RESUMEN
Polysaccharides and proteins are compatible macromolecules that can be used to obtain biopolymeric hydrogels through physical interactions. In this study, an environmentally friendly strategy is being proposed to produce gelatin-xanthan gum- cellulose hydrogels, without the addition of chemical synthetic crosslinkers. Xanthan gum was employed as an alternative crosslinking agent, and cellulose was used as a potential reinforcing agent in the polymeric matrix. Firstly, the biopolymers were mixed by the extrusion process, and glycerol was used as a plasticizer. Then, the polymeric mixture was molded by thermopressing to obtain hydrogels as laminated films. All hydrogels formulations resulted in films with smooth surfaces, without pores or cracks, resulting in amorphous polymeric matrices. The obtained hydrogels had a pH-dependent degree of swelling, the highest swelling values were obtained at pH 4 (5.3-7.9 g/g) after 24 h of immersion. Cellulose acted as a reinforcing agent for hydrogels, increasing thermal stability, tensile strength, and Young's modulus of films when employed at the higher level (7%). The strategy employed in this study to obtain nontoxic hydrogels without synthetic crosslinkers was effective, resulting in materials with promising properties to be used as pharmaceutical forms to deliver active compounds in cosmetic or pharmaceutical products.
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Celulosa , Gelatina , Gelatina/química , Hidrogeles/química , Polisacáridos Bacterianos/química , Polímeros/químicaRESUMEN
BACKGROUND: Optimising breast cancer treatment remains a challenge. Resistance to therapy is a major problem in both ER- and ER+ breast cancer. Tumour recurrence after chemotherapy and/or targeted therapy leads to more aggressive tumours with enhanced metastatic ability. Self-renewing cancer stem cells (CSCs) have been implicated in treatment resistance, recurrence and the development of metastatic disease. METHODS: In this study, we utilised in vitro, in vivo and ex vivo breast cancer models using ER+ MCF-7 and ER- MDA-MB-231 cells, as well as solid and metastatic breast cancer patient samples, to interrogate the effects of FKBPL and its peptide therapeutics on metastasis, endocrine therapy resistant CSCs and DLL4 and Notch4 expression. The effects of FKBPL overexpression or peptide treatment were assessed using a t-test or one-way ANOVA with Dunnett's multiple comparison test. RESULTS: We demonstrated that FKBPL overexpression or treatment with FKBPL-based therapeutics (AD-01, pre-clinical peptide /ALM201, clinical peptide) inhibit i) CSCs in both ER+ and ER- breast cancer, ii) cancer metastasis in a triple negative breast cancer metastasis model and iii) endocrine therapy resistant CSCs in ER+ breast cancer, via modulation of the DLL4 and Notch4 protein and/or mRNA expression. AD-01 was effective at reducing triple negative MDA-MB-231 breast cancer cell migration (n ≥ 3, p < 0.05) and invasion (n ≥ 3, p < 0.001) and this was translated in vivo where AD-01 inhibited breast cancer metastasis in MDA-MB-231-lucD3H1 in vivo model (p < 0.05). In ER+ MCF-7 cells and primary breast tumour samples, we demonstrated that ALM201 inhibits endocrine therapy resistant mammospheres, representative of CSC content (n ≥ 3, p < 0.05). Whilst an in vivo limiting dilution assay, using SCID mice, demonstrated that ALM201 alone or in combination with tamoxifen was very effective at delaying tumour recurrence by 12 (p < 0.05) or 21 days (p < 0.001), respectively, by reducing the number of CSCs. The potential mechanism of action, in addition to CD44, involves downregulation of DLL4 and Notch4. CONCLUSION: This study demonstrates, for the first time, the pre-clinical activity of novel systemic anti-cancer therapeutic peptides, ALM201 and AD-01, in the metastatic setting, and highlights their impact on endocrine therapy resistant CSCs; both areas of unmet clinical need.