Abstract 4002: Adipose tissue supernatant of overweight ER positive breast cancer patients promotes migration of MCF-7 breast cancer cells

3543 Background: Non-invasive testing in plasma using RNA biomarkers has been limited by exoribonuclease-mediated degradation of RNA. Circular RNA (circRNA) are covalently closed RNA structures that resist this degradation due to their circular structure. Therefore circRNA are more stable than their linear counterparts. CircRNA are formed by alternative backsplicing of the 3’ end of a downstream exon to the 5’ end of an upstream exon. Here, we propose a novel method for non-invasive identification of circRNA and demonstrate circularized forms of several lineage and cancer specific targets for estrogen receptor-positive breast cancer. Methods: Capture RNA sequencing on cancer tissue was previously performed to determine the relative expression of potential circRNA isoforms in breast cancer patients. These isoforms as well as those predicted by intron length were screened using a quantitative PCR-based assay on ER-positive breast cancer cells. RNA extracted from breast cancer cells are exposed to ribonuclease R to demonstrate stability of circRNA. CircRNA derived from targets with known universal expression are used as positive controls as well as for analysis on plasma. Results: We identify the circRNA isoforms with highest expression for five genes, including ESR1, that are differentially expressed in ER-positive breast cancer compared to other cancers and normal breast tissue. We determine that the circRNA corresponding to all five targets is specifically expressed in breast cancer cell lines with at least 1000-fold higher expression than in non-ER positive breast cancer cell lines. We demonstrate that the highest expressing circRNA isoforms are resistant to degradation by ribonuclease R, whereas corresponding linear mRNA is susceptible. We also demonstrate the presence and stability of positive control circRNA in plasma from patients without cancer. Conclusions: CircRNA are promising biomarkers for early non-invasive detection of cancer due to their stability in plasma. This assay reliably detects ER-positive breast cancer specific circRNA, and exoribonuclease resistance has been validated. Application of this diagnostic assay to plasma from breast cancer patients is underway.

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miR-200 affects tamoxifen resistance in breast cancer cells through regulation of MYB

Scientific Reports ◽

10.1038/s41598-019-54289-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 6

Author(s):

Yu Gao ◽

Wenzhi Zhang ◽

Chengwen Liu ◽

Guanghua Li

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Tamoxifen Resistance ◽

Breast Cancer Patients ◽

Over Expression ◽

Er Positive ◽

Emt Markers ◽

Mcf 7 ◽

Positive Breast Cancer

AbstractResistance to tamoxifen is a major clinical challenge. Research in recent years has identified epigenetic changes as mediated by dysregulated miRNAs that can possibly play a role in resistance to tamoxifen in breast cancer patients expressing estrogen receptor (ER). We report here elevated levels of EMT markers (vimentin and ZEB1/2) and reduced levels of EMT-regulating miR-200 (miR-200b and miR-200c) in ER-positive breast cancer cells, MCF-7, that were resistant to tamoxifen, in contrast with the naïve parental MCF-7 cells that were sensitive to tamoxifen. Further, we established regulation of c-MYB by miR-200 in our experimental model. C-MYB was up-regulated in tamoxifen resistant cells and its silencing significantly decreased resistance to tamoxifen and the EMT markers. Forced over-expression of miR-200b/c reduced c-MYB whereas reduced expression of miR-200b/c resulted in increased c-MYB We further confirmed the results in other ER-positive breast cancer cells T47D cells where forced over-expression of c-MYB resulted in induction of EMT and significantly increased resistance to tamoxifen. Thus, we identify a novel mechanism of tamoxifen resistance in breast tumor microenvironment that involves miR-200-MYB signaling.

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SGK3 Is an Estrogen-Inducible Kinase Promoting Estrogen-Mediated Survival of Breast Cancer Cells

Molecular Endocrinology ◽

10.1210/me.2010-0294 ◽

2011 ◽

Vol 25 (1) ◽

pp. 72-82 ◽

Cited By ~ 35

Author(s):

Yuanzhong Wang ◽

Dujin Zhou ◽

Sheryl Phung ◽

Selma Masri ◽

David Smith ◽

...

