ER-negative Breast Cancer Cell Lines Research Articles

Estrogen Receptor Is Required for Metformin-Induced Apoptosis in Breast Cancer Cells Under Hyperglycemic Conditions.

About 25% to 30% of estrogen receptor (ER)-positive breast cancer patients develop resistance to endocrine therapy. Human epidermal growth factor receptor 2 (HER2) has been shown to cooperate with several growth factors that regulate cellular energy metabolism, including the insulin-like growth factor 1 receptor (IGF-1R). As the first-line therapy for type 2 diabetes mellitus (T2DM) patients, metformin is widely known to inhibit the metabolic reprogramming of cancer cells. This study aims to investigate metformin's efficacy in inhibiting endocrine resistance related to genes regulating energy metabolism in both ER-positive and ER-negative breast cancer cell lines under hyperglycemic conditions. MDA-MB-361 (ER-positive, HER2-positive) and SKBR3 (ER-negative, HER2-positive) cancer cell lines were used to represent ER status. Cell viability and cell survival rate were measured using the colorimetric assay of Cell Counting Kit-8. All mRNA levels were quantified using real-time quantitative polymerase chain reaction preceded by reverse transcription. A P value of <.05 was considered statistically significant. Unlike MDA-MB-361, SKBR3 were found to acquire resistance upon metformin treatment in hyperglycemic conditions. Moreover, the mRNA expression of IGF-1R and its downstream signaling, such as the mammalian target of rapamycin (mTOR), was not affected by metformin. Meanwhile, the mRNA expression level of ribosomal S6 kinase 1 (S6K1) was upregulated, whereas forkhead box O1 (FOXO1) was downregulated after metformin treatment in hyperglycemic conditions. This preliminary study suggests that an alternative pathway of metformin resistance may exist in the absence of ERα. Therefore, relying solely on metformin may be inadequate to inhibit the aggressiveness of breast cancer cells.

Effects of 7-ketocholesterol on tamoxifen efficacy in breast carcinoma cell line models in vitro

Oxysterols play significant roles in many physiological and pathological processes including cancer. They modulate some of the cancer hallmarks pathways, influence the efficacy of anti-cancer drugs, and associate with patient survival. In this study, we aimed to analyze the role of 7-ketocholesterol (7-KC) in breast carcinoma cells and its potential modulation of the tamoxifen effect. 7-KC effects were studied in two estrogen receptor (ER)-positive (MCF-7 and T47D) and one ER-negative (BT-20) breast cancer cell lines. First, we tested the viability of cells in the presence of 7-KC. Next, we co-incubated cells with tamoxifen and sublethal concentrations of 7-KC. We also tested changes in caspase 3/7 activity, deregulation of the cell cycle, and changes in expression of selected genes/proteins in the presence of tamoxifen, 7-KC, or their combination. Finally, we analyzed the effect of 7-KC on cellular migration and invasion. We found that the presence of 7-KC slightly decreases the efficacy of tamoxifen in MCF-7 cells, while an increased effect of tamoxifen and higher caspase 3/7 activity was observed in the BT-20 cell line. In the T47D cell line, we did not find any modulation of tamoxifen efficacy by the presence of 7-KC. Expression analysis showed the deregulation in CYP1A1 and CYP1B1 with the opposite trend in MCF-7 and BT-20 cells. Moreover, 7-KC increased cellular migration and invasion potential regardless of the ER status. This study shows that 7-KC can modulate tamoxifen efficacy as well as cellular migration and invasion, making 7-KC a promising candidate for future studies.

Inhibitory effect of calycosin on breast cancer cell progression through downregulating lncRNA HOTAIR and downstream targets: HuR and IGF2BP1.

Breast cancer is the most commonly diagnosed cancer worldwide. Previously, we reported that calycosin, a typical isoflavone phytoestrogen, triggers apoptosis and is associated with lncRNA HOTAIR in the estrogen receptor (ER)-positive breast cancer MCF-7-cell line. In the present study, we aim to uncover the mechanism of lncRNA HOTAIR in the inhibitory effect induced by calycosin in both ER-positive and ER-negative breast cancer cell lines. Results show that calycosin significantly inhibits proliferation and triggers apoptosis in both ER-positive (MCF-7 and T47D) and ER-negative (MDA-MB-231 and SK-BR-3) breast cancer cell lines, accompanied by downregulation of lncRNA HOTAIR expression. Accordingly, knockdown of lncRNA HOTAIR promotes the anti-tumor effect of calycosin, while overexpression of lncRNA HOTAIR attenuates this effect. Meanwhile, the expression levels of HuR and IGF2BP1 are also reduced by calycosin. More importantly, calycosin facilitates the downregulation of HuR and IGF2BP1 caused by decreasing lncRNA HOTAIR expression, and the upregulation of HuR and IGF2BP1 caused by overexpression of lncRNA HOTAIR is weakened by calycosin. These results demonstrate that downregulating HuR and IGF2BP1 by suppressing lncRNA HOTAIR results in inhibited growth of breast cancer cells by calycosin. In addition, the binding of HuR and IGF2BP1 to lncRNA HOTAIR is detected by RIP assay, implying an interaction between these two proteins and lncRNA HOTAIR. Together, lncRNA HOTAIR may play a carcinogenic role in breast cancer development and has the potential to be a novel therapeutic target for breast cancer in the future, especially in isoflavone phytoestrogen therapy.

