Suberoylanilide Hydroxamic Acid,an Inhibitor of Histone Deacetylase,Enhances Radiosensitivity and Suppresses Lung Metastasis in Breast Cancer In Vitro and In Vivo

Triple-negative breast cancer (TNBC), defined by the absence of an estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression, is associated with an early recurrence of disease and poor outcome. Furthermore, the majority of deaths in breast cancer patients are from metastases instead of from primary tumors. In this study, MCF-7 (an estrogen receptor-positive human breast cancer cell line), MDA-MB-231 (a human TNBC cell line) and 4T1 (a mouse TNBC cell line) were used to investigate the anti-cancer effects of ionizing radiation (IR) combined with suberoylanilide hydroxamic acid (SAHA, an inhibitor of histone deacetylase (HDAC)) and to determine the underlying mechanisms of these effects in vitro and in vivo. We also evaluated the ability of SAHA to inhibit the metastasis of 4T1 cells. We found that IR combined with SAHA showed increased therapeutic efficacy when compared with either treatment alone in MCF-7, MDA-MB-231 and 4T1 cells. Moreover, the combined treatment enhanced DNA damage through the inhibition of DNA repair proteins. The combined treatment was induced primarily through autophagy and ER stress. In an orthotopic breast cancer mouse model, the combination treatment showed a greater inhibition of tumor growth. In addition, SAHA inhibited the migration and invasion abilities of 4T1 cells and inhibited breast cancer cell migration by inhibiting the activity of MMP-9. In an in vivo experimental metastasis mouse model, SAHA significantly inhibited lung metastasis. SAHA not only enhances radiosensitivity but also suppresses lung metastasis in breast cancer. These novel findings suggest that SAHA alone or combined with IR could serve as a potential therapeutic strategy for breast cancer.     

G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration,Invasion, and Metastasis

Triple negative breast cancer (TNBC) is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3) is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis.     

Lamin B2 promotes the progression of triple negative breast cancer via mediating cell proliferation and apoptosis

Triple negative breast cancer (TNBC) is a more common type of breast cancer with high distant metastasis and poor prognosis. The potential role of lamins in cancer progression has been widely revealed. However, the function of lamin B2 (LMNB2) in TNBC progression is still unclear. The present study aimed to investigate the role of LMNB2 in TNBC. The cancer genome atlas (TCGA) database analysis and immunohistochemistry (IHC) were performed to examine LMNB2 expression levels. LMNB2 short hairpin RNA plasmid or lentivirus was used to deplete the expression of LMNB2 in human TNBC cell lines including MDA-MB-468 and MDA-MB-231. Alterations in cell proliferation and apoptosis in vitro and the nude mouse tumorigenicity assay in vivo were subsequently analyzed. The human TNBC tissues shown high expression of LMNB2 according to the bioinformation analysis and IHC assays. LMNB2 expression was correlated with the clinical pathological features of TNBC patients, including pTNM stage and lymph node metastasis. Through in vitro and in vivo assays, we confirmed LMNB2 depletion suppressed the proliferation and induced the apoptosis of TNBC cells, and inhibited tumor growth of TNBC cells in mice, with the decrease in Ki67 expression or the increase in caspase-3 expression. In conclusion, LMNB2 may promote TNBC progression and could serve as a potential therapeutic target for TNBC treatment.     

Shuganning injection,a traditional Chinese patent medicine,induces ferroptosis and suppresses tumor growth in triple-negative breast cancer cells

