Apoptosis In Triple-negative Breast Cancer Research Articles

Abstract 2103: Small extracellular vesicles derived from macrophages show anti-tumor effects in MDA-MB-231 cells

Abstract Triple-negative breast cancer is the primary cause of death in women globally due to the absence of targeted receptors on the tumor, making it hard to treat. In addition, the rapid spread of triple-negative breast cancer makes it difficult to manage using current treatment options such as lumpectomies, mastectomies, radiation, and chemotherapy. The survival rate for patients with triple-negative breast cancer is only 8-16%. Therefore, there is a critical need to develop new and effective treatment options. The objective of this study was to investigate the potential of M1 macrophage-derived exosomes to induce cancer cell apoptosis. To test this, we isolated M1 macrophage-derived exosomes to see their effects on triple-negative breast cancer MDA-MB-231 cells. We induce polarization in RAW 264.7 macrophage cells via lipopolysaccharide (LPS). Macrophage polarization was characterized using an enzyme-linked immunosorbent assay (ELISA), real-time PCR, nitric oxide production, and bright field microscopy. We characterized exosomes using nanoparticle tracking analysis (NTA) and scanning electron microscopy (SEM). The apoptosis marker, caspase 3/7 activation, was assessed using confocal microscopy in MDA-MB-231 cells. M1 macrophage-derived exosomes were isolated, characterized, and incubated with breast cancer cells to see their anticancer effects. Data showed the induction of apoptosis in 48 hours in triple-negative breast cancer cells. We conclude that exosomes derived from M1 macrophages may have the capability to induce apoptosis in triple-negative breast cancer. Citation Format: Parth Desai, Anjali Kumari, Saqer Al Abdullah, Kristen Dellinger. Small extracellular vesicles derived from macrophages show anti-tumor effects in MDA-MB-231 cells [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 2103.

Abstract 4556: USP51-GRP78-ABCB1 axis promotes chemoresistance of triple negative breast cancer by decreasing the accumulation of doxorubicin in cells

Abstract Background: Resistance to chemotherapy remains an obstacle for triple-negative breast cancer (TNBC) patients. The core components of the ubiquitin-proteasome system have been demonstrated to regulate chemoresistance of TNBC. As a novel deubiquitinase (DUB), ubiquitin-specific peptidase 51 (USP51) plays a pivotal role in chemotherapeutic resistance in multiple malignancies. Herein, we sought to better understand how USP51 performs a cell-intrinsic role in mediating chemotherapeutic resistance in TNBC. Methods: Western blotting, RNA-sequencing and CCK8 assays were used to identify the DUBs for chemoresistance of breast cancer. Coimmunoprecipitation, GST-pulldown, protein deubiquitination and immunohistochemistry assays were then used to determine the biological phenotypes of ectopic USP51 in TNBC chemoresistance and associated signaling pathways in several different cell lines, mouse models and patient tissue samples. Ubiquitin-7-amido-4-methylcoumarin (Ub-AMC)-based deubiquitinase activity, cellular thermal shift and surface plasmon resonance (SPR) analyses were performed to investigate the activity of USP51 inhibitors. Results: To identify the critical DUBs involved in breast cancer chemoresistance, we established doxorubicin-resistant Cal51 and MDA-MB-231 TNBC cell lines by multiple dose exposure. We found ectopic USP51 in doxorubicin-resistant cells compared with their parental cells. Moreover, USP51-interfered chemoresistant tumor cells exhibited impaired cell viability as well as increased DNA damage and cell apoptosis upon treatment with doxorubicin. On the contrary, overexpression of USP51 performed the opposite effects to enhance cell viability but decrease DNA damage and apoptosis in response to doxorubicin; however, these outcomes were not shown for its catalytically inactive mutant USP51C372S. At the molecular level, GRP78 was identified as a bona fide substrate of USP51. Importantly, knockdown USP51 increased doxorubicin-induced DNA damage and apoptosis, which was rescued by overexpression of GRP78 in vitro and in vivo. In addition, the activity of ABCB1, the main efflux pump of doxorubicin, was enhanced by GRP78. Consistently, the expression of USP51, GRP78 and ABCB1 were correlated with chemoresistant phenotypes in TNBC patients. Of note, we also explored a small molecular inhibitor of USP51, WCY-4-1, which conferred chemosensitization in TNBC. Conclusion: These findings collectively indicated that ectopic USP51 enhances the activity of ABCB1 by deubiquitinating and stabilizing GRP78, which leads to decreased accumulation of doxorubicin as well as decreased DNA damage and cell apoptosis in TNBC. Our results also revealed that specific inhibition of USP51 significantly impairs doxorubicin-resistance in TNBC. Citation Format: Yang Ou, Kun Zhang, Qiuying Shuai, Chenyang Wang, Huayu Hu, Lixia Cao, Chunchun Qi, Min Guo, Zhaoxian Li, Jie Shi, Yuxin Liu, Siyu Zuo, Xiao Chen, Yanjing Wang, Mengdan Feng, Hang Wang, Yi Shi, Guang Yang, Shuang Yang. USP51-GRP78-ABCB1 axis promotes chemoresistance of triple negative breast cancer by decreasing the accumulation of doxorubicin in cells [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 4556.

