Neu Oncogene Research Articles

Overexpression of HER-2/neu oncogene and transitional cell carcinoma of bladder.

The aim of this study was to evaluate the relationship between HER-2/neu oncogene expression and grade of transitional cell carcinoma (TCC) of the bladder. In this cross-sectional study, 75 formalin-fixed paraffin-embedded specimens of primary TCC of the bladder were stained with a monoclonal antibody against HER-2/neu oncoprotein. Another section was stained by hematoxylin-eosin and the tumor grade was determined according to the World Health Organization/International Society of Urological Pathologists criteria. Tumor specimens belonged to 49 men (65.3%) and 26 women (34.7%) with a mean age of 56.3 +/- 9.1 years (range, 39 to 80 years). The tumor grades were 1, 2, and 3 in 14 (18.7%), 35 (46.7%), and 26 (34.7%) specimens, respectively. A total of 28 (37.3%) patients were positive for overexpression of HER-2/neu. There were 1 case of HER-2/neu-positive (7.1%) with a grade 1 tumor, 10 (28.6%) with grade 2, and 17 (65.4%) with grade 3; a significant relationship between HER-2/neu overexpression and grade of the bladder TCC tumors was found (P = .002). Expression of HER-2/neu oncogene has a direct relationship with the grade of the bladder TCC. Further studies with longer follow-up period and a larger sample size can determine the probable role of HER-2/neu expression as a prognostic factor in the TCC of bladder.

Development of a Conditionally Replicating Pseudorabies Virus for HER-2/neu-overexpressing Bladder Cancer Therapy

Overexpression of the HER-2/neu oncogene, a frequent molecular event in a variety of cancers including bladder cancer, is associated with tumor progression and poor prognosis. Therapeutic strategies to targeting HER-2/neu-overexpressing cancer cells have shown promise. Pseudorabies virus (PrV), a herpesvirus of swine, may be exploited as an oncolytic agent for human cancer. Herein, we generated a conditionally replicating glycoprotein E-defective PrV mutant carrying glycoprotein D and herpes simplex virus type 1 thymidine kinase genes, which are essential for viral entry and replication, under the transcriptional control of the HER-2/neu promoter. The recombinant PrV, designated YP2, selectively replicated in and lysed HER-2/neu-overexpressing human bladder, mouse bladder, and hamster oral cancer cells in vitro. Notably, YP2 retarded MBT-2 bladder tumor growth in mice by more than 50% and more than half of the mice survived for over 50 days, whereas all the control mice survived less than 30 days. Taken together, our results suggest that YP2 may have therapeutic potential for the treatment of invasive bladder cancer. Furthermore, because HER-2/neu is overexpressed in a broad spectrum of cancers, this conditionally replicating PrV may be broadly applicable.

Phosphorylation of Ser78 of Hsp27 correlated with HER-2/neu status and lymph node positivity in breast cancer

BackgroundAbnormal amplification/expression of HER-2/neu oncogene has been causally linked with tumorigenesis and metastasis in breast cancer and associated with shortened overall survival of patients. Recently, heat shock protein 27 (Hsp27) was reported to be highly expressed in HER-2/neu positive tumors and cell lines. However, putative functional links between phosphorylation of Hsp27 with HER-2/neu status and other clinicopathological features remain to be elucidated.ResultsComparative phosphoproteomic studies of HER-2/neu positive and -negative breast tumors revealed that Hsp27, one of the identified phosphoproteins, was highly phosphorylated in HER-2/neu positive tumors. The extent of Hsp27 phosphorylation at its Ser15, Ser78 and Ser82 residues were further evaluated with site-specific antibodies in tumor samples by tissue lysate array- and tissue microarray-based analyses, and in the BT474 breast cancer cell line treated with heregulin α1 (HRG α1) or the p38 MAPK inhibitor, SB203580. The tissue lysate array study indicated that only the level of pSer78 in HER-2/neu positive tumors was more than 2-fold that in HER-2/neu negative tumors. Treatment of BT474 cells with HRG α1 and SB203580 indicated that Ser78 phosphorylation was mainly regulated by the HER-2/neu-p38 MAPK pathway. Immunohistochemical staining of sections from a tissue microarray with 97 breast tumors showed that positive staining of pSer78 significantly correlated with HER-2/neu (p = 0.004) and lymph node positivity (p = 0.026).ConclusionThis investigation demonstrated the significant correlation of enhanced phosphorylation of the Ser78 residue of Hsp27 with HER-2/neu and lymph node positivity in breast cancer.

