Neu Oncogene Research Articles

Neu mutation in schwannomas induced transplacentally in Syrian golden hamsters by N-nitrosoethylurea: high incidence but low allelic representation.

Peripheral nerve tumors (PNT) and melanomas induced transplacentally on day 14 of gestation in Syrian golden hamsters by N-nitrosoethylurea were analyzed for activated oncogenes by the NIH 3T3 transfection assay, and for mutations in the neu oncogene by direct sequencing, allele-specific oligonucleotide hybridization, MnlI restriction-fragment-length polymorphism, single-strand conformation polymorphism, and mismatch amplification mutation assays. All (67/67) of the PNT, but none of the melanomas, contained a somatic missense T --> A transversion within the neu oncogene transmembrane domain at a site corresponding to that which also occurs in rat schwannomas transplacentally induced by N-nitrosoethylurea. In only 2 of the 67 individual hamster PNT did the majority of tumor cells appear to carry the mutant neu allele, in contrast to comparable rat schwannomas in which it overwhelmingly predominates. The low fraction of hamster tumor cells carrying the mutation was stable through multiple transplantation passages. In the hamster, as in the rat, specific point-mutational activation of the neu oncogene thus constitutes the major pathway for induction of PNT by transplacental exposure to an alkylating agent, but the low allelic representation of mutant neu in hamster PNT suggests a significant difference in mechanism by which the mutant oncogene acts in this species.

C-myc and Chromosome 8 Centromere Studies of Ovarian Cancer by Interphase FISH

Forty tumor specimens from patients with ovarian cancer were studied for amplification of the c-myc oncogene relative to chromosome 8 centromere number using dual-color FISH. Interphase cytogenetic analysis showed amplification of the c-myc oncogene in 40% (16/40) of tumors using the standard oncogene:centromere ratio method of analysis. Eleven of these showed moderate amplification of c-myc, and 5 samples showed high amplification. Eight of the sixteen (50%) amplified tumors were polysomic centromere 8 as were 14 of the 24 (58%) non-amplified tumors. In previously reported work with these samples, the oncogene HER-2/neu, the chromosome 17 centromere, and the tumor suppressor gene p53 had been studied. When using the standard oncogene:centromere ratio criteria, 5 samples had amplification of both the c-myc and the HER-2/neu oncogenes, 5 samples had HER-2/neu amplification but not c-myc, 11 samples had c-myc amplification but not HER-2/neu, and 19 samples had neither oncogene amplified. The p53 gene was found to be deleted in 22.5% (9/40) of samples. The loss of the p53 gene did not appear to have any clinical correlation. The presence of an extra centromere 8 also did not appear to have any clinical correlation. The Kaplan–Meier survival curve for those patients who have c-myc amplification, while not statistically significant, appears to show a trend toward poorer survival. The survival curve for patients whose tumors have HER-2/neu amplification shows no clinical significance. It is of great interest, however, that the Kaplan–Meier plot of survival for patients whose tumors have amplification of both c-myc and HER-2/neu shows a significant difference (P = 0.047). The median survival times of the doubly amplified patient group and the non-doubly amplified groups were 12 and 43 months, respectively. This is the first study of the oncogene c-myc using FISH. The results suggest that the amplification of c-myc may indicate a poorer patient survival and that the amplification of both c-myc and HER-2/neu in combination may be a better prognostic indicator of poor patient survival.

The HER-2/neu oncogene: prognostic factor, predictive factor and target for therapy

The HER-2/neu oncogene encodes a transmembrane tyrosine kinase receptor with extensive homology to the epidermal growth factor receptor. HER-2/neu has been widely studied in breast cancer. The potential value of HER-2/neu status for the prediction of disease outcome and response to therapy in breast cancer is presented in the light of a series of recently published studies showing a range of impact on the outcome of patients treated with hormonal, cytotoxic and radiation therapies. This review includes the application of serum-based HER-2/neu testing and the use of antibody-based therapies directed against the HER-2/neu protein and their potential to become a new modality for breast cancer treatment.

