RESUMEN
Therapy-related leukemia carries a poor prognosis, and leukemia after chemotherapy is a growing risk in clinic, whose mechanism is still not well understood. Ikaros transcription factor is an important regulator in hematopoietic cells development and differentiation. In the absence of Ikaros, lymphoid cell differentiation is blocked at an extremely early stage, and myeloid cell differentiation is also significantly affected. In this work, we showed that chemotherapeutic drug etoposide reduced the protein levels of several isoforms of Ikaros including IK1, IK2 and IK4, but not IK6 or IK7, by accelerating protein degradation, in leukemic cells. To investigate the molecular mechanism of Ikaros degradation induced by etoposide, immunoprecipitation coupled with LC-MS/MS analysis was conducted to identify changes in protein interaction with Ikaros before and after etoposide treatment, which uncovered KCTD5 protein. Our further study demonstrates that KCTD5 is the key stabilizing factor of Ikaros and chemotherapeutic drug etoposide induces Ikaros protein degradation through decreasing the interaction of Ikaros with KCTD5. These results suggest that etoposide may induce leukemic transformation by downregulating Ikaros via KCTD5, and our work may provide insights to attenuate the negative impact of chemotherapy on hematopoiesis.
Asunto(s)
Etopósido , Factor de Transcripción Ikaros , Factor de Transcripción Ikaros/metabolismo , Etopósido/farmacología , Humanos , Proteolisis/efectos de los fármacos , Antineoplásicos Fitogénicos/farmacologíaRESUMEN
BACKGROUND: Breast cancer is the leading cause of cancer death for women worldwide. Most of the breast cancer death are due to disease recurrence and metastasis. Increasingly accumulating evidence indicates that mitochondria play key roles in cancer progression and metastasis. Our recent study revealed that transmembrane protein PRRG4 promotes the metastasis of breast cancer. However, it is not clear whether PRRG4 can affect the migration and invasion of breast cancer cells through regulating mitochondria function. METHODS: RNA-seq analyses were performed on breast cancer cells expressing control and PRRG4 shRNAs. Quantitative PCR analysis and measurements of mitochondrial ATP content and oxygen consumption were carried out to explore the roles of PRRG4 in regulating mitochondrial function. Luciferase reporter plasmids containing different lengths of promoter fragments were constructed. Luciferase activities in breast cancer cells transiently transfected with these reporter plasmids were analyzed to examine the effects of PRRG4 overexpression on promoter activity. Transwell assays were performed to determine the effects of PRRG4-regulated pathway on migratory behaviors of breast cancer cells. RESULTS: Analysis of the RNA-seq data revealed that PRRG4 knockdown decreased the transcript levels of all the mitochondrial protein-encoding genes. Subsequently, studies with PRRG4 knockdown and overexpression showed that PRRG4 expression increased mitochondrial DNA (mtDNA) content. Mechanistically, PRRG4 via Src activated STAT3 in breast cancer cells. Activated STAT3 in turn promoted the transcription of mtDNA polymerase POLG through a STAT3 DNA binding site present in the POLG promoter region, and increased mtDNA content as well as mitochondrial ATP production and oxygen consumption. In addition, PRRG4-mediated activation of STAT3 also enhanced filopodia formation, migration, and invasion of breast cancer cells. Moreover, PRRG4 elevated migratory behaviors and mitochondrial function of breast cancer cells through POLG. CONCLUSION: Our results indicate that PRRG4 via the Src-STAT3-POLG axis enhances mitochondrial function and promotes migratory behaviors of breast cancer cells.
