RESUMEN
Despite intense research and clinical efforts, patients affected by advanced colorectal cancer (CRC) have still a poor prognosis. The discovery of colorectal (CR) cancer stem cell (CSC) as the cell compartment responsible for tumor initiation and propagation may provide new opportunities for the development of new therapeutic strategies. Given the reduced sensitivity of CR-CSCs to chemotherapy and the ability of bone morphogenetic proteins (BMP) to promote colonic stem cell differentiation, we aimed to investigate whether an enhanced variant of BMP7 (BMP7v) could sensitize to chemotherapy-resistant CRC cells and tumors. Thirty-five primary human cultures enriched in CR-CSCs, including four from chemoresistant metastatic lesions, were used for in vitro studies and to generate CR-CSC-based mouse avatars to evaluate tumor growth and progression upon treatment with BMP7v alone or in combination with standard therapy or PI3K inhibitors. BMP7v treatment promotes CR-CSC differentiation and recapitulates the cell differentiation-related gene expression profile by suppressing Wnt pathway activity and reducing mesenchymal traits and survival of CR-CSCs. Moreover, in CR-CSC-based mouse avatars, BMP7v exerts an antiangiogenic effect and sensitizes tumor cells to standard chemotherapy regardless of the mutational, MSI, and CMS profiles. Of note, tumor harboring PIK3CA mutations were affected to a lower extent by the combination of BMP7v and chemotherapy. However, the addition of a PI3K inhibitor to the BMP7v-based combination potentiates PIK3CA-mutant tumor drug response and reduces the metastatic lesion size. These data suggest that BMP7v treatment may represent a useful antiangiogenic and prodifferentiation agent, which renders CSCs sensitive to both standard and targeted therapies.
Asunto(s)
Proteína Morfogenética Ósea 7/genética , Proteína Morfogenética Ósea 7/farmacología , Neoplasias Colorrectales/patología , Mutación , Animales , Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Humanos , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The use of stem cells, including mesenchymal stem cells (MSCs), for regenerative medicine is gaining interest for the clinical benefits so far obtained in patients. This study investigates the use of adipose autologous tissue in combination with platelet-rich plasma (PRP) to improve the clinical outcome of patients affected by systemic sclerosis (SSc). METHODS: Adipose-derived mesenchymal stem cells (AD-MSCs) and PRPs were purified from healthy donors and SSc patients. The multilineage differentiation potential of AD-MSCs and their genotypic-phenotypic features were investigated. A cytokine production profile was evaluated on AD-MSCs and PRPs from both healthy subjects and SSc patients. The adipose tissue-derived cell fraction, the so-called stromal vascular fraction (SVF), was coinjected with PRP in the perioral area of SSc patients. RESULTS: Histopathological and phenotypical analysis of adipose tissue from SSc patients revealed a disorganization of its distinct architecture coupled with an altered cell composition. Although AD-MSCs derived from SSc patients showed high multipotency, they failed to sustain a terminally differentiated progeny. Furthermore, SVFs derived from SSc patients differed from healthy donors in their MSC-like traits coupled with an aberrant cytokine production profile. Finally, the administration of PRP in combination with autologous SVF improved buccal's rhyme, skin elasticity and vascularization for all of the SSc patients enrolled in this study. CONCLUSIONS: This innovative regenerative therapy could be exploited for the treatment of chronic connective tissue diseases, including SSc.
