Neu-overexpressing Breast Cancer Cells Research Articles

Inhibition of cell growth by BrMC through inactivation of Akt in HER-2/neu-overexpressing breast cancer cells.

We previously reported that chrysin (ChR) and its analogs induced cell cycle arrest and apoptosis in human estrogen receptor-positive/-negative breast cancer cells. However, it was unknown whether 8-bromo-7-methoxychrysin (BrMC), a novel synthetic ChR analog, inhibited the cell growth of human epidermal growth factor receptor 2 (HER-2)/neu-overexpressing breast cancers. In the present study, it was demonstrated that BrMC preferentially inhibited the cell viability of HER-2/neu-overexpressing MDA-MB-453 and BT-474 cells. Western blot analysis revealed that HER-2/neu expression and tyrosine phosphorylation were inhibited by BrMC in a concentration-dependent manner; whereas the proteasome inhibitor, MG-132, significantly prevented BrMC-induced HER-2/neu depletion and cell death in MDA-MB-453 cells. This directly indicated that BrMC-induced HER-2/neu depletion and cell growth inhibition was mediated by a proteasomal pathway. BrMC significantly downregulated the expression of cyclin D1, cyclin E and CDK4, followed by the suppression of protein kinase B phosphorylation and downstream effectors, GSK-3β and β-catenin. A colony formation assay also confirmed the growth-inhibitory effects of BrMC. Thus, these findings clearly demonstrate the anticancer activity of BrMC against human HER-2/neu-overexpressing breast cancer cells. Thus, these findings clearly demonstrate the anticancer activity of BrMC against human HER 2/neu-overexpressing breast cancer cells, and highlight BrMC as a promising candidate for breast cancer therapy.

Immunologically active cryogels for breast cancer therapy (P4329)

Abstract Passive immunotherapy has become an effective adjunct for the treatment of HER2/neu-overexpressing breast cancers, as patients can respond well to monoclonal antibodies such as trastuzumab (anti-HER-2/neu antibody therapy). However, patients with late-stage disease, who often become immunosuppressed are unlikely to respond, motivating the development of new prophylactic vaccines. To this end, we have developed an injectable, polymer-based cryogel vaccine containing living, attenuated HER-2/neu-overexpressing breast cancer cells. The cryogel-based vaccine mimics key aspects of bacterial infection and directly controls immune-cell trafficking and activation in the body. This system provides a sustained release of GM-CSF to recruit host dendritic cells (DCs) to the porous material, and subsequently presents breast cancer antigens and TLR9 ligand CpG oligonucleotides to activate the resident DCs. Subcutaneous injection of the cryogel vaccines provide potent prophylactic protection against mammary cancer in mice, consistent with significantly higher titers of Her-2/neu-specific antibodies compared to the humoral responses induced by bolus injection of irradiated GM-CSF-secreting breast tumor cells. The cryogel-based vaccines induced a 70-fold increase in antibody production compared to untreated mice and 100% survival was achieved in vaccinated mice. The cryogel-based vaccines represent a promising tool for the development of novel active immunotherapeutic approaches to cancer.

Enzymatic Discovery of a HER-2/neu Epitope That Generates Cross-Reactive T Cells

Patients with HER-2/neu-expressing breast cancer remain at risk for relapse following standard therapy. Vaccines targeting HER-2/neu to prevent relapse are in various phases of clinical testing. Many vaccines incorporate the HER-2/neu HLA-A2-binding peptide p369-377 (KIFGSLAFL), because it has been shown that CTLs specific for this epitope can directly kill HER-2/neu-overexpressing breast cancer cells. Thus, understanding how tumors process this epitope may be important for identifying those patients who would benefit from immunization. Proteasome preparations were used to determine if p369-377 was processed from larger HER-2/neu-derived fragments. HPLC, mass spectrometry, cytotoxicity assays, IFN-γ ELISPOT, and human breast cancer cell lines were used to assess the proteolytic fragments. Processing of p369-377 was not detected by purified 20S proteasome and immunoproteasome, indicating that tumor cells may not be capable of processing this Ag from the HER-2/neu protein and presenting it in the context of HLA class I. Instead, we show that other extracellular domain HER-2/neu peptide sequences are consistently processed by the proteasomes. One of these sequences, p373-382 (SLAFLPESFD), bound HLA-A2 stronger than did p369-377. CTLs specific for p373-382 recognized both p373-382 and p369-377 complexed with HLA-A2. CTLs specific for p373-382 also killed human breast cancer cell lines at higher levels than did CTLs specific for p369-377. Conversely, CTLs specific for p369-377 recognized p373-382. Peptide p373-382 is a candidate epitope for breast cancer vaccines, as it is processed by proteasomes and binds HLA-A2.

