Targeting triple-negative breast cancer: apoptotic and antitumor effects of Artemisia sieberi Besser extracts

lncRNA MIR503HG inhibits cell proliferation and promotes apoptosis in TNBC cells via the miR-224-5p/HOXA9 axis

Introduction: Diagnosis of triple negative breast cancer (TNBC) is associated with adverse prognosis particularly in case of chemotherapy resistance. TNBC is a heterogeneous entity and seems to consist of at least six distinct molecular subtypes (Lehman subtypes) with distinct chemotherapy sensitivity. The cytotoxic agent eribulin induces tumor cell apoptosis through depolymerization of the cell spindle apparatus. Based on clinical data it has recently been suggested that TNBC is particularly sensitive against eribulin. The goal of this analysis was to compare (i) TNBC vs. non TNBC lines and (ii) cell lines of distinct TNBC subtypes with regard to eribulin sensitivity in vitro. Methods: 17 established breast cancer cell lines comprising both TNBC (4 basal-like 1/2; 1 mesenchymal; 3 mesenchymal stem cell; 1 interleukin; 2 luminal AR; 1 unclassified) and non-TNBC (n=5) phenotypes were cultured and subjected to cell viability assay (MTT test), migration experiment (scratch assay), apoptosis analysis (Western Blot experiment for PARP cleavage) and quantitative RT-PCR analysis (for GABRP gene expression) after exposure to eribulin or control. Furthermore, gene expression of 8 genes known to induce malignant transformation (MMP7, ELF5, YBX1, RARRES1, PRNP, SOX 10, EGFR and GABRP) was analyzed via quantitative RT-PCR analysis in the triple negative cell line MDA-MB 231 after exposure to eribulin or control. Results: The effect of eribulin on the cell viability varied to a lesser extent among the TNBC compared to the non-TNBC cell lines though we could not observe a significant difference between both groups. Mentionable the TNBC cell line DU 4475 representing the interleukin phenotype displayed a significant stronger resistance to eribulin compared to all other phenotypes. A decelerated migration could be observed in the TNBC cell line MDA-MB 231 after exposure to the IC50 concentration of eribulin compared to non-treated cells. Induction of apoptosis by eribulin treatment was verified by PARP cleavage in various TNBC cell lines. GABRP known to be overexpressed especially in basal like TNBC showed a slight increase in gene expression after exposure to eribulin in various phenotypes of TNBC - most prominent in MDA-MB 231. Additionally, upregulation of ELF5 and downregulation of YBX1 and PRNP, and, to a lesser extent, of MMP7 and SOX 10 gene expression could be investigated in MDA-MB 231 after eribulin treatment. Conclusion: We did not observe a significant association with regard to eribulin sensitivity between TNBC and non-TNBC. Chemotherapy sensitivity varied to a lesser extent among TNBC cell lines compared to non-TNBC cell lines. Eribulin inhibits cell proliferation and migration, induces apoptosis in TNBC, and influences gene expression of overexpressed genes in TNBC known to participate in and induce malignant transformation. Though the current work did not explicitly specify one phenotype of TNBC for eribulin treatment regarding chemotherapy sensitivity, we identified possible target genes influenced by eribulin treatment, e. g. GABRP, and therefore need further investigation for a potential treatment approach combining eribulin with e. g. GABRP inhibitor. Citation Format: Bräutigam K, Mitzlaff K, Uebel L, Steinert G, Köster F, Polack S, Rody A, Liedtke C. Association between phenotype of triple negative breast cancer cell lines and sensitivity against eribulin mesylate in vitro. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-08-07.