HER-2/neu gene amplification in stages I-IV breast cancer detected by fluorescent in situ hybridization

PurposeApproximately 25-30% of breast and ovarian carcinomas have amplification of the HER-2/neu oncogene. The aim of the present study was to focus on HER-2/neu gene amplification in different clinical stages of breast cancer in order to (1) determine if fluorescent in situ hybridization (FISH) can be used to detect HER-2/neu gene amplification in different clinical stages of breast cancer, (2) establish whether HER-2/neu gene amplification characterizes a subset of breast cancer in each of these stages, and (3) determine whether a trend for correlation of amplification with the clinical stage of the disease can be detected using the FISH technology. MethodsA total of 40 specimens of formalin-fixed, paraffin-embedded breast cancer tissues were analyzed cytogenetically, in a blinded fashion, for HER-2/neu gene amplification using FISH and the Vysis LSI HER-2/neu Orange and CEP 17 Green DNA dual color probe. The criterion for “high amplification” was an amplification ratio of >4.0, that for “moderate amplification” a ratio between 2.1 and 4.0, and that for “low amplification” a ratio of 1.5–2.0. ResultsUsing a cutoff point of ≥ 1.5, the overall frequency of HER-2/neu gene amplification among stage I tumors was 30% (3 out of 10). Of these, one- third (1 out of 3) showed low amplification, one-third (1 out of 3) were moderately amplified, and one-third (1 out of 3) were highly amplified. The overall frequency of HER-2/neu gene amplification among stage II tumors was 0% (0 out of 10). The overall frequency of HER-2/neu gene amplification among stage III tumors was 10% (1 out of 10). The sole tumor found positive was classified as moderately amplified by our criteria. The overall frequency of HER- 2/neu gene amplification among stage IV tumors was 50% (5 out of 10). Four of the 5 tumors found positive were highly amplified. The overall frequency of gene amplification in the 40 cases studied was 22.5% (9 out of 40 tumors studied). ConclusionAlthough a linear correlation between HER-2/neu amplification and clinical stage cannot be established at this time, it is interesting to note that when stages I and II, and when stages III and IV are combined, respectively, the latter category has a higher amplification frequency than the former. Furthermore, stage IV has the highest frequency (5 out of 10) of HER-2/neu gene amplification than all three lower stages combined (4 out of 30). This is no doubt due to the high frequency of gene amplification observed in stage IV tumors, which, interestingly, also demonstrate high level amplification of HER-2/neu gene copy numbers. Although the biologic and clinical basis for gene amplification is not clear, given the observation that the most aggressive disease stage is associated with the highest frequency of gene amplification and the most high level amplification, further exploration of HER-2/neu as a prognostic marker of poor outcome using FISH is warranted.

HER-2/neu oncogene amplification by fluorescence in situ hybridization in epithelial tumors of the ovary.

HER-2/neu gene amplification and protein overexpression have been associated with prognosis in breast, lung and prostate cancers but have not been extensively studied in ovarian carcinoma. For the study, we selected 5-micron-thick, formalin-fixed, paraffin-embedded tissue sections from 74 cases of ovarian epithelial tumors of low malignant potential and ovarian carcinoma. Tumors were graded and staged and evaluated for amplification of the HER-2/neu gene by fluorescence in situ hybridization. HER-2/neu amplifications was present in 3 of 13 serous, mucinous, and endometrioid epithelial tumors of low malignant potential and in 40 of 61 epithelial carcinomas. In the carcinoma group, amplification did not correlate with stage, grade, or tumor type. Mean follow-up was 31 months; 1 patient with a low malignant potential tumor and 32 patients with carcinomas died of disease. On univariate and multivariate analysis, survival correlated with stage of disease but not with HER-2/neu amplification. HER-2/neu amplification by fluorescence in situ hybridization can be performed on tissue sections of ovarian neoplasms; amplification is uncommon in ovarian tumors of low malignant potential, but is present in 66% of ovarian epithelial carcinomas. HER-2/neu amplification did not predict outcome in ovarian epithelial neoplasia but may have an important role in tumor development.

