Inhibits Breast Cancer Stem Cells Research Articles

Abstract PO4-27-12: Increasing DAXX as a Novel Approach to Inhibit Breast Cancer Stem Cells and Estrogen Receptor-positive Tumor Recurrence

Abstract Background: Resistance to endocrine therapy (ET; tamoxifen, aromatase inhibitors, AI, or fulvestrant) in ER+ breast cancer (BC) could be due to survival of breast cancer stem cells (BCSCs). BCSCs contribute to relapse of ER+ BC. We discovered a novel and potent anti-BCSC gene, Death Associated Protein 6 (DAXX) through a pre-surgical biomarker window study combining ET plus a Notch inhibitor [MK-0752, a g-secretase inhibitor (GSI)]. We also found that ET enhances the BCSC population by decreasing DAXX protein levels. In this current study, we measured DAXX protein levels in ER+ PDX tumors and human tumor tissues treated with ET. We determined the mechanism by which ET regulates DAXX expression at the level of protein stability. The goal of this study was to identify novel therapeutic strategies to prevent ET-mediated degradation of the DAXX protein, eliminate BCSCs, and prevent ER+ recurrence. Methods: ER+ BC cell lines (MCF-7 and T47D), an ER+ PDX BCM 5097 tumor line, and human breast cancer tumor samples were used. The ER+ PDX tumor (BCM 5097) was implanted into NSG mice and passaged into female athymic, nude mice-supplemented with an estradiol capsule. After one week, the estradiol capsule was removed from 10 mice, to mimic the use of AI. The remaining 10 mice retained the estradiol capsule. Tumor area was measured weekly up to 50 weeks. Western blotting detected ERa, the stem cell factor Notch4, and DAXX levels. Immunohistochemistry (IHC) detected DAXX protein levels. ER+ human tumor samples were collected prior to and after neoadjuvant treatment with ET (N=11) or chemotherapy (N=14) and stained for the DAXX protein. Correlations were made between DAXX and Ki67. Full length and deletion mutants of DAXX were expressed in ER+ BC cells. BCSCs were measured using the mammosphere forming assay. Liquid chromatography followed by mass spectrometry were used to detect phosphorylated residues of DAXX in response to ET. Kinases that phosphorylate these residues on DAXX were identified using PhosphoSite.org. ER+ cells were treated with kinase inhibitors for AURKA (alisertib), AURKB (barasertib), CK1 (CK-IN-1), or CK2 (CX-4945) and DAXX protein was detected by western blotting. BCSC survival was measured using mammosphere forming assay. Based on results from the kinase inhibitor screen, barasertib was selected for pre-clinical testing in mice. ER+ MCF-7 xenograft tumors were treated with vehicle or barasertib in vivo up to 120 days. Results: DAXX protein levels decreased in ER+ PDX tumors in response to ET. Similar trends were detected in human tumor tissue after ET or chemotherapy alone. DAXX protein levels inversely correlated with Ki67 in ER+ human tumor samples. Analysis of DAXX deletion mutants demonstrated that amino acids 400-740 of the DAXX protein were required to inhibit BCSC survival. This region of the DAXX protein had high levels of phosphorylated residues in response to ET. Only the AURKB (barasertib) inhibitor increased DAXX protein expression and inhibited BCSCs in a DAXX-dependent manner. Barasertib inhibited growth of ER+ MCF-7 tumor xenografts compared to the vehicle control. After treatment ceased, tumor recurrence was measured up to 120 days. Regrowth of tumors was partially delayed in response to barasertib. Conclusions: ET decreased DAXX protein levels in ER+ PDX and human tumors. Downregulation of the DAXX protein by ET was through activation of AURKB and hyper-phosphorylation of DAXX which resulted in protein degradation and enhanced survival of BCSCs. Therefore, Inhibition of AURKB using barasertib partially restored DAXX expression, inhibited BCSCs, and delayed tumor recurrence. A combination approach of ET plus an AURKB inhibitor might be a novel therapeutic strategy to prevent ER+ breast cancer relapse by increasing DAXX in order to eradicate BCSCs. Support: Breast Cancer Research Foundation Citation Format: Clodia Osipo, Debra Wyatt, Kathy Albain. Increasing DAXX as a Novel Approach to Inhibit Breast Cancer Stem Cells and Estrogen Receptor-positive Tumor Recurrence [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-27-12.