Pharmacological Inhibitors of Fatty Acid Synthase (FASN)-Catalyzed Endogenous Fatty Acid Biogenesis: A New Family of Anti-Cancer Agents?

The expression and activity of Fatty Acid Synthase (FASN; the sole enzyme capable of the reductive de novo synthesis of long-chain fatty acids from acetyl-CoA, malonyl-CoA, and nicotinamide adenine dinucleotide phosphate -NADPH-) is extremely low in nearly all nonmalignant adult tissues, whereas it is significantly up-regulated or activated in many cancer types, thus creating the potential for a large therapeutic index. Since the pioneering observation that inhibition of FASN activity by the mycotoxin cerulenin preferentially kills cancer cells and retards the growth of tumors in xenografts models, numerous in vitro and in vivo studies have confirmed the potential of FASN as a target for antineoplastic intervention. Other FASN inhibitors such as the cerulenin derivative C75, the beta-lactone orlistat, the green tea polyphenol epigallocatechin-3-gallate (EGCG) and other naturally occurring flavonoids (i.e., luteolin, quercetin, and kaempferol), as well as the antibiotic triclosan, have been identified and have been shown to limit cancer cell growth by inducing apoptotic cell death. Though the exact mode of action of these FASN inhibitors is under discussion, it has been revealed that depletion of end-product fatty acids, toxic intracellular accumulation of supra-physiological concentrations of the FASN substrate malonyl-CoA and/or limited membrane synthesis and/or functioning by altered production of phospholipids partitioning into detergent-resistant membrane microdomains (lipid raft-aggregates), can explain, at least in part, the cytostatic, cytotoxic as well as the apoptotic effects occurring upon pharmacological inhibition of FASN activity in cancer cells. Moreover, several cancer-associated molecular features including nonfunctioning p53, overexpression of the Her-2/neu (erbB-2) oncogene, and hyperactivation of the PI-3'K down-stream effector protein kinase B (AKT), appear to determine an exacerbated sensitivity to FASN inhibition-induced cancer cell death. Although few of these inhibitors are expected to be "exclusively" selective for FASN, the potential of FASN as a target for antineoplastic intervention has eventually been confirmed by RNA interference (RNAi)-knockdown of FASN. Certainly, future studies should definitely elucidate the ultimate biochemical link between FASN inhibition and cancer cell death. Although the combination of FASN structural complexity and until recently the lack of X-ray crystallography data of mammalian FASN created a significant challenge in the exploitation of FASN as a valuable target for drug development, it is hoped that the improvement in the selectivity and potency of forthcoming novel FASN-targeted small molecule inhibitors by taking advantage, for instance, of the recent 4.5 A resolution X-ray crystallographic map of mammalian FASN, will direct the foundation of a new family of chemotherapeutic agents in cancer history.

DNA topoisomerase IIα (TOP2A) inhibitors up-regulate fatty acid synthase gene expression in SK-Br3 breast cancer cells: In vitro evidence for a ‘functional amplicon’ involving FAS, Her-2/neu and TOP2A genes

