Triple-negative Breast Cancer Cell Proliferation Research Articles

Functionalized siRNA-chitosan nanoformulations promote triple-negative breast cancer cell death via blocking the miRNA-21/AKT/ERK signaling axis: in-silico and in vitro studies.

Oncogenic microRNA (miRNA), especially miRNA-21 upregulation in triple-negative breast cancer (TNBC), suggests a new class of therapeutic targets. In this study, we aimed to create GE11 peptide-conjugated small interfering RNA-loaded chitosan nanoparticles (GE11-siRNA-CSNPs) for the targeting of EGFR overexpressed TNBC and selectively inhibit miRNA-21 expression. A variety of in-silico and in vitro cellular and molecular studies were conducted to investigate the binding affinities of specific targets used as well as the anticancer efficacies and mechanisms of GE11-siRNA-CSNPs in TNBC cells. An in-silico assessment reveals a distinct binding affinity of miRNA-21 with siRNA as well as between the extracellular domain of EGFR and synthesized peptides. Notably, the in vitro results showed that GE11-siRNA-CSNPs were revealed to have better cytotoxicity against TNBC cells. It significantly inhibits miRNA-21 expression, cell migration, and colony formation. The results also indicated that GE11-siRNA-CSNPs impeded cell cycle progression. It induces cell death by reducing the expression of the antiapoptotic gene Bcl-2 and increasing the expression of the proapoptotic genes Bax, Caspase 3, and Caspase 9. Additionally, the docking analysis and immunoblot investigations verified that GE1-siRNA-CSNPs, which specifically target TNBC cells and suppress miRNA-21, can prevent the effects of miRNA-21 on the proliferation of TNBC cells via controlling EGFR and subsequently inhibiting the PI3K/AKT and ERK1/2 signaling axis. The GE11-siRNA-CSNPs design, which specifically targets TNBC cells, offers a novel approach for the treatment of breast cancer with improved effectiveness. This study suggests that GE11-siRNA-CSNPs could be a promising candidate for further assessment as an additional strategy in the treatment of TNBC.

Expression and function of cytokine interleukin-22 gene in the tumor microenvironment of triple negative breast cancer

BackgroundThe tumor microenvironment (TME) and interleukin-22 (IL-22) in cytokines have recently attracted much attention due to their potential impact on tumor biology. However, the role of IL-22 in triple negative breast cancer (TNBC) TME is still poorly understood. This article investigated the gene expression and function of IL-22 in TNBC TME. MethodsTumor samples from TNBC patients were collected, and adjacent noncancerous tissues were used as controls. A functional test was performed to evaluate the impact of IL-22 for TNBC cells, including proliferation, migration, and apoptosis. ResultsIL-22 gene expression in TNBC tumor samples was markedly higher relative to adjacent non-cancerous tissues (P < 0.05). In addition, it was also observed that IL-22facilitated proliferation and migration of TNBC cells, and inhibit apoptosis. This article reveals the role of IL-22 in the TME of TNBC. The up-regulation of IL-22 gene expression in TNBC tumors and its promoting effect on cancer cell invasiveness highlight its potential as a therapeutic target in TNBC treatment strategies. ConclusionThe findings suggested that targeting IL-22 and its related pathways can offer new insights for developing effective therapies for TNBC.

PTGER3 knockdown inhibits the vulnerability of triple-negative breast cancer to ferroptosis.

Prostaglandin E receptor 3 (PTGER3) is involved in a variety of biological processes in the human body and is closely associated with the development and progression of a variety of cancer types. However, the role of PTGER3 in triple-negative breast cancer (TNBC) remains unclear. In the present study, low PTGER3 expression was found to be associated with poor prognosis in TNBC patients. PTGER3 plays a crucial role in regulating TNBC cell invasion, migration, and proliferation. Upregulation of PTGER3 weakens the epithelial-mesenchymal phenotype in TNBC and promotes ferroptosis both invitro and invivo by repressing glutathione peroxidase 4 (GPX4) expression. On the other hand, downregulation of PTGER3 inhibits ferroptosis by increasing GPX4 expression and activating the PI3K-AKT pathway. Upregulation of PTGER3 also enhances the sensitivity of TNBC cells to paclitaxel. Overall, this study has elucidated critical pathways in which low PTGER3 expression protects TNBC cells from undergoing ferroptosis, thereby promoting its progression. PTGER3 may thus serve as a novel and promising biomarker and therapeutic target for TNBC.

