Apoptosis In Triple-negative Breast Cancer Cells Research Articles

CDK4 inactivation inhibits apoptosis via mitochondria-ER contact remodeling in triple-negative breast cancer

The energetic demands of proliferating cells during tumorigenesis require close coordination between the cell cycle and metabolism. While CDK4 is known for its role in cell proliferation, its metabolic function in cancer, particularly in triple-negative breast cancer (TNBC), remains unclear. Our study, using genetic and pharmacological approaches, reveals that CDK4 inactivation only modestly impacts TNBC cell proliferation and tumor formation. Notably, CDK4 depletion or long-term CDK4/6 inhibition confers resistance to apoptosis in TNBC cells. Mechanistically, CDK4 enhances mitochondria-endoplasmic reticulum contact (MERCs) formation, promoting mitochondrial fission and ER-mitochondrial calcium signaling, which are crucial for TNBC metabolic flexibility. Phosphoproteomic analysis identified CDK4’s role in regulating PKA activity at MERCs. In this work, we highlight CDK4’s role in mitochondrial apoptosis inhibition and suggest that targeting MERCs-associated metabolic shifts could enhance TNBC therapy.

Betulinic acid and oleanolic acid modulate CD81 expression and induce apoptosis in triple-negative breast cancer cells through ROS generation.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by a lack of hormones receptors and the HER2 receptor, making it unresponsive to targeted therapy. Triterpenoids such as betulinic acid (BA) and oleanolic acid (OA) have anticancer effects by inducing apoptosis in TNBC cells. CD81 is a tetraspanin that affects the growth and metastasis of cancer cells. To examine the effect of BA and OA on the viability of TNBC cell line (MDA-MB 231) by analyzing the CD81 expression, intracellular ROS, and apoptosis. The MDA-MB 231 cells was cultured and treated by BA and OA. The viability cell was evaluated by the CCK8 assay. This study analyzed the binding of BA and OA with CD81 using molecular docking and evaluated CD81 expression, intracellular ROS, and apoptosis by flow cytometry. The result showed that BA and OA inhibited viability of MDA-MB-231 cells. BA and OA bind to CD81 in silico, with binding affinities of 9.0kcal/mol for BA and 7.2kcal/mol for OA. Flow cytometry results revealed that BA can downregulate CD81 expression. BA and OA also increased intracellular ROS levels and induced apoptosis. These findings suggest that BA and OA, especially BA, can modulate CD81 expression and promote apoptosis in TNBC cells through the generation of ROS, thereby offering a potential therapeutic strategy for the treatment of TNBC.

Pirfenidone promotes cell cycle arrest and apoptosis of triple‑negative breast cancer cells by suppressing Hedgehog/GLI1 signaling.

Breast cancer is a common malignant tumor in women and triple-negative breast cancer (TNBC) is the most challenging type of breast cancer with poor prognosis. We aimed to elucidate the effects of pirfenidone, a FDA-approved oral anti-fibrotic drug which has recently shown antitumor potential, in the progression of TNBC and the underlying mechanisms. After TNBC cells were treated with pirfenidone, cell viability was evaluated using CCK-8 assay. The EDU staining was applied for reflecting the ability of cell proliferation. Additionally, cell cycle distribution and apoptotic rate of TNBC cells exposed to pirfenidone were determined by flow cytometry. The levels of proteins associated with cell cycle, apoptosis and Hedgehog/gliomaassociated oncogene homolog (GLI)1 signaling was examined through western blot. Then, GLI1 was upregulated to analyze the proliferation, cell cycle and apoptosis of TNBC cells in the presence of pirfenidone to reveal the regulatory mechanism. Pirfenidone dose-dependently decreased the viability and proliferation of MDA-MB-231 and HCC-1937 cells. Besides, the distribution of TNBC cells in G0/G1 phase was significantly elevated by pirfenidone, accompanied by downregulated levels of CyclinD1, CDK4 and CDK6. Simultaneously, pirfenidone caused remarkably increased apoptotic MDA-MB-231 and HCC-1937 cells, coupled with downregulated BCL2 expression as well as upregulated Bax, cleaved caspase3 and cleaved PARP expression. Expression of proteins related to Hedgehog/gliomaassociated oncogene homolog (GLI)1 signaling was tested through western blot. Particularly, GLI1 and PTCH1 levels were dose-dependently inhibited after pirfenidone exposure. Interestingly, GLI1 overexpression attenuated the influences of pirfenidone on the proliferation, cell cycle and apoptosis of TNBC cells. Collectively, pirfenidone arrests the cell cycle and promotes apoptosis of TNBC cells by suppressing Hedgehog/GLI1 signaling.

