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Autophagy-mediated chemoresistance is the core mechanism for therapeutic failure and poor prognosis in breast cancer. Breast cancer chemotherapy resistance is believed to be influenced by tumor-associated macrophages (TAMs), by which C-X-C motif chemokine ligand 1 (CXCL1) is the most abundant cytokine secreted. Yet, its role in mediating autophagy-related chemoresistance is still unknown. This study aimed to explore the molecular mechanisms by which TAMs/CXCL1 induced autophagy-mediated chemoresistance in breast cancer. It was found that TAMs/CXCL1 promoted chemoresistance of breast cancer cells through autophagy activation in vitro, and CXCL1 silence could enhance the chemosensitivity of paclitaxel-resistant breast cancer cells via autophagy inhibition. A high-throughput quantitative PCR chip and subsequent target validation showed that CXCL1 induced autophagy-mediated chemoresistance by inhibiting VHL-mediated IGF1R ubiquitination. The elevated IGF1R then promoted STAT3/HMGB1 signaling to facilitate autophagy. Additionally, TAMs/CXCL1 silence improved paclitaxel chemosensitivity by suppressing autophagy in breast cancer mice xenografts, and clinical studies further linked CXCL1 to IGF1R/HMGB1 signaling, as well as shorter free survival of recurrence. Taken together, these results not only uncover the crucial role of TAMs/CXCL1 signaling in mediating breast cancer chemoresistance through enhancing autophagy, but also shed novel light on the molecular mechanism of IGF1R/STAT3/HMGB1 pathway in regulating autophagy and its impact on cancer prognosis., (© 2024. The Author(s).)

Background: Emerging studies have identified chronic psychological stress as an independent risk factor influencing breast cancer growth and metastasis. However, the effects of chronic psychological stress on pre-metastatic niche (PMN) formation and the underlying immunological mechanisms remain largely unknown., Methods: The effects and molecular mechanisms of chronic unpredictable mild stress (CUMS) on modulating tumor-associated macrophages (TAMs) and PMN formation were clarified by multiplex immunofluorescence technique, cytokine array, chromatin immunoprecipitation, the dual-luciferase reporter assay, and breast cancer xenografts. Transwell and CD8 + T cytotoxicity detection were used to analyze the mobilization and function of myeloid-derived suppressor cells (MDSCs). mCherry-labeled tracing strategy and bone marrow transplantation were applied to explore the crucial role of splenic CXCR2 +/+ MDSCs facilitating PMN formation under CUMS., Results: CUMS significantly promoted breast cancer growth and metastasis, accompanied by TAMs accumulation in the microenvironment. CXCL1 was identified as a crucial chemokine in TAMs facilitating PMN formation in a glucocorticoid receptor (GR)-dependent manner. Interestingly, the spleen index was significantly reduced under CUMS, and splenic MDSCs were validated as a key factor mediating CXCL1-induced PMN formation. The molecular mechanism study revealed that TAM-derived CXCL1 enhanced the proliferation, migration, and anti-CD8 + T cell functions of MDSCs via CXCR2. Moreover, CXCR2 knockout and CXCR2 -/- MDSCs transplantation significantly impaired CUMS-mediated MDSC elevation, PMN formation, and breast cancer metastasis., Conclusion: Our findings shed new light on the association between chronic psychological stress and splenic MDSC mobilization, and suggest that stress-related glucocorticoid elevation can enhance TAM/CXCL1 signaling and subsequently recruit splenic MDSCs to promote PMN formation via CXCR2., (© 2023. The Author(s).)