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Inmunofilinas/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Péptidos/farmacología , Proteínas Adaptadoras Transductoras de Señales , Animales , Antineoplásicos Hormonales/farmacología , Antineoplásicos Hormonales/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Mama/patología , Neoplasias de la Mama/patología , Proteínas de Unión al Calcio , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Antineoplásicos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunofilinas/uso terapéutico , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones SCID , Recurrencia Local de Neoplasia/prevención & control , Células Madre Neoplásicas/patología , Péptidos/uso terapéutico , Receptor Notch4/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas de Unión a Tacrolimus , Tamoxifeno/farmacología , Tamoxifeno/uso terapéutico , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Breast cancer specific mortality results from tumour cell dissemination and metastatic colonisation. Identification of the cells and processes responsible for metastasis will enable better prevention and control of metastatic disease, thus reducing relapse and mortality. To better understand these processes, we prospectively collected 307 patient-derived breast cancer samples (n = 195 early breast cancers (EBC) and n = 112 metastatic samples (MBC)). We assessed colony-forming activity in vitro by growing isolated cells in both primary (formation) and secondary (self-renewal) mammosphere culture, and tumour initiating activity in vivo through subcutaneous transplantation of fragments or cells into mice. Metastatic samples formed primary mammosphere colonies significantly more frequently than early breast cancers and had significantly higher primary mammosphere colony formation efficiency (0.9 % vs. 0.6 %; p < 0.0001). Tumour initiation in vivo was significantly higher in metastatic than early breast cancer samples (63 % vs. 38 %, p = 0.04). Of 144 breast cancer samples implanted in vivo, we established 20 stable patient-derived xenograft (PDX) models at passage 2 or greater. Lung metastases were detected in mice from 14 PDX models. Mammosphere colony formation in vitro significantly correlated with the ability of a tumour to metastasise to the lungs in vivo (p = 0.05), but not with subcutaneous tumour initiation. In summary, the breast cancer stem cell activities of colony formation and tumour initiation are increased in metastatic compared to early samples, and predict metastasis in vivo. These results suggest that breast stem cell activity will predict for poor outcome tumours, and therapy targeting this activity will improve outcomes for patients with metastatic disease.
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Neoplasias de la Mama/patología , Mama/patología , Transformación Celular Neoplásica/patología , Xenoinjertos/patología , Metástasis de la Neoplasia/patología , Animales , Técnicas de Cultivo de Célula/métodos , Proliferación Celular/fisiología , Progresión de la Enfermedad , Femenino , Humanos , Ratones , Estudios ProspectivosRESUMEN
Nanomedicine nowadays offers novel solutions in cancer therapy and diagnosis by introducing multimodal treatments and imaging tools in one single formulation. Nanoparticles acting as nanocarriers change the solubility, biodistribution and efficiency of therapeutic molecules, reducing their side effects. In order to successfully apply these novel therapeutic approaches, efforts are focused on the biological functionalization of the nanoparticles to improve the selectivity towards cancer cells. In this work, we present the synthesis and characterization of novel multifunctionalized iron oxide magnetic nanoparticles (MNPs) with antiCD44 antibody and gemcitabine derivatives, and their application for the selective treatment of CD44-positive cancer cells. The lymphocyte homing receptor CD44 is overexpressed in a large variety of cancer cells, but also in cancer stem cells (CSCs) and circulating tumor cells (CTCs). Therefore, targeting CD44-overexpressing cells is a challenging and promising anticancer strategy. Firstly, we demonstrate the targeting of antiCD44 functionalized MNPs to different CD44-positive cancer cell lines using a CD44-negative non-tumorigenic cell line as a control, and verify the specificity by ultrastructural characterization and downregulation of CD44 expression. Finally, we show the selective drug delivery potential of the MNPs by the killing of CD44-positive cancer cells using a CD44-negative non-tumorigenic cell line as a control. In conclusion, the proposed multifunctionalized MNPs represent an excellent biocompatible nanoplatform for selective CD44-positive cancer therapy in vitro.