Role of Harmaline in Inhibiting c-Myc, Altering Molecular Typing, and Promoting Apoptosis in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) lacks effective targeted, endocrine therapeutic agents and the development of novel agents is costly and time-consuming. The objective of this study was to identify pharmaceuticals and natural products utilized in clinical practice that have the potential to inhibit the expression of Cellular-myelocytomatosis oncogene (c-Myc), based on a review of the current literature. The aim was to assess the effect of the specified drugs on c-Myc expression in TNBC cells, determine the most potent inhibitor, and evaluate its impact on TNBC cell proliferation, invasive migration, and apoptosis, as well as the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2) at both the gene and protein levels. Explore its potential for treatment or adjuvant therapy for triple-negative breast cancer. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to quantify gene and protein expression levels. Flow cytometry was employed to measure cell proliferation and apoptosis, while the Transwell assay was utilized to assess cell invasion and migration. Harmaline emerged as the strongest inhibitor, significantly decreasing the expression of c-Myc at both the gene and protein levels in TNBC cells. It also inhibited cell proliferation, invasion, and migration while promoting apoptosis in TNBC cells. Additionally, there was a varying increase in the expression of ER and PR genes and proteins. While the expression of the HER-2 gene was elevated, there was no significant change in HER-2 protein levels. Notably, the expression of the phosphorylated HER-2 protein increased. Harmaline was found to promote apoptosis and inhibit cell proliferation, invasion, and migration in TNBC cells by targeting the inhibition of c-Myc. It also induced the re-expression of the ER, PR, and HER-2 genes, as well as the ER and PR proteins.

Naphthoquinone-derived ZSW-4B induces apoptosis in triple-negative breast cancer via AMPK signalling activation

Triple-negative breast cancer (TNBC) is the most malignant molecular subtype of breast cancer and is characterized by aggressiveness, high mortality, significant heterogeneity, and poor prognosis. AMPK plays a critical role in maintaining the cellular energy balance, and its inactivation is associated with malignant breast cancer. Here, we identified the pharmacological mechanism of the 1,4-naphthoquinone derivative ZSW-4B. MTT, colony formation, and nude mouse xenograft tumour models demonstrated that ZSW-4B selectively inhibits the proliferation of TNBC cells both in vitro and in vivo. Flow cytometry and Western blot analysis revealed that ZSW-4B induces apoptosis in TNBC cells. Phosphoproteomic analysis revealed activation of the AMPK signalling pathway by ZSW-4B. Additionally, the application of the CRISPR-Cas9 system to genetically knockout AMPK in TNBC cell lines was demonstrated to reverse the antitumour effects elicited by ZSW-4B both in vitro and in vivo. In summary, ZSW-4B inhibits TNBC by inducing cellular apoptosis through the activation of AMPK.

TROP2 promotes the proliferation of triple-negative breast cancer cells via calcium ion-dependent ER stress signaling pathway.

To explore the molecular mechanisms of tumor-associated calcium signal transduction factor 2 (TROP2) affecting the occurrence and development of triple-negative breast cancer (TNBC). The TCGA database, immunohistochemical staining, and qRT-PCR were used to analyze the expression of TROP2 in TNBC tissues and cells. The protein expressions of TROP2 and inositol 1,4,5-trisphosphate receptor (IP3R) after TROP2 knockdown were detected by western blot (WB). Cell proliferation was detected by CCK8 and colony formation assay, Annexin V-APC/PI flow cytometry was used to detect apoptosis, and intracellular calcium ion (Ca2+) was detected by flow cytometry with Fura 2-AM fluorescent probe. Finally, the morphological changes of the endoplasmic reticulum (ER) were observed by transmission electron microscopy, and the expression of ER stress (ERS)-related proteins was detected by WB and immunofluorescence staining. TROP2 was up-regulated in TNBC tumor tissues and cells. Silencing TROP2 decreased the proliferation rate and clone formation number, and increased the apoptosis rate and the Ca2+ level in TNBC cells. These phenomena were reversed after the addition of 2-APB. In addition, after TROP2 knockdown, the expressions of IP3R and ERS-related proteins were up-regulated, the ER was cystic dilated, and ERS was activated. And the addition of 2-APB significantly inhibited the activation of ERS induced by TROP2 knockdown. TROP2 regulated the proliferation and apoptosis of TNBC cells through a Ca2+-dependent ERS signaling pathway.