Dissection of triple-negative breast cancer microenvironment and identification of potential therapeutic drugs using single-cell RNA sequencing analysis

Breast cancer remains a leading cause of mortality in women worldwide. Triple-negative breast cancer (TNBC) is a particularly aggressive subtype characterized by rapid progression, poor prognosis, and lack of clear therapeutic targets. In the clinic, delineation of tumor heterogeneity and development of effective drugs continue to pose considerable challenges. Within the scope of our study, high heterogeneity inherent to breast cancer was uncovered based on the landscape constructed from both tumor and healthy breast tissue samples. Notably, TNBC exhibited significant specificity regarding cell proliferation, differentiation, and disease progression. Significant associations between tumor grade, prognosis, and TNBC oncogenes were established via pseudotime trajectory analysis. Consequently, we further performed comprehensive characterization of the TNBC microenvironment. A crucial epithelial subcluster, E8, was identified as highly malignant and strongly associated with tumor cell proliferation in TNBC. Additionally, epithelial-mesenchymal transition-associated fibroblast and M2 macrophage subclusters exerted an influence on E8 through cellular interactions, contributing to tumor growth. Characteristic genes in these three cluster cells could therefore serve as potential therapeutic targets for TNBC. The collective findings provided valuable insights that assisted in the screening of a series of therapeutic drugs, such as pelitinib. We further confirmed the anti-cancer effect of pelitinib in an orthotopic 4T1 tumor-bearing mouse model. Overall, our study sheds light on the unique characteristics of TNBC at single-cell resolution and the crucial cell types associated with tumor cell proliferation that may serve as potent tools in the development of effective anti-cancer drugs.

17β-Estradiol promotes metastasis in triple-negative breast cancer through the Calpain/YAP/β-catenin signaling axis.

β-catenin is an important regulator of malignant progression. 17β-Estradiol (E2), an important sex hormone in women, promotes the growth and metastasis of triple-negative breast cancer (TNBC). However, whether β-catenin is involved in E2-induced metastasis of TNBC remains unknown. In this study, we show that E2 induces the proliferation, migration, invasion, and metastasis of TNBC cells. E2 induces β-catenin protein expression and nuclear translocation, thereby regulating the expression of target genes such as Cyclin D1 and MMP-9. The inhibition of β-catenin reversed the E2-induced cell malignant behaviors. Additionally, E2 activated Calpain by increasing intracellular Ca2+ levels and reducing calpastatin levels. When Calpain was inhibited, E2 did not induce the proliferation, migration, invasion, or metastasis of TNBC cells. In addition, E2 promoted translocation of YAP into the nucleus by inhibiting its phosphorylation. Calpain inhibition reversed the E2-induced YAP dephosphorylation. Inhibition of YAP transcriptional activity reversed the effects of E2 on the proliferation, migration, invasion, and β-catenin of TNBC cells. In conclusion, we demonstrated that E2 induced metastasis-related behaviors in TNBC cells and this effect was mediated through the Calpain/YAP/β-catenin signaling pathway.

Abstract 4549: Combinations of ciclopirox olamine and metformin exert synergistic antitumor activity in triple-negative breast cancer