The tumor suppressor RECK interferes with HER-2/Neu dimerization and attenuates its oncogenic signaling

Our previous study demonstrates that HER-2/Neu oncogene inhibits a matrix metalloproteinase inhibitor and tumor metastasis suppressor RECK to promote metastasis. Conversely, the effect of RECK on the oncogenic function of HER-2/Neu is unknown. Ectopic expression of RECK in 293T cells and HER-2/Neu-overexpressing breast cancer cells shows that RECK and HER-2/Neu are co-localized and these two proteins can be co-immunoprecipitated. RECK inhibits HER-2/Neu receptor dimerization and autophosphorylation, which causes reduction of ERK and AKT kinase activity and down-regulation of HER-2/Neu target genes. RECK expression is reduced in 58.8% of breast cancer tissues and is associated with lymph node invasion supporting its anti-metastatic role. Collectively, we provide the first evidence that RECK can negatively regulate oncogenic activity of HER-2/Neu by inhibiting receptor dimerization. Structured summary HER-2/Neu physically interacts with HER-2/Neu by blue native page ( View interaction) HER-2/Neu physically interacts with RECK by coimmunoprecipitation ( View interaction) HER-2/Neu and RECK colocalize by fluorescence microscopy (View Interaction 1, 2) HER-2/Neu physically interacts with RECK by anti bait coimmunoprecipitation ( View interaction)

HER-2/neu transcriptionally activates Jab1 expression via the AKT/β-catenin pathway in breast cancer cells

Jab1 is a co-activator of activating protein-1 (AP-1) transcription factor and the fifth subunit of the constitutive photomorphogenesis 9 (COP9) signalosome, which has been shown to mediate nuclear exportation and ubiquitin-dependent degradation of the tumor suppressor p27(Kip1). Jab1 is overexpressed in several types of human cancer. However, de-regulation of Jab1 gene expression in cancer cells is largely unclear. In this study, we reported that expression of Jab1 was stimulated by HER-2/neu oncogene via transcriptional activation. Promoter deletion and mutation analysis indicated that HER-2/neu stimulated Jab1 via the T cell factor (TCF) binding site located at the -380/-368 region of the human Jab1 promoter. DNA affinity precipitation assay and chromatin immunoprecipitation assay verified that binding of beta-catenin and TCF-4 to this consensus site was increased by HER-2/neu. In addition, dominant-negative mutant of TCF significantly attenuated the stimulatory effect of HER-2/neu. We also demonstrated that HER-2/neu increased beta-catenin/TCF-mediated Jab1 expression via the AKT signaling pathway because chemical inhibitor or dominant-negative mutant of AKT effectively attenuated the stimulatory action of HER-2/neu. IGF-I, which is a well-known AKT activator, also up-regulated the expression of Jab1 in NIH/3T3 and MCF-7 cells. Knockdown of Jab1 by small interfering RNA (siRNA) preferentially inhibited proliferation of HER-2/neu-overexpressing breast cancer cells. Taken together, our results suggest that HER-2/neu transcriptionally activates Jab1 expression to promote proliferation of breast cancer cells.

