The transcription factor retinoic acid receptor ␤ 2 (RAR␤ 2 ) is a potent inhibitor of breast cancer cells in vitro, and studies suggest that RAR␤ expression is lost in primary breast cancer. Although RAR␤ 2 is selectively downregulated at the mRNA level in breast tumor cells, we show that expression of an RAR␤ protein is elevated in five of five breast tumor cell lines relative to normal human mammary epithelial cells. Subsequent analysis identified this protein as the translation product of the human RAR␤ 4 transcript. Unlike the previously characterized mouse RAR␤ 4 isoform, the human RAR␤ 4 retains only half of a DNA-binding domain and lacks a ligand-independent transactivation domain at its N terminus. The RAR␤ 4 protein localizes to the cytoplasm and to subnuclear compartments that resemble nuclear bodies. The structure and preliminary characterizations of human RAR␤ 4 , coupled with the observation that its expression is greatly elevated in breast tumor cell lines, support the hypothesis that RAR␤ 4 functions as a dominant-negative repressor of RAR-mediated growth suppression.Retinoids are a class of lipid-soluble micronutrients that mediate cellular signals critical for embryonic morphogenesis, cell growth, and differentiation. Retinoid signals are transduced by two families of retinoid-activated transcription factors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RARs and RXRs are members of the nuclear receptor superfamily. Both the RAR and RXR families are composed of three genes (␣, ␤, and ␥) that generate multiple RAR and RXR isoforms by using two promoters (P1 and P2) and alternative splicing (1, 2). For most retinoid-inducible genes, transcriptional activation occurs through RAR-RXR heterodimers acting at a retinoic acid-or retinoid X-response element (RAREs and RXREs, respectively) in the presence of biologically active metabolites (e.g., all-trans-retinoic acid or 9-cis-retinoic acid) of vitamin A (retinol).Based on homology with other members of the steroid hormone receptor superfamily, six distinct domains (A-F) have been identified within RARs and RXRs (3). These domains contain functional units of the transcription factor (4), including a ligand-independent transcription activation function-1, AF-1 (regions A and B); a DNA-binding domain (region C); a hinge region with roles in nuclear translocation and corepressor binding (region D); and a domain involved in dimerization, ligand binding, and ligand-dependent transcriptional activation, AF-2 (region E). The role of region F is unknown. The greatest diversity of protein sequences between RAR isoforms is generated at the N terminus by the incorporation of unique A regions with the use of alternative splicing and different promoters. Isoforms of a given RAR gene generally contain identical protein sequences B-F.There is strong evidence that of the three RAR genes, RAR␤ has the central role in the growth regulation of mammary epithelial cells. RAR␤ maps to chromosome 3p24, a region that exhibits a high frequency (45%) o...

The receptor tyrosine kinase p185c-neu can be constitutively activated by the transmembrane domain mutation Val664→ Glu, found in the oncogenic mutant p185neu. This mutation is predicted to allow intermolecular hydrogen bonding and receptor dimerization. Understanding the activation of p185c-neu has assumed greater relevance with the recent observation that achondroplasia, the most common genetic form of human dwarfism, is caused by a similar transmembrane domain mutation that activates fibroblast growth factor receptor (FGFR) 3. We have isolated novel transforming derivatives of p185c-neu using a large pool of degenerate oligonucleotides encoding variants of the transmembrane domain. Several of the transforming isolates identified were unusual in that they lacked a Glu at residue 664, and others were unique in that they contained multiple Glu residues within the transmembrane domain. The Glu residues in the transforming isolates often exhibited a spacing of seven residues or occurred in positions likely to represent the helical interface. However, the distinction between the sequences of the transforming clones and the nontransforming clones did not suggest clear rules for predicting which specific sequences would result in receptor activation and transformation. To investigate these requirements further, entirely novel transmembrane sequences were constructed based on tandem repeats of simple heptad sequences. Activation was achieved by transmembrane sequences such as [VVVEVVA]n or [VVVEVVV]n, whereas activation was not achieved by a transmembrane domain consisting only of Val residues. In the context of these transmembrane domains, Glu or Gln were equally activating, while Lys, Ser, and Asp were not. Using transmembrane domains with two Glu residues, the spacing between these was systematically varied from two to eight residues, with only the heptad spacing resulting in receptor activation. These results are discussed in the context of activating mutations in the transmembrane domain of FGFR3 that are responsible for the human developmental syndromes achondroplasia and acanthosis nigricans with Crouzon Syndrome.