Asunto(s)
Tejido Adiposo/citología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Plasma Rico en Plaquetas/fisiología , Medicina Regenerativa/métodos , Esclerodermia Sistémica/terapia , Tejido Adiposo/inmunología , Adulto , Anciano de 80 o más Años , Antígenos CD/genética , Antígenos CD/inmunología , Diferenciación Celular , Proliferación Celular , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Citocinas/genética , Citocinas/inmunología , Femenino , Expresión Génica , Humanos , Masculino , Células Madre Mesenquimatosas/inmunología , Persona de Mediana Edad , Neovascularización Fisiológica , Cultivo Primario de Células , Esclerodermia Sistémica/inmunología , Esclerodermia Sistémica/patología , Piel/patologíaRESUMEN
The tumor microenvironment supplies proinflammatory cytokines favoring a permissive milieu for cancer cell growth and invasive behavior. Here we show how breast cancer progression is facilitated by IL4 secreted by adipose tissue and estrogen receptor-positive and triple-negative breast cancer cell types. Blocking autocrine and paracrine IL4 signaling with the IL4Rα antagonist IL4DM compromised breast cancer cell proliferation, invasion, and tumor growth by downregulating MAPK pathway activity. IL4DM reduced numbers of CD44+/CD24- cancer stem-like cells and elevated expression of the dual specificity phosphatase DUSP4 by inhibiting NF-κB. Enforced expression of DUSP4 drove conversion of metastatic cells to nonmetastatic cells. Mechanistically, RNAi-mediated attenuation of DUSP4 activated the ERK and p38 MAPK pathways, increased stem-like properties, and spawned metastatic capacity. Targeting IL4 signaling sensitized breast cancer cells to anticancer therapy and strengthened immune responses by enhancing the number of IFNγ-positive CTLs. Our results showed the role of IL4 in promoting breast cancer aggressiveness and how its targeting may improve the efficacy of current therapies. Cancer Res; 77(12); 3268-79. ©2017 AACR.
Asunto(s)
Neoplasias de la Mama/patología , Fosfatasas de Especificidad Dual/metabolismo , Interleucina-4/metabolismo , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Microambiente Tumoral , Western Blotting , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Citometría de Flujo , Xenoinjertos , HumanosRESUMEN
Combined MAPK/PI3K pathway inhibition represents an attractive, albeit toxic, therapeutic strategy in oncology. Since PTEN lies at the intersection of these two pathways, we investigated whether PTEN status determines the functional response to combined pathway inhibition. PTEN (gene, mRNA, and protein) status was extensively characterized in a panel of cancer cell lines and combined MEK/mTOR inhibition displayed highly synergistic pharmacologic interactions almost exclusively in PTEN-loss models. Genetic manipulation of PTEN status confirmed a mechanistic role for PTEN in determining the functional outcome of combined pathway blockade. Proteomic analysis showed greater phosphoproteomic profile modification(s) in response to combined MEK/mTOR inhibition in PTEN-loss contexts and identified JAK1/STAT3 activation as a potential mediator of synergistic interactions. Overall, our results show that PTEN-loss is a crucial determinant of synergistic interactions between MAPK and PI3K pathway inhibitors, potentially exploitable for the selection of cancer patients at the highest chance of benefit from combined therapeutic strategies.
Asunto(s)
Quinasas Quinasa Quinasa PAM/metabolismo , Fosfohidrolasa PTEN/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Línea Celular Tumoral , Sinergismo Farmacológico , Everolimus/farmacología , Femenino , Humanos , Janus Quinasa 1/genética , Janus Quinasa 1/metabolismo , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Ratones , Ratones Desnudos , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Fosfohidrolasa PTEN/antagonistas & inhibidores , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piridonas/farmacología , Pirimidinonas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
P63 is a transcription factor belonging to the family of p53, essential for the development and differentiation of epithelia. In recent years, it has become clear that altered expression of the different isoforms of this gene can play an important role in carcinogenesis. The p63 gene encodes for two main isoforms known as TA and ΔN p63 with different functions. The role of these different isoforms in sustaining tumor progression and metastatic spreading however has not entirely been clarified. Here we show that breast cancer initiating cells express ΔNp63 isoform that supports a more mesenchymal phenotype associated with a higher tumorigenic and metastatic potential. On the contrary, the majority of cells within the tumor appears to express predominantly TAp63 isoform. While ΔNp63 exerts its effects by regulating a PI3K/CD44v6 pathway, TAp63 modulates this pathway in an opposite fashion. As a result, tumorigenicity and invasive capacity of breast cancer cells is a balance of the two isoforms. Finally, we found that tumor microenvironmental cytokines significantly contribute to the establishment of breast cancer cell phenotype by positively regulating ΔNp63 and CD44v6 expression.