BackgroundTriple-negative breast cancer (TNBC), lacking targeted therapies currently, is susceptible to ferroptosis, a recently defined form of cell death.PurposeTo evaluate the anticancer activity of Shuganning injection (SGNI), a traditional Chinese patent medicine, on TNBC cells; To elucidate the mechanism of SGNI induced ferroptosis.MethodsThe anticancer activity of SGNI was examined via in vitro cell proliferation assays and in vivo xenograft growth assay. Ferroptosis was determined by flow-cytometric analysis of lipid ROS, labile iron pool measurement, and propidium iodide exclusion assay. The dependency on heme oxygenase 1 (HO-1) of SGNI induced ferroptosis was confirmed by genetic knockdown and pharmacological inhibition of the protein.ResultsSGNI selectively inhibited the proliferation of TNBC cells compared to non-TNBC breast cancer cells and normal cells. The cell death induced by SGNI in TNBC cells showed distinct morphology from apoptosis and could not be rescued by the pan-caspase inhibitor Z-VAD(OMe)-FMK. On the other hand, SGNI induced cell death was blocked by the lipid ROS scavengers ferrostatin-1 and liproxstatin-1, the acyl-CoA synthetase long chain family member 4 inhibitor rosiglitazone, and the iron chelators 1,10-phenanthroline and deferoxamine. These data indicated that SGNI induced a ferroptotic cell death of TNBC cells. Mechanistically, SGNI induced ferroptosis was dependent on HO-1, which promotes intracellular labile iron pool accumulation, and was alleviated by HO-1 knockdown and inhibition by tin protoporphyrin IX. In line with the in vitro data, SGNI significantly inhibited the xenograft growth of TNBC cell line MD-MB-231 in nude mice.ConclusionCollectively, our study elaborates on a promising regimen for TNBC treatment through induction of ferroptosis by SGNI, a traditional Chinese patent medicine currently available in the clinic, which merits further investigation.     

Mitochondrial Protein UCP1 Inhibits the Malignant Behaviors of Triple-negative Breast Cancer through Activation of Mitophagy and Pyroptosis

Triple-negative breast cancer (TNBC) is a massive threat to women''s health due to its high morbidity, malignancy, and the refractory, effective therapeutic option of TNBC is still deficient. The mitochondrial protein showed therapeutic potential on breast cancer, whereas the mechanism and downstream pathway of mitochondrial uncoupling protein 1 (UCP1) was not fully elucidated. We found that UCP1 was negatively regulated to the process of TNBC. Overexpressing UCP1 could inhibit the proliferation and metastasis of TNBC, meanwhile inducing the mitochondrial swelling and activation of mitophagy in vitro. Mitophagy activation was then assessed to elucidate whether it was downstream of UCP1 in TNBC metastasis. GSDME is the core of pyroptosis. We found that GSDME was activated in the TNBC cells when UCP1 levels were high. It regulates TNBC cell proliferation potential instead of the apoptosis process in vitro and in vivo. Our results suggested that UCP1 could inhibit the process of TNBC by activating mitophagy and pyroptosis. Impaired activation of mitophagy weakens the regulation effect of UCP1 on metastasis of TNBC, similar to the impairment of GSDME activation on the proliferation regulation of UCP1 on TNBC. UCP1 might be a novel therapeutic target of TNBC.     

MUC4 Overexpression Augments Cell Migration and Metastasis through EGFR Family Proteins in Triple Negative Breast Cancer Cells

Introduction

Current studies indicate that triple negative breast cancer (TNBC), an aggressive breast cancer subtype, is associated with poor prognosis and an early pattern of metastasis. Emerging evidence suggests that MUC4 mucin is associated with metastasis of various cancers, including breast cancer. However, the functional role of MUC4 remains unclear in breast cancers, especially in TNBCs.

Method

In the present study, we investigated the functional and mechanistic roles of MUC4 in potentiating pathogenic signals including EGFR family proteins to promote TNBC aggressiveness using in vitro and in vivo studies. Further, we studied the expression of MUC4 in invasive TNBC tissue and normal breast tissue by immunostaining.

Results

MUC4 promotes proliferation, anchorage-dependent and-independent growth of TNBC cells, augments TNBC cell migratory and invasive potential in vitro, and enhances tumorigenicity and metastasis in vivo. In addition, our studies demonstrated that MUC4 up-regulates the EGFR family of proteins, and augments downstream Erk1/2, PKC-γ, and FAK mediated oncogenic signaling. Moreover, our studies also showed that knockdown of MUC4 in TNBC cells induced molecular changes suggestive of mesenchymal to epithelial transition. We also demonstrated in this study, for the first time, that knockdown of MUC4 was associated with reduced expression of EGFR and ErbB3 (EGFR family proteins) in TNBC cells, suggesting that MUC4 uses an alternative to ErbB2 mechanism to promote aggressiveness. We further demonstrate that MUC4 is differentially over-expressed in invasive TNBC tissues compared to normal breast tissue.