Introduction: Chronic psychological stress is a well-established risk factor for breast cancer development. Si-Ni-San (SNS) is a classical traditional Chinese medicine formula prescribed to psychological disorder patients. However, its action effects, molecular mechanisms, and bioactive phytochemicals against breast cancer are not yet clear., Objectives: This study aimed to explore the modulatory mechanism and bioactive compound of SNS in regulating estrogen metabolism during breast cancer development induced by chronic psychological stress., Methods: Mouse breast cancer xenograft was used to determine the effect of SNS on breast cancer growth and metastasis. Metabolomics analysis was conducted to discover the impact of SNS on metabolic profile changes in vivo. Multiple molecular biology experiments and breast cancer xenografts were applied to verify the anti-metastatic potentials of the screened bioactive compound., Results: SNS remarkably inhibited chronic psychological stress-induced breast cancer growth and metastasis in the mouse breast cancer xenograft. Meanwhile, chronic psychological stress increased the level of cholic acid, accompanied by the elevation of estradiol. Mechanistic investigation demonstrated that cholic acid activated farnesoid X receptor (FXR) expression, which inhibited hepatocyte nuclear factor 4α (HNF4α)-mediated estrogen sulfotransferase (EST) transcription in hepatocytes, and finally resulting in estradiol elevation. Notably, SNS inhibited breast cancer growth by suppressing estradiol level via modulating FXR/EST signaling. Furthermore, luciferase-reporting gene assay screened naringenin as the most bioactive compound in SNS for triggering EST activity in hepatocytes. Interestingly, pharmacokinetic study revealed that naringenin had the highest absorption in the liver tissue. Following in vivo and in vitro studies demonstrated that naringenin inhibited stress-induced breast cancer growth and metastasis by promoting estradiol metabolism via FXR/EST signaling., Conclusion: This study not only highlights FXR/EST signaling as a crucial target in mediating stress-induced breast cancer development, but also provides naringenin as a potential candidate for breast cancer endocrine therapy via promoting estradiol metabolism., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Production and hosting by Elsevier B.V.)

Background: Metastasis is the most common lethal cause of breast cancer-related death. Recent studies have implied that autophagy is closely implicated in cancer metastasis. Therefore, it is of great significance to explore autophagy-related molecular targets involved in breast cancer metastasis and to develop therapeutic drugs., Purpose: This study was designed to investigate the anti-metastatic effects and autophagy regulatory mechanisms of Aiduqing (ADQ) formula on breast cancer., Study Design/methods: Multiple cellular and molecular experiments were conducted to investigate the inhibitory effects of ADQ formula on autophagy and metastasis of breast cancer cells in vitro. Meanwhile, autophagic activator/inhibitor as well as CXCL1 overexpression or interference plasmids were used to investigate the underlying mechanisms of ADQ formula in modulating autophagy-mediated metastasis. Furthermore, the zebrafish xenotransplantation model and mouse xenografts were applied to validate the inhibitory effect of ADQ formula on autophagy-mediated metastasis in breast cancer in vivo., Results: ADQ formula significantly inhibited the proliferation, migration, invasion and autophagy but induced apoptosis of high-metastatic breast cancer cells in vitro. Similar results were also observed in starvation-induced breast cancer cells which exhibited elevated metastatic ability and autophagy activity. Mechanism investigations further approved that either CXCL1 overexpression or autophagic activator rapamycin can significantly abrogated the anti-metastatic effects of ADQ formula, suggesting that CXCL1-mediated autophagy may be the crucial pathway of ADQ formula in suppressing breast cancer metastasis. More importantly, ADQ formula suppressed breast cancer growth, autophagy, and metastasis in both the zebrafish xenotransplantation model and the mouse xenografts., Conclusion: Our study not only revealed the novel function of CXCL1 in mediating autophagy-mediated metastasis but also suggested ADQ formula as a candidate drug for the treatment of metastatic breast cancer., (Copyright © 2021. Published by Elsevier GmbH.)