Asunto(s)
Neoplasias de la Mama/patología , Receptores de Hialuranos/fisiología , Proteínas de la Membrana/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Anciano , Anciano de 80 o más Años , Animales , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos , Transición Epitelial-Mesenquimal , Femenino , Humanos , Ratones , Persona de Mediana Edad , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal/fisiología , Microambiente TumoralRESUMEN
Thyroid hormone is a pleiotropic factor that controls many cellular processes in multiple cell types such as cancer stem cells (CSC). Thyroid hormone concentrations in the blood are stable, but the action of the deiodinases (D2-D3) provides cell-specific regulation of thyroid hormone activity. Deregulation of deiodinase function and thyroid hormone status has been implicated in tumorigenesis. Therefore, we investigated the role of thyroid hormone metabolism and signaling in colorectal CSCs (CR-CSC), where deiodinases control cell division and chemosensitivity. We found that increased intracellular thyroid hormone concentration through D3 depletion induced cell differentiation and sharply mitigated tumor formation. Upregulated BMP4 expression and concomitantly attenuated Wnt signaling accompanied these effects. Furthermore, we demonstrate that BMP4 is a direct thyroid hormone target and is involved in a positive autoregulatory feedback loop that modulates thyroid hormone signaling. Collectively, our findings highlight a cell-autonomous metabolic mechanism by which CR-CSCs exploit thyroid hormone signaling to facilitate their self-renewal potential and suggest that drug-induced cell differentiation may represent a promising therapy for preventing CSC expansion and tumor progression.
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Proteína Morfogenética Ósea 4/fisiología , Neoplasias Colorrectales/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Transducción de Señal/fisiología , Hormonas Tiroideas/fisiología , Vía de Señalización Wnt/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Humanos , Masculino , Ratones , Persona de Mediana Edad , Células Madre Neoplásicas/citologíaRESUMEN
Approximately 5-10% of metastatic colorectal cancers harbor a BRAF-V600E mutation, which is correlated with resistance to EGFR-targeted therapies and worse clinical outcome. Vice versa, targeted inhibition of BRAF-V600E with the selective inhibitor PLX 4032 (Vemurafenib) is severely limited due to feedback re-activation of EGFR in these tumors. Mounting evidence indicates that upregulation of the ErbB-3 signaling axis may occur in response to several targeted therapeutics, including Vemurafenib, and NRG-1ß-dependent re-activation of the PI3K/AKT survival pathway has been associated with therapy resistance.Here we show that colon CSCs express, next to EGFR and ErbB-2, also significant amounts of ErbB-3 on their membrane. This expression is functional as NRG-1ß strongly induces AKT/PKB and ERK phosphorylation, cell proliferation, clonogenic growth and promotes resistance to Vemurafenib in BRAF-V600E mutant colon CSCs. This resistance was completely dependent on ErbB-3 expression, as evidenced by knockdown of ErbB-3. More importantly, resistance could be alleviated with therapeutic antibody blocking ErbB-3 activation, which impaired NRG-1ß-driven AKT/PKB and ERK activation, clonogenic growth in vitro and tumor growth in xenograft models. In conclusion, our findings suggest that targeting ErbB-3 receptors could represent an effective therapeutic approach in BRAF-V600E mutant colon cancer.