Abstract P3-10-06: High levels of miR-18a is associated with increased proliferation but suppression of EMT phenotype in ER negative breast cancer

Abstract Introduction: miRNA-based regulation has been implicated in tumor evolution and progression. miR-18a belonging to the miR-17-92 polycistronic cluster has also been reported to have oncogenic effects across multiple cancer types including breast cancer. We have previously demonstrated the epigenetic regulation of ER by miR-18a and its high levels as a poor prognostic marker in ER-positive breast tumors (Nair et al, Cancer Med. 2016). Here, we have examined the effects of high expression of miR-18a in the ER-negative subtype of breast cancer. Methods: 275 surgically excised specimens of primary breast cancers were analyzed. Samples were segregated into ER-positive and ER-negative tumors based on ER positivity as determined by Immunohistochemistry. Relative abundance of hsa-miR-18a-5p in these samples was assessed using a TaqMan qRT-PCR and used to correlate with a probability distribution of proliferation that was derived by fitting a binomial logistic regression model using 4 genes - FOXM1, UBE2C, BIRC5 &amp; ANLN. miR-18a was inhibited using synthetic inhibitors in ER-negative breast cancer cell lines - MDA-MB-468 AND MDA-MB-231. Migratory ability was assessed using wound-healing assays. The expression of miR-18a was further analyzed in ER-negative breast cancer samples from the TCGA (n= 116) and the METABRIC cohort (n=107). ER-negative tumors with higher than the third quartile and lower than the first quartile expression of miR-18a were segregated into tumors with high and low expression, respectively. Functional enrichment of differentially expressed genes (DEGs) between these groups was performed using the G: profiler to identify the deregulated pathways. Result: Evaluation of the levels of miR-18a in 275 breast tumor samples showed that the microRNA was highly expressed (p&amp;lt;0.0001) in the ER-negative tumors (n=105) when compared to ER-positive samples (n=170). The ER-negative tumors were further stratified based on the 3rd quartile expression of miR-18a into high (n=27) and low miR-18a (n=78) expressing tumors. The high miR-18a expressing tumors were associated with a higher proliferation score (p=0.005). Inhibition of miR-18a levels in ER-negative breast cancer increased migration ability up to 33 % in MDA-MB-468 (p=0.009) and up to 26 % in MDA-MB-231 (p=0.0003). Analysis of ER-negative tumors of TCGA and METABRIC cohort revealed that the high miR-18a expressing tumors expressed low levels of EMT master regulators-ZEB1 and ZEB2 and low levels of Matrix metalloproteinases -MMP2, MMP3, MMP10, MMP11, MMP13, and MMP17 (p&amp;lt;0.01). Functional enrichment of differentially expressed genes (DEGs) demonstrated suppression of pathways related to cell motility and migration, ECM activation, Wnt signaling, and Focal adhesion-PI3K-Akt signaling pathways (p&amp;lt;0.01). Conclusion: In ER-positive tumors, high levels of miR-18a are associated with activated EMT, increased migratory ability, and poor prognosis. On the contrary, in ER-negative breast tumors, high levels of miR-18a is associated with a higher rate of proliferation but lower migratory ability and suppression of signaling pathways necessary for an epithelial to mesenchymal transition. Citation Format: Madhumathy G Nair, Chandrakala M, Apoorva D, Snijesh VP, Jyothi S Prabhu, Savitha Rajarajan, Aruna Korlimarla, Rakesh S Ramesh, Srinath BS, Sridhar TS. High levels of miR-18a is associated with increased proliferation but suppression of EMT phenotype in ER negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-10-06.

Truncated WT1 Protein Isoform Expression Is Increased in MCF-7 Cells with Long-Term Estrogen Depletion.