Fatty acid synthase (FAS), the key metabolic multi-enzyme that is responsible for the terminal catalytic step in the de novo fatty acid biosynthesis, plays an active role in the development, maintenance, and enhancement of the malignant phenotype in a subset of breast carcinomas. We recently described that a molecular bi-directional cross-talk between FAS and the Her-2/neu (erbB-2) oncogene is taking place at the level of transcription, translation, and activity in breast cancer cells. Because Her-2/neu has been linked with altered sensitivity to cytotoxic drugs, we envisioned that FAS gene expression may represent a novel predictive molecular factor for breast cancer response to chemotherapy in a Her-2/neu-related manner. We herein evaluated whether chemotherapy-induced cell damage acts in an epigenetic fashion by inducing changes in the transcriptional activation of FAS gene in breast cancer cells. To evaluate this option, FAS- and Her-2/neu-overexpressing SK-Br3 breast cancer cells were transiently transfected with a FAS promoter-reporter construct (FAS-Luciferase) harboring all the elements necessary for high level expression in cancer cells. SK-Br3 cells cultured in the presence of topoisomerase IIalpha (TOP2A) inhibitors doxorubicin and etopoxide (VP-16) demonstrated a 2- to 3-fold increase in FAS promoter activity when compared with control cells growing in drug-free culture conditions. We failed to observe any significant activation of FAS promoter following exposure to the anti-metabolite 5-fluorouracil, the alkylating drug cisplatin, or the microtubule interfering-agents paclitaxel and vincristine. Moreover, the up-regulatory effects of TOP2A inhibitors on the transcriptional activation of FAS gene expression were not significantly decreased when the FAS promoter was damaged at the sterol regulatory element binding protein (SREBP)-binding site. Considering that FAS inhibition produces profound inhibition of DNA replication and S-phase progression in cancer cells, we finally asked whether a cross-talk between TOP2A and FAS could exhibit a Her-2/neu-related bi-directional nature. TOP2A protein levels were decreased during treatment with the anti-Her-2/neu antibody trastuzumab while, concomitantly, FAS promoter activity and FAS protein expression were significantly reduced. Of note, when the expression levels of TOP2A protein were analyzed following exposure of SK-Br3 cells to increasing concentrations of the novel slow-binding FAS inhibitor C75, a dose-dependent reduction in TOP2A expression was observed. Although FAS gene is not physically located in the Her-2/neu-TOP2A amplicon, our present findings strongly suggest that a tight functional association between FAS, Her-2/neu and TOP2A genes is taking place in a subset of breast carcinoma cells.

Mechanisms of acquired androgen independence during arsenic-induced malignant transformation of human prostate epithelial cells.

BackgroundProstate cancer progression often occurs with overexpression of growth factors and receptors, many of which engage the Ras/mitogen-activated protein MAP kinase (MAPK) pathway.ObjectivesIn this study we used arsenic-transformed human prostate epithelial cells, which also show androgen-independent growth, to study the possibility that chronic activation of Ras/MAPK signaling may contribute to arsenic-induced prostate cancer progression.MethodsControl and chronic arsenic–transformed prostate epithelial cells (CAsE-PE) were compared for Ras/MAPK signaling capacities using reverse transcription–polymerase chain reaction and Western blot analyses.ResultsWe found activation of HER-2/neu oncogene in transformed CAsE-PE cells, providing molecular evidence of androgen independence in the transformed cells. CAsE-PE cells displayed constitutively increased expression of unmutated K-Ras (6-fold), and the downstream MAP kinases A-Raf and B-Raf (2.2-fold and 3.2-fold, respectively). There was also increased expression of phosphorylated MEK1/2 and Elk1 in the transformant cells. The MEK1/2 inhibitor, U0126, blocked PSA overexpression in CAsE-PE cells.ConclusionThus, arsenic-induced malignant transformation and acquired androgen independence are linked to Ras signaling activation in human prostate epithelial cells. Chronic activation of this pathway can sensitize the androgen receptor to subphysiologic levels of androgen. This may be important in arsenic carcinogenesis and provide a mechanism that may be common for prostate cancer progression driven by diverse agents.

Establishment and characterization of new mammary adenocarcinoma cell lines derived from double transgenic mice expressing GFP and neu oncogene.