Conclusions

MUC4 mucin expression is associated with TNBC pathobiology, and its knockdown reduced aggressiveness in vitro, and tumorigenesis and metastasis in vivo. Overall, our findings suggest that MUC4 mucin promotes invasive activities of TNBC cells by altering the expression of EGFR, ErbB2, and ErbB3 molecules and their downstream signaling.     

5′-AMP-activated Protein Kinase (AMPK) Supports the Growth of Aggressive Experimental Human Breast Cancer Tumors

Rapid tumor growth can establish metabolically stressed microenvironments that activate 5′-AMP-activated protein kinase (AMPK), a ubiquitous regulator of ATP homeostasis. Previously, we investigated the importance of AMPK for the growth of experimental tumors prepared from HRAS-transformed mouse embryo fibroblasts and for primary brain tumor development in a rat model of neurocarcinogenesis. Here, we used triple-negative human breast cancer cells in which AMPK activity had been knocked down to investigate the contribution of AMPK to experimental tumor growth and core glucose metabolism. We found that AMPK supports the growth of fast-growing orthotopic tumors prepared from MDA-MB-231 and DU4475 breast cancer cells but had no effect on the proliferation or survival of these cells in culture. We used in vitro and in vivo metabolic profiling with [¹³C]glucose tracers to investigate the contribution of AMPK to core glucose metabolism in MDA-MB-231 cells, which have a Warburg metabolic phenotype; these experiments indicated that AMPK supports tumor glucose metabolism in part through positive regulation of glycolysis and the nonoxidative pentose phosphate cycle. We also found that AMPK activity in the MDA-MB-231 tumors could systemically perturb glucose homeostasis in sensitive normal tissues (liver and pancreas). Overall, our findings suggest that the contribution of AMPK to the growth of aggressive experimental tumors has a critical microenvironmental component that involves specific regulation of core glucose metabolism.     

Anti-metastatic activity of MPT0G211, a novel HDAC6 inhibitor,in human breast cancer cells in vitro and in vivo

Triple-negative breast cancer (TNBC) is associated with an increased risk of metastasis and a poor prognosis. The invasive ability of TNBC relies on actin reorganization and is regulated by histone deacetylase 6 (HDAC6). The present study aimed to examine the effect of MPT0G211, a novel HDAC6 inhibitor, on cell migration and microtubule association in both in vitro and in vivo models of TNBC. Here MPT0G211 more selectively and potently targeted and inhibited HDAC6, compared with tubastatin A, another selective HDAC6 inhibitor. In vitro, MPT0G211 decreased the migration of the TNBC cell line MDA-MB-231, particularly when administered together with paclitaxel, and increased heat shock protein 90 (Hsp90) acetylation, leading to the dissociation of Hsp90 from aurora-A and proteasomal degradation. Furthermore, MPT0G211 significantly disrupted F-actin polymerization by increasing cortactin acetylation and downregulating slingshot protein phosphatase 1 (SSH1) and active cofilin expression. In vivo, MPT0G211 treatment significantly ameliorated TNBC metastasis. In conclusion, our results demonstrate that MPT0G211 reduces TNBC cell motility by promoting cortactin acetylation and aurora-A degradation, and inhibiting the cofilin–F-actin pathway via HDAC6 activity attenuation. MPT0G211 therefore demonstrates therapeutic potential for invasive TNBC.     

Antiproliferative and Apoptotic Effects of pH-Responsive Veratric Acid-Loaded Polydopamine Nanoparticles in Human Triple Negative Breast Cancer Cells