Mechanisms of Yiai Fuzheng formula in the treatment of triple-negative breast cancer based on UPLC-Q-Orbitrap-HRMS, network pharmacology, and experimental validation

Ethnopharmacological relevanceYiai Fuzheng formula (YAFZF), as a Traditional Chinese Medicine (TCM) prescription, has been used widely at Zhongnan Hospital of Wuhan University for its therapeutic effects and high safety on triple-negative breast cancer (TNBC). ObjectiveIn this study, we employed ultra-high-performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap-HRMS), network pharmacology, and experimental validation to elucidate the underlying action mechanism of YAFZF in the treatment of TNBC. MethodsThe key active ingredients in YAFZF were analyzed using UPLC-Q-Orbitrap-HRMS, and then the potential components, target genes and signalling pathways of YAFZF were predicted using the network pharmacological method. We then used molecular docking to visualize the combination characteristics between major active components and macromolecules in the crucial pathway. In vitro experiments were conducted to investigate the inhibitory effects of YAFZF treatment on the cell viability, invasion, and migration of 4T1 and MDA-MB-231 cells. The xenograft TNBC models were constructed using female Balb/c mice, and their body weights, tumour volumes, and weights were monitored during YAFZF treatment. Quantitative real-time PCR (qRT-PCR), Hematoxylin-eosin (HE), immunohistochemistry (IHC) staining, Western blot (WB), and terminal deoxynucleotidyl transferase (TdT)-dUTP nick-end labeling (TUNEL) staining were used for further experimental validation. ResultsBased on UPLC-Q-Orbitrap-HRMS and network pharmacology analysis, 6 major bioactive components and 153 intersecting genes were obtained for YAFZF against TNBC. Functional enrichment analysis identified that the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signalling pathway might be the mechanism of action of YAFZF in the treatment of TNBC. Molecular docking results suggested that the main active compounds in YAFZF had strong binding energies with the proteins in the PI3K/Akt pathway. In vitro experiments showed that YAFZF inhibited the cell viability, invasion, and migration abilities of TNBC cells. Animal experiments confirmed that YAFZF treatment suppressed tumour cell proliferation and increased apoptotic cells. PCR, HE, WB, and IHC results indicated that YAFZF could suppress xenograft tumour metastases by inhibiting the PI3K/AKT/mTOR pathway regulating the epithelial-mesenchymal transition (EMT) process. ConclusionYAFZF therapy showed its potential for reducing proliferation, invasion, and migration abilities, increasing apoptosis of TNBC cells. Furthermore, YAFZF treated TNBC by inhibiting xenograft tumour distant metastases via the regulation of EMT by the PI3K/Akt/mTOR pathway, suggesting that it may be useful as an adjuvant treatment.

Eurochevalierines A-I, Sesquiterpene Alkaloid Hybrids with Anti-Triple Negative Breast Cancer Activity from Penicillium sp. HZ-5.

Nine new sesquiterpene alkaloids, eurochevalierines A-I (1-9), were separated from the rice cultures of the endophytic fungus Penicillium sp. HZ-5 originated from the fresh leaf of Hypericum wilsonii N. Robson. The structures' illumination was conducted by single-crystal X-ray diffraction, extensive spectroscopic analysis, alkaline hydrolysis reaction, and Snatzke's method. Importantly, the antitumor activities screen of these isolates indicated that 1 could suppress triple negative breast cancer (TNBC) cell proliferation and induce apoptosis, with an IC50 value of 5.4 μM, which is comparable to the positive control docetaxel (DXT). Flow cytometry experiments mentioned that compound 1 significantly reduced mitochondrial membrane potential (MMP) of TNBC cells. In addition, 1 could activate caspase-3 and elevated the levels of reactive oxygen species (ROS) and expressions of suppressive cytokines and chemokines. Further Western blot analysis showed that 1 could selectively induce mitochondria-dependent apoptosis in TNBC cells via the BAX/BCL-2 pathway. Remarkably, these finding provide a new natural product skeleton for the treatment of TNBC.