Abstract Introduction: Triple-negative breast cancer (TNBC) represents a challenging subtype of breast cancer because of its limited treatment options. Recent research has highlighted the potential benefits of combining pharmacological agents to enhance therapeutic outcomes. Studies show that Ciclopirox olamine (CPX), an off-patent anti-fungal drug, induces cell cycle G1-phase arrest and activates the intrinsic apoptosis pathway by downregulating the anti-apoptotic proteins. Meanwhile, metformin, a safe and first-line drug for the treatment of type II diabetes, has been shown to induce cell cycle S-phase arrest and activates the extrinsic apoptosis pathway via induction of endogenous TRAIL in TNBC cells. However, whether the combinations of CPX and metformin would exert potent antitumor activity against TNBC remains unclear. Methods: Cell proliferation (MTS) assays and LIVE/DEAD cell imaging were used to determine the combined inhibitory effects on TNBC cells. IC50s and the combination index (CI) were calculated. Flow cytometry assays were carried out to analyze if the combinations are more potent to block cell cycle progression. Apoptotic-specific ELISA and flow cytometry analysis of Annexin V-PI staining were performed to assess if the combinations are significantly more effective to induce apoptosis. Western blot assays were used to examine if the combinations markedly induced the expression of TRAIL and cleavages of PARP and caspases. Tumor xenograft models will be established to test the in vivo antitumor activity of CPX and metformin either alone or in combinations. Results: Data indicated that both CPX and metformin independently inhibited the proliferation of TNBC cells. A synergistic effect was observed when CPX and metformin were used in combination, resulting in a more potent inhibition of TNBC cell proliferation. The combinatorial treatment effectively suppressed colony formation of TNBC cells. Synergy between CPX and metformin was partially linked to the enhanced activation of the AMPK-mTOR pathway, as the combination significantly enhanced the phosphorylation of AMPKα. The combinations also induced enhanced apoptosis in TNBC cells through both the intrinsic and extrinsic pathways. The combinatorial effects of CPX and metformin on in vivo tumor growth are under investigation. Conclusions: Combinations of CPX and metformin show synergistic effects on inhibiting TNBC cell proliferation. Our studies suggest a potential therapeutic strategy for TNBC, with the combinations of these agents exhibiting an enhanced antitumor activity. Further investigation is warranted to validate the results and explore their clinical applications. Key words: Triple-negative breast cancer, Ciclopirox olamine, Metformin, Combination, Apoptosis Citation Format: Yakun Wu, Hui Lyu, Congcong Tan, Margaret E. Larsen, Shile Huang, Bolin Liu. Combinations of ciclopirox olamine and metformin exert synergistic antitumor activity in 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 4549.

Abstract 5697: MILIP drives triple-negative breast cancer through complexing with transfer RNAs to promote protein production

Abstract Despite the progress in endocrine therapy, targeted therapy and immunotherapy, the treatment of triple-negative breast cancer (TNBC), one of the most aggressive types of breast cancer, is challenging. This is closely associated with the lack of effective molecular targets in TNBC cells for therapeutic intervention. Here we report that the long noncoding RNA (lncRNA) MILIP drives TNBC cell survival, proliferation and tumorigenicity through complexing with transfer RNAs (tRNAs) to promote protein production, and thus represents a potential therapeutic target in TNBC. MILIP was expressed at high levels in TNBC cells that commonly harbor loss-of-function mutations of the tumor suppressor p53, yet MILIP silencing diminished TNBC cell viability and retarded TNBC xenograft growth, indicating that MILIP functions distinctively in TNBC beyond its well-established role in repressing p53 in other types of cancers. Mechanistical investigations revealed that MILIP interacted with eukaryotic translation elongation factor 1 alpha 1 (eEF1α1) and formed an RNA-RNA duplex with tRNALeu and tRNASer, type II tRNAs, through their variable loops, thus facilitating the binding of eEF1α1 to these tRNAs. Disrupting the interaction between MILIP and eEF1α1 or tRNAs diminished protein production and cell viability, with therapeutic potential revealed where targeting MILIP using Gapmers inhibited TNBC growth and cooperated with the clinically available protein synthesis inhibitor omacetaxine mepesuccinate in vivo. Collectively, these results identified MILIP as an RNA translation elongation factor to promote protein production in TNBC cells and demonstrated that experimental therapy with MILIP Gapmers inhibits TNBC growth, indicating MILIP targeting is a potential avenue for developing improved TNBC treatment through blocking protein synthesis. Citation Format: Yuchen Feng, Simin Zheng, Xiaohong Zhao, Yuanyuan Zhang, Ran Xu, Liang Xu, Thomas Preiss, Lei Jin, Jinnan Gao, Xu Dong Zhang. MILIP drives triple-negative breast cancer through complexing with transfer RNAs to promote protein production [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 5697.