The retinoic acid receptor beta2 (RARbeta2) protein is a putative tumor suppressor that inhibits proliferation and can induce apoptosis when introduced into breast, cervical, lung, and pancreatic cancer cell lines. To determine if RARbeta2 suppresses proliferation of mammary-derived cancer cells in vivo, we transduced MDA-MB-435 breast cancer cells with the LXSN retroviral vector containing RARbeta2 and implanted LXSN vector- or RARbeta2-transduced cells into the mammary fat pads of nude and severe combined immune deficiency (SCID) mice. We analyzed the xenografts for several tumor parameters, including tumor size, inflammation, vascularity, mitoses, tumor recurrence at the primary site following resection, and metastases. We found that 19 of 52 mice inoculated with vector-transduced cells developed metastases in multiple organs while only one of 55 mice receiving RARbeta2-transduced cells displayed evidence of metastases (p < 0.000001, combined experiments, two-tailed Fisher's exact test). Moreover, RARbeta2-tumor cell recipient mice had a lower incidence of post-resection tumor recurrence (8/55 vs. 25/52, p = 0.0004), 34% less necrosis (in three of four experiments, p = 0.001), and 39% fewer mitoses in tumor tissue (p < 0.000001). Our findings suggest that RARbeta2 may play a role in inhibiting the metastatic cascade in a mouse mammary gland xenograft tumor model and is a potential candidate for therapeutic intervention in human breast cancer.

Retinoic acid receptors (RARs) are essential for the differentiation and maintenance of normal epithelium. In studies of RARs in breast cancer, there are striking differences in the expression of certain protein isoforms of the RAR␤ gene between cells derived from normal human mammary glands and those derived from breast tumors. While the protein isoforms RAR␤2 and RAR␤4 consist of the longest open reading frames of the RAR␤2 and RAR␤4 mRNAs, respectively, we find that a fraction of scanning ribosomes bypass these upstream RAR␤2 and RAR␤4 protein start codons and initiate translation downstream. This downstream translation initiation site is identical in the RAR␤2 and RAR␤4 transcripts and generates a third RAR␤ protein isoform, here termed RAR␤ (formerly human RAR␤4). RAR␤ lacks protein domains found in the N terminus of RAR␤2 and RAR␤4, including one of two zinc fingers required for DNA binding. However, RAR␤ retains the ability to heterodimerize with RXR␣ and interact with transcription cofactors. In reporter gene assays, RAR␤ repressed retinoic acid-activated transcription of co-transfected RAR␤2, RAR␤4, and RAR␣. This repression required the presence of acidic amino acids within the AF2 domain. These findings demonstrate an antagonistic role for RAR␤ in signaling by retinoic acid.Vitamin A derivatives, called retinoids, are required for epithelial cell development and differentiation. Retinoid signals are transduced largely through ligand activation of two families of retinoid-activated transcription factors belonging to the nuclear receptor superfamily, retinoic acid receptors (RARs) 1 and retinoid X receptors (RXRs). Receptors of both the RAR and RXR subfamilies share conserved protein regions designated A through F. The A and B regions include a ligand-independent transactivation domain (AF-1) whereas region C encompasses the DNA-binding domain. The D region acts as a conformational hinge between the DNA-binding domain and the remainder of the protein. The ligand binding, dimerization, and liganddependent transactivation (AF-2) domains are all found within regions E and F. RAR-RXR heterodimers bind to cis-acting DNA elements in gene promoter regions called retinoic acid response elements (RAREs) and transactivate target genes in the presence of a retinoid ligand. Both the RAR and RXR families are comprised of three genes (␣, ␤, and ␥) that generate multiple isoforms via the usage of two promoters, P1 and P2, and alternative splicing (1).As the primary effectors of retinoid signaling, the RARs and RXRs themselves appear to be targets for disruption in tumorigenesis (2), including loss of heterozygosity (3), gene rearrangements (4, 5), mutations (6), and aberrant mRNA expression (7,8). Retinoic acid receptor ␤ (RAR␤) in particular has been extensively studied in human carcinomas, and a body of evidence indicates that it may play a role in tumor suppression. Such findings include the loss of heterozygosity of its genetic locus (3p24) in primary tumors of the breast (3) and the loss of RAR␤ mRNA expression i...