Transcriptional coregualtor NUPR1 maintains tamoxifen resistance in breast cancer cells

AbstractTo support cellular homeostasis and mitigate chemotherapeutic stress, cancer cells must gain a series of adaptive intracellular processes. Here we identify that NUPR1, a tamoxifen (Tam)-induced transcriptional coregulator, is necessary for the maintenance of Tam resistance through physical interaction with ESR1 in breast cancers. Mechanistically, NUPR1 binds to the promoter regions of several genes involved in autophagy process and drug resistance such as BECN1, GREB1, RAB31, PGR, CYP1B1, and regulates their transcription. In Tam-resistant ESR1 breast cancer cells, NUPR1 depletion results in premature senescence in vitro and tumor suppression in vivo. Moreover, enforced-autophagic flux augments cytoplasmic vacuolization in NUPR1-depleted Tam resistant cells, which facilitates the transition from autophagic survival to premature senescence. Collectively, these findings suggest a critical role for NUPR1 as a transcriptional coregulator in enabling endocrine persistence of breast cancers, thus providing a vulnerable diagnostic and/or therapeutic target for endocrine resistance.

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Autophagy Triggers Tamoxifen Resistance in Human Breast Cancer Cells by Preventing Drug-Induced Lysosomal Damage

Cancers ◽

10.3390/cancers13061252 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1252

Author(s):

Chiara Actis ◽

Giuliana Muzio ◽

Riccardo Autelli

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Endocrine Resistance ◽

Tamoxifen Resistance ◽

Major Complication ◽

Autophagic Flux ◽

Messenger Rnas ◽

Mcf7 Cells ◽

Drug Induced

Endocrine resistance is a major complication during treatment of estrogen receptor-positive breast cancer. Although autophagy has recently gained increasing consideration among the causative factors, the link between autophagy and endocrine resistance remains elusive. Here, we investigate the autophagy-based mechanisms of tamoxifen resistance in MCF7 cells. Tamoxifen (Tam) triggers autophagy and affects the lysosomal compartment of MCF7 cells, such that activated autophagy supports disposal of tamoxifen-damaged lysosomes by lysophagy. MCF7 cells resistant to 5 µM tamoxifen (MCF7-TamR) have a higher autophagic flux and an enhanced resistance to Tam-induced lysosomal alterations compared to parental cells, which suggests a correlation between the two events. MCF7-TamR cells overexpress messenger RNAs (mRNAs) for metallothionein 2A and ferritin heavy chain, and they are re-sensitized to Tam by inhibition of autophagy. Overexpressing these proteins in parental MCF7 cells protects lysosomes from Tam-induced damage and preserves viability, while inhibiting autophagy abrogates lysosome protection. Consistently, we also demonstrate that other breast cancer cells that overexpress selected mRNAs encoding iron-binding proteins are less sensitive to Tam-induced lysosomal damage when autophagy is activated. Collectively, our data demonstrate that autophagy triggers Tam resistance in breast cancer cells by favoring the lysosomal relocation of overexpressed factors that restrain tamoxifen-induced lysosomal damage.

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SUN-735 Functional Analysis of Testis-Specific Noncoding Genes in Estrogen-Dependent Transcription

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.934 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Ramesh Choudhari ◽

Barbara Yang ◽

Enrique Ivan Ramos ◽

Mina Zilaie ◽

Laura A Sanchez-Michael ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Therapeutic Potential ◽

Critical Role ◽

Genomic Analysis ◽

Breast Cancers ◽

Breast Cancer Patients ◽

Mcf 7

Abstract Emerging studies have shown that germ cell (GC)-specific genes play critical roles in several cancers. The expression of these genes is tightly regulated and restricted to testis; however, many of them escape regulation and become aberrantly expressed in tumors. Interestingly, our genomic analysis suggests that several of these genes are long noncoding RNAs (lncRNAs) and are located at regions previously considered to be gene deserts in the human genome. In this regard, we used an integrated genomic approach to identify GC-lncRNA genes that are overexpressed in breast cancer. Further, by incorporating gene expression analysis from RNA-seq data from MCF-7 and T47D breast cancer cells, we generated a comprehensive list of estrogen-regulated GC-lncRNA genes. We hypothesize that GC-lncRNA genes regulate estrogen-dependent signaling in breast cancer. The selected genes: (a) CAERRC (Chromatin Associated Estrogen-Regulated RNA in Cancer, (b) LncRNA568, (c) LncRNA16 are primate-specific, and exclusively expressed in testis. All of them are regulated by estrogen, and their expression predicts poor outcome in ERα+ breast cancer patients. They have now been fully annotated (transcription start and stop site, 5’ cap, polyA tail, and exon/intron structure), and cloned. Further, we have created gene-specific KO MCF-7 cell lines using CRISPR to study their molecular roles. Our data suggest that these genes regulate estrogen-dependent gene expression and tumor growth in breast cancer cells. Genome-wide analysis of ERα binding and gene expression data indicate that they play a critical role in the estrogen-dependent transcription. Collectively, our results suggest that GC-genes, including CAERRC, LncRNA568, and LncRNA16, are excellent targets with prognostic and therapeutic potential in ER+ breast cancers.