Background: Metastasis represents the leading cause of death in patients with breast cancer. Traditional Chinese medicine is particularly appreciated for metastatic diseases in Asian countries due to its benefits for survival period prolongation and immune balance modulation. However, the underlying molecular mechanisms remain largely unknown. This study aimed to explore the antimetastatic effect and immunomodulatory function of a clinical formula Aiduqing (ADQ)., Methods: Naive CD4 + T cells, regulatory T cells (Tregs), and CD8 + T cells were sorted by flow cytometry. Then, breast cancer cells and these immune cells were co-cultured in vitro or co-injected into mice in vivo to simulate their coexistence. Flow cytometry, ELISA, qPCR, double luciferase reporter gene assay, and chromatin immunoprecipitation assay were conducted to investigate the immunomodulatory and antimetastatic mechanisms of ADQ., Results: ADQ treatment by oral gavage significantly suppressed 4T1-Luc xenograft growth and lung metastasis in the orthotopic breast cancer mouse model, without noticeable hepatotoxicity, nephrotoxicity, or hematotoxicity. Meanwhile, ADQ remodeled the immunosuppressive tumor microenvironment (TME) by increasing the infiltration of tumor-infiltrating lymphocytes (TILs) and cytotoxic CD8 + T cells, and decreasing the infiltration of Tregs, naive CD4 + T cells, and tumor-associated macrophages (TAMs). Molecular mechanism studies revealed that ADQ remarkably inhibited CXCL1 expression and secretion from TAMs and thus suppressed the chemotaxis and differentiation of naive CD4 + T cells into Tregs, leading to the enhanced cytotoxic effects of CD8 + T cells. Mechanistically, TAM-derived CXCL1 promoted the differentiation of naive CD4 + T cells into Tregs by transcriptionally activating the NF-κB/FOXP3 signaling. Lastly, mouse 4T1-Luc xenograft experiments validated that ADQ formula inhibited breast cancer immune escape and lung metastasis by suppressing the TAM/CXCL1/Treg pathway., Conclusions: This study not only provides preclinical evidence supporting the application of ADQ in inhibiting breast cancer metastasis but also sheds novel insights into TAM/CXCL1/NF-κB/FOXP3 signaling as a promising therapeutic target for Treg modulation and breast cancer immunotherapy. Video Abstract., (© 2021. The Author(s).)

Depression and anxiety are common comorbidities in breast cancer patients. Whether depression and anxiety are associated with breast cancer progression or mortality is unclear. Herein, based on a systematic literature search, 17 eligible studies involving 282,203 breast cancer patients were included. The results showed that depression was associated with cancer recurrence [1.24 (1.07, 1.43)], all-cause mortality [1.30 (1.23, 1.36)], and cancer-specific mortality [1.29 (1.11, 1.49)]. However, anxiety was associated with recurrence [1.17 (1.02, 1.34)] and all-cause mortality [1.13 (1.07, 1.19)] but not with cancer-specific mortality [1.05 (0.82, 1.35)]. Comorbidity of depression and anxiety is associated with all-cause mortality [1.34 (1.24, 1.45)] and cancer-specific mortality [1.45 (1.11, 1.90)]. Subgroup analyses demonstrated that clinically diagnosed depression and anxiety, being female and of younger age (<60 years), and shorter follow-up duration (≤5 years) were related to a poorer prognosis. Our study highlights the critical role of depression/anxiety as an independent factor in predicting breast cancer recurrence and survival. Further research should focus on a favorable strategy that works best to improve outcomes among breast cancer patients with mental disorders.

