Abstract
Despite the successful combination of therapies improving survival of estrogen receptor α (ER+) breast cancer patients with metastatic disease, mechanisms for acquired endocrine resistance remain to be fully elucidated. The RNA binding protein HNRNPA2B1 (A2B1), a reader of N(6)-methyladenosine (m6A) in transcribed RNA, is upregulated in endocrine-resistant, ER+ LCC9 and LY2 cells compared to parental MCF-7 endocrine-sensitive luminal A breast cancer cells. The miRNA-seq transcriptome of MCF-7 cells overexpressing A2B1 identified the serine metabolic processes pathway. Increased expression of two key enzymes in the serine synthesis pathway (SSP), phosphoserine aminotransferase 1 (PSAT1) and phosphoglycerate dehydrogenase (PHGDH), correlates with poor outcomes in ER+ breast patients who received tamoxifen (TAM). We reported that PSAT1 and PHGDH were higher in LCC9 and LY2 cells compared to MCF-7 cells and their knockdown enhanced TAM sensitivity in these-resistant cells. Here we demonstrate that stable, modest overexpression of A2B1 in MCF-7 cells increased PSAT1 and PHGDH and endocrine resistance. We identified four miRNAs downregulated in MCF-7-A2B1 cells that directly target the PSAT1 3′UTR (miR-145-5p and miR-424-5p), and the PHGDH 3′UTR (miR-34b-5p and miR-876-5p) in dual luciferase assays. Lower expression of miR-145-5p and miR-424-5p in LCC9 and ZR-75-1-4-OHT cells correlated with increased PSAT1 and lower expression of miR-34b-5p and miR-876-5p in LCC9 and ZR-75-1-4-OHT cells correlated with increased PHGDH. Transient transfection of these miRNAs restored endocrine-therapy sensitivity in LCC9 and ZR-75-1-4-OHT cells. Overall, our data suggest a role for decreased A2B1-regulated miRNAs in endocrine resistance and upregulation of the SSP to promote tumor progression in ER+ breast cancer.
Introduction
Breast cancer (BCa) is the most common malignant disease among women worldwide (Ciriello et al. 2015) and the second most common cause of cancer mortality in women in the United States (Siegel et al. 2023). Approximately 70% of primary breast tumors express estrogen receptor alpha (ERα) (Ring et al. 2004). The current adjuvant treatments for ERα+ BCa target ERα either by competitively inhibiting the binding of estrogens to ERα using selective ER modulators (SERMs), e.g. tamoxifen (TAM) for premenopausal patients (Rani et al. 2019) and reducing the level of estrogens available to bind ERα by inhibiting aromatase activity with aromatase inhibitors for postmenopausal women (Visvanathan et al. 2019). A major limitation of current adjuvant endocrine therapies (ET) for ERα+ BCa patients is the development of acquired resistance in ~30-40% of patients- even up to 30 years after initial therapy (Dowsett et al. 2005). Although combination targeted therapies with fulvestrant, e.g. everolimus, alpelisib, palbociclib, ribociclib, and abemaciclib, have demonstrated a significantly longer progression-free survival advantage of ~11 months (Samuel Eziokwu et al. 2020) in ~ 39.9% of patients (Lin et al. 2021a), these data indicate the number of patients who still die of metastatic disease is high. A variety of mechanisms have been implicated in the progression of endocrine resistance (Clarke et al. 2015, Fan & Jordan 2019), including PI3K mutations, epigenetic and metabolism changes, and altered expression of noncoding (nc) RNAs, e.g. miRNAs (Egeland et al. 2015, Muluhngwi & Klinge 2015), but the complete understanding of endocrine-resistant metastatic spread remains to be determined (Rani et al. 2019, Petri & Klinge 2020).
miRNAs are critical for posttranscriptional regulation of gene expression, but miRNA regulation and targets in oncogenesis and metastasis remain to be fully elucidated. miRNA biogenesis has been reviewed (Klinge 2015). The processing of primary miRNAs (pri-miRs) to precursor miRNAs (pre-miRs) is facilitated by the RNA binding protein HNRNPA2B1 (A2B1) (Alarcon et al. 2015b). A2B1 acts as a reader of N(6)-methyladenosine (m6A), a common modification of mRNAs and pri-miRNAs (Alarcon et al. 2015a). Previously, we reported higher A2B1 in tamoxifen (TAM) and fulvestrant-resistant, ERα+ LCC9 and LY2 cells derived from TAM- and fulvestrant-sensitive, ERα+ MCF-7 BCa cells (Petri et al. 2021). Transient overexpression of A2B1 in MCF-7 cells reduced cell sensitivity to 4-hydroxytamoxifen (4-OHT) and fulvestrant, mimicking the endocrine resistance of LCC9 and LY2 cells (Klinge et al. 2019). Using nontargeted miRNA sequencing (miRNA seq), we identified miRNAs regulated by A2B1 in MCF-7 cells and used MetaCore pathway analysis to identify A2B1-regulated gene ontology (GO) pathways. One of the GO pathways identified as A2B1-regulated in MCF-7 cells was ‘serine family amino acid metabolic processes.’
Serine is a nonessential amino acid that fuels glycine synthesis and nucleotide metabolism (De Marchi et al. 2017). The serine biosynthetic pathway (SSP) contributes to tumorigenesis and progression of BCa, although the mechanisms by which serine supports tumor progression and metastasis remain to be fully elucidated (Geck & Toker 2016). This pathway converts 3-phosphoglyerate into serine in a series of three reactions starting with the rate-limiting step catalyzed by D-3-phosphoglycerate dehydrogenase (PHGDH) (Supplementary Fig. 1, see section on supplementary materials given at the end of this article), an enzyme upregulated by MYC, which is overexpressed in ER+ BCa (Fallah et al. 2017). MYC is suppressed by p53 (Ou et al. 2015), which is mutated in triple-negative BCa (TNBC) (Ji et al. 2022). Inducing expression of PHGDH in mice generated with a PHGDH tetO allele allowing for tissue-specific, doxycycline-inducible PHGDH expression resulted in accelerated breast tumor growth (Sullivan et al. 2019). The second rate-limiting step in the SSP is a transamination reaction that converts 3-phospho-hydroxypyruvate to 3-phospho-serine and is catalyzed by phosphoserine aminotransferase (PSAT1). Dephosphorylation of 3-phospho-serine to serine is catalyzed by phosphoserine phosphatase (PSPH). The expression of PSAT1 is higher in TAM-resistant breast tumors and was reported to enhance cancer cell proliferation, metastasis, and TAM resistance (Martens et al. 2005a, De Marchi et al. 2017, Gao et al. 2017). Although PHGDH and PSAT1 are increased in breast tumors, the mechanism for this upregulation and the roles for these proteins and serine synthesis in endocrine resistance and/or metastasis are not yet understood. We reported that increased PSAT1 expression was positively correlated with higher tumor grade in TNBC tumor samples (Metcalf et al. 2020a). In addition, we found that both PSAT1 and PHGDH transcript expression correlated with reduced relapse-free survival in ER+ BCa patients treated with TAM (Metcalf et al. 2020b). We recently reported higher levels of PHGDH and PSAT1 in LCC9 and LY2 TAM-resistant cells compared to the parental MCF-7 cell line (Metcalf et al. 2020b). PSAT1 overexpression in MCF-7 TAM-sensitive cells and transient PSAT1 knockdown in LCC9 TAM-resistant cells altered TAM responses, i.e. inhibiting and enhancing TAM-induced cell growth inhibition (Metcalf et al. 2020b). These data suggest a role for elevated PSAT1 in TAM resistance (Metcalf et al. 2020b). Downregulation or pharmacological inhibition of PHGDH increased TAM sensitivity in LCC9 cells. We also demonstrated that shPSAT1 reduced MDA-MB-231 TNBC cell migration and lung tumor metastasis in vivo after tail vein injection in mice (Metcalf et al. 2020a). However, the knockdown of PHGDH had no impact on these parameters, suggesting that PSAT1 increases migratory potential in TNBC cells, independent of its role in serine synthesis (Metcalf et al. 2020a). It is important to note that a number of metabolic enzymes have cellular roles in addition to their metabolic activities (Pan et al. 2021, Xu et al. 2021).
The aim of this study was to determine if miRNAs decreased with A2B1 overexpression in MCF-7 cells, computationally associated with mRNA targets in the gene ontology/biological process (GO:BP) ‘serine family amino acid metabolic processes’ (Klinge et al. 2019), play a role in the increased expression of PSAT and PHGDH detected in TAM-resistant cell lines. We tested the hypothesis that A2B1 downregulates miRNAs that directly target PHGDH and PSAT1, resulting in increased protein levels. We identified A2B1-regulated miRNAs, miR-145-5p and miR-424-5p, that directly target PSAT1, and miR-34b-5p and miR-876-5p, that directly target PHGDH. Transfection of miR-145-5p and miR-424-5p reduced endogenous PSAT1 protein expression in some BCa cell lines; however, the effects of transfected miR-34-5p and miR-875-5p on PHGDH protein expression were cell line selective. Functionally, we demonstrated that the A2B1-downregulated miRs increased endocrine sensitivity in endocrine-resistant LCC9 cells.
