Oncogenic p95HER2 regulates Na+–HCO3− cotransporter NBCn1 mRNA stability in breast cancer cells via 3′UTR-dependent processes*

2016 ◽  
Vol 473 (21) ◽  
pp. 4027-4044 ◽  
Author(s):  
Andrej Gorbatenko ◽  
Christina W. Olesen ◽  
Nathalie Loebl ◽  
Haraldur H. Sigurdsson ◽  
Carolina Bianchi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Rna Binding ◽  
Growth Factor Receptor ◽  
Luciferase Reporter ◽  
Central Position ◽  
Reporter Expression ◽  
General Role ◽  
Mcf 7

The Na+–HCO3– cotransporter NBCn1 (SLC4A7) is up-regulated in breast cancer, important for tumor growth, and a single nucleotide polymorphism (SNP), rs4973768, in its 3′ untranslated region (3′UTR) correlates with increased breast cancer risk. We previously demonstrated that NBCn1 expression and promoter activity are strongly increased in breast cancer cells expressing a constitutively active oncogenic human epidermal growth factor receptor 2 (HER2) (p95HER2). Here, we address the roles of p95HER2 in regulating NBCn1 expression via post-transcriptional mechanisms. p95HER2 expression in MCF-7 cells reduced the rate of NBCn1 mRNA degradation. The NBCn1 3′UTR down-regulated luciferase reporter expression in control cells, and this was reversed by p95HER2, suggesting that p95HER2 counteracts 3′UTR-mediated suppression of NBCn1 expression. Truncation analyses identified three NBCn1 3′UTR regions of regulatory importance. Mutation of putative miRNA-binding sites (miR-374a/b, miR-200b/c, miR-29a/b/c, miR-488) in these regions did not have significant impact on 3′UTR activity. The NBCn1 3′UTR interacted directly with the RNA-binding protein human antigen R (HuR), and HuR knockdown reduced NBCn1 expression. Conversely, ablation of a distal AU-rich element increased 3′UTR-driven reporter activity, suggesting complex regulatory roles of these sites. The cancer-associated SNP variant decreased reporter expression in T-47D breast cancer cells, yet not in MCF-7, MDA-MB-231 and SK-BR-3 cells, arguing against a general role in regulating NBCn1 expression. Finally, p95HER2 expression increased total and plasma membrane NBCn1 protein levels and decreased the rate of NBCn1 protein degradation. Collectively, this is the first work to demonstrate 3′UTR-mediated NBCn1 regulation, shows that p95HER2 regulates NBCn1 expression at multiple levels, and substantiates the central position of p95HER2–NBCn1 signaling in breast cancer.

scholarly journals Unravelling the RNA-Binding Properties of SAFB Proteins in Breast Cancer Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Elaine Hong ◽  
Andrew Best ◽  
Hannah Gautrey ◽  
Jas Chin ◽  
Anshuli Razdan ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
High Throughput Sequencing ◽  
Rna Binding ◽  
Open Reading Frames ◽  
Rna Recognition Motif ◽  
Binding Properties ◽  
Intergenic Regions ◽  
Mcf 7

Scaffold attachment factor B1 (SAFB1) and SAFB2 proteins are oestrogen (ER) corepressors that bind to and modulate ER activity through chromatin remodelling or interaction with the basal transcription machinery. SAFB proteins also have an internal RNA-recognition motif but little is known about the RNA-binding properties of SAFB1 or SAFB2. We utilised crosslinking and immunoprecipitation (iCLIP) coupled with high-throughput sequencing to enable a transcriptome-wide mapping of SAFB1 protein-RNA interactions in breast cancer MCF-7 cells. Analysis of crosslinking frequency mapped to transcript regions revealed that SAFB1 binds to coding and noncoding RNAs (ncRNAs). The highest proportion of SAFB1 crosslink sites mapped to ncRNAs, followed by intergenic regions, open reading frames (ORFs), introns, and 3′ or 5′ untranslated regions (UTR). Furthermore, we reveal that SAFB1 binds directly to RNA and its binding is particularly enriched at purine-rich sequences not dissimilar to the RNA-binding motifs for SR proteins. Using RNAi, we also show, for the first time, that single depletion of either SAFB1 or SAFB2 leads to an increase in expression of the other SAFB protein in both MCF-7 and MDA-MD231 breast cancer cells.

