RESUMEN
BACKGROUND: Angiogenesis is a critical step in the growth of pancreatic neuroendocrine tumors (PNETs) and may be a selective target for PNET therapy. However, PNETs are robustly resistant to current anti-angiogenic therapies that primarily target the VEGFR pathway. Thus, the mechanism of PNET angiogenesis urgently needs to be clarified. METHODS: Dataset analysis was used to identify angiogenesis-related genes in PNETs. Immunohistochemistry was performed to determine the relationship among Neuropilin 2 (NRP2), VEGFR2 and CD31. Cell proliferation, wound-healing and tube formation assays were performed to clarify the function of NRP2 in angiogenesis. The mechanism involved in NRP2-induced angiogenesis was detected by constructing plasmids with mutant variants and performing Western blot, and immunofluorescence assays. A mouse model was used to evaluate the effect of the NRP2 antibody in vivo, and clinical data were collected from patient records to verify the association between NRP2 and patient prognosis. RESULTS: NRP2, a VEGFR2 co-receptor, was positively correlated with vascularity but not with VEGFR2 in PNET tissues. NRP2 promoted the migration of human umbilical vein endothelial cells (HUVECs) cultured in the presence of conditioned medium PNET cells via a VEGF/VEGFR2-independent pathway. Moreover, NRP2 induced F-actin polymerization by activating the actin-binding protein cofilin. Cofilin phosphatase slingshot-1 (SSH1) was highly expressed in NRP2-activating cofilin, and silencing SSH1 ameliorated NRP2-activated HUVEC migration and F-actin polymerization. Furthermore, blocking NRP2 in vivo suppressed PNET angiogenesis and tumor growth. Finally, elevated NRP2 expression was associated with poor prognosis in PNET patients. CONCLUSION: Vascular NRP2 promotes PNET angiogenesis by activating the SSH1/cofilin/actin axis. Our findings demonstrate that NRP2 is an important regulator of angiogenesis and a potential therapeutic target of anti-angiogenesis therapy for PNET.
RESUMEN
BACKGROUND: Although chemotherapy represents a predominant anti-cancer therapeutic modality, drug treatment efficacy is often limited due to the development of resistant tumor cells. The pregnane X receptor (PXR) affects chemotherapeutic effects by regulating targets involved in drug metabolism and transportation, but the regulatory mechanism is poorly understood. METHODS: Oxaliplatin (L-OHP) content in tumor cells was analyzed by mass cytometry. The roles of PXR on cancer cell proliferation, apoptosis and tumor growth with L-OHP-treated were investigated by MTS, colony formation, flow cytometry and xenograft tumor assays. Luciferase reporter, Chromatin-immunoprecipitation and Site-directed mutagenesis were evaluated the mechanisms. The PXR and multidrug resistance-related protein 3 (MRP3) expressions were examined by western blot, RT-PCR or immunohistochemistry of TMA. Kaplan-Meier and Cox regression were adopted to analyze the prognostic value of PXR in colorectal cancer (CRC). RESULTS: PXR over-expression significantly increased oxaliplatin (L-OHP) transport capacity with a reduction of its content and repressed the effects of L-OHP on tumour cell proliferation and apoptosis. Conversely, PXR knockdown augments L-OHP-mediated cellular proliferation and apoptosis. Moreover, PXR significantly reduced the therapeutic effects of L-OHP on tumor growth in nude mice. Further studies indicated a positive correlation between PXR and MRP3 expression and this finding was confirmed in two independent cohorts. Significantly increased MRP3 expression was also found in PXR over-expressing cell lines. Mechanistically, PXR could directly bind to the MRP3 promoter, activating its transcription. The PXR binding sites were determined to be at -796 to -782bp (CTGAAGCAGAGGGAA) and the key binding sites were the "AGGGA" (-787 to -783bp) on the MRP3 promoter. Accordingly, blockade of MRP3 diminishes the effects on drug resistance of PXR. In addition, PXR expression is significantly associated with poor overall survival and represents an unfavorable and independent factor for male or stage I + II CRC patient prognosis. CONCLUSIONS: PXR is a potential biomarker for predicting outcome and activates MRP3 transcription by directly binding to its promoter resulting in an increased L-OHP efflux capacity, and resistance to L-OHP or platinum drugs in CRC. Our work reveals a novel and unique mechanism of drug resistance in CRC.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/mortalidad , Resistencia a Antineoplásicos/genética , Receptores de Esteroides/genética , Activación Transcripcional , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , Ratones , Estadificación de Neoplasias , Compuestos Organoplatinos/farmacología , Oxaliplatino , Receptor X de Pregnano , Pronóstico , Modelos de Riesgos Proporcionales , Receptores de Esteroides/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Because colorectal cancer (CRC) stem-like cells (CCS-like cells) contribute to poor patient prognosis, these cells are a potential target for CRC therapy. However, the mechanism underlying the maintenance of CCS-like cell properties remains unclear. Here, we found that patients with advanced stage CRC expressed high levels of polycomb group protein enhancer of zeste homologue 2 (EZH2). High expression of EZH2 in tumor tissues correlated with poor patient prognosis. Conversely, silencing EZH2 reduced CRC cell proliferation. Surprisingly, EZH2 was more highly expressed in the CCS-like cell subpopulation than in the non-CCS-like cell subpopulation. EZH2 knockdown significantly reduced the CD133+/CD44+ subpopulation, suppressed mammosphere formation, and decreased the expression of self-renewal-related genes and strongly impaired tumor-initiating capacity in a re-implantation mouse model. Gene expression data from 433 human CRC specimens from TCGA database and in vitro results revealed that EZH2 helped maintain CCS-like cell properties by activating the Wnt/ß-catenin pathway. We further revealed that p21cip1-mediated arrest of the cell cycle at G1/S phase is required for EZH2 activation of the Wnt/ß-catenin pathway. Moreover, the specific EZH2 inhibitor EPZ-6438, a clinical trial drug, prevented CRC progression. Collectively, these findings revealed EZH2 maintaining CCS-like cell characteristics by arresting the cell cycle at the G1/S phase. These results indicate a new approach to CRC therapy.