Implication of IZUMO2 in the cell-in-cell phenomenon: A potential therapeutic target for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is characterized by the loss of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. The aggressive clinicopathological features and resistance to currently available therapeutics of the disease warrant an urgent need for the development of novel alternate therapeutic options. We have previously reported adiponectin-expressing regulatory T cells (A-Tregs), which can induce apoptosis in TNBC through the cell-in-cell phenomenon. In this study, we aimed to elucidate the molecule that allows TNBC cells to engulf A-Tregs. A monoclonal antibody, which repressed the engulfment of A-Tregs by TNBC cells, was developed. Immunoprecipitation followed by mass spectrometry and small interfering RNAs-mediated gene silencing was performed to characterize the antigen. We successfully generated a monoclonal antibody, designated G1D7, which abrogated the engulfment of A-Tregs by TNBC and subsequent A-Treg-mediated apoptosis. G1D7 detected the immunoglobulin-like type I membrane protein IZUMO2, a molecule related to IZUMO1 that is essential for cell-cell membrane binding and fusion of sperm to oocyte. The findings highlight the importance of IZUMO2 on TNBC cells in facilitating the cell-in-cell phenomenon by A-Tregs.

Abstract LB_C06: Utilizing a novel HDAC inhibitor bocodepsin (OKI-179) to overcome doxorubicin resistance in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic treatment options and a high rate of metastatic recurrence. TNBC is defined by lack of estrogen and progesterone receptor expression with non-amplified HER2, and accounts for 10-15% of all breast cancer cases. While pembrolizumab, sacituzumab govitecan and PARP inhibitors are beneficial for a subset of TNBC patients, chemotherapy including doxorubicin continues to be the primary treatment. Bocodepsin is a novel, orally bioavailable clinical stage Class 1-targeting HDAC inhibitor with activity in preclinical models of TNBC. The purpose of this study was to investigate OKI-005 (in vitro) and bocodepsin (in vivo) in combination with doxorubicin (DOX) to overcome resistance. Procedures: TNBC cell lines were exposed to a no drug, DOX, OKI-005 or combination for 72 hours and cell viability was assessed via CellTiter-Glo. Synergy scores were calculated from three independent replicates using Synergy Finder+. Apoptosis was assessed following 48 hours of drug exposure by flow cytometry. Immunoblots were performed following 24 hours of drug treatment to evaluate mechanism of action. In vitro senescence-associated beta-galactosidase was performed after 6 days drug treatment. For in vivo studies, 5 million MDA-MB-231 cells were injected subcutaneously in athymic nude mice. Mice were treated with either no drug, 1.5 mg/kg DOX IP QW, 80 mg/kg bocodepsin PO QD, or the combination. Results: Bliss scores calculated from Synergy Finder+ using the CellTiter-Glo data showed synergy for the combination of OKI-005 and DOX in all TNBC cell lines. The combination resulted in statistically significant increased apoptosis measured by Annexin V compared to single agent drug treatment in CAL-51, MDA-MB-231 and CAL-120 cell lines. Immunoblot analysis demonstrated an increase in the pro-apoptotic protein BIM and decrease in anti-apoptotic protein BCL-XL with the combination. The cyclin dependent kinase inhibitor p21 was increased by OKI-005 independently of p53 and doxorubicin for the cell lines MDA-MB-231, Hs 578T, and CAL-120. The combination resulted in increased cleaved caspase-3 for all cell lines evaluated. Senescence-associated beta-galactosidase showed an increase in senescent cells for both DOX and OKI-005, which was eradicated in combination. DOX and bocodepsin were further tested in vivo, where significant tumor growth inhibition was observed in combination compared to DOX (p=0.0016) and bocodepsin (p=0.0068). Conclusion: The addition of OKI-005 or bocodepsin to doxorubicin resulted in improved anti-proliferative and anti-cancer activity with evidence of increased apoptosis and decreased senescence. As a result, bocodepsin is a promising combination partner for doxorubicin to overcome doxorubicin resistance and promote apoptosis in TNBC. Bocodepsin has a favorable toxicity profile compared to other HDAC inhibitors and this work supports the potential clinical investigation of doxorubicin and bocodepsin in patients with metastatic TNBC. Citation Format: Stephen G Smoots, Anna R Schreiber, Marilyn M Jackson, Evan D Dus, Stacey M Bagby, Adrian T A. Dominguez, Cameron A Binns, Jennifer R Diamond, Todd M Pitts. Utilizing a novel HDAC inhibitor bocodepsin (OKI-179) to overcome doxorubicin resistance in triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C06.