Keyword(s):

Breast Cancer ◽

Protein Kinase ◽

Cell Survival ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Dependent Manner ◽

Binding Regions ◽

Er Positive ◽

Mcf 7 ◽

Positive Breast Cancer

Serum- and glucocorticoid-inducible kinase 3 (SGK3) is a protein kinase of the AGC family of protein kinase A, protein kinase G, and protein kinase C and functions downstream of phosphatidylinositol 3-kinase (PI3K). Recent study revealed that SGK3 plays a pivotal role in Akt/protein kinase B independent signaling downstream of oncogenic PI3KCA mutations in breast cancer. Here we report that SGK3 is an estrogen receptor (ER) transcriptional target and promotes estrogen-mediated cell survival of ER-positive breast cancer cells. Through a meta-analysis on 22 microarray studies of breast cancer in the Oncomine database, we found that the expression of SGK3 is significantly higher (5.7-fold, P < 0.001) in ER-positive tumors than in ER-negative tumors. In ER-positive breast cancer cells, SGK3 expression was found to be induced by 17β-estradiol (E2) in a dose- and time-dependent manner, and the induction of SGK3 mRNA by E2 is independent of newly synthesized proteins. We identified two ERα-binding regions at the sgk3 locus through chromatin immunoprecipitation with massively parallel DNA sequencing. Promoter analysis revealed that ERα stimulates the activity of sgk3 promoters by interaction with these two ERα-binding regions on E2 treatment. Loss-of-function analysis indicated that SGK3 is required for E2-mediated cell survival of MCF-7 breast carcinoma cells. Moreover, overexpression of SGK3 could partially protect MCF-7 cells against apoptosis caused by antiestrogen ICI 182,780. Together, our study defines the molecular mechanism of regulation of SGK3 by estrogen/ER and provides a new link between the PI3K pathway and ER signaling as well as a new estrogen-mediated cell survival mechanism mediated by SGK3 in breast cancer cells.

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Identification of estrogenically synthesized LRP16 as an ERα coactivator and its role in ERα-positive breast cancer

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.10676 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 10676-10676

Author(s):

W. Han ◽

Y. Zhao ◽

Z. Wu ◽

Y. Mu ◽

L. Yu ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Northern Blot ◽

Breast Cancer Cells ◽

Tumor Diameter ◽

Er Positive ◽

Mcf 7 ◽

Positive Breast Cancer

10676 Background: Aberrant ERα activity is linked to genesis and malignant progression of breast cancer through direct target gene activation or repression. A complex network of coregulatory proteins is largely believed to determine the transcriptional activity of ERα. LRP16 was identified previously to be an estrogen (E2) responsive gene, but its function involving in conferring estrogen signalling pathway is not clear. Methods: Endogenous LRP16 expression in MCF-7 cells was stably suppressed by retrovirus-mediated small interference RNA (siRNA). The effects of LRP16 expression on E2-stimulated growth and invasive ability of MCF-7 cells were determined in vitro and in vivo assays. The effects of LRP16 expression on ERα transactivation were determined by luciferase assays. The interaction of LRP16 and ERα was examined by GST pull-down and coimmunopricipitation (CoIP) assays. Northern blot and Western blot were used to detect the mRNA and protein levels of ER target genes in LRP16-inhibited MCF-7 cells. The LRP16 expression levels in primary breast cancer were detected by Northern blot. Results: Fristly, LRP16 expression was characterized to be dependent on estrogen activities. Then, LRP16 was identified to be an estrogen-independent ERα cofactor in ER-positive breast cancer cells and demonstrate that LRP16 is an essential coactivator to ERα-mediated transactivation in an estrogen-dependent manner. Suppression of LRP16 expression in ER-positive breast cancer cells specifically inhibits the transcription of ER upregulated genes, results in the increase of E-cadherin expression through ER mediation. In vitro and in vivo data demonstrate that suppression of LRP16 inhibits the ability of estrogen-stimulated proliferation and invasiveness of ER-positive breast cancer cells. The pathological and clinical characteristics of human breast cancer includining ER/PR-positiveness, tumor diameter and the involvement of axillary lymphoid nodes were tightly linked with the LRP16 gene expression level. Conclusions: These results establish a mechanistic link between estrogen receptor status, its coactivator LRP16, and progression of ER-positive breast cancers, and may provide a novel antiestrogenic target for the therapy of ER positive breast cancer. No significant financial relationships to disclose.

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Hyperglycaemia-induced chemoresistance in breast cancer cells: role of the estrogen receptor

Endocrine Related Cancer ◽

10.1530/erc-15-0507 ◽

2015 ◽

Vol 23 (2) ◽

pp. 125-134 ◽

Cited By ~ 7

Author(s):

L Zeng ◽

H A Zielinska ◽

A Arshad ◽

J P Shield ◽

A Bahl ◽

...