Vaccination with the extracellular domain of p185neu prevents mammary tumor development in neu transgenic mice.

The HER2/neu oncogene product, p185(HER2/neu), is overexpressed on the surface of many human breast cancers. Strains of transgenic mice have been developed that express the rat neu oncogene in mammary epithelial cells and develop spontaneous mammary tumors that overexpress p185neu. This model provides an ideal system for testing interventions to prevent tumor development. In this study, we immunized neu-transgenic mice with a vaccine consisting of the extracellular domain of p185neu (NeuECD). Immunized mice developed Neu-specific humoral immune responses, as measured by circulating anti-Neu antibodies in their sera, and cellular immune responses, as measured by lymphocyte proliferation to NeuECD in vitro. In addition, the subsequent development of mammary tumors was significantly lower in immunized mice than in controls and vaccine treatment was associated with a significant increase in median survival.

Inhibition of aberrant proliferation and induction of apoptosis in HER-2/neu oncogene transformed human mammary epithelial cells by N-(4-hydroxyphenyl)retinamide.

Epithelial cells from non-cancerous mammary tissue in response to exposure to chemical carcinogens or transfection with oncogenes exhibit hyperproliferation and hyperplasia prior to the development of cancer. Aberrant proliferation may, therefore, represent a modifiable early occurring preneoplastic event that is susceptible to chemoprevention of carcinogenesis. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR), has exhibited preventive efficacy in several in vitro and in vivo breast cancer models, and represents a promising chemopreventive compound for clinical trials. Clinically relevant biochemical and cellular mechanisms responsible for the chemopreventive effects of HPR, however, are not fully understood. Experiments were performed on preneoplastic human mammary epithelial 184-B5/HER cells derived from reduction mammoplasty and initiated for tumorigenic transformation by overexpression of HER-2/neu oncogene, to examine whether HPR inhibits aberrant proliferation of these cells and to identify the possible mechanism(s) responsible for the inhibitory effects of HPR. Continuous 7-day treatment with HPR produced a dose-dependent, reversible growth inhibition. Long-term (21 day) treatment of 184-B5/HER cells with HPR inhibited anchorage-dependent colony formation by approximately 80% (P < 0.01) relative to that observed in the solvent control. A 24 h treatment with cytostatic 400 nM HPR produced a 25% increase (P = 0.01) in G0/G1 phase, and a 36% decrease (P = 0.01) in S phase of the cell cycle. HPR treatment also induced a 10-fold increase (P = 0.02) in the sub-G0 (apoptotic) peak that was down-regulated in the presence of the antioxidant N-acetyl-L-cysteine. Treatment with HPR resulted in a 30% reduction of cellular immunoreactivity to tyrosine kinase, whereas immunoreactivity to p185HER remained essentially unaltered. HPR exposure resulted in time-dependent increase in cellular metabolism of the retinoid as evidenced by increased formation of the inert metabolite N-(4-methoxyphenyl)-retinamide (MPR) and progressive increase in apoptosis. Thus, HPR-induced inhibition of aberrant proliferation may be caused, in part, by its ability to inhibit HER-2/neu-mediated proliferative signal transduction, retard cell cycle progression and upregulate cellular apoptosis.

Genetic mapping of the rat Lcn2 gene to chromosome 3

The expression of rat 24p3, encoded by the Lcn2 gene, has been associated with rat mammary carcinomas initiated by the neu oncogene (Stoesz and Gould, 1995). In this study, we assign the Lcn2 gene to rat chromosome band 3q12 by genetic linkage analysis.