Abstract LB_C07: Utilizing a BCL-2 inhibitor (venetoclax) to overcome doxorubicin resistance in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of the progesterone receptor, estrogen receptor, and human epidermal growth factor-2 over-expression. TNBC comprises 10-15% of all breast cancer cases and compared to other subtypes of breast cancer, TNBC is associated with a higher risk for metastatic recurrence and the chemotherapy agent doxorubicin (dox) remains a mainstay of treatment. Senescence is a cellular phenomenon where cells are committed to an arrested state, however, senescent cells can secrete pro-tumorigenic factors which can help to promote tumor progression and invasion. Senescence is one potential mechanism of resistance to doxorubicin in breast cancer. Avoiding senescence-mediated resistance and promoting apoptosis is one strategy to improve response to dox in TNBC. Venetoclax is an orally bioavailable FDA approved BCL-2 inhibitor with activity in preclinical models of TNBC. The purpose of this study was to investigate venetoclax in combination with dox to overcome senescence-mediated resistance and induce apoptosis. Experimental Procedures: TNBC cell lines were exposed to a no drug, dox, venetoclax or combination at increasing doses for 72 hours and cell viability was assessed via CellTiter-Glo. Synergy scores were calculated from three independent replicates using Synergy Finder+. Apoptosis at 48 hours was analyzed by flow cytometry using Annexin V on cells treated in combination and with single agents. In vitro cell survival following drug treatment for 24 hours was assessed via clonogenic assay. In vitro senescence-associated β-gal was performed after 6 days drug treatment. Athymic nude mice were subcutaneously injected with 5 million MDA-MB-231 cells for in vivo studies. Mice were treated with either no drug, 60 mg/kg venetoclax PO QD, 1.5 mg/kg dox IP QW, or the combination. Results: The combination of venetoclax and dox resulted in synergy based on CellTiter-Glo data, which was used to calculate Bliss scores using Synergy Finder+ in CAL-51, MDA-MB-231, and CAL-120 TNBC cell lines. The dox and venetoclax combination caused a statistically significant increase in apoptosis measured by Annexin V staining when compared to single agents and control in all cell lines. The combination resulted in a decrease in cellular survival in all cell lines tested as measured by the clonogenic assay. Senescence-associated beta-galactosidase showed an increase in senescent cells for both dox and venetoclax treated cells, which was eradicated in combination. Dox and venetoclax were further tested in vivo, where significant tumor growth inhibition was observed in combination compared to doxorubicin (p = 0.0031) and venetoclax alone (p = 0.0038) after 48 days of treatment. Conclusion: The addition of venetoclax to doxorubicin resulted in decreased cellular proliferation, increased apoptosis, and decreased senescence. As a result, venetoclax is a promising combination partner for doxorubicin to overcome doxorubicin resistance and promote apoptosis in TNBC. Citation Format: Evan D Dus, Stephen G Smoots, Anna R Schreiber, Marilyn M Jackson, Stacey M Bagby, Adrian T A Dominguez, Cameron A Binns, Todd M Pitts, Jennifer R Diamond. Utilizing a BCL-2 inhibitor (venetoclax) to overcome doxorubicin resistance in triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C07.

Long non‑coding RNA PTCSC3 suppresses triple‑negative breast cancer by downregulating long non‑coding RNA MIR100HG.

