Endocrine nuclear receptors and long non‑coding RNAs reciprocal regulation in cancer (Review).

Nuclear receptors (NRs) are ligand-activated transcription factors involved in main cellular processes including homeostasis, metabolism, growth, differentiation and development ( 1 ). These receptors interact with specific DNA sequences in the promoter and enhancer regions of their target genes modulating their transcription by co-binding to a series of co-factors/coregulators belonging to transcription initiation machinery ( 2 ). The NR superfamily can be divided into three classes based on the type of ligand: Endocrine, metabolic and orphan NRs ( 3 ). The endocrine NR subfamily includes estrogen receptors (ERs) αlpha (ERα or ESR1) and ERβ (ERβ or ESR2), androgen receptors (AR), progesterone receptors, glucocorticoid receptors, mineralocorticoid receptors, vitamin D receptors (VDRs), retinoic acid receptors (RARs), as well as thyroid hormone receptors (THRs) ( 1 ). Numerous factors contribute to the regulation of NRs, especially the recruitment of coregulatory proteins, post-translational modifications and interactions with other transcription factors. Dysregulated NR signaling can promote a large series of pathological processes. In particular, mutation or aberrant expression of NR and/or of their coregulators may influence both the development and progression of several human diseases including cancer ( 4 ). Moreover, epigenetic regulation, especially by activation of non-coding RNA molecules (ncRNAs), represents an element of complexity in the modulation of NR-dependent gene expression. Long non-coding (lnc) RNAs are non-coding molecules longer than 200 bp, able to interact with mRNA, DNA, protein and microRNA (miRNA or miR) ( 5 ). They have different regulatory functions in humans: i) Regulation of histone modifications at the chromatin level by interaction with histone-modified complexes or enzymes; ii) transcriptional regulation and post-transcriptional regulation; iii) miRNA sponge; iv) RNA stability; v) Protein relocalization and vi) post-translation modifications ( 6 ). During cancer evolution, lncRNAs may regulate cell proliferation, apoptosis, migration, invasion, stem-like phenotype and remodeling of the tumor microenvironment (TME) ( 7 ). Previous studies have shown that lncRNAs can function as positive or negative regulators of NR-dependent gene expression, and in turn, NRs can regulate different lncRNAs. This crosstalk appears to be particularly important in the modulation of oncogenic processes ( 8 , 9 ). The steroid receptor RNA activator (SRA) was identified as the first lncRNA able to bind and increase the activity of steroid receptors ( 10 ) ( Fig. 1 ). SRA is able to interact with numerous proteins acting as a 'scaffold' for the assembly of other coregulatory proteins that direct NR-dependent transcription ( 11 - 14 ). In addition to the role of signaling by NR, numerous studies revealed that SRA is involved in other physiological functions: i) Modulation of adipogenesis via direct binding to and promoting the transcriptional activity of peroxisome proliferator activated receptor γ, ii) regulation of steroidogenesis and adrenal biology by binding and activation of SF-1 as well as dosage-sensitive sex reversal-adrenal hypoplasia protein, iii) promotion of muscle differentiation by increasing the activity of myogenic differentiation 1 protein ( 12 ), iv) induction of proliferation and migration of vascular smooth muscle cells by stimulating the phosphorylation of mitogen-activated protein kinase, extracellular signal-regulated kinases and cAMP response element-binding protein CREB ( 15 ), v) promotion of melanin pigmentation process by modulation of p38 and phosphoribosyl-anthranilate isomerase gene expression ( 16 ); and vi) modulation of insulin signaling and regulation of β-oxidation ( 17 ). Instead, aberrant SRA expression and mutant variants have been identified in several pathological conditions, not only in hormone-driven tumors ( Fig. 1 ) ( 12 ). SRA promotes cervical cancer cell migration and invasion via upregulation of matrix metalloproteinase-9, matrix metalloproteinase-2, vascular endothelial growth factor A (VEGFA) expression and NOTCH receptor 1 signaling pathways ( 18 ). Park et al ( 19 ) revealed that SRA is involved in the proliferation, migration and invasion of endometrial cancer cells by increasing the expression of EIF4E-BP1 and Wnt/β-catenin signaling activity. SRA mediates p38 activation, cell invasion along with proliferation, regulates epithelial-mesenchymal transition (EMT) and distant metastasis in melanoma ( 20 ) and induces tumor progression as well as drug resistance in colorectal cancer by oxidative phosphorylation pathway genes ( 21 ). In hormone-driven cancers, SRA is directly involved in the mechanisms underlying breast tumorigenesis and tumor progression ( 22 ), and their isoforms are capable of enhancing AR activities in prostate cancer cells ( 23 ). More recently, Kim et al ( 24 ) demonstrated that SRA is able to regulate cell migration, proliferation, and invasion of ovarian cancer cells by modulation of EMT and expression of NOTCH-related genes. Given the importance of NRs in hormone-driven cancers, a deeper understanding of epigenetic regulatory mechanisms in human tumors may offer new scenarios for new diagnostic and predictive biomarkers. In the present review, it was proposed to schematize the crosstalk existing between the main endocrine NR and the lncRNAs in tumor initiation and progression.