Silk fibroin mediated delivery of liposomal emodin to breast cancer cells

The efficacy of a drug is dependent on its mode of delivery and its potency at the tumor site. In this study, the drug delivery and efficacy of silk fibroin coated liposomes (SF-ELP), encapsulating a receptor tyrosine kinase inhibitor, emodin, on Her2/ neu over-expressing breast cancer cells, was investigated. This study demonstrates that SF-ELP was more efficacious in suppressing the growth of Her2/ neu over-expressing breast cancer cells MDA-MB-453 and BT-474 as compared to uncoated emodin loaded liposomes (ELP). Reduced levels of phosphorylated Her2/ neu correlated with growth inhibition observed in the MDA-MB-453 cells, treated with both ELP and SF-ELP. ELP treatment of MDA-MB-453 breast cancer cells resulted in inhibition of the PI3K pathway whereas SF-ELP treatment inhibited both the PI3K and MAPK pathways, which contributed to the enhanced growth inhibitory effects of Her2/ neu over-expressing breast cancer cells. Coating of ELP with silk fibroin did not alter the target specificity of emodin, on the other hand the emodin efficacy was enhanced. Higher uptake of emodin delivered by SF-ELP lead to increased cell death as compared to emodin delivery via ELP. Silk fibroin coating around the liposome imparts an extra layer that emodin has to extravasate in order to release from the encapsulating liposome. This increases retention of the drug in the cell for a longer time and protects emodin from quick release and metabolism. Longer intracellular retention may lead to the longer availability of emodin for down-modulation of various Her2/ neu pathways. This study demonstrates that silk fibroin coating enhanced emodin delivery in Her2/ neu over-expressing breast cancer cells thereby increasing the overall efficacy of the drug.

Trastuzumab mimotope vaccination of HER-2/neu transgenic mice results in prolonged tumor-free survival and reduced tumor load

12510 Background: Passive immunotherapy with the humanized monoclonal antibody trastuzumab (Herceptin) to date is the most effective treatment for patients with HER-2/neu overexpressing breast cancer. In previous studies we could show that active immunization with peptide mimotopes, i.e. structural mimics of the epitope recognized by trastuzumab, leads to formation of antibodies again recognizing HER-2/neu in mice. Functional in vitro analyses of the induced antibodies demonstrated “trastuzumab-like” properties, such as receptor internalization, inhibition of signaling, and antibody-mediated cytotoxicity against HER-2/neu overexpressing breast cancer cells. The aim of the present study was to test the effects of trastuzumab mimotope vaccination in vivo, namely in a HER-2/neu transgenic mouse model. Methods: We used BALB-neuT mice, which carry the activated neu oncogene on the BALB/c background. These mice constitute the most aggressive animal model for HER-2/neu driven carcinogenesis, as all females uniformly develop mammary carcinomas at the age of 12 weeks. One group of mice was immunized with the previously described trastuzumab mimotope - KLH conjugate, a control group with the carrier protein KLH alone, and a further control group was left naïve. Mice were palpated weekly to monitor tumor development and size, and blood samples were taken at regular intervals to follow up the induced immune responses. Results: Trastuzumab mimotope immunizations lead to delayed tumor development and thus to an increase in tumor-free survival. When tumors occurred, they were smaller and grew more slowly than in the control mice. In contrast, control KLH immunizations did not affect tumor growth kinetics as compared to the naïve mice. Serum analysis demonstrated that all immunized animals had mounted an anti-KLH immune response, so we accredit the observed tumor-inhibitory effects in the mimotope group to the biologic properties of induced anti-HER-2/neu antibodies. Conclusion: These results indicate that epitope-specific vaccination with mimotopes elicits trastuzumab-like antibodies that are effective in vivo against HER-2/neu overexpressing tumor cells also in HER-2/neu expressing organisms. [Table: see text]

Combinatorial synergy induced by adenoviral-mediated mda-7 and Herceptin in Her-2+ breast cancer cells