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Compuestos Férricos/química , Receptores de Hialuranos/metabolismo , Nanopartículas/química , Línea Celular Tumoral , Química Farmacéutica/métodos , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Compuestos Férricos/administración & dosificación , Humanos , Magnetismo/métodos , Nanomedicina/métodos , Nanopartículas/administración & dosificación , Células Neoplásicas Circulantes/metabolismo , Células Madre Neoplásicas/metabolismo , Distribución Tisular/fisiología , GemcitabinaRESUMEN
INTRODUCTION: Although oestrogen is essential for the development of the normal breast, adult mammary stem cells are known to be oestrogen receptor alpha (ER) negative and rely on paracrine signals in the mammary epithelium for mediation of developmental cues. However, little is known about how systemic oestrogen regulates breast cancer stem cell (CSC) activity. METHODS: Here, we tested the effects of oestrogen on CSC activity in vitro and in vivo and investigated which paracrine signalling pathways locally mediate oestrogen effects. RESULTS: CSC-enriched populations (ESA+CD44+CD24low) sorted from ER positive patient derived and established cell lines have low or absent ER expression. However, oestrogen stimulated CSC activity demonstrated by increased mammosphere and holoclone formation in vitro and tumour formation in vivo. This effect was abrogated by the anti-oestrogen tamoxifen or ER siRNA. These data suggest that the oestrogen response is mediated through paracrine signalling from non-CSCs to CSCs. We have, therefore, investigated both epidermal growth factor (EGF) and Notch receptor signals downstream of oestrogen. We demonstrate that gefitinib (epidermal growth factor receptor (EGFR) inhibitor) and gamma secretase inhibitors (Notch inhibitor) block oestrogen-induced CSC activity in vitro and in vivo but GSIs more efficiently reduce CSC frequency. CONCLUSIONS: These data establish that EGF and Notch receptor signalling pathways operate downstream of oestrogen in the regulation of ER negative CSCs.
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Neoplasias de la Mama/tratamiento farmacológico , Receptores ErbB/metabolismo , Estrógenos/farmacología , Regulación Neoplásica de la Expresión Génica , Células Madre Neoplásicas/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Receptores Notch/metabolismo , Animales , Western Blotting , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Transformación Celular Neoplásica , Receptores ErbB/genética , Femenino , Citometría de Flujo , Humanos , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Comunicación Paracrina , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Estrógenos/antagonistas & inhibidores , Receptores de Estrógenos/genética , Receptores Notch/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Breast cancer stem-like cells (CSCs) are an important therapeutic target as they are purported to be responsible for tumor initiation, maintenance, metastases, and disease recurrence. Interleukin-8 (IL-8) is upregulated in breast cancer compared with normal breast tissue and is associated with poor prognosis. IL-8 is reported to promote breast cancer progression by increasing cell invasion, angiogenesis, and metastases and is upregulated in HER2-positive cancers. Recently, we and others have established that IL-8 via its cognate receptors, CXCR1 and CXCR2, is also involved in regulating breast CSC activity. Our work demonstrates that in metastatic breast CSCs, CXCR1/2 signals via transactivation of HER2. Given the importance of HER2 in breast cancer and in regulating CSC activity, a pathway driving the activation of these receptors would have important biological and clinical consequences, especially in tumors that express high levels of IL-8 and other CXCR1/2-activating ligands. Here, we review the IL-8 signaling pathway and the role of HER2 in maintaining an IL-8 inflammatory loop and discuss the potential of combining CXCR1/2 inhibitors with other treatments such as HER2-targeted therapy as a novel approach to eliminate CSCs and improve patient survival.
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Neoplasias de la Mama/metabolismo , Interleucina-8/metabolismo , Células Madre Neoplásicas/metabolismo , Transducción de Señal , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Femenino , Humanos , Inflamación/metabolismo , Inflamación/patología , Terapia Molecular Dirigida , Células Madre Neoplásicas/efectos de los fármacos , Receptor ErbB-2/metabolismo , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/metabolismo , Transducción de Señal/efectos de los fármacosAsunto(s)
Neoplasias de la Mama/etnología , Carcinoma Ductal de Mama/etnología , Resistencia a Antineoplásicos , Células Madre Neoplásicas/efectos de los fármacos , Población Negra , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/tratamiento farmacológico , Carcinoma Ductal de Mama/genética , Doxorrubicina/efectos adversos , Femenino , Humanos , Proyectos Piloto , Población BlancaRESUMEN
Since the discovery that neural tissue contains a population of stem cells that form neurospheres in vitro, sphere-forming assays have been adapted for use with a number of different tissue types for the quantification of stem cell activity and self-renewal. One tissue type widely used for stem cell investigations is mammary tissue, and the mammosphere assay has been used in both normal tissue and cancer. Although it is a relatively simple assay to learn, it can be difficult to master. There are methodological and analytical aspects to the assay which require careful consideration when interpreting the results. We describe here a detailed mammosphere assay protocol for the assessment of stem cell activity and self-renewal, and discuss how data generated by the assay can be analysed and interpreted.