Materials and methods
Chemicals
4-hydroxytamoxifen (4-OHT) was purchased from Sigma-Aldrich. Fulvestrant was purchased from Tocris Bioscience (Ellisville, MO, USA). Both were dissolved in DMSO, which was used as the vehicle control in experiments.
Cell culture and treatments
MCF-7, T47D, and ZR-75-1 BCa cells were purchased from American Type Culture Collection (Manassas, VA, USA) and were used within nine passages. MCF-7 cells were maintained in modified IMEM (Gibco, ThermoFisher Scientific) with 10% fetal bovine serum (FBS) and 1% Pen/Strep (P/S). T47D cells were maintained in RPMI 1640 (GenClone Cat# 25-506, Genesee Scientific, El Cajon, CA, USA) supplemented with 5% FBS, 6 μg/mL insulin (Sigma-Aldrich), and 1% P/S. ZR-75-1 cells were maintained with RPMI 1640 (GenClone Cat# 25-506, Genesee Scientific) with 10% FBS and 1% P/S. ZR-75-1 cells were exposed to 100 nM 4-OHT for ~20 weeks to create ZR-75-1-4-OHT cells which were maintained in half ZR-75-1 media and half Improved MEM (Richter’s media) (Corning) with 5% dextran-coated charcoal-stripped FBS and 1% P/S. LCC9 and LY2 tamoxifen/fulvestrant endocrine-resistant BCa cells were generously provided by Dr Robert Clarke, Georgetown University Medical Center (Davidson et al. 1986, Brunner et al. 1997, Crawford et al. 2010) and maintained with modified IMEM (Gibco) with 5% FBS and 1% P/S. MCF-7-A2B1 cells were stably transfected and G418-selected with pcDNA3.1+C-DYK into which A2B1 was cloned (GenScript, Piscataway, NJ, USA) as described (Klinge et al. 2019). All cell lines were verified by short tandem repeat (STR) genotyping (Genetica, LabCorp, Burlington, NC, USA). STR profiles were compared with publicly available profiles using Cellosaurus STR (ExPASy).
Transient transfection
Silencer™Select siHNRNPA2B1 (#4390824) and Negative Control No. 1 (#4390843) were purchased from Thermo Fisher Scientific. Prior to transfection, cells were grown in phenol red-free Opti-MEM (cat. # 11058021, Thermo Fisher Scientific) for ~18–24 h. Lipofectamine RNAiMAX reagent (cat # 13778075 Thermo Fisher Scientific) was used for transfection per the manufacturer’s protocol. LCC9, LY2, T47D, ZR-75-1 and ZR-75-1-4-OHT cells were transiently transfected for 48 h or 72 h with pre-miR-145-5p, pre-miR-424-5p, pre-miR-34b-5p, or pre-miR-876-5p (Pre-miR™s, Ambion, Austin, TX, USA), and anti-miR-145-5p, anti-miR-424-5p, anti-miR-34b-5p, or anti-miR-876-5p (Anti-miR™s, Ambion) using Lipofectamine3000 (Ref # L3000-015, ThermoFisher) in Opti-MEM® Reduced Serum Medium (Invitrogen). Negative controls were Anti-miR™ negative control #1 (Ambion) and Pre-miR™ negative control #1 (Ambion).
RNA extraction and quantitative real-time PCR
RNA and miRNA were extracted from cells using the RNeasy and miRNeasy Mini Kits, respectively (Qiagen). RNA concentration and quality and reverse transcription for cDNA used reagents and followed previously published (Klinge et al. 2019). Quantitative real-time PCR (qPCR) for PHGDH and PSAT1 was performed using TaqMan assays (ThermoFisher). 18S rRNA and GAPDH were used as normalizers. qPCR for miR-145-5p and miR424-5p used TaqMan assays and were normalized to RNU6B (Thermo Fisher Scientific). qPCR was performed using an ABI Viia 7 Real-Time PCR system (LifeTechnologies) with each reaction run in triplicate. The comparative threshold cycle (CT) method was used to determine ΔCT, ΔΔCT, and fold-change, log 2, relative to control (Schmittgen & Livak 2008).
MTT assays
Cells were ‘serum starved’ for 48 h in IMEM supplemented with 5% DCC-FBS (dextran-coated charcoal-stripped FBS). Cells were treated every 48 h with vehicle control (DMSO), E2 (estradiol), 4-OHT, or fulvestrant (ICI 182,780) at the concentrations and number of days indicated in figure legends. MTT assays used CellTiter (Promega). Each treatment was in quadruplicate within each experiment.
Western blotting
Whole cell extracts (WCE) were prepared as described (Radde et al. 2016) and separated on 10% SDS-PAGE, transferred to PVDF membranes (Bio-Rad Laboratories) and immunoblotted with antibodies: HNRNPA2B1 (B1 epitope-specific: IBL # 18941, IBL America, Minneapolis, MN, USA), PHGDH (cat # HPA021241, Sigma-Aldrich), PSAT1 (cat # 10501-1-AP, Proteintech, Rosemont, IL, USA), α-tubulin (Thermo Fisher Scientific # MS-81-P1), and GAPDH (Santa Cruz Biotechnology, cat # sc-365062). Membranes were washed with TBS-Tween followed by incubation with anti-mouse (#7076S) or anti-rabbit (#7074S) (Cell Signaling Technology) secondary antibodies. Membranes were incubated with Clarity Western ECL (Bio-Rad) and imaged on a Bio-Rad ChemiDoc™ XRS+ System with Image Lab™ Software (Bio-Rad). Blots were stained with Ponceau S (Romero-Calvo et al. 2010, Moritz 2017) or Amido Black (Goldman et al. 2016) for additional quantification. We observed that the protein levels of GAPDH and α-tubulin varied between some of the breast cancer cell lines and thus we used Ponceau S or Amido Black for quantification where indicated in the Figure legends.
Immunohistochemistry
A tissue microarray (TMA) including samples from 48 patients with histologically confirmed estrogen receptor-positive BCa was purchased from Reveal Biosciences (San Diego, CA, USA). The tissue sections were dried at 57℃ for 30 min and subsequently dewaxed with xylene and decreasing concentrations of ethanol. Antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) at 100℃ for 2.5 h. Slides were washed with 1× PBS and blocked with 10% goat serum for 1 h at room temperature followed by an additional wash with 1× PBS. Each section was incubated with the polyclonal primary rabbit antibody against PSAT1 (cat # 10501-1-AP, Proteintech) or HNRNPA2B1 (IBL # 18941, IBL America) at 1:200 dilutions overnight at 4℃. After washing with PBS (3 × 5 min). Slides were subsequently incubated with 1:500 dilution of horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Invitrogen, Cat No. 32260) at room temperature for 30 min. After another wash in PBS (3 × 5 min), each section was immersed in 500 μL of diaminobenzidine (DAB) working solution at room temperature for 1.5 min. Finally, the slides were counterstained with hematoxylin and mounted in Crystal Mount medium. Images were captured using a Nikon Eclipse Ci microscope with a dedicated Nikon DS-Fi3 microscope camera (Tokyo, Japan) for higher magnification. Semiquantitative measures were performed by two independent observers. Cytoplasmic expression of PSAT1 was evaluated based on the intensity of staining.
Statistical analysis
Prism™ software (version 9, GraphPad Inc.) was used to perform data transformations and plotting, linear and nonlinear regression, and statistical analyses. Where two data sets are compared, Student’s two-tailed t-test was used. Where more than two data sets were compared, one way ANOVA followed by Tukey’s multiple comparison post hoc test or two-way ANOVA followed by Bonferroni post hoc test were performed. P-values are provided in figures, with P < 0.05 being considered significant.
Results
PSAT1 is increased in breast tumors from patients with ERα+ BCa
Higher PSAT1 mRNA levels and protein expression was reported in primary breast tumors compared to normal breast tissue (De Marchi et al. 2017). Lower PSAT1 and PHGDH transcript expression is associated with better prognosis in BCa patients (Mascia et al. 2022) (Supplementary Fig. 2A and B). IHC was performed to examine PSAT1 staining in a TMA-containing primary breast tumors paired with normal breast tissue or lymph node metastases (LNM) from the same patient (Fig. 1A, B, C and D). Strong cytoplasmic PSAT1 staining was identified in the epithelial cells of normal breast tissue (Nl. Br.), invasive ductal carcinomas (IDC), and in LNM. PSAT1 expression intensity was higher in the ER+ IDC tumors than the paired normal ER+ breast tissue (8.3% of stained cells with an intensity score of 3) (Fig. 1A and B) and was lower in LNM compared to normal ER+ breast tissue (60% of stained cells with an intensity score of 1) (Fig. 1B and D). Similarly, PSAT1 staining intensity was lower in ER+, PR+, HER2− LNM compared to IDC from the same molecular subtype (Fig. 1E). These results show that PSAT1 expression is higher in ER+ invasive breast tumors compared to normal breast tissue, although the increased PSAT1 expression does not extend to associated LNM.
IHC staining for PSAT1 in ER+ invasive breast carcinoma (IDC) tumors paired with normal breast or lymph node (LN) metastasis from the same patient. Shown are representative images from a stained TMA. The tumor in (A) is paired with normal breast (nl. Br.) tissue (B) from the same patient. The tumor in (C) is paired with its LN metastasis in (D). Bar in 200 μm. Images were taken under 20× and 40× magnification. (E) Quantification based on 100% staining and intensity of cells stained are indicated as the percent total number of that sample in the TMA.