Background: Triple-negative breast cancer (TNBC) accounts for 10-15% of all breast cancer cases. A major area of innovation in TNBC is identifying potential treatment targets, especially in TNBC cells which survive chemotherapy. Our previous study showed that TNBC cells displayed deregulated kinase-dependent signaling cascades, and uniquely divergent phospho-circuits could be distinguished between TNBC vs non-TNBC cell lines [2014 SABCS abstract 1672, poster P2-05-09]. We further hypothesized that specific dysfunctional phospho-signaling network played a key role in the early adaptive changes in DNA damage response of TNBC cells exposed to DNA damaging chemotherapeutic agents. Methods: TNBC cell lines MDA-MB-231 and MDA-MB-436 were treated with 5-fluorouracil (5-Fu), carboplatin and doxorubicin at their respective half maximal inhibitory concentrations (ic50s). MiSeq gene sequencing of the untreated vs treated TNBC cells was performed to investigate whether exposure to chemotherapy agents for 3-day's duration would induce additional adaptive genetic mutation. Apoptosis and cell-cycle distribution of the untreated and treated TNBC cells were analyzed with flow cytometry. The functional phospho-signature of each TNBC cell sample was analyzed using a high throughput experimental platform that monitors the level of activity of myriad kinases at once. This technique used over 450 phospho-sensing probes, including over 150 controls in an aqueous-based assay to simultaneously and directly measure the phospho-catalytic activity of phosphorylating enzymes in cell lysates. The kinome activities of the untreated vs treated TNBC cell lines were compared respectively, and the most significantly deranged and functionally altered phospho-signaling cascades and their related kinases were identified as the early adaptive changes of the survived TNBC cells after the 3-day exposure to DNA damage chemotherapies. Results: Using the two TNBC cell lines treated with the three chemotherapies, we made 8 cell line samples, including 6 treated and 2 untreated as the control. MiSeq gene sequencing showed no significant additional adaptive genetic mutations in the treated TNBC cells after the 3-day short-term exposure to 5-Fu, carboplatin and doxorubicin. 36 phospho-signatures were generated and validated for repeatability and robustness. The kinase activity signature of each TNBC sample was analyzed and compared to each other using unsupervised hierarchical clustering. The phospho-sensing assay revealed that phospho-signaling cascades related to CHK1/2 and IKK kinases were differentially altered in the untreated vs treated TNBC cell lines, which, when respectively inhibited by AZD7762 and IKK16, successfully increased growth inhibition and cell death of TNBCs. Conclusions: We identified specific phospho-fingerprints of the early adaption of TNBC cell lines and combinatorial targeted therapies that improve treatment outcome. Our next goal is to identify specific phosphorylation cascades in a broader range of cell lines and tumor tissues, to explore the actionable, kinase-dependent mechanisms critical to the DNA damage-induced adaptive reprogramming of TNBCs and early changes driving drug-resistance. Citation Format: Pan B, Olow A, Sun Q, Mori M, Lee PRE, Hartog M, Wang C, Wolf D, Yau C, van 't Veer L, Coppé J-P. Functional detection and inhibition of the targetable oncogenic kinome of chemotherapy-treated triple-negative breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-08-03.

Introduction: Triple-negative breast cancer (TNBC) represents a highly aggressive subtype of breast cancer, accounting for approximately 15% of all cases of invasive breast cancer globally. The absence of a well-defined cell membrane receptor has significantly limited the therapeutic options available for treating this malignancy. Consequently, there is a compelling need to explore novel receptors that could potentially revolutionize the treatment strategies employed for TNBC. Recent scientific research has unveiled SLC22A17, a cell surface receptor known for its involvement in iron transport, as a substantial contributor to the development of various cancer types. Thus, the primary objective of this study is to comprehensively evaluate the significance of SLC22A17 and its potential as a therapeutic target in TNBC. Experimental Procedures: In this study, we meticulously examined the expression patterns of SLC22A17 across distinct subtypes of breast cancer utilizing immunohistochemistry. Computational tools were employed to delve into the role of SLC22A17 within TNBC datasets. To assess the impact of SLC22A17 on TNBC, we employed various methods, including siRNA-mediated silencing, to investigate its role in cell proliferation, apoptosis, epithelial-mesenchymal transition (EMT), and migration. Additionally, we conducted western blot analyses to assess the expression levels of SLC22A17 and cellular signaling molecules, such as Akt/mTOR and JAK/STAT, within the context of TNBC cells. Results: Our analysis revealed a significant upregulation of SLC22A17 in both TNBC tissues and cell lines, establishing a direct correlation with patient survival outcomes. Silencing the expression of SLC22A17 resulted in reduced cell proliferation, migration, EMT, increased apoptosis, and enhanced autophagy. These effects were accompanied by the inhibition of Akt/mTOR and STAT3 signaling pathways. Moreover, we observed the involvement of the inflammatory cytokine TNF-α, which induced the expression of SLC22A17 in TNBC cell lines and governed various hallmark features of TNBC. Notably, the suppression of SLC22A17 mitigated these processes. Conclusion: In conclusion, this investigation suggests the pivotal role played by SLC22A17 in TNBC and posits its potential as a novel and promising therapeutic target for the management of this formidable disease. Citation Format: Ajaikumar B. Kunnumakkara, Krishan Kumar Thakur. SLC22A17 as a promising target for triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 573.

