Inhibits Triple-negative Breast Cancer Metastasis Research Articles

Baohuoside I chemosensitises breast cancer to paclitaxel by suppressing extracellular vesicle/CXCL1 signal released from apoptotic cells.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and chemotherapy is the cornerstone treatment for TNBC. Regrettably, emerging findings suggest that chemotherapy facilitates pro-metastatic changes in the tumour microenvironment. Extracellular vesicles (EVs) have been highly implicated in cancer drug resistance and metastasis. However, the effects of the EVs released from dying cancer cells on TNBC prognosis and corresponding therapeutic strategies have been poorly investigated. This study demonstrated that paclitaxel chemotherapy elicited CXCL1-enriched EVs from apoptotic TNBC cells (EV-Apo). EV-Apo promoted the chemoresistance and invasion of co-cultured TNBC cells by polarizing M2 macrophages through activating PD-L1 signalling. However, baohuoside I (BHS) remarkably sensitized the co-cultured TNBC cells to paclitaxel chemotherapy via modulating EV-Apo signalling. Mechanistically, BHS remarkably decreased C-X-C motif chemokine ligand 1 (CXCL1) cargo within EV-Apo and therefore attenuated macrophage M2 polarization by suppressing PD-L1 activation. Additionally, BHS decreased EV-Apo release by diminishing the biogenesis of intraluminal vesicles (ILVs) within multivesicular bodies (MVBs) of TNBC cells. Furthermore, BHS bound to the LEU104 residue of flotillin 2 (FLOT2) and interrupted its interaction with RAS oncogene family member 31 (RAB31), leading to the blockage of RAB31-FLOT2 complex-driven ILV biogenesis. Importantly, BHS remarkably chemosensitised paclitaxel to inhibit TNBC metastasis in vivo by suppressing EV-ApoCXCL1-induced PD-L1 activation and M2 polarization of tumour-associated macrophages (TAMs). This pioneering study sheds light on EV-ApoCXCL1 as a novel therapeutic target to chemosensitise TNBC, and presents BHS as a promising chemotherapy adjuvant to improve TNBC chemosensitivity and prognosis by disturbing EV-ApoCXCL1 biogenesis.

PH-responsive coacervate with adrenergic nerve blockade function inhibits triple-negative breast cancer metastasis by blocking nerve-tumor-immune loop

Chronic stress, a common comorbidity of breast cancer, is shown to promote tumor progression by affecting tumor microenvironment. Metastasis is the leading cause of death in triple-negative breast cancer (TNBC), and clarifying the specific role of chronic stress on TNBC metastasis will facilitate the development of new targets and improve its prognosis. In this study, a reciprocal nerve-tumor-immune loop is firstly depicted to promote TNBC metastasis under chronic stress and can be interrupted by propranolol (Pro), which shows rapid clinical translation potential due to “new uses for old drugs”. However, low bioavailability and insufficient efficacy of Pro when applied systemically may hinder its clinical translation in cancer patients. Based on this, an injectable and pH-responsive Pro-loaded coacervate (Pro@Coa) is further developed, which can accumulate and sustainably release Pro within the tumor. For the first time, the study demonstrates that Pro@Coa not only inhibits TNBC metastasis by interrupting the reciprocal loop but also suppresses distant tumor growth by inducing systemic immunity. Compared to traditional free Pro administration, Pro@Coa improves treatment efficiency and has no side effects on normal tissues. Overall, this work delineates a nerve-tumor-immune loop that promotes TNBC metastasis under chronic stress and provides a reference for applying Pro@Coa for safer and more effective treatment of TNBC metastasis, which is more promising for clinical translation.

Oncolytic adenovirus encoding apolipoprotein A1 suppresses metastasis of triple-negative breast cancer in mice

