one of the most recognizable molecular alterations in breast tumors known to be associated with a poor prognosis. In the study described here, we explored the effect of HER2 overexpression on the sensitivity of breast cancer cells to the growth-inhibitory effects of 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), a synthetic triterpenoid, both in vitro and in vivo in a xenograft model of breast cancer. Both cell growth and colony formation in the soft agar assay, a hallmark of the transformation phenotype, were preferentially suppressed in HER2-overexpressing cell lines at low concentrations of CDDO, whereas growth-inhibitory effects at high concentrations did not correlate with the expression level of HER2. CDDO dose-dependently inhibited phosphorylation of HER2 in HER2-overexpressing cells and diminished HER2 kinase activity in vitro. CDDO induced the transactivation of the nuclear receptor peroxisome proliferator-activated receptor-gamma in both vector control and HER2-transfected MCF7 cells. Dose-response studies showed that the growth inhibition seen at lower concentrations of CDDO correlated with induction of the tumor suppressor gene caveolin-1, which is known to inhibit breast cancer cell growth. CDDO also reduced cyclin D1 mRNA and protein expression. In vivo studies with liposomally encapsulated CDDO showed complete abrogation of the growth of the highly tumorigenic MCF7/HER2 cells in a xenograft model of breast cancer. These findings provide the first in vitro and in vivo evidence that CDDO effectively inhibits HER2 tyrosine kinase activity and potently suppresses the growth of HER2-overexpressing breast cancer cells and suggest that CDDO has a therapeutic potential in advanced breast cancer.

C201 抗HER2抗体標識量子ドットを用いたマウス腫瘍内での単粒子イメージング

Quantum dot (Qdot) is nanometer-sized crystals which improved brightness, resistance against photobleaching compared with organic dyes and fluorescent proteins. Therefore, Qdot is thought to become new adjuncts of fluorescent bioprobes for medical applications, especially for cancer imaging 1, 2. We have used Qdot conjugated with, monoclonal anti-HER2 (human epithelial growth factor type 2 ) antibody (Trastuzumab) for molecular imaging of breast cancer cells. We utilized poly ethylene glycol (PEG) coated Qdot-Trastuzumab complex to label a cell membrane of the HER2 overexpressing breast cancer cells. Using this complex, we established the in vivo fluorescent cancer imaging method. After injection of the Qdot-Trastuzumab complex to the human breast cancer xenograft mouse model, the accumulation of Qdot to the tumor tissue was clearly observed at subcellular resolution with the original 3D intravital microscopic system. Furthermore, We report the tracking of single particles of quantum dots conjugated with trastuzumab in living mice. This suggests that we can eventually develop a novel cancer imaging and drug tracking system.

Conformational HER-2/neu B-cell Epitope Peptide Vaccine Designed to Incorporate Two Native Disulfide Bonds Enhances Tumor Cell Binding and Antitumor Activities

Cancer vaccines designed to elicit an antibody response that target antigenic sites on a tumor antigen must closely mimic the three-dimensional structure of the corresponding region on the antigen. We have designed a complex immunogen derived from the extracellular domain of human HER-2/neu-(626-649) that represents a three-dimensional epitope. We have successfully introduced two disulfide bonds into this sequence, thereby recapitulating the natural disulfide pairings observed in the native protein. To evaluate the immunogenicity of the doubly cyclized disulfide-linked peptide versus the free uncyclized peptide we examined the induction of antibody responses in both inbred and outbred mice strains, with both constructs eliciting high titered antibodies. The disulfide-paired specific antibodies exhibited enhanced cross-reactivity to HER-2/neu expressed on BT-474 cell line as determined by flow cytometry. The antitumor activities of the disulfidepaired specific antibodies did not improve the in vitro growth inhibition of human breast cancer cells overexpressing HER-2, but showed superior antitumor responses in the context of ADCC and interferon-gamma induction. Inbred mice (FVB/n) vaccinated with the disulfide-paired epitope exhibited a statistically significant reduction in the development of exogenously administered tumors in vivo compared with mice receiving either the free uncyclized or the promiscuous T-cell epitope (MVF) control peptide (p = 0.001). This study demonstrates the feasibility and importance of designing conformational epitopes that mimic the tertiary structure of the native protein for eliciting biologically relevant anti-tumor antibodies. Such approaches are a prerequisite to the design of effective peptide vaccines.

