Remodeling Ca2+ dynamics by targeting a promising E-box containing G-quadruplex at ORAI1 promoter in triple-negative breast cancer

Abstract

ORAI1 is an intrinsic component of store-operated calcium entry (SOCE) that strictly regulates Ca²⁺ influx in most non-excitable cells. ORAI1 is overexpressed in a wide variety of cancers, and its signal transduction has been associated with chemotherapy resistance. There is extensive proteomic interaction of ORAI1 with other channels and effectors, resulting in various altered phenotypes. However, the transcription regulation of ORAI1 is not well understood. We have found a putative G-quadruplex (G4) motif, ORAI1-Pu, in the upstream promoter region of the gene, having regulatory functions. High-resolution 3-D NMR structure elucidation suggests that ORAI1-Pu is a stable parallel-stranded G4, having a long 8-nt loop imparting dynamics without affecting the structural stability. The protruded loop further houses an E-box motif that provides a docking site for transcription factors like Zeb1. The G4 structure was also endogenously observed using Chromatin Immunoprecipitation (ChIP) with anti-G4 antibody (BG4) in the MDA-MB-231 cell line overexpressing ORAI1. Ligand-mediated stabilization suggested that the stabilized G4 represses transcription in cancer cell line MDA-MB-231. Downregulation of transcription further led to decreased Ca²⁺ entry by the SOCE pathway, as observed by live-cell Fura-2 Ca²⁺ imaging.

Keywords: Ca(2+) signaling; Cancer; G-quadruplex; ORAI1.

Graphical Abstract

ORAI1 expression is downregulated in the presence of a stable G4 structure at the predicted ORAI1-Pu motif, which is formed at a regulatory docking site for transcription factors and RNA polymerase II. The G4 structure might act as a dynamic switch, as it is a transient structure and is regulated by various factors within a cell. Ligand-mediated stabilization of ORAI1-Pu via TMPyP4/ BRACO-19 indicated gain-of-function in ORAI1 expression. In MDA-MB-231, we observed greater fold endogenous binding of Zeb1 at the ORAI-Pu motif, compared to BG4, which binds to stable G4 structures. In MDA-MB-231, the ORAI1-Pu locus might be in a partially unfolded state or unfolded state, leading to over-expression of ORAI1. However, further studies can reveal the physiological dynamics of the structure and its functional effects on ORAI expression. Overexpression of ORAI1 increases its transport to the plasma membrane and increases SOCE on interaction with activated STIM at physiological conditions where there is loss of Ca²⁺ in the endoplasmic reticulum (ER) calcium store. A spike in intracellular Ca²⁺ cascades its effect on deregulated cellular activity and enhances various hallmarks of cancer, epithelialmesenchymal transition (EMT), drug resistance, and immune evasion. Etc.

Figure 1. Structure determination of the ORAI1-Pu G4.

(A) Schematic representation of the ORAI1-Pu topology with residue numbering; anti-G residues of the G-core are colored grey, loop and overhang residues are represented by circles colored in blue and red, respectively. (B) CD spectrum of the ORAI1-Pu sequence. (C-E) 2D NOESY spectral regions of ORAI1-Pu (mixing time 300 ms). (C) H1-H1 spectral region with corresponding 1D imino proton spectrum shown on top with resonance assignments. (D) H8/H6(ω₂)-H1'(ω₁) spectral region. (E) H8(ω₂)-H1(ω₁) spectral region. (F) 1D ¹⁵N-filtered HMQC spectra of ORAI1-Pu sequences site-specifically labelled at G2, G6, G17, G21, G23, G9, G14, and G15 (10% ¹⁵N enrichment). Assignments of guanine H1 (left) and H8 resonances (right) with G imino and G H8 spectral regions of unlabelled ORAI1-Pu shown on top. NMR spectra were acquired in a 10 mM potassium phosphate buffer, pH 7.0, at 40 °C.

Figure 2. Representative structure of the ORAI1-Pu G4.

(A) Top view and (B) side view; anti-G residues are colored grey, loop residues are colored blue and overhang residues are colored red. (C) View onto the 5'-tetrad with stacked T1 and (D) view onto the 3'-tetrad with stacked G24.

Figure 3. ORAI1-Pu G4 as a potential promoter element.