YAP1-induced RBM24 promotes the tumorigenesis of triple-negative breast cancer through the β-catenin pathway.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and refractory to current treatments. RBM24 is an RNA-binding protein and shows the ability to regulate tumor progression in multiple cancer types. However, its role in TNBC is still unclear. In this study, we analyzed publicly available profiling data from TNBC tissues and cells. Loss- and gain-of-function experiments were performed to determine the function of RBM24 in TNBC cells. The mechanism for RBM24 action in TNBC was investigated. RBM24 was deregulated in TNBC tissues and TNBC cells with depletion of SIPA1, YAP1, or ARID1A, three key regulators of TNBC. Compared to MCF10A breast epithelial cells, TNBC cells had higher levels of RBM24. Knockdown of RBM24 inhibited TNBC cell proliferation, colony formation, and tumorigenesis, while overexpression of RBM24 promoted aggressive phenotype in TNBC cells. YAP1 overexpression induced the expression of RBM24 and the RBM24 promoter-driven luciferase reporter. YAP1 was enriched at the promoter region of RBM24. Overexpression of RBM24 increased β-catenin-dependent transcriptional activity. Most importantly, knockdown of CTNNB1 rescued RBM24 aggressive phenotype in TNBC cells. Collectively, the YAP1/RBM24/β-catenin axis plays a critical role in driving TNBC progression. RBM24 may represent a novel therapeutic target for TNBC treatment.

Abstract 5945: Exploring the antiproliferative potential of KHS101 on triple negative breast cancer cell lines

Abstract Breast cancer is the second leading cause of cancer deaths in the world. Among its subtypes, triple-negative breast cancer (TNBC) is highly aggressive, particularly affecting African American (AA) women, with limited treatment choices. HSP60 protein promotes tumorigenesis and is being studied as a possible biomarker to treat cancers such as TNBC. The small molecule KHS101 specifically binds and inhibits the mitochondrial chaperone HSP60. In this study, we hypothesized that KHS101 would inhibit the proliferation of TNBC cells and therefore may have a therapeutic potential.We studied the antiproliferative effects of KHS101 on TNBC cell lines (MDA-MB-231, MDA-MB-468, HCC1806) through MTT cell proliferation and colony forming assays at varying doses and time points (24, 48, 72, and 96 hours). We performed cell cycle analysis to understand the cell cycle response and apoptotic events in treated or control cells. Additionally, the gene expression and frequently altered pathways resulting from KHS101 treatment were analyzed by RNA sequencing of cells treated with vehicle (DMSO) or with various concentrations of KHS101(1, 2.5, 5, and 7.5 uM) for 72 hours. We analyzed the data using GraphPad Prism and Microsoft Excel, with statistical significance set at p ≤ 0.05. TNBC cell lines exhibited significant dose-response and antiproliferative effects with increased KHS101 doses and treatment durations. Notably, AA breast cancer cells (MDA-MB-468) displayed heightened cytotoxicity and reduced proliferation compared to Caucasian American (CA) breast cancer cells (MDA-MB-231 and HCC1806). Cell cycle analysis revealed G2/M phase arrest, consistent with RNA sequencing data showing downregulation of the activation of ATM/ATR response in KHS101-treated cell lines compared to the controls. Gene set enrichment analysis indicated downregulation of the cell cycle control genes in MDA-MB-231 cells after 72 hours of KHS101 treatment (7.5 µM). Reactome pathway analysis in KHS101-treated MDA-MB-231 cells showed upregulation of IFN-gamma signaling and degradation of extracellular matrix. Similarly, Kegg pathway analysis showed upregulation in cell adhesion molecules, cytokine-cytokine receptor interaction, antigen processing and presentation, and Natural Killer cell-mediated cytotoxicity in KHS101-treated cancer cells compared to the controls. KHS101 demonstrates varying cytotoxic effects on TNBC cells, with potential implications for antitumor immune responses. Our findings highlight the distinct susceptibility of AA breast cancer cells and suggest KHS101 as a promising avenue for further research in understanding its mechanism of action and validating therapeutic potential in TNBC. Citation Format: Reshma Gurung, Alehegne W. Yirsaw, Muhammad G. Khodary, Temesgen Samuel, Maninder Sandey, Clayton C. Yates, Deepa Bedi. Exploring the antiproliferative potential of KHS101 on triple negative breast cancer cell lines [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 5945.

Abstract 5435: CUEDC2 involved in the inhibitory effect of dandelion on triple-negative breast cancer stem cells