Background: Breast cancer is the most common malignancy in women and metastasis is the leading cause of breast cancer-related deaths. Our previous studies have shown that XIAOPI formula, a newly approved drug by the State Food and Drug Administration of China (SFDA), can dramatically inhibit breast cancer metastasis by modulating the tumor-associated macrophages/C-X-C motif chemokine ligand 1 (TAMs/CXCL1) pathway. However, the bioactive compound accounting for the anti-metastatic effect of XIAOPI formula remains unclear., Purpose: This study was designed to separate the anti-metastatic bioactive compound from XIAOPI formula and to elucidate its action mechanisms., Study Design/methods: TAMs/CXCL1 promoter activity-guided fractionation and multiple chemical structure identification approaches were conducted to screen the bioactive compound from XIAOPI formula. Breast cancer cells and TAMs were co-cultured in vitro or co-injected in vivo to simulate their coexistence. Multiple molecular biology experiments, zebrafish breast cancer xenotransplantation model and mouse breast cancer xenografts were applied to validate the anti-metastatic activity of the screened compound., Results: Bioactivity-guided fractionation identified baohuoside I (BHS) as the key bioactive compound of XIAOPI formula in inhibiting TAMs/CXCL1 promoter activity. Functional studies revealed that BHS could significantly inhibit the migration and invasion as well as the expression of metastasis-related proteins in both human and mouse breast cancer cells, along with decreasing the proportion of breast cancer stem cells (CSCs). Furthermore, BHS could suppress the M2 phenotype polarization of TAMs and therefore attenuate their CXCL1 expression and secretion. Notably, mechanistic investigations validated TAMs/CXCL1 as the crucial target of BHS in suppressing breast cancer metastasis as exogenous addition of CXCL1 significantly abrogated the anti-metastatic effect of BHS on breast cancer cells. Moreover, BHS was highly safe in vivo as it exhibited no observable embryotoxicity or teratogenic effect on zebrafish embryos. More importantly, BHS remarkably suppressed breast cancer metastasis and TAMs/CXCL1 activity in both zebrafish breast cancer xenotransplantation model and mouse breast cancer xenografts., Conclusion: This study not only provides novel insights into TAMs/CXCL1 as a reliable screening target for anti-metastatic drug discovery, but also suggests BHS as a promising candidate drug for metastatic breast cancer treatment., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Breast cancer stem cells (BCSCs) are considered to be the root of breast cancer occurrence and progression. However, the characteristics and regulatory mechanisms of BCSCs metabolism have been poorly revealed, which hinders the development of metabolism-targeted treatment strategies for BCSCs elimination. Herein, we demonstrated that the downregulation of Caveolin-1 (Cav-1) usually occurred in BCSCs and was associated with a metabolic switch from mitochondrial respiration to aerobic glycolysis. Meanwhile, Cav-1 could inhibit the self-renewal capacity and aerobic glycolysis activity of BCSCs. Furthermore, Cav-1 loss was associated with accelerated mammary-ductal hyperplasia and mammary-tumor formation in transgenic mice, which was accompanied by enrichment and enhanced aerobic glycolysis activity of BCSCs. Mechanistically, Cav-1 could promote Von Hippel-Lindau (VHL)-mediated ubiquitination and degradation of c-Myc in BCSCs through the proteasome pathway. Notably, epithelial Cav-1 expression significantly correlated with a better overall survival and delayed onset age of breast cancer patients. Together, our work uncovers the characteristics and regulatory mechanisms of BCSCs metabolism and highlights Cav-1-targeted treatments as a promising strategy for BCSCs elimination.

Background: Recent findings suggested that premetastatic niche (PMN) is a prerequisite in mediating cancer metastasis. Previously we demonstrated that XIAOPI formula could inhibit breast cancer lung metastasis via inhibiting tumor associated macrophages (TAMs)-secreted CXCL1. Herein, we aimed to explore the effects of XIAOPI formula on preventing breast cancer PMN formation and its underlying molecular mechanisms., Methods: CXCL1 expression of TAMs was detected by qPCR and Western blotting assay. The influences of XIAOPI formula on the proliferation of TAMs and 4 T1 in the co-culture system were tested by CCK8 or EdU staining. Transwell experiment was applied to determine the effects of XIAOPI formula on the invasion ability of HSPCs and 4 T1. Breast cancer xenografts were built by inoculating 4 T1 cells into the mammary pads of Balb/c mice and lung metastasis was monitored by luciferase imaging. Immune fluorescence assay was used to test the epithelial-mesenchymal transition process and PMN formation in the lung tissues. The effects of XIAOPI formula on TAMs phenotype, hematopoietic stem/progenitor cells (HSPCs) and myeloid-derived suppressor cells (MDSCs) were