Yifeng Zheng

Network-pharmacology-based validation of TAMS/CXCL-1 as key mediator of XIAOPI formula preventing breast cancer development and metastasis

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.

Caveolin-1: An Oxidative Stress-Related Target for Cancer Prevention

Aberrant oxidative metabolism is one of the hallmarks of cancer. Reactive species overproduction could promote carcinogenesis via inducing genetic mutations and activating oncogenic pathways, and thus, antioxidant therapy was considered as an important strategy for cancer prevention and treatment. Caveolin-1 (Cav-1), a constituent protein of caveolae, has been shown to mediate tumorigenesis and progression through oxidative stress modulation recently. Reactive species could modulate the expression, degradation, posttranslational modifications, and membrane trafficking of Cav-1, while Cav-1-targeted treatments could scavenge the reactive species. More importantly, emerging evidences have indicated that multiple antioxidants could exert antitumor activities in cancer cells and protective activities in normal cells by modulating the Cav-1 pathway. Altogether, these findings indicate that Cav-1 may be a promising oxidative stress-related target for cancer antioxidant prevention. Elucidating the underlying interaction mechanisms between oxidative stress and Cav-1 is helpful for enhancing the preventive effects of antioxidants on cancer, for improving clinical outcomes of antioxidant-related therapeutics in cancer patients, and for developing Cav-1 targeted drugs. Herein, we summarize the available evidence of the roles of Cav-1 and oxidative stress in tumorigenesis and development and shed novel light on designing strategies for cancer prevention or treatment by utilizing the interaction mode between Cav-1 and oxidative stress.

Direct inhibition of ACTN4 by ellagic acid limits breast cancer metastasis via regulation of β-catenin stabilization in cancer stem cells

BackgroundPharmacology-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.MethodsThe 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.ResultsEA 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.ConclusionsTaken together, our results suggest that ACTN4 plays an important role in breast CSCs-related metastasis and is a novel therapeutic target of EA treatment.

Inflammasome and Cancer

The current chapter focuses on the role of inflammasome in cancer prevention and development. Emerging evidence suggested that inflammasome is closely correlated with elevated levels of IL-1β and IL-18, activation of NF-κB signaling, enhanced mitochondrial oxidative stress, and activation of autophagic process in cancer. Meanwhile, inflammasome component NOD-like receptors (NLRs) are also involved in carcinogenesis and closely correlated to chemoresponse and prognosis. Although several lines indicated the duplex role of inflammasome in cancer development, the phenomenon might be attributed to NLR difference, cell and tissue type, cancer stage, and specific experimental conditions. Designation of inflammasome targeting strategy has become a novel tool for cancer prevention or treatment.

Astragaloside IV enhances taxol chemosensitivity of breast cancer via caveolin-1-targeting oxidant damage: zheng et al.

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.

Network Pharmacology-Based Validation of Caveolin-1 as a Key Mediator of Ai Du Qing Inhibition of Drug Resistance in Breast Cancer

Chinese formulas have been paid increasing attention in cancer multidisciplinary therapy due to their multi-targets and multi-substances property. Here, we aim to investigate the anti-breast cancer and chemosensitizing function of Ai Du Qing (ADQ) formula made up of Hedyotis diffusa, Curcuma zedoaria (Christm.) Rosc., Astragalus membranaceus (Fisch.) Bunge, and Glycyrrhiza uralensis Fisch. Our findings revealed that ADQ significantly inhibited cell proliferation in both parental and chemo-resistant breast cancer cells, but with little cytotoxcity effects on the normal cells. Besides, ADQ was found to facilitate the G2/M arresting and apoptosis induction effects of paclitaxel. Network pharmacology and bioinformatics analysis further demonstrated that ADQ yielded 132 candidate compounds and 297 potential targets, and shared 22 putative targets associating with breast cancer chemoresponse. Enrichment analysis and experimental validation demonstrated that ADQ might improve breast cancer chemosensitivity via inhibiting caveolin-1, which further triggered expression changes of cell cycle-related proteins p21/cyclinB1 and apoptosis-associated proteins PARP1, BAX and Bcl-2. Besides, ADQ enhanced in vivo paclitaxel chemosensitivity on breast cancer. Our study not only uncovers the novel function and mechanisms of ADQ in chemosensitizing breast cancer at least partly via targeting caveolin-1, but also sheds novel light in utilizing network pharmacology in Chinese Medicine research.

CXCL1 derived from tumor-associated macrophages promotes breast cancer metastasis via activating NF-κB/SOX4 signaling

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.

Betulinic acid chemosensitizes breast cancer by triggering ER stress-mediated apoptosis by directly targeting GRP78

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.

Abstract 1924: Caveolin-1 inhibits mammary carcinogenesisviasuppressing c-myc-induced metabolism reprogramming in breast cancer stem cells

Purpose: Caveolin-1(CAV1), a membrane constituent protein, exhibits tumor suppressor activities in multiple malignancies, whereas the underlying mechanisms remain unclear. The current study aimed to determine the significance of CAV-1 in regulating cancer metabolism and its relation to breast cancer stem cells (CSCs). Experimental Design: The anti-carcinogenic function of CAV1 was evaluated by in vitro cell model, CAV1 knockout mice and MMTV-PyMT spontaneous breast cancer xenografts. Glycolysis activity and mitochondrial metabolism were assessed by immunoblotting, oxygen consumption and mitotacker staining analysis. Mammosphere formation, tumorigenic ability, reattachment differentiation and signaling pathway analysis were applied to study the regulation effects of CAV1 on breast CSCs. The clinical significance of CAV1 was also analyzed by human tissue microarray. Results: In both mammary transformed cells and spontaneous breast cancer xenografts, CAV1 is found significantly downregulated and positively correlated to increased glycolysis activity and impaired mitochondrial metabolism. Cav1 knockout results in increased ductal hyperplasia, associating with increased stem cell population and glycolysis metabolism. Breast CSCs exhibits glycolytic phenotype and decreased CAV1 expression compared to normal mammary stem cells, and CAV1 overexpression significantly limits CSCs’ self-renewal via inhibiting c-myc induced glycolysis. Clinical investigation suggests that high CAV1 expression is revealed with better overall survival and decreased CSCs population. Conclusions: CAV1 loss facilitates mammary carcinogenesis via enhancing glycolytic activity in breast CSCs, and CAV1 based therapy might become a novel strategy for breast cancer prevention. Note: This abstract was not presented at the meeting. Citation Format: Zhiyu Wang, Neng Wang, Shengqi Wang, Yifeng Zheng. Caveolin-1 inhibits mammary carcinogenesis via suppressing c-myc-induced metabolism reprogramming in breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1924. doi:10.1158/1538-7445.AM2017-1924