Background The wt1 gene codes for a transcription factor that presents several protein isoforms with diverse biological properties, capable of positively and negatively regulating genes involved in proliferation, differentiation, and apoptosis. WT1 protein is overexpressed in more than 90% of breast cancer; however, its role during tumor progression is still unknown. Methodology. In this work, we analyzed the expression of WT1 isoforms in several breast cancer cells with different tumor marker statuses and an in vitro assay using MCF-7 cells cultured with long-term estrogen depletion (MCF-7 LTED cells) with the finality to mimic the process of switching from hormone-dependent to hormone-independent. Moreover, growth kinetics, sensitivity to tamoxifen, and relative expression analysis of ER and Her2/neu were performed. Results Initially, the expression of 52-54 kDa protein isoform of WT1 in the breast cancer cell line ER (+) was detected by western blot and was absent in ER (-), and the 36-38 kDa protein isoform of WT1 was detected in all cell lines analyzed. The analysis of alternative splicing by RT-PCR shows that the 17AA (+)/KTS (-) isoform of WT1 was the most frequent in the four cell lines analyzed. In vitro, the MCF-7 cells in the estrogen depletion assay show an increase in the expression of the 52-54 kDa isoform of WT1 in the first 48 hours, and this was maintained until week 13, and later, this expression was decreased, and the 36-38 kDa isoform of WT1 did not show change during the first 48 hours but from week 1 showed an increase of expression, and this remained until week 27. Growth kinetic analysis showed that MCF-7 LTED cells presented a 1.4-fold decrease in cellular proliferation compared to MCF-7 cells cultured under normal conditions. In addition, MCF-7 LTED cells showed a decrease in sensitivity to the antiproliferative effect of tamoxifen (p ≤ 0.05). Samples collected until week 57 analyzed by qRT-PCR showed an increase in the relative expression of the Her2/neu and ER. Conclusions Modulation of protein isoforms showed differential expression of WT1 isoforms dependent on estrogen receptor. The absence of 52-54 kDa and the presence of the 36-38 kDa protein isoform of WT1 were detected in ER-negative breast cancer cell lines classified as advanced stage cells. Long-term estrogen depletion assay in MCF-7 cells increased the expression of the 36-38 kDa isoform and reduced the 52-54 kDa isoform, and these cells show an increase in the expression of tumor markers of ER and Her2/neu. MCF-7 LTED cells showed low proliferation and insensitivity to tamoxifen compared to MCF-7 cells in normal conditions. These results support the theory about the relationship of the 36-38 kDa isoform of WT1 and the absence of ER function in advanced breast cancer.

RANK-C Expression Sensitizes ER-Negative, EGFR-Positive Breast Cancer Cells to EGFR-Tyrosine Kinase Inhibitors (TKIs).

Background: We have previously shown that overexpression of RANK-c in ER-negative breast cancer cell lines attenuates aggressive properties of cancer cells, partially through a RANK-c/EGFR interaction. EGFR inhibition through TKIs in breast cancer has been tested in triple-negative disease settings with limited clinical benefit for patients. Here we test if expression of RANK-c in ER-negative breast cancer cells in conjunction with treatment with TK inhibitors (erlotinib or gefitinib) can affect survival and colony-forming capacity of cancer cells. Methods: Stably expressing MDA-MB-231-RANK-c and SKBR3-RANK-c cells were employed to test proliferation and colony formation in the presence of TKIs. In addition, Western blot analysis was performed to dissect EGFR related signaling cascades upon TK inhibition in the presence of RANK-c. Results: Interestingly the two RANK-c expressing, ER-negative cells lines presented with a distinct phenotype concerning TKI sensitivity upon treatment. MDA-MB-231-RANK-c cells had a higher sensitivity upon gefitinib treatment, while erlotinib decreased the proliferation rate of SKBR3-RANK-c cells. Further, colony formation assays for MDA-MB-231-RANK-c cells showed a decrease in the number and size of colonies developed in the presence of erlotinib. In addition, RANK-c seems to alter signaling through EGFR after TKI treatment in a cell type-specific manner. Conclusions: Our results indicate that ER-negative breast cancer cells that express RANK-c alter their sensitivity profile against tyrosine kinase inhibitors (erlotinib and gefitinib) in a cell type-specific and culture substrate-dependent manner.

The analog of cGAMP, c-di-AMP, activates STING mediated cell death pathway in estrogen-receptor negative breast cancer cells.

Immune adaptor protein like STING/MITA regulate innate immune response and plays a critical role in inflammation in the tumor microenvironment and regulation of metastasis including breast cancer. Chromosomal instability in highly metastatic cells releases fragmented chromosomal parts in the cytoplasm, hence the activation of STING via an increased level of cyclic dinucleotides (cDNs) synthesized by cGMP-AMP synthase (cGAS). Cyclic dinucleotides 2' 3'-cGAMP and it's analog can potentially activate STING mediated pathways leading to nuclear translocation of p65 and IRF-3 and transcription of inflammatory genes. The differential modulation of STING pathway via 2' 3'-cGAMP and its analog and its implication in breast tumorigenesis is still not well explored. In the current study, we demonstrated that c-di-AMP can activate type-1 IFN response in ER negativebreast cancer cell lines which correlate with STING expression. c-di-AMP binds to STING and activates downstream IFN pathways in STING positive metastatic MDA-MB-231/MX-1cells. Prolonged treatment of c-di-AMP induces cell death in STING positive metastatic MDA-MB-231/MX-1 cells mediated by IRF-3. c-di-AMP induces IRF-3 translocation to mitochondria and initiates Caspase-9 mediated cell death and inhibits clonogenicity of triple-negative breast cancer cells. This study suggests that c-di-AMP can activate and modulates STING pathway to induce mitochondrial mediated apoptosis in estrogen-receptor negative breast cancer cells.