determined by flow cytometry., Results: It was found that XIAOPI formula could inhibit the proliferation and polarization of M2 phenotype macrophages, and reduce CXCL1 expression in a dose-dependent manner. However, M1 phenotype macrophages were not significantly affected by XIAOPI formula. TAMs/CXCL1 signaling was subsequently found to stimulate the recruitment of c-Kit + /Sca-1 + HSPCs and their differentiation into CD11b + /Gr-1 + MDSCs, which were symbolic events accounting for PMN formation. Moreover, XIAOPI formula was effective in inhibiting HSPCs activation and suppressing the proliferation and metastasis of breast cancer cells 4 T1 induced by HSPCs and TAMs co-culture system, implying that XIAOPI was effective in preventing PMN formation in vitro. Breast cancer xenograft experiments further demonstrated that XIAOPI formula could inhibit breast cancer PMN formation and subsequent lung metastasis in vivo. The populations of HSPCs in the bone marrow and MDSCs in the lung tissues were all remarkably declined by XIAOPI formula treatment. However, the inhibitory effects of XIAOPI formula could be relieved by CXCL1 overexpression in the TAMs., Conclusions: Taken together, our study provided preclinical evidence supporting the application of XIAOPI formula in preventing breast cancer PMN formation, and highlighted TAMs/CXCL1 as a potential therapeutic strategy for PMN targeting therapy. Video Abstract.

XIAOPI formula is a national approved drug prescribed to patients with high breast cancer risk. Previously we demonstrated that XIAOPI formula could inhibit breast cancer metastasis via suppressing CXCL1 expression, and postulated that "autophagy in cancer" might be one of its most core anti-cancer mechanisms. However, whether XIAOPI formula could be simultaneously applied with chemodrugs and their synergistic mechanisms are still remained unknown. In the present study, XIAOPI formula at non-cytotoxic doses could synergistically enhance the chemosensitivity of breast cancer cells MDA-MB-231 and MCF-7. We found that rapamycin-induced autophagy could reduce the chemosensitivity of breast cancer cells to XIAOPI formula, and the autophagy suppression and chemosensitizing activity of this formula was CXCL1-dependent. The evidence came from that XIAOPI formula was associated with a lower expression of CXCL1 combined with either rapamycin or taxol alone. Besides, the inhibitory effect of XIAOPI formula on the LC3-II and ABCG2 signals was weakened following CXCL1 over-expression, whereas P62 upregulation induced by XIAOPI formula was re-declined. A high throughput - qPCR (HT-qPCR) assay identified HMGB1 as the main autophagic target of XIAOPI formula in chemosensitizing breast cancer. and furhter validation suggested XIAOPI formula exerted chemosensitivity mainly via CXCL1/HMGB1 autophagic axis. Finally, we generated both mice and zebrafish xenotransplantation models bearing MDA-MB-231 breast cancer cells, and found that XIAOPI formula safely enhanced in vivo taxol chemosensitivity on breast cancer. Taken together, XIAOPI formula is a potential adjuvant drug via inhibiting CXCL1/HMGB1-mediated autophagy for breast cancer treatment with good safety., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Targeting aberrant metabolism is a promising strategy for inhibiting cancer growth and metastasis. Research is now geared towards investigating the inhibition of glycolysis for anticancer drug development. Betulinic acid (BA) has demonstrated potent anticancer activities in multiple malignancies. However, its regulatory effects on glycolysis and the underlying molecular mechanisms are still unclear. BA inhibited invasion and migration of highly aggressive breast cancer cells. Moreover, BA could suppress aerobic glycolysis of breast cancer cells presenting as a reduction of lactate production, quiescent energy phenotype transition, and downregulation of aerobic glycolysis-related proteins. In this study, glucose-regulated protein 78 (GRP78) was also identified as the molecular target of BA in inhibiting aerobic glycolysis. BA treatment led to GRP78 overexpression, and GRP78 knockdown abrogated the inhibitory effect of BA on glycolysis. Further studies demonstrated that overexpressed GRP78 activated the endoplasmic reticulum (ER) stress sensor PERK. Subsequent phosphorylation of eIF2 α led to the inhibition of β -catenin expression, which resulted in the inhibition of c-Myc-mediated glycolysis. Coimmunoprecipitation assay revealed that BA interrupted the binding between GRP78 and PERK, thereby initiating the glycolysis inhibition cascade. Finally, the lung colonization model validated that BA inhibited breast cancer metastasis in vivo , as well as suppressed the expression of aerobic glycolysis-related proteins. In conclusion, our study not only provided a promising drug for aerobic glycolysis inhibition but also revealed that GRP78 is a novel molecular link between glycolytic metabolism and ER stress during tumor metastasis., Competing Interests: The authors declare no conflicts of interest.