Triple negative breast cancer (TNBC), an aggressive subtype of breast cancer, lacks the three major receptors for predicting outcome or targeting therapy. Hence, our aim was to evaluate the potential of estrogen receptor beta (ERβ) as a possible endocrine therapy target in TNBC. The expression and prognostic effect of ERβ isoforms were analyzed using TCGA breast tumor data, and the expression of ERβ isoform mRNA and protein in TNBC cell lines was assayed. Endogenous ERβ2 and ERβ5 were knocked down with siRNA, and ERβ2, ERβ5, and ERβ1 were upregulated using a doxycycline-inducible lentiviral system. Cell proliferation, migration and invasion, and specific gene expressions were evaluated. ERβ2 and ERβ5 were the predominant endogenous forms of ERβ in TNBC tumors and cell lines. High ERβ2 predicted worse clinical outcome. Knockdown of endogenous ERβ2/ERβ5 in cell lines suppressed proliferation, migration and invasion, and downregulated proto-oncogene survivin expression. ERβ2/ERβ5 upregulation did the reverse, increasing survivin and these cell activities. ERβ1 was barely detectable in TNBC cell lines, but its upregulation reduced survivin, increased tumor suppressor expression (E-cadherin and cystatins), and suppressed proliferation, migration and invasion in both ligand-independent and dependent manners, suggesting the possible translational benefit of ERβ ligands. ERβ2/ERβ5 and ERβ1 exhibit sharply contrasting activities in TNBC cells. Our findings imply that delineating the absolute amounts and relative ratios of the different ERβ isoforms might have prognostic and therapeutic relevance, and could enable better selection of optimal approaches for treatment of this often aggressive form of breast cancer.

Background: Breast cancer constitutes 30% of all new cancer cases in women. Despite steady decrease in breast cancer mortality, the lack of therapeutic targets is still a major problem. While the hormone-receptor positive (ER+) and human epidermal growth factor receptor 2-positive (HER2+) breast cancers respond to current targeted therapies, no targeted therapy is available for the treatment of triple negative breast cancer (TNBC), which lacks the expression of ER alpha (ER), progesterone receptor (PR), and HER2-receptor. Hence, there is an urgent need for effective targets against this sub-type of breast cancer. Previous studies in our laboratory used gene expression profiling of human breast cancers to identify kinases overexpressed in ER-negative breast cancers. One of these highly expressed kinases is the maternal embryonic leucine-zipper kinase (MELK). MELK is a serine/threonine protein kinase known to have a role in cell cycle progression, apoptosis and DNA repair. The purpose of this study was to test the hypothesis that MELK is required for the growth and migration of TNBC. Methods: RNA and protein was isolated from a panel of TNBC and ER-positive breast cancer cell lines and MELK expression was quantified by qPCR and immunoblotting. To determine whether MELK regulates cell growth, ER-positive and TNBC cell lines were transfected with siRNA targeting MELK and cell number was measured by manual counting. Anchorage-independent growth was measured using soft agar assays. Effect on migration and invasion was determined using Boyden Chamber assays. Immunostaining with actin-phalloidin was performed on MELK knockdown cells to determine effect of MELK loss on the cytoskeleton. Results: MELK mRNA and protein levels were significantly higher in TNBC cell lines compared to ER-positive breast cancers. Knockdown of MELK suppressed growth (≥50% growth inhibition) in six TNBC cell lines but had no effect on growth of six ER positive cell lines. Colony formation was also greatly reduced in TNBC cell lines but was not affected in ER positive cell lines upon siRNA knockdown of MELK. In