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ER-positive breast cancer cells are poised for RET-mediated endocrine resistance

10.1101/098848 ◽

2017 ◽

Author(s):

Sachi Horibata ◽

Edward J. Rice ◽

Hui Zheng ◽

Lynne J. Anguish ◽

Scott A. Coonrod ◽

...

Keyword(s):

Breast Cancer ◽

Tyrosine Kinase ◽

Cancer Cells ◽

Signaling Pathway ◽

Breast Cancer Cells ◽

Endocrine Resistance ◽

Breast Cancers ◽

Kinase Signaling ◽

Tyrosine Kinase Signaling ◽

Kinase Signaling Pathway

AbstractThe RET tyrosine kinase signaling pathway is involved in the development of endocrine resistant ER+ breast cancer. However, the expression of the RET receptor itself has not been directly linked to clinical cases of resistance, suggesting that additional factors are involved. We show that both ER+ endocrine resistant and sensitive breast cancers have functional RET tyrosine kinase signaling pathway, but that endocrine sensitive breast cancer cells lack RET ligands that are necessary to drive endocrine resistance. Transcription of one RET ligand, GDNF, is necessary and sufficient to confer resistance in the ER+ MCF-7 cell line. In patients, RET ligand expression predicts responsiveness to endocrine therapies and correlates with survival. Collectively, our findings show that ER+ tumor cells are “poised” for RET mediated endocrine resistance, expressing all components of the RET signaling pathway, but endocrine sensitive cells lack high expression of RET ligands that are necessary to initiate the resistance phenotype.

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Histone methyltransferases regulate the transcriptional expression of ERα and the proliferation of tamoxifen-resistant breast cancer cells

Breast Cancer Research and Treatment ◽

10.1007/s10549-019-05517-0 ◽

2020 ◽

Vol 180 (1) ◽

pp. 45-54 ◽

Cited By ~ 3

Author(s):

Seung-Su Kim ◽

Min-Ho Lee ◽

Mi-Ock Lee

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Chromatin Immunoprecipitation ◽

Breast Cancer Cells ◽

Methylation Status ◽

Endocrine Resistance ◽

Breast Cancers ◽

Histone Methyltransferases ◽

Expression Levels ◽

Tamoxifen Resistant

Abstract Purpose Although tamoxifen remains the frontline treatment for ERα-positive breast cancers, resistance to this drug limits its clinical efficacy. Most tamoxifen-resistant patients retain ERα expression which may support growth and progression of breast cancers. Therefore, we investigated epigenetic regulation of ERα that may provide a rationale for targeting ERα in these patients. Methods Expression levels of the mixed-lineage leukemia (MLL) family of proteins in tamoxifen-resistant breast cancer cells and publicly available breast cancer patient data sets were analyzed. Histone methylation levels in ERα promoter regions were assessed using chromatin immunoprecipitation. Expression levels of ERα and its target gene were analyzed using western blotting and real-time qPCR. Cell-cycle was analyzed by flow cytometry. Results The expression of MLL3 and SET-domain-containing 1A (SET1A) were increased in tamoxifen-resistant breast cancers. An MLL3 chromatin immunoprecipitation-sequencing data analysis and chromatin immunoprecipitation experiments for MLL3 and SET1A suggested that these proteins bound to enhancer or intron regions of the ESR1 gene and regulated histone H3K4 methylation status. Depletion of MLL3 or SET1A downregulated the expression level of ERα and inhibited the growth of tamoxifen-resistant breast cancer cells. Additional treatment with fulvestrant resulted in a synergistic reduction of ERα levels and the growth of the cells. Conclusions The enhanced expression of MLL3 and SET1A in tamoxifen-resistant breast cancer cells supported the ERα-dependent growth of these cells by increasing ERα expression. Our results suggest that targeting these histone methyltransferases might provide an attractive strategy to overcome endocrine resistance.