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Neoplasias de la Mama/patología , Glándulas Mamarias Humanas/patología , Células Madre Neoplásicas/patología , Ensayo de Tumor de Célula Madre , Animales , Carcinoma Intraductal no Infiltrante/patología , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Glándulas Mamarias Animales/patologíaRESUMEN
Given the environmental issues caused by the extensive use of conventional petroleum-based packaging, this work proposes functional films based on commercial κ-carrageenan (κc), poly(vinyl alcohol) (PVA), and gallic acid (GA) prepared by the "casting" method. Metallic ions in the κc composition stabilized the films, supporting processability and suitable mechanical properties. However, the incorporated GA amount (6.25 and 10 wt%) in the films created from an aqueous κc solution at 3.0 % wt/v (κc3) prevented crystalline domains in the resulting materials. The κc3/GA6.25 and κc3/GA10 films had less tensile strength (8.50 ± 0.61 and 10.28 ± 0.65 MPa) and high elongation at break (2.36 ± 0.16 and 1.19 ± 0.17 %) compared to the other samples, respectively. Low κc contents (κc2.5/GA6.25 and κc2.5/GA10) promoted stiff films and less permeability to water vapor (5.36 ± 0.51 and 3.76 ± 0.02 [×10-12 g(Pa × m × s)-1], respectively. The κc/GA weight ratio also influenced the film wettability, indicating water contact angles (WCAs) between 55 and 74°. The surface wettability implies a low oil permeability and high water swelling capacity of up to 1600 %. The κc/GA also played an essential role in the film's antimicrobial action against Staphylococcus aureus and Escherichia coli. Thus, the κc3/GA10 film showed suitable physical, chemical, and biological properties, having the potential to be applied as food coatings.
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Ácido Gálico , Alcohol Polivinílico , Carragenina/química , Alcohol Polivinílico/química , Resistencia a la Tracción , Permeabilidad , Escherichia coli , Embalaje de Alimentos/métodosRESUMEN
BACKGROUND: Breast cancer is a leading cause of death in premenopausal women. Progesterone drives expansion of luminal progenitor cells, leading to the development of poor-prognostic breast cancers. However, it is not known if antagonising progesterone can prevent breast cancers in humans. We suggest that targeting progesterone signalling could be a means of reducing features which are known to promote breast cancer formation. METHODS: In healthy premenopausal women with and without a BRCA mutation we studied (i) estrogen and progesterone levels in saliva over an entire menstrual cycle (n = 20); (ii) cancer-free normal breast-tissue from a control population who had no family or personal history of breast cancer and equivalently from BRCA1/2 mutation carriers (n = 28); triple negative breast cancer (TNBC) biopsies and healthy breast tissue taken from sites surrounding the TNBC in the same individuals (n = 14); and biopsies of ER+ve/PR+ve stage T1-T2 cancers and healthy breast tissue taken from sites surrounding the cancer in the same individuals (n = 31); and (iii) DNA methylation and DNA mutations in normal breast tissue (before and after treatment) from clinical trials that assessed the potential preventative effects of vitamins and antiprogestins (mifepristone and ulipristal acetate; n = 44). RESULTS: Daily levels of progesterone were higher throughout the menstrual cycle of BRCA1/2 mutation carriers, raising the prospect of targeting progesterone signalling as a means of cancer risk reduction in this population. Furthermore, breast field cancerization DNA methylation signatures reflective of (i) the mitotic age of normal breast epithelium and (ii) the proportion of luminal progenitor cells were increased in breast cancers, indicating that luminal progenitor cells with elevated replicative age are more prone to malignant transformation. The progesterone receptor antagonist mifepristone reduced both the mitotic age and the proportion of luminal progenitor cells in normal breast tissue of all control women and in 64% of BRCA1/2 mutation carriers. These findings were validated by an alternate progesterone receptor antagonist, ulipristal acetate, which yielded similar results. Importantly, mifepristone reduced both the TP53 mutation frequency as well as the number of TP53 mutations in mitotic-age-responders. CONCLUSIONS: These data support the potential usage of antiprogestins for primary prevention of poor-prognostic breast cancers. TRIAL REGISTRATION: Clinical trial 1 Mifepristone treatment prior to insertion of a levonorgestrel releasing intrauterine system for improved bleeding control - a randomized controlled trial, clinicaltrialsregister.eu, 2009-009014-40 ; registered on 20 July 2009. Clinical trial 2 The effect of a progesterone receptor modulator on breast tissue in women with BRCA1 and 2 mutations, clinicaltrials.gov, NCT01898312 ; registered on 07 May 2013. Clinical trial 3 A pilot prevention study of the effects of the anti- progestin Ulipristal Acetate (UA) on surrogate markers of breast cancer risk, clinicaltrialsregister.eu, 2015-001587-19 ; registered on 15 July 2015.
