The role of transforming growth factor alpha production and ErbB-2 overexpression in induction of tumorigenicity of lung epithelial cells

Abstract

Summary Over-expression of erbB-2 is associated with shortened survival of patients with lung adenocarcinomas. We demonstrated that human lung epithelial cells, overexpressing erbB-2, formed tumours in nude mice only when high levels of transforming growth factor (TGF)-a were produced (E6T cells). To define the role that TGF-a production played in induction of tumorigenicity, a non-tumorigenic TGF-a-negative clone of ErbB-2 overexpressing cells (E2 cells) was transfected with an expression vector for TGF-a (E2a cells). Transfected clones produced TGF-a at 11-25% of the level produced by the E6T cell line. Tumorigenic E6T cells transfected with a TGF-a antisense vector (E6TA cells) expressed only 6% of the TGF-a level of the parental cells. Clones of E6T, E6TA, E2 and E2a were inoculated into athymic nude mice to measure tumorigenic potential. E6T cells formed tumours with a 70% efficiency. E2, E6TA and E2a cells failed to form tumours. The levels of EGFR were similar in non-tumorigenic E2 and tumorigenic E6T cells but higher in E2a and E6TA cells, and ErbB-2 were greatly overexpressed in an E2a clone. In vitro, ErbB-2 co-immunoprecipitated with EGFR in lysates of unstimulated E6T and E2a TGF-a-producing cells, indicating that the lower TGF-a levels were sufficient to induce in vitro heterodimerization. These studies suggest that induction of the tumorigenic phenotype depends on achieving a threshold level of TGF-a sufficient to activate downstream signalling by ErbB-2 containing active heterodimers.

HER2/neu: an increasingly important therapeutic target. Part 2: Distribution of HER2/neu overexpression and gene amplification by organ, tumor site and histology

“…• [179,180], as acquisition of HER2/neu overexpression follows disruption of p53 function and precedes acquisition of ras mutations [181]. Surprisingly, overexpression is associated with lower stage and grade of tumor [182].…”

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

“…We previously demonstrated the presence of constitutive ErbB-1/ErbB-2 heterodimers in tumorigenic E6T cells [13]. Since receptor dimerization is ligand-dependent [4], and determines which downstream signaling pathways will be activated [7], we examined the presence and relative abundance of ligands for ErbB-1 in both the tumorigenic E6T and the non-tumorigenic E6TA cells.…”

“…Since receptor dimerization is ligand-dependent [4], and determines which downstream signaling pathways will be activated [7], we examined the presence and relative abundance of ligands for ErbB-1 in both the tumorigenic E6T and the non-tumorigenic E6TA cells. Earlier studies had shown that EGF is not produced by BEAS-2B or its ErbB-2-overexpressing derivatives [15], while TGFa secretion by E6T cells (243 pg/ml per 10 6 cells) was reduced by 93% (17 pg/ml per 10 6 cells) by expression of an antisense construct in E6TA cells [13]. We initially examined the mRNA expression of the ErbB-1 ligands AR and BTC by RT-PCR.…”

“…They were grown in serum-free LHC-8 medium containing geneticin (200 mg/ml) (Gibco-BRL, Gaithersburg, MD). E6TA cells were prepared by introducing a TGFa antisense expression vector (pLTRTGF-aHYG) into E6T cells, as previously described [13]. These cells were grown in serumfree LHC-8 medium containing hygromycin B (200 mg/ml) (Boehringer Mannheim, Indianapolis, IN) as well as geneticin as for E6T.…”

“…We showed that human lung epithelial cells, overexpressing ErbB-2, formed tumors in nude mice only when high levels of TGFa were produced. Inhibition of TGFa expression using an antisense strategy resulted in inhibition of tumor growth in nude mice and blocked the induction of constitutive ErbB-1/ErbB-2 heterodimers in the malignant cells [13]. We hypothesized that induction of a tumorigenic phenotype depended on achieving a threshold level of TGFa secretion.…”