Long non-coding RNA (lncRNA) PTCSC3 is characterized as a tumor suppressor in thyroid cancer and glioma. The present study aimed to investigate the role of PTCSC3 in triple-negative breast cancer (TNBC). A total of 82 patients with TNBC were enrolled in the present study. The results showed that PTCSC3 was downregulated, while lncRNA MIR100HG was upregulated in tumor tissues compared with that in adjacent non-cancerous tissues of patients with TNBC. The follow-up study showed that low expression levels of PTCSC3 and high expression levels of MIR100HG were closely associated with poor survival of patients with TNBC. The expression levels of MIR100HG were decreased with the clinic stages of TNBC, while the expression levels of MIR100HG showed the opposite trend. Correlation analysis showed that the expression levels of PTCSC3 and MIR100HG were significantly correlated in both tumor tissues and adjacent non-cancerous tissues. The overexpression of PTCSC3 inhibited the expression level of MIR100HG in TNBC cells, while the expression level of PTCSC3 was unaffected. Cell Counting Kit-8 and Annexin V-FITC Apoptosis flow cytometry assays showed that overexpression of PTCSC3 led to inhibition, while overexpression of MIR100HG led to the promotion of TNBC cells viability and inhibited apoptosis of TNBC cells. In addition, overexpression of MIR100HG attenuated the effects of PTCSC3 overexpression on cancer cell viability. However, the overexpression of PTCSC3 did not affect cancer cell migration and invasion. Western-blot analysis showed that PTCSC3 suppressed viability and promoted apoptosis of TNBC cells through the Hippo signaling pathway. Thus, the present study demonstrated that lncRNA PTCSC3 inhibits cancer cell viability and promotes cancer cell apoptosis in TNBC by downregulating MIR100HG.

Novel delivery of sorafenib by natural killer cell-derived exosomes-enhanced apoptosis in triple-negative breast cancer.

Aim: We investigated the delivery of sorafenib (SFB) to breast cancer spheroids by natural killer cell-derived exosomes (NK-Exos). Methods: SFB-NK-Exos were constructed by electroporation. Their antitumor effects were evaluated by methyl thiazolyl tetrazolium, acridine orange/ethidium bromide, 4',6-diamidino-2-phenylindole, annexin/propidium iodide, scratch and migration assay, colony formation, RT-PCR, western blot and lipophagy tests. Result: The loading efficacy was 46.66%. SFB-NK-Exos-treated spheroids showed higher cytotoxic effects (33%) and apoptotic population (44.9%). Despite the reduction of SFB concentration in the SFB-NK-Exos formulation, similar cytotoxic effects to thoseof free SFB were observed. Increased intracellular trafficking, sustained release of the drug and selective inhibitory effects demonstrated efficient navigation. Conclusion: This is the first report for SFB loading into NK-Exos, which led to significant cytotoxic intensification against cancer cells.

Small-molecule inhibitor of Fam20C in combination with paclitaxel suppresses tumor growth by LIF-JAK2/STAT3-modulated apoptosis in triple-negative breast cancer

BackgroundTriple negative breast cancer (TNBC) is a strongly proliferative, relapse prone, high-mortality subtype of breast cancer. Due to the lack of effective therapeutic targets, the current clinical treatment is still dominated by chemotherapeutic drugs, such as paclitaxel (PTX). Moreover, Fam20C kinase inhibitor (3R) has recently been reported to inhibit TNBC growth. Thus, the objectives of this study were to verify whether the combination therapy with 3R could enhance the efficacy of PTX treatment on TNBC, and to further elucidate their potential synergistic mechanisms. MethodsThe inhibitory effect on TNBC cell viability was verified by the MTT assay, and the effect of drug combination on apoptosis was analyzed by flow cytometry. The mechanism of action of the drug combination was validated based on transcriptome analyses and western blot (WB) analysis. Xenograft mouse models were used to verify the therapeutic efficacy of PTX+3R in vivo by immunohistochemistry and WB analysis. Significant findingsFam20C inhibitor (3R) enhanced the inhibitory effect of PTX on the viability and proliferation of MDA-MB-231 and BT-549 cells, and their combination significantly promoted cell apoptosis. In addition, we found that 3R reduced the expression of leukemia inhibitor factor (LIF). 3R and PTX limit the phosphorylation activation of signal transducer and activator of translation 3 (STAT3). Finally, immunohistochemical staining showed that combined treatment with 3R and PTX inhibited tumor growth in an ectopic tumor model of MDA-MB-231 cells in nude mice.

Methanol extract of Pergularia daemia (Forssk.) Chiov. leaves induce apoptosis in triple-negative breast cancer through intrinsic pathway