addition, knockdown of MELK reduced migration of three TNBC cell lines (MDAMB231, MDAMB468 and Hcc70) but had no effect on the ER-positive cell line (MCF7). Decreased staining of actin filaments was observed in cell lines where migration was reduced upon MELK knockdown suggesting a role of MELK in formation of actin cytoskeleton. Current studies are focused on understanding how MELK regulates the cell growth and migration in TNBC. Conclusions: MELK is an important growth regulator of TNBC, but not of ER positive breast cancers. Our results indicate that MELK promotes cell migration in TNBC cells. These findings suggest that MELK is a promising target for the treatment of TNBC. Supported by a Susan G. Komen for the Cure Promise Grant (KG081694), and the John Charles Cain Award. Citation Format: Nidhi Batra, Corey Speers, Ivan Uray, Abhijit Mazumdar, Anna Tsimelzon, Susan Hilsenbeck, Gordon Mills, Powel Brown. Maternal embryonic leucine zipper kinase is critical for the growth and migration of triple negative breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3299. doi:10.1158/1538-7445.AM2014-3299

Background: Triple-negative breast cancer (TNBC) accounts for approximately 10 to 15% of all breast cancer cases. The management of TNBC cases will significantly improve once molecular mechanisms specific to TNBC cells will be identified and treated accordingly. Evidence suggests that TNBC cells display deregulated kinase-dependent signaling cascades that differ from non-triple-negative breast cancer cells. We hypothesized that uniquely divergent phospho-circuits could be distinguished between TNBC and non-TNBC cell lines. By revealing such unique, dysfunctional phospho-signaling network, our long-term objective is to identify kinases that underpin triple-negative breast cancer development, and can be inhibited using targeted therapy. Methods: The kinome activity of TNBC and non-TNBC cell lines was identified (HCC70, MDA-MB-231, MDA-MB-436 compared to AU565, MCF-7, T47D). The functional phospho-signature of each breast cancer cell was analyzed using a high throughput experimental platform that monitors the level of activity of myriad kinases at once. This technique uses 242 phospho-sensing probes and 78 controls in an aqueous-based assay to simultaneously and directly measure the phospho-catalytic activity of phosphorylating enzymes in cell lysates. We mapped the most significantly deranged phospho-signaling cascades and the related kinases. Results: Using 6 cell lines tested under various conditions, we generated 72 phospho-signatures, out of a total of 23,040 data points. After validating the repeatability and robustness of the assay, the kinase activity signature of each breast cancer cell line was analyzed and compared to each other using unsupervised hierarchical clustering. The phospho-sensing assay revealed the heterogeneity of kinase activity networks among breast cancer cells. These data also established that phospho-signaling cascades related to AKT, ERK, and SRC kinases were differentially altered in TNBC and non-TNBC cell lines. Conclusions: We successfully identified unique phospho-circuits of TNBC and non-TNBC cell lines. Our goal is now to test whether specific kinase inhibitors can efficiently kill or prevent the growth of TNBC cell lines in culture and animal models. We will expand our approach into a high-content, functional kinome-screening platform to characterize the phospho-fingerprint of breast cancer cells and tissues, and explore the druggable, kinase-dependent mechanisms critical to triple-negative breast tumors. Citation Format: Bo Pan, Miki Mori, Pei Rong Evelyn Lee, Marij Hartog, Qiang Sun, Laura van 't Veer, Jean-Philippe Coppe. Functional mapping of the oncogenic kinome activity of triple-negative breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-05-09.