Veratric acid (VA) is plant-derived phenolic acid known for its therapeutic potential, but its anticancer effect on highly invasive triple-negative breast cancer (TNBC) is yet to be evaluated. Polydopamine nanoparticles (nPDAs) were chosen as the drug carrier to overcome VA's hydrophobic nature and ensure a sustained release of VA. We prepared pH-sensitive nano-formulations of VA-loaded nPDAs and subjected them to physicochemical characterization and in vitro drug release studies, followed by cell viability and apoptotic assays on TNBC cells (MDA-MB-231 cells). The SEM and zeta analysis revealed spherical nPDAs were uniform size distribution and good colloidal stability. In vitro drug release from VA-nPDAs was sustained, prolonged and pH-sensitive, which could benefit tumor cell targeting. MTT and cell viability assays showed that VA-nPDAs (IC50=17.6 μM) are more antiproliferative towards MDA-MB-231 cells than free VA (IC50=437.89 μM). The induction of early and late apoptosis by VA-nPDAs in the cancer cells was identified using annexin V and dead cell assay. Thus, the pH response and sustained release of VA from nPDAs showed the potential to enter the cell, inhibit cell proliferation, and induce apoptosis in human breast cancer cells, indicating the anticancer potential of VA.     

The deubiquitinase USP28 stabilizes the expression of RecQ family helicases and maintains the viability of triple negative breast cancer cells

Triple-negative breast cancer (TNBC) lacks significant expression of the estrogen receptor, the progesterone receptor, and of human epidermal growth factor receptor. It is the most aggressive and malignant of all breast cancers, and for which, there are currently no effective targeted therapies. We have shown previously that the RecQ helicase family member RECQL5 is essential for the proliferation and survival of TNBC cells; however, the mechanism of its involvement in cell viability has not been shown. Here, we report that the expression of RecQ family helicases, including RECQL5, is regulated by the deubiquitinase USP28. We found using genetic depletion or a small molecule inhibitor that like RECQL5, USP28 is also essential for TNBC cells to proliferate in vitro and in vivo. Compromising the function of USP28 by shRNA knockdown or the inhibitor caused TNBC cells to arrest in S/G2 phases, concurrent with DNA-damage checkpoint activation. We further showed that the small molecule inhibitor of USP28 displayed anti-tumor activity against xenografts derived from TNBC cells. Our results suggest that USP28 could be a potential therapeutic target for triple negative breast cancer.     

Ephrin receptor A10 monoclonal antibodies and the derived chimeric antigen receptor T cells exert an antitumor response in mouse models of triple-negative breast cancer

Expression of the receptor tyrosine kinase ephrin receptor A10 (EphA10), which is undetectable in most normal tissues except for the male testis, has been shown to correlate with tumor progression and poor prognosis in several malignancies, including triple-negative breast cancer (TNBC). Therefore, EphA10 could be a potential therapeutic target, likely with minimal adverse effects. However, no effective clinical drugs against EphA10 are currently available. Here, we report high expression levels of EphA10 in tumor regions of breast, lung, and ovarian cancers as well as in immunosuppressive myeloid cells in the tumor microenvironment. Furthermore, we developed anti-EphA10 monoclonal antibodies (mAbs) that specifically recognize cell surface EphA10, but not other EphA family isoforms, and target tumor regions precisely in vivo with no apparent accumulation in other organs. In syngeneic TNBC mouse models, we found that anti-EphA10 mAb clone #4 enhanced tumor regression, therapeutic response rate, and T cell–mediated antitumor immunity. Notably, the chimeric antigen receptor T cells derived from clone #4 significantly inhibited TNBC cell viability in vitro and tumor growth in vivo. Together, our findings suggest that targeting EphA10 via EphA10 mAbs and EphA10-specific chimeric antigen receptor–T cell therapy may represent a promising strategy for patients with EphA10-positive tumors.     

Effect arylamine N-acetyltransferase 1 on morphology,adhesion, migration,and invasion of MDA-MB-231 cells: role of matrix metalloproteinases and integrin αV

ABSTRACT

Reducted arylamine N-acetyltransferase (NAT1) in breast cancers is associated with poor patient survival. NAT1 has also been associated with changes in cancer cell survival and invasion both in vitro and in vivo. Here, we report the effects of NAT1 in cancer cell invasion by addressing its role in adherence, migration, and invasion in vitro. The NAT1 gene was deleted in MDA-MB-231, HT-29 and HeLa cells using CRISPR/Cas9 gene editing. Loss of NAT1 increased adherence to collagen in all three cell-lines but migration was unaffected. NAT1 deletion decreased invasion and induced changes to cell morphology. These effects were independent of matrix metalloproteinases but were related to integrin ITGαV expression. The data suggest NAT1 is important in adhesion and invasion through integrin expression.     