BackgroundDysregulation of cholesterol metabolism is associated with the metastasis of triple-negative breast cancer (TNBC). Apolipoprotein A1 (ApoA1) is widely recognized for its pivotal role in regulating cholesterol efflux and maintaining cellular cholesterol homeostasis. However, further exploration is needed to determine whether it inhibits TNBC metastasis by affecting cholesterol metabolism. Additionally, it is necessary to investigate whether ApoA1-based oncolytic virus therapy can be used to treat TNBC.MethodsIn vitro experiments and mouse breast cancer models were utilized to evaluate the molecular mechanism of ApoA1 in regulating cholesterol efflux and inhibiting breast cancer progression and metastasis. The gene encoding ApoA1 was inserted into the adenovirus genome to construct a recombinant adenovirus (ADV-ApoA1). Subsequently, the efficacy of ADV-ApoA1 in inhibiting the growth and metastasis of TNBC was evaluated in several mouse models, including orthotopic breast cancer, spontaneous breast cancer, and human xenografts. In addition, a comprehensive safety assessment of Syrian hamsters and rhesus monkeys injected with oncolytic adenovirus was conducted.ResultsThis study found that dysregulation of cholesterol homeostasis is critical for the progression and metastasis of TNBC. In a mouse orthotopic model of TNBC, a high-cholesterol diet promoted lung and liver metastasis, which was associated with keratin 14 (KRT14), a protein responsible for TNBC metastasis. Furthermore, studies have shown that ApoA1, a cholesterol reverse transporter, inhibits TNBC metastasis by regulating the cholesterol/IKBKB/FOXO3a/KRT14 axis. Moreover, ADV-ApoA1 was found to promote cholesterol efflux, inhibit tumor growth, reduce lung metastasis, and prolonged the survival of mice with TNBC. Importantly, high doses of ADV-ApoA1 administered intravenously and subcutaneously were well tolerated in rhesus monkeys and Syrian hamsters.ConclusionsThis study provides a promising oncolytic virus treatment strategy for TNBC based on targeting dysregulated cholesterol metabolism. It also establishes a basis for subsequent clinical trials of ADV-ApoA1 in the treatment of TNBC.

ICAM-1-suPAR-CD11b Axis Is a Novel Therapeutic Target for Metastatic Triple-Negative Breast Cancer.

Accumulating evidence demonstrates that circulating tumor cell (CTC) clusters have higher metastatic ability than single CTCs and negatively correlate with cancer patient outcomes. Along with homotypic CTC clusters, heterotypic CTC clusters (such as neutrophil-CTC clusters), which have been identified in both cancer mouse models and cancer patients, lead to more efficient metastasis formation and worse patient outcomes. However, the mechanism by which neutrophils bind to CTCs remains elusive. In this study, we found that intercellular adhesion molecule-1 (ICAM-1) on triple-negative breast cancer (TNBC) cells and CD11b on neutrophils mediate tumor cell-neutrophil binding. Consequently, CD11b deficiency inhibited tumor cell-neutrophil binding and TNBC metastasis. Furthermore, CD11b mediated hydrogen peroxide (H2O2) production from neutrophils. Moreover, we found that ICAM-1 in TNBC cells promotes tumor cells to secrete suPAR, which functions as a chemoattractant for neutrophils. Knockdown of uPAR in ICAM-1+ TNBC cells reduced lung-infiltrating neutrophils and lung metastasis. Bioinformatics analysis confirmed that uPAR is highly expressed in TNBCs, which positively correlates with higher neutrophil infiltration and negatively correlates with breast cancer patient survival. Collectively, our findings provide new insight into how neutrophils bind to CTC to facilitate metastasis and discover a novel potential therapeutic strategy by blocking the ICAM-1-suPAR-CD11b axis to inhibit TNBC metastasis.

Abstract 2478: The interplay between Apurinic/apyrimidinic Endonuclease 1 (APE1) and DNA G-quadruplex (G4) in regulating triple-negative breast cancer (TNBC) metastasis

