During hormone depletion or tamoxifen treatment of breast cancer cells the estrogen receptor apoprotein supports cell cycling through the retinoic acid receptor α1 apoprotein

Introduction: Current hormonal adjuvant therapies for breast cancer including tamoxifen treatment and estrogen depletion are overall tumoristatic and are severely limited by the frequent recurrence of the tumors. Regardless of the resistance mechanism, development and progression of the resistant tumors requires the persistence of a basal level of cycling cells during the treatment for which the underlying causes are unclear. Methods: In estrogen-sensitive breast cancer cells the effects of hormone depletion and treatment with estrogen, tamoxifen, all-trans retinoic acid (ATRA), fulvestrant, estrogen receptor alpha (ER) siRNA or retinoic acid receptor alpha (RAR alpha) siRNA were studied by examining cell growth and cycling, apoptosis, various mRNA and protein expression levels, mRNA profiles and known chromatin associations of RAR. RAR alpha subtype expression was also examined in breast cancer cell lines and tumors by competitive PCR. Results: Basal proliferation persisted in estrogen-sensitive breast cancer cells grown in hormone depleted conditioned media without or with 4-hydroxytamoxifen (OH-Tam). Downregulating ER using either siRNA or fulvestrant inhibited basal proliferation by promoting cell cycle arrest, without enrichment for ErbB2/3+ overexpressing cells. The basal expression of RAR alpha 1, the only RAR alpha isoform that was expressed in breast cancer cell lines and in most breast tumors, was supported by apo-ER but was unaffected by OH-Tam; RAR-beta and -gamma were not regulated by apo-ER. Depleting basal RAR alpha 1 reproduced the antiproliferative effect of depleting ER whereas its restoration in the ER depleted cells partially rescued the basal cycling. The overlapping tamoxifen-insensitive gene regulation by apo-ER and apo-RAR alpha 1 comprised activation of mainly genes promoting cell cycle and mitosis and suppression of genes involved in growth inhibition; these target genes were generally insensitive to ATRA but were enriched in RAR binding sites in associated chromatin regions. Conclusions: In hormone-sensitive breast cancer, ER can support a basal fraction of S-phase cells (i) without obvious association with ErbB2/3 expression, (ii) by mechanisms unaffected by hormone depletion or OH-Tam and (iii) through maintenance of the basal expression of apo-RAR alpha 1 to regulate a set of ATRA-insensitive genes. Since isoform 1 of RAR alpha is genetically redundant, its targeted inactivation or downregulation should be further investigated as a potential means of enhancing hormonal adjuvant therapy.