A retrospective FISH study of HER-2/neu oncogene amplification in relapsed and non-relapsed node-negative breast cancer

We previously reported the results of 29 relapsed and 31 non-relapsed cases of node-negative breast cancer studied for HER-2/neu oncogene amplification using FISH and the Vysis HER-2/neu and chromosome 17 control probes (Vysis, Downers Grove, IL). Previous research performed in this laboratory on HER-2/neu amplification in stages I to IV breast cancer indicated that the most aggressive disease stages were associated with the higher frequency of oncogene amplification and the highest level of amplification. These data support the hypothesis that HER-2/neu is a prognostic marker of poor outcome. Thus, a retrospective study of HER-2/neu amplification in relapsed and non-relapsed node-negative breast cancer was initiated to explore further the above question. Out of a total of 46 relapsed cases studied, 13 (28.3%) were found to be amplified. The corresponding frequency of amplification for the controls was 4 (8.7%) of 46 non-relapsed cases. The difference was statistically significant with a p value of <0.05. Although these results still need to be confirmed and extended in other centers, they are quite remarkable and suggest an association between HER-2/neu amplification and disease relapse in patients with node-negative breast cancer. Our results also demonstrate that FISH is a sensitive technique for studying oncogene amplification in archival pathological specimens. (This study was partially funded by Vysis, Inc., Downers Grove, IL).

HER-2/neu oncogene amplification in cervical cancer studied by fluorescent in situ hybridization.

Oncogene amplification, such as HER-2/neu (C-erbB-2), is a manifestation of genetic instability often associated with the genesis and progression of cancer, including cervical cancer. Oncogene overexpression is traditionally studied using immunohistochemistry. We previously reported studies of oncogene amplification in breast cancer using fluorescent in situ hybridization (FISH), where the data support the hypothesis that HER-2/neu is a prognostic marker of poor outcome. To explore further the possible significance of HER-2/neu oncogene amplification in cervical cancer, we conducted a pilot study of 24 cervical cancer cases. The HER-2/neu FISH probe (Vysis, Inc., Downers Grove, IL) was used to measure gene amplification, with a chromosome 17 centromeric probe as an internal control. Out of 24 cases studied, 23 were informative. Of the 23 informative cases, 2 (8.7%) were found to be amplified. The rest (21 out of 23 or 91.3%) were nonamplified. Both amplified cases were invasive adenocarcinoma. Although the sample size of this pilot study may be somewhat small, the data obtained so far clearly demonstrated that detection of oncogene amplification in cervical cancer is not only feasible but is very sensitive, and suggest that further exploration using a larger sample size may be warranted.

HER-2/neu Oncogene Amplification; A Factor for Predicting Response of Platinum-based Combination Chemotherapy in Ovarian Cancers

HER-2/neu Oncogene Amplification; A Factor for Predicting Response of Platinum-based Combination Chemotherapy in Ovarian Cancers

Trisomy 8 in cervical cancer

Cervical cancer is a malignancy which typically occurs at the transformation zone between squamous and glandular epithelium. The vast majority fall into two histologic types: squamous cell and adenocarcinoma. We have previously reported extensively on abnormal chromosome 8 copy number in various cancers, which appears to be an ubiquitous phenomenon. In the present pilot project, we studied chromosome 8 copy number together with a chromosome 17 control using formalin-fixed paraffin-embedded archival cervical cancer tissues. HER-2/neu oncogene amplification was also studied in this sample, as reported in a previous abstract presented at the 1998 Annual Meeting of the American Society of Human Genetics. From a total of 23 cases successfully analyzed, 12 (52%) were found to be trisomic for chromosome 8, and 3 (13%) were found to be trisomic for chromosome 17. Of the trisomic 8 cases, 2 were also trisomic for chromosome 17, which implies that the tumors are either triploid or doubly trisomic. However 10 rumors were trisomic for chromosome 8, but were disomic for chromosome 17. Among the 12 trisomic 8 cases, 1 was also amplified for HER-2/neu whereas 11 were nonamplified. Clinicopathologic parameters of the trisomic and amplified cases will be compared to those of the disomic nonamplified cases. In view of the present results, further exploration using a larger study sample size may be warranted. (This study was partially funded by Vysis, Inc., Downers Grove, IL).

