Expression and activation of erbB-2 and epidermal growth factor receptor in lung adenocarcinomas.

ErbB-2 and EGFR (epidermal growth factor receptor) are expressed in lung adenocarcinomas and associated with a poor prognosis. Immunocytochemical analysis revealed erbB-2 and EGFR coexperession as a characteristic feature of most lung adenocarcinomas, and at levels of receptor expression present in bronchial epithelial cells. In primary lung tumours and cell lines, erbB-2 detected using Western blot analysis demonstrated low-level phosphotyrosine staining of the 185 kDa band, as compared with breast cancer cell lines. A549 and A427 lung adenocarcinoma cells treated with neu differentiation factor (NDF) showed increased erbB-2 phosphotyrosine staining, but to a much lesser extent than breast cancer cells. The lung cells were examined for expression of the potential autocrine growth factors NDF and transforming growth factor alpha (TGF-alpha) by Northern blot analysis. Both NDF and TFG-alpha mRNA were abundantly expressed in the A549 cells. NDF mRNA was highest during active cell proliferation and decreased in confluent cells or after treatment with the growth-inhibitory steroid dexamethasone. Primary tumours and cell lines expressed EGFR, showing higher basal level phosphotyrosine staining than erbB-2. Treatment with NDF and EGF (epidermal growth factor) stimulated cell growth, and in A549 cells the presence of both factors provided an additive increase in cell growth. The growth stimulus that ligand-activated erbB-2 and EGFR provides to lung adenocarcinoma cells may establish a background of continued cell proliferation over which other critical transforming events may occur.     

Production of epidermal growth factor related ligands in tumorigenic and benign human lung epithelial cells.

We recently demonstrated that human lung epithelial cells, overexpressing ErbB-2, formed tumors in nude mice only when high levels of transforming growth factor alpha (TGFalpha) were produced. Cells transfected with a TGFalpha antisense vector failed to form tumors in nude mice. In order to further evaluate the importance, for tumorigenicity, of TGFalpha and its stimulation of ErbB family signalling, the production of other EGF family growth factors by these human lung epithelial cells was studied. We demonstrate for the first time that both tumorigenic and non-tumorigenic human lung epithelial cells produced, in addition to TGFalpha, amphiregulin, betacellulin, heparin-binding EGF and heregulin. These data suggest that human lung epithelial cells have the potential for multifactorial modulation of ErbB receptor family signalling through control of ligand as well as receptor production. In this system, the probable importance of TGFalpha-stimulated signaling for tumorigenicity is supported by its 13-fold higher production in tumorigenic as compared with non-tumorigenic cells and the 2-fold or lower differences observed in production of the other epidermal growth factor (EGF) family ligands.     

Oncogenic potential of erbB-2 in human mammary epithelial cells.

Introduction of the normal erbB-2 gene into immortalized human mammary epithelial cells (184B5) by transfection conferred a growth advantage to these cells both in vitro and in vivo. The 184B5 cells overexpressing erbB-2 formed colonies in semi-solid medium, frequently induced transient nodules in athymic mice and produced progressive tumors in vivo at a low frequency. Those tumors which did arise from erbB-2-transfected cells displayed substantially higher levels of normal gp185erb-2 protein when compared to the original transfectants, consistent with their selection for increased erbB-2 expression. Introduction of genes encoding genetically altered erbB-2 molecules into 184B5 cells increased their colony-forming efficiency and converted the cells to a tumorigenic phenotype at a high frequency. When the biological and biochemical properties of human mammary carcinoma cell lines known to overexpress erbB-2 were compared to the transfected 184B5 lines, they behaved most like those overexpressing the normal erbB-2 protein. Results indicate that overexpression of normal erbB-2 may directly contribute to the transformation of human mammary epithelium if sufficient levels of erbB-2 protein are expressed or if the erbB-2 gene is genetically altered.     

Targeted inhibition of tumour cell growth by a bispecific single-chain toxin containing an antibody domain and TGF alpha.