UV radiation resistance-associated gene (UVRAG) promotes cell proliferation,migration, invasion by regulating cyclin-dependent kinases (CDK) and integrin-β/Src signaling in breast cancer cells

Breast cancer is a highly heterogeneous group of human cancer with distinct genetic, biological and clinicopathological features. Triple-negative breast cancer (TNBC) is the most aggressive and metastatic type of breast cancer and associated with poor patient survival. However, the role of UV Radiation Resistance-Associated Gene (UVRAG) in TNBC remains unknown. Here, we report that UVRAG is highly upregulated in all TNBC cells and its knockdown leads to the inhibition of cell proliferation, colony formation and progression of cell cycle, which is associated with and reduced expression of cell cycle related protein expression, including Cyclin A2, B1, D1, cdc2 and cdk6 in TNBC cells. Inhibition of UVRAG also suppressed cell motility, migration and invasion of TNBC cells by inhibition of Integrin β1 and β3 and Src activity. Our findings suggest for the first time that UVRAG expression contributes to proliferation, cell cycle progression, motility/migration and invasion of TNBC cells. Thus, targeting UVRAG could be a potential strategy in breast cancer especially against TNBC.

     

Metabotropic Glutamate Receptor-1 Contributes to Progression in Triple Negative Breast Cancer

TNBC is an aggressive breast cancer subtype that does not express hormone receptors (estrogen and progesterone receptors, ER and PR) or amplified human epidermal growth factor receptor type 2 (HER2), and there currently exist no targeted therapies effective against it. Consequently, finding new molecular targets in triple negative breast cancer (TNBC) is critical to improving patient outcomes. Previously, we have detected the expression of metabotropic glutamate receptor-1 (gene: GRM1; protein: mGluR1) in TNBC and observed that targeting glutamatergic signaling inhibits TNBC growth both in vitro and in vivo. In this study, we explored how mGluR1 contributes to TNBC progression, using the isogenic MCF10 progression series, which models breast carcinogenesis from nontransformed epithelium to malignant basal-like breast cancer. We observed that mGluR1 is expressed in human breast cancer and that in MCF10A cells, which model nontransformed mammary epithelium, but not in MCF10AT1 cells, which model atypical ductal hyperplasia, mGluR1 overexpression results in increased proliferation, anchorage-independent growth, and invasiveness. In contrast, mGluR1 knockdown results in a decrease in these activities in malignant MCF10CA1d cells. Similarly, pharmacologic inhibition of glutamatergic signaling in MCF10CA1d cells results in a decrease in proliferation and anchorage-independent growth. Finally, transduction of MCF10AT1 cells, which express c-Ha-ras, using a lentiviral construct expressing GRM1 results in transformation to carcinoma in 90% of resultant xenografts. We conclude that mGluR1 cooperates with other factors in hyperplastic mammary epithelium to contribute to TNBC progression and therefore propose that glutamatergic signaling represents a promising new molecular target for TNBC therapy.     

Extracellular vesicles extracted from bone marrow mesenchymal stem cells carrying MicroRNA-342-3p inhibit the INHBA/IL13Rα2 axis to suppress the growth and metastasis of breast cancer

Increasing focus has come to the role of extracellular vesicles (EVs) in various cancers. Hence, we designed this study to explore the mechanism whereby microRNA-342-3p (miR-342-3p)-containing EVs derived from BMSCs might affect breast cancer. MCF-7 breast cancer cell line was co-incubated with the EVs isolated from rat BMSCs, followed by alteration of miR-342-3p and INHBA expression. Microarray-based analyses predicted a possible regulatory mechanism involving miR-342-3p, INHBA, and IL13Rα2 in breast cancer, which was verified by luciferase reporter, RNA pull-down, and RIP assays. Besides, in order to evaluate the effects of miR-342-3p on the biological features of breast cancer cells in vitro and in vivo, we employed the scratch assay, Transwell assay, CCK-8 assay, and nude mouse tumorigenicity assay. miR-342-3p carried by BMSC-EVs was transferred into breast cancer cells through co-culture, which inhibited the proliferation and metastasis of breast cancer cells in vitro. miR-342-3p downregulated the expression of INHBA, which further repressed the expression of IL13Rα2. Finally, the in vivo experimental results revealed the inhibitory role of miR-342-3p in tumor growth and metastasis in nude mice. To sum up, BMSC-EVs carrying miR-342-3p could prevent breast cancer growth and metastasis by downregulating the INHBA/IL13Rα2 axis, highlighting a potential target for anti-cancer treatment for breast cancer.     