Most traditional anticancer medications are ineffective in treating triple negative breast cancer (TNBC), which has enhanced resistance to chemotherapeutic agents. Herbs continue to be a key element in the discovery of anticancer medications. In the present study, we assessed the cytotoxicity of methanol extract of leaves of Pergularia daemia (MLPD) in MDA-MB-231 triple-negative breast cancer (TNBC) cell line. The cytotoxicity of MLPD was assessed using MTT assay which revealed significant concentration-dependent cytotoxicity with an IC50 value of 35.95±3.57 µg/mL. Morphological evaluation of MDA-MB-231 cells treated with MLPD showed cytotoxic changes like vacuole formation, altered morphology, and reduction in the number of cells with apoptotic bodies formation. Staining techniques, such as acridine orange and ethidium bromide (AO/EB) showed early apoptosis characterized by yellow-green fluorescence and crescent-shaped nucleus. Hoechst 33258 staining of MDA-MB-231 cells treated with MLPD showed nuclear marginalization indicative of nuclear apoptotic changes. To assess the mitochondrial membrane potential (MMP), we did JC-1 staining and the results revealed green fluorescence indicative of mitochondrial dependent apoptotic pathway. Comet assay confirmed significant DNA damaging property in MDA-MB-231 cells treated with MLPD. Western blotting analysis showed significant downregulation of Bcl-2 by 0.52±0.03 folds with no significant change in caspase-8 expression. Hence, the present study demonstrated that MLPD possessed potent cytotoxic effect against MDA-MB-231 TNBC cells through mitochondria mediated intrinsic pathway of apoptosis.

SCN5A promotes the growth and lung metastasis of triple-negative breast cancer through EF2-kinase signaling

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer often associated with early metastasis, poor prognosis, and shortened survival. The main causes of its high mortality level are attributed to significant genetic heterogenity, rapid development of chemoresistance, and a limited number of targeted therapeutics. Therefore, as described herein, we investigated the role of the SCN5A gene encoding the voltage-gated sodium ion channel NaV1.5 in vitro and in vivo in TNBC models. Cell proliferation, colony formation, invasion, western blot and in vivo analyzes were performed. We found that SCN5A gene/NaV1.5 and its neonatal spliced isoform nNaV1.5 were markedly upregulated in TNBC cells and that they promoted cell proliferation, colony formation, motility/migration, and invasion. Therapeutic targeting of NaV1.5/nNaV1.5 via systemic injection of specific siRNAs incorporated in single-lipid nanoparticles suppressed tumor growth and lung metastasis in orthotopic TNBC models. We further demonstrated that targeting of NaV1.5/nNaV1.5 expression associated with eukaryotic elongation factor-2 kinase (EF2K) expression. This approach led to the inhibition EF2K's oncogenic signaling and its clinically significant downstream targets, including integrin β1/Src/focal adhesion kinase, c-myc, cyclin D1, phosphoinositide 3-kinase/Akt, p-ERK, p-IF2α and p-PI3K. Targeting of NaV1.5/nNaV1.5 also induced robust apoptosis in TNBCs in vivo. Inhibition of both isoforms of SCN5A in TNBC cells enhanced the effect of first-line chemotherapeutics such as paclitaxel. In conclusion, our results provide the first evidence that expression of SCN5A has an important role in the growth of primary and lung-metastatic TNBCs and that targeting of NaV1.5/nNaV1.5 by systemically administered therapeutics is a potential molecularly targeted strategy to control TNBC progression and may enhance the efficacy of currently used chemotherapeutics.

BPTF promotes the progression of distinct subtypes of breast cancer and is a therapeutic target.

To assess the biomarker and functional role of the chromatin remodeling factor, bromodomain PHD finger transcription factor (BPTF), in breast cancer progression. BPTF copy number was assessed using fluorescence in situ hybridization. BPTF expression was regulated in breast cancer cells by shRNA/siRNA-mediated gene silencing and BPTF cDNA overexpression. The effects of regulating BPTF expression were examined on key oncogenic signaling pathways and on breast cancer cell proliferation, apoptosis, and cell cycle progression, as well as in xenograft models. The consequences of pharmacological bromodomain inhibition, alone or in combination with other targeted agents, on breast cancer progression were assessed in culture and in xenograft models. BPTF copy number was gained in 34.1% and separately amplified in 8.2% of a breast cancer tissue cohort. Elevated BPTF copy number was significantly associated with increasing patient age and tumor grade and observed in both ER-positive and triple-negative breast cancer (TNBC) subtypes. BPTF copy number gain and amplification were also observed in The Cancer Genome Atlas (TCGA) breast cancer cohort. Stable shRNA-mediated silencing of BPTF significantly inhibited cell proliferation and induced apoptosis in TNBC and ER-positive human breast cancer cell lines. BPTF knockdown suppressed signaling through the phosphoinositide 3 kinase (PI3K) pathway, including reduced expression of phosphorylated AKT (Ser473), phosphorylated GSK-β (Ser9), and CCND1. These findings were confirmed following transient BPTF knockdown by a distinct siRNA in TNBC and ER-positive breast cancer cells. Stable suppression of BPTF expression significantly inhibited the in vivo growth of TNBC cells. Conversely, BPTF cDNA overexpression in TNBC and ER-positive breast cancer cells enhanced breast cancer cell proliferation and reduced apoptosis. BPTF targeting with the bromodomain inhibitor bromosporine, alone or in combination with the PI3K pathway inhibitor gedatolisib, produced significant anti-tumor effects against TNBC cells in vitro and in vivo. These studies demonstrate BPTF activation in distinct breast cancer subtypes, identify pathways by which BPTF promotes breast cancer progression, and suggest BPTF as a rational target for breast cancer therapy.