Nobiletin Inhibits Breast Cancer Stem Cell by Regulating the Cell Cycle: A Comprehensive Bioinformatics Analysis and In Vitro Experiments

Inhibiting breast cancer stem cell (BCSC) signaling pathways is a strategic method for successfully treating breast cancer. Nobiletin (NOB) is a compound widely found in orange peel that exhibits a toxic effect on various types of cancer cells, and inhibits the signaling pathways that regulate the properties of BCSCs; however, the effects of NOB on BCSCs remain elusive. The purpose of this study was to determine the target genes of NOB for inhibiting BCSCs using in vitro three-dimensional breast cancer cell culture (mammospheres) and in silico approaches. We combined in vitro experiments to develop mammospheres and conducted cytotoxicity, next-generation sequencing, and bioinformatics analyses, such as gene ontology, the Reactome pathway enrichment, network topology, gene set enrichment analysis, hub genes selection, genetic alterations, prognostic value related to the mRNA expression, and mRNA and protein expression of potential NOB target genes that inhibit BCSCs. Here, we show that NOB inhibited BCSCs in mammospheres from MCF-7 cells. We also identified CDC6, CHEK1, BRCA1, UCHL5, TOP2A, MTMR4, and EXO1 as potential NOB targets inhibiting BCSCs. NOB decreased G0/G1, but increased the G2/M cell population. These findings showed that NOB is a potential therapeutic candidate for BCSCs treatment by regulating cell cycle.

Construction and in vitro evaluation of pH-sensitive nanoparticles to reverse drug resistance of breast cancer stem cells

Breast cancer is a major threat to safety and health of women. The breast cancer stem cells (BCSCs) have multi-drug resistance to chemotherapy drugs, which leads to chemotherapy failure. We proposed a strategy of delivery of tumor-killing drugs and a resistance reversal agent, to enhance inhibition of BCSCs. Here, schisandrin B (SchB)/AP NPs are constructed using acid-grafted-poly (β-amino ester) (ATRA-g-PBAE, AP) grafted polymer nanoparticle encapsulated SchB, with pH-sensitive release function. This drug delivery system has good pharmacological properties and can increase the SchB release with the decrease of pH. The NPs showed cytotoxic effects in reversing ATRA resistance to BCSCs. Lysosomal escape was achieved when the nanoparticles were taken up by BCSCs. In addition, we found that NPs may reverse MDR by inhibiting the expression of P-glycoprotein (P-gp) and affecting the energy supply of drug efflux. This study provides a nanodelivery therapy strategy that reverses BCSCs multidrug resistance (MDR) and demonstrates that it did so by interfering with cancer cell energy metabolism. Therefore, the co-delivery strategy of ATRA and SchB provides a new option for the treatment of breast cancer.

Targeting ABCA12-controlled ceramide homeostasis inhibits breast cancer stem cell function and chemoresistance.

Cancer stem cells (CSCs) drive tumor growth, metastasis, and chemoresistance. While emerging evidence suggests that CSCs have a unique dependency on lipid metabolism, the functions and regulation of distinct lipid species in CSCs remain poorly understood. Here, we developed a stem cell factor SOX9-based reporter for isolating CSCs in primary tumors and metastases of spontaneous mammary tumor models. Transcriptomic analyses uncover that SOX9high CSCs up-regulate the ABCA12 lipid transporter. ABCA12 down-regulation impairs cancer stemness and chemoresistance. Lipidomic analyses reveal that ABCA12 maintains cancer stemness and chemoresistance by reducing intracellular ceramide abundance, identifying a CSC-associated function of ABCA subfamily transporter. Ceramide suppresses cancer stemness by inhibiting the YAP-SOX9 signaling pathway in CSCs. Increasing ceramide levels in tumors enhances their sensitivity to chemotherapy and prevents the enrichment of SOX9high CSCs. In addition, SOX9high and ABCA12high cancer cells contribute to chemoresistance in human patient-derived xenografts. These findings identify a CSC-suppressing lipid metabolism pathway that can be exploited to inhibit CSCs and overcome chemoresistance.

Unravelling the Therapeutic Potential of Antibiotics in Hypoxia in a Breast Cancer MCF-7 Cell Line Model.