Effect and mechanism of Compound Shougong Powder in attenuating triple-negative breast cancer progression by reversing epithelial-to-mesenchymal transition

The study investigated the effect of Compound Shougong Powder(CSGP) on the biological functions of triple-negative breast cancer(TNBC) cells and whether its mechanism of action was related to the epithelial-mesenchymal transition(EMT) signaling pathway. TNBC cells(MDA-MB-231 and BT-549) were treated with different concentrations of CSGP-containing serum. MTS assay was used to detect the effect of CSGP on the proliferation of TNBC cells. The EdU staining was used to detect the effect of CSGP on the proliferation of TNBC cells. Flow cytometry was used to examine the impact of CSGP on apoptosis of TNBC cells. Wound-healing and Transwell assays were used to evaluate the effects of different concentrations of CSGP on the migration and invasion capabilities of TNBC cells. RNA sequencing technology was utilized to elucidate its mechanism. Subsequently, qRT-PCR was performed to measure the mRNA expression levels of E-cadherin, N-cadherin, Slug, Snail, Vimentin, Twist, Zinc finger E-box-Binding homeobox 1(Zeb1), and Zinc finger E-box-Binding homeobox 2(Zeb2). Western blot was used to assess the protein expression levels of Slug, Vimentin, and E-cadherin. After intervention with CSGP, the proliferation of MDA-MB-231 and BT-549 cells significantly decreased, while the apoptosis rate markedly increased. The expression levels of the epithelial marker protein E-cadherin significantly increased, while the expression levels of the EMT-related transcription factors Slug and Vimentin showed a decrease. In conclusion, CSGP inhibits the EMT, thereby suppressing the malignant progression of TNBC.

MW-19, a dihydropyrazole derivative, induces human triple-negative breast cancer cell apoptosis by targeting apoptosis-related pathways.

Previous studies have indicated that heterocyclic substituted dihydropyrazole derivatives, particularly MW-19, potentially exert anticancer activity invitro; however, the underlying mechanism remains unknown. The present study was designed to investigate the mechanisms underlying MW-19 activity in triple-negative breast cancer cells. A sulforhodamine B assay was performed to evaluate cell proliferation inhibition rates, and the antitumor effect of MW-19 was evaluated in mice with HCC-1806 xenografts. Apoptosis was analyzed by Hoechst 33342 and annexin V/propidium iodide staining. Expression of pro- and antiapoptotic proteins and mRNA were analyzed by western blotting and reverse transcription-quantitative (RT-q) PCR, respectively. We found that MW-19 significantly inhibited HCC-1806 cell proliferation in a dose- and time-dependent manner, and significantly inhibited MDA-MB-231 cell migration. Importantly, oral administration of MW-19 significantly inhibited HCC-1806 tumor growth in BALB/c-nu/nu mice. Moreover, MW-19 treatment induced marked apoptosis and G2/M arrest in the sensitive cell line, HCC-1806. RT-qPCR analysis showed that levels of proapoptotic genes (Bax, caspase-3, caspase-7, and Fas) were considerably increased in the MW-19 group relative to the control group, while those of antiapoptotic factors (Bcl-2, C-MYC) were dramatically decreased. Consistently, Bax, caspase-3, and caspase-7 were significantly induced after MW-19 treatment, while levels of phosphorylated (p-)AKT, p-PI3K, p-ERK, and the antiapoptotic protein, Bcl-2, were clearly diminished, and the P38 MAPK signaling pathway was activated. Furthermore, P38 pharmacological inhibitors abrogated MW-19-induced apoptosis. Together, our findings indicate that MW-19 exerts antitumor effects by targeting PI3K/AKT and ERK/P38 signaling pathways.

TROP2 regulates cisplatin sensitivity of triple-negative breast cancer cells by regulating endoplasmic reticulum stress.