Abstract Background: Cancer stem cells (CSCs) exert a substantial influence on the initiation, progression, and metastasis of triple-negative breast cancer (TNBC). Dandelion has exhibited encouraging outcomes in impeding the proliferation of TNBC cells. Nevertheless, the extent of investigation regarding the efficacy of dandelion in targeting CSCs remains restricted. Purpose: The aim of this study was to investigate the impact of dandelion extract (DE) on CSCs properties. Methods: In vitro experiments were conducted using TNBC cell lines. Mice bearing TNBC tumors were established for in vivo investigations. Biochemical analysis and cell biological assay were carried to evaluate the effect of dandelion and the underlying mechanisms. Results: DE treatment significantly decreased the proportion of ALDH+ cells, mammosphere formation ability, and CSCs-related markers. CUEDC2 is highly expressed in ALDH+ TNBC cells, and its overexpression increased the expression of markers related to CSCs. Conversely, the tumor growth was impeded in CUEDC2 KO mice and the markers related to CSCs also were also reduced. CUEDC2 overexpression reversed the inhibitory effect of DE on TNBC CSCs properties. Mechanistically, CUEDC2 related pathway involved in the inhibitory effect of DE on TNBC CSCs properties. Conclusion: CUEDC2 played a crucial role in maintaining the stem cell properties of TNBC. DE inhibit the self-renewal of TNBC stem cells by regulating the CUEDC2 related signaling pathway. Keywords: Dandelion; CUEDC2; Triple-negative breast cancer; Cancer stem cell. Citation Format: Xinxin Deng, Yanna Jiao, Huifeng Hao, Zhengwang Guo, Dong Xue, Shuyan Han. CUEDC2 involved in the inhibitory effect of dandelion on triple-negative breast cancer stem 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 5435.

Abstract 3328: Oridonin derivative compounds target STATs to inhibit triple-negative breast cancer

Abstract Breast cancer remains the second leading cause of cancer related deaths in women. Despite the development of targeted treatments and immunotherapies, triple-negative breast cancer (TNBC) continues to evade effective treatment. TNBC lacks sufficient expression of druggable markers, ER, PR, and Her2, and exhibits especially aggressive characteristics including later diagnosis, lower survival rate, and higher rates of metastasis and reoccurrence. Currently, cytotoxic chemotherapy and radiation remain the standard of care for TNBC. Our work utilizes compounds from nature to develop therapies for TNBC with improved efficacy and safety. Oridonin, a natural kaurene-type diterpenoid enriched in the medicinal herb Rabdosia rubescens, is a promising anticancer agent, but exhibits limited bioavailability and potency. Our team successfully developed oridonin derivative CYD0618 compound that exhibits significantly improved solubility and anticancer activity. This study aims to characterize the mechanism of action of CYD0618 on TNBC cells. We found that CYD0618 reduces the proliferation of TNBC cells in vitro with IC50s at the nanomolar level. We also found CYD0618 has high potency against TNBC in vivo. Within a xenograft mouse model, CYD0618 suppresses the growth of TNBC tumors and extends animal survival. Using Western blot to probe for putative targets, we found CYD0618 reduces activation of STAT3, a transcription factor critically involved in proliferation, survival, and transformation of epithelial cells. Because STAT3 is constitutively active in ~70% of human cancers, there has been a tremendous effort to develop STAT3 targeting therapies; however, none has achieved FDA approval. In addition to STAT3, we show CYD0618 also suppresses other STAT transcription factors. Compared to selective STAT3 degradation by PROTAC treatment, poly-STAT targeting by CYD0618 exhibits superior activity against TNBC cells. Our study identifies CYD0618 as a novel poly-STAT inhibitor with promising activity against TNBC. Citation Format: Gabrielle Vontz, Jun Li, Zhipin Liang, Ruixia Ma, Pingyuan Wang, Haiying Chen, Jia Zhou, Qiang Shen. Oridonin derivative compounds target STATs to inhibit 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 3328.

Mammary tissue-derived extracellular matrix hydrogels reveal the role of irradiation in driving a pro-tumor and immunosuppressive microenvironment

Radiation therapy (RT) is essential for triple negative breast cancer (TNBC) treatment. However, patients with TNBC continue to experience recurrence after RT. The role of the extracellular matrix (ECM) of irradiated breast tissue in tumor recurrence is still unknown. In this study, we evaluated the structure, molecular composition, and mechanical properties of irradiated murine mammary fat pads (MFPs) and developed ECM hydrogels from decellularized tissues (dECM) to assess the effects of RT-induced ECM changes on breast cancer cell behavior. Irradiated MFPs were characterized by increased ECM deposition and fiber density compared to unirradiated controls, which may provide a platform for cell invasion and proliferation. ECM component changes in collagens I, IV, and VI, and fibronectin were observed following irradiation in both MFPs and dECM hydrogels. Encapsulated TNBC cell proliferation and invasive capacity was enhanced in irradiated dECM hydrogels. In addition, TNBC cells co-cultured with macrophages in irradiated dECM hydrogels induced M2 macrophage polarization and exhibited further increases in proliferation. Our study establishes that the ECM in radiation-damaged sites promotes TNBC invasion and proliferation as well as an immunosuppressive microenvironment. This work represents an important step toward elucidating how changes in the ECM after RT contribute to breast cancer recurrence.