Accumulating evidence suggests that caveolin-1 (CAV-1) is a stress-related oncotarget and closely correlated to chemoresistance. Targeting CAV-1 might be a promising strategy to improve chemosensitivity for breast cancer treatment. Astragaloside IV (AS-IV), a bioactive compound purified from Astragalus membranaceus, has been shown to exhibit multiple bioactivities, including anticancer. However, the involved molecular targets are still ambiguous. In this study, we investigated the critical role of CAV-1 in mediating the chemosensitizing effects of AS-IV to Taxol on breast cancer. We found that AS-IV could enhance the chemosensitivity of Taxol with minimal direct cytotoxicity on breast cancer cell lines MCF-7 and MDA-MB-231, as well as the nontumor mammary epithelial cell line MCF-10A. AS-IV was further demonstrated to aggravate Taxol-induced apoptosis and G2/M checkpoint arrest. The phosphorylation of mitogen-activated protein kinase (MAPK) signaling extracellular signal-regulated kinase (ERK) and c-Jun N-terminal Kinase (JNK), except p38, was also abrogated by a synergistic interaction between AS-IV and Taxol. Moreover, AS-IV inhibited CAV-1 expression in a dose-dependent manner and reversed CAV-1 upregulation induced by Taxol administration. Mechanism study further demonstrated that AS-IV treatment triggered the eNOS/NO/ONOO - pathway via inhibiting CAV-1, which led to intense oxidant damage. CAV-1 overexpression abolished the chemosensitizing effects of AS-IV to Taxol by inhibiting oxidative stress. In vivo experiments further validated that AS-IV increased Taxol chemosensitivity on breast cancer via inhibiting CAV-1 expression, followed by activation of the eNOS/NO/ONOO - pathway. Taken together, our findings not only suggested the potential of AS-IV as a promising candidate to enhance chemosensitivity, but also highlighted the significance of CAV-1 as the target to reverse cancer drug resistance., (© 2018 Wiley Periodicals, Inc.)

Background: Breast cancer remains the most common female malignancy and metastasis is the leading cause of death in breast cancer patients. Oldenlandia diffusa has been empirically and extensively used as an adjuvant therapy for metastatic breast cancer patients in Traditional Chinese Medicine (TCM) with proven efficacy. However, its anti-metastasis mechanism has been poorly revealed., Methods: Multiple molecular biology experiments as well as network pharmacology, bioinformatics analysis were conducted to investigate the anti-metastasis mechanism of Oldenlandia diffusa in breast cancer., Results: We demonstrated that ethanol extract of Oldenlandia diffusa (EEOD) significantly inhibited proliferation and induced apoptosis of high-metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-453, while having no obvious cytotoxic effect on multiple nonmalignant cells. Furthermore, EEOD remarkably suppressed the migration and invasion capacities of the above breast cancer cells by modulating the matrix metalloproteinases (MMPs) and the epithelial-mesenchymal transition (EMT) pathway. More importantly, EEOD also significantly inhibited breast cancer metastasis in zebrafish xenotransplantation model in vivo. Network pharmacology and bioinformatics analysis further demonstrated that EEOD yielded 12 candidate compounds and 225 potential targets, and shared 85 putative targets associated with breast cancer metastasis. Mechanistically, RNA sequencing and experimental validation results suggested that EEOD might inhibit breast cancer