All-trans retinoic acid (RA) is the main active metabolite of vitamin A ( 140 ). RA signaling is involved in different biological processes, including embryonic development along with organogenesis, cell growth, proliferation, cell differentiation, together with metabolism regulation ( 141 ). RA translocates in the nucleus by binding to an intracytoplasmic transporter, cellular RA binding protein type II. Retinoids bind to two different NRs, RARs (RAR-α and RAR-β) and retinoid X receptors (RXRs). RARs function as heterodimers with RXRs. In turn, RXRs can be involved in the formation of heterodimers with other different NRs, such as vitamin D 3 , thyroid hormone and PPARs ( 142 ). RAR/RXR and RXR/RXR are localized in the nucleus bound to retinoid hormone response elements (RAREs) in the promoter regions of retinoid-responsive genes thereby inducing the transcription of numerous target genes ( 143 ). There are few examples in the literature regarding crosstalk of RAR signaling and lncRNAs in cancer. In acute promyelocytic leukemia (APL) RA/RARs signaling can induce the expression of the lncRNA HOXA-AS2 antisense, which suppresses the TRAIL pathway and reduces expression of caspases ( 144 ). Similarly, RA/RARs signaling induces H19 expression in APL cells and its upregulation perturbs telomerase activity, mediated by hTERC-hTR interaction, during tumor evolution ( 145 ). Conversely, the lncRNA RA early transcript 1K pseudogene (RAET1K) can reduce RA/RARs signaling. In lung cancer RAET1K interferes with the protective role of RA, modulating miR-135a-5p, which in turn inhibits Cyclin E1 (CCNE1) expression, a cyclin required for G1-S transition. RA promotes the expression of this miRNA, by blocking lung cancer cells in the G1 phase and thereby tumor cells proliferation and progression. RAET1K silencing can repress CCNE1 expression and inhibit cell cycle progression from the G1 to the S phase ( 146 ). A recent study also highlighted crosstalk between HOTAIR and RA/RARs signaling in acute myeloid leukemia. Different acute myeloid leukemia cell lines (HL-60, NB4, U937 and THP-1) have induced myeloid differentiation by all-trans RA (ATRA). All four cell lines exhibited a higher expression level of HOTAIR after the ATRA treatment suggesting its role in the regulation of RA/RARs pathway and consequently in myeloid cell differentiation ( 147 ). The lncRNA RNA HOTAIR myeloid 1 (HOTAIRM1) can mediate ATRA-induced differentiation of ALK cells ( 148 ). Upregulation of HOTAIRM1 expression is able to enhance ATRA-induced PML-RARA degradation by affecting autophagic flux and thereby controlling myeloid cell differentiation in APL cells ( 149 ). lncRNA NEAT1 is also involved in APL cell differentiation induced by ATRA. C/EBPα binds and transactivates NEAT1 whereas PML/RARα suppresses this process suggesting that PML/RARα could contribute to the pathogenesis of APL by suppressing C/EBPα targets ( 150 ). In glioblastoma multiforme cells ATRA treatment dysregulated different lncRNAs' expression. In particular, ATRA dose-dependently decreased the expression of GAS5 within the microenvironment of the U87MG cell line ( 151 ). RAET1K is necessary for retinoid-induced differentiation in different cells by binding RXRs. RAET1K is upregulated in tumor tissues and is associated with a poor prognosis in patients with lung adenocarcinoma. Aberrant RAET1K expression upregulated cyclin E1 by targeting miR-135, thereby promoting tumor progression ( 146 ). Currently, Wang et al ( 152 ) asserted that lncHOXA10 is significantly upregulated in gastric cancer tissues and cell lines, and can promote proliferation, migration as well as invasion of cells. lncHOXA10 can suppress RAR-β expression, involved in the modulation of apoptosis, while ATRA can rescue the expression of RAR-β, inhibiting lncHOXA10 in gastric