Asunto(s)
Neoplasias del Colon/patología , Resistencia a Antineoplásicos/fisiología , Células Madre Neoplásicas/metabolismo , Neurregulina-1/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Receptor ErbB-3/metabolismo , Animales , Antineoplásicos/farmacología , Proliferación Celular , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Citometría de Flujo , Humanos , Inmunohistoquímica , Indoles/farmacología , Ratones , Ratones Desnudos , Células Madre Neoplásicas/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfonamidas/farmacología , Vemurafenib , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Cancer stem cells represent a population of immature tumor cells found in most solid tumors. Their peculiar features make them ideal models for studying drug resistance and sensitivity. In this study, we investigated whether cancer stem cells isolation and in vitro sensitivity assay are feasible in a clinical setting. METHODS: Cancer stem cells were isolated from effusions or fresh cancer tissue of 23 patients who progressed after standard therapy failure. Specific culture conditions selected for immature tumor cells that express markers of stemness. These cells were exposed in vitro to chemotherapeutic and targeted agents. RESULTS: Cancer stem cells were extracted from liver metastases in 6 cases (25%), lung nodules in 2 (8%), lymph node metastases in 3 (12.5%) and pleural/peritoneal/pericardial effusion in 13 (54%). Cancer stem cells were successfully isolated in 15 patients (63%), including 14 with lung cancer (93.3%). A sensitivity assay was successfully performed in 7 patients (30.4%), with a median of 15 drugs/combinations tested (range 5-28) and a median time required for results of 51 days (range 37-95). CONCLUSION: The approach used for the STELLA trial allowed isolation of cancer stem cells in a consistent proportion of patients. The low percentage of cases completing the full procedure and the long median time for obtaining results highlights the need for a more efficient procedure. TRIAL REGISTRATION: ClinalTrials.gov NCT01483001.
Asunto(s)
Antineoplásicos/farmacología , Separación Celular/métodos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Células Madre Neoplásicas/efectos de los fármacos , Anciano , Anciano de 80 o más Años , Neoplasias Colorrectales/patología , Combinación de Medicamentos , Estudios de Factibilidad , Femenino , Humanos , Neoplasias Hepáticas/patología , Neoplasias Pulmonares/patología , Metástasis Linfática/patología , Masculino , Persona de Mediana Edad , Células Madre Neoplásicas/patología , Derrame Pleural/patología , Factores de TiempoRESUMEN
Bone morphogenetic proteins (BMPs), members of the TGF-ß superfamily, have numerous biological activities including control of growth, differentiation, and vascular development. Using an in vitro co-culture endothelial cord formation assay, we investigated the role of a BMP7 variant (BMP7v) in VEGF, bFGF, and tumor-driven angiogenesis. BMP7v treatment led to disruption of neo-endothelial cord formation and regression of existing VEGF and bFGF cords in vitro. Using a series of tumor cell models capable of driving angiogenesis in vitro, BMP7v treatment completely blocked cord formation. Pre-treatment of endothelial cells with BMP7v significantly reduced their cord forming ability, indicating a direct effect on endothelial cell function. BMP7v activated the canonical SMAD signaling pathway in endothelial cells but targeted gene knockdown using shRNA directed against SMAD4 suggests this pathway is not required to mediate the anti-angiogenic effect. In contrast to SMAD activation, BMP7v selectively decreased ERK and AKT activation, significantly decreased endothelial cell migration and down-regulated expression of critical RTKs involved in VEGF and FGF angiogenic signaling, VEGFR2 and FGFR1 respectively. Importantly, in an in vivo angiogenic plug assay that serves as a measurement of angiogenesis, BMP7v significantly decreased hemoglobin content indicating inhibition of neoangiogenesis. In addition, BMP7v significantly decreased angiogenesis in glioblastoma stem-like cell (GSLC) Matrigel plugs and significantly impaired in vivo growth of a GSLC xenograft with a concomitant reduction in microvessel density. These data support BMP7v as a potent anti-angiogenic molecule that is effective in the context of tumor angiogenesis.