Asunto(s)
Proliferación Celular/genética , Neoplasias Colorrectales/patología , Proteína Potenciadora del Homólogo Zeste 2/fisiología , Células Madre Neoplásicas/patología , Vía de Señalización Wnt , Adulto , Anciano , Anciano de 80 o más Años , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Células Madre Neoplásicas/metabolismo , Vía de Señalización Wnt/genética , Adulto Joven , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Interleukin-12 (IL-12) is a potent immunomodulatory cytokine with unknown direct effect on the property of cancer stem cells (CSCs). In this study, we investigated the capacity of IL-12 to regulate the self-renewal and differentiation of human colon CSCs in vitro, as well as the effect of IL-12 on the growth of tumors initiated by CSCs in mice. After over-expression of IL-12 with lentiviral transfection, CSCs exhibited reduced invasiveness and tumorsphere formation in association with increased apoptosis in vitro. After injection into NOD/SCID mice, tumors initiated by CSCs transfected with IL-12 showed markedly reduced rate of growth. Mechanistic studies revealed that over-expression of IL-12 reduced the expression of IL-4 and STAT6 in CSCs. Thus, our study demonstrates a potentially beneficial role of IL-12 in directly limiting the malignant phenotype of CSCs.
Asunto(s)
Neoplasias del Colon/patología , Interleucina-12/fisiología , Células Madre Neoplásicas/fisiología , Adulto , Anciano , Animales , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Progresión de la Enfermedad , Femenino , Humanos , Interleucina-12/genética , Interleucina-4/fisiología , Masculino , Ratones , Ratones SCID , Persona de Mediana Edad , Factor de Transcripción STAT6/fisiología , Transducción de Señal , TransfecciónRESUMEN
INTRODUCTION: Planar imaging of (188)Re-labeled vascular endothelial growth factor (VEGF)(189) exon 6-encoded peptide (QKRKRKKSRYKS) with single photon emission computed tomography (SPECT) in tumor-bearing nude mice and effects of the transfecting truncated KDR gene on its imaging were investigated, so as to provide a basis for further applying the peptide to tumor-targeted radionuclide treatment. METHODS: QKRKRKKSRYKS, coupling with mercaptoacetyltriglycine (MAG(3)) chelator was labeled with (188)Re; then in vivo distribution, planar imaging with SPECT and blocking experiment in tumor-bearing nude mice were analyzed. Recombinant adenovirus vectors carrying the truncated KDR gene were constructed to transfect tumor tissues to evaluate the effects of truncated KDR on the in vivo distribution and tumor planar imaging of (188)Re-MAG(3)-QKRKRKKSRYKS in tumor-bearing nude mice. RESULTS: The labeled peptide exhibited a sound receptor binding activity. Planar imaging with SPECT demonstrated significant radioactivity accumulation in tumor 1 h after injection of the labeled peptide and disappearance of radioactivity 3 h later. Significant radioactivity accumulation was also observed in the liver, intestines and kidneys but was not obvious in other tissues. An hour after injection of the labeled peptide, the percentage of the injected radioactive dose per gram (%ID/g) of tumor and tumor/contralateral muscle tissues ratio were 1.98+/-0.38 and 2.53+/-0.33, respectively, and increased to 3.08+/-0.84 and 3.61+/-0.59 in the group transfected with the truncated KDR gene, respectively, and radioactivity accumulation in tumor with planar imaging also increased significantly in the transfection group. CONCLUSION: (188)Re-MAG(3)-QKRKRKKSRYKS can accumulate in tumor tissues, which could be increased by the transfection of truncated KDR gene. This study provides a basis for further applying the peptide to tumor targeted radionuclide imaging and treatment.