Objective To explore the expression of tiny RNA-25 (microRNA-25, miR-25) in the plasma、tissues of triple-negative breast cancer (TNBC) patients and cell lines, to investigate the potential molecular mechanisms of miR-25 on migration and invasion of TNBC. Methods Real-time fluorescent quantitative PCR was used to detect the expression of miR-25 in the plasma of TNBC patients. Linked omics web platform was used to analyse miR-25 level in samples of TNBC and non-TNBC. Real-time fluorescent quantitative PCR was also used to detect the miR-25 level in TNBC cell lines. The wound healing and transwell assay was applied to assess the effects on migration and invasion of TNBC cell lines which transfected with miR-25 inhibitor or the negative control. The luciferase reporter assay was used to validate the relationship between miR-25 and the sphingosine-1-phosphate phosphatase 1 (SGPP1) in HEK293T cell. The wound healing and transwell assay was used to detect the migration and invasion ability of TNBC cell lines when cotransfected with pCMV6-SGPP1 and miR-25. Furthermore, Western blot was performed to detect the SGPP1 level in TNBC cell lines. Results The expression of miR-25 was significantly elevated in the plasma of 86 TNBC patients compared with the healthy controls (P value was 0.031). LinkedOmics web platform analysis showed that miR-25 expression was significantly higher in TNBC samples than in non-TNBC samples with Luminal A or Luminal B (P value was<0.001 and 0.006). The level of miR-25 was also elevated in TNBC cell lines HS578T, HCC1806, MDA-MB-231 and BT549(P value was 0.006, 0.01, 0.029 and 0.046). The MDA-MB-231 and HS578T cells which transfected with miR-25 inhibitor exhibited a significant slower wound healing rate than control (P value was 0.035 and 0.001). At the same time, when transfected with miR-25 inhibitor, MDA-MB-231 and HS578T both exhibited a decreased invasion ability compared with the control group(P value was 0.002 and 0.001). LinkedOmics web platform analysis showed that sphingosine-1-phosphate phosphatase 1 (SGPP1) gene level was negatively correlated with miR-25 in the tissues of TNBC patients (P value was 0.037). The luciferase reporter assay validated that SGPP1 was a directed target of miR-25. The western blot assay indicated that the SGPP1 level was increased in MDA-MB-231 and HS578T after transfection with miR-25 inhibitor. Over-expression of SGPP1 could abrogate the positive effects of miR-25 on migration and invasion when pCMV6-SGPP1 was cotransfected with miR-25 (P value was all 0.002). Conclusions MiR-25 was elevated in both plasma and tissues of TNBC patients and also increased in TNBC cell lines. Transfection of MDA-MB-231 and HS578T cells with miR-25 inhibitor resulted in reduced migration and invasion. Moreover, SGPP1 was identified as a novel target of miR-25. The ability of miR-25 to promote TNBC cell migration and invasion is attributable to its effect on SGPP1 suppression. Key words: Triple negative breast neoplasms; microRNAs; Membrane proteins; Phosphoric monoester hydrolases; Neoplasm metastasis

Triple-negative breast cancer (TNBC) has a poorer outcome compared with that of other subtypes of breast cancer, and the discovery of dysregulated microRNA (miRNA) and their role in tumor progression has provided a new avenue for elucidating the mechanism involved in TNBC. Previous studies have revealed that aberrant expression of miRNA-589 (miR-589) was frequently observed in various types of cancer. However, the expression and function of miR-589 in TNBC has not been well illustrated. In the present study, the expression level of miR-589 was explored in TNBC tissues and TNBC cell lines by quantitative polymerase chain reaction (qPCR). The results revealed that the expression of miR-589 was decreased in TNBC tissues and cell lines compared with that in normal tissues and breast cell lines. Furthermore, miR-589 overexpression decreased the TNBC cell proliferation, migration and invasion, whereas miR-589 silencing generated the opposite results in vitro. Bioinformatic algorithms predicted a direct target site for miR-589 in the 3′-untranslated region of metastasis-associated protein 2 (MTA2), which was confirmed with a dual-luciferase reporter assay and western blot analysis. Results of the qPCR and western blot analysis illustrated that miR-589 negatively regulated MTA2 expression with regard to mRNA and protein levels in the TNBC cell lines. MTA2 silencing reversed the promotion function of miR-589 inhibitor in the TNBC cell line. Finally, miR-589 could inhibit the process of epithelial-mesenchymal transition via MTA2. In summary, the present study revealed the biological function and molecular mechanism of miR-589 in the progression of TNBC. MiR-589 inhibition in the progression of TNBC may be a potential therapeutic target for TNBC.