RESUMEN
Metastatic breast cancer is incurable and often due to breast cancer stem cell (CSC)-mediated self-renewal. We previously determined that the aryl hydrocarbon receptor (AhR) agonist aminoflavone (AF) inhibits the expression of the CSC biomarker α6-integrin (ITGA6) to disrupt the formation of luminal (hormone receptor-positive) mammospheres (3D breast cancer spheroids). In this study, we performed miRNA-sequencing analysis of luminal A MCF-7 mammospheres treated with AF to gain further insight into the mechanism of AF-mediated anti-cancer and anti-breast CSC activity. AF significantly induced the expression of >70 microRNAs (miRNAs) including miR125b-2-3p, a predicted stemness gene regulator. AF-mediated miR125b-2-3p induction was validated in MCF-7 mammospheres and cells. miR125b-2-3p levels were low in breast cancer tissues irrespective of subtype compared to normal breast tissues. While miR125b-2-3p levels were low in MCF-7 cells, they were much lower in AHR100 cells (MCF-7 cells made unresponsive to AhR agonists). The miR125b-2-3p mimic decreased, while the antagomiR125b-2-3p increased the expression of stemness genes ITGA6 and SOX2 in MCF-7 cells. In MCF-7 mammospheres, the miR125b-2-3p mimic decreased only ITGA6 expression although the antagomiR125b-2-3p increased ITGA6, SOX2 and MYC expression. AntagomiR125b-2-3p reversed AF-mediated suppression of ITGA6. The miR125b-2-3p mimic decreased proliferation, migration, and mammosphere formation while the antagomiR125b-2-3p increased proliferation and mammosphere formation in MCF-7 cells. The miR125b-2-3p mimic also inhibited proliferation, mammosphere formation, and migration in AHR100 cells. AF induced AhR- and miR125b2-3p-dependent anti-proliferation, anti-migration, and mammosphere disruption in MCF-7 cells. Our findings suggest that miR125b-2-3p is a tumor suppressor and AF upregulates miR125b-2-3p to disrupt mammospheres via mechanisms that rely at least partially on AhR in luminal A breast cancer cells.
RESUMEN
Breast cancer stem cells (BCSCs) promote endocrine therapy (ET) resistance, also known as endocrine resistance in hormone receptor (HR) positive breast cancer. Endocrine resistance occurs via mechanisms that are not yet fully understood. In vitro, in vivo and clinical data suggest that signaling cascades such as Notch, hypoxia inducible factor (HIF), and integrin/Akt promote BCSC-mediated endocrine resistance. Once HR positive breast cancer patients relapse on ET, targeted therapy agents such as cyclin dependent kinase inhibitors are frequently implemented, though secondary resistance remains a threat. Here, we discuss Notch, HIF, and integrin/Akt pathway regulation of BCSC activity and potential strategies to target these pathways to counteract endocrine resistance. We also discuss a plausible link between elevated BCSC-regulatory gene levels and reduced survival observed among African American women with basal-like breast cancer which lacks HR expression. Should future studies reveal a similar link for patients with luminal breast cancer, then the use of agents that impede BCSC activity could prove highly effective in improving clinical outcomes among African American breast cancer patients.
Asunto(s)
Antineoplásicos Hormonales/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Fulvestrant/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Antineoplásicos Hormonales/efectos adversos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Fulvestrant/efectos adversos , Humanos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Células Madre Neoplásicas/patología , Proteínas Proto-Oncogénicas c-akt/genética , Receptor ErbB-2/genética , Receptores de Estrógenos/antagonistas & inhibidores , Receptores de Estrógenos/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Nearly 40 000 women die annually from breast cancer in the United States. Clinically available targeted breast cancer therapy is largely ineffective in triple negative breast cancer (TNBC), characterized by tumors that lack expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her2). TNBC is associated with a poor prognosis. Previous reports show that aryl hydrocarbon receptor (AhR) partial agonist 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) selectively inhibits the growth of breast cancer cells, including those of the TNBC subtype. We previously demonstrated that 5F 203 induced the expression of putative tumor suppressor gene cytoglobin (CYGB) in breast cancer cells. In the current study, we determined that 5F 203 induces apoptosis and caspase-3 activation in MDA-MB-468 TNBC cells and in T47D ER+ PR + Her2 - breast cancer cells. We also show that caspases and CYGB promote 5F 203-mediated apoptosis in MDA-MB-468 cells. 5F 203 induced lysosomal membrane permeabilization (LMP) and cathepsin B release in MDA-MB-468 and T47D cells. In addition, silencing CYGB attenuated the ability of 5F 203 to induce caspase-3/-7 activation, proapoptotic gene expression, LMP, and cathepsin B release in MDA-MB-468 cells. Moreover, 5F 203 induced CYGB protein expression, proapoptotic protein expression, and caspase-3 cleavage in MDA-MB-468 cells and in MDA-MB-468 xenograft tumors grown orthotopically in athymic mice. These data provide a basis for the development of AhR ligands with the potential to restore CYGB expression as a novel strategy to treat TNBC.