DTMP imbalance through thymidylate 5′-phosphohydrolase activity induces apoptosis in triple-negative breast cancers

Immunotherapy has a number of advantages over traditional anti-tumor therapy but can cause severe adverse reactions due to an overactive immune system. In contrast, a novel metabolic treatment approach can induce metabolic vulnerability through multiple cancer cell targets. Here, we show a therapeutic effect by inducing nucleotide imbalance and apoptosis in triple negative breast cancer cells (TNBC), by treating with cytosolic thymidylate 5'-phosphohydrolase (CT). We show that a sustained consumption of dTMP by CT could induce dNTP imbalance, leading to apoptosis as tricarboxylic acid cycle intermediates were depleted to mitigate this imbalance. These cytotoxic effects appeared to be different, depending on substrate specificity of the 5′ nucleotide or metabolic dependency of the cancer cell lines. Using representative TNBC cell lines, we reveal how the TNBC cells were affected by CT-transfection through extracellular acidification rate (ECAR)/oxygen consumption rate (OCR) analysis and differential transcription/expression levels. We suggest a novel approach for treating refractory TNBC by an mRNA drug that can exploit metabolic dependencies to exacerbate cell metabolic vulnerability.

Novel BH4-BCL-2 Domain Antagonists Induce BCL-2-Mediated Apoptosis in Triple-Negative Breast Cancer.

Targeting the challenging tumors lacking explicit markers and predictors for chemosensitivity is one of the major impediments of the current cancer armamentarium. Triple-negative breast cancer (TNBC) is an aggressive and challenging molecular subtype of breast cancer, which needs astute strategies to achieve clinical success. The pro-survival B-cell lymphoma 2 (BCL-2) overexpression reported in TNBC plays a central role in deterring apoptosis and is a promising target. Here, we propose three novel BH4 mimetic small molecules, SM396, a covalent binder, and two non-covalent binders, i.e., SM216 and SM949, which show high binding affinity (nM) and selectivity, designed by remodeling the existing BCL-2 chemical space. Our mechanistic studies validate the selectivity of the compounds towards cancerous cells and not on normal cells. A series of functional assays illustrated BCL-2-mediated apoptosis in the tumor cells as a potent anti-cancerous mechanism. Moreover, the compounds exhibited efficacious in vivo activity as single agents in the MDA-MB-231 xenograft model (at nanomolar dosage). Overall, these findings depict SM216, SM396, and SM949 as promising leads, pointing to the clinical translation of these compounds in targeting triple-negative breast cancer.

Eupalinolide O Induces Apoptosis in Human Triple-Negative Breast Cancer Cells via Modulating ROS Generation and Akt/p38 MAPK Signaling Pathway.

Background Triple-negative breast cancer (TNBC) is a subtype of breast cancer with limited therapeutic options. Eupalinolide O (EO) was reported to inhibit tumor growth. This study is aimed at exploring the role of EO on TNBC both in vivo and in vitro. Methods. In in vitro experiments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assay were conducted to measure the impact of EO on TNBC cell growth at different concentrations and time points. Flow cytometry was conducted to evaluate cell apoptosis. Mitochondrial membrane potential (MMP) loss, caspase-3 activity, and reactive oxygen species (ROS) generation were assessed. The expressions of apoptosis-related mRNAs and Akt/p38 MAPK signaling pathway-related proteins were measured. In in vivo experiments, by injecting TNBC cells into the nude mice to induce xenograft tumor, mice were treated with EO for 20 days. Then, in vivo bioluminescence imaging system was utilized to monitor the growth and distribution of TNBC cells. Tumor volume and weight were also recorded. Hematoxylin-eosin (HE) staining and ELISA assay were applied to observe tumor tissue morphology and ROS levels. Furthermore, western blotting was conducted to observe the expression of apoptosis-related proteins and Akt/p38 MAPK signaling pathway-associated proteins. Results EO inhibited the cell viability and proliferation of TNBC cells but not normal epithelial cells. Furthermore, EO induced apoptosis, decreased MMP, and elevated caspase-3 activity and ROS content in TNBC cells. Meanwhile, the expression of apoptosis-related mRNAs and Akt/p38 MAPK pathway-related proteins was regulated by EO treatment. Besides, in vivo experiments demonstrated EO not only suppressed tumor growth, Ki67 expression, ROS generation, and Akt phosphorylation but also upregulated caspase-3 expression and p-38 phosphorylation. Conclusion EO may induce cell apoptosis in TNBC via regulating ROS generation and Akt/p38 MAPK pathway, indicating EO may be a candidate drug for TNBC.