A new murine cell line, named GFPneu, was established from a mammary adenocarcinoma arising in double transgenic MMTVneu x CMV-GFP mice. Breast tumours develop in 100% of females after 2 months latency, as a result of the over-expression of the activated rat neu oncogene in the mammary glands. All tissues, and in particular the breast tumours, express the GFP protein. This cell line was tumorigenic when inoculated into nude mice and the derived tumours showed the same histological features as the primaries from which they were isolated. Their histopathology reproduces many characteristics of human breast adenocarcinomas, in particular their ability to metastasize. The GFP marker allows us to visualize the presence of lung metastases in fresh tissues immediately, to confirm the histopathology. From a lung metastatic fluorescent nodule, we derived a further cell line, named MTP-GFP, which we also characterized. These two cell lines could be useful to study the role played by the neu oncogene in the maintenance of the transformed phenotype, in the metastatic process, to test novel therapeutic strategies to inhibit primary tumour growth and to observe the generation of distant metastases.

Experimental research of effectiveness of siRNA-Her-2/neu on drug sensitivity of Her-2/neu-over-expressing lung adenocarcinoma cell line

Objective: Her-2/neu protein overexpression has been demonstrated in lung adenocarcinoma. Its overexpression often indicates a poor prognosis and resistance to chemotherapeutic agents. The objectives of this paper is to explore the effectiveness of double-stranded short inhibitory RNAs (siRNA) targeting Her-2/neu oncogene on the drug sensitivity of Her-2/neu-overexpressing lung adenocarcinoma cells. Methods: Lung adenocarcinoma cell line calu-3 was transfected with siRNAs formulated LipofectAMINE 2000, and Her-2/neu protein and P-gp were determined by flow cytometry (FCM). The chemosensitivity of transfected cells to cisplatin (CDDP) was measured by MTT. Cell apoptosis detection kit (Annexin V method) was used to examine the drug induced apoptosis rate. Results: siRNA targeting Her-2/neu greatly reduced the cell surface expression of Her-2/neu protein and had no effect on P-gp. Consequently the inhibitory rate of CDDP in combination with siRNA targeting Her-2/neu was (67.1±2.3)%, while the inhibitory rates were (48.1±3.5)%, (46.3±5.9)% and (50.2±2.9)% in untreated control, empty vector and unrelated siRNA groups, respectively. The FCM results showed that the apoptosis rate of cells treated with CDDP combined with siRNAs-Her-2/neu was elevated when compared with unrelated siRNA group and empty vector group. Conclusion: Sequence specific siRNA targeting Her-2/neu was capable of enhancing the chemosensitivity of calu-3 cell to cisplatin.

Topoisomerase II α gene amplification in gastric carcinomas: correlation with the HER2 gene. An immunohistochemical, immunoblotting, and multicolor fluorescence in situ hybridization study

Topoisomerase IIalpha (topoIIalpha) is an enzyme required for DNA replication and a molecular target for drugs called anthracyclines. The topoIIalpha gene (TOP2A) is located close to the HER-2/neu oncogene (HER2). We assessed gastric cancers to (1) clarify the relationship between gene amplification and protein overexpression of topoIIalpha and HER2; (2) evaluate the correlation between gene amplification and protein overexpression of topoIIalpha; and (3) examine the relationship between the results of immunohistochemistry and Western blot analysis for topoIIalpha. In a combined analysis of immunohistochemistry and fluorescence in situ hybridization on 552 formalin-fixed and paraffin-embedded gastric cancer tissues, 38 cases were found to have HER2 amplification. Further examination by fluorescence in situ hybridization revealed amplification of TOP2A in 13 of the 38 cases. No aberrations in the TOP2A gene were observed in cases without HER2 overexpression, except for one containing a gene deletion. The TopoIIalpha protein-labeling index was not correlated with TOP2A amplification. Fluorescence in situ hybridization was performed on nuclear imprint specimens obtained from 9 cases using simultaneous probes for TOP2A, HER2, and centromere 17. Of these 9 cases, 3 displayed coamplification of TOP2A and HER2, and only 1 of the 3 cases revealed a high expression of topoIIalpha in Western blot. Although patients having gastric adenocarcinoma with TOP2A amplification could be considered suitable for clinical trials, information involving anthracycline therapy is not firmly understood in regards to the status of TOP2A amplification or protein overexpression. Therefore, results of the current study will provide further insight for the clinical application of anthracycline in gastric cancers.