Asunto(s)
Apoptosis/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/agonistas , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Citoglobina/metabolismo , Receptores de Hidrocarburo de Aril/agonistas , Receptores de Hidrocarburo de Aril/metabolismo , Tiazoles/farmacología , Neoplasias de la Mama Triple Negativas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Caspasa 3/metabolismo , Catepsina B/metabolismo , Línea Celular Tumoral , Citoglobina/genética , Femenino , Humanos , Ligandos , Ratones , Ratones Desnudos , Transfección , Neoplasias de la Mama Triple Negativas/patología , Carga Tumoral/efectos de los fármacos , Proteínas Supresoras de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
More than 40% of patients with luminal breast cancer treated with endocrine therapy agent tamoxifen demonstrate resistance. Emerging evidence suggests tumor initiating cells (TICs) and aberrant activation of Src and Akt signaling drive tamoxifen resistance and relapse. We previously demonstrated that aryl hydrocarbon receptor ligand aminoflavone (AF) inhibits the expression of TIC gene α6-integrin and disrupts mammospheres derived from tamoxifen-sensitive breast cancer cells. In the current study, we hypothesize that tamoxifen-resistant (TamR) cells exhibit higher levels of α6-integrin than tamoxifen-sensitive cells and that AF inhibits the growth of TamR cells by suppressing α6-integrin-Src-Akt signaling. In support of our hypothesis, TamR cells and associated mammospheres were found to exhibit elevated α6-integrin expression compared with their tamoxifen-sensitive counterparts. Furthermore, tumor sections from patients who relapsed on tamoxifen showed enhanced α6-integrin expression. Gene expression profiling from the TCGA database further revealed that basal-like breast cancer samples, known to be largely unresponsive to tamoxifen, demonstrated higher α6-integrin levels than luminal breast cancer samples. Importantly, AF reduced TamR cell viability and disrupted TamR mammospheres while concomitantly reducing α6-integrin messenger RNA and protein levels. In addition, AF and small interfering RNA against α6-integrin blocked tamoxifen-stimulated proliferation of TamR MCF-7 cells and further sensitized these cells to tamoxifen. Moreover, AF reduced Src and Akt signaling activation in TamR MCF-7 cells. Our findings suggest elevated α6-integrin expression is associated with tamoxifen resistance and AF suppresses α6-integrin-Src-Akt signaling activation to confer activity against TamR breast cancer.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Flavonoides/farmacología , Integrina alfa6/genética , Receptores de Hidrocarburo de Aril/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/genética , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Ligandos , Células MCF-7 , Células Madre Neoplásicas/efectos de los fármacos , Proteína Oncogénica v-akt/genética , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Transducción de Señal , Tamoxifeno/efectos adversos , Familia-src Quinasas/genéticaRESUMEN
Quassinoids often exhibit antioxidant and antiproliferative activity. Emerging evidence suggests that these natural metabolites also display chemopreventive actions. In this study, we investigated the potential for the quassinoid glaucarubulone glucoside (Gg), isolated from the endemic Jamaican plant Castela macrophylla (Simaroubaceae), to display potent cytotoxicity and inhibit human cytochrome P450s (CYPs), particularly CYP1A enzymes, known to convert polyaromatic hydrocarbons into carcinogenic metabolites. Gg reduced the viability of MCF-7 breast adenocarcinoma cells (IC50 = 121 nm) to a greater extent than standard of care anticancer agents 5-fluorouracil, tamoxifen (IC50 >10 µm) and the tamoxifen metabolite 4-hydroxytamoxifen (IC50 = 2.6 µm), yet was not cytotoxic to non-tumorigenic MCF-10A breast epithelial cells. Additionally, Gg induced MCF-7 breast cancer cell death. Gg blocked increases in reactive oxygen species in MCF-10A cells mediated by the polyaromatic hydrocarbon benzo[a]pyrene (B[a]P) metabolite B[a]P 1,6-quinone, yet downregulated the expression of genes that promote antioxidant activity in MCF-7 cells. This implies that Gg exhibits antioxidant and cytoprotective actions in non-tumorigenic breast epithelial cells and pro-oxidant, cytotoxic actions in breast cancer cells. Furthermore, Gg inhibited the activities of human CYP1A according to non-competitive kinetics and attenuated the ability of B[a]P to induce CYP1A gene expression in MCF-7 cells. These data indicate that Gg selectively suppresses MCF-7 breast cancer cell growth without impacting non-tumorigenic breast epithelial cells and blocks B[a]P-mediated CYP1A induction. Taken together, our data provide a rationale for further investigations of Gg and similar plant isolates as potential agents to treat and prevent breast cancer. Copyright © 2017 John Wiley & Sons, Ltd.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Citotoxinas/uso terapéutico , Glaucarrubina/análogos & derivados , Extractos Vegetales/uso terapéutico , Simaroubaceae/química , Antioxidantes/uso terapéutico , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Femenino , Expresión Génica/efectos de los fármacos , Glaucarrubina/uso terapéutico , Humanos , Jamaica , Células MCF-7/efectos de los fármacos , Cuassinas/uso terapéuticoRESUMEN
Traditional chemotherapies debulk tumors but fail to produce long-term clinical remissions due to their inability to eradicate tumor-initiating cells (TICs). This necessitates therapy with activity against the TIC niche. Αlpha6-integrin (α6-integrin) promotes TIC growth. In contrast, aryl hydrocarbon receptor (AhR) signaling activation impedes the formation of mammospheres (clusters of cells enriched for TICs). We investigated the ability of AhR agonist Aminoflavone (AF) and AF pro-drug (AFP464) to disrupt mammospheres derived from breast cancer cells and a M05 mammary mouse model of breast cancer respectively. We further examined the capacity of AF and AFP464 to exhibit anticancer activity and modulate the expression of 'stemness' genes including α6-integrin using immunofluorescence, flow cytometry and qRT-PCR analysis. AF disrupted mammospheres and prevented secondary mammosphere formation. In contrast, AF did not disrupt mammospheres derived from AhR ligand-unresponsive MCF-7 cells. AFP464 treatment suppressed M05 tumor growth and disrupted corresponding mammospheres. AF and AFP464 reduced the expression and percentage of cells that stained for 'stemness' markers including α6-integrin in vitro and in vivo respectively. These data suggest AFP464 thwarts bulk breast tumor and TIC growth via AhR agonist-mediated α6-integrin inhibition.