Antibiotics inhibit breast cancer stem cells (CSCs) by suppressing mitochondrial biogenesis. However, the effectiveness of antibiotics in clinical settings is inconsistent. This inconsistency raises the question of whether the tumor microenvironment, particularly hypoxia, plays a role in the response to antibiotics. Therefore, the goal of this study was to evaluate the effectiveness of five commonly used antibiotics for inhibiting CSCs under hypoxia using an MCF-7 cell line model. We assessed the number of CSCs through the mammosphere formation assay and aldehyde dehydrogenase (ALDH)-bright cell count. Additionally, we examined the impact of antibiotics on the mitochondrial stress response and membrane potential. Furthermore, we analyzed the levels of proteins associated with therapeutic resistance. There was no significant difference in the number of CSCs between cells cultured under normoxic and hypoxic conditions. However, hypoxia did affect the rate of CSC inhibition by antibiotics. Specifically, azithromycin was unable to inhibit sphere formation in hypoxia. Erythromycin and doxycycline did not reduce the ratio of ALDH-bright cells, despite decreasing the number of mammospheres. Furthermore, treatment with chloramphenicol, doxycycline, and tetracycline led to the overexpression of the breast cancer resistance protein. Our findings suggest that hypoxia may weaken the inhibitory effects of antibiotics on the breast cancer model.

Abstract 3978: The growth inhibitory effect of MDV3100 on hormone receptor-positive, HER2-negative breast cancer cell lines with low DYRK2 expression

Abstract Introduction: Tumor progression is the main cause of death in patients with breast cancer. Accumulating evidence suggests that dual-specificity tyrosine-regulated kinase 2 (DYRK2) functions as a tumor suppressor by regulating cell survival, differentiation, proliferation and apoptosis. Recently, we reported for the first time that DYRK2 inhibits breast cancer stem cells and tumor cell proliferation through transcriptional downregulation of KLF4 and CDK14, respectively.Our previous study showed that Androgen receptor (AR) was a DYRK2-dependent transcriptional activator. In this context, we hypothesized that MDV3100, an inhibitor of AR, may be effective for breast cancer with low DYRK2 expression. Methods: We established stable DYRK2-depleted cells. MCF-7 cells, hormone receptor-positive, HER2-negative breast cancer cell line, were transfected with pSuper-puro vector (pSuper control) or pSuper-puro DYRK2 shRNAs (shDYRK2) along with puromycin to isolate stable cell lines.These manipulated cells were compared with the control cells by various assays. We analyzed the activity of AR by Androgen receptor reporter assay. MTS assays were used to assess growth inhibition after treatment with MDV3100. Results: Androgen receptor reporter assay showed that knocking down DYRK2 induced AR transcription activity in MCF-7 cells.In MCF-7 DYRK2-depleted cells, MTS assays revealed that the treatment by MDV3100 was more effective than that in MCF-7 control cells. Conclusion: These findings demonstrate that reduced DYRK2 expression in hormone receptor-positive, HER2-negative breast cancer increases the activity of AR and significantly inhibits the growth by treatment with MDV3100. Citation Format: Yoshimi Imawari, Rei Mimoto, Kiyotsugu Yoshida. The growth inhibitory effect of MDV3100 on hormone receptor-positive, HER2-negative breast cancer cell lines with low DYRK2 expression. [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 3978.

Targeting hypoxia-induced CBS expression inhibits breast cancer stem cells through the induction of ferroptosis

Targeting hypoxia-induced CBS expression inhibits breast cancer stem cells through the induction of ferroptosis

Abstract P3-11-01: Increasing DAXX Expression in ER+ Breast Cancer Cells to Overcome Endocrine Therapy Resistance

