Abstract 3074: The role of orphan nuclear receptors in breast cancer cell proliferation

Abstract Nuclear receptors (NRs) are ligand-dependent transcription factors which are involved in the proliferation/development of normal and neoplastic cells. The family of NR genes consists of 48 elements and the endogenous ligands binding the majority of NRs are known. Indeed, it is generally assumed that NRs function as sensors for fat-soluble hormones, vitamins and dietary lipids. For instance, RARs (Retinoic Acid Receptors) bind to and are trans-activated by retinoic acids, the active metabolites of vitamin A, while ERs (Estrogen Receptors) and PR (Progesterone Receptor) interact with estradiol and progesterone, respectively. In spite of all the available information, there are at least 15 NRs (ONRs, Orphan Nuclear Receptors), which are still devoid of a recognized endogenous ligand. The dysregulation of NRs has been associated with different types of cancer. NRs act in hierarchical, integrated networks to promote tissue- and cell-specific functions. In breast cancer, the role of ERs and PR has been thoroughly investigated, while the function of other members of the NR family is largely unknown. In the present study, we define the expression levels of NR mRNAs in normal and breast cancer tissues, using public databases. The results obtained indicate that the majority of NRs are deregulated in mammary tumors. We focus our attention on ONRs, as many of them are differentially expressed in normal and tumor samples. To get functional information, we use the siRNA technology to assess the effects of ONR silencing on the growth/motility of breast cancer cells. To this purpose, we employ 40 cell lines, which are representative of the different tumor subtypes. We choose at least five cell lines among the ones with high, medium and low expression of the ONR under investigation. We transiently silence the selected NR with the transfection of at least 3 selective siRNAs. Cell proliferation is evaluated after 2, 4 and 6-days from the transfection of the siRNAs using the sulphorodamine assay. We identify NR2F6 as the only ONR whose silencing inhibits cell proliferation. In contrast, NR2F2 silencing increases cell proliferation. We confirm the results obtained on NR2F6 with the use of the CRISPR/CAS9 and the shRNA technologies. In line with our results, NR2F6 expression is higher, while NR2F2 expression is lower, in breast tumors than in normal tissue. Interestingly, the highest levels of NR2F6 protein are observed in ER- and in HER2-positive breast cancer cell-lines. By converse, Triple-Negative cells are endowed with the lowest levels of this ONR. To identify the gene-networks modulated by NR2F2 and NR2F6, we performed RNA-sequencing experiments in control and silenced breast cancer cells, which provide evidence on the underlying molecular mechanisms of action. In conclusion, our data indicate that NR2F2 and NR2F6 represent novel targets for the development of therapeutic strategies in the context of the personalized treatment of breast cancer. Citation Format: Gabriela Paroni, Maria Azzurra Caricasulo, Adriana Zanetti, Andrea Cavallaro, Luca Guarrera, Mineko Terao, Enrico Garattini. The role of orphan nuclear receptors in breast cancer cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3074.