Linc00707 regulates autophagy and promotes the progression of triple negative breast cancer by activation of PI3K/AKT/mTOR pathway

Triple-negative breast cancer (TNBC) is a pathological subtype of breast cancer (BC) with high malignancy, strong invasiveness and poor prognosis. Long non-coding RNA (LncRNA) plays an important role during tumorigenesis. We identified that Linc00707 was upregulated in TNBC tissues by TCGA database and RT-qPCR assay, compared with normal breast tissues and other subtypes of BC. Linc00707 promoted TNBC cells proliferation, migration and invasion. Furthermore, we found that knockdown of Linc00707 influenced autophagy via PI3K/AKT/mTOR signaling pathway in TNBC cells. Linc00707 affected the progress of TNBC cells through affecting autophagy. Further mechanistic experiments confirmed that Linc00707 could competitively bind with miR-423-5p to up-regulate MARCH2 expression, ultimately promoting TNBC progression and autophagy through PI3K/AKT/mTOR pathway. In conclusion, we demonstrate that Linc00707 is a key molecule in tumor progression and may be an effective target for patients with TNBC.

CARM1 drives triple-negative breast cancer progression by coordinating with HIF1A.

Coactivator-associated arginine methyltransferase 1 (CARM1) promotes the development and metastasis of estrogen receptor alpha (ERα)-positive breast cancer. The function of CARM1 in triple-negative breast cancer (TNBC) is still unclear and requires further exploration. Here, we report that CARM1 promotes proliferation, epithelial-mesenchymal transition (EMT), and stemness in TNBC. CARM1 is upregulated in multiple cancers and its expression correlates with breast cancer progression. Genome-wide analysis of CARM1 showed that CARM1 is recruited by hypoxia-inducible factor 1 subunit alpha (HIF1A) and occupy the promoters of CDK4, Cyclin D1, β-catenin, HIF1A, MALAT1, and SIX1 critically involved in cell cycle, HIF-1 signaling pathway, Wnt signaling pathway, VEGF signaling pathway, thereby modulating the proliferation and invasion of TNBC cells. We demonstrated that CARM1 is physically associated with and directly interacts with HIF1A. Moreover, we found that ellagic acid, an inhibitor of CARM1, can suppress the proliferation and metastasis of TNBC by directly inhibiting CDK4 expression. Our research has determined the molecular basis of CARM1 carcinogenesis in TNBC and its effective natural inhibitor, which may provide new ideas and drugs for cancer therapy.

Design, optimization, characterization, and in vitro evaluation of metformin-loaded liposomes for triple negative breast cancer treatment

Recently, metformin (Met) has shown to have antineoplastic properties in cancer treatment by improving hypoxic tumor conditions, and causing reduction in the synthesis of biomolecules, which are vital for cancer growth. However, as an orally administered drug, Met has low bioavailability and rapid renal clearance. Thus, the goal of this study was to vectorize Met inside liposomes in the context of triple negative breast cancer (TNBC), which currently lacks treatment options when compared to other types of breast cancer. Vectorization of Met inside liposomes was done using Bangham method by implementing double design of experiment methodology to increase Met drug loading (minimum-run resolution V characterization design and Box-Behnken design), as it is generally extremely low for hydrophilic molecules. Optimization of Met-loaded liposome synthesis was successfully achieved with drug loading of 190 mg/g (19% w/w). The optimal Met-liposomes were 170 nm in diameter with low PdI (< 0.1) and negative surface charge (-20 mV), exhibiting sustained Met release at pH 7.4. The liposomal Met delivery system was stable over several months, and successfully reduced TNBC cell proliferation due to the encapsulated drug. This study is one the first reports addressing liposome formulation through thin-film hydration using two design of experiment methods aiming to increase drug loading of Met.