The melanoma differentiation-associated gene-7 (mda-7) is a member of the interleukin-10 cytokine family and a novel tumor suppressor gene. Adenoviral-mediated mda-7 (Ad-mda7) gene transfer has tumor-specific growth inhibitory and proapoptotic effects in a broad spectrum of cancer cells. In breast cancer cells, adenoviral-induced mda-7 expression triggers antiproliferative effects by downregulation of survival signals, such as Bcl-2 and Akt. The anti-human epidermal growth factor receptor-2 (Her-2) monoclonal antibody, Trastuzumab (Herceptin), increases the sensitivity of Her-2/neu-overexpressing breast cancer cells to chemotherapeutic agents and radiotherapy. In this study, we evaluate the effects of treatment with Ad-mda7 and Herceptin combination therapy in a panel of Her-2/neu-overexpressing cell lines, and in established tumors in nude mice. Compared to individual treatments, the combination of Ad-mda7 and Herceptin elicits supra-additive antitumor activity in Her-2/neu-overexpressing tumor cell lines: increased cell death, cell cycle block and apoptosis. The Ad-mda7 and Herceptin interaction was shown to be synergistic by isobologram analysis. Ad-mda7 does not alter cell surface Her-2/neu levels, but the combination of Ad-mda7+Herceptin results in increased expression of cell surface E-cadherin with concomitant translocation of beta-catenin from the nucleus to the cell membrane. In vivo, the combination of Ad-mda7 and Herceptin showed significantly increased antitumor activity (P<0.003) against Her-2/neu-overexpressing tumors. These data suggest that the combination of Ad-mda7 with Herceptin may be a novel therapy for breast cancer patients whose tumors overexpress Her-2/neu. The observed synergistic effect may improve treatment options for otherwise poorly responsive, Her-2-positive, breast cancer patients.

Microarray-Assisted Pathway Analysis Identifies Mitogen-Activated Protein Kinase Signaling as a Mediator of Resistance to the Green Tea Polyphenol Epigallocatechin 3-Gallate in Her-2/neu–Overexpressing Breast Cancer Cells

Overexpression of the epidermal growth factor receptor family member Her-2/neu in breast cancer leads to autophosphorylation of the receptor and induction of multiple downstream signaling pathways, including the Akt kinase to nuclear factor-kappaB (NF-kappaB) cascade that is associated with poor prognosis. Previously, we showed that the green tea polyphenol epigallocatechin 3-gallate (EGCG) inhibits growth of NF639 Her-2/neu-driven breast cancer cells via reducing receptor autophosphorylation and downstream Akt and NF-kappaB activities. Interestingly, upon prolonged culture in the presence of EGCG, cells resistant to the polyphenol could be isolated. Here, we report that resistant cells have lost tyrosine phosphorylation on the Her-2/neu receptor. Surprisingly, they displayed elevated NF-kappaB activity, and inhibition of this activity sensitized cells to EGCG. Data from microarray studies of the original and resistant NF639 populations of cells were subjected to Gene Set Enrichment Analysis pathway assessment, which revealed that the mitogen activated protein kinase (MAPK) pathway was activated in the resistant cells. Treatment of the resistant cells with the MAPK inhibitor U0216 reduced growth in soft agar and invasive phenotype, whereas the combination of EGCG and U0216 resulted in cells with a cobblestone epithelial phenotype. Thus, activation of the MAPK pathway mediates resistance to EGCG.

Degradation of HER2/ neu by apigenin induces apoptosis through cytochrome c release and caspase-3 activation in HER2/ neu-overexpressing breast cancer cells

We have shown that exposure of the HER2/ neu-overexpressing breast cancer cells to apigenin resulted in induction of apoptosis by depleting HER2/ neu protein and, in turn, suppressing the signaling of the HER2/HER3-PI3K/Akt pathway. Here, we examined whether inhibition of this pathway played a role in the anti-tumor effect. The results revealed that treatment with apigenin induced apoptosis through cytochrome c release and caused a rapid induction of caspase-3 activity and stimulated proteolytic cleavage of DFF-45. Furthermore, apigenin downregulated cyclin D1, D3 and Cdk4 and increased p27 protein levels. Colony formation in the soft agar assay, a hallmark of the transformation phenotype, was preferentially suppressed in HER2/ neu-overexpressing breast cancer cells in the presence of apigenin. In addition, a structure–activity relationship study indicated that (1) the position of B ring; and (2) the existence of the 3′, 4′-hydroxyl group on the 2-phenyl group were important for the depletion of HER2/ neu protein by flavonoids. These results provided new insights into the structure–activity relationship of flavonoids.