Atractylenolide I Inhibits Triple-Negative Breast Cancer Cell Proliferation and Promotes Apoptosis via Blocking the Janus Kinase 2/Signal Transducer and Activator of Transcription 3 Signaling Pathway

Bioactive agent Atractylenolide I (AT-1) has been shown to possess therapeutic value for treating various malignancies. The purpose of the current study is to clarify the potential effect of AT-1 on the development of triple-negative breast cancer (TNBC) and to investigate relevant signaling pathways involved in its mechanism. MTT assay was used to assess the effect of different concentrations of AT-1 on cell survival rate in MCF-10A normal breast epithelial cell line and MDA-MB-231 TNBC cell line. Exogenous IL-6 and WP1066 respectively acted as the agonist and the inhibitor of JAK2/STAT3. Determination of MDA-MB-231 cell viability, proliferation and apoptosis employed MTT, colony formation assay and TUNEL. Western blotting was conducted to measure the expression of proliferation- and apoptosis-related proteins. The viability of MCF-10A cells was unaffected by AT-1, whereas in MDA-MB-231 cells the proliferation level was decreased and the apoptosis level was increased after AT-1 treatment. IL-6 partially restored the expression of AT-1-blocked JAK2/STAT3, and WP1066 inhibited JAK2/STAT3 expression in combination with IL-6 and AT-1. Furthermore, compared with the AT-1 group, co-incubation of IL-6 and AT-1 partially restored the proliferative capacity and reduced the apoptosis of MDA-MB-231 cells, while WP1066 reversed these effects in combination with IL-6 and AT-1. AT-1 suppressed proliferation and promoted apoptosis in TNBC cells likely through inhibiting the activation of JAK2/STAT3 signaling pathway. The findings from this study may provide guidance for future studies on AT-1 and theoretical basis of AT-1 pharmacological activities.

Rutin induces endoplasmic reticulum stress-associated apoptosis in human triple-negative breast carcinoma MDA-MB-231 cells – In vitro and in silico docking studies

Rutin is a bioactive compound that possesses anti-tumor activities through triggering apoptosis. Triple-negative breast cancer (TNBC) is insensitive to targeted anti-tumoral drugs, and drug resistance in TNBC poses a challenge for a successful cure. The accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER) results in cellular stress that initiates a specialized response designated as the unfolded protein response. This study aimed to find potential ER stress targets in triple-negative breast cancer. The viability of cells was evaluated using an MTT assay. Cell migration and proliferation were done by wound scratch and colony formation assay. Cell cycle detection, measurement of ER stress, mitochondrial membrane potential disruption, and cell death identification was performed using flow cytometry. The interaction of rutin with ER stress proteins is predicted using in silico docking. The pattern of gene expression was determined by qRT-PCR. The elevated rate of cell viability, cell cycle arrest, ER stress, MMP, and apoptotic induction was observed in combination treatment. Rutin exhibited the highest glide score with ASK1 and JNK. The results of qRT-PCR showed that rutin induced apoptosis through upregulation of ASK1 and JNK. The present study provides strong evidence supporting an important role of the ER stress response in mediating rutin-induced apoptosis in triple-negative breast cancer.

Gypensapogenin H suppresses tumor growth and cell migration in triple-negative breast cancer by regulating PI3K/AKT/NF-κB/MMP-9 signaling pathway

Due to its aggressiveness and high metastasis rates, triple-negative breast cancer (TNBC) is a ubiquitous and deadly disease for the majority of women globally. Gypensapogenin H (GH) is a novel dammarane-type triterpene isolated from hydrolyzate of total saponins from Gynostemma pentaphyllum. Our previous work demonstrated that GH promoted apoptosis in TNBC. In the present study, xenograft TNBC models (xenotransplantation of MDA-MB-231 cells in nude mice) were used to evaluate the efficacy of GH in vivo. We preliminarily predicted the mechanism of GH inhibiting breast cancer tumors at the gene level through transcriptome screening. Through western blot analysis of tumor tissue, we found that GH could inhibit tumor proliferation and migration by regulating the PI3K/AKT/NF-κB/MMP-9 signaling pathway in vivo. We also analyzedthe results at the cell level in vitro, which were consistent with those in vivo. In summary, GH inhibited TNBC growth in vivo and suppressed TNBC cell migration in vitro. Our findings could help understand the mechanism of action of GH and suggest that GH would be a promising agent for TNBC therapy.