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Fatty Acid Synthase Confers Tamoxifen Resistance to ER+/HER2+ Breast Cancer

Cancers ◽

10.3390/cancers13051132 ◽

2021 ◽

Vol 13 (5) ◽

pp. 1132

Author(s):

Javier A. Menendez ◽

Adriana Papadimitropoulou ◽

Travis Vander Steen ◽

Elisabet Cuyàs ◽

Bharvi P. Oza-Gajera ◽

...

Keyword(s):

Breast Cancer ◽

Fatty Acid ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Promoter Activity ◽

Fatty Acid Synthase ◽

Endocrine Resistance ◽

Gene Promoter ◽

Her2 Positive ◽

Her2 Breast Cancer

The identification of clinically important molecular mechanisms driving endocrine resistance is a priority in estrogen receptor-positive (ER+) breast cancer. Although both genomic and non-genomic cross-talk between the ER and growth factor receptors such as human epidermal growth factor receptor 2 (HER2) has frequently been associated with both experimental and clinical endocrine therapy resistance, combined targeting of ER and HER2 has failed to improve overall survival in endocrine non-responsive disease. Herein, we questioned the role of fatty acid synthase (FASN), a lipogenic enzyme linked to HER2-driven breast cancer aggressiveness, in the development and maintenance of hormone-independent growth and resistance to anti-estrogens in ER/HER2-positive (ER+/HER2+) breast cancer. The stimulatory effects of estradiol on FASN gene promoter activity and protein expression were blunted by anti-estrogens in endocrine-responsive breast cancer cells. Conversely, an AKT/MAPK-related constitutive hyperactivation of FASN gene promoter activity was unaltered in response to estradiol in non-endocrine responsive ER+/HER2+ breast cancer cells, and could be further enhanced by tamoxifen. Pharmacological blockade with structurally and mechanistically unrelated FASN inhibitors fully impeded the strong stimulatory activity of tamoxifen on the soft-agar colony forming capacity—an in vitro metric of tumorigenicity—of ER+/HER2+ breast cancer cells. In vivo treatment with a FASN inhibitor completely prevented the agonistic tumor-promoting activity of tamoxifen and fully restored its estrogen antagonist properties against ER/HER2-positive xenograft tumors in mice. Functional cancer proteomic data from The Cancer Proteome Atlas (TCPA) revealed that the ER+/HER2+ subtype was the highest FASN protein expressor compared to basal-like, HER2-enriched, and ER+/HER2-negative breast cancer groups. FASN is a biological determinant of HER2-driven endocrine resistance in ER+ breast cancer. Next-generation, clinical-grade FASN inhibitors may be therapeutically relevant to countering resistance to tamoxifen in FASN-overexpressing ER+/HER2+ breast carcinomas.

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Tioconazole and Chloroquine Act Synergistically to Combat Doxorubicin-Induced Toxicity via Inactivation of PI3K/AKT/mTOR Signaling Mediated ROS-Dependent Apoptosis and Autophagic Flux Inhibition in MCF-7 Breast Cancer Cells

Pharmaceuticals ◽

10.3390/ph14030254 ◽

2021 ◽

Vol 14 (3) ◽

pp. 254

Author(s):

Afnan H. El-Gowily ◽

Samah A. Loutfy ◽

Ehab M. M. Ali ◽

Tarek M. Mohamed ◽

Mohammed A. Mansour

Keyword(s):

Breast Cancer ◽

Combination Therapy ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Mtor Pathway ◽

