A wide variety of human tumors contain an amplified or overexpressed erbB-2 gene, which encodes a growth factor receptor-like protein. When erbB-2 complementary DNA was expressed in NIH/3T3 cells under the control of the SV40 promoter, the gene lacked transforming activity despite expression of detectable levels of the erbB-2 protein. A further five- to tenfold increase in its expression under influence of the long terminal repeat of Moloney murine leukemia virus was associated with activation of erbB-2 as a potent oncogene. The high levels of the erbB-2 product associated with malignant transformation of NIH/3T3 cells were observed in human mammary tumor cells that overexpressed this gene. These findings demonstrate a new mechanism for acquisition of oncogenic properties by genes encoding growth factor receptor-like proteins and provide a functional basis for the role of their overexpression in the development of human malignancies.

The cellular gene encoding the receptor for epidermal growth factor (EGF) has considerable homology to the oncogene of avian erythroblastosis virus. In a human mammary carcinoma, a DNA sequence was identified that is related to v-erbB but amplified in a manner that appeared to distinguish it from the gene for the EGF receptor. Molecular cloning of this DNA segment and nucleotide sequence analysis revealed the presence of two putative exons in a DNA segment whose predicted amino acid sequence was closely related to, but different from, the corresponding sequence of the erbB/EGF receptor. Moreover, this DNA segment identified a 5-kilobase transcript distinct from the transcripts of the EGF receptor gene. Thus, a new member of the tyrosine kinase proto-oncogene family has been identified on the basis of its amplification in a human mammary carcinoma.

Amplification of the erbB/EGF receptor and a structurally related gene, designated erbB‐2, have previously been detected in a variety of human tumors. In a series of human mammary tumor cell lines, analysis of transcripts of these genes revealed elevated levels of one or the other in more than 60% of tumors analyzed. Eight cell lines demonstrated erbB‐2 mRNA levels ranging from 4‐ to 128‐fold above those of normal controls. erbB‐2 expression was evaluated in comparison to the expression level of actin observed in these cell lines. There was no evidence of an aberrantly sized erbB‐2 transcript in any of these lines. Immunoblot analysis indicated elevation in levels of the 185‐kd product of the erbB‐2 gene expressed by these cells. In four lines erbB‐2 gene amplification in the absence of an apparent gene rearrangement was demonstrated. In a representative cell line of this type, SK‐BR‐3, the amplified erbB‐2 gene copies were located in an aberrant chromosomal location. Four additional cell lines, which demonstrated 4‐ to 8‐fold overexpression of erbB‐2 mRNA, did not exhibit gene amplification. In a representative cell line of this type ZR‐75‐1, an apparently normal chromosomal location was found for the erbB‐2 gene. Our findings indicate that overexpression of the erbB‐2 gene in mammary tumor cell lines is frequent and associated with different genetic abnormalities.

Recombinant expression of a chimeric EGFR/ErbB-3 receptor in NIH 3T3 fibroblasts allowed us to investigate cytoplasmic events associated with ErbB-3 signal transduction upon ligand activation. An EGFR/ErbB-3 chimera was expressed on the surface of NIH 3T3 transfectants as two classes of receptors possessing epidermal growth factor (EGF) binding affinities comparable to those of the wild-type EGF receptor (EGFR). EGF induced autophosphorylation in vivo of the chimeric receptor and DNA synthesis of EGFRI ErbB-3 transfectants with a dose response similar to that of EGFR transfectants. However, the ErbB-3 and EGFR cytoplasmic domains exhibited striking differences in their interactions with several known tyrosine kinase substrates. We demonstrated strong association of phosphatidylinositol 3-kinase activity with the chimeric receptor upon ligand activation comparable in efficiency with that of the platelet-derived growth factor receptor, while the EGFR exhibited a 10-to 20-fold-lower efficiency in phosphatidylinositol 3-kinase recruitment. By contrast, both phospholipase Cy and GTPase-activating protein failed to associate with or be phosphorylated by the ErbB-3 cytoplasmic domain under conditions in which they coupled with the EGFR. In addition, though certain signal transmitters, including Shc and GRB2, were recruited by both kinases, EGFR and ErbB-3 elicited tyrosine phosphorylation of distinct sets of intracellular substrates. Thus, our findings show that ligand activation of the ErbB-3 kinase triggers a cytoplasmic signaling pathway that hitherto is unique within this receptor subfamily.Signal transduction by receptor tyrosine kinases (RTKs) involves tyrosine phosphorylation and/or physical association of a number of cytosolic enzymes by the activated receptor. Initially identified as immediate targets of the platelet-derived growth factor (PDGF) receptor (PDGFR) and the epidermal growth factor (EGF) receptor (EGFR), these substrates include phospholipase C-y (PLCy), GTPaseactivating protein (GAP), and phosphatidylinositol 3-kinase (PtdIns 3-kinase) (2,11,13,24, 36,39,41,58,64). Their coding sequences share regions of homology with the noncatalytic portion of c-src, termed SH2 and SH3, that are involved in the assembly of signaling complexes with the activated receptor. Thereby, SH2 domains bind to discrete phosphotyrosine (P-Tyr)-containing peptide sequences present in RTKs, whereas SH3 domains appear to associate with proline-rich motifs in proteins regulating the activity of low-molecular-mass G proteins (5,14,26,43,49,52,69

Gli family members mediate constitutive Hedgehog signaling in the common skin cancer, basal cell carcinoma (BCC). Snail/Snai1 is rapidly induced by Gli1 in vitro, and is coexpressed with Gli1 in human hair follicles and skin tumors. In the current study, we generated a dominant-negative allele of Snail, SnaZFD, composed of the zinc-finger domain and flanking sequence. In promoter-reporter assays, SnaZFD blocked the activity of wild-type Snail on the E-cadherin promoter. Snail loss-of-function mediated by SnaZFD or by one of several short hairpin RNAs inhibited transformation of RK3E epithelial cells by Gli1. Conversely, enforced expression of Snail promoted transformation in vitro by Gli1, but not by other genes that were tested, including Notch1, ErbB2, and N-Ras. As observed for Gli1, wild-type Snail repressed E-cadherin in RK3E cells and induced blebbing of the cytoplasmic membrane. Induction of a conditional Gli1 transgene in the basal keratinocytes of mouse skin led to rapid upregulation of Snail transcripts and to cell proliferation in the interfollicular epidermis. Established Gli1-induced skin lesions exhibited molecular similarities to BCC, including loss of E-cadherin. The results identify Snail as a Gli1-inducible effector of transformation in vitro, and an early Gli1-responsive gene in the skin.