Triple-negative breast cancer (TNBC) is a kind of breast cancer with a high metastasis rate and poor prognosis. As a transmembrane glycoprotein, tumor-associated calcium signal transducer 2 (TROP2) plays a certain role in the cancers. This study aimed to explore the potential mechanism of TROP2 affecting cisplatin (CDDP) resistance in TNBC from endoplasmic reticulum stress (ERS). MDA-MB-231 and CDDP-resistant cell lines MDA-MB-231/CDDP were used in this study, and the expression of TROP2 was detected by western blotting. After transfecting with the interference sequence of siRNA targeting TROP2, cell proliferation and apoptosis were detected by the cell counting kit-8, colony formation, and flow cytometry, and the expression of ERS-marker proteins was detected by western blotting. Furthermore, the effects of ERS in TROP2 on drug resistance of TNBC cells were explored by using ERS inhibitor 4-phenylbutyric acid (4-PBA). Results found that TROP2 expression in MDA-MB-231/CDDP was significantly upregulated compared with MDA-MB-231. The expression of TROP2 in MDA-MB-231/CDDP was significantly decreased after transfection with siRNA-TROP2, and the proliferation of MDA-MB-231 and MDA-MB-231/CDDP cells was significantly decreased after further induction with CDDP. TROP2 significantly affected TNBC cell cloning, apoptosis, and the expression of ERS-related marker proteins, while 4-PBA reversed the promoting effects of siRNA-TROP2 on apoptosis and ERS, as well as the inhibitory effects on cell proliferation, suggesting that TROP2 affected the resistance of TNBC cells to CDDP through ERS. In conclusion, TROP2 inhibited apoptosis of TNBC cells, improved the cell cloning ability, and regulated the sensitivity of TNBC cells to CDDP through ERS.

Lysophosphatidic Acid Receptor 1 (LPA1) Antagonists as Potential Migrastatics for Triple Negative Breast Cancer.

Metastasis is responsible for about 90 % of cancer deaths. Anti-metastatic drugs, termed as migrastatics, offer a distinctive therapeutic approach to address cancer migration and invasion. However, therapeutic exploitation of metastasis-specific targets remains limited, and the effective prevention and suppression of metastatic cancer continue to be elusive. Lysophosphatidic acid receptor 1 (LPA1) is activated by an endogenous lipid molecule LPA, leading to a diverse array of cellular activities. Previous studies have shown that the LPA/LPA1 axis supports the progression of metastasis for many types of cancer. In this study, we report the synthesis and biological evaluation of fluorine-containing triazole derivatives as potent LPA1 antagonists, offering potential as migrastatic drugs for triple negative breast cancer (TNBC). In particular, compound 12 f, the most potent and highly selective in this series with an IC50 value of 16.0 nM in the cAMP assay and 18.4 nM in the calcium mobilization assay, inhibited cell survival, migration, and invasion in the TNBC cell line. Interestingly, the compound did not induce apoptosis in TNBC cells and demonstrated no cytotoxic effects. These results highlight the potential of LPA1 as a migrastatic target. Consequently, the LPA1 antagonists developed in this study hold promise as potential migrastatic candidates for TNBC.

Abstract PO5-27-07: A novel ROR1 inhibitor CPD86 suppresses Triple-Negative Breast Cancer cells via regulation of AKT/GSK3β pathway

Abstract Breast cancer is the second most commonly diagnosed cancer in women in the United States. The most aggressive subtype is triple negative breast cancer (TNBC), which accounts for 15-20% of new cases on average each year. Histologically, it is characterized by the absence of estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor 2 (HER2), which are typically targeted by hormone therapies such as tamoxifen, fulvestrant, or letrozole. Standard of care for TNBC includes traditional chemotherapy and radiation, which are detrimental to both cancerous and normal cells. Therefore, the lack of all hormone receptors in TNBC calls for the investigation of novel anti-cancer compounds that specifically target TNBC with minimal damage to non-cancerous tissues. Receptor orphan tyrosine kinase-like receptor 1 (ROR1) is an oncoprotein that is overexpressed in several human malignancies such as lung, breast, prostate and pancreatic cancers, but not in normal tissues. Inhibition of ROR1 signaling has shown to repress proliferation and induce apoptosis of cancer cells. Through in-silico docking and isothermal titration calorimetry, we identified a novel compound CPD86 that interacts with and inhibits ROR1. We hypothesize that CPD86 selectively targets TNBC cells by reducing the phosphorylation of AKT/GSK3β via the inhibition of ROR1. Our in vitro results suggest that CPD86 inhibits cell viability and induces intrinsic apoptosis of TNBC cells at a half-maximal inhibitory concentration of 2-5 µM, but not in normal breast epithelial cells with minimal ROR1 expression. CPD86 also represses TNBC cell migration and invasion while leaving non-malignant cells unharmed. This study highlights that ROR1 inhibitors could be developed as a potential therapeutic for TNBC and the data would serve as proof-of-concept justification for evaluation in other ROR1-upregulated cancers. Citation Format: Tram Ta, Victoria Reed, Nikhil Chandra, Nick Dwyer, Norman Fultang, Shradheya Gupta, Indrakant Singh, Bela Peethambaran. A novel ROR1 inhibitor CPD86 suppresses Triple-Negative Breast Cancer cells via regulation of AKT/GSK3β pathway [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-27-07.

