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. 2016 Nov 22;7(52):87064–87080. doi: 10.18632/oncotarget.13500

Figure 1. Evidence that regulation of breast cancer cell growth by RA implicates, in addition to the RARA transcriptional function, another RARA function.

Figure 1

A. Analysis of human gene expression microarrays shows that the transcription of RARA-target genes is repressed under ‘physiological’ (‘physio’) RA conditions, but can be reactivated by ‘supraphysiological’ (‘supra’) RA (10-6 M) in T47DCtrl cells. B. T47DCtrl xenograft tumors are growth-inhibited in nude mice fed a RA-enriched diet relative to a control diet (***p<0.001) (top, left), consistent with CYP26A1 transcript induction in both mice liver and tumors (*p<0.05, **p<0.01) (top, right). 3D T47DCtrl growth (assessed by EdU incorporation) is promoted by low ‘supraphysiological’ RA (10-9 M) (*p<0.05), but inhibited by high RA (10-7 M) (*p<0.05). C. ‘Supraphysiological’ RA promotes 2D T47DCtrl cell growth (assessed by colony formation) at concentrations below the threshold required for induction of CYP26A1 and RARB2 transcripts (measured by qRT-PCR), but inhibits growth above this threshold (CYP26A1: RA 10-7M = **p<0.01, RA 10-6M = *p<0.05; RARB2: RA 10-7M = ***p<0.001, RA 10-6M = **p<0.01; growth: RA≤10-9 M = *p<0.05, RA≥10-7M = ***p<0.001; relative to ‘physio’ RA). D. In T47D403 cells, ‘supraphysiological’ RA does not re-activate RARA-target gene expression as it does in T47DCtrl cells. E. The RA-enriched diet promotes T47D403 xenograft tumor growth (***p<0.001) (top, left). This diet induces CYP26A1 transcript in mice liver but not in xenograft tumors (**p<0.01) (top, right). Both low and high ‘supraphysiological’ RA promote 3D T47D403 growth (*p<0.05) (bottom). F. ‘Supraphysiological’ RA induces CYP26A1 (RA 10-7M = *p<0.05, RA 10-6M = **p<0.01) and RARB2 (RA 10-7M = ***p<0.001, RA 10-6M = **p<0.01) significantly less relative to T47DCtrl cells. At all concentrations, RA promotes 2D T47D403 growth (***p<0.001, relative to ‘physio’ RA). G. The RARA agonist AM580 recapitulates the effects of RA on cell growth (T47DCtrl: **p<0.01 and T47D403: ***p<0.01, relative to untreated) and RARB2 transcription (T47DCtrl: AM580≥10-8M = **p<0.01, relative to untreated; T47D403: **p<0.01, relative to T47DCtrl). H. Stable knock down of wild type RARA in T47D403 cells (left) counteracts RA-induced cell growth (assessed by colony formation assay, right) (**p<0.01). Student's t-test was used for in vitro studies and ANOVA for in vivo studies. RU= relative units.