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with limited treatment options. The nuclear export protein XPO1 has emerged as a potential therapeutic target in cancer, but its role in TNBC hasn’t been fully characterized. OBJECTIVES: This study screens, nominates, and investigates the potential of repurposing selinexor, an FDA-approved XPO1 inhibitor, as a novel therapeutic option for TNBC. METHODS: We utilized a computational drug repurposing pipeline to predict breast cancer patient tumor responses to hundreds of drugs. We then stratified patient tumor’s predicted sensitivities by breast cancer subtype and focused on drugs preferentially sensitive towards TNBC. We identified XPO1 inhibitors as the top candidate drug and its efficacy validated in an independent patient dataset and across various TNBC cell line. Additionally, we conducted RNA-sequencing after longitudinal XPO1 inhibition in a panel of TNBC cell lines to explore the mechanism contributing to the preferential sensitivity of TNBC cells to XPO1-inhibition mediated cell death. Further mechanistic studies were conducted to validate our RNA-sequencing findings. RESULTS: Selinexor was found to significantly reduce the viability of a variety of TNBC cell lines. Mechanistically, we reveal that selinexor induces TNBC cell death by inhibiting the NF-kB pathway through nuclear retention of NFKBIA, a key regulator of this pathway. This effect was consistent across multiple TNBC cell lines of differing TNBC subtypes, confirming the broad applicability of XPO1 inhibition in TNBC treatment. CONCLUSION: This study identifies XPO1 inhibitors as a promising therapeutic candidate for TNBC patients by inhibiting the XPO1-mediated export of NFKBIA and blocking the NF-kB pathway. These findings pave the way for more personalized treatment strategies and provide a potential alternative to conventional chemotherapy. IMPLICATIONS FOR PRACTICE: Repurposing selinexor for the treatment of TNBC could accelerate the availability of targeted therapies for patients with this aggressive cancer. New mechanistic insight into the causes leading to TNBC cell specific sensitivity to XPO1-inhibition mediated cell death warrant further clinical trials to evaluate the safety and efficacy of selinexor in TNBC patients. Citation Format: Amy L. Paulson, Robert F. Gruener, Adam M. Lee, R. Stephanie Huang. Discovery of XPO1 as a therapeutic target in TNBC: Mechanistic insights and preclinical validation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5032.

This study aimed to analyze the specific expression of hsa_circ_0007823 in triple-negative breast cancer (TNBC) and explore the roles and related molecular mechanisms of hsa_circ_0007823 in TNBC. Relative hsa_circ_0007823 levels in TNBC tissues and cell lines were examined by reverse transcription-quantitative polymerase chain reaction. The value of hsa_circ_0007823 levels was evaluated in patients' clinicopathological characteristics and prognostic prediction. A dual-luciferase reporter assay was used to determine the relationship between hsa_circ_0007823, miR-182-5p, and FOXO1. The effect of circ_0007823 overexpression on the growth of TNBC cells was investigated in vitro and in vivo. Lower levels of hsa_circ_0007823 were found in TNBC tissues and cell lines and were closely associated with lymph node metastasis, poorer overall and disease-free survival rates. MiR-182-5p was significantly up-regulated, whereas FOXO1 was down-regulated in TNBC cell lines. The miR-182-5p inhibition up-regulated FOXO1 in TNBC cells. Dual-luciferase reporter assays showed that hsa_circ_0007823, miR-182-5p, and FOXO1 interacted with each other. Overexpression of circ_0007823 significantly inhibited the viability, migration, and invasion of TNBC cell lines, but promoted apoptosis. In vivo experiments showed that circ_0007823 overexpression inhibited tumor growth and down-regulated miR-182-5p and up-regulated FOXO1. Hsa_circ_0007823 overexpression could suppress the growth, invasion, and migration of TNBC cells, and inhibit tumor growth by regulating miR-182-5p/FOXO1.