Abstract Triple-negative breast cancer (TNBC) is the most aggressive breast cancer (BC) among all the BC subtypes due to its exceptionally high metastasis rate. TNBC patients with metastasis have a short overall survival as the endocrine therapy and chemotherapy are inefficient in TNBC treatment. Hence, efforts are still needed to figure out the molecular mechanism driving TNBC metastasis and find promising therapeutic targets for TNBC treatment. A newly published study revealed that a secondary DNA structure G-quadruplex (G4) is highly enriched in TNBC signature genes’ promoters, and our lab was the first one to uncover that DNA repair protein Apurinic/apyrimidinic endonuclease 1 (APE1) binds to G4 and regulates its stability in cells, so we are aimed to investigate the interaction between APE1 and G4 in TNBC and their roles in regulating TNBC metastasis. We found that knocking out or knocking down APE1 in TNBC cell lines, MDA-MB-231 and BT-549, significantly impaired the cell migration ability. RNA-seq analysis and Gene Ontology analysis revealed that APE1 knockout downregulated expression of genes such as CXCL1, VEGFA, PIK3R3, BMI1, etc. which are mostly involved in promoting cell migration. Further, APE1 ChIP-seq analysis showed that APE1 was highly enriched in CXCL1, VEGFA, PIK3R3 and BMI1 gene promoters, and these regions contained potential G4 folding sequences, suggesting a potential interaction between APE1 and G4 in these gene promoters. Consisted with this, our study with the enzyme linked immunosorbent assays (ELISA) showed that recombinant APE1 bound with high affinity to CXCL1 and VEGFA G4 secondary structures as compared to double-strand DNA oligos in vitro, suggesting that APE1’s enrichment to the metastasis-related gene promoters attributes to the G4 structures in those promoter regions. Promoter-directed ChIP assay further revealed that downregulating APE1 inhibited transcription factor (TF) c-JUN’s occupancy to VEGFA promoters. Interestingly, we discovered that the G4-targeted small molecule TMPyP4 blocked APE1-G4 binding in vitro and reduced APE1’s occupancy at gene promoters in cells. Treatment of cells with TMPyP4 significantly downregulated CXCL1, VEGFA, PIK3R3, and BMI1 gene expression as well as inhibiting cell migration. In orthotopic TNBC metastasis mouse model, mice treating with TMPyP4 significantly reduced the frequency and volume of metastatic tumors in lung tissues. Overall, our study revealed that G4 in gene promoters recruited APE1 which further promoted transcription factors’ binding to promoters and enhanced metastasis-related gene expression. Blocking APE1-G4 interaction by TMPyP4 is a potential therapeutic approach to inhibit TNBC metastasis. Citation Format: Yingling Chen, Suravi Pramanik, Mason Tarpley, Achyuth Kalluchi, Bhopal Mohapatra, Kyle Hewitt, Michael J. Rowley, Vimla Band, Kishor Bhakat. The interplay between Apurinic/apyrimidinic Endonuclease 1 (APE1) and DNA G-quadruplex (G4) in regulating triple-negative breast cancer (TNBC) metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2478.

Sanyin Formula Enhances the Therapeutic Efficacy of Paclitaxel in Triple-Negative Breast Cancer Metastases through the JAK/STAT3 Pathway in Mice.

Sanyin formula (SYF) is used as a complementary treatment for triple-negative breast cancer (TNBC). The purpose of this study was to identify the potential functional components and clarify the underlying molecular mechanisms of SYF in TNBC. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used to identify the main components of SYF extracts. Network pharmacology and bioinformatic analyses were carried out to identify potential candidate targets of SYF in TNBC. Cell proliferation was determined with a Celigo imaging cytometer. Wound-healing and Transwell assays were adopted to evaluate cell migration. A Transwell cell-invasion assay was performed with Matrigel-coated membranes. In vivo bioluminescence imaging (BLI) and pathological analyses illustrated the effect of SYF on cancer cell metastasis in tumour-bearing mice. The inhibitory mechanism of SYF was investigated via quantitative PCR (qPCR) and Western blotting. We found that 3,4-dihydroxyphenyllactic acid, kaempferol, p-coumaric acid, and vanillic acid may be the active components of SYF. Molecular docking confirmed that kaempferol, p-coumaric acid, vanillic acid, and 3,4-dihydroxyphenyllactic acid bound stably to proteins such as AKR1C3, MMPs, and STAT3. SYF extract suppressed TNBC cell proliferation, migration, invasion, and metastasis by inhibiting JAK/STAT3 signalling and then regulating downstream genes, such as MMP-2/MMP-9. SYF regulates the expression of genes involved in cell proliferation, migration, and invasion by regulating the JAK/STAT3 signalling pathway and finally inhibits tumour cell metastasis in TNBC. The present study clarifies the mechanism by which SYF inhibits TNBC metastasis and lays an experimental foundation for the continued clinical development of SYF targeting the JAK/STAT3 pathway.

Depolarization or hyperpolarization: Emerging role of altered bioelectricity in breast cancer metastasis.

Depolarization or hyperpolarization: Emerging role of altered bioelectricity in breast cancer metastasis.

Vitamin C Inhibits Triple-Negative Breast Cancer Metastasis by Affecting the Expression of YAP1 and Synaptopodin 2.