Overexpression of the epidermal growth factor receptor (EGFR) and ErbB-2 has been observed in a variety of human tumours, making these receptors promising targets for directed tumour therapy. Since many tumour cells express both ErbB-2 and EGFR and these receptors synergise in cellular transformation, therapeutic reagents simultaneously binding to ErbB-2 and EGFR might offer advantages for tumour therapy. We have previously described the potent anti-tumoral activity of a bispecific antibody toxin that contains ErbB-2- and EGFR-specific single-chain Fv (scFv) domains. Here we report the construction and functional characterisation of a novel bispecific recombinant toxin, scFv(FRP5)-TGF alpha-ETA. The fusion protein consists of the antigen-binding domain of the ErbB-2-specific MAb, FRP5, and the natural EGFR ligand, TGF alpha, inserted at different positions in truncated Pseudomonas exotoxin A. ScFv(FRP5)-TGF alpha-ETA protein displayed binding to EGFR and ErbB-2, thereby inducing activation of the receptors, which was dependent on the cellular context and the level of EGFR and ErbB-2 expression. The bispecific molecule was cytotoxic in vitro for tumour cells expressing various levels of the target receptors. In vivo scFv(FRP5)-TGF alpha-ETA potently inhibited the growth of established A431 tumour xenografts in nude mice.     

Epidermal growth factor receptor vIII enhances tumorigenicity in human breast cancer

Epidermal growth factor receptor vIII (EGFRvIII) is a tumor-specific, ligand-independent, constitutively active variant of the EGFR. Its expression has been detected in gliomas and various other human malignancies. To more fully characterize the function and potential biological role of EGFRvIII in regulating cell proliferation and in tumorigenesis, we transfected EGFRvIII cDNA into a nontumorigenic, interleukin 3 (IL-3)-dependent murine hematopoietic cell line (32D cells). We observed 32D cells expressing high levels of EGFRvIII (32D/EGFRvIII P5) to be capable of abrogating the IL-3-dependent pathway in the absence of ligands. In contrast, the parental cells, 32D/EGFR, 32D/ErbB-4, and 32D/ErbB-2+ErbB-3 cells, all depended on IL-3 or EGF-like ligands for growth. 32D/EGFRvIII P5 cells subjected to long-term culture conditions in the absence of IL-3 revealed further elevation of EGFRvIII expression levels. These results suggested that the IL-3-independent phenotype is mediated by EGFRvIII. The level of expression is a critical driving force for the IL-3-independent phenotype. Dose-response analysis revealed 32D/EGFRvIII cells to require 500-fold higher concentrations (50 ng/ml) of EGF to further stimulate the EGF-mediated proliferation than in the 32D/EGFR cells (100 pg/ml). Similar effects were also observed in beta-cellulin-mediated proliferation. Moreover, 32D cells expressing high levels of EGFRvIII formed large tumors in nude mice, even when no exogenous EGF ligand was administered. In contrast, no tumors grew in mice injected with 32D/EGFR, 32D/ErbB-4, and 32D/ErbB-2+ErbB-3 cells or low-expressing clone 32D/EGFRvIII C2 cells or the parental 32D cells. The changes of the ligand specificity support the notion for an altered conformation of EGFRvIII to reveal an activated ligand-independent oncoprotein with tumorigenic activity analogous to v-erbB. These studies clearly demonstrate that EGFRvIII is capable of transforming a nontumorigenic, IL-3-dependent murine hematopoietic cell line (32D cells) into an IL-3-independent and ligand-independent malignant phenotype in vitro and in vivo. To delineate the biological significance of EGFRvIII in human breast cancer, we expressed EGFRvIII in the MCF-7 human breast cancer cell line. Expression of EGFRvIII in MCF-7 cells produced a constitutively activated EGFRvIII receptor. Expression of EGFRvIII in MCF-7 cells also elevated ErbB-2 phosphorylation, presumably through heterodimerization and cross-talk. These MCF-7/EGFRvIII transfectants exhibited an approximately 3-fold increase in colony formation in 1% serum with no significant effect observed at higher percentages of serum. A similar result was also seen in anchorage-dependent assays. Furthermore, EGFRvIII expression significantly enhanced tumorigenicity of MCF-7 cells in athymic nude mice with P < 0.001. Collectively, these results provide the first evidence that EGFRvIII could play a pivotal role in human breast cancer progression.     