Recent studies found that TNBC had a highly activated profile in the insulin-like growth factor 1 receptor (IGF-1R)/insulin receptor (InsR) pathway. Patients in whom the IGF-1R/InsR pathway was activated had a worse prognosis than did those in whom the pathway was not activated. We also previously found that the tumor-initiating cells in mouse TNBCs had a highly activated IGF-1R pathway. On the basis of these findings, we tested an IGF-1R/InsR dual kinase inhibitor, KW-2450, in TNBC. Results: To investigate the antitumor effects of KW-2450 in TNBC, we first confirmed the high IGF-1R and low InsR expression in TNBC cell lines (e.g., SUM149, MDA-MB-231, MDA-MB-468). An in vitro growth inhibition assay revealed that KW-2450 inhibited cell growth (all IC50 &amp;lt;0.5 μM), and an agar assay for colony formation confirmed this antitumor effect in TNBC cells. We also tested KW-2450's inhibitory effect against cancer stem cell (CSC) activity. With KW-2450 treatment of SUM149 and MDA-MB-231 cells, proportions of CSCs, profiled as CD44+CD24−, were significantly reduced in a dose-dependent manner. Indeed, KW-2450 dose-dependently inhibited mammosphere-forming, a hallmark of CSC activity. Cell cycle analysis revealed that KW-2450 induced mitotic accumulation and apoptosis in TNBC cells. Interestingly, MDA-MB-468 cells were the most susceptible to death, and their sensitivity to KW-2450 was associated with the high activation level of the mitotic checkpoint, the levels of which were determined by accumulation of cyclin B1 (on Western blot) and of phospho-histone H3–positive cells, a mitosis marker (on FACS). In contrast, SUM149 and MDA-MB-231 cells, which are relatively unsusceptible to death from KW-2450, exited from mitosis (as indicated by an accumulation of 8N; octaploidy) without significant cell death. This variable sensitivity to KW-2450 was also observed in in vivo studies. We confirmed that MDA-MB-468 tumors that were treated with 80 mg/kg of KW-2450 in vivo were stable and had many more mitotic cells than did those treated with vehicle control, which suggests that mitotic accumulation is a key process for this antitumor effect. Since it is known that any of the MAPKs (e.g., JNKs, p38 kinase, ERKs) become activated at the mitotic phase in mammalian cells, we next investigated whether the activation levels of MAPKs play critical roles in either mitotic progression or mitotic death in TNBC. Western blot analysis revealed that KW-2450 activated ERKs, but not JNKs or p38 kinase, in KW-2450–insensitive MDA-MB-231 and SUM149 cells, suggesting that ERK activation may promote mitotic progression but not mitotic death. Indeed, the combination of a MEK inhibitor (AZD6244), which inhibits ERK activation, and KW-2450 significantly reduced the 8N fraction and increased cell death in SUM149 and MDA-MB-231 cells. Conclusion: KW-2450 had significant antitumor effects in vitro and in vivo in TNBC cells. KW-2450–induced cell death, accompanied by mitotic accumulation, depended on mitotic checkpoint activity. TNBC cells refractory to KW-2450 were sensitized to KW-2450 by the addition of a MEK inhibitor. This novel combination therapy targeting IGF-1R/InsR and MEK in TNBCs, whose mitotic checkpoints are commonly abrogated, needs to be developed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-04.