Citation: Endocrine-Related Cancer 30, 11; 10.1530/ERC-23-0148
Stable HNRNPA2B1-overexpressing MCF-7 cells have increased PSAT1 and PHGDH protein expression
We recently reported higher expression of PSAT1 and PHGDH in LCC9 and LY2 TAM-resistant cell lines derived from parental luminal A, endocrine-sensitive MCF-7 human BCa cells (Metcalf et al. 2020b ). We also demonstrated that stable overexpression of A2B1 in MCF-7 cells (MCF-7-A2B1 cells) results in TAM resistance (Petri et al. 2021). We examined the protein expression of PSAT1 and PHGDH in TAM-resistant MCF-7-A2B1, LCC9, and LY2 cells (Fig. 2A). MCF-7-A2B1 cells showed higher expression of PSAT1 and PHGDH protein compared to MCF-7 cells with values similar to those observed in LCC9 and LY2 cells (Fig. 2B and C). We investigated the effect of A2B1 knockdown (Supplementary Fig. 3A) (Petri et al. 2021) on the expression of PSAT1 and PHGDH transcript and protein in A2B1 cells and observed that siA2B1 reduced PSAT1 and PHGDH transcripts (Supplementary Fig. 3B and C) and protein (Supplementary Fig. 3D and E), suggesting a regulatory role for A2B1 in PSAT1 and PHGDH expression.
PSAT1 and PHGDH expression in breast cancer cell lines. (A) WCE were prepared from MCF-7, MCF-7-A2B1, LCC9, and LY2 cells and probed for PSAT1, then stripped and reprobed for PHGDH and for α-tubulin. (B and C) Individual values and the mean ± s.e.m. for PSAT1/α-tubulin and PHGDH/α-tubulin. *P ≤ 0.01 , one-way ANOVA, Tukey’s post hoc multiple comparisons test. (D) WCE were prepared from the indicated breast cancer cell lines and probed for PSAT1, then stripped and reprobed for PHGDH. The blot was stained with Ponceau S and with Amido Black for protein normalization. Shown here is one representative western blot of four separate blots. (E and F) Values of individual cell lysates from separate cell lysates as biological replicates. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons post hoc test. *P < 0.05; **P < 0.01, **P < 0.001; ****P < 0.0001.
Citation: Endocrine-Related Cancer 30, 11; 10.1530/ERC-23-0148
To determine the expression of SSP enzyme proteins PSAT1 and PHGDH in luminal A BCa cell lines unrelated to MCF-7 cells, we selected T47D (a p53 mutant cell line) and ZR-75-1 (no p53 or other established BCa-associated mutations) (Kenny et al. 2007). PSAT1 and PHGDH protein levels were higher in T47D and ZR-75-1 than in MCF-7 cells, but lower than in LCC9 and LY2 TAM-resistant BCa cell lines (Fig. 2D, E and F). To develop a TAM-resistant ZR-75-1 cell line (ZR-75-1-4OHT), we grew ZR-75-1 cells in medium containing 100 nM 4-OHT for > 8 mos. These cells have higher basal proliferation and are TAM- and fulvestrant-resistant compared to ZR-75-1 parental cells (Supplementary Fig. 4A and B). ZR-75-1-4-OHT cells have higher PSAT1 and PHGDH protein expression compared to the parental ZR-75-1 cell line (Fig. 2D, E, and F). Compared to MCF-7, all the BCa cell lines showed higher PSAT1 transcript abundance (Supplementary Fig. 5A); however, only MCF-7A2B1 cells showed higher PHGDH transcript abundance (Supplementary Fig. 5B). The difference between PHGDH protein and mRNA transcript abundance in these BCa cell lines suggest posttranscriptional modulation of PHGDH protein expression.
A2B1 expression decreases miRNAs predicted to target PSAT1 and PHGDH
Gene ontology identified ‘serine family amino acid metabolic processes’ as a pathway regulated by A2B1 overexpression in MCF-7 cells (Klinge et al. 2019). To determine if A2B1 regulates PSAT1 and PHGDH expression by downregulating miRNAs that target the 3′UTR of PSAT1 and PHGDH, we searched miRTarBase for miRNAs that target each transcript. There were no verified miRNAs targeting PSAT1 or PHGDH in that database, despite the reports that miR-340 (Yan et al. 2015), miR-424 (Fang et al. 2018), miR-195-5p (Wang et al. 2023), and miR-145-5p (Ding et al. 2022) directly target PSAT1. For PHGDH, miR-107 and miR-103a-3p were reported to directly target PHGDH (Zhang et al. 2021). When we compared miRNAs predicted to target PSAT1 and PHDGH with the A2B1-regulated miRNAs in MCF-7 cells (Klinge et al. 2019) using mirDIP 4.1 (Tokar et al. 2018), we identified 21 miRNAs predicted to target the 3′UTR of PSAT1 that were downregulated after transfection of MCF-7 cells with A2B1 (Table 1). Four miRNAs had either ‘very high’ (miR-4790-3p) or ‘high’ (miR-424-5p, miR-145-5p, and miR-6794-3p) confidence according to mirDIP prediction algorithms (Tokar et al. 2018). We selected miR-145-5p and miR-424-5p for further analysis since they had ‘high’ confidence and a high integration score (0.68 for miR-145-5p and 0.75 for miR-424-5p) (Tokar et al. 2018). Further, miR-145-5p and miR-424-5p have been reported to regulate other targets in BCa (Petri & Klinge 2020). We found 19 A2B1-downregulated miRs predicted to target PHGDH mRNA with mirDIP (Table 2). Three miRNAs (miR-34b-5p, miR-6801-5p, and miR-937-3p) were rated with ‘high’ confidence (Tokar et al. 2018). We selected miR-34b-5p and miR-876-5p for their relevance in BCa. miR-34b-5p was reported to inhibit BCa progression by targeting ARHGAP1 (Dong et al. 2020) and miR-876-5p inhibited BCa cell proliferation and invasion by targeting TFAP2A (Xu et al. 2019). Figure 3A shows the alignment of miR-145-5p, miR-424-5p, miR-34b-5p, and miR-876-5p with their miRNA response elements (MREs) in the 3′UTR of PSAT1 or PHGDH. We examined whether the expression of the selected miRNAs is associated with overall survival in BCa using Kaplan–Meier plotter (Gyorffy et al. 2010). These data suggest that low expression of miR-145-5p and miR-424-5p is associated with reduced overall survival (OS) of all BCa patients (Fig. 3B and C). The association of reduced miR-34b-5p with OS was not significant and high expression of miR-876-5p was associated with lower OS (Fig. 3D and E).
A2B1-downregulated miRs are predicted to target PSAT1 and PHGDH mRNA. miR-424-5p, miR-145-5p, miR-34b-5p, and miR-876-5p are downregulated in MCF-7-A2B1 cells (Klinge et al. 2019) (Tables 1 and 2). (A) The seed element and MRE for selected miRNA in the 3′UTR of PSAT1 and PHGDH. (B–E) Kaplan–Meier plotter was used to examine the role of miR-145-5p, miR-424-5p, miR-34b-5p, and miR-876-5p expression in overall survival of breast cancer patients. Low expression of miR-145 and miR-424 is significantly associated with lower overall survival (OS).
Citation: Endocrine-Related Cancer 30, 11; 10.1530/ERC-23-0148
A2B1-downregulated miRNA predicted to target the 3′UTR of PSAT1 mRNA. Using mirDIP 4.1 prediction tools we identified human miRNAs downregulated in MCF-7 cells overexpressing A2B1 (miRNA-seq. FC > 0.50, P < 0.05). The miRNA selected are predicted to bind to the MRE in the 3′UTR of PSAT1mRNA with very high, high, or medium confidence. Also shown are the integration score, number of sources that identify PSAT1 as a target of corresponding miRNA, and the sources that have identified the relationship.