Ellagic Acid-Gold Nanoparticle Complex as Anticancer Agent in Triple-Negative Breast Cancer Cells

Triple-negative breast cancer (TNBC) is the most aggressive sub-type of breast cancer, mainly affecting premenopausal women. As it neither expresses hormone receptors nor overexpresses HER2 receptors, it is difficult to tackle this deadly disease effectively. It is a well-known fact that phytochemicals have remarkable therapeutic potential in the prevention and treatment of different disorders. One of the well-known phytochemicals, ellagic acid (EA) has shown better efficacy in the prevention and treatment of cancer in many preclinical and clinical settings. Therefore, in this study, ellagic acid gold nanoparticles (EA-AuNp) were synthesized and characterized to passively target TNBC cells through EPR effect. EA-AuNp were prepared using the direct reduction method and characterized by UV–Visible spectroscopy, DLS, XRD, EDX, TEM and FT-IR spectra. We found that it significantly suppressed the TNBC cell proliferation (IC[Formula: see text] 12 [Formula: see text]g/ml) compared to EA alone (IC[Formula: see text] 36 [Formula: see text]g/ml). Flow cytometry and live and dead assays revealed that EA-AuNp induced more cell death in TNBC cells compared to EA alone. Further, western blot analysis substantiated that EA-AuNp induced apoptosis by regulating the expression of Bcl-2, survivin, caspase 3 and caspase 9. In conclusion, EA-AuNp has high anti-cancer potential and additional preclinical and clinical studies are highly recommended.

YB-1 Targeted by miR-509-3-5p Affects Migration and Invasion of Triple‑Negative Breast Cancer by Regulating Cellular Epithelial‑Mesenchymal Transition.

The epithelial-mesenchymal transition (EMT) process is closely linked to metastasis of breast cancer. This article elucidates the role of Y-box binding protein-1 (YB-1) on the migration and invasion of triple-negative breast cancer (TNBC) cells by regulating EMT, and the related mechanism. The expression data of YB-1 and miR-509-3-5p in TNBC samples and normal samples were downloaded from the GEO database. The proliferation, migration, invasion, and EMT of TNBC cells were detected by CCK-8 assay, colony formation assay, wound-healing assay, transwell assay, and immunoblotting analyses. The targeted binding of YB-1 and miR-509-3-5p was validated by luciferase reporter experiment. A xenograft mouse model was constructed to investigate the influence of the miR-509-3-5p/YB-1 axis on TNBC tumor growth in vivo. YB-1 was overexpressed, while miR-509-3-5p was underexpressed in TNBC tumor tissues and various cell lines. Silencing YB-1 depressed cell viability, proliferation, motility, and EMT in vitro, and miR-509-3-5p upregulation exerted the same effects. YB-1 was targeted by miR-509-3-5p. The suppressive effects on the phenotypes of TNBC cells caused by overexpressed miR-509-3-5p were attenuated by YB-1 upregulation. In addition, miR-509-3-5p overexpression restrained TNBC tumor growth and downregulated the YB-1-mediated EMT process in vivo. YB-1 targeted by miR-509-3-5p affects motility of TNBC cells by regulating cellular EMT.

The deubiquitinase BRCC3 increases the stability of ZEB1 and promotes the proliferation and metastasis of triple-negative breast cancer cells.

Triple negative breast cancer (TNBC) has a high recurrence rate, metastasis rate and mortality rate. The aim of this study is to identify new targets for the treatment of TNBC. Clinical samples are used for screening deubiquitinating enzymes (DUBs). MDA-MB-231 cells and a TNBC mouse model are used for in vitro and in vivo experiments, respectively. Western blot analysis is used to detect the protein expressions of DUBs, zinc finger E-box binding homeobox 1 (ZEB1), and epithelial-mesenchymal transition (EMT)-related markers. Colony formation and transwell assays are used to detect the proliferation, migration and invasion of TNBC cells. Wound healing assay is used to detect the mobility of TNBC cells. Immunoprecipitation assay is used to detect the interaction between breast cancer susceptibility gene 1/2-containing complex subunit 3 (BRCC3) and ZEB1. ZEB1 ubiquitination levels, protein stability, and protein degradation are also examined. Pathological changes in the lung tissues are detected via HE staining. Our results show a significant positive correlation between the expressions of BRCC3 and ZEB1 in clinical TNBC tissues. Interference with BRCC3 inhibits TNBC cell proliferation, migration, invasion and EMT. BRCC3 interacts with ZEB1 and interferes with BRCC3 to inhibit ZEB1 expression by increasing ZEB1 ubiquitination. Interference with BRCC3 inhibits TNBC cell tumorigenesis and lung metastasis in vivo. In all, this study demonstrates that BRCC3 can increase the stability of ZEB1, upregulate ZEB1 expression, and promote the proliferation, migration, invasion, EMT, and metastasis of TNBC cells, providing a new direction for cancer therapy.