Abstract Background: New treatment paradigms are needed to overcome resistance to endocrine therapy (ET; tamoxifen or aromatase inhibitors, AI) in ER+ breast cancer (BC). ET resistance is due to survival of breast cancer stem cells (BCSCs) that contribute to relapse of ER+ BC. Notch signaling drives BCSCs. In order to identify Notch specific biomarkers for the purpose of patient selection for anti-Notch therapy, we conducted a pre-surgical biomarker window study combining ET plus MK-0752, a -secretase inhibitor (GSI). Death Associated Protein 6 (DAXX) was discovered to be a novel Notch1 target gene and necessary for GSI-mediated inhibition of BCSCs. Subsequently, we found that DAXX alone was sufficient to inhibit BCSCs. In this current study, we investigated the mechanism by which high DAXX expression inhibited growth of ET resistant ER+ BC cells in vitro and in vivo. Methods: Isogenic ER+ BC cell lines (parental MCF-7, ET resistant MCF-7/5C, parental T47D, and ET resistant T47D-ED) were used. Cells were cultured in estrogen-deprived medium for more than 1 year to mimic AI use. DAXX was depleted using siRNA or overexpressed using a pCMV-expression vector. Bulk cell proliferation was analyzed in response to estrogen depletion or increasing concentrations of 17-estradiol. BCSC survival was measured using the mammosphere-forming assay. Tumor onset and burden were measured by injecting DAXX-expressing or depleted mammospheres into mammary fat pads of female, athymic nude mice. Recurrence of an ER+ PDX tumor (BCM 5097) was measured after withdrawal of estrogen. RNA sequencing identified enriched genes and pathways that required DAXX. Based on these results, cell death was assessed using Annexin V/7-AAD flow cytometry, PARP-1 and Caspase 8 cleavage, phosphorylation of JNK, and expression of apoptotic protein regulators, BIM, BAX, Bcl-2, and Bcl-xL. JNK signaling was inhibited using SB600125. Results: Estradiol stimulated proliferation of parental, ER+ MCF-7 and T47D cells. In contrast, estradiol inhibited proliferation of isogenic ET resistant MCF-7/5C and T47D-ED cells in a concentration and ER-dependent manner. Estradiol induced the DAXX protein in both ET sensitive and resistant cells. DAXX was required for BCSC survival in ET sensitive cells. However, once ER+ cells acquired resistance to ET, DAXX was necessary and sufficient to inhibit both bulk cell proliferation and BCSC survival, suggesting that increasing DAXX might be a novel approach to overcome ET resistance. In mice, high DAXX expression significantly inhibited tumor onset and burden of ET resistant tumors compared to DAXX-depleted tumors. Low DAXX expression was significantly associated with recurrence of an ER+ PDX tumor (BCM 5097) after withdrawal of estrogen. RNA sequencing revealed that DAXX activated an anti-neoplastic gene signature, including transcription factors that regulate cell death genes including the Bcl2-family. DAXX was required for high BIM expression and low levels of Bcl-xL. DAXX was necessary and sufficient to induce apoptosis, PARP-1 cleavage, and phosphorylation of JNK in ET resistant cells. A selective JNK inhibitor, SB600125 rescued DAXX-mediated inhibition of ET resistant bulk cell proliferation and BCSC survival, suggesting that high DAXX expression activates JNK signaling to regulate apoptotic proteins to induce cell death of BCSCs-derived from ET resistant BC. Conclusions: Expressing high DAXX levels is a potent method to inhibit ET-resistant BC cell proliferation and BCSC survival. The mechanism by which DAXX inhibits ET-resistant BC is through activation of JNK signaling, regulation of pro-apoptotic genes, and induction of apoptosis. The translational impact of this research is to identify novel agents that can increase DAXX expression and test them pre-clinically and in clinical trials for patients with ET-resistant breast cancer. Citation Format: Clodia Osipo, Kathy S. Albain, Daniel Peiffer, Debra Wyatt. Increasing DAXX Expression in ER+ Breast Cancer Cells to Overcome Endocrine Therapy Resistance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-01.

Tetrandrine Inhibits Cancer Stem Cell Characteristics and Epithelial to Mesenchymal Transition in Triple-Negative Breast Cancer via SOD1/ROS Signaling Pathway.

Targeting the stemness of triple-negativebreastcancer (TNBC)is a potential therapeutic approach for treating TNBC.Tetrandrine, a natural plant alkaloid, has several anticancer effects. Here, we aimed to evaluate the efficacy of tetrandrine in cancer stemness and epithelial to mesenchymal transition (EMT) in TNBC, and to explore the underlying mechanisms. The effects of tetrandrine on cell growth, cell viability, cell stemness capacity, cell migration, and cell invasion, as well as the molecules involved in these processes, were investigated in a cell culture system. An in vivo xenograft tumor and lung metastasis study was performed using nude mice to verify the effects and mechanisms of tetrandrine. Tetrandrine exhibited antiproliferative and cell cycle arrest activities in TNBC cell lines, significantly reduced aldehyde dehydrogenase and CD44[Formula: see text]CD24[Formula: see text] characteristic subpopulation, and successfully prevented mammosphere formation. It suppressed migration and invasion, enhanced anoikis, and regulated the expression of proteins involved in the EMT, including E-cadherin, Vimentin, and Occludin, in both TNBC cells and MDA-MB-231 spheroid cells. Further studies revealed that tetrandrine downregulated the expression of superoxide dismutase 1 (SOD1) and catalase and induced reactive oxygen species (ROS) production, which subsequently contributed to the inhibition of cell EMT and stemness. The in vivo studies also showed that tetrandrine inhibited tumor growth and metastasis of both adherent normal cells, and flow cytometry sorted specific CD44[Formula: see text]CD24[Formula: see text] breast cancer stem cells, which could be rescued by SOD1 overexpression. The results of this study suggest that tetrandrine could effectively inhibit breast cancer stem cell characteristics and the EMT process via the SOD1/ROS signaling pathway. Therefore, tetrandrine can be considered a promising anti-TNBC agent.