Keyword(s):

Breast Cancer ◽

Estrogen Receptor ◽

Cancer Cells ◽

Cancer Patients ◽

Breast Cancer Cells ◽

Diabetic Patients ◽

Breast Cancer Patients ◽

Patients With Diabetes ◽

The Impact ◽

Positive Breast Cancer

Breast cancer patients with diabetes respond less well to chemotherapy; in keeping with this we determined previously that hyperglycaemia-induced chemoresistance in estrogen receptor (ERα) positive breast cancer cells and showed that this was mediated by fatty acid synthase (FASN). More recent evidence suggests that the effect of metabolic syndrome and diabetes is not the same for all subtypes of breast cancer with inferior disease-free survival and worse overall survival only found in women with ERα positive breast cancer and not for other subtypes. Here we examined the impact of hyperglycaemia on ERα negative breast cancer cells and further investigated the mechanism underlying chemoresistance in ERα with a view to identifying strategies to alleviate hyperglycaemia-induced chemoresistance. We found that hyperglycaemia-induced chemoresistance was only observed in ERα breast cancer cells and was dependent upon the expression of ERα as chemoresistance was negated when the ERα was silenced. Hyperglycaemia-induced an increase in activation and nuclear localisation of the ERα that was downstream of FASN and dependent on the activation of MAPK. We found that fulvestrant successfully negated the hyperglycaemia-induced chemoresistance, whereas tamoxifen had no effect. In summary our data suggests that the ERα may be a predictive marker of poor response to chemotherapy in breast cancer patients with diabetes. It further indicates that anti-estrogens could be an effective adjuvant to chemotherapy in such patients and indicates the importance for the personalised management of breast cancer patients with diabetes highlighting the need for clinical trials of tailored chemotherapy for diabetic patients diagnosed with ERα positive breast cancers.

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Narciclasine Targets STAT3 via Distinct Mechanisms in Tamoxifen-Resistant Breast Cancer Cells

10.21203/rs.3.rs-680769/v1 ◽

2021 ◽

Author(s):

Chao Lv ◽

Yun Huang ◽

Rui Huang ◽

Qun Wang ◽

Hongwei Zhang ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Sh2 Domain ◽

Breast Cancers ◽

Stat3 Phosphorylation ◽

Er Positive ◽

Tamoxifen Resistant ◽

Mcf 7 ◽

Positive Breast Cancer

Abstract Background: Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in multiple malignant tumors. Compared with regular estrogen receptor (ER)-positive breast cancers, the patients with tamoxifen-resistant breast cancers often exhibit higher level of STAT3 phosphorylation. Narciclasine (Nar) possesses strong inhibiting effects against a variety of cancer cells, however, the underlying antitumor target(s)/mechanism(s) remains barely understood. Methods: Targets prediction of narciclasine was performed by combining connectivity map (CMAP) and drug affinity responsive target stability (DARTS) strategy. Molecular and biochemical methods were used to elucidate the distinct mechanisms of narciclasine targeting STAT3. The narciclasine nano-delivery system was synthesized by thin film hydration method. Xenograft models were established to determine antitumor activity of narciclasine and its liposome in vivo.Results: In this study, we successfully identified the STAT3 was the direct target of Nar through the combination strategies of CMAP and DARTS. In ER-positive breast cancer cells, Nar could suppress phosphorylation, activation, dimerization, and nuclear translocation of STAT3 by directly binding with the STAT3 SH2 domain. Additionally, Nar could also specifically promote total STAT3 degradation via proteasome pathway and reduce the STAT3 protein stability in tamoxifen-resistant breast cancer cells (MCF-7/TR). This distinct mechanism of Nar targeting STAT3 was mainly attributed to the various levels of reactive oxygen species (ROS) in regular and tamoxifen-resistant ER-positive breast cancer cells. Meanwhile, Nar loaded nanoparticles could markedly decrease the protein levels of STAT3 in tumor sites, resulting in significant MCF-7/TR xenograft tumor regression without obvious toxicity. Conclusions: Our findings successfully highlight the STAT3 as the direct therapeutic target of Nar in ER-positive breast cancer cells, especially Nar leaded STAT3 degradation as a promising strategy for the tamoxifen-resistant breast cancer treatment.