Antitumor Agents. 239. Isolation, Structure Elucidation, Total Synthesis, and Anti-Breast Cancer Activity of Neo-tanshinlactone from Salvia miltiorrhiza

Neo-tanshinlactone (1) was isolated and synthesized for the first time and evaluated in vitro against several human cancer cell lines. Compound 1 showed significant inhibition against two ER+ human breast cancer cell lines and was 10-fold more potent and 20-fold more selective as compared to tamoxifen citrate. Compound 1 also potently inhibited an ER-, HER-2 overexpressing breast cancer cell line. Therefore, this novel compound merits further development as an anti-breast cancer drug candidate.

The HER2/Grb2/Akt pathway regulates the DNA binding activity of AP-1 in breast cancer cells

We showed that the HER2/Grb2/Akt pathway induces all-trans retinoic acid (ATRA) resistance in breast cancer cells by suppressing the DNA binding activity of retinoic acid receptors (RAR). AP-1 activation was shown to induce ATRA resistance. Here, we determined whether AP-1 binding activity is correlated with ATRA resistance in HER2-overexpressing cells. Inhibition of HER2/Grb2/Akt decreased AP-1 binding activity in HER2-transfected cells, but increased AP-1 activity in cells that are naturally HER2-overexpressing. Since HER2/Grb2/Akt inhibition sensitized both cell types to ATRA, our results indicate that, unlike RAR, AP-1 binding activity is not correlated with ATRA sensitivity in HER2-overexpressing breast cancer cells.

319 Factors that govern the cell death response induced by inhibition of the molecular chaperone heat shock protein 90

The ginger rhizome is rich in bioactive compounds, including [6]-gingerol, [8]-gingerol, and [10]-gingerol; however, to date, most research on the anti-cancer activities of gingerols have focused on [6]-gingerol. In this study, we compared [10]-gingerol with [8]-gingerol and [6]-gingerol in terms of their ability to inhibit the growth of human and mouse mammary carcinoma cells. A colorimetric assay based on the enzymatic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide revealed that [10]-gingerol was more potent than [6]-gingerol and at least as potent as [8]-gingerol for the inhibition of triple-negative human (MDA-MB-231, MDA-MB-468) and mouse (4T1, E0771) mammary carcinoma cell growth. Further investigation of [10]-gingerol showed that it suppressed the growth of estrogen receptor-bearing (MCF-7, T47D) and HER2-overexpressing (SKBR3) breast cancer cells. The inhibitory effect of [10]-gingerol on the growth of MDA-MB-231 cells was associated with a reduction in the number of rounds of cell division and evidence of S phase-cell cycle arrest, as well as induction of apoptosis due to mitochondrial outer membrane permeabilization and the release of proapoptotic mitochondrial cytochrome c and SMAC/DIABLO into the cytoplasm. Surprisingly, killing of MDA-MB-231 cells by [10]-gingerol was not affected by a pan-caspase inhibitor (zVAD-fmk) or an anti-oxidant (N-acetylcysteine), suggesting that the cytotoxic effect of [10]-gingerol did not require caspase activation or the accumulation of reactive oxygen species. These findings suggest that further investigation of [10]-gingerol is warranted for its possible use in the treatment of breast cancer.

Enhancement of the p27Kip1‐mediated antiproliferative effect of trastuzumab (Herceptin) on HER2‐overexpressing tumor cells

