A dual role of miR-22 modulated by RelA/p65 in resensitizing fulvestrant-resistant breast cancer cells to fulvestrant by targeting FOXP1 and HDAC4 and constitutive acetylation of p53 at Lys382

Abstract

Antiestrogen resistance is a major challenge encountered during the treatment of estrogen receptor alpha positive (ERα+) breast cancer. A better understanding of signaling pathways and downstream transcription factors and their targets may identify key molecules that can overcome antiestrogen resistance in breast cancer. An aberrant expression of miR-22 has been demonstrated in breast cancer; however, its contribution to breast cancer resistance to fulvestrant, an antiestrogen drug, remains unknown. In this study, we demonstrated a moderate elevation in miR-22 expression in the 182R-6 fulvestrant-resistant breast cancer line we used as a model system, and this elevation was positively correlated with the expression of the miRNA biogenesis enzymes AGO2 and Dicer. The level of phosphorylated HER2/neu at Tyr877 was also upregulated in these cells, whereas the level of RelA/p65 phosphorylated at Ser536 (p-p65) was downregulated. Knockdown of HER2/neu led to an induction of p-p65 and a reduction in miR-22 levels. Luciferase assays identified two NF-κB binding motifs in the miR-22 promoter that contributed to transcriptional repression of miR-22. Activation of RelA/p65, triggered by LPS, attenuated miR-22 expression, but this expression was restored by sc-514, a selective IKKβ inhibitor. Inhibition of miR-22 suppressed cell proliferation, induced apoptosis and caused cell cycle S-phase arrest, whereas enhancing expression of p21Cip1/Waf1 and p27Kip1. Surprisingly, ectopic expression of miR-22 also suppressed cell proliferation, induced apoptosis, caused S-phase arrest, and promoted the expression of p21Cip1/Waf1 and p27Kip1. Ectopic overexpression of miR-22 repressed the expression of FOXP1 and HDAC4, leading to a marked induction of acetylation of HDAC4 target histones. Conversely, inhibition of miR-22 promoted the expression of both FOXP1 and HDAC4, without the expected attenuation of histone acetylation. Instead, p53 acetylation at lysine 382 was unexpectedly upregulated. Taken together, our findings demonstrated, for the first time, that HER2 activation dephosphorylates RelA/p65 at Ser536. This dephosphoryalted p65 may be pivotal in transactivation of miR-22. Both increased and decreased miR-22 expression cause resensitization of fulvestrant-resistant breast cancer cells to fulvestrant. HER2/NF-κB (p65)/miR-22/HDAC4/p21 and HER2/NF-κB (p65)/miR-22/Ac-p53/p21 signaling circuits may therefore confer this dual role on miR-22 through constitutive transactivation of p21.

TET2 directs mammary luminal cell differentiation and endocrine response

“…Unlike other TET proteins, TET2 does not have a putative DNA-binding domain 6 and can be recruited to specific chromatin regions through interaction with DNA-binding transcription factors 10 , 26 – 28 . To identify key transcription regulatory elements and putative transcription factors that may serve as co-activators of TET2 to mediate gene expression of Foxa1, Gata3 , and Esr1 , we performed promoter analysis (Genomatix MetInspector) and found that Forkhead box protein P1 (FOXP1) binding motif was the most enriched in the promoter regions of all three FOXA1, GATA3, and ESR1 genes (high matrix consensus score greater than 0.99) and might serve as a potential transcription factor to modulate expression of these genes (Supplementary Data 2 ).…”

“…Previous studies have revealed that TET2 can be a direct target of the Let-7adf cluster in LPS-activated macrophages 33 , it can be suppressed by miR-29 in prostate cancer cells 9 , and it is down-regulated by miR-22 in breast cancer cells 34 . miR-22, a microRNA that is associated with oncogenic signaling and overexpressed in high-grade breast tumors with poor clinical outcomes 26 , 34 , 35 , directly targets TET family members, including TET2, which in turn leads to hypermethylation of the mir-200 promoter and induction of breast cancer stemness phenotype and metastasis 34 . The associated TET2 regulatory mechanism was further elaborated by another study that showed TET2 could complex with RARβ to epigenetically activate a cohort of gene targets involved in cell differentiation, including RUNX1, BMP6, IKZF1 and CAV1 , and Mir-200c 10 .…”

“…Unlike TET1 and TET3, which contain CXXC DNA binding domain, TET2 binds to DNA through tissue-specific DNA-binding factors 10 , 27 , 38 – 40 to activate transcription of respective target genes. Wang et al has recently reported that TET2 coordinates with MLL3 at enhancers to facilitate the recruitment of transcription factors 26 . It has also been implicated that TET2 can function independently of its catalytic activity though binding to specific enhancers and facilitate transcription factor recruitment 30 , 41 ; as a result, differential stoichiometry between TET2, target enhancers/binding regions, and the associated transcription factors may contribute to the context-dependent TET2 functioning.…”

Genome-wide identification of methylated CpG sites in nongenital cutaneous warts

“…The survival and growth of multiple myeloma is regulated by the HDAC4-RelB-p52 complex, and the disruption of the latter blocks the growth of these cells [ 46 ]. Moreover, HDAC4 degradation by certain chemotherapeutic agents results in the apoptosis of head-and-neck cancer cells that are resistant to TRAIL, while miR-22-driven HDAC4 repression helped to resensitize fulvestrant-resistant breast cancer cells [ 47 , 48 ]. Likewise, eptoposide resistance in human A549 lung cancer cells was conferred by STAT1-HDAC4 upregulation, and HDAC4 inhibition has been reported to induce apoptosis in non-small cell lung cancer PC-9 cells [ 49 , 50 ].…”

Perspectives on the Role of Histone Modification in Breast Cancer Progression and the Advanced Technological Tools to Study Epigenetic Determinants of Metastasis

“…Both HDAC3 and HDAC6 could increase the expression of survivin and siRNA treatment of HDAC3 and HDAC6 showed a similar effect of SAHA and induced autophagy in MCF7 and MDA-MB-231 cancer cells (Lee et al, 2016). miRNA-22, miRNA-10b and miRNA-125a-5p was suggested to interact with HDAC4 and contributed an impact on the drug-resistance in breast cancer cells (Ahmad et al, 2015;Hsieh et al, 2015a;Wang et al, 2018). Similar to HDAC4, HDAC5 also displays a regulatory effect on tumor resistance in breast cancer, which is related to its regulation of cell proliferation, cell differentiation and autophagy (Hsieh et al, 2015b;Li et al, 2016;Cao et al, 2017;Huang et al, 2017;Cao et al, 2018;Xue et al, 2019).…”