| miRNA | Hours post TF | logFC | P | Integration score | Number of sources | Score class | Sources |
|---|---|---|---|---|---|---|---|
| miR-4790-3p | 48 | −1.66 | 4.79E-02 | 0.33 | 7 | Very high | bitargeting_May_2021|DIANA|MBStar|MirAncesTar|miranda_May_2021|miRDB_v6|mirzag |
| miR-424-5p | 48 | −1.18 | 8.96E-03 | 0.75 | 14 | High | BCmicrO|DIANA|MBStar|MirAncesTar|miranda_May_2021|miRcode|miRDB_v6|mirmap_May_2021|MiRNATIP|miRTar2GO|MirTar2|mirzag|MultiMiTar|TargetScan_v7_2 |
| miR-145-5p | 48 | −1.85 | 1.17E-02 | 0.68 | 10 | High | BCmicrO|CoMeTa|MirAncesTar|miranda_May_2021|miRcode|miRDB_v6|MiRNATIP|mirzag|RNA22|TargetScan_v7_2 |
| miR-6794-3p | 48 | −2.57 | 4.35E-02 | 0.13 | 2 | High | MirAncesTar|mirzag |
| miR-17-5p | 48 | −0.77 | 1.06E-02 | 0.38 | 8 | Medium | BCmicrO|bitargeting_May_2021|MBStar|MirAncesTar|miranda_May_2021|mirbase|MiRNATIP|miRTar2GO |
| miR-20a-5p | 48 | −0.64 | 4.35E-02 | 0.37 | 9 | Medium | bitargeting_May_2021|DIANA|MBStar|MirAncesTar|miranda_May_2021|mirbase|MiRNATIP|miRTar2GO|RNA22 |
| miR-3125 | 48 | −3.38 | 3.27E-03 | 0.32 | 10 | Medium | bitargeting_May_2021|DIANA|MBStar|MirAncesTar|miranda_May_2021|mirmap_May_2021|MiRNATIP|mirzag|PITA_May_2021|RNA22 |
| miR-224-5p | 48 and 72 | −2.86 | 1.63E-02 | 0.31 | 8 | Medium | BCmicrO|DIANA|MBStar|MirAncesTar|miranda_May_2021|miRDB_v6|MiRNATIP|miRTar2GO |
| miR-193a-3p | 48 | −0.82 | 1.72E-02 | 0.27 | 4 | Medium | BCmicrO|MirAncesTar|miRcode|miRTar2GO |
| miR-5008-3p | 48 and 72 | −2.63 | 2.93E-03 | 0.20 | 6 | Medium | MBStar|MirAncesTar|miranda_May_2021|miRDB_v6|MiRNATIP|mirzag |
| miR-520g-3p | 48 | −2.63 | 2.83E-02 | 0.17 | 5 | Medium | BCmicrO|MBStar|MirAncesTar|miranda_May_2021|MiRNATIP |
| miR-934 | 48 | −1.46 | 3.52E-03 | 0.17 | 4 | Medium | BCmicrO|DIANA|MirAncesTar|mirzag |
| miR-2053 | 72 | −3.82 | 1.46E-03 | 0.15 | 5 | Medium | DIANA|MBStar|MirAncesTar|miranda_May_2021|mirCoX |
| miR-497-3p | 48 | −0.66 | 4.08E-02 | 0.13 | 5 | Medium | DIANA|MirAncesTar|miranda_May_2021|mirmap_May_2021|mirzag |
| miR-518d-5p | 48 and 72 | −2.37 | 4.98E-02 | 0.13 | 5 | Medium | BCmicrO|bitargeting_May_2021|MBStar|MirAncesTar|miranda_May_2021 |
| miR-520c-5p | 48 and 72 | −2.37 | 4.98E-02 | 0.13 | 5 | Medium | BCmicrO|bitargeting_May_2021|MBStar|MirAncesTar|miranda_May_2021 |
| miR-1283 | 48 and 72 | −2.27 | 2.51E-02 | 0.10 | 5 | Medium | BCmicrO|MBStar|MirAncesTar|miranda_May_2021|MiRNATIP |
| miR-486-5p | 48 and 72 | −1.24 | 2.52E-02 | 0.10 | 4 | Medium | DIANA|MirAncesTar|MiRNATIP|mirzag |
| miR-4720-5p | 72 | −3.05 | 7.52E-03 | 0.08 | 4 | Medium | bitargeting_May_2021|MirAncesTar|miranda_May_2021|RNA22 |
| miR-3118 | 72 | −3.99 | 2.77E-04 | 0.08 | 3 | Medium | MBStar|MirAncesTar|miranda_May_2021 |
| let-7i-3p | 48 | −0.76 | 1.18E-02 | 0.04 | 2 | Medium | MBStar|MirAncesTar |
A2B1-downregulated miRNA predicted to target the 3′UTR of PHGDH mRNA. Using mirDIP 4.1 prediction tools we identified human miRNAs downregulated in MCF-7 cells overexpressing A2B1 (miRNA-seq. FC > 0.50, P < 0.05). The miRNA selected are predicted to bind to the MRE in the 3′UTR of PHGDH mRNA with very high, high, or medium confidence. Also shown are the integration score, number of sources that identify PHGDH as a target of corresponding miRNA, and the sources that have identified the relationship.
| miRNA | Hours post TF | logFC | P | Integration score | Number of sources | Score class | Sources |
|---|---|---|---|---|---|---|---|
| miR-34b-5p | 48 | −2.20 | 2.90E-02 | 0.38 | 11 | High | bitargeting_May_2021|DIANA|MirAncesTar|miranda_May_2021|mirbase|mirmap_May_2021|MiRNATIP|miRTar2GO|PITA_May_2021|RNA22|rnahybrid_May_2021 |
| miR-6801-5p | 72 | −3.01 | 1.42E-06 | 0.20 | 8 | High | bitargeting_May_2021|MirAncesTar|miranda_May_2021|mirmap_May_2021|mirzag|PITA_May_2021|RNA22|rnahybrid_May_2021 |
| miR-937-3p | 48 | −2.17 | 3.69E-02 | 0.21 | 6 | High | BCmicrO|bitargeting_May_2021|MirAncesTar|miranda_May_2021|miRTar2GO|PITA_May_2021 |
| miR-150-5p | 72 | −2.83 | 2.39E-02 | 0.22 | 9 | Medium | BCmicrO|bitargeting_May_2021|DIANA|MirAncesTar|miranda_May_2021|miRcode|mirCoX|mirmap_May_2021|RNA22 |
| miR-34c-5p | 48 | −2.36 | 8.62E-03 | 0.27 | 6 | Medium | BCmicrO|MirAncesTar|miranda_May_2021|mirbase|miRTar2GO|RNA22 |
| miR-876-5p | 72 | −3.05 | 1.83E-02 | 0.19 | 6 | Medium | BCmicrO|DIANA|MirAncesTar|mirbase|mirzag|RNA22 |
| miR-193a-3p | 48 | −0.82 | 1.72E-02 | 0.14 | 4 | Medium | BCmicrO|bitargeting_May_2021|miranda_May_2021|miRcode |
| miR-5088-5p | 72 | −2.95 | 1.80E-02 | 0.13 | 6 | Medium | bitargeting_May_2021|MirAncesTar|miranda_May_2021|mirmap_May_2021|MiRNATIP|RNA22 |
| miR-3663-5p | 48 | −2.96 | 2.38E-02 | 0.09 | 5 | Medium | bitargeting_May_2021|MirAncesTar|miranda_May_2021|MiRNATIP|RNA22 |
| miR-518d-5p | 48 and 72 | −2.37 | 4.98E-02 | 0.09 | 4 | Medium | BCmicrO|mirbase|mirmap_May_2021|RNA22 |
| miR-6789-5p | 72 | −3.05 | 5.93E-03 | 0.09 | 5 | Medium | bitargeting_May_2021|MirAncesTar|mirmap_May_2021|PITA_May_2021|RNA22 |
| miR-6794-3p | 48 | −2.57 | 4.35E-02 | 0.09 | 5 | Medium | bitargeting_May_2021|MirAncesTar|MiRNATIP|PITA_May_2021|RNA22 |
| miR-1271-3p | 72 | −3.37 | 2.15E-03 | 0.08 | 4 | Medium | bitargeting_May_2021|MirAncesTar|miranda_May_2021|RNA22 |
| miR-5682 | 48 | −2.37 | 4.98E-02 | 0.07 | 4 | Medium | bitargeting_May_2021|MirAncesTar|miranda_May_2021|RNA22 |
| miR-6733-3p | 48 | −1.74 | 3.49E-02 | 0.06 | 3 | Medium | bitargeting_May_2021|MirAncesTar|RNA22 |
| miR-6856-3p | 72 | −3.38 | 1.74E-03 | 0.06 | 3 | Medium | MirAncesTar|miranda_May_2021|mirmap_May_2021 |
| miR-4767 | 48 | −1.63 | 3.97E-02 | 0.06 | 4 | Medium | MirAncesTar|mirmap_May_2021|PITA_May_2021|RNA22 |
| miR-7975 | 48 | −2.00 | 1.86E-02 | 0.06 | 3 | Medium | bitargeting_May_2021|miranda_May_2021|RNA22 |
| miR-6872-5p | 72 | −2.86 | 1.28E-03 | 0.04 | 2 | Medium | bitargeting_May_2021|RNA22 |
MCF-7-A2B1, LCC9, and LY2 cells have lower miR-145-5p, miR-424-5p, miR-34b-5p, and miR-876-5p expression compared to MCF-7 cells
To confirm the downregulation of miR-145-5p, miR-424-5p, miR-34b-5p, and miR-876-5p by A2B1 in MCF-7 cells, RT-qPCR was performed. The expression of miR-145-5p and miR-424-5p was significantly reduced in MCF-7-A2B1 cells compared to parental MCF-7 cells (Fig. 4A and B), verifying that modest, stable A2B1 overexpression reduces their expression as seen initially in miRNA-seq data from A2B1-transiently transfected MCF-7 cells (Klinge et al. 2019). In contrast, miR-34b-5p expression was higher in MCF-7-A2B1 cells while miR-876-5p expression was unaltered in MCF-7-A2B1 cells compared to MCF-7 cells, suggesting differences between A2B1 transient and stable A2B1 regulation of these miRNAs (Fig. 4C and D). TAM-resistant LCC9 and LY2 cells have higher endogenous A2B1 compared to MCF-7 cells (Klinge et al. 2019). If A2B1 downregulates miR-145-5p, miR-424-5p, miR-34b-5p, and miR-876-5p, we expect lower expression of these miRNAs in LCC9 and LY2 cells relative to MCF-7 cells. As predicted, compared to MCF-7 cells, LCC9 and LY2 cells have a significantly lower abundance of miR-145-5p, miR-424-5p, and miR-876-5p (Fig. 4E, F and H). However, miR-34b-5p transcript abundance was lower only in the LY2 cells (Fig. 4G), suggesting factors in addition to A2B1 regulate miR-34-5p abundance in LCC9 cells. Taken together, these data show reduced miR-145-5p, miR-424-5p, and miR-876-5p in both LCC9 and LY2 TAM-resistant cells and that miR-34b-5p is lower in LY2 cells compared to ET-sensitive MCF-7 cells.