Statin induces inhibition of triple negative breast cancer (TNBC) cells via PI3K pathway

Background: Breast cancer is the most diagnosed female cancer and the second leading cause of cancer death in the United States. Triple negative breast cancer (TNBC), a special subtype, defined as breast cancer lacking estrogen, progesterone and HER-2 receptors, showed clinically aggressive features and was associated with poor prognosis. TNBC is resistant to endocrine or HER-2 targeted therapies, and only conventional chemotherapeutic regimens were accepted as the treatment guidelines. Therefore, searching for novel pharmaceutical agents for TNBC is urgent and a hot spot in present clinical research. Teriflunomide, an orally available immunomodulatory drug, approved for treatment of multiple sclerosis (MS) by FDA, has demonstrated the potential application in cancer therapy, such as chronic lymphocytic leukemia (CLL), prostate cancer and melanoma. Therefore, we assessed the therapeutic value of teriflunomide in TNBC cells. Methodology/Principal Findings: In this study, we showed that teriflunomide treatment resulted in a dose- and time-dependent inhibition of proliferation in three TNBC cell lines: MDA-MB-231, MDA-MB-468 and BT549. Meanwhile, the agent could also induce loss of clonogenic survival in dose-dependent fashion in TNBC cells. The analysis of cell cycle distribution by flow cytometry revealed that teriflunomide for 48 h entrapped TNBC cells in S-phase with concomitant reduction in both G1- and G2/M-phase. Furthermore, by Annexin-V/PI staining, we showed high doses of teriflunomide for 2 days led to significant necrosis and minor apoptosis in TNBC cells. Additionally, the effect of teriflunomide on TNBC cell migration and invasion was also tested using Boyden chamber assays. Short-term treatment of teriflunomide decreased the cell motility and invasiveness considerably in a concentration-dependent manner. When evaluated for underlying mechanisms, teriflunomide was found to modulate multiple cell signaling pathways in three TNBC cell lines. First, teriflunomide inhibited expression of proteins linked to cell proliferation, such as cyclin D1 and c-Myc. Second, teriflumomide delayed cell cycle transition by up-regulating cyclin A, along with p27 down-regulation and unchanged cyclin B1. Third, teriflunomide regulated the cell survival proteins, such as up-regulation of BAX and down-regulation of Bcl-Xl, by activation of MAPK pathway. Fourth, teriflunomide suppressed the marker signals involved in epithelial–mesenchymal transition(EMT) and invasion and inhibited activation of FAK/Src complex. Fifth, teriflunomide down-regulated growth factor receptors involved in TNBC growth maintenance, such as EGFR, IGF1R and FGFR4. Conclusion/Significance: Teriflunomide, although an anti-inflammatory agent, is a potent inhibitor of TNBC cells through modulation of multiple signaling pathways and may be of therapeutic benefit for TNBC in clinical practice. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-21.

Emerging evidence has suggested that long noncoding RNAs (lncRNA) involved in the development and progression of cancer. Triple negative breast cancer (TNBC) was an aggressive type of breast cancer with high rates of cancer recurrence and metastasis. The pathogenesis of TNBC is largely unknown. Recent studies suggested that lncRNA HCP5 plays an important role in carcinogenesis. The purpose of this study was to examine the function and mechanism of HCP5 in TNBC. We observed that HCP5 was upregulated in TNBC cell lines and specimens. HCP5 knockdown induced TNBC cell apoptosis, and inhibited cell proliferation and orthotopic xenograft tumor growth. RNA sequencing and antibody array suggested that HCP5 achieves its functions through regulating apoptosis pathway. Bioinformatics, luciferase and RIP experiments proved that both HCP5 and BIRC3 could competitively bind to miR‐219a‐5p. Increased BIRC3 and decreased miR‐219a‐5p were observed in TNBC tissues and cell lines. We then performed gain‐ and loss‐of‐function studies as well as rescue experiments in TNBC cells. The decrease of proliferation and migration due to HCP5 knockdown could be rescued when miR‐219a‐5p inhibitor or BIRC3 was transfected and vice versa. Our study suggested that lncRNA HCP5 promotes TNBC progression as a ceRNA to regulate BIRC3 by sponging miR‐219a‐5p. In a word, we revealed a new signaling pathway to mediate TNBC, and provided HCP5 as a new target for improving treatment of TNBC.