Asunto(s)
Proteína Morfogenética Ósea 7/uso terapéutico , Células Endoteliales/metabolismo , Glioblastoma/irrigación sanguínea , Neovascularización Patológica/tratamiento farmacológico , Tejido Adiposo/citología , Animales , Proteína Morfogenética Ósea 7/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Colágeno/farmacología , Combinación de Medicamentos , Células Endoteliales/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Laminina/farmacología , Masculino , Ratones Desnudos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Neovascularización Patológica/patología , Neovascularización Fisiológica/efectos de los fármacos , Proteoglicanos/farmacología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Smad/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The cancer stem cell (CSC) model is describing tumors as a hierarchical organized system and CSCs are suggested to be responsible for cancer recurrence after therapy. The identification of specific markers of CSCs is therefore of paramount importance. Here, we show that high levels of lipid droplets (LDs) are a distinctive mark of CSCs in colorectal (CR) cancer. This increased lipid content was clearly revealed by label-free Raman spectroscopy and it directly correlates with well-accepted CR-CSC markers as CD133 and Wnt pathway activity. By xenotransplantation experiments, we have finally demonstrated that CR-CSCs overexpressing LDs retain most tumorigenic potential. A relevant conceptual advance in this work is the demonstration that a cellular organelle, the LD, is a signature of CSCs, in addition to molecular markers. A further functional characterization of LDs could lead soon to design new target therapies against CR-CSCs.
Asunto(s)
Neoplasias Colorrectales/patología , Células Madre Neoplásicas/patología , Espectrometría Raman/métodos , Animales , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/metabolismo , Humanos , Gotas Lipídicas , Ratones , Células Madre Neoplásicas/metabolismo , Vía de Señalización WntRESUMEN
Cancer stem cells drive tumor formation and metastasis, but how they acquire metastatic traits is not well understood. Here, we show that all colorectal cancer stem cells (CR-CSCs) express CD44v6, which is required for their migration and generation of metastatic tumors. CD44v6 expression is low in primary tumors but demarcated clonogenic CR-CSC populations. Cytokines hepatocyte growth factor (HGF), osteopontin (OPN), and stromal-derived factor 1α (SDF-1), secreted from tumor associated cells, increase CD44v6 expression in CR-CSCs by activating the Wnt/ß-catenin pathway, which promotes migration and metastasis. CD44v6(-) progenitor cells do not give rise to metastatic lesions but, when treated with cytokines, acquire CD44v6 expression and metastatic capacity. Importantly, phosphatidylinositol 3-kinase (PI3K) inhibition selectively killed CD44v6 CR-CSCs and reduced metastatic growth. In patient cohorts, low levels of CD44v6 predict increased probability of survival. Thus, the metastatic process in colorectal cancer is initiated by CSCs through the expression of CD44v6, which is both a functional biomarker and therapeutic target.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Reprogramación Celular , Neoplasias del Colon/patología , Receptores de Hialuranos/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Carcinogénesis/patología , Neoplasias del Colon/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Ratones SCID , Metástasis de la Neoplasia , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Pronóstico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal , Resultado del Tratamiento , Proteínas Wnt/metabolismoRESUMEN
It is widely accepted by the scientific community that cancer, including colon cancer, is a "stem cell disease". Until a few years ago, common opinion was that all neoplastic cells within a tumor contained tumorigenic growth capacity, but recent evidences hint to the possibility that such a feature is confined to a small subset of cancer-initiating cells, also called cancer stem cells (CSCs). Thus, malignant tumors are organized in a hierarchical fashion in which CSCs give rise to more differentiated tumor cells. CSCs possess high levels of ATP-binding cassette (ABC) transporters and anti-apoptotic molecules, active DNA-repair, slow replication capacities and they produce growth factors that confer refractoriness to antineoplastic treatments. The inefficacy of conventional therapies towards the stem cell population might explain cancer chemoresistance and the high frequency of relapse shown by the majority of tumors. Nowadays, in fact all the therapies available are not sufficient to cure patients with advanced forms of colon cancer since they target differentiated cancer cells which constitute most of the tumor mass and spare CSCs. Since CSCs are the entities responsible for the development of the tumor and represent the only cell population able to sustain tumor growth and progression, these cells represent the elective target for innovative therapies.