Vaccination Strategy Determines the Emergence and Dominance of CD8+ T-Cell Epitopes in a FVB/N Rat HER-2/neuMouse Model of Breast Cancer

The HER-2/neu oncogene has >25 HLA epitopes, yet only one FVB/N mouse CD8(+) T-cell epitope has been mapped to date. This epitope has been termed the immunodominant epitope for the FVB/N mouse, but we propose that the vaccination strategy determines the dominance of epitopes. Using a series of overlapping peptides, we have mapped another CD8(+) T-cell epitope that emerges in the FVB/N mouse following vaccination with Listeria monocytogenes-based vaccines that express fragments of HER-2/neu. Following the identification of this novel H-2K(q)-restricted epitope, we sought to compare the T-cell response to this epitope with the previously identified PDSLRDLSVF epitope. This newly identified epitope and the previously identified epitope lie within fragments contained in different vaccines, the PDSLRDLSVF epitope in Lm-LLO-EC2 and the newly identified PYNYLSTEV epitope in Lm-LLO-EC1; thus, it has been possible to compare the responses of these epitopes independent of any competing response between the epitopes. CTL analysis of individual peptide-pulsed target cells and intracellular cytokine stain for IFN-gamma produced by splenocytes from Lm-LLO-EC1 compared with Lm-LLO-EC2 vaccinated FVB/N mice shows that there is no difference between the responses generated to either of these epitopes. We also show that the avidity of the CD8(+) T cells for either of these epitopes is similar based on the concentration of peptide necessary to mediate similar levels of lysis of target cells. In addition, HER-2/neu DNA vaccination followed by CTL analysis further showed that both of these peptides can emerge as epitopes.

Challenge with mammary tumor cells expressing MHC class II and CD80 prevents the development of spontaneously arising tumors in MMTV-neu transgenic mice

The HER-2/Neu oncogene has been implicated in human and mouse breast cancer. Indeed, transgenic MMTV-neu mice expressing this oncogene from the mammary tumor virus long terminal repeat develop spontaneous mammary tumors and die within 1 year of life. We have expressed the class II transactivator (CIITA) and/or the costimulatory molecule CD80 (B7.1) in a mammary carcinoma cell line (MCNeuA) derived from these mice. Class II transactivator directs the expression of MHC class II and the machinery for antigen processing and presentation by this pathway. When injected into MMTV-neu mice, tumor cells expressing CD80 or CD80 and CIITA, were rejected completely. In addition, following the rejection of dual expressing cells, 75% of the mice were protected against the development of subsequent spontaneous tumors. Cells expressing only CD80 or CIITA were not as effective as antitumor vaccines in preventing the development of spontaneous tumors. Thus, converting cancer cells into antigen presenting cells could represent an effective immunotherapy for breast cancer.

Neu-Induced Retroviral Rat Mammary Carcinogenesis: A Novel Chemoprevention Model for Both Hormonally Responsive and Nonresponsive Mammary Carcinomas