Estrogen-induced hypomethylation and overexpression of YAP1 facilitate breast cancer cell growth and survival

Increased expression of Yes-associated protein-1 (YAP1) was shown to correlate with reduced survival in breast cancer (BC) patients. However, the exact mechanism of YAP1 regulation in BC cells remains ambiguous. Genomic sequence search showed that the promoter region of the YAP1 gene contains CpG Islands, hence the likelihood of epigenetic regulation by DNA methylation. To address this possibility, the effect of estrogen (17β estradiol; E2) on YAP1 gene expression and YAP1 promoter methylation status was evaluated in BC cells. The functional consequences of E2 treatment in control and YAP1-silenced BC cells were also investigated. Our data showed that E2 modulates YAP1 expression by hypomethylation of its promoter region via downregulation of DNA methyltransferase 3B (DNMT3B); an effect that seems to facilitate tumor progression in BC cells. Although the effect of E2 on YAP1 expression was estrogen receptor (ER) dependent, E2 treatment also upregulated YAP1 expression in MDA-MB231 and SKBR3 cells, which are known ER-negative BC cell lines but expresses ERα. Functionally, E2 treatment resulted in increased cell proliferation, decreased apoptosis, cell cycle arrest, and autophagic flux in MCF7 cells. The knockdown of the YAP1 gene reversed these carcinogenic effects of E2 and inhibited E2-induced autophagy. Lastly, we showed that YAP1 is highly expressed and hypomethylated in human BC tissues and that increased YAP1 expression correlates negatively with DNMT3B expression but strongly associated with ER expression. Our data provide the basis for considering screening of YAP1 expression and its promoter methylation status in the diagnosis and prognosis of BC.

The Psychiatric Drug Lithium Increases DNA Damage and Decreases Cell Survival in MCF-7 and MDA-MB-231 Breast Cancer Cell Lines Expos ed to Ionizing Radiation.

Breast cancer is the most common cancer among women. Radiation therapy is used for treating almost every stage of breast cancer. A strategy to reduce irradiation side effects and to decrease the recurrence of cancer is concurrent use of radiation and radiosensitizers. We studied the effect of the antimanic drug lithium on radiosensitivity of estrogen-receptor (ER)-positive MCF-7 and ER-negative, invasive, and radioresistant MDA-MB-231 breast cancer cell lines. MCF-7 and MDA-MB-231 breast cancer cell lines were treated with 30 mM and 20 mM concentrations of lithium chloride (LiCl), respectively. These concentrations were determined by MTT viability assay. Growth curves were depicted and comet assay was performed for control and LiCl-treated cells after exposure to X-ray. Total and phosphorylated inactive levels of glycogen synthase kinase-3beta (GSK-3β) protein were determined by ELISA assay for control and treated cells. Treatment with LiCl decreased cell proliferation after exposure to X-ray as indicated by growth curves of MCF-7 and MDA-MB-231 cell lines within six days following radiation. Such treatment increased the amount of DNA damages represented by percent DNA in Tails of comets at 0, 1, 4, and even 24 hours after radiation in both studied cell lines. The amount of active GSK-3β was increased in LiCl-treated cells in ER-positive and ER-negative breast cancer cell lines. Treatment with LiCl that increased the active GSK-3β protein, increased DNA damages and decreased survival independent of estrogen receptor status in breast cancer cells exposed to ionizing radiation.

Effect of CCT137690 on long non-coding RNA expression profiles in MCF-7 and MDA-MB-231 cell lines.