metastasis by attenuating the expression of caveolin-1 (Cav-1) as overexpression of Cav-1 could weaken the anti-metastasis efficacy of EEOD., Conclusions: Overall, our findings proved that EEOD could inhibit breast cancer metastasis by attenuating the expression of Cav-1, highlighting the use of EEOD as an adjunctive therapy for metastatic breast cancer patients. This study also provides novel insights into network pharmacology and bioinformatics analysis as effective tools to illuminate the scientific basis of TCM., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Emerging evidence has suggested that targeting glycolysis may be a promising strategy for cancer treatment. Betulinic acid (BA) is a natural pentacyclic terpene that has been reported to be active in inhibiting various malignancies. Here, we showed that BA could inhibit aerobic glycolysis activity in breast cancer cell lines MCF-7 and MDA-MB-231 by hampering lactate production, glucose uptake and extracellular acidification rate (ECAR), as well as suppressing aerobic glycolysis-related proteins including c-Myc, lactate dehydrogenase A (LDH-A) and p-PDK1/PDK1 (pyruvate dehydrogenase kinase 1). Mechanistic studies validated Caveolin-1 (Cav-1) as one of key targets of BA in suppressing aerobic glycolysis, as BA administration resulted in Cav-1 upregulation, whereas silencing Cav-1 abrogated the inhibitory effect of BA on aerobic glycolysis. Further investigations demonstrated that BA suppressed aerobic glycolysis in breast cancer cells by regulating the Cav-1/NF-κB/c-Myc pathway. More meaningfully, BA significantly inhibited breast cancer growth and glycolytic activity in both the transgenic MMTV-PyVT +/- breast cancer spontaneous model and the zebrafish breast cancer xenotransplantation model without any detectable side effects in vivo. Taken together, our study sheds novel insights into BA as a promising candidate drug for suppressing aerobic glycolysis, highlighting Cav-1 as a potential molecular target of BA and aerobic glycolysis regulation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Tumor-associated macrophages (TAMs) have been implicated in the promotion of breast cancer growth and metastasis, and multiple TAM-secreted cytokines have been identified associating with poor clinical outcomes. However, the therapeutic targets existing in the loop between TAMs and cancer cells are still required for further investigation. Here in, cytokine array validated that C-X-C motif chemokine ligand 1 (CXCL1) is the most abundant chemokine secreted by TAMs, and CXCL1 can promote breast cancer migration and invasion ability, as well as epithelial-mesenchymal transition in both mouse and human breast cancer cells. QPCR screening further validated SOX4 as the highest responsive gene following CXCL1 administration. Mechanistic study revealed that CXCL1 binds to SOX4 promoter and activates its transcription via NF-κB pathway. In vivo breast cancer xenografts demonstrated that CXCL1 silencing in TAMs results in a significant reduction in breast cancer growth and metastatic burden. Bioinformatic analysis and clinical investigation finally suggested that high CXCL1 expression is significantly correlated with breast cancer lymph node metastasis, poor overall survival and basal-like subtype. Taken together, our results indicated that TAMs/CXCL1 promotes breast cancer metastasis via NF-κB/SOX4 activation, and CXCL1-based therapy might become a novel strategy for breast cancer metastasis prevention.