HER-2/neu oncogene amplification in prostate cancer

Oncogene amplification is a manifestation of genetic instability, which has been implicated in the pathogenesis of many cancers. Work performed in this laboratory has focused on HER-2/neu amplification in breast cancer, ovarian cancer, cervical cancer and rhabdomyosarcoma, all of which revealed HER-2/neu amplification in at least a proportion of tumors. In the present project, we studied HER-2/neu oncogene amplification in prostate carcinoma, 244,000 new cases of which are diagnosed yearly, and which affects a significant number of men. Fluorescent in situ hybridization (FISH) using the Vysis HER-2/neu probe with a chromosome 17 centromere control probe was performed on formalin-fixed paraffin-embedded archival prostate cancer tissues. Out of a total of 85 cases studied, 7 cases (8.2%) were found to be amplified. Of those 7 amplified cases, 2 were also trisomic for chromosome 17. Amplification is defined as a signal ratio of HER-2/neu to chromosome 17 of >1.5, after Mark et al. (1998). Clinicopathologic characteristics of the amplified cases versus the nonamplified cases will be compared. Although the results of the present study still need to be confirmed and extended by additional cases in other centers, the data thus far do indicate that a small subset of prostate cancer is characterized by HER-2/neu gene amplification, as in other cancers. (This study was partially funded by Vysis, Inc., Downers Grove, IL).

Sequential immunotyping and genotyping of tumor cells in bone marrow of cancer patients: a model study.

Epithelial cells can be detected in bone marrow or peripheral stem cell preparations of patients with various kinds of cancer and their presence in bone marrow is of prognostic significance. Characterization of these cells has been hampered by their low frequency. Here we present a method that may allow sequential immunophenotyping and genotyping of epithelial cells in bone marrow. To simulate in vivo situations, cells from the colon cancer cell line HT29 were seeded into bone marrow and were first detected by the Fab fragment of the A45-B/B3 anticytokeratin antibody. Expression of Ki67, p53, Her-2/ neu (c-erbB2), and 17-1A could be detected on A45-B/B3-stained cells by immunofluorescence using a fluorescein-labeled anti-mouse immunoglobulin specific for the Fc part of mouse immunoglobulins. Reactivity for all antigens except for Ki67 persisted after A45-B/B3 labeling even when a scoring step for the presence of epithelial cells was performed before proceeding with the immunophenotyping. After immunophenotyping, numerical chromosomal aberrations and amplifications of the Her-2/neu oncogene could be assessed by fluorescence in situ hybridization in the same A45-B/B3-stained cells. This combination of immunophenotyping and genotyping may help in establishing the role of epithelial cells in bone marrow or peripheral stem cell harvests for tumor relapse and formation of metastases.

The HER-2/neu oncogene in breast cancer: prognostic factor, predictive factor, and target for therapy.

The HER-2/neu oncogene encodes a transmembrane tyrosine kinase receptor with extensive homology to the epidermal growth factor receptor. HER-2/neu has been widely studied in breast cancer. In this review, the association of HER-2/neu gene and protein abnormalities studied by Southern and slot blotting, immunohistochemistry, enzyme immunoassays, and fluorescence in situ hybridization with prognosis in breast cancer is studied in depth by review of a series of 47 published studies encompassing more than 15,000 patients. The relative advantages of gene amplification assays and frozen/fresh tissue immunohistochemistry over paraffin section immunohistochemistry are discussed. The significance of HER-2/neu overexpression in ductal carcinoma in situ and the HER-2/neu status in uncommon female breast conditions and male breast cancer are also considered. The potential value of HER-2/neu status for the prediction of response to therapy in breast cancer is presented in the light of a series of recently published studies showing a range of impact on the outcome of patients treated with hormonal, cytotoxic, and radiation therapies. The evidence that HER-2/neu gene and protein abnormalities in breast cancer predict resistance to tamoxifen therapy and relative sensitivity to chemotherapy regimens including adriamycin is presented. The review will also evaluate the status of serum-based testing for circulating the HER-2/neu receptor protein and its ability to predict disease outcome and therapy response. In the final section, the review will briefly present preliminary data concerning the use of antibody-based therapies directed against the HER-2/neu protein and their potential to become a new modality for breast cancer treatment. The recently presented phase III clinical trial evidence that systemic administration of anti-HER2 antibodies (Herceptin), alone and in combination with cytotoxic chemotherapy in patients with HER-2/neu overexpressing primary tumors, can increase the time to recurrence and overall response rates in metastatic breast cancer is reviewed.