Clinically relevant animal models of mammary carcinogenesis are crucial for the development and evaluation of new breast cancer chemopreventive agents. The neu-induced retroviral rat mammary carcinogenesis model is based on the direct in situ transfer of the activated neu oncogene into the mammary epithelium using a replication-defective retroviral vector. The resulting mammary carcinomas in intact Wistar-Furth rats exhibit a mixed hormonal response in the same proportion as has been observed in women. In intact rats, approximately 50% of mammary carcinomas can be prevented by tamoxifen treatment. In ovariectomized animals, the mammary carcinomas are hormonally nonresponsive and cannot be prevented by tamoxifen. We evaluated the efficacy of retinoic X receptor-selective retinoids (rexinoids) in this novel model of mammary carcinogenesis. The rexinoids LG100268 and bexarotene (LG1069, Targretin) were highly efficacious in the prevention of neu-induced mammary carcinomas. Dietary LG100268 at 100 mg/kg diet decreased tumor multiplicity by 32% (P = 0.0114) in intact rats and 50% (P < 0.0001) in ovariectomized rats. Bexarotene treatment at a dose of 250 mg/kg diet was associated with reductions in tumor multiplicity of 84% (P < 0.0001) and 86% (P < 0.0001) in intact and ovariectomized animals, respectively. In addition to tumor multiplicity, proliferation and apoptosis were modulated by bexarotene treatment independently of estrogen signaling. The neu-induced retroviral rat mammary carcinogenesis model represents a valuable addition to existing rodent chemoprevention models. The model is useful for assessing the efficacy of chemopreventive agents, specifically those compounds that target hormonally nonresponsive tumors.

Targeting Fatty Acid Synthase in Breast and Endometrial Cancer: An Alternative to Selective Estrogen Receptor Modulators?

There is an urgent need to identify and develop a new generation of therapeutic agents and systemic therapies targeting the estradiol (E2)/estrogen receptor (ER) signaling in breast cancer. In this regard, new information on the mechanisms of E2/ER function and/or cross talk with other prosurvival cascades should provide the basis for the development of other ideal anti-E2 therapies with the intent to enhance clinical efficacy, reduce side effects or both. Our very recent assessment of the mechanisms by which cancer-associated increased lipogenesis and its inhibition alters the E2/ER signaling discovered that fatty acid synthase (FASN), the enzyme catalyzing the terminal steps in the de novo biosynthesis of long-chain fatty acids, differentially modulates the state of sensitivity of breast and endometrial cancer cells to E2-stimulated ER transcriptional activation and E2-dependent cell growth and survival: 1) pharmacological inhibition of FASN activity induced a dramatic augmentation of E2-stimulated ER-driven gene transcription, whereas interference (RNAi)-mediated silencing of FAS gene expression drastically lowered E2 requirements for optimal activation of ER transcriptional activation in breast cancer cells; conversely, pharmacological and RNAi-induced inhibition of FASN worked as an antagonist of E2- and tamoxifen-dependent ER transcriptional activity in endometrial adenocarcinoma cells; 2) pharmacological and RNAi-induced inhibition of FASN synergistically enhanced E2-mediated down-regulation of ER protein and mRNA expression in breast cancer cells, whereas specific FASN blockade resulted in a marked down-regulation of E2-stimulated ER expression in endometrial cancer cells; and 3) FASN inhibition decreased cell proliferation and cell viability by promoting apoptosis in hormone-dependent breast and endometrial cancer cells. In this review we propose that, through a complex mechanism involving the regulation of MAPK/ER cross talk as well as critical E2-related proteins including the Her-2/neu (erbB-2) oncogene and the cyclin-dependent kinase inhibitors p21(WAF1/CIP1) and p27(Kip1), a previously unrevealed connection exists between FASN and the genomic and nongenomic ER activities in breast and endometrial cancer cells. From a clinical perspective, we suggest that if chemically stable FASN inhibitors or cell-selective systems able to deliver RNAi targeting FASN gene demonstrate systemic anticancer effects of FASN inhibition in vivo, additional preclinical studies to characterize their anti-breast cancer actions should be of great interest as the specific blockade of FASN activity may also provide a protective means against endometrial carcinoma associated with tamoxifen-based breast cancer therapy.

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]

A DNA vaccine targeting angiomotin inhibits angiogenesis and suppresses tumor growth

Endogenous angiogenesis inhibitors have shown promise in preclinical trials, but clinical use has been hindered by low half-life in circulation and high production costs. Here, we describe a strategy that targets the angiostatin receptor angiomotin (Amot) by DNA vaccination. The vaccination procedure generated antibodies that detected Amot on the endothelial cell surface. Purified Ig bound to the endothelial cell membrane and inhibited endothelial cell migration. In vivo, DNA vaccination blocked angiogenesis in the matrigel plug assay and prevented growth of transplanted tumors for up to 150 days. We further demonstrate that a combination of DNA vaccines encoding Amot and the extracellular and transmembrane domains of the human EGF receptor 2 (Her-2)/neu oncogene inhibited breast cancer progression and impaired tumor vascularization in Her-2/neu transgenic mice. No toxicity or impairment of normal blood vessels could be detected. This work shows that DNA vaccination targeting Amot may be used to mimic the effect of angiostatin.