Monoclonal antibodies directed to the erbB-2 receptor inhibit in vivo tumour cell growth.

Four monoclonal antibodies (MAbs) specific for the extracellular domain of the human erbB-2/HER2 protein (FRP5, FSP16, FWP51 and FSP77) have been isolated (Harwerth et al., J. Biol. Chem., 267, 15160-15167, 1992). In this paper we describe the effects of erbB-2 specific MAb administration on the tumorigenic growth of human erbB-2 transformed NIH3T3 cells implanted into athymic nude mice. Two antibodies, FWP51 and FSP77, inhibited the onset of tumour growth, while the administration of FRP5 and FSP16 did not affect tumour growth. In addition, administration of MAbs FWP51 and FSP77 led to a retardation in the growth of established tumours. Treatment was not curative in that tumours regrew within two weeks of the final treatment. The administration of a combination of MAbs FWP51 and FSP77 which react with two distinct regions on the erbB-2 molecule was more effective than treatment with either MAb alone. The two growth-inhibitory antibodies were also effective in the treatment of tumours established from SKOV3 cells, a human ovarian tumour cell line with high levels of the erbB-2 protein. The effect of the MAbs on the anchorage-independent growth of erbB-2 transformed cells and on erbB-2 receptor turnover was also measured.     

Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumorigenicity in ovarian cancer cells

Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness.     

Constitutive overexpression of a 89 kDa heat shock protein gene in the HBL100 human mammary cell line converted to a tumorigenic phenotype by the EJ/T24 Harvey-ras oncogene

The non tumorigenic human mammary cell line HBL100 has been transformed by the EJ/T24 human bladder carcinoma Harvey(Ha)-ras oncogene. Six cell lines were established from transformed colonies. They all expressed a high level of the ras oncogene and were tumorigenic in athymic nude mice. During an in vivo passage in animals, tumour cells presenting a growth advantage were selected, and some of the tumours revealed an amplification of the transfected ras sequences. Using this model of human cell transformation, we have isolated a cDNA clone corresponding to a heat shock protein gene (hsp89 alpha). This gene, normally transcribed at a higher rate in response to serum stimulation, was found to be constitutively overexpressed in ras-transformed HBL100 cells. In contrast, a closely related hsp gene (hsp89 beta), remained sensitive to serum stimulation, in both untransformed and ras-transformed HBL100 cells. Thus, the regulation of the expression of the hsp89 genes, upon serum stimulation, involves ras-dependent and ras-independent pathways. Constitutive overexpression of the murine homolog of the hsp89 alpha was observed in NIH3T3 cells transformed by the three ras oncogenes, but not with some other oncogenes. Therefore, alteration of the hsp89 alpha gene expression is not a general characteristic of transformed cells, but seems to be linked to ras transformation.     

Clinical relevance of erbB-1 and -2 oncogenes in oral carcinomas

To gain a better understanding of molecular changes in oral squamous cell carcinomas, we tested fresh tumour specimens from 110 patients for erbB-1 and -2 oncogene aberrations using the competitive differential polymerase chain reaction. The significance of established tumour characteristics such as TNM stage, differentiation and oncogene aberrations for tumour progression were analyzed. ErbB-2 amplification with a gene copy number > 1.6 in tumour tissue and erbB-1 deletion with a gene copy number < 0.4 in tumour-surrounding mucosa are of clinical relevance and indicate an early tumour recurrence or metastasis (p < 0.05). In T1/T2 tumours an erbB-2 gene dosage study allows differentiation between tumours with high or low risk for early progression. In a multivariate statistical analysis T stage (p < 0.01) and erbB-2 amplification in tumour material (p < 0.05) were independent prognostic variables.     

Androgenic regulation of growth factor and growth factor receptor expression in the CWR22 model of prostatic adenocarcinoma.