H3K27ac-induced RHOXF2 activates Wnt2/β-catenin pathway by binding to HOXC13 to aggravate the malignant progression of triple negative breast cancer

Triple negative breast cancer (TNBC) is insensitive to conventional targeted therapy and endocrine therapy, and is characterized by high invasiveness and high recurrence rate. This study aimed to explore the role and mechanism of RHOXF2 and HOXC13 on the malignant progression of TNBC. RT-qPCR and western blot were used to detect RHOXF2 and HOXC13 expression in TNBC cells. The proliferation, colony formation, invasion, migration, apoptosis and cell cycle of TNBC cells after transfection were analyzed by CCK-8 assay, colony formation assay, transwell assay, wound healing assay and flow cytometry analysis. Co-Immunoprecipitation and GST pull-down assays were used to analyze the combination between RHOXF2 and HOXC13. ChIP-PCR and luciferase reporter gene assay were used to examine the regulation of H3K27ac on RHOXF2. Besides, the expression of Ki67 and cleaved Caspase3 in tumor tissues of nude mice was determined by immunofluorescence. Results revealed that RHOXF2 and HOXC13 expression was increased in TNBC cells. RHOXF2 knockdown suppressed the proliferation, invasion and migration, as well as induced G0/G1 cell cycle arrest and apoptosis of TNBC cells. Besides, RHOXF2 could bind to HOXC13 and RHOXF2 knockdown suppressed HOXC13 expression in TNBC cells. Furthermore, HOXC13 overexpression reversed the impacts of RHOXF2 downregulation on the proliferation, invasion, migration, G0/G1 cell cycle arrest and apoptosis of TNBC cells. In addition, RHOXF2 silencing limited the tumor volume in nude mice, which was reversed by HOXC13 overexpression. Moreover, RHOXF2 knockdown interfered with Wnt2/β-catenin pathway in vitro and in vivo by binding to HOXC13. Importantly, H3K27ac acetylation could activate the expression of RHOXF2 promoter region. In conclusion, RHOXF2 activated by H3K27ac functioned as a tumor promoter in TNBC via mediating Wnt2/β-catenin pathway by binding to HOXC13, which provided promising insight into exploration on TNBC therapy.

Biotin-stapled mixed micelles of lapatinib: Unleashing apoptosis power in triple-negative breast cancer

The treatment options currently offered for triple-negative breast cancer (TNBC) encounter numerous obstacles, including disappointing overall survival rates, unavoidable side effects, and the development of drug resistance. Additionally, there is a growing need for nutrients, like vitamins (e.g., biotin), to sustain the unregulated growth of TNBC cells. Hence, targeted therapy (LPT B-MM) for enhancing the treatment of TNBC was explored which encompassed hydrophobized [using α-TOS (α-Tocopheryl succinate] Pluronic-based mixed micelles having inherent sodium-dependent multivitamin transporter (SMVT) targeting (biotin-stapled) ability. LPT B-MM had optimal size (210.72± 25.88 nm), PDI (0.272± 0.023), and enhanced drug loading capacity (∼13.31± 2.12%). Transmission electron microscopy validated the spherical shape of these micelles. In vitro release studies demonstrated drug release in a sustained manner without the risk of hemolysis. Importantly, LPT B-MM displayed heightened cellular uptake, increased cytotoxicity, a lower IC50 value, elevated reactive oxygen species, induced mitochondrial membrane depolarization, and a greater apoptosis index in TNBC cell lines compared to free LPT. This study highlights the considerable potential of LPT B-MM to induce apoptosis, showcasing its promise in enhancing payload capabilities and enabling SMVT specific targeting within the context of TNBC. LPT B-MM demonstrated the synergistic effects of LPT and α-TOS in inducing apoptosis in TNBC cells, leading to enhanced therapeutic efficacy. This novel combination therapy offers a promising strategy for improving treatment outcomes in TNBC patients and represents a significant advancement in the field of TNBC therapy.