The her-2/neu oncogene stimulates the transcription of N-acetylglucosaminyltransferase V and expression of its cell surface oligosaccharide products.

Malignant transformation is associated with changes in the glycosylation of cell surface proteins. For example, the N-linked oligosaccharides containing the [GlcNAc beta(1,6)Man] branch are increased after transformation of many cell types by a number of tumor viruses and oncogenes which induce the expression of N-acetylglucosaminyl-transferase V (GlcNAc-T V), the enzyme that adds this branch. A large percentage of human breast carcinomas have increased N-linked beta(1,6) branches on glycoproteins, while up to 30% of breast carcinomas have amplified the oncogene her-2/neu (erb-B2). We tested the hypothesis that expression of her-2/neu stimulates GlcNAc-T V gene expression and increases the beta(1,6) branching of N-linked oligosaccharides. We found that neu-transformed NIH3T3 cells have a threefold increase in GlcNAc-T V enzyme activity and increased beta(1,6) branching on a specific set of glycoproteins. Promoter/reporter experiments showed that her-2/neu stimulates transcription from the human GlcNAc-T V promoter and that the her-2/neu response element was located about 400 bp 5' of the transcription initiation site and includes three Ets transcription factor binding sequences. Co-transfections with dominant-negative Raf and Ets expression plasmids demonstrated that the transcriptional activation of the GlcNAc-T V promoter by neu is mediated by the Ras-Raf-Ets signal transduction pathway.

Interleukin 12-mediated prevention of spontaneous mammary adenocarcinomas in two lines of Her-2/neu transgenic mice.

The ability of interleukin (IL)-12 to prevent tumors when administered to individuals with a genetic risk of cancer was studied in two lines of transgenic mice expressing rat HER-2/neu oncogene in the mammary gland. Female BALB/c (H-2(d)) mice carrying the activated HER-2/ neu oncogene show no morphological abnormalities of the mammary gland until 3 wk of age. They then progress through atypical hyperplasia to in situ lobular carcinoma and at 33 wk of age all 10 mammary glands display invasive carcinomas. Adult FVB mice (H-2(q)) carrying the HER-2/neu protooncogene develop mammary carcinomas with a longer latency (38-49 wk) and a lower multiplicity (mean of 2.6 tumors/mice). Treatment with IL-12 (5 daily intraperitoneal injections, 1 wk on, 3 wk off; the first course with 50 ng IL-12/day, the second with 100 ng IL-12/day) begun at 2 wk of age in BALB/c mice and at 21 wk of age in FVB mice markedly delayed tumor onset and reduced tumor multiplicity. Analogous results were obtained in immunocompetent and permanently CD8(+) T lymphocyte-depleted mice. In both transgenic lines, tumor inhibition was associated with mammary infiltration of reactive cells, production of cytokines and inducible nitric oxide synthase, and reduction in microvessel number, in combination with a high degree of hemorrhagic necrosis.

The HER‐2/ neu Oncogene in Breast Cancer: Prognostic Factor, Predictive Factor, and Target for Therapy