Vitamin C supplementation has been shown to decrease triple-negative breast cancer (TNBC) metastasis. However, the molecular mechanism whereby vitamin C inhibits metastasis remains elusive. It has been postulated that vitamin C reduces the levels of HIF-1α, the master regulator of metastasis, by promoting its hydroxylation and degradation. Here, we show that vitamin C at 100 µM, a concentration achievable in the plasma in vivo by oral administration, blocks TNBC cell migration and invasion in vitro. The protein level of HIF-1α remains largely unchanged in cultured TNBC cells and xenografts, partially due to its upregulated transcription by vitamin C, suggesting that HIF-1α unlikely mediates the action of vitamin C on metastasis. Vitamin C treatment upregulates the expression of synaptopodin 2 and downregulates the expression of the transcription coactivator YAP1, both genes in the Hippo pathway. The changes in SYNPO2 and YAP1 expression were subsequently validated at mRNA and protein levels in cultured TNBC cells and xenografts. Further experiments showed that vitamin C treatment inhibits F-actin assembly and lamellipodia formation, which correlates with the changes in SYNPO2 and YAP1 expression. Overall, these results suggest that vitamin C inhibits TNBC metastasis by affecting the expression of SYNPO2 and YAP1. Vitamin C may thus have a potential role in the prevention and treatment of TNBC metastasis.

Shikonin inhibits triple-negative breast cancer-cell metastasis by reversing the epithelial-to-mesenchymal transition via glycogen synthase kinase 3β-regulated suppression of β-catenin signaling

Triple-negative breast cancer (TNBC) is characterized by elevated metastasis, low survival, and poor response to therapy. Although many specific and effective agents for treating TNBC have been investigated, promising therapeutic options remain elusive. Here, we screened the inhibitory activities of three main components of Lithospermum erythrorhizon Sieb. et Zucc (shikonin, acetylshikonin, and β,β-dimethylacrylshikonin) on TNBC cells. The results revealed that shikonin potently decreased the viabilities of TNBC MDA-MB-231 and 4T1 cells but showed less cytotoxicity to normal mammary epithelial MCF-12A cells. Additionally, shikonin reversed the epithelial-to-mesenchymal transition (EMT) in MDA-MB-231 and 4T1 cells. Shikonin depressed cell migration and invasion, upregulated E-cadherin levels, downregulated N-cadherin, vimentin, and Snail levels, and reorganized the cytoskeletal proteins F-actin and vimentin. Shikonin reversed EMT by inhibiting activation of β-catenin signaling through attenuating β-catenin expression, nuclear accumulation, binding to T-cell factor consensus oligos, and transcription of its targeted EMT-related genes. Moreover, shikonin upregulated glycogen synthase kinase 3β (GSK-3β) levels, leading to enhanced phosphorylation and decreased levels of β-catenin. Furthermore, shikonin administration significantly inhibited lung metastasis of MDA-MB-231 cells in NOD/SCID mice accompanied by low systemic toxicity. Histological analysis confirmed that shikonin elevated levels of E-cadherin, phosphorylated β-catenin, and GSK-3β, and decreased levels of vimentin and β-catenin in pulmonary metastatic foci. These results indicated that shikonin potently inhibits TNBC metastasis by targeting the EMT via GSK-3β-regulated suppression of β-catenin signaling, which highlights the importance of shikonin as a potential candidate for novel anticancer therapeutics against TNBC.

Electrophilic Nitroalkenes Inhibit Triple Negative Breast Cancer Metastasis

Triple negative breast cancer (TNBC), which lacks estrogen receptor (ER), progesterone receptor (PR) and Her2/Neu, is an aggressive and therapy‐resistant metastatic cancer. A high level of tumor necrosis factor‐α (TNFα) expression and activation in TNBC patients is associated with distant tumor metastasis. Current therapy for TNBC is limited, and there is an unmet need for novel therapeutic agents. Electrophilic fatty acid nitroalkenes are endogenous signaling mediators that modulate cell differentiation and proliferation via post‐translational modification (PTM) of functionally‐significant nucleophilic amino acids (Cys, His) of transcriptional regulatory proteins such as NFκB, PPARγ, and Keap1/Nrf2. Recent studies have revealed that an exemplary electrophilic nitroalkene, nitro‐oleic acid (NO2‐OA), inhibits TNFα‐induced TNBC cell migration. NFκB plays an important role in tumor development and progression, and its signaling actions are constitutively activated in ER‐negative breast cancer cell lines and primary tumors. Notably, NO2‐OA causes polyubiquitination and degradation of NFκB RelA protein via protein nitroalkylation in TNBC cells. Therefore, it is hypothesized that electrophilic nitroalkenes have therapeutic potential that inhibit TNBC metastasis through down‐regulation of TNFα‐NFκB signaling. We demonstrate that NO2‐OA significantly inhibits two TNFα‐induced NFκB‐regulated genes involved in tumor metastasis, ICAM‐1 and uPA, in TNBC cells. The TNBC xenograft study indicates that NO2‐OA reduces MDA‐MB‐231 tumor xenograft growth. Baseline mortality rate for vehicle treated mice was 28.6 percent due to metastasis phenotype. We show that the survival curve increased with NO2‐OA dose level, correlating with better survival of TNBC xenograft mice. Moreover, ICAM‐1 and uPA expression levels in NO2‐OA treated tumors are lower compared to vehicle treated tumors. This study serves as a prelude to the clinical study of electrophilic nitroalkenes as therapeutic agents that may display high selectivity for inhibition of TNBC progression and metastasis.

