The cDNAs coding for two novel mouse molybdo-flavoproteins, AOH1 and AOH2 (aldehyde oxidase homolog 1 and 2), were isolated. The AOH1 and AOH2 cDNAs code for polypeptides of 1336 amino acids. The two proteins have similar primary structure and show striking amino acid identity with aldehyde oxidase and xanthine oxidoreductase, two other molybdo-flavoenzymes. AOH1 and AOH2 contain consensus sequences for a molybdopterin-binding site and two distinct 2Fe-2S redox centers. In its native conformation, AOH1 has a molecular weight consistent with a homotetrameric structure. Transfection of the AOH1 and AOH2 cDNAs results in the production of proteins with phenanthridine but not hypoxanthine oxidizing activity. Furthermore, the AOH1 protein has benzaldehyde oxidizing activity with electrophoretic characteristics identical to those of a previously identified aldehyde oxidase isoenzyme (Holmes, R. S. (1979) Biochem. Genet. 17, 517-528). The AOH1 transcript is expressed in the hepatocytes of the adult and fetal liver and in spermatogonia. In liver, the AOH1 protein is synthesized in a gender-specific fashion. The expression of AOH2 is limited to keratinized epithelia and the basal layer of the epidermis and hair folliculi. The selective cell and tissue distribution of AOH1 and AOH2 mRNAs is consistent with the localization of the respective protein products.

Aldehyde oxidase was purified to homogeneity from bovine liver and primary structural information obtained by sequencing a series of cleavage peptides permitted the cloning of the corresponding cDNA. The cDNA is 4,630 base pairs long, and it consists of a 102-base pair 5'-untranslated region followed by a 4017-base pair coding region and a 511-base pair 3'-untranslated region. The open reading frame predicts a 1339-amino acid polypeptide with a calculated molecular weight of 147,441, which is consistent with the size of the aldehyde oxidase monomeric subunit. The aldehyde oxidase polypeptide contains consensus sequences for iron-sulfur centers and a molybdopterin binding site. The amino acid sequence deduced from the cDNA shows significant similarity with that of xanthine dehydrogenases from various sources. The primary structure of bovine aldehyde oxidase is remarkably similar (approximately 86%) to that of the translation product of a cDNA recently isolated by Wright et al. (Wright, R. M., Vaitaitis, G. M., Wilson, C. M., Repine, T. B., Terada, L. S., and Repine, J. E. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10690-10694) and reported to represent human xanthine dehydrogenase. With the help of a monospecific antibody raised against the purified protein and the isolated cDNA, the tissue distribution of the bovine aldehyde oxidase protein and corresponding mRNA was determined. Aldehyde oxidase is expressed at high levels in liver, lung, and spleen, and, at a much lower level, in many other organs.

We report the cloning of the AOH1 and AOH2 genes, which encode two novel mammalian molybdo-flavoproteins. We have purified the AOH1 protein to homogeneity in its catalytically active form from mouse liver. Twenty tryptic peptides, identified or directly sequenced by mass spectrometry, confirm the primary structure of the polypeptide deduced from the AOH1 gene. The enzyme contains one molecule of FAD, one atom of molybdenum, and four atoms of iron per subunit and shows spectroscopic features similar to those of the prototypic molybdo-flavoprotein xanthine oxidoreductase. The AOH1 and AOH2 genes are 98 and 60 kilobases long, respectively, and consist of 35 coding exons. The AOH1 gene has the potential to transcribe an extra leader non-coding exon, which is located downstream of exon 26, and is transcribed in the opposite orientation relative to all the other exons. AOH1 and AOH2 map to chromosome 1 in close proximity to each other and to the aldehyde oxidase gene, forming a molybdo-flavoenzyme gene cluster. Conservation in the position of exon/ intron junctions among the mouse AOH1, AOH2, aldehyde oxidase, and xanthine oxidoreductase loci indicates that these genes are derived from the duplication of an ancestral precursor.

Forty-two cell lines recapitulating mammary carcinoma heterogeneity were profiled for all-trans retinoic acid (ATRA) sensitivity. Luminal and ER+ (estrogen-receptor-positive) cell lines are generally sensitive to ATRA, while refractoriness/low sensitivity is associated with a Basal phenotype and HER2 positivity. Indeed, only 2 Basal cell lines (MDA-MB157 and HCC-1599) are highly sensitive to the retinoid. Sensitivity of HCC-1599 cells is confirmed in xenotransplanted mice. Short-term tissue-slice cultures of surgical samples validate the cell-line results and support the concept that a high proportion of Luminal/ER+ carcinomas are ATRA sensitive, while triple-negative (Basal) and HER2-positive tumors tend to be retinoid resistant. Pathway-oriented analysis of the constitutive gene-expression profiles in the cell lines identifies RARα as the member of the retinoid pathway directly associated with a Luminal phenotype, estrogen positivity and ATRA sensitivity. RARα3 is the major transcript in ATRA-sensitive cells and tumors. Studies in selected cell lines with agonists/antagonists confirm that RARα is the principal mediator of ATRA responsiveness. RARα over-expression sensitizes retinoid-resistant MDA-MB453 cells to ATRA anti-proliferative action. Conversely, silencing of RARα in retinoid-sensitive SKBR3 cells abrogates ATRA responsiveness. All this is paralleled by similar effects on ATRA-dependent inhibition of cell motility, indicating that RARα may mediate also ATRA anti-metastatic effects. We define gene sets of predictive potential which are associated with ATRA sensitivity in breast cancer cell lines and validate them in short-term tissue cultures of Luminal/ER+ and triple-negative tumors. In these last models, we determine the perturbations in the transcriptomic profiles afforded by ATRA. The study provides fundamental information for the development of retinoid-based therapeutic strategies aimed at the stratified treatment of breast cancer subtypes.