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Neoplasias de la Mama , Neoplasias de la Mama Triple Negativas , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Epigénesis Genética , Femenino , Humanos , Mifepristona , Mutación , Progesterona , Receptores de Estrógenos/genética , Receptores de Progesterona/genética , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
There is increasing evidence that breast cancers contain tumor-initiating cells with stem cell properties. The importance of estrogen in the development of the mammary gland and in breast cancer is well known, but the influence of estrogen on the stem cell population has not been assessed. We show that estrogen reduces the proportion of stem cells in the normal human mammary gland and in breast cancer cells. The embryonic stem cell genes NANOG, OCT4, and SOX2 are expressed in normal breast stem cells and at higher levels in breast tumor cells and their expression decreases upon differentiation. Overexpression of each stem cell gene reduces estrogen receptor (ER) expression, and increases the number of stem cells and their capacity for invasion, properties associated with tumorigenesis and poor prognosis. These results indicate that estrogen reduces the size of the human breast stem cell pool and may provide an explanation for the better prognosis of ER-positive tumors.
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Mama/citología , Mama/efectos de los fármacos , Estrógenos/farmacología , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Adulto , Antineoplásicos Hormonales/farmacología , Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular Tumoral , Femenino , Expresión Génica/genética , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Glándulas Mamarias Humanas/efectos de los fármacos , Persona de Mediana Edad , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Esferoides Celulares/efectos de los fármacos , Tamoxifeno/farmacología , Adulto JovenRESUMEN
The identification and characterization of normal and breast cancer stem cells have provided a new vision of breast tumorigenesis. Cancer stem cells may be responsible for breast tumor initiation, progression and development of resistance to therapy. Most breast cancers express the estrogen receptor, and several studies have linked long-term estrogen exposure to enhanced breast cancer risk; however, estrogen receptor-positive tumors usually present a better prognosis than estrogen receptor-negative ones. The finding that estrogen reduces the pool of human breast stem cells may explain the more differentiated phenotype observed in estrogen receptor-positive tumors. In this article, our current understanding of the complex role of estrogen in human breast stem cells is discussed in the context of breast malignancy.
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Diferenciación Celular , Estrógenos/metabolismo , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Humanos , Receptores de Estrógenos/metabolismo , Células Madre/metabolismoRESUMEN
It is unclear how genetic aberrations impact the state of nascent tumour cells and their microenvironment. BRCA1 driven triple negative breast cancer (TNBC) has been shown to arise from luminal progenitors yet little is known about how BRCA1 loss-of-function (LOF) and concomitant mutations affect the luminal progenitor cell state. Here we demonstrate how time-resolved single-cell profiling of genetically engineered mouse models before tumour formation can address this challenge. We found that perturbing Brca1/p53 in luminal progenitors induces aberrant alveolar differentiation pre-malignancy accompanied by pro-tumourigenic changes in the immune compartment. Unlike alveolar differentiation during gestation, this process is cell autonomous and characterised by the dysregulation of transcription factors driving alveologenesis. Based on our data we propose a model where Brca1/p53 LOF inadvertently promotes a differentiation program hardwired in luminal progenitors, highlighting the deterministic role of the cell-of-origin and offering a potential explanation for the tissue specificity of BRCA1 tumours.
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Proteína BRCA1/genética , Transformación Celular Neoplásica/genética , Neoplasias Mamarias Experimentales/genética , Fenobarbital/metabolismo , Análisis de la Célula Individual/métodos , Células Madre/patología , Animales , Proteína BRCA1/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Transformación Celular Neoplásica/metabolismo , Femenino , Humanos , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Mutación , Células Madre/fisiología , Microambiente Tumoral/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Nanotechnology-based approaches hold substantial potential to avoid chemoresistance and minimize side effects. In this work, we have used biocompatible iron oxide magnetic nanoparticles (MNPs) called MF66 and functionalized with the antineoplastic drug doxorubicin (DOX) against MDA-MB-231 cells. Electrostatically functionalized MNPs showed effective uptake and DOX linked to MNPs was more efficiently retained inside the cells than free DOX, leading to cell inactivation by mitotic catastrophe, senescence and apoptosis. Both effects, uptake and cytotoxicity, were demonstrated by different assays and videomicroscopy techniques. Likewise, covalently functionalized MNPs using three different linkers-disulfide (DOX-S-S-Pyr, called MF66-S-S-DOX), imine (DOX-I-Mal, called MF66-I-DOX) or both (DOX-I-S-S-Pyr, called MF66-S-S-I-DOX)-were also analysed. The highest cell death was detected using a linker sensitive to both pH and reducing environment (DOX-I-S-S-Pyr). The greatest success of this study was to detect also their activity against breast cancer stem-like cells (CSC) from MDA-MB-231 and primary breast cancer cells derived from a patient with a similar genetic profile (triple-negative breast cancer). In summary, these nanoformulations are promising tools as therapeutic agent vehicles, due to their ability to produce efficient internalization, drug delivery, and cancer cell inactivation, even in cancer stem-like cells (CSCs) from patients.