Ononin triggers ferroptosis-mediated disruption in the triple negative breast cancer both in vitro and in vivo

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that is difficult to treat due to a lack of targeted therapies. In this study, we aimed to investigate whether a natural flavonoid compound called ononin could be effective in treating TNBC by triggering ferroptosis in MDA-MB-231 and 4 T1 cell lines, and MDA-MB-231-xenograft nude mice model. Ononin inhibited TNBC through ferroptosis, which was determined by MTT assay, flow cytometry, RT-PCR, immunofluorescence, transmission electron microscopy, histological analysis, western blot and bioluminescence assay. Our results showed that treatment with ononin led to increased levels of malondialdehyde and reactive oxygen species and decreased activity of superoxide dismutase, which are indicatives of ferroptosis. We also found that ononin downregulated two key markers of ferroptosis, SLC7A11 and Nrf2, at both the transcriptional and translational level. Additionally, the administration of ononin resulted in a notable decrease in tumor size and weight in the mouse model. Furthermore, it was observed to enhance the rate of apoptosis in TNBC cells. Importantly, ononin did not induce any histological changes in the kidney, liver, and heart. Taken together, our findings suggest that ononin could be a promising therapeutic strategy for TNBC, and that it works by disrupting the Nrf2/SLC7A11 axis through ferroptosis. These results are encouraging and may lead to the development of new treatments for this challenging cancer subtype.

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 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.

DDX58 deficiency leads to triple negative breast cancer chemotherapy resistance by inhibiting Type I IFN-mediated signalling apoptosis.

Triple-negative breast cancer (TNBC) is characterized by its aggressive nature and absence of specific therapeutic targets, necessitating the reliance on chemotherapy as the primary treatment modality. However, the drug resistance poses a significant challenge in the management of TNBC. In this study, we investigated the role of DDX58 (DExD/H-box helicase 58), also known as RIG-I, in TNBC chemoresistance. The relationship between DDX58 expression and breast cancer prognosis was investigated by online clinical databases and confirmed by immunohistochemistry analysis. DDX58 was knockout by CRISPR-Cas9 system (DDX58-KO), knockdown by DDX58-siRNA (DDX58-KD), and stably over expressed (DDX58-OE) by lentivirus. Western blotting, immunofluorescence and qPCR were used for related molecules detection. Apoptosis was analyzed through flow cytometry (Annexin V/7AAD apoptosis assay) and Caspase 3/7 activity assay. Patients with lower expression of DDX58 led to lower rate of pathological complete response (pCR) and worse prognosis by online databases and hospital clinical data. DDX58-KD cells showed multiple chemo-drugs resistance (paclitaxel, doxorubicin, 5-fluorouracil) in TNBC cell lines. Similarly, DDX58-KO cells also showed multiple chemo-drugs resistance in a dosage-dependent manner. In the CDX model, tumours in the DDX58-KO group had a 25% reduction in the tumour growth inhibition rate (IR) compared to wild-type (WT) group after doxorubicin (Dox) treatment. The depletion of DDX58 inhibited proliferation and promoted the migration and invasion in MDA-MB-231 cells. The findings of our research indicated that DDX58-KO cells exhibit a reduction in Dox-induced apoptosis both in vivo and in vitro. Mechanistically, Dox treatment leads to a significant increase in the expression of double-stranded RNAs (dsRNAs) and activates the DDX58-Type I interferon (IFN) signaling pathway, ultimately promoting apoptosis in TNBC cells. In the process of TNBC chemotherapy, the deficiency of DDX58 can inhibit Dox-induced apoptosis, revealing a new pathway of chemotherapy resistance, and providing a possibility for developing personalized treatment strategies based on DDX58 expression levels.

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.