Tumor Growth Inhibition ◽

Autophagic Flux ◽

Ki 67 ◽

Mcf 7

Cancer is a complex devastating disease with enormous treatment challenges, including chemo- and radiotherapeutic resistance. Combination therapy demonstrated a promising strategy to target hard-to-treat cancers and sensitize cancer cells to conventional anti-cancer drugs such as doxorubicin. This study aimed to establish molecular profiling and therapeutic efficacy assessment of chloroquine and/or tioconazole (TIC) combination with doxorubicin (DOX) as anew combination model in MCF-7 breast cancer. The drugs are tested against apoptotic/autophagic pathways and related redox status. Molecular docking revealed that chloroquine (CQ) and TIC could be potential PI3K and ATG4B pathway inhibitors. Combination therapy significantly inhibited cancer cell viability, PI3K/AkT/mTOR pathway, and tumor-supporting autophagic flux, however, induced apoptotic pathways and altered nuclear genotoxic feature. Our data revealed that the combination cocktail therapy markedly inhibited tumor proliferation marker (KI-67) and cell growth, along with the accumulation of autophagosomes and elevation of LC3-II and p62 levels indicated autophagic flux blockage and increased apoptosis. Additionally, CQ and/or TIC combination therapy with DOX exerts its activity on the redox balance of cancer cells mediated ROS-dependent apoptosis induction achieved by GPX3 suppression. Besides, Autophagy inhibition causes moderately upregulation in ATGs 5,7 redundant proteins strengthened combinations induced apoptosis, whereas inhibition of PI3K/AKT/mTOR pathway with Beclin-1 upregulation leading to cytodestructive autophagy with overcome drug resistance effectively in curing cancer. Notably, the tumor growth inhibition and various antioxidant effects were observed in vivo. These results suggest CQ and/or TIC combination with DOX could act as effective cocktail therapy targeting autophagy and PI3K/AKT/mTOR pathways in MCF-7 breast cancer cells and hence, sensitizes cancer cells to doxorubicin treatment and combat its toxicity.

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Transcriptome profiles of stem-like cells from primary breast cancers allow identification of ITGA7 as a predictive marker of chemotherapy response

British Journal of Cancer ◽

10.1038/s41416-021-01484-w ◽

2021 ◽

Author(s):

Noha Gwili ◽

Stacey J. Jones ◽

Waleed Al Amri ◽

Ian M. Carr ◽

Sarah Harris ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Predictive Marker ◽

Therapy Resistance ◽

Differentially Expressed ◽

Molecular Profile ◽

Chemotherapy Response ◽

Breast Cancers

Abstract Background Breast cancer stem cells (BCSCs) are drivers of therapy-resistance, therefore are responsible for poor survival. Molecular signatures of BCSCs from primary cancers remain undefined. Here, we identify the consistent transcriptome of primary BCSCs shared across breast cancer subtypes, and we examine the clinical relevance of ITGA7, one of the genes differentially expressed in BCSCs. Methods Primary BCSCs were assessed using immunohistochemistry and fluorescently labelled using Aldefluor (n = 17). Transcriptomes of fluorescently sorted BCSCs and matched non-stem cancer cells were determined using RNA-seq (n = 6). ITGA7 expression was examined in breast cancers using immunohistochemistry (n = 305), and its functional role was tested using siRNA in breast cancer cells. Results Proportions of BCSCs varied from 0 to 9.4%. 38 genes were significantly differentially expressed in BCSCs; genes were enriched for functions in vessel morphogenesis, motility, and metabolism. ITGA7 was found to be significantly downregulated in BCSCs, and low expression significantly correlated with reduced survival in patients treated with chemotherapy, and with chemoresistance in breast cancer cells in vitro. Conclusions This study is the first to define the molecular profile of BCSCs from a range of primary breast cancers. ITGA7 acts as a predictive marker for chemotherapy response, in accordance with its downregulation in BCSCs.

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Spot 14: A Marker of Aggressive Breast Cancer and a Potential Therapeutic Target

Endocrinology ◽

10.1210/en.2006-0463 ◽

2006 ◽

Vol 147 (9) ◽

pp. 4048-4055 ◽

Cited By ~ 31

Author(s):

William B. Kinlaw ◽

Jennifer L. Quinn ◽

Wendy A. Wells ◽

Christopher Roser-Jones ◽

Joel T. Moncur

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Therapeutic Target ◽

Milk Fat ◽

Breast Cancer Cells ◽

De Novo ◽

Lipid Synthesis ◽

Breast Cancers ◽

Spot 14 ◽

Aggressive Breast Cancer

Spot 14 (S14) is a nuclear protein that communicates the status of dietary fuels and fuel-related hormones to genes required for long-chain fatty acid synthesis. In mammary gland, S14 is important for both epithelial proliferation and milk fat production. The S14 gene is amplified in some breast cancers and is strongly expressed in most. High expression of S14 in primary invasive breast cancer is conspicuously predictive of recurrence. S14 mediates the induction of lipogenesis by progestin in breast cancer cells and accelerates their growth. Conversely, S14 knockdown impairs de novo lipid synthesis and causes apoptosis. We found that breast cancer cells do not express lipoprotein lipase (LPL) and hypothesize that they do not have access to circulating lipids unless the local environment supplies it. This may explain why primary breast cancers with low S14 do not survive transit from the LPL-rich mammary fat pad to areas devoid of LPL, such as lymph nodes, and thus do not appear as distant metastases. Thus, S14 is a marker for aggressive breast cancer and a potential target as well. Future effort will center on validation of S14 as a therapeutic target and producing antagonists of its action.