Long non-coding RNAs (lncRNAs) are involved in a range of biological processes, such as cellular differentiation, migration, apoptosis, invasion, proliferation, and transcriptional regulation. The aberrant expression of lncRNAs plays a significant role in several cancer types. Aurora kinases are increasingly expressed in various malignancies; accordingly, the inhibition of these enzymes may represent a novel approach for the treatment of various cancers. CCT137690, an Aurora kinase inhibitor, displays an anti-proliferative activity in human cancer cell lines. The aim of the present study was to investigate the anti-proliferative and cytotoxic effects of CCT137690 on estrogen receptor (ER)-positive human breast cancer cell line (MCF-7) and ER-negative human breast cancer cell line (MDA-MB-231). In addition, this study was targeted toward determining the changes induced in lncRNA expression levels following the initiation of Aurora kinase inhibitor treatment. The cytotoxic effects of CCT137690 were determined by means of the xCELLigence system. Furthermore, the anti-proliferative role of CCT137690 in breast cancer was investigated by checking the changes in lncRNA expression profiles using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The half-maximal inhibitory concentrations (IC50) of CCT137690 were determined as 4.5 µM (MCF-7) and 7.27 µM (MDA-MB-231). Several oncogenic lncRNAs (e.g., PRINS, HOXA1AS, and NCRMS) were downregulated in both ER-negative and ER-positive cell lines. On the other hand, tumor suppressor lncRNAs (e.g., DGCR5 and IGF2AS) were upregulated in the ER-positive cell line. After CCT137690 treatment, HOXA11AS and PCAT-14 lncRNAs were downregulated in the ER-positive cell lines. In addition, MER11C, SCA8, BC200, HOTAIR, PCAT-1, UCA1, SOX2OT, and HULC lncRNAs were downregulated in the ER-negative cell lines. The results of the present study indicated that Aurora kinase inhibitor CCT137690 could be a potential anti-cancer agent for breast cancer treatment.

Simvastatin induces breast cancer cell death through oxidative stress up-regulating miR-140-5p.

Statins, a class of hyperlipidemic drugs, are widely used cholesterol lowering drugs that selectively inhibit 3-hydroxy-3-methylglutaryl CoA reductase, which is the rate-limiting enzyme in cholesterol biosynthesis, leading to decreasing of cholesterol biosynthesis. Statins exert anti-tumoral effects on various cancer, including breast cancer. However, the molecular mechanisms for the actions were not fully elucidated. The purpose of this study was to elucidate the effects of statins on proliferation and apoptosis in the ER-negative breast cancer cell line MDA-MB-231. Our results showed that simvastatin increased the expression of miR-140-5p in a dose dependent manner via activating transcription factor NRF1, reduced cell proliferation and induced apoptosis, and we also found that SLC2A1 was a new target of miR-140-5p. In conclusion, data in this study shed light on the potential anti-tumoral effects of simvastatin in breast cancer and presents a highly promising therapeutic option, using drug and miRNA for combined treating cancers.

Inactivation of Rab27B-dependent signaling pathway by calycosin inhibits migration and invasion of ER-negative breast cancer cells

Previous studies report the upregulation of the secretory Rab27B small GTPase in human breast cancer, which could promote invasive growth and metastasis in estrogen receptor (ER)-positive breast cancer cells. However, there is limited evidence for its role in ER-negative breast cancer, along with the signaling pathways. Consistent with previous studies, we here confirmed that Rab27B is upregulated in breast tumor tissue in comparison with normal breast tissue. In addition, in ER-negative breast cancer cell line MDA-MB-231, when the levels of Rab27B expression were further elevated by transduction with recombinant lentivirus vector, migration and invasion assays demonstrated that cell migration and invasion was significantly stimulated. Moreover, Rab27B overexpression increased levels of β-catenin, followed by upregulation of vascular endothelial growth factor (VEGF). Our findings reveal a key function for the Rab27B-mediated modulation of β-catenin and VEGF in ER-negative breast cancer cell metastasis. Notably, the suppressed expression of Rab27B, β-catenin and VEGF was found in calycosin-treated MDA-MB-231 cells, accompanied with decreased invasive and migratory potential of these cells. What's more, these inhibitory effects of calycosin were all attenuated by Rab27B overexpression. The results demonstrated that calycosin-induced inhibition of migration and invasion in ER-negative breast cancer cells may be associated with the inactivation of Rab27B-dependent signaling, and suggest that antagonism of this pathway by calycosin may offer alternative therapeutic strategy for the aggressive breast cancer.

CREBBP/EP300 Bromodomain Inhibition Affects the Proliferation of AR-Positive Breast Cancer Cell Lines.

Inhibitors that prevent the binding of bromodomains to acetylated histones hold therapeutic potential. However, the effects of targeting most of the 60 different bromodomains found in the human proteome remain unexplored. Here, we investigate the molecular mechanisms responsible for the antiproliferative properties of CREBBP/EP300 bromodomain inhibition in ER-negative breast cancer cell lines. We show using genetic and chemical approaches that CREBBP/EP300 bromodomains are critical to support the proliferation of the triple-negative breast cancer cell line MDA-MB-453. Analysis of the transcriptional pathways affected by CREBBP/EP300 bromodomain inhibitors reveals that the expression of genes associated with super-enhancers is downregulated, which in turn are occupied by very high levels of androgen receptor (AR) in MDA-MB-453 cells. Treatment of MDA-MB-453 with CREBBP/EP300 bromodomain inhibitors downregulates the expression of an AR-dependent signature distinctive of breast cancer tumors that express AR and causes a decrease in H3K27ac levels at AR-binding sites. In accordance, in prostate cancer cell lines that express AR CREBBP/EP300 bromodomain inhibitors downregulate the expression of genes bound by AR and associated with super-enhancers. In summary, we report that triple-negative breast cancer cell lines that express AR are particularly sensitive to CREBBP/EP300 bromodomain inhibitors and consequently these inhibitors hold potential to treat this type of cancer. IMPLICATIONS: AR-dependent cancer cell lines are sensitive to CREBBP/EP300 bromodomain inhibitors.