cancer cells ( 152 ). RAR-β is also modulated by lncRNA HAND2-AS1 in human bladder cancer. HAND2-AS1 inhibits cell proliferation and promotes apoptosis in bladder cancer cells by sponging miR-146. The last of these can promote cell proliferation by targeting RAR-β and, in turn, HAND2-AS1 can suppress cell proliferation via releasing RAR-β from miR-146 ( 153 ). More recently, Fu et al ( 154 ) described that another lncRNA, lymphocytic leukemia 2 (DLEU2), can modulate RAR-β in colorectal cancer cells. DLEU2 induced promoter methylation of RAR-β to downregulate its expression, and its upregulation induced the MAPK signaling pathway promoting colorectal cancer progression ( 154 ). VDR is a transcription factor that besides interacting with hormonally active vitamin D3, regulates the expression of more than 900 genes involved in numerous different cellular processes. Both vitamin D/VDR signaling and lncRNAs affect numerous genomic and non-genomic processes, the dysregulation of which can be associated with a wide range of diseases including cancer. The principal evidence is associated with skin cancers. Several studies confirmed the protective effect of VDR in cancer initiation and progression ( 155 ), especially in keratinocyte carcinoma ( 156 ). Jiang and Bikle ( 157 ) profiled 90 well-annotated mouse lncRNAs from cultured mouse keratinocytes after deleting VDR. They detected that H19, HOTTIP, mHOTAIR, Malat1, SRA and Nespas were significantly increased, whereas H19 as, Kcnq1ot1 and lincRNA-p21 were decreased ( 157 ). Subsequently, other VDR-associated lncRNAs in BC ( 158 ) as well as in lung cancer ( 159 ) have been identified. VDR, MALAT1 and LINC00511 were significantly upregulated in tumor cells in BC compared with non-cancerous tissues. In addition, VDR and another lncRNA SNHG16 were associated in both tumor and non-tumor breast tissues ( 158 ). In lung cancer, the coordinate expression of VDR and the lncRNAs MALAT1, SNHG16, SNHG6, LINC00346 and LINC00511 has also been validated in lung cancer tissues ( 159 ). In oral squamous cell carcinoma (OSCC) Jin et al ( 160 ) evaluated crosstalk between vitamin D/VDR signaling and lncRNAs selecting 46 pairs of tumor tissue and adjacent non-tumor tissue as well as two OSCC cell lines. In the latter, they observed 1045 lncRNAs differentially expressed after the 1,25(OH) 2 D treatment. In particular lung cancer-associated transcript 1 (LUCAT1) expression was reduced by the 1,25(OH) 2 D treatment in vitro and it appeared strongly overexpressed in the OSCC tumor tissues. LUCAT1 silencing in the OSCC cell lines reduced cell growth in association with a reduction in ERK1/2 phosphorylation ( 160 ). The oncogenic lncRNA colon cancer-associated transcript 2 (CCAT2) has been described as overexpressed in ovarian cancer. Wang et al ( 161 ) described that 1,25(OH) 2 D inhibited CCAT2 expression leading to decreased binding of transcription factor 4 (TCF4) to the MYC promoter in ovarian cancer cell lines. This produced inhibition of proliferation, migration and invasive features of ovarian cells ( 161 ). In addition, the lncRNA TOPORS-AS1, overexpressed in ovarian cancer cells and involved in suppression of cell proliferation, migration and invasion, is upregulated by VDR. Crosstalk is essential to interrupt the Wnt/β-catenin signaling thereby defining an improved prognosis in patients with ovarian cancer ( 162 ). Finally, the relation between the lncRNA H19 and VDR has been described in colon cancer. VDR can inhibit H19 expression through the regulation of the c-Myc/Mad-1 network. In turn, H19 upregulation blocked VDR expression by upregulating miRNA 675-5p ( 163 ). Human GR (h-GR) belongs to the nuclear hormone receptor superfamily and is encoded by nuclear receptor subfamily 3C1 gene on chromosome 5q31.3 ( 164 ). In the absence of