scholarly journals Knockdown of ICB-1 gene enhanced estrogen responsiveness of ovarian and breast cancer cells

2010 ◽  
Vol 17 (1) ◽  
pp. 147-157 ◽  
Author(s):  
Anna Konwisorz ◽  
Anette Springwald ◽  
Martina Haselberger ◽  
Regina Goerse ◽  
Olaf Ortmann ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Ovarian Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Chromosome 1 ◽  
Ovarian Cancer Cells ◽  
General Role ◽  
Estrogen Response ◽  
Mcf 7

ICB-1 chromosome 1 open reading frame 38 (C1orf38) is a human gene initially described by our group to be involved in differentiation processes of cancer cells. Recently, we have reported ICB-1 as a novel estrogen target gene and identified an estrogen response element in its promoter. In this study, we examined the role of ICB-1 in regulation of proliferation of breast and ovarian cancer cells. We knocked down its expression in estrogen-dependent MCF-7 breast cancer cells and hormone-unresponsive SK-OV-3 ovarian cancer cells by stable transfection with a specific shRNA plasmid followed by G-418 selection. Knockdown of ICB-1 enabled a considerable estrogen response of SK-OV-3 cells in terms of proliferation. This transformation of SK-OV-3 cells into an estrogen-responsive phenotype was accompanied by upregulation of estrogen receptor α (ERα) expression and a significant decrease of ERβ expression on the mRNA level. Expression of ERα-dependent genes progesterone receptor, pS2, fibulin 1c, and c-fos was elevated in SK-OV-3 cells stably expressing ICB-1 shRNA. In MCF-7 cells, ICB-1 knockdown exerted similar effects on gene expression, supporting a general role of ICB-1 in estrogen responsiveness. Our data suggest that differentiation-associated gene ICB-1 might exert antagonistic actions on cellular estrogen response, which can result in inhibition of estradiol-triggered proliferation. The molecular mechanisms mediating this inhibitory effect of ICB-1 on estrogen signaling are suggested to be limitation of ERα transcript levels but sustaining high levels of ERβ, reducing both activation of ERα target genes and cellular proliferation. The identification of ICB-1 as a new player in endocrine-related cancer encourages further studies on the significance of this gene in cancer development and therapy.

scholarly journals MiR-218-5p promotes breast cancer progression via LRIG1

2021 ◽  
Author(s):  
Mingping Qian ◽  
Hui Xu ◽  
Hongming Song ◽  
Hao Xi ◽  
Lin Fang
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Cancer Cells ◽  
Human Breast Cancer ◽  
Breast Cancer Cells ◽  
Luciferase Reporter ◽  
Human Breast ◽  
Egfr Expression ◽  
Breast Tissues ◽  
Mcf 7

Abstract Background : MiR-218-5p is a small non-coding RNA acting as either oncogenes or tumor suppressor genes in human cancer. The expression levels of some miRNAs in human breast cancer plays a potential role in disease pathogenesis. Methods : Thirty pairs of invasive ductal carcinoma and adjacent specimens were included in the study. Breast tissues cell lines MCF-7 and MDA-MB-231 were identified as a breast cancer research cell line. MiR-218-5p mimics, miR-218-5p inhibitor, or negative controls were transfected. Specific antibodies were probed with LRIG1, ErbB2, and EGFR. Proliferation, migration, cell cycle and apoptosis, dual-luciferase reporter assay and immunohistochemistry were used to analyze miR-218-5p、LRIG1 and so on. Results : It was shown that miR-218-5p expression was higher in 30 breast cancer specimens than adjacent normal breast tissues. In human breast cancer cells MCF-7 and MDA-MB-231, restoring miR-218-5p promoted cell proliferation and migration and inhibited cell apoptosis and cell cycle arrest in the G1 stage. Luciferase assays indicated miR-218-5p could bind with its putative target site in the 3'-untranslated region (3'-UTR) of LRIG1. RT-qPCR, western blot, and immunocytochemistry analyses all indicated miR-218-5p overexpression results in LRIG1 downregulation at the mRNA and protein levels. ErbB2 and EGFR were found to be downstream effectors of miR-218-5p. Conclusion : MiR-218-5p promotes ErbB2 and EGFR expression by inhibiting LRIG1 in breast cancer cells, which suggests miR-218-5p and LRIG1 may act as an oncogene in breast cancer and it could be used as a therapeutic target for breast cancer treatments. Keywords: Breast cancer; miR-218-5p; LRIG1; Oncogene

scholarly journals hsa-miR-33-5p as a Therapeutic Target Promotes Apoptosis of Breast Cancer Cells via Selenoprotein T