Examination of miR-424-5p, miR-145-5p, miR-34b-5p, and miR-876-5p expression in MCF-7-A2B1, LCC9, and LY2 cells. qPCR was performed on independent samples of miRNA isolated from MCF-7, MCF-7-A2B1, LCC9, and LY2 breast cancer cells. Values were normalized by RNU48. Data in A and B were evaluated by Student’s unpaired t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data in E, F, G, and H were evaluated by one-way ANOVA followed by Tukey’s multiple comparison test.
Citation: Endocrine-Related Cancer 30, 11; 10.1530/ERC-23-0148
Validation of PSAT1 and PHDGH as bona fide targets of A2B1-regulated miRNAs
To determine whether PSAT1 and PHGDH are bona fide targets of miR-145-5p and miR-424-5p and miR-35b-5p and miR-876-5p, respectively, we tested the ability of each miRNA to repress luciferase activity from reporter vectors containing the 3′UTR of PSAT1 or PHGDH. Cotransfection of pre-miR-145-5p and pre-miR-424-5p repressed luciferase activity from the reporter containing the PSAT1 3′UTR (Fig. 5A). Similarly, cotransfection of pre-miR-34b-5p and pre-miR-876-5p inhibited luciferase reporter activity from the construct containing the PHGDH 3′UTR (Fig. 5B). Cotransfection of pre-miR-145-5p with a luciferase reporter containing the miR-145-5p MRE from the 3′UTR of PSAT1 also repressed luciferase activity; however, miR-145-5p did not inhibit luciferase activity from the reporter containing the MRE for miR-424-5p (Fig. 5C). Conversely, miR-424-5p inhibited luciferase activity from the miR-424-5p MRE in PSAT1 but not the miR-145-5p MRE in PSAT1 (Fig. 5D). While miR-34b-5p and miR-876-5p inhibited luciferase activity when cotransfected with the reporter containing the respective MRE from the PHGDH 3′UTR, miRNA inhibition was not significantly blocked when cotransfected with a nontarget MRE reporter (MRE 424) (Fig. 5E and F). These data demonstrate the specificity of the miR-MRE recognition and direct miR-MRE interaction for miR-145-5p and miR-424-5p for targeting PSAT1; however, the ability of miR-34b-5p and miR-876-5p to directly target their putative MREs in the 3′UTR of PHGDH is not fully supported. Together, these data validate that PSAT1 is a bona fide direct target of miR-145-5p and miR-424-5p.
A2B1-downregulated miRNAs regulate luciferase report activity. (A and B) HEK-293T cells were transiently transfected with pEZX-MT06 containing the full-length 3′UTR of PSAT1 or PHGDH, pre-miR-Control (Cont), pre-miR-145-5p, pre-miR-424-5p, pre-mR-846-5p, or pre-miR-34b-5p, as indicated. (C–F) HEK-293T cells were transiently transfected with pmirGLO containing the indicated MRE sequence from PSAT1 or PHGDH and the indicated pre-miR-Control or miR as in A and B. After 48 h, dual luciferase assays were performed with the FF–renilla ratios normalized to the control transfection. n = 5 separate experiments. Statistical analysis used one-way ANOVA followed by Tukey’s multiple comparison test: *P ≤ 0.05, **P < 0.01, ****P ≤ 0.001.
Citation: Endocrine-Related Cancer 30, 11; 10.1530/ERC-23-0148
miR-145-5p and miR-424-5p regulate endogenous expression of PSAT1 in ER+ BCa cells
To investigate if miR-145-5p and miR-424-5p regulate endogenous PSAT1 expression in TAM-resistant BCa cells with high PSAT1, we transfected pre-miR-145-5p or pre-miR-424-5p into LCC9 cells. Increased miR-145-5p and miR-424-5p were detected in the transfected LCC9 cells (Supplementary Fig. 6A and B). Only the transfection of pre-miR-424-5p significantly reduced PSAT1 mRNA abundance (Fig. 6A); however, PSAT1 protein abundance was decreased 48 h post transfection of either pre-miR-145-5p or pre-miR-424-5p (Fig. 6B, C, and D). Further, this decrease was inhibited by cotransfection of anti-miR-145-5p or anti-miR-424-5p (Fig. 6B, C, and D). The effect on PSAT1 protein abundance was lost at 72 h post transfection with pre-miR-145-5p and pre-miR-424-5p, suggesting a transient miRNA activity (Supplementary Fig. 6C, D, and E). We transfected pre-miR-34b or pre-miR-876b, putatively targeting PHGDH (Fig. 4), into LCC9 cells and increased miR-34b and miR-876-5p were detected (Supplementary Fig. 7A and B). Transfection of LCC9 cells with pre-miR-34b-5p or pre-miR-876-5p did not reduce PSAT1 protein 48 h or 72 h post transfection (Supplementary Fig. 7C, D, E, and F), demonstrating specificity. Thus, both miR-145-5p and miR-424-5p specifically regulate endogenous PSAT1 protein abundance in TAM-resistant LCC9 cells. Transfection of pre-miR-145-5p and pre-miR-424-5p (Supplementary Fig. 8A and B) did not alter PSAT1 protein expression in LY2 cells at 48 h or 72 h (Supplementary Fig. 8C, D, E, F, G, and H), suggesting cell-specific miRNA-mediated regulation of PSAT1 abundance.
miR-145-5p and miR-424-5p target PSAT1 in LCC9, ZR-751, and ZR-75-1-4-OHT cells. (A) LCC9 cells were transfected with pre-miR-control pre-miR-145-5p, or pre-miR-424-5p and RT-qPCR was performed on RNA isolated 48 h after transfection. Values were normalized by 18S. Values are the mean ± s.e.m. of three independent, biological replicate experiments. (B) WCE were prepared from LCC9 cells 48 h post transfection with pre-miR-control pre-miR-145-5p, pre-miR-424-5p or cotransfection with anti-miR-145-5p or anti-miR-424-5p, and probed with a PSAT1 antibody. The blots were stained with Ponceau S as a protein loading control. (C and D) Analysis of western blots from three independent samples. Values were normalized to LCC9 miR control. Values are normalized to GAPDH or Ponceau S. (E) WCE were prepared from ZR-75-1 cells 72 h post transfection with pre-miR-145-5p, pre-miR-424-5p, or cotransfection with anti-miR-145-5p or anti-miR-424-5p and probed with a PSAT1 antibody. (F and G) Analysis of western blot performed on 3 independent samples. Values were normalized to ZR-75-1 miRNA control. (H) WCE were prepared from ZR-75-1 4-OHT cells 72 h post transfection with pre-miR-145-5p, pre-miR-424-5p, or cotransfection with anti-miR-145-5p or anti-miR-424-5p and probed with a PSAT1 antibody. (I and J) Analysis of western blots performed on three independent samples. Values are normalized to GAPDH or Ponceau S. Statistical analysis used one-way ANOVA followed by Tukey’s multiple comparison test: *P ≤ 0.05, **P < 0.01,***P ≤ 0.001.
Citation: Endocrine-Related Cancer 30, 11; 10.1530/ERC-23-0148
Since T47D cells showed higher PSAT1 protein and mRNA abundance relative to MCF-7 (Fig. 2D), we also investigated the effects of miR-145-5p and miR-424-5p on endogenous PSAT1 expression. We transfected pre-miR-145-5p or pre-miR-424-5p into T47D cells. Increased miR-145-5p and miR-424-5p were detected in the transfected cells (Supplementary Fig. 9A and B). miR-424-5p decreased PSAT1 protein and the reduction in PSAT1 by miR-424-5p was abrogated by cotransfection anti-miR-424-5p 48 h, but not 72 h, post transfection (Supplementary Fig. 9C, E, F, and H). In contrast, transfection with pre-miR-145-5p did not significantly reduce PSAT1 protein in T47D cells 48 h or 72 h post transfection (Supplementary Fig. 9C, D, F, and G). These observations support the previous data showing that miR-424-5p targets PSAT1 in LCC9 cells 48 h post transfection and suggest that cell-line-specific factors modulate the ability of miR-145-5p to target PSAT1.