Quercetin regulates β-catenin signaling and reduces the migration of triple negative breast cancer.

Triple negative breast cancer (TNBC) is characterized by a lack in estrogen, progesterone, and epidermal growth factor 2 receptors. TNBC exhibits most of the characteristics of basal-like and claudin-low breast cancer subtypes. The main contributor in the mortality of TNBC is due to the higher invasive and migratory ability of these tumor cells. Some plant flavonoids inhibit the epithelial mesenchymal transition (EMT) of tumor cells and suppress cancer metastasis. In this study, we aimed to determine whether the flavonoid quercetin is effective in modulating the molecular signaling associated with EMT in TNBC. Our data indicated that quercetin can induce the expression of E-cadherin and also downregulate vimentin levels in TNBC. The ability of quercetin to modulate these EMT markers resulted in a mesenchymal-to-epithelial transition (MET). Quercetin-induced MET was linked with the alteration of nuclear localization of β-catenin and modulation of β-catenin target genes such as cyclin D1 and c-Myc. Furthermore, we observed that quercetin induced the anti-tumor activity of doxorubicin by inhibiting the migratory ability of TNBC cells. These results suggested that quercetin may inhibit TNBC metastasis and also improve the therapeutic efficacy of existing chemotherapeutic drugs.

Abstract 5766: The Hsp90 inhibitor PU-H71 is a potent suppressor of metastasis in triple-negative breast cancer models

Abstract Background: Triple-negative breast cancer (TNBC) characterized by estrogen and progesterone receptor and HER-2-negativity is a particularly aggressive tumor with currently no effective targeted therapies. Our previous findings demonstrated that Hsp90 inhibition by the purine-scaffold inhibitor PU-H71 is a promising therapeutic approach in this tumor type. In TNBC cells, a multitude of malignancy driving proteins, including EGFR, HER3, Akt, PDK1, Bcl-xL and Raf-1, were in a complex with Hsp90 and were preferentially targeted for degradation by PU-H71 (Abstract 5637, AACR-Annual Meeting 2008/2009). In TNBC xenografts, PU-H71 exhibited potent and durable anti-tumor activity inducing complete responses without toxicity to the host. PU-H71 also inhibited the invasiveness of TNBC cells through a mechanism involving downregulation and inhibition of several pro-metastatic proteins such as NF-kB, p-Akt, ERK2, TYK2 and PKC, suggesting a potential effect of PU-H71 on TNBC metastasis. Study design: In order to determine whether in vivo PU-H71 also inhibits TNBC metastasis, we analyzed the effect of the Hsp90 inhibitor on lung colonization and metastastic body formation in immunocompromised mice. A lung tropic MDA-MB231 derivative expressing luciferase was used to establish the experimental models of metastasis. Animals were treated with 75mg/kg PU-H71 three times/week and lung metastases were monitored periodically by bioluminiscence imaging (IVIS system). Results: At completion of treatment both bioluminescent imaging and histological analyses demonstrated significant reduction of the metastastatic burden in mice treated with PU-H71 compared to the vehicle treated group. Importantly, flow cytometry analysis demonstrated that circulating hematopoietic precursors related to metastatic spread were reduced after treatment with PU-H71 and several hematopoietic cell types recruited to metastatic sites such as CD11b+/Gr1+ were reduced after the treatment. In addition, analysis of metastatic lungs demonstrated that CD24low/CD44+ cells were attracted to metastatic lung in MDA-MB231 bearing mice and strongly reduced after PU-H71 treatment. Conclusion: These data demonstrate that in addition to inhibition of primary tumors, PU-H71 leads to potent impairment of lung metastasis in an in vivo model of TNBC, supporting the use of PU-H71 for clinical trials involving patients with advanced and metastatic disease. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5766.