ligands, the GR is sequestered in the cytoplasm by chaperone proteins. Glucocorticoids, such as cortisol, prednisolone and dexamethasone bind to GR leading to both its dimerization and translocation into the nucleus, where it performs its activity as a transcription regulator ( 164 ). DNA-bound GR recruits coregulator complexes forming transcription regulatory complexes that can function in both activation and suppression of transcription. Some lncRNAs have been described as regular and can be regulated by glucocorticoid/GR signaling. GAS5 lncRNA is able to directly interact with the DNA binding domain of GR acting as a molecular decoy by competing with the glucocorticoid-response element for GR binding, thereby inhibiting GR transcription ( 165 ). GAS5 is also able to accumulate in cells that have been starved of growth factors by suppressing GR activity, and GAS5 sensitizes cells to apoptosis ( 165 ). In the same way, overexpression of GAS5 leads to growth arrest and apoptosis in human breast cell lines ( 165 ). Thyroid hormone action is predominantly mediated by THRs, which are encoded by the THRA and THRB genes ( 166 ). THRs act as ligand-activated nuclear transcription factors to regulate different physiologic processes through direct gene regulation and with ncRNAs relation ( 167 , 168 ). Current evidence indicates a crosstalk between the thyroid hormone/THR pathway and lncRNAs in liver cancer. Brain cytoplasmic RNA 1 (BCYRN1/BC200) is abnormally overexpressed in several tumor types, with a significant overexpression in HCC tissues. Using the Disease-Related Human lncRNA Profiler to identify lncRNAs regulated by thyroid hormone/THR signaling in liver cells, BC200 was identified as a lncRNA downregulated by the thyroid hormone ( 169 ). In HCC cells, the taurine upregulated gene 1 (TUG1) is overexpressed and can induce cell proliferation, invasion, metastatic progression as well as apoptosis by distal-less homeobox 2 (DLX2) activation ( 170 ). Alpha-fetoprotein (AFP) is increased in the majority of patients with HCC and TUG1 is involved in THR/AFP signaling. Thyroid hormones suppress TUG1 expression, leading to the downregulation of AFP ( 171 ).

NRs function as a transcriptional signaling network that mediates gene regulatory actions to maintain cellular homeostasis in response to hormonal and environmental factors. The dysregulation of NR signaling is known to contribute to the evolution of hormone-dependent tumors, such as breast and prostate cancer, which are also therapeutic targets. However, NRs signaling affects cancer also because the therapeutic response associated with their target drugs is often altered by changes in the expression and function of a large number of coregulators. They could modulate receptor sensitivity, and modify their protein-protein interaction, thus altering transcription, as well as regulation of chromatin accessibility. Coregulators include coactivators that generally associate with agonist-bound NRs to stimulate gene expression and corepressors that are usually bound to unliganded or antagonist-bound NRs to suppress gene expression. lncRNAs are emerging as new genetic/epigenetic coregulators of NRs. In the last 20 years, lncRNAs have been extensively studied due to their potential role in cancer development and progression. However, the functional characterization of lncRNAs remains complex, interacting with different molecules involved in the regulation of key processes during cell development and diseases. lncRNAs can modulate NRs activity, and in turn, they can be regulated by NRs. Therefore, understanding the mechanisms by which such coregulators interact and modulate NR activity and vice versa could offer new opportunities to develop improved prognostic in addition to diagnostic approaches, along with new therapeutic targets.