Abstract P3-09-06: CXCL1 chemokine stimulates migration and invasion in ER-negative breast cancer through activation of the ERK/MMP-2/9 signaling axis

Abstract Chemokine (C-X-C motif) ligand 1 (CXCL1), a member of the CXC chemokine family, has been reported to be a critical factor in inflammatory diseases and tumor progression. However, its functions and molecular mechanism in estrogen receptor α (ER) negative breast cancer (BC) has little been known. In this study, we discovered that CXCL1 is overexpression in ER-negative breast cancer tissues and cell lines compared with ER-positive patient tissues and cell lines. Treatment with recombinant human CXCL1 protein promotes ER-negative BC cell migration and invasion in dose-dependent manner and stimulates the activation of p-ERK1/2, but not p-STAT3 or p-AKT. Whereas, knockdown of CXCL1 in BC cells attenuates these effects. Moreover, CXCL1 induces the expression of MMP2/9 via ERK1/2 pathway. The blockage of ERK by its antagonists (U0126) can abolish the effect of CXCL1 on MMP2/9 expression. Furthermore, immunohistochemical (IHC) analysis revealsa strong positive correlation between CXCL1 and p-ERK1/2 expressions in BC tissues. In conclusion, our study illustrates that CXCL1 is highly expressed in ER-negative BC and stimulates cell migration and invasion through ERK/MMP2/9 pathway, which may serve as a potential therapeutictarget in ER-negative breast cancer. Citation Format: Liu S, Yang C, Guo S, Chen R. CXCL1 chemokine stimulates migration and invasion in ER-negative breast cancer through activation of the ERK/MMP-2/9 signaling axis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-09-06.

Estrogen receptor signaling regulates the expression of the breast tumor kinase in breast cancer cells

BackgroundBRK is, a non-receptor tyrosine kinase, overexpressed in approximately 85% of human invasive ductal breast tumors. It is not clear whether BRK expression correlates with breast cancer subtypes, or the expression has prognostic or diagnostic significance. Herein, we investigated the correlation of BRK with any breast cancer subtypes and clinicopathological significance of BRK expression in breast cancer.MethodsIn this study, we examined BRK expression in 120 breast tumor samples and 29 breast cancer cell lines to explore the positive correlation between BRK and the expression of ERα. We used immunohistochemistry, RT-PCR, and immunoblotting to analyse our experimental samples.ResultWe demonstrate that estrogen induces BRK gene and protein expression in ER+ breast cancer cells. Over-expression of ERα in the ER-negative breast cancer cell line increased BRK expression, and knock-down of ESR1 in MCF7 cells reduced BRK levels. Further, we provide evidence that BRK is regulated by ERα signaling and the presence of ER antagonists (tamoxifen and fulvestrant) reduce the expression of BRK in ER-positive breast cancer cells. Finally, we demonstrate that the overall survival of ER-positive breast cancer patients is poor when their cancers express high levels of BRK.ConclusionOur data indicate that BRK is a prognostic marker for ER+ breast cancers and provide a strong rationale for targeting BRK to improve patients’ survival.

RANK-c attenuates aggressive properties of ER-negative breast cancer by inhibiting NF-κB activation and EGFR signaling.

The RANK/RANKL axis emerges as a key regulator of breast cancer initiation, progression, and metastasis. RANK-c is a RANK receptor isoform produced through alternative splicing of the TNFRSF11A (RANK) gene and a dominant-negative regulator of RANK-induced nuclear factor-κB (NF-κB) activation. Here we report that RANK-c transcript is expressed in 3.2% of cases in The Cancer Genome Atlas breast cancer cohort evenly between ER-positive and ER-negative cases. Nevertheless, the ratio of RANK to RANK-c (RANK/RANK-c) is increased in ER-negative breast cancer cell lines compared to ER-positive breast cancer cell lines. In addition, forced expression of RANK-c in ER-negative breast cancer cell lines inhibited stimuli-induced NF-κB activation and attenuated migration, invasion, colony formation, and adhesion of cancer cells. Further, RANK-c expression in MDA-MB-231 cells inhibited lung metastasis and colonization in vivo. The RANK-c-mediated inhibition of cancer cell aggressiveness and nuclear factor-κB (NF-κB) activation in breast cancer cells seems to rely on a RANK-c/TNF receptor-associated factor-2 (TRAF2) protein interaction. This was further confirmed by a mutated RANK-c that is unable to interact with TRAF2 and abolishes the ability to attenuate NF-κB activation, migration, and invasion. Additional protein interaction characterization revealed epidermal growth factor receptor (EGFR) as a novel interacting partner for RANK-c in breast cancer cells with a negative effect on EGFR phosphorylation and EGF-dependent downstream signaling pathway activation. Our findings further elucidate the complex molecular biology of the RANKL/RANK system in breast cancer and provide preliminary data for RANK-c as a possible marker for disease progression and aggressiveness.