2021 ◽  
Vol 8 ◽  
Author(s):  
Wei Zhuang ◽  
Jianhui Liu ◽  
Wenjin Li
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Therapeutic Target ◽  
Cell Apoptosis ◽  
Breast Cancer Cells ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Selenoprotein T ◽  
Cancer Tissues ◽  
Mcf 7

Objective: Increasing evidence suggests that microRNA (miRNA) participates in regulating tumor cell apoptosis. We aimed to observe the effect of hsa-miR-33-5p on the apoptosis of breast cancer cells and to explore its regulatory relationship with selenoprotein T (SelT).Methods: RT-qPCR was used to examine the expression of hsa-miR-33-5p and SelT both in breast cancer tissues and cells. MCF-7 and MDA-MB-231 cells were transfected with hsa-miR-33-5p mimics or si-SelT. Then, a flow cytometry assay was carried out to examine the apoptosis of cells. Furthermore, SelT and apoptosis-related proteins including caspase-3, caspase-8, caspase-9, Bax, and Bcl-2 were detected via RT-qPCR and western blot. A luciferase reporter assay was utilized for assessing whether SelT was targeted by hsa-miR-33-5p.Results: Downregulated hsa-miR-33-5p was found both in breast cancer tissues and cells. After its overexpression, MCF-7 cell apoptosis was significantly promoted. Furthermore, our data showed that miR-33-5p elevated apoptosis-related protein expression in MCF-7 cells. Contrary to hsa-miR-33-5p, SelT was upregulated both in breast cancer tissues and cells. SelT expression was significantly inhibited by hsa-miR-33-5p overexpression. The luciferase reporter assay confirmed that SelT was a direct target of hsa-miR-33-5p. SelT overexpression could ameliorate the increase in apoptosis induced by hsa-miR-33-5p mimics.Conclusion: Our findings revealed that hsa-miR-33-5p, as a potential therapeutic target, could accelerate breast cancer cell apoptosis.

scholarly journals Signal Transducer and Activator of Transcription 5b, c-Src, and Epidermal Growth Factor Receptor Signaling Play Integral Roles in Estrogen-Stimulated Proliferation of Estrogen Receptor-Positive Breast Cancer Cells

2008 ◽  
Vol 22 (8) ◽  
pp. 1781-1796 ◽  
Author(s):  
Emily M. Fox ◽  
Teresa M. Bernaciak ◽  
Jie Wen ◽  
Amanda M. Weaver ◽  
Margaret A. Shupnik ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cyclin D1 ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Transcriptional Activity ◽  
Growth Factor Receptor ◽  
Tamoxifen Resistance ◽  
Cancer Proliferation ◽  
Epidermal Growth ◽  
Mcf 7

Abstract 17β-Estradiol (E2) acts through the estrogen receptor α (ERα) to stimulate breast cancer proliferation. Here, we investigated the functional relationship between ERα and signal transducer and activator of transcription (STAT)5b activity in ER+ MCF-7 and T47D human breast cancer cells after specific knockdown of STAT5b. STAT5b small interfering RNA (siRNA) inhibited E2-induced bromodeoxyuridine (BrdU) incorporation in both cell lines, as well as the E2-induced increase in MCF-7 cell number, cyclin D1 and c-myc mRNA, and cyclin D1 protein expression, indicating that STAT5b is required for E2-stimulated breast cancer proliferation. E2 treatment stimulated STAT5b tyrosine phosphorylation at the activating tyrosine Y699, resulting in increased STAT5-mediated transcriptional activity, which was inhibited by a Y669F STAT5b mutant. E2-induced STAT5-mediated transcriptional activity was inhibited by overexpressing a kinase-defective epidermal growth factor receptor (EGFR), or the EGFR tyrosine kinase inhibitor tyrphostin AG1478, indicating a requirement for EGFR kinase activity. Both E2-induced STAT5b tyrosine phosphorylation and STAT5-mediated transcription were also inhibited by the ER antagonist ICI 182,780 and the c-Src inhibitor PP2, indicating additional requirements for the ER and c-Src kinase activity. EGFR and c-Src kinase activities were also required for E2-induced cyclin D1 and c-myc mRNA. Together, these studies demonstrate positive cross talk between ER, c-Src, EGFR, and STAT5b in ER+ breast cancer cells. Increased EGFR and c-Src signaling is associated with tamoxifen resistance in ER+ breast cancer cells. Here we show that constitutively active STAT5b not only increased basal DNA synthesis, but also conferred tamoxifen resistance. Because STAT5b plays an integral role in E2-stimulated proliferation and tamoxifen resistance, it may be an effective therapeutic target in ER+ breast tumors.