Scutellaria barbata D.Don extract regulates Ezrin-mediated triple negative breast cancer progress via suppressing the RhoA /ROCK1 signaling.

Triple-negative breast cancer (TNBC) lacks effective therapeutic targets. Scutellaria barbata D.Don (SB) has been revealed to have anti-breast cancer (BC) effect, but the effect of SB extract in TNBC is still unclear. Herein, this research delves into the underlying mechanism. SB was extracted by solvent extraction, and the main components were identified using an Agilent 6,520 HPLC-Chip/Q-TOF (Chip/Q-TOF) MS system. In vitro cell experiments were conducted. The effects of SB extract alone, SB extract plus EGF, GSK alone, GSK plus Ezrin overexpression, or SB extract plus Ezrin overexpression on cell viability, invasion, migration, and apoptosis were examined by cell function experiments. The apoptosis- and RhoA/ROCK1 pathway-related protein levels were analyzed by western blot assay. Mass spectrometry analysis exhibited that SB extract mainly contains long-chain fatty acids and ursolic acid. SB extract mitigated TNBC cell biological phenotypes, apoptosis- and RhoA/ROCK1 pathway-related marker expressions, which were reversed by EGF. The further results found that GSK obviously weakens TNBC cell biological behaviors, apoptosis- and RhoA/ROCK1 signaling-related protein levels, while oe-Ezrin treatment reverses the effect of GSK on TNBC cells. Moreover, SB extract regulated Ezrin-mediated function of TNBC cells by impeding the RhoA/ROCK1 pathway. Our findings demonstrated that SB extract regulated Ezrin-mediated proliferation, migration, invasion, and apoptosis of TNBC cells via suppressing the RhoA /ROCK1 signaling. Our results offer the experimental foundation for further investigation of the anti-cancer role of SB in TNBC cells. SB extract inhibits the biological phenotypes of TNBC cells.SB extract inhibits the biological behaviors of TNBC cells through the RhoA/ROCK1 pathway.SB extract modulates Ezrin-mediated TNBC cell proliferation, migration, invasion, and apoptosis via restraining the RhoA/ROCK1 signaling.

Activation of epidermal growth factor receptors in triple-negative breast cancer cells by morphine; analysis through Raman spectroscopy and machine learning

Triple negative breast cancer (TNBC) is a very aggressive form of breast cancer, and the analgesic drug morphine has been shown to promote the proliferation of TNBC cells. This article investigates whether morphine causes activation of epidermal growth factor receptors (EGFR), the roles of μ-opioid and EGFR receptors on TNBC cell proliferation and migration. While examining the changes with molecular techniques, we also aimed to investigate the analysis ability of Raman spectroscopy and machine learning-based approach. Effects of morphine on the proliferation and migration of MDA.MB.231 cells were evaluated by MTT and scratch wound-healing tests, respectively. Morphine-induced phosphorylation of the EGFR was analyzed by western blotting in the presence and absence of μ-receptor antagonist naltrexone and the EGFR-tyrosine kinase inhibitor gefitinib. Morphine-induced EGFR phosphorylation and cell migration were significantly inhibited by pretreatments with both naltrexone and gefitinib; however, morphine-increased cell proliferation was inhibited only by naltrexone. While morphine-induced changes were observed in the Raman scatterings of the cells, the inhibitory effect of naltrexone was analyzed with similarity to the control group. Principal component analysis (PCA) of the Raman confirmed the epidermal growth factor (EGF)-like effect of morphine and was inhibited by naltrexone and partly by gefitinib pretreatments. Our in vitro results suggest that combining morphine with an EGFR inhibitor or a peripherally acting opioidergic receptor antagonist may be a good strategy for pain relief without triggering cancer proliferation and migration in TNBC patients. In addition, our results demonstrated the feasibility of the Raman spectroscopy and machine learning-based approach as an effective method to investigate the effects of agents in cancer cells without the need for complex and time-consuming sample preparation. The support vector machine (SVM) with linear kernel automatically classified the effects of drugs on cancer cells with ∼95% accuracy.