The oncogenic activity of the overexpressed HER2 tyrosine kinase receptor requires its localization in the plasma membrane. The antitumor effect of anti-HER2 antibodies (Abs) is mainly dependent on receptor downregulation and comprises p27Kip1-mediated G1 cell cycle arrest. However, one major limitation of anti-HER2 therapy is the reversibility of tumor growth inhibition after discontinuation of treatment caused by the mitogenic signaling associated with cell surface receptor re-expression. We found that the level of p27Kip1 upregulation, inhibition of Cdk2 activity and magnitude of G1 arrest induced by the humanized Ab trastuzumab (Herceptin, HCT) on BT474 and SKBr3 HER2-overexpressing breast cancer cells correlates with the level of cell surface receptor. Thus, continuous exposure of cells to HCT for 72 hr results in downregulation of the cell surface receptor and a concurrent increase in the level of p27Kip1 protein. Discontinuation of Ab exposure after the first 8 hr results in failure to upregulate p27Kip1 and arrest of cell cycle progression. We show that the lysosomotropic amine chloroquine (CQ) augments receptor internalization in HER2-overexpressing cells either pretreated or continuously treated with HCT and leads to an increased and sustained inhibitory effect. The enhanced CQ-dependent loss of functional HER2 from the cell surface resulted in sustained inactivation of the serine/threonine kinase Akt, upregulation of p27Kip1 protein and inhibition of cyclin E/Cdk2 activity. Potentiation of the inhibitory effect of HCT by CQ was directly related to loss of HER2 from the plasma membrane since prevention of Ab-mediated receptor endocytosis by engagement of the receptor with immobilized HCT abrogated the effect of CQ.

Rational combinations of trastuzumab with chemotherapeutic drugs used in the treatment of breast cancer.

Trastuzumab, a humanized anti-HER2 antibody, increases the clinical benefit of first-line chemotherapy in patients with metastatic breast cancers that overexpress HER2. We characterized interactions between trastuzumab and chemotherapeutic agents commonly used in the treatment of breast cancer. Multiple drug effect/combination index isobologram analysis was used to study the efficacy of chemotherapeutic drug plus trastuzumab combinations tested against four HER2-overexpressing breast cancer cell lines (SK-BR-3, BT-474, MDA-MB-361, and MDA-MB-453). Combination index values were derived from parameters of the median effect plots, and statistical tests were used to determine whether the mean combination index values at multiple effect levels were statistically significantly different from a combination index value of 1.0. Values less than 1.0 indicate synergistic interactions, values greater than 1.0 indicate antagonistic interactions, and values equal to 1.0 indicate additive interactions. At a wide range of clinically achievable drug concentrations, synergistic interactions were observed in all four breast cancer cell lines for trastuzumab plus carboplatin (mean combination index values ranged from 0.32 [P<.001] to 0.53 [P<.001]), 4-hydroxycyclophosphamide (mean combination index values ranged from 0.38 [P<.001] to 0.73 [P =.010]), docetaxel (mean combination index values ranged from 0.30 [P<.001] to 0.62 [P<.001]), and vinorelbine (mean combination index values ranged from 0.24 [P<.001] to 0.78 [P<.034]). Additive interactions were observed in all four cell lines with trastuzumab plus doxorubicin, epirubicin, and paclitaxel. Interactions between trastuzumab and gemcitabine were synergistic at low concentrations of gemcitabine and antagonistic at high concentrations. A synergistic interaction was observed with a three-drug combination of docetaxel plus carboplatin plus trastuzumab in SK-BR-3 cells (mean combination index value = 0.09; P<.001). Consistent synergistic interactions of trastuzumab plus carboplatin, 4-hydroxycyclophosphamide, docetaxel, or vinorelbine across a wide range of clinically relevant concentrations in HER2-overexpressing breast cancer cells indicate that these are rational combinations to test in human clinical trials.

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.

In vitro comparative evaluation of trastuzumab (Herceptin®) combined with paclitaxel (Taxol®) or docetaxel (Taxotere®) in HER2-expressing human breast cancer cell lines