Combination therapy of Ad-mda7 and trastuzumab increases cell death in Her-2/neu–overexpressing breast cancer cells

BackgroundOverexpression of the tumor suppressor gene melanoma differentiation-associated gene-7 (mda-7) induces apoptosis in many cancer cells, and adenoviral-mediated overexpression of mda-7 downregulates β-catenin and PI 3-kinase signaling in breast cancer cells. Trastuzumab (Herceptin) improves the efficacy of chemotherapeutics against Her-2/neu–overexpressing breast cancer cells. We sought to evaluate the impact of combination therapy of a recombinant adenovirus vector encoding for human mda-7 (Ad-mda7) and Herceptin on Her-2/neu–overexpressing breast cancer. MethodsThe MCF-7-Her-18 cell line was subjected to treatment with Ad-mda7 with and without Herceptin. Western blot analysis was performed with antibodies to β-catenin, Akt, and phosphorylated Akt (p-Akt). The same treatment groups were utilized in a nude mouse model in vivo. Treatment was initiated when the tumors reached 100 mm3 in size. ResultsIn Western blotting, the combination of Ad-mda7+Herceptin showed decreased levels of β-catenin, Akt and p-Akt compared with Ad-mda7 or Herceptin alone (P<.05). The in vivo analysis revealed a marked decrease in tumor size with the Ad-mda7+Herceptin combination (P<.05). ConclusionsThese studies demonstrate the growth inhibitory effect of Ad-mda7+Herceptin on Her-2/neu–overexpressing breast cancer cells in vitro and in vivo. This combination appears to inhibit the pathways involving β-catenin and Akt, which play important roles in the growth of breast cancer cells.

675. Combination Therapy of Ad-mda7 and Herceptin Increases Cell Death in Her-2/Neu Positive Breast Cancer Cells

Adenoviral mediated transduction of the tumor suppressor melanoma differentiation-associated gene-7 (Ad-mda7) induces apoptosis in a broad spectrum of cancer cells, but not in normal cells. Transgenic MDA-7 protein expression downregulates β-catenin and PI3K signaling in breast cancer cells. Breast cancers overexpressing Her-2/neu are resistant to conventional chemotherapeutic agents, resulting in significantly lower overall survival rates. Herceptin (Trastuzumab), a monoclonal antibody directed against Her-2/neu, improves the efficacy of chemotherapeutics against Her-2/neu-overexpressing breast cancer cells. In this study, we evaluated the impact of combination therapy of Ad-mda7 and Herceptin on Her-2/neu-positive and negative breast cancer cell lines.

Inhibition of tumor cell growth by antibodies induced after vaccination with peptides derived from the extracellular domain of Her-2/neu.

The anti Her-2/neu monoclonal antibody Trastuzumab has strong inhibiting effects on tumor growth in vitro and in vivo and is therefore used for immunotherapy in breast cancer patients. Due to necessity of frequent applications, however, cost intensiveness of Trastuzumab treatment and its limited duration of affectivity, an active immunization inducing a perhaps preventive and long-term immunity to Her-2/neu remains a desirable goal. We attempted to induce anti Her-2/neu antibodies by peptide vaccination and to test their efficacy in inhibiting tumor cell growth in vitro. By computer aided analyses, 7 putative B cell epitopes of Her-2/neu were defined and synthesized. These peptide epitopes were coupled to tetanus toxoid and used for immunization in BALB/c mice. Among these peptides, immunizations with 2 single peptides or a combination of 2 peptides induced anti-peptide antibody levels, primarily of the IgG1 isotype. These antibodies were also directed against the native Her-2/neu antigen, as shown in precipitation assays and ELISA with cell lysates of the Her-2/neu overexpressing breast cancer cell line SK-BR-3. Isolated IgG fractions from immune sera incubated with SK-BR-3 cells led to a moderate inhibition of the tumor cell growth in vitro, as well as to complement dependent cell lyses comparable to that achieved by incubation with Trastuzumab. Moreover, peptide immunization in rabbits generated anti-Her 2-neu IgG that, in contrast to mouse sera, were able to mediate a 31-46% lysis of SK-BR-3 cells in ADCC experiments. We conclude from our data that immunization with Her-2/neu peptides successfully induced humoral immune response with anti-tumor activity in an animal model.