Timely DNA Vaccine Combined with Systemic IL-12 Prevents Parotid Carcinomas before a Dominant-Negative p53 Makes Their Growth Independent ofHER-2/neuExpression

Double transgenic mice overexpressing the transforming rat HER-2/neu oncogene and the mutated p53, with both dominant-negative and a gain-of-function properties, display early aggressive and metastasizing parotid tumors. Multiple acinar and ductal hyperplasia foci overexpressing the HER-2/neu gene product are evident at wk 5 and progress to poorly differentiated carcinoma by wk 7. Mice die before wk 18 with invasive carcinomas and multiple metastases that no longer express HER-2/neu. A combination of repeated electroporations of plasmids coding for the extracellular and transmembrane domains of the rat HER-2/neu receptor with systemic IL-12 administrations started when the parotids that present diffuse hyperplasia protected all female and 50% of the male mice until the close of the experiment at wk 40. This combined treatment began when multifocal in situ carcinomas that were already present cured 33% of the females and 25% of the males. The most prominent immunologic features associated with the antitumor protection were the production of high titers of anti-HER-2/neu Abs and the nonappearance of cell-mediated cytotoxic reactivity. In conclusion, anti-HER-2/neu vaccination combined with systemic IL-12 control parotid carcinomas as far as p53 mutation makes their growth independent of HER-2/neu expression.

Antitumor activity of an Ets protein, PEA3, in breast cancer cell lines MDA‐MB‐361DYT2 and BT474M1

Polyomavirus enhancer activator 3 (PEA3) is a member of the Ets family of transcription factors. We demonstrated in a previous study that, by downregulating the HER-2/neu oncogene at the transcriptional level, PEA3 can inhibit the growth and development into tumors of HER-2/neu-overexpressing ovarian cancer cells. Here, we establish stable clones of the human breast cancer cell line MDA-MB-361DYT2 that express PEA3 under the control of a tetracycline-inducible promoter. Ectopic expression of PEA3 in this cell line inhibited cell growth and resulted in cell cycle accumulation in the G1 phase. We demonstrate that expression of PEA3 in an orthotopic breast cancer model inhibited tumor growth and prolonged the survival of tumor-bearing mice. In a parallel experiment with another breast cancer cell line, BT474M1, we were unable to obtain stable PEA3-inducible transfectants, suggesting that PEA3 may exert a strong growth inhibition effect in this cell line. Indeed, PEA3 coupled with the liposome SN2 demonstrated therapeutic effects in mice bearing tumors induced by BT474M1. These results provide evidence for the antitumor activity of PEA3 in human breast cancers.

Novel Approach of Human Epidermal Growth Factor Receptor 2 Detection in Noninvasive and Invasive Transitional Cell Carcinoma of the Bladder