BackgroundTrastuzumab (Herceptin®) has clinical indication in association with paclitaxel (Taxol®) for the treatment of human epidermal growth factor receptor 2 (HER2)-expressing breast cancer. Synergistic interactions have been reported with taxane derivatives in HER2-expressing breast cancer cells. However, no direct comparison of the potential interest in combining trastuzumab with either paclitaxel or docetaxel (Taxotere®) has been reported. Materials and methodsThe present study was designed to evaluate in a comparative way the interaction of trastuzumab with paclitaxel or docetaxel in HER2-overexpressing human breast cancer cell lines. HER2 expression was documented in MCF-7, MDA-MB453 and SK-BR3 cell lines using immunocytochemistry with purified mouse anti-human monoclonal antibody. Cytotoxicity assays were performed using the sulforhodamine B assay and in vitro interactions between trastuzumab and taxanes were analyzed using the median-effect principle. ResultsTrastuzumab cytotoxicity was confirmed to be directly related to HER2 expression level. At the IC50, the combination of trastuzumab with either paclitaxel or docetaxel led to synergism in all cell lines. However, considering mean values calculated in the IC30–IC70 range of concentrations, trastuzumab interacted additively with docetaxel in SK-BR3 and MDA-MB453 cell lines while additive and synergistic interactions were achieved with paclitaxel in SK-BR3 and MDA-MB453, respectively. On the same basis, trastuzumab yielded synergistic interaction with both taxanes in the MCF-7 cell line. ConclusionsThe present study shows that at least additive interactions are observed when trastuzumab is combined with either paclitaxel or docetaxel in weak to moderate or more than moderate HER2-expressing cells. Some interesting results were achieved in cells displaying weak HER2 expression which could suggest some further potential interest in trastuzumab.

External control of Her2 expression and cancer cell growth by targeting a Ras-linked coactivator.

Overproduction of the Her2 oncoprotein has been found in approximately 30% of breast tumors, and patients who have Her2 excesses typically have more aggressive disease. Here we show that the expression of the Her2 gene can be decreased by inhibiting the interaction of the two cancer-linked proteins, DRIP130/CRSP130/Sur-2 (a Ras-linked subunit of human mediator complexes) and ESX (an epithelial-restricted transcription factor). Disruption of the interaction by a short cell-permeable peptide reduced the expression of the Her2 gene and specifically impaired the growth and viability of Her2-overexpressing breast cancer cells. The association of ESX with DRIP130 is mediated by a small hydrophobic face of an 8-aa helix in ESX, suggesting a therapeutic approach to incapacitating the Her2 gene by small organic molecules.

Combined biological therapy of breast cancer using monoclonal antibodies directed against HER2/neu protein and vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is one of the most important endothelial mitogens involved in the development and differentiation of the vascular system. Vascular endothelial growth factor is a highly conserved, homodimeric glycoprotein with multiple isoforms. The most abundant isoform, VEGF165, binds to vascular endothelial growth factor receptors-1 (Flt-1) and 2 (KDR/Flk-1) with picomolar affinity. Recently, correlation between microvessel density and engineered expression of VEGF in human breast xenografts was observed. A role of VEGF in breast cancer progression is evident from clinical studies showing elevated serum VEGF in invasive breast cancers. Vascular endothelial growth factor in breast tumor cytosols is correlated with microvessel density, and VEGF165 content correlates with disease-free and overall survival in primary breast cancers. Preliminary data indicate a transcriptional upregulation of VEGF in HER2-overexpressing breast cancer cells. We hypothesize that the upregulation of VEGF in HER2-overexpressing breast cancers contributes to the aggressive phenotype observed in HER2-positive cases and that the "angiogenic switch" associated with HER2 can be attenuated by trastuzumab. Although tumor angiogenesis in breast cancer is complex, the VEGF/vascular endothelial growth factor receptor (VEGFR) system provides a useful model for testing new angiogenesis inhibitors that target this pathway. The VEGF/VEGFR system provides a number of opportunities for therapeutic intervention in breast cancer. Understanding the biology of this system is paramount to fully exploiting VEGF as a therapeutic target in breast cancer. We hypothesize that new therapeutic molecules targeting VEGF and/or its receptors, such as recombinant humanized monoclonal anti-VEGF antibody (rhuMAb VEGF), may have unique activity against HER2-overexpressing breast cancers.

Ansamycin antibiotics inhibit Akt activation and cyclin D expression in breast cancer cells that overexpress HER2.