The HER-2/neu oncogene encodes a transmembrane tyrosine kinase receptor with extensive homology to the epidermal growth factor receptor. HER-2/neu has been widely studied in breast cancer. In this review, the association of HER-2/neu gene and protein abnormalities studied by Southern and slot blotting, immunohistochemistry, enzyme immunoassays, and fluorescence in situ hybridization with prognosis in breast cancer is studied in depth by review of a series of 47 published studies encompassing more than 15,000 patients. The relative advantages of gene amplification assays and frozen/fresh tissue immunohistochemistry over paraffin section immunohistochemistry are discussed. The significance of HER-2/neu overexpression in ductal carcinoma in situ and the HER-2/neu status in uncommon female breast conditions and male breast cancer are also considered. The potential value of HER-2/neu status for the prediction of response to therapy in breast cancer is presented in the light of a series of recently published studies showing a range of impact on the outcome of patients treated with hormonal, cytotoxic, and radiation therapies. The evidence that HER-2/neu gene and protein abnormalities in breast cancer predict resistance to tamoxifen therapy and relative sensitivity to chemotherapy regimens including adriamycin is presented. The review will also evaluate the status of serum-based testing for circulating the HER-2/neu receptor protein and its ability to predict disease outcome and therapy response. In the final section, the review will briefly present preliminary data concerning the use of antibody-based therapies directed against the HER-2/neu protein and their potential to become a new modality for breast cancer treatment. The recently presented phase III clinical trial evidence that systemic administration of anti-HER2 antibodies (Herceptin&reg;), alone and in combination with cytotoxic chemotherapy in patients with HER-2/neu overexpressing primary tumors, can increase the time to recurrence and overall response rates in metastatic breast cancer is reviewed.

Tyrosine kinase inhibitors, emodin and its derivative repress HER-2/neu-induced cellular transformation and metastasis-associated properties.

We have previously shown that emodin suppresses tyrosine kinase activity of HER-2/neu-encoded p185neu receptor tyrosine kinase. In this study, we examine the relationship between the chemical structure and the activity of emodin and nine derivatives, and identified that one methyl, one hydroxy, and one carbonyl functional groups are critical for the biological activities of emodin. We also found that one of the derivatives 10-(4-acetamidobenzylidene)-9-anthrone (DK-V-47) is more effective than emodin in repressing the tyrosine phosphorylation of p185neu and in inhibiting the proliferation and transformation of HER-2/neu-overexpressing human breast cancer cells. Using mutation-activated HER-2/neu transformed 3T3 cells, we also investigated whether emodin and DK-V-47 can inhibit malignant transformation induced solely by the HER-2/neu oncogene. We found that DK-V-47 is more potent than emodin in suppressing transformation phenotypes of activated HER-2/neu transformed 3T3 cells including anchorage-dependent and -independent growth, metastasis-associated properties. These results clearly indicate that the inhibition of p185neu tyrosine kinase by both emodin and DK-V-47 is capable of suppressing the HER-2/neu associated transformed phenotypes including the ability to induce metastatic potential. Our results also support the chemotherapeutic implications of the use of either emodin or DK-V-47 to target HER-2/neu-overexpressing cancer cells.

Involvement of tyrosine phosphorylation of p185cerbB2neu in tumorigenicity induced by x‐rays and the neu oncogene in human breast epithelial cells

Ionizing radiation is the exogenous agent best proven to induce breast cancer. c-erbB2/neu amplification and overexpression are known to occur in breast cancer and are correlated with aggressive tumor growth and poor prognosis. We have developed simian virus 40–immortalized cell lines from normal human breast epithelial cells (HBECs) with luminal and stem-cell characteristics. In this study, we examined whether x-rays and a mutated neu oncogene are capable of inducing tumorigenicity in these cells. The results indicated that x-rays were effective in converting immortal non-tumorigenic HBECs to weakly tumorigenic cells that then could be transformed to highly tumorigenic cells by the neu oncogene. The in vitro growth of these tumorigenic cells was significantly faster than that of the parental non-tumorigenic cells in growth factor– and hormone-supplemented or -depleted media. The neu oncogene, however, had no tumorigenic effect on immortal non-tumorigenic cells. The expression of p185c-erbB2/neu was elevated in neu-transduced immortal or weakly tumorigenic cell lines. However, only in the latter was p185c-erbB2/neu found to be phosphorylated at tyrosine residues. Thus, x-rays appear to induce a genetic alteration that confers weak tumorigenicity on immortal HBECs and interacts with p185c-erbB2/neu directly or indirectly to give rise to fast-growing tumors. Mol. Carcinog. 21:225–233, 1998. © 1998 Wiley-Liss, Inc.