The role of heregulin-alpha as a motility factor and amphiregulin as a growth factor in wound healing.

Wound healing is a complex process of which growth and motility are essential features. The aim of this study was to search for keratinocyte-derived secreted factors that may play a role in these mechanisms, and their corresponding receptors. Growth and motility factors were purified from conditioned medium from cultured primary keratinocytes. Receptor and growth factor expression profiles were investigated by immunohistochemical, western blotting, and in situ hybridization analysis on cultured keratinocytes and tissue sections derived from chronic wounds. The most potent autocrine growth factor for keratinocytes, which it was possible to purify and sequence from keratinocyte-conditioned medium, is amphiregulin. Its receptor HER-1 is up-regulated on the membranes of keratinocytes lining the edge of the wound. From the same keratinocyte-conditioned medium, heregulin-alpha was purified as a potent motility factor for keratinocytes. Its receptor is HER-3, which is up-regulated on the membranes of keratinocytes lining the edge of the wound and on keratinocytes that had migrated towards the centre of the wound. HER-4 - another receptor for heregulin-alpha - is weakly present in occasional cells near the edge of the wound. The co-receptor for HER-3 and HER-4 is HER-2/neu, which is also present in epidermal cells but not overexpressed. This study shows that heregulin-alpha is a potent motility factor for normal epithelial cells and that it plays a central role in the process of wound healing of stratified epithelia. Heregulin-alpha has already been shown to be the motility factor leading to migration of HER-2/neu-overexpressing breast cancer cells. The role of amphiregulin as a growth factor and of heregulin-alpha as a motility factor for keratinocytes in epidermal and mucosal wound healing parallels their motility and growth induction in carcinogenesis.

AKT2 is frequently upregulated in HER-2/neu-positive breast cancers and may contribute to tumor aggressiveness by enhancing cell survival.

Amplification or overexpression of the HER-2/neu gene in breast cancers is associated with aggressive behavior and resistance to therapeutic regimens. The molecular mechanisms that contribute to therapeutic resistance/survival of HER-2/neu-overexpressing tumor cells have not been well defined. To determine if phosphatidylinositol 3-kinase/AKT signaling contributes to cell survival in HER-2/neu-positive breast cancers, we performed immunohistochemical analyses to evaluate expression of HER-2/neu and AKT in a series of 52 breast carcinomas. Elevated expression of HER-2/neu was found to correlate with overexpression of AKT2 protein and activation of AKT kinase. HER-2/neu-overexpressing breast cancer cell lines were resistant to apoptosis induced by UV treatment and hypoxia, which was suppressed in the presence of the phosphatidylinositol 3-kinase inhibitors LY294002 and wortmannin, indicating a link between AKT activation and stress resistance in HER-2/neu-overexpressing cells. These observations suggest that AKT signaling augments resistance to stress-induced apoptosis in breast cancer cells overexpressing HER-2/neu.