Ansamycin antibiotics, such as 17-allylaminogeldanamycin (17-AAG), bind to Hsp90 and regulate its function, resulting in the proteasomal degradation of a subset of signaling proteins that require Hsp90 for conformational maturation. HER2 is a very sensitive target of these drugs. Ansamycins cause RB-dependent G1 arrest that is associated with loss of D-cyclins via a PI3 kinase, Akt dependent pathway. Downregulation of D-cyclin was due, in part, to loss of Akt expression in response to drug. Moreover, in HER2 overexpressing breast cancer cells, 17-AAG caused rapid inhibition of Akt activity prior to any change in Akt protein. Ansamycins caused rapid degradation of HER2 and a concomitant loss in HER3 associated PI3 kinase activity. This led to a loss of Akt activity, dephosphorylation of Akt substrates, and loss of D-cyclin expression. Introduction into cells of a constitutively membrane bound form of PI3 kinase prevented the effects of the drug on Akt activity and D-cyclins. Thus, in breast cancer cells with high HER2, Akt activation by HER2/HER3 heterodimers is required for D-cyclin expression. In murine xenograft models, non-toxic doses of 17-AAG markedly reduced the expression of HER2 and phosphorylation of Akt and inhibited tumor growth. Thus, pharmacological inhibition of Akt activation is achievable with ansamycins and may be useful for the treatment of HER2 driven tumors.

Combined biological therapy of breast cancer using monoclonal antibodies directed against HER2/ neu protein and vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is one of the most important endothelial mitogens involved in the development and differentiation of the vascular system. Vascular endothelial growth factor is a highly conserved, homodimeric glycoprotein with multiple isoforms. The most abundant isoform, VEGF165, binds to vascular endothelial growth factor receptors-1 (Flt-1) and 2 (KDR/Flk-1) with picomolar affinity. Recently, correlation between microvessel density and engineered expression of VEGF in human breast xenografts was observed. A role of VEGF in breast cancer progression is evident from clinical studies showing elevated serum VEGF in invasive breast cancers. Vascular endothelial growth factor in breast tumor cytosols is correlated with microvessel density, and VEGF165 content correlates with disease-free and overall survival in primary breast cancers. Preliminary data indicate a transcriptional upregulation of VEGF in HER2-overexpressing breast cancer cells. We hypothesize that the upregulation of VEGF in HER2-overexpressing breast cancers contributes to the aggressive phenotype observed in HER2-positive cases and that the “angiogenic switch” associated with HER2 can be attenuated by trastuzumab. Although tumor angiogenesis in breast cancer is complex, the VEGF/vascular endothelial growth factor receptor (VEGFR) system provides a useful model for testing new angiogenesis inhibitors that target this pathway. The VEGF/VEGFR system provides a number of opportunities for therapeutic intervention in breast cancer. Understanding the biology of this system is paramount to fully exploiting VEGF as a therapeutic target in breast cancer. We hypothesize that new therapeutic molecules targeting VEGF and/or its receptors, such as recombinant humanized monoclonal anti-VEGF antibody (rhuMAb VEGF), may have unique activity against HER2-overexpressing breast cancers. Semin Oncol 29 (suppl 11):29-37. Copyright 2002, Elsevier Science (USA). All rights reserved.

Dynamic chromatin remodeling on the HER2 promoter in human breast cancer cells

Deregulation of the HER2 oncogene occurs in 30% of human breast cancers and correlates with poor prognosis and increased propensity for metastasis. Since the molecular basis of HER2 overexpression in human cancers is not known, we sought to determine whether chromatin remodeling pathways are involved in the regulation of HER2 expression. We report that compared with breast cancer cells expressing a low level of HER2, HER2-overexpressing breast cancer cells contained significantly higher levels of acetylated and phosphorylated histone H3, and acetylated histone H4 associated with the HER2 promoter. Decreased recruitment of histone deacetylases in the promoter is also noted in the HER2-overexpressing cell. The association of acetylated histone H4 with HER2 gene chromatin and HER2 expression in breast cancer cells was upregulated by an inhibitor of histone deacetylases. Treatment with histone deacetylase inhibitor also reduced the association of histone deacetylase-1 and -2 with the HER2 promoter. In addition, the tumor promoters 12- O-tetradecanoylphorbol-13-acetate and okadaic acid stimulated the association of phosphorylated histone H3 on serine 10 with the HER2 promoter and also stimulated HER2 expression. These findings identify histone acetylation and histone phosphorylation as novel regulatory modifications that target HER2 gene chromatin, and suggest that elevated levels of these chromatin-relaxing components in the vicinity of the HER2 gene promoter may constitute an important non-genomic mechanism of HER2 overexpression in human breast cancer.