scholarly journals miR-186 Regulates the Initiation and Development of Breast Cancer via SHP-1 Methylation

2020 ◽  
Author(s):  
Kun Wang ◽  
Chunxia Zhou ◽  
Di Wang ◽  
Bin Zhang ◽  
Yongjian Zheng ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Promoter Region ◽  
Breast Cancer Cells ◽  
Methylation Status ◽  
Luciferase Reporter ◽  
Molecular Diagnostic ◽  
Double Labeling ◽  
Reporter Assays ◽  
Mcf 7

Abstract Background Change in the methylation status of genomic DNA, especially in CpG islands in the promoter region, is considered to be an early event in tumor initiation, leading to silencing of gene expression, subsequent abnormalities in gene structure and function, and malignant transformation of the cell. Due to the abnormal expression of miR-186 and SHP-1 in breast cancer tissues and cells, we propose that miR-186 is closely related to the methylation of SHP-1Method Using 5-azacytidine as a de-methylation agent and Validating with Setylation-specific polymerase chain reaction (MSP) after treatment. Measurement of the viability of breast cancer cells using the CCK-8 method Measurement of the apoptotic rate of breast cancer cells using annexin V-FITC/PI double labeling. Cell metastasis were measured by wound healing assay. Luciferase reporter assays was used to confirm the target of MiR-186. SHP-1 and miR-186 expression was measured by RT-PCR and western blot.Results In the present study, we found that SHP-1 expression was reduced to various degrees in all 5 cell lines (UACC-812, MDA-MB-213, MDA-MB-468, SK-RB-3 and MCF-7). 5-azacytidine can remove the methylation from the SHP-1 promoter region. Apoptosis was observed in MCF-7 cells after demethylation of the SHP-1 gene promoter region by 5-azacytidine, and the effect was time- and concentration-dependent. Luciferase reporter assays showed that miR-186 promotes methylation through binding with the 3’ UTR of the SHP-1 promoter region.Western blot showed miR-186 regulates the initiation and development of tumor cells through the SHP-1-JAK-STAT axis. In animal models, low expression of miR-186 can cause significantly limited tumor growth.Conclusion The low SHP-1 expression may be an important factor in the initiation of breast cancer, and that miR-186 could serve as an excellent molecular diagnostic marker and a possible therapeutic target.

scholarly journals Identification of a Novel AU-Rich Element in the 3′ Untranslated Region of Epidermal Growth Factor Receptor mRNA That Is the Target for Regulated RNA-Binding Proteins

2001 ◽  
Vol 21 (6) ◽  
pp. 2070-2084 ◽  
Author(s):  
L. A. Balmer ◽  
D. J. Beveridge ◽  
J. A. Jazayeri ◽  
A. M. Thomson ◽  
C. E. Walker ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Mrna Stability ◽  
Breast Cancer Cells ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Growth Factor Receptor ◽  
Human Breast ◽  
Shift Analysis