(A) The representative schematic diagram for the reporter luciferase construct. The promoter sequence of ORAI1 (249 bp upstream and 41 bp downstream sequence of ORAI1 from TSS) was cloned with or without the wild-type G4 (ORAI1-Pu) scaffold into the Kpn-I and Hind-III restriction sites. Kpn-I and Hind-III restriction sites are upstream of the hRluc gene, and the sequence is cloned at the promoter region involved in the expression of the reporter gene hRluc. The deletion clone without the ORAI1-Pu sequence is called ORAI1-Pu-null. Abbreviations include ampicillin resistance gene (AmpR), Pause (RNA pol pause signal), oriC (origin of replication), hCL1 and hPEST (protein-destabilizing sequences), and hRluc (Renilla luciferase gene). (B) In MDA-MB-231, the dual luciferase assay was run in increasing concentrations of TMPyP4 using the wildtype and ORAI1-Pu-Null constructs. After comparing the activity of firefly luciferase to that of Renilla luciferase, the relative luciferase activity was computed and displayed as a fold change. (C) Representation of effects of G4 ligand. There is stabilization of ORAI1-Pu. q-PCR of mRNA post-treatment and quantitative analysis by ΔΔCt method shows a loss in ORAI1 expression with TMPyP4 (25μM & 50μM) or BRACO-19 (25μM & 50μM) treatment. (* represents p-value < 0.05, ** is p-value <0.01, *** is p-value <0.001, **** is p-value <0.0001).

Figure 4. Transcriptional landscape of ORAI1 promoter mediated by G4.

(A) Primer design region for ChIP assay encompassing the ORAI1-Pu sequence at the ORAI1 promoter. (B) Endogenous characterization of G4 formation by BG4 at ORAI1-Pu and RNA polymerase II binding affinity by subsequent fold enrichment on chromatin pull-down assay, with IgG as negative control. (C) Representation of the region with E-box motif. Transcriptional regulation of ORAI1-Pu by acting as a docking platform for various transcription factors (SNAI2, Zeb1, E2F1, and c-Myc) as observed by fold enrichment in ChIP assay. IgG was used as a negative control. (* represents p-value < 0.05, ** is p-value <0.01, *** is p-value <0.001, **** is p-value <0.0001).

Figure 5. Downregulation of SOCE activity by G4 binding ligands at ORAI1-Pu.

(A) and (C) Representative Ca²⁺ imaging traces for control nuclease free water i.e. NFW (n=94) and treatment (A) BRACO-19 (n=84) or (C) TMPyP4 (n=75) where ‘’n” denotes the no. of cells in that trace. Cells were stimulated with 2 μM thapsigargin (Tg) in Ca²⁺ free buffer and restored with 1 mM extracellular Ca²⁺. In (B) and (D), the extent of SOCE was calculated from 350 NFW and (B) 229 BRACO-19 or (D) 333 TMPyP4 treated MDA-MB-231 cells, which were imaged from 4 independent experiments (“n = x, y” where “x” denotes a total number of cells imaged and “y” denotes the number of traces recorded). Data presented are mean ± S.E.M. For statistical analysis, an unpaired student’s t-test was performed. Here, **** means p< 0.0001.

Figure 6. Overview/TOC.

ORAI1 expression is downregulated in the presence of a stable G4 structure at the predicted ORAI1-Pu motif, which is formed at a regulatory docking site for transcription factors and RNA polymerase II. The G4 structure might act as a dynamic switch, as it is a transient structure and is regulated by various factors within a cell. Ligand-mediated stabilization of ORAI1-Pu via TMPyP4/ BRACO-19 indicated gain-of-function in ORAI1 expression. In MDA-MB-231, we observed greater fold endogenous binding of Zeb1 at the ORAI-Pu motif, compared to BG4, which binds to stable G4 structures. In MDA-MB-231, the ORAI1-Pu locus might be in a partially unfolded state or unfolded state, leading to over-expression of ORAI1. However, further studies can reveal the physiological dynamics of the structure and its functional effects on ORAI expression. Overexpression of ORAI1 increases its transport to the plasma membrane and increases SOCE on interaction with activated STIM, at physiological conditions, where there is a loss of Ca²⁺ in the endoplasmic reticulum (ER) calcium store. A spike in intracellular Ca²⁺ cascades its effect on deregulated cellular activity and enhances various hallmarks of cancer, epithelial-mesenchymal transition (EMT), drug resistance, and immune evasion.