RICH1 inhibits breast cancer stem cell traits through activating kinases cascade of Hippo signaling by competing with Merlin for binding to Amot-p80

Cancer stem cells (CSCs) are regarded as the root of tumor recurrence and distant metastasis, as well as the major cause of resistance to conventional cancer therapies. Elucidating the mechanism of regulating CSCs is of great significance for the development of CSCs-targeting therapy strategies. YAP/TAZ are identified as key regulators of CSCs-related traits on breast cancer cells; however, the upstream regulatory mechanism of Hippo kinases cascade involved in regulating YAP/TAZ remains elusive. In this study, we found that the low expression of RICH1 in breast cancer was associated with poor prognosis. Depletion of RICH1 promoted the stemness and disrupted the normal epithelial architecture of MCF10A cells. Besides, RICH1 inhibited the migration and invasion of breast cancer cells and sensitized these cells to chemotherapeutic drugs. Mechanistically, RICH1 activated the kinases cascade of Hippo signaling via displacing Amot-p80 from the complex with Merlin. Further studies revealed that the deletion of the BAR domain of RICH1 abolished the function of attenuating the binding of Amot-p80 and Merlin, illustrating that the competitive binding to Amot-p80 with Merlin was mediated by the BAR domain of RICH1. In conclusion, our work elucidated the role and molecular mechanism of RICH1 in stemness regulation of breast cancer, and might provide opportunities for CSCs-targeting therapy.

Abstract P127: Mitochondrial matrix protease ClpP agonists suppress breast cancer stem cell function by downregulating multiple stem cell regulatory mechanisms

Abstract Background: We previously reported that small molecule ONC201 induces mitochondrial structural and functional damage, leading to death in breast cancer cells. Subsequent studies demonstrated that ONC201 and the related analogs TR compounds are agonists of mitochondrial caseinolytic protease P (ClpP), an essential protein for maintenance of mitochondrial protein homeostasis. Recent studies have shown that cancer stem cells (CSCs) preferentially use mitochondrial oxidative metabolism for energy production. Here, we report that ClpP agonists inhibit breast CSCs by unique mechanisms targeting pathways vital to maintain CSC function. Methods: ONC201 and other ClpP agonists (TR-57, 65), other mitochondria-targeting drugs, such as oligomycin, metformin, CPI-613 were used. MDA-MB231 cell line was used as a primary in vitro model system. CLPP knockout (KO) cells were generated by CRISPR/Cas9 technology. Seahorse XF analyzer was used for cellular respiration analysis. Luminescence-based assays were used for cell viability and metabolism assays. Protein expression was examined by Western blotting. Aldefluor assay and SORE6 (OCT4/SOX2 response element)-reporter gene were used to monitor CSC fraction. Mammosphere formation assay was used to evaluate CSC function in vitro. In vivo limiting dilution analysis was used to evaluate tumor initiation capability of cells injected into mammary fat pad of athymic nude female mice. Results: Seahorse XF analyzer showed that mammospheres are more dependent on OxPhos than glycolysis compared with cells grown in 2D, supporting the hypothesis that breast CSCs rely on OxPhos. ClpP agonists reduced the CSC fraction in both Aldefluor and SORE6 reporter assays. ClpP agonists inhibited mammosphere formation in CLPP WT, but not in CLPP KO cells, demonstrating the on-target effects on CSC function. In in vivo assays, tumor formation was significantly (p<0.001) inhibited in the ClpP agonist-treated group compared with the control group, and the effect was CLPP-dependent. Altogether, these findings support that ClpP agonists inhibit CSC in breast cancers. We found that ClpP agonists downregulate multiple pathways and proteins critical for CSC maintenance including mevalonate pathway, HIF1a, EPAS1, YAP, and Myc. We also observed that other mitochondria targeting drugs such as oligomycin, metformin downregulate these signaling pathways and proteins to some extent. Importantly, however, ClpP agonists showed significantly greater impact in mammosphere formation and cell growth assays, compared with other mitochondria-targeting drugs. Further studies revealed that ClpP agonists uniquely deplete NAD+/NADH and promote reactive oxygen species, both of which are shown as key factors to maintain pluripotency of stem cells. Moreover, ClpP agonists uniquely inhibited enzymes involved with glutamine catabolism and proline biosynthesis, vital to amino acids and nucleotide synthesis. Conclusion: ClpP agonists inhibit cell growth and tumor initiation in breast cancer cells by targeting multiple pathways essential to maintain CSC function. Citation Format: Yoshimi E. Greer, Lidia Hernandez, Donna Voeller, Raj Chari, Binwu Tang, Christina M. Annunziata, Sam Gilbert, Lalage Wakefield, Edwin Iwanowicz, Lee M. Graves, Stanley Lipkowitz. Mitochondrial matrix protease ClpP agonists suppress breast cancer stem cell function by downregulating multiple stem cell regulatory mechanisms [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P127.