Stress-induced cellular defense machinery has a critical role in mediating cancer drug resistance, and targeting stress-related signaling has become a novel strategy to improve chemosensitivity. Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid with potent anticancer bioactivities in multiple malignancies, whereas its underlying mechanisms remain unclear. Here in, we found that BA has synergistic effects with taxol to induce breast cancer cells G2/M checkpoint arrest and apoptosis induction, but had little cytotoxicity effects on normal mammary epithelial cells. Drug affinity responsive target stability (DARTS) strategy further identified glucose-regulated protein 78 (GRP78) as the direct interacting target of BA. BA administration significantly elevated GRP78-mediated endoplasmic reticulum (ER) stress and resulted in the activation of protein kinase R-like ER kinase (PERK)/eukaryotic initiation factor 2a/CCAAT/enhancer-binding protein homologous protein apoptotic pathway. GRP78 silencing or ER stress inhibitor salubrinal administration was revealed to abolish the anticancer effects of BA, indicating the critical role of GRP78 in mediating the bioactivity of BA. Molecular docking and coimmunoprecipitation assay further demonstrated that BA might competitively bind with ATPase domain of GRP78 to interrupt its interaction with ER stress sensor PERK, thereby initiating the downstream apoptosis cascade. In vivo breast cancer xenografts finally validated the chemosensitizing effects of BA and its biofunction in activating GRP78 to trigger ER stress-mediated apoptosis. Taken together, our study not only uncovers GRP78 as a novel target underlying the chemosensitizing effects of BA, but also highlights GRP78-based targeting strategy as a promising approach to improve breast cancer prognosis.

Background: Pharmacology-based target identification has become a novel strategy leading to the discovery of novel pathological biomarkers. Ellagic acid (EA), a dietary polyphenol compound, exhibits potent anticancer activities; however, the underlying mechanisms remain unclear. The current study sought to determine the role and regulation of ACTN4 expression in human breast cancer metastasis and EA-based therapy., Methods: The anti-metastasis ability of EA was validated by MMTV-PyMT mice and in vitro cell models. Drug affinity responsive target stability (DARTS) was utilized to identify ACTN4 as the direct target of EA. The metastatic regulated function of ACTN4 were assessed by cancer stem cells (CSCs)-related assays, including mammosphere formation, tumorigenic ability, reattachment differentiation, and signaling pathway analysis. The mechanisms of ACTN4 on β-catenin stabilization were investigated by western blotting, co-immunoprecipitation and ubiquitination assays. The clinical significance of ACTN4 was based on human tissue microarray (TMA) analysis and The Cancer Genome Atlas (TCGA) database exploration., Results: EA inhibited breast cancer growth and metastasis via directly targeting ACTN4 in vitro and in vivo, and was accompanied by a limited CSC population. ACTN4 knockdown resulted in the blockage of malignant cell proliferation, colony formation, and ameliorated metastasis potency. ACTN4-positive CSCs exhibited a higher ESA + proportion, increased mammosphere-formation ability, and enhanced in vivo tumorigenesis ability. Mechanism exploration revealed that interruption of ACTN4/β-catenin interaction will result in the activation of β-catenin proteasome degradation. Increased ACTN4 expression was directly associated with the advanced cancer stage, an increased incidence of metastasis, and poor overall survival period., Conclusions: Taken together, our results suggest that ACTN4 plays an important role in breast CSCs-related metastasis and is a novel therapeutic target of EA treatment.

Network pharmacology has become a powerful means of understanding the mechanisms underlying the action of Chinese herbs in cancer treatment. This study aims to validate the preventive effects and molecular mechanisms of a clinical prescription XIAOPI formula against breast cancer. In vivo breast cancer xenograft data showed that XIAOPI delayed breast cancer development and efficiently inhibited lung metastasis, accompanied by prolonged survival benefits and decreased cancer stem cell subpopulations. However, similar phenomenon were not observed in a cell model. The herb-ingredient-target network analysis further identified a total of 81 genes closely correlated with the breast cancer chemoprevention effects of XIAOPI. Cytokine array analysis further validated CXCL-1 as the key target of XIAOPI both in vitro and in vivo. Evaluation of the mechanism demonstrated that CXCL-1 administration significantly abrogated the metastatic inhibition effects of XIAOPI on breast cancer migration, invasion, stem cells subpopulations, epithelial-mesenchymal transition(EMT), or mammosphere formation abilities. Overall, our study provides experimental evidence and molecular mechanisms that may facilitate the safe and effective use of herbal medicine for the prevention of breast cancer growth or metastasis, and may lead to CXCL-1-based therapeutic strategies for mammary malignancies.