RESUMEN
Estrogen-receptor-positive breast tumors are treated with anti-estrogen (AE) therapies but frequently develop resistance. Cancer stem cells (CSCs) with high aldehyde dehydrogenase activity (ALDH+ cells) are enriched following AE treatment. Here, we show that the interleukin-1ß (IL-1ß) signaling pathway is activated in ALDH+ cells, and data from single cells reveals that AE treatment selects for IL-1 receptor (IL1R1)-expressing ALDH+ cells. Importantly, CSC activity is reduced by an IL1R1 inhibitor in AE-resistant models. Moreover, IL1R1 expression is increased in the tumors of patients treated with AE therapy and predicts treatment failure. Single-cell gene expression analysis revealed that at least two subpopulations exist within the ALDH+ population, one proliferative and one quiescent. Following AE therapy the quiescent population is expanded, which suggests CSC dormancy as an adaptive strategy that facilitates treatment resistance. Targeting of ALDH+IL1R1+ cells merits testing as a strategy to combat AE resistance in patients with residual disease.
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Familia de Aldehído Deshidrogenasa 1/metabolismo , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Antagonistas de Estrógenos/farmacología , Células Madre Neoplásicas/enzimología , Receptores de Interleucina-1/metabolismo , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis de la Neoplasia , Células Madre Neoplásicas/efectos de los fármacos , Análisis de la Célula Individual , Transcriptoma/genéticaRESUMEN
Estrogen receptor (ER) positive breast cancer is frequently sensitive to endocrine therapy. Multiple mechanisms of endocrine therapy resistance have been identified, including cancer stem-like cell (CSC) activity. Here we investigate SFX-01, a stabilised formulation of sulforaphane (SFN), for its effects on breast CSC activity in ER+ preclinical models. SFX-01 reduced mammosphere formation efficiency (MFE) of ER+ primary and metastatic patient samples. Both tamoxifen and fulvestrant increased MFE and aldehyde dehydrogenase (ALDH) activity of patient-derived xenograft (PDX) tumors, which was reversed by combination with SFX-01. SFX-01 significantly reduced tumor-initiating cell frequency in secondary transplants and reduced the formation of spontaneous lung micrometastases by PDX tumors in mice. Mechanistically, we establish that both tamoxifen and fulvestrant induce STAT3 phosphorylation. SFX-01 suppressed phospho-STAT3 and SFN directly bound STAT3 in patient and PDX samples. Analysis of ALDH+ cells from endocrine-resistant patient samples revealed activation of STAT3 target genes MUC1 and OSMR, which were inhibited by SFX-01 in patient samples. Increased expression of these genes after 3 months' endocrine treatment of ER+ patients (n = 68) predicted poor prognosis. Our data establish the importance of STAT3 signaling in CSC-mediated resistance to endocrine therapy and the potential of SFX-01 for improving clinical outcomes in ER+ breast cancer.