MiR-142-3p targets HMGA2 and suppresses breast cancer malignancy

MicroRNAs (miRNAs) have the ability to regulate gene expression programs in cells. Hence, altered expression of miRNAs significantly contributes to breast cancer development and progression. Here, we demonstrate that the miRNA miR-142-3p directly targets the 3′ untranslated region of HMGA2, which encodes an onco-embryonic protein that is overexpressed in most cancers, including breast cancer. Down regulation of miR-142-3p predicting poor patient survival in grade 3 breast cancer (P-value = 0.045). MiR-142-3p downregulates HMGA2 mRNA and protein levels. Higher miR-142-3p and lower HMGA2 expressed are found in breast cancer versus normal breast tissue (P-value<0.05), and their levels inversely correlate in breast cancers (P-value = 1.46 × 10−4). We demonstrate that miR-142-3p induces apoptosis and G2/M cell cycle arrest in breast cancer cells. In addition, it inhibits breast cancer stem cell properties and decreases SOX2, NANOG, ALDH and c-Myc expression. MiR-142-3p also decreases cell proliferation through inhibition of the ERK/AKT/STAT3 signaling pathways. Finally, pathway analyses of patient samples suggest that these mechanisms also acting in the tumors of breast cancer patients. Thus, our work identifies HMGA2 as a direct miR-142-3p target and indicates that miR-142-3p is an important suppressor of breast cancer oncogenesis. This identifies miR-142-3p may candidate as a therapeutic molecule for breast cancer treatment.

An Extract of Taro (Colocasia esculenta) Mediates Potent Inhibitory Actions on Metastatic and Cancer Stem Cells by Tumor Cell-Autonomous and Immune-Dependent Mechanisms.

The taro plant, Colocasia esculenta, contains bioactive proteins with potential as cancer therapeutics. Several groups have reported anti-cancer activity in vitro and in vivo of taro-derived extracts (TEs). We reported that TE inhibits metastasis in a syngeneic murine model of Triple-Negative Breast Cancer (TNBC).Purpose:We sought to confirm our earlier studies in additional models and to identify novel mechanisms by which efficacy is achieved.Methods:We employed a panel of murine and human breast and ovarian cancer cell lines to determine the effect of TE on tumor cell viability, migration, and the ability to support cancer stem cells. Two syngeneic models of TNBC were employed to confirm our earlier report that TE potently inhibits metastasis. Cancer stem cell assays were employed to determine the ability of TE to inhibit tumorsphere-forming ability and to inhibit aldehyde dehydrogenase activity. To determine if host immunity contributes to the mechanism of metastasis inhibition, efficacy was assessed in immune-compromised mice.Results:We demonstrate that viability of some, but not all cell lines is inhibited by TE. Likewise, tumor cell migration is inhibited by TE. Using 2 immune competent, syngeneic models of TNBC, we confirm our earlier findings that tumor metastasis is potently inhibited by TE. We also demonstrate, for the first time, that TE directly inhibits breast cancer stem cells. Administration of TE to mice elicits expansion of several spleen cell populations but it was not known if host immune cells contribute to the mechanism by which TE inhibits tumor cell dissemination. In novel findings, we now show that the ability of TE to inhibit metastasis relies on immune T-cell-dependent, but not B cell or Natural Killer (NK)-cell-dependent mechanisms. Thus, both tumor cell-autonomous and host immune factors contribute to the mechanisms underlying TE efficacy. Our long-term goal is to evaluate TE efficacy in clinical trials. Most of our past studies as well as many of the results reported in this report were carried out using an isolation protocol described earlier (TE). In preparation for a near future clinical trial, we have now developed a strategy to isolate an enriched taro fraction, TE-method 2, (TE-M2) as well as a more purified subfraction (TE-M2F1) which can be scaled up under Good Manufacturing Practice (GMP) conditions for evaluation in human subjects. We demonstrate that TE-M2 and TE-M2F1 retain the anti-metastatic properties of TE.Conclusions:These studies provide further support for the continued examination of biologically active components of Colocasia esculenta as potential new therapeutic entities and identify a method to isolate sufficient quantities under GMP conditions to conduct early phase clinical studies.