The effects of androgen manipulation on epidermal growth factor (EGF) receptor, p185erbB-2 and transforming growth factor-alpha (TGF-alpha) levels were examined in prostatic adenocarcinoma. Male nude mice were inoculated with the CWR22 androgen-dependent human prostatic tumor or an androgen-independent (CWR22R) derivative. Mice with CWR22 tumors were castrated and subsequently killed at 3, 7, 21, 28 or 42 days post-castration. Other CWR22-bearing mice received s.c. testosterone pellets at 21 days post-castration and were killed 7 days later. EGF receptor, p185erbB-2 and TGF-alpha levels were examined by immuno-histochemistry. Strong EGF receptor and p185erbB-2 immunostaining was detected in CWR22 tumors from intact controls. EGF receptor immunostaining decreased by 65% to 70% at 21 to 42 days post-castration. Testosterone treatment at 21 to 28 days post-castration resulted in a 2-fold increase in EGF receptor immunostaining. p185erbB-2 immunostaining within CWR22 tumors did not decrease following castration and, in fact, was slightly increased at 7 days post-castration. The effects of castration on EGF receptor and p185erbB-2 levels were confirmed by Western blot analysis. Fewer than 10% of CWR22 tumor cells demonstrated strong TGF-alpha immunostaining, and androgen manipulation did not effect TGF-alpha immunostaining. In contrast, 30% of androgen-independent CWR22R tumor cells were strongly immunostained for TGF-alpha. Our findings indicate that EGF receptor levels, but not p185erbB-2 levels, are strongly dependent on testosterone in CWR22 tumors. The co-localization of TGF-alpha and the EGF receptor in CWR22R tumors suggests that these factors may constitute an autocrine pathway that regulates androgen-independent growth.     

Growth factor production by human colon carcinoma cell lines

Conditioned media collected under serum-free conditions over 24 to 48 h from 18 human colon adenocarcinoma cell lines were analyzed for transforming growth factor, types alpha and beta (TGF-alpha and -beta), and platelet-derived growth factor in assays for anchorage-independent growth and radioreceptor competition. Detectable levels of TGF-alpha, TGF-beta, and platelet-derived growth factor were produced by 17, 16, and 6 cell lines, respectively. Three liters of conditioned medium from highly tumorigenic (HT-29, DLD-1, and SW620) and nontumorigenic (SKCO-1) colon cell lines and from nonneoplastic rat kidney (NRK-52E) and small intestinal (IEC-6) epithelial cells were purified by high-performance liquid chromatography and assayed for TGF-alpha- and TGF-beta-like activity. The highly tumorigenic colon cell lines produced 10- to 45-fold (soft agar), 19- to 90-fold (radioreceptor), and 4- to 35-fold (radioimmunoassay) more TGF-alpha activity compared to the nonneoplastic rat intestinal (IEC) epithelial cells. NRK-52E did not produce detectable TGF-alpha activity. Radioimmunoassay analysis of peak fractions revealed only TGF-alpha immunoreactivity; epidermal growth factor was not detected. Levels of TGF-beta-like material in the colon carcinoma populations were comparable (HT-29) or elevated (DLD-1, SW620) only 3- to 4-fold (soft agar) or 1- to 3-fold (radioreceptor binding) compared to IEC cells or NRK-52E. Growth factor production is an ubiquitous property of colon carcinoma cell lines maintained in vitro and is consistent with this class of molecule, playing a contributory role in regulating cell growth.     

Additive effects of c-erbB-2, c-Ha-ras, and transforming growth factor-alpha genes on in vitro transformation of human mammary epithelial cells.