In recent years over expression of HER2 has been identified in malignant tumors of organs other than breast. Studies of bladder carcinoma that analyzed HER2 protein expression and gene amplification with a variety of nonstandardized methods have shown heterogeneous results. The results reported vary from 2% to 74% of protein over expression, to 4% to 59% of gene amplification of HER2, possibly due to differences in study design, material selection or laboratory methodology. In the present study archival tissue from 87 patients with noninvasive papillary (25) and invasive (62) TCC was analyzed for amplification of the HER2 gene and over expression of its encoded protein. HER2 protein expression was detected by immunohistochemistry using the HercepTest. Routinely processed paraffin embedded tissue was investigated for HER2 gene amplification using CISH and FISH. Of the invasive 37 (58%) and of the noninvasive 19 (76%) transitional cell carcinomas investigated showed over expression of the HER2 protein (3+ and 2+) using a standardized immunohistochemical method. HER2 gene amplification assays performed on positive cases evaluated by immunohistochemistry were obtained in 81% and 43% of 3+ and 2+ HER2 protein over expressing invasive, respectively, and in 21% of noninvasive papillary bladder tumors. HER2 gene amplification detection results using CISH and FISH showed a concordance of 100%. The occurrence of aneusomy of chromosome 17 in association with HER2 gene amplification was investigated. Validation of the HER2 oncogene in bladder cancer may allow for the potential use of Herceptin(R) antibody therapy. Therefore, the appropriate treatment approach has to be based on reliable and standardized analysis. Our results indicate that CISH could provide an accurate and practical alternative to FISH for the clinical diagnosis of HER2 oncogene amplification in bladder cancer.

HER-2/neu Represses the Metastasis Suppressor RECK via ERK and Sp Transcription Factors to Promote Cell Invasion

Matrix metalloproteinase (MMP) inhibitory proteins may negatively regulate MMP activity to suppress tumor metastasis. In this study, we demonstrate that the HER-2/neu oncogene inhibits the expression of the MMP inhibitor RECK to promote cell invasion. RECK was inhibited via transcriptional repression in B104-1-1 cells, which express constitutively active HER-2/neu. Overexpression of HER-2/neu in NIH/3T3 or HaCaT cells also suppressed RECK expression. Deletion and mutation assays showed that HER-2/neu repressed RECK via the Sp1-binding site localized in the -82/-71 region from the translational start site. DNA affinity precipitation and chromatin immunoprecipitation assays indicated that binding of Sp1 and Sp3 to this consensus site was increased in B104-1-1 cells. We also found that HER-2/neu inhibited RECK via the ERK signaling pathway. Sp1 proteins phosphorylated at Thr453 and Thr739 by ERK bound preferentially to the RECK promoter, and this binding was reversed by HER-2/neu and ERK inhibitors. Furthermore, our data indicate that HER-2/neu obviously increased HDAC1 binding to the Sp1-binding site localized in the -82/-71 region of the RECK promoter. The histone deacetylase inhibitor trichostatin A reversed HER-2/neu-induced inhibition of RECK. HER-2/neu activation was associated with increased MMP-9 secretion and activation. Re-expression of RECK in HER-2/neu-overexpressing cells inhibited MMP-9 secretion and cell invasion. Taken together, our results suggest that HER-2/neu induces the binding of Sp proteins and HDAC1 to the RECK promoter to inhibit RECK expression and to promote cell invasion. Restoration of RECK provides a novel strategy for the inhibition of HER-2/neu-induced metastasis.

HER-2/neu Expression in Primary Breast Cancer With Sentinel Lymph Node Metastasis

Amplification of the protein product of the HER-2/neu oncogene in primary breast cancer specimens is associated with an adverse prognosis. We hypothesized that overexpression of HER-2/neu would predict metastases to the sentinel lymph nodes (SLNs). A retrospective review of a prospective nonrandomized evaluation of 1055 clinically node-negative breast cancer patients undergoing 1063 SLN biopsies was performed. HER-2/neu analysis was performed by immunohistochemistry and, in selected cases, by fluorescence in situ hybridization. Clinical, demographic, surgical, radiological, and pathologic data were analyzed by using generalized estimating equations logistic regression models. Two hundred thirty-two (23.6%) of 985 operations in which the SLN was found at operation resulted in positive nodes. In a multivariate analysis, size (P < .0001) and HER-2/neu overexpression (P = .026) were independent predictors of SLN metastasis. Size is a known predictor of SLN metastasis in the modern SLN era, as it was in the pre-SLN eras. HER-2/neu was found to be significantly predictive of SLN metastasis in our study. We anticipate a future when even the relatively minor procedure of SLN biopsy might be avoided with the predictive information gained from studying the pathology and molecular markers of primary breast cancers.