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Anti-Her-2/neu antibody is known to induce apoptosis in HER-2/neu overexpressing breast cancer cells. However, exact regulatory mechanisms mediating and controlling this phenomenon are still unknown. In the present study, we have investigated the effect of anti-Her-2/neu antibody on apoptosis of HER-2/neu overexpressing human breast cancer cell lines SK-BR-3, HTB-24, HTB-25, HTB-27, HTB-128, HTB-130 and HTB-131 in relation to p53 genotype and bcl-2 status. SK-BR-3, HTB-24, HTB-128 and HTB-130 cells exhibited mutant p53, whereas wild type p53 was found in HTB-25, HTB-27 and HTB-131 cells. All seven cell lines weakly expressed bcl-2 protein (10–20%). Anti-Her-2/neu antibody, irrespective of p53 and bcl-2 status, induced apoptosis in all 7 cell lines dose- and time-dependently and correlated with Her-2/neu overexpression. In addition, incubation of cell lines with anti-Her-2/neu antibody did not alter p53 or bcl-2 expression. Anti-HER-2/neu antibody did not induce apoptosis in HER-2/neu negative HBL-100 and HTB-132 cell lines. Our results indicate that within the panel of tested breast cancer cell lines, anti-Her-2/neu antibody-induced apoptosis was independent from the presence of intact p53. © 2001 Cancer Research Campaign http://www.bjcancer.com

Targeting HER2: Recent developments and future directions for breast cancer patients

Overexpression of the HER2/ neu oncogene (also known as c-erbB2) is a frequent molecular event in multiple human cancers, including breast and ovarian cancer. Patients with cancer that overexpress HER2/ neu are associated with unfavorable prognosis, shorter relapse time, and low survival rate. Treatments that target HER2/ neu expression in cancer cells have been shown to be useful strategies to significantly reverse the malignancy induced by HER2/ neu overexpression. The humanized anti-HER2/ neu antibody, trastuzumab (Herceptin; Genentech, Inc, South San Francisco, CA) has proven to be effective in clinical trials in patients with metastatic breast cancer. In addition, tyrosine kinase inhibitors such as emodin can also target the HER2/ neu oncogenic activity. Emodin treatment inhibits HER2/ neu tyrosine kinase activity and preferentially suppresses the transformation of HER2/ neu-overexpressing breast cancer cells. Emodin also sensitizes HER2/ neu-over-expressing cancer cells to chemotherapeutic agents, including cisplatin, doxorubicin, etoposide, and paclitaxel. Alternatively, HER2/ neu overexpression can be repressed by attenuating the promoter activity of the HER2/ neu gene. We have identified a number of potent transcriptional regulators, including the ets family member PEA3 and the adenovirus type 5 EIA, which are able to repress HER2/ neu gene expression. Expression of these transcriptional regulators resulted in downregulation of HER2/ neu promoter activity and reversed the transformed phenotype of the cancer cells in vitro. In vivo studies show that these HER2/ neu repressors can act therapeutically as tumor suppressor genes for tumors that overexpress HER2/ neu. These preclinical studies clearly indicate that transcriptional repressors that downregulate HER2/ neu can be effective regimens for cancer treatment in a gene therapy format. More importantly, the tumor-free survival rate of treated animals is dramatically increased under nontoxic doses compared with untreated animals. A phase I clinical trial using EIA-liposome in breast and ovarian patients has recently been completed. Following treatment, we observed downregulation of the HER2/ neu protein accompanied by EIA expression in both cancer and noncancer cells. Numbers of tumor cells in the pleural effusion or ascites were found to be dramatically reduced after treatment. Furthermore, apoptosis was strongly induced in the tumor cells. A phase II study has been started to further evaluate therapeutic efficacy and tumor suppression mechanisms of El A. These studies show the clinical potential of targeting HER2/neu in cancer therapy.

Anti-Her-2/neu antibody induces apoptosis in Her-2/neu overexpressing breast cancer cells independently from p53 status.