Similarly, ZR-75-1-4-OHT cells showed higher PSAT1 protein expression compared to parental ZR-75-1 or MCF-7 cells (Fig. 2D). We transfected ZR-75-1 and ZR-75-1-4-OHT cells with pre-miR-145-5p or pre-miR-424-5p (Supplementary Fig. 10A, B, C, and D). At 48 h post transfection with either pre-miR-145-5p or miR-424-5p, PSAT1 protein was unchanged (Supplementary Fig. 11). However, 72 h transfection of ZR-75-1 or ZR-75-1-4-OHT cells with either pre-miR-145-5p or miR-424-5p reduced PSAT1 protein abundance and this reduction was blocked by cotransfection with the respective anti-miR (Fig. 6E, F, G, H, I, and J). These data demonstrate that miR-145-5p and miR-424-5p specifically reduce PSAT1 protein in these cells.
miR-34b-5p and miR-876-5p regulate endogenous expression of PHGDH in T47D cells
To investigate the effects of miR-34b-5p and miR-876-5p overexpression on endogenous PHGDH, LCC9, T47D, ZR-75-1, and ZR-75-1-4-OHT cells were transfected with either pre-miR-34b-5p or pre-miR-876-5p and increased miRNA abundance was detected with RT-qPCR (Supplementary Figs. 7A, B, 12A, B, C, D, E, F, G, and H). Both miR-34b-5p and miR-876-5p reduced PHGDH mRNA in LCC9 cells (Fig. 7A). We detected a significant decrease in PHGDH protein in LCC9 cells 72 h post transfection with pre-miR-876-5p and although cotransfection of anti-miR-876-5p did not affect this inhibition, the level of PHGDH protein was similar to the control with cotransfection (Fig. 7B, C, and D). In T47D cells, which are less 4-OHT-sensitive compared to MCF-7 cells (Petri et al. 2021), PHGDH was reduced by pre-miR-34-5p in T47D cells and this reduction was blocked by anti-miR-34b-5p cotransfection demonstrating specificity (Fig. 7E and F). We did not detect any change in PHGDH protein abundance in ZR-75-1 or ZR-75-1-4-OHT cells transfected with pre-miR-34b-5p or pre-miR-876-5p at 48 h or 72 h post transfection (Supplementary Fig. 13).
miR-34b-5p and miR-876-5p target PHGDH in T47D cells. (A) qPCR was performed on independent samples of RNA isolated from LCC9 cells 48 h post transfection (TF) with pre-miR-34b-5p and pre-miR-876-5p. Values were normalized by 18S. Data are the average of 3 individual experiments. (B) WCE were prepared from LCC9 cells 72 h post transfection with pre-miR-34b-5p, pre-miR-876-5p, or cotransfection with pre-miRs and anti-miRs and probed with a PHGDH antibody. (C and D) Analysis of western blot performed on 3 independent samples. Values were normalized to LCC9 media control. (E) WCE were prepared from T47D cells 72 h post transfection with pre-miR-34b-5p, pre-miR-876-5p, or cotransfection with pre-miRs and anti-miRs and probed with a PHGDH antibody. (F and G) Analysis of western blot performed on three independent samples. Values were normalized to T47D media control. Individual values are the mean ± s.e.m. for PHGDH/Ponceau S. ***P ≤ 0.001, **P ≤ 0.01 one-way ANOVA, Tukey’s multiple comparison test. Values were normalized to GAPDH. Statistical analysis used one-way ANOVA followed by Tukey’s multiple comparison test: *P ≤ 0.05, **P < 0.01, ***P ≤ 0.001.
Citation: Endocrine-Related Cancer 30, 11; 10.1530/ERC-23-0148
Taken together, these results demonstrate the inhibition of PHGDH protein expression by miR-876-5p in LCC9 cells, and by miR-34b-5p in T47D cells. The inability of miR-34b-5p and miR-876-5p to suppress PHGDH expression in ZR-75-1 and ZR-75-1-4-OHT cells suggests that cell-specific factors mediate the effect of miRNA inhibition of PHGDH protein synthesis.
Transfection of miRNAs targeting PSAT1 and PHGDH reduces LCC9 and ZR-75-1-4-OHT cell viability, increases ET sensitivity, and results in E2 growth inhibition
Since inhibition of the SSP by inhibiting PHGDH activity increased TAM sensitivity of LCC9 (Metcalf et al. 2020b ), we examined the effects of the miRs targeting PSAT1 and PHGDH on the cell viability of LCC9 and ET-resistant ZR-75-1-4-OHT cells (Fig. 8). Cells were transfected with pre-miRs for miR-145-5p, miR-424-5p, miR-34b-5p, and miR-876-5p and then treated with 100 nM or 1 μM 4-OHT, 100 nM fulvestrant, or 10 nM E2. The concentration of 4-OHT models the concentrations detected in BCa tissues when patients received 5 or 20 mg tamoxifen citrate/day (Kisanga et al. 2004). Cotransfection of the four miRs reduced the viability of ZR-75-1-4-OHT cells and increased sensitivity to 4-OHT and fulvestrant in LCC9 and ZR-75-1-OHT cells (Fig. 8). The miR-transfected cells were also growth-inhibited by 10 nM E2, suggesting again in sensitivity to E2 as an antagonist, as seen in MCF-7 cells that adapted under long-term estrogen deprivation (LTED) (Lewis-Wambi & Jordan 2009). These data suggest that the reduction of miR-145-5p, miR-424-5p, miR-34b-5p, and miR-876-5p detected in TAM-resistant LCC9 BCa cells promotes resistance to antiestrogens and rescuing the miRNA expression restores antiestrogen sensitivity in these ER+ BCa cells.
Transfection with miRs targeting PSAT1 and PHGDH restores growth inhibition by 4-OHT and fulvestrant in endocrine-resistant LCC9 cells. (A) and ZR-75-1-4-OHT (B) cells were transfected with miR Control (−) or pre-mir-145-5p, pre-miR-424-5p, pre-miR-34b-5p, and pre-miR-876-5p for 48 h prior to treatment: DMSO control, 100 nM 4-OHT, 1 μM 4-OHT, 100 nM fulvestrant, or 10 nM E2. Treatment was for 5 days. Values are the mean ± s.e.m. from three and four replicate experiments in A and B, respectively, where data from B include data from two plates/experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 two-way ANOVA, Sidak’s multiple comparisons test with single pooled variance.
Citation: Endocrine-Related Cancer 30, 11; 10.1530/ERC-23-0148
Discussion
Endocrine therapies are the first line of treatment for patients with ER+ BCa resulting in a significant reduction of BCa-related mortality (Robertson et al. 2021). However, acquired resistance to endocrine therapies continues to present major challenges in the treatment of ER+ BCa (Clarke et al. 2015) and the mechanisms of ER+ endocrine resistance are not completely understood. In this study, we tested the hypothesis that four miRNAs downregulated by the m6A reader RNA binding protein A2B1, miR-145-5p, miR-424-5p, miR-34b-5p, and miR-876-5p, are regulators of key enzymes in the SSP and that overexpression of these A2B1-regulated miRNAs increases the sensitivity ER+ endocrine-resistant LCC9 cells to TAM and fulvestrant.
Using computational analysis, we identified four miRNA that were downregulated by A2B1 and predicted to target PSAT1 (miR-145-5p and miR-424-5p) or PHGDH (miR-34b-5p and miR-876-5p). We demonstrated for the first time that PSAT1 is a bona fide direct target of miR-145-5p and miR-424-5p, miRs that are reduced in TAM-R BCa cell lines LCC9, LY2, and MCF-7-A2B1 cells. In addition, miR-145-5p and miR-424-5p reduced PSAT1 protein in T47D and ZR-75-1 and TAM-resistant ZR-75-1-4-OHT BCa cells, demonstrating that this targeting is not specific for MCF-7 and its TAM-resistant derivative cell lines LCC9 and LY2. We found that the ability of miR-145-5p and miR-424-5p to reduce endogenous PSAT1 protein varied between cell lines.
Serine is central to the biosynthesis of many molecules (Locasale 2013) and increased serine biosynthesis is a metabolic change that has been reported in cancer cells (Davis et al. 1970, Locasale 2013). We reported that overexpression of two key enzymes of the serine synthetic pathway, PSAT1 and PHGDH, contributed to TAM resistance in ER+ BCa (Metcalf et al. 2020b). Serine is also an allosteric activator of PKM2 (Chaneton et al. 2012), an isoform of pyruvate kinase that is highly expressed in breast tumors and negatively associates with reduced overall and disease-free survival (Zhu et al. 2017). PHGDH was demonstrated to be a gene essential for breast tumorigenesis (Possemato et al. 2011) and levels of PHGDH, PSPH, SHMT, and PSAT1 (Supplementary Fig. 1) are elevated in breast tumors and correlate with reduced OS (Geck & Toker 2016). Conversely, low PSAT1 was correlated with higher progression-free survival in TAM-treated BCa patients with ERα + primary breast tumors (Martens et al. 2005b). The higher intensity of PSAT1 expression in the intraductal carcinoma of patients with ER+ BCa compared to normal breast tissue, suggests that serine biosynthesis is more active in ER+ tumors; however, due to the lack of treatment and disease progression data from the TMA, it is unclear if the increased activity results from acquired therapy resistance. Examination of RNA seq data from MCF-7 versus LCC9 cells revealed that PHGDH, PSAT1, and PSPH transcript expression was significantly higher in LCC9 endocrine-resistant cells compared to MCF-7 cells (Muluhngwi et al. 2017).