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Cytotoxic Effects of Hellebrigenin and Arenobufagin Against Human Breast Cancer Cells

Frontiers in Oncology ◽

10.3389/fonc.2021.711220 ◽

2021 ◽

Vol 11 ◽

Author(s):

Yu Zhang ◽

Bo Yuan ◽

Baolin Bian ◽

Haiyu Zhao ◽

Anna Kiyomi ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Cancer Cell Line ◽

Therapeutic Strategies ◽

Expression Level ◽

Cycle Arrest ◽

Er Positive ◽

Mcf 7 ◽

Positive Breast Cancer

Development of new therapeutic strategies for breast cancer is urgently needed due to the sustained emergence of drug resistance, tumor recurrence and metastasis. To gain a novel insight into therapeutic approaches to fight against breast cancer, the cytocidal effects of hellebrigenin (Helle) and arenobufagin (Areno) were investigated in human estrogen receptor (ER)-positive breast cancer cell line MCF-7 and triple-negative breast cancer cell line MDA-MB-231. Helle exhibited more potent cytotoxicity than Areno in both cancer cells, and MCF-7 cells were more susceptible to both drugs in comparison with MDA-MB-231 cells. Apoptotic-like morphological characteristics, along with the downregulation of the expression level of Bcl-2 and Bcl-xL and the upregulation of the expression level of Bad, were observed in Helle-treated MCF-7 cells. Helle also caused the activation of caspase-8, caspase-9, along with the cleavage of poly(ADP-ribose) polymerase in MCF-7 cells. Helle-mediated necrosis-like phenotype, as evidenced by the increased propidium iodide (PI)-positive cells was further observed. G2/M cell cycle arrest was also induced by Helle in the cells. Upregulation of the expression level of p21 and downregulation of the expression level of cyclin D1, cyclin E1, cdc25C and survivin were observed in MCF-7 cells treated with Helle and occurred in parallel with G2/M arrest. Autophagy was triggered in MCF-7 cells and the addition of wortmannin or 3-MA, two well-known autophagy inhibitors, slightly but significantly rescued the cells. Furthermore, similar alterations of some key molecules associated with the aforementioned biological phenomena were observed in MDA-MB-231 cells. Intriguingly, the numbers of PI-positive cells in Helle-treated MCF-7 cells were significantly reduced by wortmannin and 3-MA, respectively. In addition, Helle-triggered G2/M arrest was significantly corrected by wortmannin, suggesting autophagy induction contributed to Helle-induced cytotoxicity of breast cancer cells by modulating necrosis and cell cycle arrest. Collectively, our results suggested potential usefulness of both Helle and Areno in developing therapeutic strategies to treat patients with different types of breast cancer, especially ER-positive breast cancer.

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Pim1 Kinase Inhibitors Exert Anti-Cancer Activity Against HER2-Positive Breast Cancer Cells Through Downregulation of HER2

Frontiers in Pharmacology ◽

10.3389/fphar.2021.614673 ◽

2021 ◽

Vol 12 ◽

Author(s):

Bo-Wei Wang ◽

Chih-Hao Huang ◽

Liang-Chih Liu ◽

Fang-Ju Cheng ◽

Ya-Ling Wei ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Cancer Patients ◽

Breast Cancer Cells ◽

Breast Cancer Patients ◽

Her2 Positive ◽

Her2 Positive Breast Cancer ◽

Pim1 Kinase ◽

Anti Cancer ◽

Positive Breast Cancer

The proviral integration site for moloney murine leukemia virus 1 (Pim1) is a serine/threonine kinase and able to promote cell proliferation, survival and drug resistance. Overexpression of Pim1 has been observed in many cancer types and is associated with the poor prognosis of breast cancer. However, it remains unclear whether Pim1 kinase is a potential therapeutic target for breast cancer patients. In this study, we found that Pim1 expression was strongly associated with HER2 expression and that HER2-overexpressing breast cancer cells were more sensitive to Pim1 inhibitor-induced inhibitions of cell viability and metastatic ability. Mechanistically, Pim1 inhibitor suppressed the expression of HER2 at least in part through transcriptional level. More importantly, Pim1 inhibitor overcame the resistance of breast cancer cells to HER2 tyrosine kinase inhibitor lapatinib. In summary, downregulation of HER2 by targeting Pim1 may be a promising and effective therapeutic approach not only for anti-cancer growth but also for circumventing lapatinib resistance in HER2-positive breast cancer patients.

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Up-Regulation of Glioma-Associated Oncogene Homolog 1 Expression by Serum Starvation Promotes Cell Survival in ER-Positive Breast Cancer Cells

Cellular Physiology and Biochemistry ◽

10.1159/000430156 ◽

2015 ◽

Vol 36 (5) ◽

pp. 1862-1876 ◽

Cited By ~ 3

Author(s):

Juan Xu ◽

Gaoxiang Huang ◽

Zongjing Zhang ◽

Jieying Zhao ◽

Mingzhuo Zhang ◽

...

