v-erbA oncogene function in neoplasia correlates with its ability to repress retinoic acid receptor action.

The v-erbA oncoprotein of avian erythroblastosis virus is an aberrant version of a thyroid hormone receptor and functions in neoplasia by blocking erythroid differentiation and by modifying the growth properties of fibroblasts. v-erbA has been proposed to represent a novel dominant negative oncogene, acting in the cancer cell by interfering with the actions of its normal cell homologs, the thyroid hormone receptors. We report here that v-erbA can actually interfere with the actions of a variety of members of the steroid/retinoid receptor family and that the ability of v-erbA to act in neoplasia best correlates not with suppression of c-erbA action, but with interference with the retinoic acid receptor response. We suggest that v-erbA may act in neoplasia by promiscuously interfering with a retinoid-mediated differentiation process.     

V-erba homodimers mediate the potent dominant negative activity of v-erba on everted repeats

The oncoprotein v-erbA is a mutated form of TRalpha1 that is unable to bind thyroid hormone (T3). V-erbA homodimerizes or heterodimerizes with retinoid X receptor (RXR) on core motifs arranged as direct, everted, or inverted repeats (DRs, ERs, or IRs). We created a series of v-erbA mutants in order to obtain a better understanding of the role of v-erbA homodimers versus v-erbA-RXR heterodimers in the dominant negative activity of v-erbA on ERs (the most potent v-erbA response elements). We found that one of these mutants, v-erbA mutant E325A, is able to homodimerize but unable to heterodimerize with RXR on ERs. Our data also suggest that v-erbA homodimers interact preferentially with the corepressor NCoR over SMRT and that the interaction with corepressors is stronger with v-erbA homodimers over v-erbA-RXR heterodimers. Furthermore, functional studies showed that v-erbA homodimers rather than v-erbA-RXR heterodimers mediate the dominant negative activity of v-erbA on ERs.     

Ontogeny of the v-erbA oncoprotein from the thyroid hormone receptor: an alteration in the DNA binding domain plays a role crucial for v-erbA function.

The avian erythroblastosis virus v-erbA oncogene is imprecisely derived from a cellular gene (c-erbA) encoding a thyroid hormone receptor: the v-erbA protein has sustained both small terminal deletions and internal amino acid sequence changes relative to c-erbA. We report here that one of these missense differences between v- and c-erbA proteins, located in a zinc finger DNA binding domain, has dramatic effects on the biological activities of the encoded protein. Back mutation of the viral coding sequence to resemble c-erbA at this site severely impairs erythroid transformation and produces subtle changes in DNA binding by the encoded protein, suggesting that differences in DNA binding by the viral and cellular proteins may be involved in the activation of v-erbA as an oncogene.     

Sequence-specific DNA binding by the v-erbA oncogene protein of avian erythroblastosis virus.

The v-erbA oncogene, a transduced copy of a thyroid hormone receptor, plays an important role in establishment of the transformed cell phenotype induced by avian erythroblastosis virus. The ability of thyroid hormone receptors to bind to specific sites on chromatin and to thereby modify the expression of adjacent target genes is a crucial element in their mechanism of action in the normal cell. The v-erbA protein also bound at high affinity to a set of DNA fragments recognized by the rat thyroid hormone receptor, but the relative affinity of the v-erbA protein for the different binding sites was distinct from that previously reported for the thyroid hormone receptors.     

A single point mutation in erbA restores the erythroid transforming potential of a mutant avian erythroblastosis virus (AEV) defective in both erbA and erbB oncogenes.

We have characterized the v-erbA and v-erbB oncogenes of td359, a transformation-defective mutant of avian erythroblastosis virus (AEV) unable to transform erythroblasts, and the revertant r12, obtained after in vivo passage of the mutant. Molecular cloning, sequencing, construction of chimeric viruses and testing of their oncogenic capacities revealed that both oncogenes of td359 are mutated and biologically defective. The r12 virus, although still containing a mutant v-erbB gene, recovered its erythroid transforming potential by acquiring a highly active gag-erbA gene. These results demonstrate that two co-operating oncogenes, an active v-erbA and a defective v-erbB, can transform a cell type not transformed by either oncogene alone. Furthermore, a single amino acid substitution inactivated the td359 v-erbA protein and we show that its reversion led to the reactivation of the protein. This lesion is located in the same region as several previously described inactivating mutations of glucocorticoid receptors, suggesting that the structure/function relationship of the virally transduced form of the c-erbA/thyroid hormone receptor is closely similar to that of steroid hormone receptors.     

Expression of the v-erbA product, an altered nuclear hormone receptor, is sufficient to transform erythrocytic cells in vitro

We investigated the effect of the v-erbA oncogene product, an altered thyroid hormone receptor, in chicken erythrocyte progenitor cells. Bone marrow cells were infected with a retrovirus vector (XJ12) carrying the v-erbA gene in association with the neoR gene. XJ12-infected erythrocyte progenitor cells gave rise to G418-resistant clones. Some were composed of blast cells identified as transformed CFU-Es blocked in their differentiation. These cells could be grown in culture for at least 25 generations and required anemic chicken serum as a source of erythropoietic growth factors. XJ12 can infect erythrocyte progenitor cells in vivo but is not sufficient to induce erythroleukemia. These data suggest that the activation of a nuclear hormone receptor might represent one step toward the development of neoplasms.     

Unliganded T3R, but not its oncogenic variant, v-erbA, suppresses RAR-dependent transactivation by titrating out RXR.

V-erbA is thought to be an antagonist of thyroid hormone receptor (T3R) function. Here we show that unliganded T3R, but not v-erbA, suppresses retinoic acid (RA)-dependent induction of the RAR-beta 2 promoter by competing for the common dimerization partner, the retinoid X receptor (RXR). Firstly, T3R suppression can be alleviated by co-transfection of RXR. Secondly, T3R, but not v-erbA, competes with RAR for RXR and causes the dissociation of a preformed RAR/RXR-RARE ternary complex in vitro. A single point mutation located in the dimerization interface of v-erbA (Pro349 to Ser) abolishes the transdominant phenotype when introduced at the respective position in T3R. The hypertransforming v-erbA variant r12, in which this mutation is reversed (Ser349 to Pro) suppresses RA-induced differentiation in chicken erythroid progenitors, while v-erbA does not. Our data thus suggest that unliganded T3R and v-erbA act as dominant suppressors through mechanistically distinct pathways.     

Expression of the v-erbA oncogene in chicken embryo fibroblasts stimulates their proliferation in vitro and enhances tumor growth in vivo

In contrast to uninfected chicken embryo fibroblasts (CEFs), CEFs infected with a retroviral vector that carries the v-erbA gene of avian erythroblastosis virus displayed new properties. These included limited anchorage-independent growth in soft agar, growth without latency in serum-supplemented medium, ability to overcome quiescence induced by serum deprivation, growth at low cell density, and an extended life span in vitro. Furthermore, when explanted in vivo onto the chorioallantoic membrane of chicken embryo, the transformed CEFs expressing v-erbA in addition to v-erbB exhibited a high proliferative rate, giving rise to fibrosarcoma tumors that were ten times larger than those developed from transformed CEFs expressing v-erbB alone. All these data show that CEFs expressing the v-erbA oncogene display activated growth and suggest that the v-erbA product interferes with the mechanisms regulating the growth and/or differentiation of primary CEFs.