The expression of PSAT1 and PHGDH is regulated by many transcription factors including Sp1 (Jun et al. 2008), NFYB (Jun et al. 2008), p53 (Simabuco et al. 2018), ATF3 (Li et al. 2021b, Luo et al. 2022), ATF4 (Ye et al. 2012, Gao et al. 2017), NRF2 (DeNicola et al. 2015), and YAP (Li et al. 2021b)). In addition, PSAT1 expression is downregulated by miRNAs in cancers: miR-195-5p (ovarian), miR-145-5p (colon), and miR-195-5p (TNBC), respectively (Ding et al. 2022, Wang et al. 2023). The processing of primary miRNAs (pri-miRNAs) to precursor miRNAs (pre-miRNAs) is facilitated by the DROSHA/DGCR8 microprocessor complex in the nucleus. For some pri-miRNAs, this process is facilitated by A2B1, an RNA binding protein that recognizes the RNA modification N6-methyladenosine (m6A) and associates with DGCR8 in the DROSHA complex to regulate pri-miRNA processing (Alarcon et al. 2015a) or recruit negative miRNA regulators. We reported that overexpression of A2B1 in ER+ MCF-7 cells dysregulates the expression of miRNAs (Klinge et al. 2019). We have also reported that stable overexpression of A2B1 confers an endocrine-resistant phenotype in MCF-7 cells (Petri et al. 2021). Here we found that, similar to the LCC9 and LY2 TAM-resistant cell lines, MCF-7-A2B1 cells had higher PSAT1 and PHGDH expression compared to MCF-7 cells. These results suggest a role for A2B1 in the processing of miRNAs that target enzymes in the serine biosynthetic pathway contributing to the progression from endocrine sensitivity to endocrine resistance.
miR-145-5p has been suggested to have anticancer effects in various cancers, e.g. gastric cancer (Zhou et al. 2020), cervical cancer (He et al. 2020), and ovarian cancer (Pan et al. 2022). Overexpression of miR-145-5p in ovarian cancer cells reduced PSAT1 mRNA (Pan et al. 2022). Cotransfection of miR-145-5p with a dual-luciferase reporter vector containing the PSAT1 3′UTR decreased luciferase intensity implicating PSAT1 as a direct target of miR-145-5p. In addition, overexpression of miR-145-5p in ER+ BCa cells decreased PSAT1 mRNA and PSAT1 protein. Taken together with previous reports, our new data suggest a common inverse relationship between miR-145-5p and PSAT1 in some cancers. Although miR-145-5p was negatively correlated with CCAT2 and AKT3 which have higher expression in TAM-resistant cells (Moradi et al. 2022), this is the first evidence of a negative correlation between miR-145-5p and PSAT1 in endocrine-resistant BCa cell lines.
The activity of miR-424-5p varies between cancer types. For example, miR-424-5p levels were higher in laryngeal squamous cell carcinoma and promoted cancer progression by targeting the tumor suppressor CADM1, suggesting onco-miRNA activity (Li et al. 2019). However, miR-424-5p plays a tumor-suppressive role in other cancers by targeting oncogenes including ACSL4 in ovarian cancer (Ma et al. 2021) and SOCS6 in pancreatic cancer (Wu et al. 2013). More specifically, miR-424-5p was downregulated in colorectal cancer and played a tumor-suppressive role by targeting AKT3 and PSAT1 (Fang et al. 2018). miR-424-5p reduced BCa cell viability in MDA-MB-231 TNBC cells by targeting PD-L1 (Dastmalchi et al. 2020). No previous studies have investigated the effects of miR-424-5p on endocrine resistance in BCa. Importantly, we demonstrated that miR-424-5p directly targets PSAT1 mRNA and decreases PSAT1 protein in endocrine-resistant BCa cells. Additionally, we demonstrated that cotransfection of miR-145-5p, miR-424-5p, miR-34b-5p, and miR-876-5p increases sensitivity of endocrine-resistant LCC9 cells to 4-OHT and fulvestrant.
There are no reports of miR-34b-5p activity in BCa; however, mir-34b-5p was downregulated in papillary thyroid cancer (Rogucki et al. 2022), colon cancer (Wang et al. 2022), cervical cancer (Ye et al. 2022), and lung cancer (Mizuno et al. 2017). In colon cancer cells, HuR was a target of miR-34b-5p (Wang et al. 2022). HuR is a key posttranscriptional regulator in BCa progression and higher expression of HuR correlated to aggressive BCa phenotypes (Liao et al. 2023). HuR stimulated the expression of BCa oncogenes including CCL2 (Lee et al. 2017), cyclin D1 (CCND1) (Yuan et al. 2011), MMP-9 (Heinonen et al. 2007), and vascular endothelial growth factor (Ortega et al. 2008). These studies demonstrate an anti-oncogenic role for miR-34b-5p. Our study is the first to identify PHGDH as a direct miR-34b-5p target. There is some evidence that miR-876-5p plays a role in decreasing cell proliferation in BCa (Dong et al. 2019a, Xiu et al. 2022). While we confirmed PHGDH as a bona fide target of miR-876-5p, the ability of miR-34b-5p to reduce endogenous PHGDH protein expression was less conclusive. We have shown that miRNA activity is dependent on cell type and length of time post transfection, but there may be other regulatory factors influencing miRNA modulation of PSAT1 and PHGDH protein expression, including regulation of miRNA by other noncoding RNA.
There are a number of noncoding RNAs, including circular RNAs (circRNAs), long noncoding RNAs (lncRNAs) that have critical regulatory roles in tumorigenesis and drug resistance in cancer (Hua et al. 2019, Wu et al. 2019, Yu et al. 2020). Mechanistically, lncRNAs and circRNAs bind directly to miRNA seed regions and act as competing endogenous RNAs (ceRNA) to ‘sponge’ miRNAs, resulting in downregulation of miRNAs and upregulation of miRNA targets (Zheng et al. 2016, Li et al. 2017). Several of these ceRNA–miRNA relationships have been identified with miR-145-5p, miR-424-5p, miR-34b-5p, and miR-876-5p in cancer (Table 3). In MDA-MB-231 cells, lncRNA ST8SIA6-AS1 sponged miR-145-5p which increased CDCA3 expression and inactivated p53/p21 signaling (Qiao et al. 2022). Upregulation of TGFBR2 in MCF-7 and MDA-MB-231 cells due to LINC00052 suppression of miR-145-5p resulted in increased cell proliferation, migration, and invasion (Dong et al. 2021). LINC00473 is elevated in BCa tissue and cell lines and LINC00473 acted as ceRNA of miR-424-5p in BCa cells, blocking its inhibition of CCNE1 and increasing EMT, cell migration and cell invasion (Zhang & Yang 2023). Inhibition of GRK5 by miR-876-5p in TNBC cells was abrogated by LINC01315-miR-876-5p sponging, promoting TNBC progression (Xiu et al. 2022). The cross-regulatory network between ceRNAs and miRNAs offers a possible explanation for the incomplete decrease in PSAT1 and PHGDH protein expression observed in this study. Future research is necessary to understand the ceRNA–miRNA–mRNA mechanisms involved in ER+ BCa progression and endocrine resistance.
Summary of competing endogenous RNA (ceRNA) interactions with miR-145-5p, miR-424-5p, miR-34b-5p, or miR-876-5p in various types of cancer. The table summarizes evidence of ceRNAs and their respective interactions with miR-145-5p, miR-424-5p, miR-34b-5p, or miR-876-5p in cancer. Two databases were searched for ceRNA expression in breast cancer cell lines (LncExpDB – https://ngdc.cncb.ac.cn/lncexpdb/ and circBase – http://www.circbase.org/).