ABSTRACT The epidermal growth factor receptor (EGF-R) plays an important role in the growth and progression of estrogen receptor-negative human breast cancers. EGF binds with high affinity to the EGF-R and activates a variety of second messenger pathways that affect cellular proliferation. However, the underlying mechanisms involved in the regulation of EGF-R expression in breast cancer cells are yet to be described. Here we show that the EGF-induced upregulation of EGF-R mRNA in two human breast cancer cell lines that overexpress EGF-R (MDA-MB-468 and BT-20) is accompanied by stabilization (>2-fold) of EGF-R mRNA. Transient transfections using a luciferase reporter identified a novel EGF-regulated ∼260-nucleotide (nt)cis-acting element in the 3′ untranslated region (3′-UTR) of EGF-R mRNA. This cis element contains two distinct AU-rich sequences (∼75 nt), EGF-R1A with two AUUUA pentamers and EGF-R2A with two AUUUUUA extended pentamers. Each independently regulated the mRNA stability of the heterologous reporter. Analysis of mutants of the EGF-R2A AU-rich sequence demonstrated a role for the 3′ extended pentamer in regulating basal turnover. RNA gel shift analysis identified cytoplasmic proteins (∼55 to 80 kDa) from breast cancer cells that bound specifically to the EGF-R1A and EGF-R2A cis-acting elements and whose binding activity was rapidly downregulated by EGF and phorbol esters. RNA gel shift analysis of EGF-R2A mutants identified a role for the 3′ extended AU pentamer, but not the 5′ extended pentamer, in binding proteins. These EGF-R mRNA-binding proteins were present in multiple human breast and prostate cancer cell lines. In summary, these data demonstrate a central role for mRNA stabilization in the control of EGF-R gene expression in breast cancer cells. EGF-R mRNA contains a novel complex AU-rich 260-nt cis-acting destabilizing element in the 3′-UTR that is bound by specific and EGF-regulatedtrans-acting factors. Furthermore, the 3′ extended AU pentamer of EGF-R2A plays a central role in regulating EGF-R mRNA stability and the binding of specific RNA-binding proteins. These findings suggest that regulated RNA-protein interactions involving this novel cis-acting element will be a major determinant of EGF-R mRNA stability.

scholarly journals Effects of miR-107 on the Chemo-drug sensitivity of breast cancer cells

Open Medicine ◽  
2019 ◽  
Vol 14 (1) ◽  
pp. 59-65 ◽  
Author(s):  
Yong Luo ◽  
Tebo Hua ◽  
Xia You ◽  
Jinfeng Lou ◽  
Xuxiong Yang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Flow Cytometry ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Target Gene ◽  
Drug Sensitivity ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Qrt Pcr ◽  
Mcf 7

AbstractBackgroundA growing body of evidence indicates that aberrant expression of miR-107 plays a core role in cancers. This study aims to demonstrate the function of miR-107 and its roles in chemo-drug resistance in breast cancer cells.MethodologyCCK-8 assays were carried out to test the effect of miR-107 mimics on the proliferation of MCF-7 cells. The apoptosis level of each group was detected by flow cytometry. miR-107 level, mRNA levels of Bcl-2/Bax and TRIAP1 were detected by quantitative real-time Polymerase Chain Reaction (qRT-PCR) analysis. Protein levels of Bcl-2/Bax, p-Akt/Akt in MCF-7 cells were detected by using Western Blot. Lastly, the dual luciferase reporter gene assay system was used to confirm interaction between miR-107 and its target gene TRIAP1.ResultsCCK-8 assays indicated that miR-107 mimics augmented Taxol-induced cell viability inhibition. Flow cytometry showed that miR-107 mimics augmented Taxol-induced elevation of cell apoptosis. qRT-PCR analysis revealed that miR-107 mimics inhibited the mRNA expression of Bcl-2 and induced the mRNA level of Bax. Western Blotting indicated that miR-107 mimics inhibited the expression of proteins Bcl-2 and p-Akt, and induced the expression of Bax, while showing no significant effects on Akt. The relative luciferase activity revealed that oncogene TRIAP1 is a potential target gene of miR-107.ConclusionsmiR-107 plays a role in regulating chemo-drug sensitivity in mammary cancer cell by targeting TRIAP1.

scholarly journals miR-17-5p Combined with Epirubicin Inhibits the Progression of Breast Cancer Cells

2020 ◽  
Author(s):  
Lingling Zhao ◽  
Zhongjian Zhu ◽  
Jiang Du ◽  
Yuanyu Zhao ◽  
Dandan Fan
Keyword(s):  
Breast Cancer ◽  
Western Blot ◽  
Cancer Cells ◽  
Mtt Assay ◽  
Breast Cancer Cells ◽  
Luciferase Reporter ◽  
Rt Pcr ◽  
Expression Levels ◽  
Cancer Tissues ◽  
Mcf 7