Asunto(s)
Antineoplásicos/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/agonistas , Neoplasias de la Mama/tratamiento farmacológico , Flavonoides/farmacología , Integrina alfa6/metabolismo , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Profármacos/farmacología , Receptores de Hidrocarburo de Aril/agonistas , Transporte Activo de Núcleo Celular , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Integrina alfa6/genética , Ligandos , Células MCF-7 , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones Endogámicos BALB C , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fenotipo , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal/efectos de los fármacos , Esferoides Celulares , Factores de TiempoRESUMEN
Breast tumors often show profound sensitivity to exogenous oxidative stress. Investigational agent 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) induces aryl hydrocarbon receptor (AhR)-mediated DNA damage in certain breast cancer cells. Since AhR agonists often elevate intracellular oxidative stress, we hypothesize that 5F 203 increases reactive oxygen species (ROS) to induce DNA damage, which thwarts breast cancer cell growth. We found that 5F 203 induced single-strand break formation. 5F 203 enhanced oxidative DNA damage that was specific to breast cancer cells sensitive to its cytotoxic actions, as it did not increase oxidative DNA damage or ROS formation in nontumorigenic MCF-10A breast epithelial cells. In contrast, AhR agonist and procarcinogen benzo[a]pyrene and its metabolite, 1,6-benzo[a]pyrene quinone, induced oxidative DNA damage and ROS formation, respectively, in MCF-10A cells. In sensitive breast cancer cells, 5F 203 activated ROS-responsive kinases: c-Jun-N-terminal kinase (JNK) and p38 mitogen activated protein kinase (p38). AhR antagonists (alpha-naphthoflavone, CH223191) or antioxidants (N-acetyl-l-cysteine, EUK-134) attenuated 5F 203-mediated JNK and p38 activation, depending on the cell type. Pharmacological inhibition of AhR, JNK, or p38 attenuated 5F 203-mediated increases in intracellular ROS, apoptosis, and single-strand break formation. 5F 203 induced the expression of cytoglobin, an oxidative stress-responsive gene and a putative tumor suppressor, which was diminished with AhR, JNK, or p38 inhibition. Additionally, 5F 203-mediated increases in ROS production and cytoglobin were suppressed in AHR100 cells (AhR ligand-unresponsive MCF-7 breast cancer cells). Our data demonstrate 5F 203 induces ROS-mediated DNA damage at least in part via AhR, JNK, or p38 activation and modulates the expression of oxidative stress-responsive genes such as cytoglobin to confer its anticancer action.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Daño del ADN/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Receptores de Hidrocarburo de Aril/metabolismo , Tiazoles/farmacología , Apoptosis/efectos de los fármacos , Mama/efectos de los fármacos , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Células MCF-7 , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
A series of cinnamylideneacetophenones were synthesized via a modified Claisen-Schmidt condensation reaction and evaluated for cytotoxicity against breast cancer cells using the Alamar Blue™ assay. Derivatives 17 and 18 bearing a 2-nitro group on the B ring, exhibited sub-micromolar cytotoxicity in MCF-7 cells (IC50=71 and 1.9 nM), respectively. Derivative 17 also displayed sub-micromolar (IC50=780 nM) cytotoxicity in MDA-MB-468 cells. Additionally, 17 and 18 displayed significantly less cytotoxicity than the chemotherapeutic doxorubicin in non-tumorigenic MCF-10A cells. This study provides evidence supporting the continued development of nitro-substituted cinnamylideneacetophenones as small molecules to treat breast cancer.