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Neoplasias de la Mama/terapia , Isotiocianatos/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Anticarcinógenos/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Células Madre Neoplásicas/metabolismo , Transducción de Señal/genética , Sulfóxidos , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Despite the effectiveness of endocrine therapies to treat estrogen receptor-positive (ER+) breast tumours, two thirds of patients will eventually relapse due to de novo or acquired resistance to these agents. Cancer Stem-like Cells (CSCs), a rare cell population within the tumour, accumulate after anti-estrogen treatments and are likely to contribute to their failure. Here we studied the role of p21-activated kinase 4 (PAK4) as a promising target to overcome endocrine resistance and disease progression in ER + breast cancers. PAK4 predicts for resistance to tamoxifen and poor prognosis in 2 independent cohorts of ER + tumours. We observed that PAK4 strongly correlates with CSC activity in metastatic patient-derived samples irrespective of breast cancer subtype. However, PAK4-driven mammosphere-forming CSC activity increases alongside progression only in ER + metastatic samples. PAK4 activity increases in ER + models of acquired resistance to endocrine therapies. Targeting PAK4 with either CRT PAKi, a small molecule inhibitor of PAK4, or with specific siRNAs abrogates CSC activity/self-renewal in clinical samples and endocrine-resistant cells. Together, our findings establish that PAK4 regulates stemness during disease progression and that its inhibition reverses endocrine resistance in ER + breast cancers.
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Antineoplásicos Hormonales/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Células Madre Neoplásicas/patología , Receptores de Estrógenos/metabolismo , Tamoxifeno/farmacología , Quinasas p21 Activadas/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Progresión de la Enfermedad , Regulación hacia Abajo , Resistencia a Antineoplásicos , Antagonistas del Receptor de Estrógeno/farmacología , Femenino , Fulvestrant/farmacología , Expresión Génica , Humanos , Células MCF-7 , Metaanálisis como Asunto , Metástasis de la Neoplasia , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Pronóstico , Bibliotecas de Moléculas Pequeñas/farmacología , Quinasas p21 Activadas/antagonistas & inhibidores , Quinasas p21 Activadas/biosíntesis , Quinasas p21 Activadas/genéticaRESUMEN
Despite significant advancements in the diagnosis and treatment of osteosarcoma, the molecular mechanisms underpinning disease progression remain unclear. This work presents strong clinical and experimental evidence demonstrating that Notch signaling contributes to osteosarcoma progression. First, using a cohort of 12 patients, Notch genes were upregulated in tumors compared with adjacent normal tissue, and high tumor expression of Notch1 intercellular domain (NICD1) and the Notch target gene Hes1 correlated with poor chemotherapy response. Data mining of publicly available datasets confirmed that expression of Notch pathway genes is related to poor prognosis in osteosarcoma. On the basis of in vitro analysis, Notch signaling promoted osteosarcoma proliferation, enhanced chemoresistance, facilitated both migration and invasion, and upregulated stem cell-like characteristics. Xenograft models demonstrated that Notch signaling promotes primary tumor growth and pulmonary metastasis, and Notch inhibition is effective in reducing tumor size and preventing metastasis. Mechanistically, activated Notch signaling induces the expression of ephrinB1 and enhances the tumor-promoting ephrin reverse signaling. Overall, these findings provide functional evidence for Notch pathway genes as candidate biomarkers to predict prognosis in patients with osteosarcoma, and suggest a mechanistic rationale for the use of Notch inhibitors to treat osteosarcoma. IMPLICATIONS: The study provides preclinical evidence for Notch pathway as a molecular marker to predict osteosarcoma prognosis and as a therapeutic target against osteosarcoma. In addition, we identified a novel mechanism that ephrin reverse signaling acts as a key mediator of Notch pathway.
Asunto(s)
Efrinas/genética , Osteosarcoma/genética , Receptores Notch/genética , Factor de Transcripción HES-1/genética , Adolescente , Adulto , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Niño , Progresión de la Enfermedad , Femenino , Xenoinjertos , Humanos , Masculino , Ratones , Osteosarcoma/patología , Receptor Notch1/genética , Receptores de la Familia Eph/genética , Transducción de Señal , Adulto JovenRESUMEN
Increased cancer stem cell content during development of resistance to tamoxifen in breast cancer is driven by multiple signals, including Sox2-dependent activation of Wnt signalling. Here, we show that Sox2 increases and estrogen reduces the expression of the transcription factor Sox9. Gain and loss of function assays indicate that Sox9 is implicated in the maintenance of human breast luminal progenitor cells. CRISPR/Cas knockout of Sox9 reduces growth of tamoxifen-resistant breast tumours in vivo. Mechanistically, Sox9 acts downstream of Sox2 to control luminal progenitor cell content and is required for expression of the cancer stem cell marker ALDH1A3 and Wnt signalling activity. Sox9 is elevated in breast cancer patients after endocrine therapy failure. This new regulatory axis highlights the relevance of SOX family transcription factors as potential therapeutic targets in breast cancer.