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Vitamin A is an essential diet-derived nutrient that has biological activity affected through an active metabolite, all-trans retinoic acid (atRA). Retinol-binding protein type 1 (RBP1) is an intracellular chaperone that binds retinol and retinal with high affinity, protects retinoids from non-specific oxidation, and delivers retinoids to specific enzymes to facilitate biosynthesis of RA. RBP1 expression is reduced in many of the most prevalent cancers, including breast cancer. Here, we sought to understand the relationship between RBP1 expression and atRA biosynthesis in mammary epithelial cells, as well as RBP1 expression and atRA levels in human mammary tissue. We additionally aimed to investigate the impact of RBP1 expression and atRA on the microenvironment as well as the potential for therapeutic restoration of RBP1 expression and endogenous atRA production. Using human mammary ductal carcinoma samples and a series of mammary epithelial cell lines representing different stages of tumorigenesis, we investigated the relationship between RBP1 expression as determined by QPCR and atRA via direct liquid chromatography-multistage-tandem mass spectrometry-based quantification. The functional effect of RBP1 expression and atRA in epithelial cells was investigated via the expression of direct atRA targets using QPCR, proliferation using Ki-67 staining, and collagen deposition via picrosirius red staining. We also investigated the atRA content of stromal cells co-cultured with normal and tumorigenic epithelial cells. Results show that RBP1 and atRA are reduced in mammary tumor tissue and tumorigenic epithelial cell lines. Knock down of RBP1 expression using shRNA or overexpression of RBP1 supported a direct relationship between RBP1 expression with atRA. Increases in cellular atRA were able to activate atRA direct targets, inhibit proliferation and inhibit collagen deposition in epithelial cell lines. Conditions encountered in tumor microenvironments, including low glucose and hypoxia, were able to reduce RBP1 expression and atRA. Treatment with either RARα agonist AM580 or demethylating agent Decitabine were able to increase RBP1 expression and atRA. Cellular content of neighboring fibroblasts correlated with the RA producing capacity of epithelial cells in co-culture. This work establishes a direct relationship between RBP1 expression and atRA, which is maintained when RBP1 expression is restored therapeutically. The results demonstrate diseases with reduced RBP1 could potentially benefit from therapeutics that restore RBP1 expression and endogenous atRA.

The accurate estimation and eradication of Human Immunodeficiency Virus (HIV) viral reservoirs is limited by the incomplete reactivation of cells harboring the latent replication-competent virus. We investigated whether the in vitro and in vivo addition of retinoic acid (RA) enhances virus replication and improves the detection of latent virus. Peripheral blood mononuclear cells (PBMCs) from naive and anti-retroviral therapy (ART)-treated SIV-infected rhesus macaques (RMs) were cultured in vitro with anti-CD3/CD28 + IL-2 in the presence/absence of RA. Viral RNA and p27 levels were quantified using RT-qPCR and ELISA, respectively. Viral reservoirs were estimated using the Tat/Rev-Induced Limited Dilution Assay (TILDA) and Quantitative Viral Outgrowth Assay (QVOA). In vitro and in vivo measures revealed that there was also an increase in viral replication in RA-treated versus without RA conditions. In parallel, the addition of RA to either CD3/CD28 or phorbol myristate acetate (PMA)/ionomycin during QVOA and TILDA, respectively, was shown to augment reactivation of the replication-competent viral reservoir in anti-retroviral therapy (ART)-suppressed RMs as shown by a greater than 2.3-fold increase for QVOA and 1 to 2-fold increments for multi-spliced RNA per million CD4 + T cells. The use of RA can be a useful approach to enhance the efficiency of current protocols used for in vitro and potentially in vivo estimates of CD4 + T cell latent reservoirs. In addition, flow cytometry analysis revealed that RA improved estimates of various viral reservoir assays by eliciting broad CD4 T-cell activation as demonstrated by elevated CD25 and CD38 but reduced CD69 and PD-1 expressing cells.