The hydro-ethanolic extract of Acacia seyal (Mimosaceae) stem barks induced death in an ER-negative breast cancer cell line by the intrinsic pathway of apoptosis and inhibited cell migration

BackgroundDespite the significant developments occurring in the treatment of cancer, it still remains the second deadly disease, responsible for 8.2 million deaths every year. Various natural substances have been studied for active molecules of tumor suppression in the past and the tropical flora, by its diversity, continues to provide new antitumor drugs. Acacia seyal is a plant used in Cameroonian traditional system to treat cancer. It exhibited cytotoxic effects towards human breast adenocarcinoma cells. The present work was therefore designed to elucidate the underlying mechanisms by which A. seyal extract induced its cytotoxic effect. MethodsThe cell death mechanism (apoptosis or necrosis) and cell cycle analyses were assessed using flow cytometry. The levels of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), caspases activities as well as Bcl-2 and Bcl-xL protein contents were assessed in MDA-MB-231 cells. Afterwards, cell migration/invasion was also assessed. ResultsThe A. seyal extract induced apoptosis in MDA-MB-231 cells, while it failed to do so in MCF-7 cells. It induced cell cycle arrest in G2/M phase. Further it induced a decrease in ΔΨm, an increase in ROS levels and caspases activities as well as a down regulation in Bcl-2 and Bcl-xL protein contents in MDA-MB-231 cells. Moreover, A. seyal extract exhibited anti-migration, anti-invasion activities in MDA-MB-231 cells. ConclusionThese results demonstrate that A. seyal extract induced its antitumor effects mainly by interference in metastasis related events, by triggering apoptosis through a ROS-mediated mitochondrial pathway.

Abstract B158: Exploiting IGF1R pathway activation as a therapeutic strategy for E-cadherin deficient breast cancers

Abstract Insulin-like growth factor I (IGF1) plays an important role in breast cancer initiation and progression due to its regulation of cell proliferation, migration, and invasion. These characteristics make IGF1R an attractive therapeutic target; however, only a subset of patients show beneficial response to IGF1R inhibitors in clinical trials. To identify putative biomarkers of response to IGF1, we performed a proteomic screen in 21 breast cancer cell lines stimulated with IGF1. Using a novel integrated computational and experimental approach, we identified levels of E-cadherin (CDH1), a major component of the adherens junction (AJ), as a repressor of IGF1R signaling. CDH1 is genetically lost in invasive lobular breast cancer (ILC), a subtype of breast cancer that accounts for ~10-15% of total cases. E-cadherin is also downregulated in estrogen receptor (ER)-negative breast cancers that have undergone epithelial to mesenchymal transition. Consistent with the proteomic screen, knockdown of E-cadherin enhanced IGF1-induced activation of IGF1R and downstream Akt and ERK1/2 phosphorylation, and this enhanced cell cycle progression. Disruption of the AJ alone with an E-cadherin monoclonal antibody also enhanced IGF1R signaling. Further, we have shown a direct interaction of IGF1R and E-cadherin using in situ proximity ligation assay indicating a physical regulation of E-cadherin on IGF1R signaling. Supporting the clinical relevance of our observations, we found increased expression of IGF1 ligand and increased IGF1R phosphorylation in E-cadherin deficient ER+ ILC tumors in The Cancer Genome Atlas (TCGA) compared to ER+ invasive ductal carcinomas (IDC). We also discovered a correlation between mRNA expression and the activation of the IGF gene signature in (1) a cohort of ER+ ILC tumors compared to ER+ IDC and (2) a subset of ER-negative tumors within TCGA. We have previously published that IGF1R inhibitors are effective in inhibiting growth of ER-negative breast cancer cell lines and xenograft models in combination with chemotherapy. Thus, we focused our studies on understanding the utility of IGF1R pathway inhibition in ER+ ILC as a therapeutic strategy. Our data indicate that IGF1R and Akt pathway inhibitors are effective in inhibiting growth in ER+ ILC cell lines and ex vivo tumor culture, and synergize with standard-of-care anti-ER targeted therapy. Therefore, we hypothesize that loss of E-cadherin potentiates IGF1R signaling to enhance breast cancer progression and that loss of E-cadherin expression in ILC and ER-negative tumors may highlight those susceptible to IGF1R inhibition. Citation Format: Alison M. Nagle, Cemal Erdem, Kevin Levine, Christos Sotiriou, Otto Metzger, D Lans Taylor, Timothy Lezon, Steffi Oesterreich, Adrian V. Lee. Exploiting IGF1R pathway activation as a therapeutic strategy for E-cadherin deficient breast cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B158.