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SDF-1α regulation in breast cancer cells contacting bone marrow stroma is critical for normal hematopoiesis

Blood ◽

10.1182/blood-2006-01-017459 ◽

2006 ◽

Vol 108 (10) ◽

pp. 3245-3252 ◽

Cited By ~ 41

Author(s):

Anabella L. Moharita ◽

Marcelo Taborga ◽

Kelly E. Corcoran ◽

Margarette Bryan ◽

Prem S. Patel ◽

...

Keyword(s):

Breast Cancer ◽

Bone Marrow ◽

Cancer Cells ◽

Connexin 43 ◽

Breast Cancer Cells ◽

Critical Role ◽

Contact Inhibition ◽

Enzyme Linked Immunosorbent Assay ◽

Hematopoietic Regulation

Abstract Breast cancer cells (BCCs) show preference for the bone marrow (BM). An animal model showed 2 populations of BCCs in the BM with regard to their cycling states. An in vitro model of early BC entry into BM showed normal hematopoiesis. Here, we show a critical role for BCC-derived SDF-1α in hematopoietic regulation. The studies used a coculture of BM stroma and BCCs (cell lines and stage II BCCs). Northern blots and enzyme-linked immunosorbent assay (ELISA) showed gradual decreases in SDF-1α production in BCCs as they contact BM stroma, indicating partial microenvironmental effects caused by stroma on the BCCs. SDF-1 knock-down BCCs and increased exogenous SDF-1α prevented contact inhibition between BCCs and BM stroma. Contact inhibition was restored with low SDF-1α levels. Long-term culture-initiating assays with CD34+/CD38–/Lin– showed normal hematopoiesis provided that SDF-1α levels were reduced in BCCs. Gap junctions (connexin-43 [CX-43]) were formed between BCCs and BM stroma, with concomitant interaction between CD34+/CD38–/Lin– and BM stroma but not with the neighboring BCCs. In summary, SDF-1α levels are reduced in BCCs that contact BM stroma. The low levels of SDF-1α in BCCs regulate interactions between BM stroma and hematopoietic progenitors, consequently facilitating normal hematopoiesis.

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Cxcl10 chemokine induces migration of ING4-deficient breast cancer cells via a novel crosstalk mechanism between the Cxcr3 and Egfr receptors

Molecular and Cellular Biology ◽

10.1128/mcb.00382-21 ◽

2021 ◽

Author(s):

Emily Tsutsumi ◽

Jeremiah Stricklin ◽

Emily A. Peterson ◽

Joyce A. Schroeder ◽

Suwon Kim

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Cancer Progression ◽

Breast Cancer Cells ◽

Critical Role ◽

Disease Free Survival ◽

Intact Cells ◽

Free Survival ◽

Egfr Receptors

The chemokine Cxcl10 has been associated with poor prognosis in breast cancer, but the mechanism is not well understood. Our previous study have shown that CXCL10 was repressed by the ING4 tumor suppressor, suggesting a potential inverse functional relationship. We thus investigated a role for Cxcl10 in the context of ING4 deficiencies in breast cancer. We first analyzed public gene expression datasets and found that patients with CXCL10 -high/ ING4 -low expressing tumors had significantly reduced disease-free survival in breast cancer. In vitro , Cxcl10 induced migration of ING4 -deleted breast cancer cells, but not of ING4 -intact cells. Using inhibitors, we found that Cxcl10-induced migration of ING4 -deleted cells required Cxcr3, Egfr, and the Gβγ subunits downstream of Cxcr3, but not Gαi. Immunofluorescent imaging showed that Cxcl10 induced early transient colocalization between Cxcr3 and Egfr in both ING4 -intact and ING4 -deleted cells, which recurred only in ING4 -deleted cells. A peptide agent that binds to the internal juxtamembrane domain of Egfr inhibited Cxcr3/Egfr colocalization and cell migration. Taken together, these results presented a novel mechanism of Cxcl10 that elicits migration of ING4 -deleted cells, in part by inducing a physical or proximal association between Cxcr3 and Egfr and signaling downstream via Gβγ. These results further indicated that ING4 plays a critical role in the regulation of Cxcl10 signaling that enables breast cancer progression.

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