Keyword(s):

Breast Cancer ◽

Cell Survival ◽

Cancer Cells ◽

Microarray Analysis ◽

Breast Cancer Cells ◽

Serum Starvation ◽

Over Expression ◽

Er Positive ◽

Mcf 7 ◽

Positive Breast Cancer

Background/Aims: Cancer cells are resistant to ischemia and starvation. Glioma-associated oncogene homolog 1 (Gli1) is a positive transcriptional activator of Hedgehog (Hh) pathway and plays an essential role in the development of cancers, including breast cancer. However, how Gli1 promotes cell survival remains elusive. The main purpose of this study is to investigate the pro-survival effect of Gli1 under serum starvation and its molecular mechanism in ER-positive breast cancer cells. Methods: Gene expression was determined by quantitative real-time PCR (QRT-PCR) and Western blot. The survival of Gli1 stably transfected ER-positive breast cancer cell lines (Gli1-MCF-7 and Gli1-T47D cells) and their untransfected control cells was estimated by WST-8 assay. Microarray analysis was performed to screen downstream Hh/Gli1 target genes in Gli1-overexpressed MCF-7 cells. Transcriptional activities of NF-kappaB were measured by luciferase assays. ChIP analysis was performed to explore whether cIAP2 was a direct target gene of Gli1. Results: Serum starvation significantly up-regulated the expression of Gli1 gene through activating PI3K/AKT pathway. Over-expression of Gli1 markedly promoted cell survival under serum starvation. Microarray analysis revealed that 338 genes were differentially expressed in Gli1-MCF-7 cells compared with those in the control cells. Among these genes, cellular inhibitor of apoptosis 2 (cIAP2), coding an anti-apoptosis and pro-survival protein, was significantly up-regulated not only by Hh/Gli1 pathway, but also by serum starvation. However, ChIP assay revealed no binding of Gli1 to cIAP2 promoter at the region of -1792 to -1568bp. Moreover, over-expression of Gli1 resulted in enhanced trans-activation of transcriptional factor NF-κB. Suppression of NF-κB signaling with NF-κB inhibitor Bay11-7082, significantly reduced the expression of cIAP2 and the cell survival under serum starvation. Conclusion: Serum starvation significantly up-regulated the expression of Gli1, which in turn increased its key target cIAP2 expression and enhanced NF-κB/cIAP2 pathway, resulting in promoting cell survival under serum starvation. These findings may provide new insights into the pro-survival mechanisms of Gli1 in breast cancer.

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Paeoniflorin Enhances the Sensitivity of ER-Positive Breast Cancer Cells to Tamoxifen through Promoting Sirtuin 4

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2022/6730559 ◽

2022 ◽

Vol 2022 ◽

pp. 1-11

Author(s):

Pei Zhang ◽

Nan Wu ◽

Zhi-Jun Song ◽

Zheng-Fu Tai

Keyword(s):

Breast Cancer ◽

Cell Proliferation ◽

Protein Expression ◽

Cancer Cells ◽

Cell Lines ◽

Breast Cancer Cells ◽

Expression Ratio ◽

Er Positive ◽

Mcf 7 ◽

Positive Breast Cancer

Tamoxifen is an effective drug for treating patients with advanced estrogen receptor-positive (ER+) breast cancer (BC), but not for all ER + BC patients. Drug tolerance is the biggest obstacle. In this study, we designed an experiment to investigate whether paeoniflorin affects the ER + BC cell’s sensitivity to tamoxifen in the T47D and MCF-7 cell lines. Herein, we found that paeoniflorin inhibited cell proliferation without inducing apoptosis. However, it enhanced tamoxifen-induced apoptosis in both cell lines. Immunoblotting revealed that paeoniflorin significantly increased the already elevated Bax/Bcl2 protein expression ratio and the caspase 3 activity levels, both induced by tamoxifen. Paeoniflorin was also found to increase SIRT4 expression, and deletion of SIRT4 could significantly reverse the inhibition of cell proliferation induced by paeoniflorin and significantly decrease paeoniflorin-enhanced apoptosis induced by tamoxifen. Moreover, protein expression detection revealed that paeoniflorin enhanced the tamoxifen-induced inhibition of STAT3 activation. Besides, the deletion of SIRT4 could significantly increase STAT3 activation in the T47D and MCF-7 cells. In conclusion, paeoniflorin suppressed STAT3 activation to enhance the sensitivity of ER-positive breast cancer cells to tamoxifen through promoting SIRT4 expression.

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