Protection by α-tocopherol but not ascorbic acid from hydrogen peroxide induced cell death in normal human breast epithelial cells in culture

α-Tocopherol and ascorbic acid have been suggested to play a role in breast cancer prevention due to their antioxidative capacity. Increased exposure to endogenous and exogenous sex steroids is a known risk factor for breast cancer. We have studied the effects of α-tocopherol and ascorbic acid on hydrogen peroxide induced cell death in sex hormone treated normal breast epithelial cells in culture. We found that α-tocopherol but not ascorbic acid alone protected the cells. The effect of α-tocopherol increased when ascorbic acid was added to the cultures. The hydrogen peroxide degradation rate decreased in cultures treated with α-tocopherol alone and in combination with ascorbic acid compared to cells grown in medium or with ascorbic acid only. Oestradiol and progesterone treatment did not influence the results. Possible beneficial effects of combining various antioxidants, endogenous as well as exogenous, on human breast tissue need to be investigated further both in vivo and in vitro.     

Picroside III,an Active Ingredient of Picrorhiza scrophulariiflora,Ameliorates Experimental Colitis by Protecting Intestinal Barrier Integrity

This study aims to explore the protective effects of Picroside III, an active ingredient of Picrorhiza scrophulariiflora, on the intestinal epithelial barrier in tumor necrosis factor-α (TNF-α) induced Caco-2 cells and dextran sulfate sodium (DSS) induced colitis in mice. Results show that Picroside III significantly alleviated clinical signs of colitis including body weight loss, disease activity index increase, colon shortening, and colon tissue damage. It also increased claudin-3, ZO-1 and occludin expressions and decreased claudin-2 expression in the colon tissues of mice with colitis. In vitro, Picroside III also significantly promoted wound healing, decreased the permeability of cell monolayer, upregulated the expressions of claudin-3, ZO-1 and occludin and downregulated the expression of claudin-2 in TNF-α treated Caco-2 cells. Mechanism studies show that Picroside III significantly promoted AMP-activated protein kinase (AMPK) phosphorylation in vitro and in vivo, and blockade with AMPK could significantly attenuate the upregulation of Picroside III in ZO-1 and occludin expressions and the downregulation of claudin-2 expression in TNF-α treated Caco-2 cells. In conclusion, this study demonstrates that Picroside III attenuated DSS-induced colitis by promoting colonic mucosal wound healing and epithelial barrier function recovery via the activation of AMPK.     

Steroid Receptor Coactivator-3 (SRC-3/AIB1) as a Novel Therapeutic Target in Triple Negative Breast Cancer and Its Inhibition with a Phospho-Bufalin Prodrug

Triple negative breast cancer (TNBC) has the poorest prognosis of all types of breast cancer and currently lacks efficient targeted therapy. Chemotherapy is the traditional standard-of-care for TNBC, but is frequently accompanied by severe side effects. Despite the fact that high expression of steroid receptor coactivator 3 (SRC–3) is correlated with poor survival in estrogen receptor positive breast cancer patients, its role in TNBC has not been extensively investigated. Here, we show that high expression of SRC–3 correlates with both poor overall survival and post progression survival in TNBC patients, suggesting that SRC–3 can serve as a prognostic marker for TNBC. Furthermore, we demonstrated that bufalin, a SRC–3 small molecule inhibitor, when introduced even at nM concentrations, can significantly reduce TNBC cell viability and motility. However, because bufalin has minimal water solubility, its in vivo application is limited. Therefore, we developed a water soluble prodrug, 3-phospho-bufalin, to facilitate its in vivo administration. In addition, we demonstrated that 3-phospho-bufalin can effectively inhibit tumor growth in an orthotopic TNBC mouse model, suggesting its potential application as a targeted therapy for TNBC treatment.