MCF-10A cells are a spontaneously immortalized untransformed human mammary epithelial cell line. We have previously shown that overexpression of a human point-mutated c-Ha-ras proto-oncogene, the rat c-neu (c-erbB-2) proto-oncogene, or the human transforming growth factor-alpha (TGF-alpha) gene in MCF-10A cells leads to in vitro transformation of such cells. To ascertain whether the introduction of two of these genes into MCF-10A human mammary epithelial cells induces a completely tumorigenic phenotype, we infected MCF-10A Ha-ras and MCF-10A TGF-alpha cells with a recombinant retroviral vector containing the human c-erbB-2 proto-oncogene and the hygromycin-resistance gene. Ten MCF-10A TGF-alpha/c-erbB-2 (MCF-10A TE) and 10 MCF-10A Ha-ras/c-erbB-2 (MCF-10A HE) hygromycin-resistant clones were randomly selected and expanded into cell lines. MCF-10A TE and MCF-10A HE clones expressed a 10-fold to 40-fold increase in p185 erbB-2 protein levels compared with parental uninfected cells. These cells exhibited a fourfold increase in their growth rate in serum-free medium and showed a strongly reduced mitogenic response to exogenous epidermal growth factor or TGF-alpha compared with MCF-10A cells. Moreover, both MCF-10A TE and MCF-10A HE clones exhibited a fivefold to 20-fold higher cloning efficiency in soft agar than MCF-10A Ha-ras, MCF-10A c-erbB-2, or MCF-10A TGF-alpha clones. However, neither MCF-10A TE nor MCF-10A HE cells were able to grow as tumors in vivo when they were injected into nude mice. These results suggest that c-Ha-ras, c-erbB-2, and TGF-alpha genes have an additive effect on the in vitro transformation of an immortalized human mammary epithelial cell line, but that additional genetic changes such as activation of other proto-oncogenes or inactivation of a tumor suppressor gene may be necessary to elicit a fully tumorigenic phenotype.     

Critical role for D-type cyclins in cellular transformation induced by E6/E7 of human papillomavirus type 16 and E6/E7/ErbB-2 cooperation

Recently, we reported that E6/E7 of human papillomavirus (HPV) type 16 cooperates with the ErbB-2 receptor to induce cellular transformation of human normal oral epithelial (NOE) and mouse normal embryonic fibroblast (NEF) cells. Furthermore, we demonstrated that cyclin D1 is essential for this transformation induced by E6/E7 and E6/E7/ErbB-2 cooperation using cyclin D1 antisense and knockout (D1(-/-)) cells. To determine the role of all D-type cyclins (D1, D2 and D3) in E6/E7/ErbB-2 cooperation, we examined the effects of E6/E7, ErbB-2 alone and E6/E7/ErbB-2 together in NEF, NEF-D1(-/-), NEF-D2(-/-) and NEF-D3(-/-) cells. We confirm that NEF-E6/E7 and NEF-E6/E7/ErbB-2, but not NEF-ErbB-2 cells, induce colony formation in soft agar and tumor formation in nude mice. We report that E6/E7, ErbB-2 and E6/E7/ErbB-2 together all fail to induce neoplastic transformation of D1(-/-) and D2(-/-) cells in vitro and in vivo. In contrast, E6/E7/ErbB-2 together but neither E6/E7 nor ErbB-2 alone provoke cellular transformation of D3(-/-) cells. Nevertheless, D3(-/-)E6/E7/ErbB-2 cells resulted in up to a 60 and 50% decrease in colony and tumor formation in soft agar and nude mice, respectively, compared with NEF-E6/E7/ErbB-2 cells. Furthermore, using cyclin D2 small interfering RNA we inhibited tumor and colony formation of the human NOE-E6/E7-ErbB-2-transformed cell line; in contrast, cyclin D3 small interfering RNA repressed approximately 50% of colony and 40% of tumor formation of E6/E7/ErbB-2 cooperation in this cell line. These data suggest that cyclins D1, D2 and D3 (to a lesser extent) are important downstream mediators of the cellular transformation induced by E6/E7 and E6/E7/ErbB-2 cooperation in normal cells. Our data imply that anti-D-type cyclin therapies are important in the treatment of human cancers expressing high-risk HPV or HPV/ErbB-2.     