Abstract Background: Breast cancer is the most common malignant tumor for women, which has been ranked first in women’ s cancer-related death. The objectives of the study are to uncover the underlying mechanisms of combination therapy of epirubicin with miR-17-5p in breast cancer. Methods: The expression levels of miR-17-5p were determined by quantitative RT-PCR. The survival rate of MCF-7 cell was detected respectively by MTT assay. The expression levels of miR-17-5p in MCF-7 cell was tested respectively with Ep via quantitative RT-PCR. miR-17-5p was to be transected with miR-17-5p mimic and negative control of miR-17-5p mimic (NC). Quantitative RT-PCR, MTT assay, flow cytometry assay, western blot for the proliferation and apoptosis related proteins were performed to determine the function of miR-17-5p in breast cancer cells. The bioinformatic programs TargetScan was used to predict the targets for miR-17-5p. Luciferase reporter gene assay system was used to validate and determine the targets of miR-17-5p. The relation between targets protein levels in breast cancer cells was investigated by western blot. Results: The expression levels of miR-17-5p was associated with the breast cancer tissues. The levels of miR-17-5p was down-regulated in breast cancer tissues and cells. Ep could inhibit viability of cancer cells in a concentration dependent manner and promote the expression of miR-17-5p in breast cancer cell lines. Over-expression of miR-17-5p induced cell apoptosis and upregulated the expression of p53, p21 and p27. miR-17-5p co-cultured with Ep is better than the other groups. The relative luciferase activity revealed that STAT3 was a potential target gene of miR-17-5p. Conclusions: Our work will prove that epirubicin regulated the expression of miR-17-5p to strengthen this effect of epirubicin and inhibited the progression of breast cancer.

scholarly journals OR09-06 HNRNPA2B1 Mediates Endocrine-Sensitivity and Alters PSAT1 in Breast Cancer Cells

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Carolyn M Klinge ◽  
Kellianne M Piell ◽  
Bran F Clem ◽  
Belinda J Petri
Keyword(s):  
Breast Cancer ◽  
Growth Inhibition ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Rna Binding ◽  
Cell Junction ◽  
Breast Cancer Patients ◽  
Mirna Regulation ◽  
Luminal A ◽  
Mcf 7

Abstract Higher expression of the RNA binding protein HNRNPA2B1 (Heterogeneous Nuclear Ribonucleoprotein A2/B1), a reader of the N(6)-methyladenosine (m6A) mark in transcribed RNA, is found in endocrine-resistant, estrogen receptor (ERα)+ LCC9 and LY2 breast cancer cells derived from MCF-7 endocrine-sensitive luminal A cells (1). HNRNPA2B1 interacts with DGCR8 in the DROSHA complex to promote processing of pri-miRNAs to pre-miRNAs. We identified HNRNPA2B1-regulated miRNAs by RNA seq and target pathways, including serine family amino acid metabolic processes, TGFβ signaling, response to estrogen, and cell junction by MetaCore enrichment pathway analysis (1). Stable 4.5-fold overexpression of HNRNPA2B1 in MCF-7 cells (MCF-7-A2B1 cells) results in ablation of growth inhibition by 4-hydroxytamoxifen (4-OHT) and fulvestrant. This was not due to loss or decrease of ERα; in fact, ERα was increased ~ 1.6-fold. Conversely, transient knockdown of HNRNPA2B1 expression in LCC9 and LY2 cells sensitized the cells to growth inhibition by 4-OHT and fulvestrant, without changing ESR1 expression. MCF-7-A2B1 cells showed increased migration, reduced E-cadherin and increased vimentin, suggestive of EMT; however, they exhibited reduced clonogenic survival. Follow-up on the identification of HNRNPA2B1-miRNA regulation of the serine pathway revealed higher expression of phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) transcripts and proteins in LCC9, LY2, and MCF-7-A2B1 compared to MCF-7 cells. We identified two miRNAs, miR-424-5p and miR-145-5p downregulated in MCF-7-A2B1 cells that directly targeted the PSAT1 3’UTR in dual luciferase assays. Lower miR-424-5p and miR-145-5p in endocrine-resistant LCC9 and LY2 correlate with increased PSAT1 and higher PSAT1 is associated with reduced overall and metastasis-free survival in breast cancer patients. Overall, our data suggest a role for increased HNRNPA2B1 in tamoxifen-resistance. Reference: (1) Klinge CM, Piell KM, Tooley CS, Rouchka EC. HNRNPA2/B1 is upregulated in endocrine-resistant LCC9 breast cancer cells and alters the miRNA transcriptome when overexpressed in MCF-7 cells. Sci. Rep. 2019; 9:9430.

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