| miRNA | Evidence of ceRNA activity by lncRNA or circRNA | ceRNA expressed in breast cancer cell lines (database) |
|---|---|---|
| miR-145-5p | CircZNF609 acted as ceRNA of miR-145-5p in MCF-7 and MDA-MB-231 cells, upregulating the expression of oncogene p70S6K1 expression and increasing cell proliferation (Wang et al. 2018). | |
| CircZNF236 absorbed miR-145-5p in SCC-15 and CAL-27 and increased proliferation, invasion, and migration of oral squamous cell carcinoma (OSCC) cells (Lu et al. 2023). | (circBASE) | |
| circGOLPH3 was upregulated in OSCC cells and bound miR-145-5p, decreased its abundance and prevented miR-145-5p suppression of KDM2A which promotes tumorigenesis and metastasis (Cheng et al. 2023). | ||
| Circ_0015756 indirectly regulated PSAT1 by binding miR-145-5p, resulting in increased migration and invasion of ovarian cancer (OC) cells (Pan et al. 2022). | ||
| Circ0001955 was increased in a genome-wide study of colorectal cancer (CRC) patients and was negatively correlated with miR-145-5p (Kadkhoda et al. 2022). | ||
| Circ 0058063 is increased in OSCC cells and bound to miR-145-5p, upregulating the downstream target SERPIN1, contributing to cancer cell proliferation (Yu et al. 2022) | ||
| circMYOF bound to miR-145-5p, and prevented the inhibition by miR-145-5p of OTX1 in the exosomes of laryngeal squamous cell carcinoma (LSCC) patients, increasing cell migration and invasion ability (Li et al. 2022). | (circBase) | |
| LncRNA PVT1 acted as ceRNA of miR-145-5p in MDA-MB-231 cells and enhanced glycolysis and cell proliferation (Qu et al. 2023) | (LncExpDB) | |
| miR-424-5p | LncRNA HCG28 inhibited miR-424-5p suppression of SOX9 expression in cholangiocarcinoma tissues and cell lines which increased cell proliferation (Ni et al. 2023). | (LncExpDB) |
| Circ-HSP90A upregulated PD-L1 by binding to miR-424-5p and reduced the effects of immunotherapy in non-small-cell lung cancer (NSCLC) cells (Lei et al. 2023). | ||
| Depletion of circRBMS3, which regulated eIF4B and YRDC by acting as ceRNA of miR-424-5p, inhibited malignancy in an osteosarcoma xenograft mouse model (Gong et al. 2023). | ||
| The upregulation of lncRNA AGAP2-AS1 targeted and reduced miR-424-5p in keratinocytes, leading to an upregulation of miR-424-5p target AKT3 and activation of the AKT/mTOR pathway in keratinocyte proliferation (Xian et al. 2022). | (LncExpDB) | |
| circACTN4 (circ_0050898) acted as ceRNA of miR-424-5p in an intrahepatic cholangiocarcinoma (ICC) xenograft mouse model, increasing YAP1, an miR-424-5p target and activated Wnt/β-catenin signaling (Chen et al. 2022). | ||
| LINC00355 induced EMT in bladder cancer cells by inhibiting miR-424-5p which increased HMGA2 expression (Li et al. 2021a ). | (LncExpDB) | |
| miR-34b-5p | lncRNA SNHG3, upregulated in CRC patients, sponged miR-34b-5p in CRC cells which increased miR-34b-5p target HuR. Depleting lncRNA SNHG3 abundance inhibited cell proliferation (Zhao et al. 2022). | (LncExpDB) |
| lncRNA OIP5-AS1, which is elevated in colon cancer (CC), targeted and inhibited miR-34b-5p in CC cells, preventing miR inhibition of HuR expression and driving CC malignancy (Wang et al. 2022). | (LncExpDB) | |
| High expression of lncRNA PVT1 in diffuse large B-cell lymphoma (DLBCL) patients correlated to poor prognosis. lncPVT1 acted as ceRNA of miR-34b-5p in BLBCL cells, increasing Foxp. Compared to nontransfected tumor cells, cells with shRNA knockdown of PVT1 injected into mice resulted in slower tumor growth (Tao et al. 2022) | (LncExpDB) | |
| Upregulation of LINC00355 increased ABCB1 by inhibiting miR-34b-5p in bladder cancer cells which promoted bladder cancer cell resistance to cisplatin (Luo et al. 2021). | (LncExpDB) | |
| LINC02418 inhibited miR-34-5p suppression of BCL2 in CRC cells, promoting cell proliferation (Tian et al. 2020). | (LncExpDB) | |
| circBFAR acted as a ceRNA of mir-34b-5p, inhibiting its suppression of MET expression in pancreatic ductal carcinoma (PDAC) cells and promoted proliferation, migration, and invasion (Guo et al. 2020b ). | (circBASE) | |
| LncNEAT1 inhibited miR-34b-5p suppression of GL1 in DLBCL cells, and increased cell proliferation (Qian et al. 2020). | (LncExpDB) | |
| miR-875-5p | circCDR1 acted as a ceRNA of miR-876-5p, inhibiting miR suppression of SLC7A11 in OSCC cells, which induced cell proliferation, migration, and invasion (Cui et al. 2023). | |
| circ_0001686 inhibited miR-876-5p suppression of SPIN1 in esophagus cancer cells, inhibiting apoptosis and radiosensitivity (Yu et al. 2023). | ||
| LncRNA HOXC-AS2 decreased miR-876-5p and inhibited miR suppression of HKDC1 in endometrial cancer cells, which increased cell proliferation (Guo et al. 2022). | (LncExpDB) | |
| ciRS-7 inhibited miR-876-5p suppression of tumor antigen MAGE-A family members in esophageal squamous cell carcinoma cells and induced cell proliferation, migration, and invasion (Sang et al. 2018). | ||
| circRPS16 decreased miR-876-5p and inhibited miR suppression of SPINK1 in hepatocellular carcinoma (HCC) cells, which increased cell proliferation, invasion, and cell cycle (Lin et al. 2021b ). | (circBASE) | |
| CircVAPA acted as ceRNA of miR-876-5p, which inhibited miR suppression of WNT5A in NSCLC cells and increased cell viability and proliferation (Zhao et al. 2021). | (circBASE) | |
| lncRNA SNGHG14 inhibited miR-876-5p suppression of SSR2 in HCC cells which induced cell proliferation, migration, and invasion (Liao et al. 2021). | ||
| circHIPK3 inhibited miR-875-5p suppression of PIK3R1 in gastric cancer cells and increased cell proliferation, migration, invasion, and glutaminolysis (Li et al. 2020a ). | (circBASE) | |
| LncRNA PITPNA-AS1 acted as a ceRNA of miR-875-5p, reducing miR suppression of c-MET in cervical cancer cells and increased cell proliferation and cell cycle (Guo et al. 2020a ). | (LncExpDB) | |
| MCM3AP-AS1 acted as a ceRNA of miR-876-5p, reducing miR suppression of WNT5A in prostate cancer cells, which increased cell proliferation (Wu et al. 2020). | (LncExpDB) | |
| lncRNA MINCR inhibited miR-876-5p suppression of GSPT1 in glioma cells and increased cell migration and invasion (Li et al. 2020b ). | (LncExpDB) | |
| LncRNA PITPNA-AS1 acted as a ceRNA of miR-875-5p, reducing miR suppression of WNT5a in HCC cells and promoted EMT (Sun et al. 2019). | (LncExpDB) | |
| LncRNA HOXC-AS2 decreased miR-876-5p and inhibited miR suppression of ZEB in glioma cells, which increased EMT (Dong et al. 2019b ). | (LncExpDB) |
Conclusion
Mechanisms for endocrine resistance in BCa are diverse and depend on a number of influences (reviewed in (Clarke et al. 2015, Hanker et al. 2020). Here we identified the downregulation of miR-145-5p, miR-424-5p, miR-34b-5p, and miR-876-5p by A2B1 in ER+ endocrine-resistant BCa cells. We show that miR-145-5p and miR-424-5p directly target PSAT1 and that miR-34b-5p and miR-876-5p directly target PHGDH, key enzymes in the serine biosynthetic pathway. Overexpression of miR-145-5p and miR-424-5p decreased PSAT1 expression in LCC9, T47D, ZR-75-1, and ZR-75-1 4-OHT cells, demonstrating a role for these miRNAs in endogenous PSAT1 regulation. We also observed a cell-line-specific role for miR-34b-5p and miR-876-5p regulating PHGDH. Further, overexpression of all four miRs restored tamoxifen and fulvestrant sensitivity in endocrine-resistant LCC9 and ZR-75-1-4-OHT cells and sensitized them to inhibition by E2, a property reported in LTED MCF-7 cells (Lewis-Wambi & Jordan 2009). In addition, conjugated equine estrogens protected postmenopausal women in the Women’s Health Initiative trial were noted to have a prolonged decrease in breast cancer incidence due to estrogen-induced apoptosis (Jordan 2015, Abderrahman & Jordan 2022). Future studies are needed to identify ceRNA that influences A2B1-regulated miRNAs that target PSAT1 and PHGDH and their cell line expression. Examining the role of miRNAs in regulating serine synthesis in ER+ endocrine resistance further elucidates the function of the noncoding RNA cross-regulatory network in the deregulation of gene expression in BCa toward the goal of improving therapeutics for endocrine-resistant disease in BCa patients.
Supplementary materials
This is linked to the online version of the article at https://doi.org/10.1530/ERC-23-0148.
Declaration of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.
Funding
This work was supported by NIH R21CA212952, a grant from Jewish Heritage Fund for Excellence, and by a grant from the Office of the Assistant Secretary of Defense for Health Affairs, in the amount of $1,313,309.00, through the Breast Cancer Research Program under Award No. HT9425-23-1-0017 to CMK. B.F.C. was supported by a research grant from the Office of the Assistant Secretary of Defense for Health Affairs, in the amount of $1,015,291.00, through the Breast Cancer Research Program under Award No. HT9425-23-1-0018. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the Department of Defense. B.J.P. was supported by a fellowship from NIH T32 ES011564 and R21 ES031510 S1. A.E.W. and A.D.H. were supported by fellowships from NIH T35 DK072923. This work was supported in part by NIH P30ES030283. We thank Dr Robert Clarke, The Hormel Institute, University of Minnesota, for the gift of the LCC9 and LY2 cell lines.
Author contribution statement
Belinda J. Petri: investigation, formal analysis, and writing; Kellianne M. Piell: investigation and editing; Ali E. Wilt: investigation and editing; Alexa D. Howser: investigation and editing; Laura Winkler: investigation and editing; Mattie R. Whitworth, investigation; Bailey L. Valdes, investigation; Norman L. Lehman: formal analysis and editing; Brian F. Clem: formal analysis and editing; Carolyn M. Klinge: conceptualization, supervision, formal analysis, writing, and editing.
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