Cancer cell line specific co-factors modulate the FOXM1 cistrome.

ChIP-seq has been commonly applied to identify genomic occupation of transcription factors (TFs) in a context-specific manner. It is generally assumed that a TF should have similar binding patterns in cells from the same or closely related tissues. Surprisingly, this assumption has not been carefully examined. To this end, we systematically compared the genomic binding of the cell cycle regulator FOXM1 in eight cell lines from seven different human tissues at binding signal, peaks and target genes levels. We found that FOXM1 binding in ER-positive breast cancer cell line MCF-7 are distinct comparing to those in not only other non-breast cell lines, but also MDA-MB-231, ER-negative breast cancer cell line. However, binding sites in MDA-MB-231 and non-breast cell lines were highly consistent. The recruitment of estrogen receptor alpha (ERα) caused the unique FOXM1 binding patterns in MCF-7. Moreover, the activity of FOXM1 in MCF-7 reflects the regulatory functions of ERα, while in MDA-MB-231 and non-breast cell lines, FOXM1 activities regulate cell proliferation. Our results suggest that tissue similarity, in some specific contexts, does not hold precedence over TF-cofactors interactions in determining transcriptional states and that the genomic binding of a TF can be dramatically affected by a particular co-factor under certain conditions.

Abstract 2913: The SOX11 transcription factor is critical for basal-like breast cancer growth and migration and is associated with poor survival

Abstract Introduction: Basal-like breast cancers (BLBCs) are aggressive breast cancers that are associated with poor survival and have distinctive mRNA expression profiles compared to non-BLBC tumors. Through an integrated screen of transcription factors (TFs) we identified SOX11 as uniquely critical for growth in BLBC, but not essential in estrogen-receptor (ER)-positive breast cancer cells. In this study we examine the role of SOX11 regulating growth and other BLBC phenotypes, including increased migration, invasion and poor survival. Methods: SOX11 was identified through an integrated primary screen comparing mRNA expression of TFs, DNA sequences of differentially expressed BLBC genes, and differential binding of nuclear proteins to oligonucleotide sequences of transcription factor binding sites, and a secondary siRNA screen of growth in a panel of breast cancer cell lines. Further growth studies were performed using siRNA to SOX11 and measuring 2D-growth, or 3D-growth of cell lines grown in soft-agar. Immuno-fluorescent staining of phosphorylated histone H3 (pH3) was used to measure the effect of SOX11 depletion on proliferation. Migration and invasion assays were performed with membranes coated in Matrigel (invasion) or uncoated (migration). Survival analyses were performed with patients dichotomized by median SOX11 expression level, and log rank statistical comparisons as well as a multivariable Cox Proportional Hazards model to investigate the prognostic value of SOX11. Results: SOX11 depletion resulted in reduced growth of ER-negative breast cancer cell lines, including ER-negative/ Her2-positive cells; whereas, growth of ER-positive breast cancer cells or ER-negative, non-transformed breast cells was not affected. In MDA-MB-468 cells, SOX11 depletion resulted in a 60% reduction of pH3-positive cells. Additionally, depletion of SOX11 in MDA-MB-231 or MDA-MB-468 cells reduced migration and invasion 40-60%, while over-expression of SOX11 in MCF7 cells increased migration nearly 12-fold. High SOX11 is correlated with worse prognosis, whether analyzed for all patients or subsets of patients divided by ER or HER2 status. In the multivariable analysis accounting for tumor size, grade, lymph node status, and PAM50 class, high SOX11 expression was associated with increased risk of breast cancer related death with a hazard ratio of 1.42 and p-value &amp;lt; 0.001. Conclusions: SOX11 is a critical regulator of multiple BLBC phenotypes, including growth, migration, invasion, and expression of signature BLBC genes. Growth reduction of BLBC cells following SOX11 depletion is a result of decreased proliferation. High SOX11 expression was also found to be an independent prognostic indicator of poor survival in women with breast cancer. These results identify SOX11 as a potential target for the treatment of BLBC, the most aggressive form of breast cancer. Citation Format: Jonathan Shepherd, Abhijit Mazumdar, Anna Tsimelzon, Susan G. Hilsenbeck, Powel H. Brown. The SOX11 transcription factor is critical for basal-like breast cancer growth and migration and is associated with poor survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2913.