Identification of key genes of hesperidin in inhibition of breast cancer stem cells by functional network analysis

Breast cancer therapy with classical chemotherapy is unable to eradicate breast cancer stem cells (BCSCs). Loss of p53 function causes growth and differentiation in cancer stem cells (CSCs); therefore, p53-targeted compounds can be developed for BCSCs-targeted drugs. Previously, hesperidin (HES), a citrus flavonoid, showed anticancer activities and increased efficacy of chemotherapy in several types of cancer in vitro and in vivo. This study was aimed to explore the key protein and molecular mechanism of hesperidin in the inhibition of BCSCs using bioinformatics and in vitro study. Bioinformatics analysis revealed about 75 potential therapeutic target proteins of HES in BCSCs (TH), in which TP53 was the only direct target protein (DTP) with a high degree score. Furthermore, the results of GO enrichment analysis showed that TH was taken part in the biological process of regulation of apoptosis and cell cycle. The KEGG pathway enrichment analysis also showed that TH is involved in several pathways, including cell cycle, p53 signaling pathway. In vitro experiment results showed that HES inhibited cell proliferation, mammosphere, and a colony formation, and migration in on MCF-7 3D cells (mammospheres). HES induced G0/G1 cell cycle arrest and apoptosis in MCF-7 cells 3D. In addition, HES treatment reduced the mRNA level of p21 but increased the mRNA level of cyclin D1 and p53 in the mammosphere. HES inhibits BCSCs in mammospheres. More importantly, this study highlighted p53 as a key protein in inhibition of BCSCs by HES. Future studies on the molecular mechanism are needed to validate the results of this study.

MicroRNA-128-3p overexpression inhibits breast cancer stem cell characteristics through suppression of Wnt signalling pathway by down-regulating NEK2.

Emerging evidence has reported that dysregulation of microRNAs (miRNAs) participated in the development of diverse types of cancers. Our initial microarray‐based analysis identified differentially expressed NEK2 related to breast cancer and predicted the regulatory microRNA‐128‐3p (miR‐128‐3p). Herein, this study aimed to characterize the tumour‐suppressive role of miR‐128‐3p in regulating the biological characteristics of breast cancer stem cells (BCSCs). CD44＋CD24−/low cells were selected for subsequent experiments. After verification of the target relationship between miR‐128‐3p and NEK2, the relationship among miR‐128‐3p, NEK2 and BCSCs was further investigated with the involvement of the Wnt signalling pathway. The regulatory effects of miR‐128‐3p on proliferation, migration, invasion and self‐renewal in vitro as well as tumorigenicity in vivo of BCSCs were examined via gain‐ and loss‐of‐function approaches. Highly expressed NEK2 was found in breast cancer based on GSE61304 expression profile. Breast cancer stem cells and breast cancer cells showed a down‐regulation of miR‐128‐3p. Overexpression of miR‐128‐3p was found to inhibit proliferation, migration, invasion, self‐renewal in vitro and tumorigenicity in vivo of BCSCs, which was further validated to be achieved through inhibition of Wnt signalling pathway by down‐regulating NEK2. In summary, this study indicates that miR‐128‐3p inhibits the stem‐like cell features of BCSCs via inhibition of the Wnt signalling pathway by down‐regulating NEK2, which provides a new target for breast cancer treatment.