Anti-Her-2/neu antibody is known to induce apoptosis in HER-2/neu overexpressing breast cancer cells. However, exact regulatory mechanisms mediating and controlling this phenomenon are still unknown. In the present study, we have investigated the effect of anti-Her-2/neu antibody on apoptosis of HER-2/neu overexpressing human breast cancer cell lines SK-BR-3, HTB-24, HTB-25, HTB-27, HTB-128, HTB-130 and HTB-131 in relation to p53 genotype and bcl-2 status. SK-BR-3, HTB-24, HTB-128 and HTB-130 cells exhibited mutant p53, whereas wild type p53 was found in HTB-25, HTB-27 and HTB-131 cells. All seven cell lines weakly expressed bcl-2 protein (10–20%). Anti-Her-2/neu antibody, irrespective of p53 and bcl-2 status, induced apoptosis in all 7 cell lines dose- and time-dependently and correlated with Her-2/neu overexpression. In addition, incubation of cell lines with anti-Her-2/neu antibody did not alter p53 or bcl-2 expression. Anti-HER-2/neu antibody did not induce apoptosis in HER-2/neu negative HBL-100 and HTB-132 cell lines. Our results indicate that within the panel of tested breast cancer cell lines, anti-Her-2/neu antibody-induced apoptosis was independent from the presence of intact p53. © 2001 Cancer Research Compaign http://www.bjcancer.com

Targeting HER-2/neu-overexpressing breast cancer cells by an antisense iron responsive element-directed gene expression

Overexpression of HER-2/neu proto-oncogene is found in many human cancers including 20–30% of breast cancer and is a predictor of poor prognosis. To target breast cancer cells that overexpress HER-2/neu mRNA, we previously described a novel strategy that combines the principle of antisense (AS) and translational inhibitory activity conferred by an iron-responsive element (IRE) (AS-IRE). Here, we showed that three potential AS-IREs, i.e. AS-IRE1, 4, and 5, derived from HER-2/neu antisense sequence could bind endogenous iron regulatory protein (IRP) and, when placed in 5′ untranslated region (5′UTR) of a reporter gene, the gene expression could be translationally repressed by recombinant IRP in vitro. Using AS-IRE4 as our model, we demonstrated that it is regulated by iron, and importantly, such regulation is impaired in HER-2/neu-overexpressing breast cancer cells. Furthermore, we showed that AS-IRE4 could preferentially direct the expression of a reporter gene in HER-2/neu-overexpressing breast cancer cells. Interestingly, when AS-IRE4 was placed in 5′UTR of Bax gene, a pro-apoptotic protein in the Bcl-2 protein family, we observed a preferential cell killing in breast cancer cells that overexpress HER-2/neu. Taken together, our results suggest that AS-IRE behaves as a functional IRE and it may direct therapeutic gene expression to preferentially target HER-2/neu-overexpressing breast cancer cells.

HER2/neu antisense targeting of human breast carcinoma.

Overexpression of the HER2/neu oncogene is observed in approximately 30% of human breast carcinoma specimens. HER2/neu overexpression is a negative prognostic factor in breast cancer patients. Cancer cells that overexpress HER2/neu may also be less sensitive to chemotherapy. In order to further define mechanisms by which HER2/neu overexpression drives neoplastic cell growth and chemoresistance, antisense oligonucleotides (ODNs) have been utilized to selectively down-regulate HER2/neu expression in human breast cancer cells. Such antisense ODNs suppress HER2/neu mRNA and protein levels in a dose-dependent, sequence-specific manner. Down-regulation of HER2/neu expression in HER2/neu overexpressing breast cancer cells inhibits cell cycle progression in G0/G1 and results in apoptotic cell death. In tissue culture studies, combined treatment of HER2/ neu overexpressing breast cancer cells with HER2/neu antisense ODNs and conventional chemotherapeutic agents results in synergistic inhibition of cancer cell growth and activation of apoptotic cell death mechanisms. These studies have been extended to demonstrate synergistic antitumor effects following systemic treatment with antisense ODNs plus doxorubicin in nude mice bearing human breast carcinoma xenografts. Collectively these findings demonstrate that HER2/neu overexpression stimulates anti-apoptotic cell survival mechanisms and suggest that HER2/neu antisense ODNs may be of use in cancer therapeutics.