The c-Src tyrosine kinase associates with the catalytic domain of ErbB-2: implications for ErbB-2 mediated signaling and transformation

c-Src associates with and is activated by the ErbB-2 receptor tyrosine kinase, but is unable to bind the EGFR. Although c-Src has been found to interact directly and specifically with the ErbB-2 receptor, the significance of this interaction is unclear. Using both chimeric receptor and site-directed mutagenesis approaches, the region of interaction of c-Src on ErbB-2 was identified. Significantly, EGFR could be converted into a receptor capable of binding c-Src by replacement of a catalytic domain of ErbB-2. We further demonstrated that MDCK cells that express mutant EGFR that are competent in c-Src recruitment lose epithelial polarity in organoid cultures, whereas cells overexpressing the wild-type EGFR retain a polarized phenotype. ErbB-2-dependent activation of c-Src results in disruption of epithelial cell-cell contacts leading to cell dispersal that correlates with the re-localization of phospho-MAPK to focal adhesions. Taken together, these observations suggest that recruitment of c-Src to these closely related EGFR family members plays a critical role in modulating cell polarity.     

裸小鼠高致瘤性K562-n细胞系的生物学特性研究

目的：探讨高致瘤性K562－n细胞系在胸腺缺陷裸小鼠体内致瘤的细胞生物学机理。方法：通过极限稀释法建立K562－n细胞系致瘤率不同的克隆株，并以K562细胞为对照，对各克隆株进行裸小鼠体内外生物学特性的比较研究，结果：高致瘤性的T562－B、K562－C、K562－E克隆株（致瘤率≥5／6），其软琼脂培养集落形成率，特别是大集落形成率，较对照K562细胞显著增高，（P＜0．01），而无致瘤性或低致瘤性的K562－A、K562－D克隆株（致瘤率≤2／6），其集落形成率与对照无显著差别（P＞0．05）；超微结构观察提示高致瘤性克隆株，常染色质成分增多，异染色质少见，胞浆内微丝增多且排列紊乱；流式细胞仪细胞周期分布分析显示高致瘤性的克隆株S期细胞比例增加。实验结果还表明裸小鼠NK细胞对高致瘤性克隆株的杀伤活性显著低于对照的K562细胞（P＜0．01），而低致瘤性克隆株与对照无显著差别（P＞0．05）；组织病理观察显示低瘤性的克隆株及对照K562细胞，所成裸小鼠肿瘤组织内有大量的炎性细胞浸润，大量肿细胞被杀伤，血供丰富区尤甚，而高致瘤性克隆株所致肿瘤组织内炎性细胞浸润少见，肿瘤组织增殖旺盛，结论：K562－n细胞系的裸小鼠高致瘤机理在于：1．软琼脂集落形成率增高，2．细胞增殖和DNA合成活跃，涉及细胞周期调节因素和细胞骨架成分的改变，3．对裸小鼠NK细胞杀伤的耐受性增强，所接种宿主的免疫力受到抑制，形成肿瘤组织内炎性细胞浸润减少。     

Human herpesvirus-6: tumorigenicity and tumor infiltrating lymphocytes.

We previously reported that human herpes-virus-6 (HHV-6) genome (strain GS) and a cloned subfragment (pZVH14) transfected NIH 3T3 cells, induced foci of transformation with a frequency significantly above the background level. The transformed cells produced tumors in nude mice and immunocompetent (Swiss) mice. In the current study, nude mice tumors were passed into Swiss mice and more aggressive tumors (G-2TS and 14-2TS derived from HHV-6 genome and pZVH14 DNA, respectively) were produced. 14-2TS tumors caused lung metastasis upon intravenous injection. In the case of subcutaneously growing aggressive tumors, tumor infiltrating lymphocytes (TIL) were isolated and characterized as T cells but lacked tumor specific killing as monitored by 51Cr-release assays. TIL lost activity at day 52 in a 4 h assay (but not in a 19 h assay) against the autologous tumor, but not a Maloney virus induced tumor (Yac-1). These studies indicate that an established nontumorigenic fibroblast cell line, when transfected with pZVH14 DNA of HHV-6, acquires both tumorigenic and metastatic potential and tumor bearing hosts mount immune response against such tumors.