Combination of Docetaxel with Thymoquinone in Nanoemulsion Impedes the Migration of Breast Cancer Stem Cells

Objectives: Metastatic breast cancer is an advanced stage that is highly dependent on migration signaling pathways, though it remains unclear what mechanisms control the maintenance of tumorigenic cells that drives the migration and invasion processes. This in vitro study examined the intercellular cross-talk in MCF-7 and MDA-MB-231 breast cancer cells when treated with the simultaneous administration of docetaxel (DOC) and thymoquinone (TQ) loaded in nanoemulsion (NE) delivery system. Method: The cell cycle and breast cancer stem cells (BCSC) population were evaluated using flow cytometric analysis, alongside a DNA fragmentation assessment. Further molecular investigations were performed utilizing quantitative polymerase chain reaction (Q-PCR) technique. Results: The DOC+TQ/NE formulation inhibits the proliferation of human cancer cells by the G2/M phase and S-phase cell cycle arrest in MCF-7 and MDA-MB-231 cells, receptively, and it stimulates apoptosis through induction of DNA damage. Moreover, the DOC+TQ/NE causes a significant reduction in the cellular migration activity in both cell lines as detected by the wound-healing assay. This observation could possibly be mediated through the inhibition of BCSC, accompanied by marked down-regulation of SNAIL-1 and TWIST-1 expression. Conclusion: Overall, the results demonstrate that the co-delivery of DOC and TQ within NE formulation might be considered as a promising and effective therapeutic approach for breast cancer that could prevent the intercellular signaling function of metastasis.

Caveolin-1 inhibits breast cancer stem cells via c-Myc-mediated metabolic reprogramming

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.

Bioinformatics and In Vitro Studies Reveal the Importance of p53, PPARG and Notch Signaling Pathway in Inhibition of Breast Cancer Stem Cells by Hesperetin.

Purpose: The failure of chemotherapy in breast cancer is caused by breast cancer stem cells (BCSCs), a minor population of cells in bulk mammary tumors. Previously, hesperetin, a citrus flavonoid, showed cytotoxicity in several cancer cells and increased cytotoxicity of doxorubicin and cisplatin. Hesperetin also inhibited osteogenic and adipocyte differentiation, however, a study of the effect of hesperetin on BCSCs has not yet been performed. Methods: In this study, we combined bioinformatics and in vitro works. A bioinformatic approach was performed to identify molecular targets, key proteins, and molecular mechanisms of hesperetin targeted at BCSCs, and genetic alterations among key genes. In addition, an in vitro study was carried out to measure the effects of hesperetin on BCSCs using the spheroids model of MCF-7 breast cancer cells (mammospheres). Results: Using a bioinformatics approach, we identified P53, PPARG, and Notch signaling as potential targets of hesperetin in inhibition of BCSCs. The in vitro study showed that hesperetin exhibits cytotoxicity on mammospheres, inhibits mammosphere and colony formation, and inhibits migration. Hesperetin modulates the cell cycle and induces apoptosis in mammospheres. Moreover, hesperetin treatment modulates the expression of p53, PPARG, and NOTCH1. Conclusion: Taken together, hesperetin has potential for the treatment of BCSC by targeting p53, PPARG and Notch signaling. Further investigation of the molecular mechanisms involved is required for the development of hesperetin as a BCSC-targeted drug.

Corrigendum to “Targeting LRP8 inhibits breast cancer stem cells in triple-negative breast cancer” [Canc. Lett. 438 (2018, Dec 1) 165–173

Corrigendum to “Targeting LRP8 inhibits breast cancer stem cells in triple-negative breast cancer” [Canc. Lett. 438 (2018, Dec 1) 165–173

Dihydrotanshinone-Induced NOX5 Activation Inhibits Breast Cancer Stem Cell through the ROS/Stat3 Signaling Pathway.

Cancer stem cells (CSCs) are known to mediate metastasis and recurrence and are therefore a promising therapeutic target. In this study, we found that dihydrotanshinone (DHTS) inhibits CSC formation. DHTS inhibited mammosphere formation in a dose-dependent manner and showed significant tumor growth inhibition in a xenograft model. This compound reduced the CD44high/CD24low- and aldehyde dehydrogenase- (ALDH-) expressing cell population and the self-renewal-related genes Nanog, SOX2, OCT4, C-Myc, and CD44. DHTS induced NOX5 activation by increasing calcium, and NOX5 activation induced reactive oxygen species (ROS) production. ROS production reduced the nuclear phosphorylation levels of Stat3 and secreted IL-6 levels in the mammospheres. DHTS deregulated the dynamic equilibrium from non-stem cancer cells to CSCs by dephosphorylating Stat3 and decreasing IL-6 secretion and inhibiting CSC formation. These novel findings showed that DHTS-induced ROS deregulated the Stat3/IL-6 pathway and induced CSC death. NOX5 activation by DHTS inhibits CSC formation through ROS/Stat3/IL-6 signaling, and DHTS may be a promising potential therapeutic agent against breast CSCs.