Ginsenoside CK induces apoptosis in triple-negative breast cancer cells by targeting glutamine metabolism

Breast cancer (BC) has replaced lung cancer as the most common cancer worldwide. Ginsenoside CK (CK) can effectively inhibit triple-negative breast cancer (TNBC), the occurrence and development of which are associated with glutamine addiction. However, the connection between CK and glutamine metabolism in TNBC proliferation and the mechanism of cell death induction remains unclear. Here, we found that high glutamine-addicted TNBC cells were particularly sensitive to CK treatment. CK exerted antitumour activity against TNBC by suppressing glutamine consumption and glutamate production via downregulation of glutaminase 1 (GLS1) expression. CK treatment further decreased cellular ATP production, reduced the utilisation of amino acids associated with glutamine metabolism, and induced glutathione (GSH) depletion and reactive oxygen species (ROS) accumulation, consequently triggering apoptosis in TNBC. Furthermore, CK decreased GLS1 expression in SUM159 xenograft mouse mammary tumours and significantly inhibited tumour growth with few side effects. Together, our data provide a powerful theoretical basis for the application of CK as a glutamine metabolic inhibitor in TNBC treatment.

JAC1 targets YY1 mediated JWA/p38 MAPK signaling to inhibit proliferation and induce apoptosis in TNBC

Triple negative breast cancer (TNBC) is a type of breast cancer with poor prognosis, and has no ideal therapeutic target and ideal medicine. Downregulation of JWA is closely related to the poor overall survival in many cancers including TNBC. In this study, we reported at the first time that JWA gene activating compound 1 (JAC1) inhibited the proliferation of TNBC in vitro and in vivo experimental models. JAC1 specifically bound to YY1 and eliminated its transcriptional inhibition of JWA gene. The rescued JWA induced G1 phase arrest and apoptosis in TNBC cells through the p38 MAPK signaling pathway. JAC1 also promoted ubiquitination and degradation of YY1. In addition, JAC1 disrupted the interaction between YY1 and HSF1, and suppressed the oncogenic role of HSF1 in TNBC through p-Akt signaling pathway. In conclusion, JAC1 suppressed the proliferation of TNBC through the JWA/P38 MAPK signaling and YY1/HSF1/p-Akt signaling. JAC1 maybe a potential therapeutic agent for TNBC.

Investigating the mechanism of Xian-ling-lian-xia-fang for inhibiting vasculogenic mimicry in triple negative breast cancer via blocking VEGF/MMPs pathway

BackgroundXian-ling-lian-xia-fang (XLLXF), a Chinese medicine decoction, is widely used in the treatment of triple negative breast cancer (TNBC). However, the underlying mechanism of XLLXF in TNBC treatment has not been totally elucidated.MethodsHere, network pharmacology and molecular docking were used to explore the mechanism of Traditional Chinese medicine in the treatment of TNBC. Then, biological experiments were integrated to verify the results of network pharmacology.ResultsNetwork pharmacology showed that the candidate active ingredients mainly included quercetin, kaempferol, stigmasterol, and β-sitosterol through the “XLLXF–active ingredients–targets” network. Vascular endothelial growth factor A (VEGFA) and matrix metalloproteinase (MMP) 2 were the potential therapeutic targets obtained through the protein–protein interaction (PPI) network. Molecular docking confirmed that quercetin, kaempferol, stigmasterol, and β-sitosterol could stably combine with VEGFA and MMP2. Experimental verification showed that XLLXF could inhibit proliferation, colony ability, and vasculogenic mimicry (VM) formation and promote cell apoptosis in TNBC. Laser confocal microscopy found that XLLXF impaired F-actin cytoskeleton organization and inhibited epithelial mesenchymal transition. Animal experiments also found that XLLXF could inhibit tumor growth and VM formation in TNBC xenograft model. Western blot analysis and immunohistochemical staining showed that XLLXF inhibited the protein expression of VEGFA, MMP2, MMP9, Vimentin, VE-cadherin, and Twist1 and increased that of E-cadherin, tissue inhibitors of metalloproteinase (TIMP)-1, and TIMP-3 in vitro and in vivo.ConclusionsIntegrating the analysis of network pharmacology and experimental validation revealed that XLLXF could inhibit VM formation via downregulating the VEGF/MMPs signaling pathway.