RESUMEN
Breast cancer is the most common malignant cancer in women. Triple-negative breast cancer (TNBC) has a poorer prognosis than other subtypes and is challenging to treat. MUC1 is a therapeutic target in breast and pancreatic cancer. We developed a novel humanized antibody that specifically binds MUC1 expressed in breast cancer cells and conjugated a humanized MUC1 (HzMUC1) antibody to monomethyl auristatin (MMAE). HzMUC1-MMAE showed an anti-proliferative effect on HER2 positive trastuzumab-resistant breast cancer. Immunoprecipitation indicated that HzMUC1 recognized native MUC1 in TNBC cells. Confocal microscopy showed that HzMUC1 bound MUC1 on the surface of TNBC cells, and the conjugates exhibited the same binding ability to HCC70 as unconjugated HzMUC1 by cell-based ELISA. Treatment of TNBC cells with HzMUC1-MMAE reduced growth of MUC1-positive cells and induced G2/M cell cycle arrest and apoptosis. In a mouse model of breast cancer, HzMUC1-MMAE significantly reduced the growth of tumors established by subcutaneous injection of HCC70 TNBC cells. Therefore, HzMUC1-ADC has therapeutic potential for TNBC.
RESUMEN
BACKGROUND: Pancreatic cancer is one of the most aggressive malignancies without effective targeted therapies. MUC1 has emerged as a potential common target for cancer therapy because it is overexpressed in a variety of different cancers including the majority of pancreatic cancer. However, there are still no approved monoclonal antibody drugs targeting MUC1 have been reported. Recently, we generated a humanized MUC1 antibody (HzMUC1) specific to the interaction region between MUC1-N and MUC1-C. In this study, we generated the antibody drug conjugate (ADC) by conjugating HzMUC1 with monomethyl auristatin (MMAE), and examined the efficacy of HzMUC1-MMAE against the MUC1-positive pancreatic cancer in vitro and in vivo. METHODS: Western blot and immunoprecipitation were used to detect MUC1 in pancreatic cancer cells. MUC1 localization in pancreatic cancer cells was determined by confocal microscopy. HzMUC1 was conjugated with the monomethyl auristatin (MMAE), generating the HzMUC1-MMAE ADC. Colony formation assay and flow cytometry were used to assess the effects of the HzMUC1-MMAE cell viability, cell cycle progression and apoptosis. Capan-2 and CFPAC-1 xenograft model were used to test the efficacy of HzMUC1-MMAE against pancreatic cancer. RESULTS: HzMUC1 antibody binds to MUC1 on the cell surface of pancreatic cancer cells. HzMUC1-MMAE significantly inhibited cell growth by inducing G2/M cell cycle arrest and apoptosis in pancreatic cancer cells. Importantly, HzMUC1-MMAE significantly reduced the growth of pancreatic xenograft tumors by inhibiting cell proliferation and enhancing cell death. CONCLUSION: Our results indicate that HzMUC1-ADC is a promising novel targeted therapy for pancreatic cancer. HzMUC1-ADC should also be an effective drug for the treatment of different MUC1-positive cancers.
RESUMEN
Background: Cytogenetic abnormalities are considered initiating events in the pathogenesis of multiple myeloma (MM) and are assumed to be of clinical significance. Methods: Fluorescence in situ hybridization (FISH) was used to analyze chromosomal architecture in 101 patients with MM. We evaluated overall patient survival and assessed the cytotoxicity of imatinib against MM cells using a CCK8 assay. Results: ABL gene amplification was detected in 67 patients (66.3%). However, ABL gene amplification was not associated with clinical features, cytogenetic abnormalities (c-Myc amplification, IGH rearrangement, RB1 deletion, p53 deletion, or 1q21 amplification), or overall survival. ABL amplification in MM cell lines (LP-1 and U266) was revealed by FISH. Furthermore, the ABL protein was easily detectable in MM cell lines and some tumor cells by western blotting. A CCK8 assay indicated limited cytotoxicity of imatinib against MM cells. Conclusions: Our study firstly discussed ABL gene amplification was prevalent in MM cells, and we believe that the ABL gene would potentially be a useful target in the treatment of combination strategy for MM with ABL amplification in the future.
RESUMEN
Acute myeloid leukemia (AML) is a hematologic malignant disease largely affecting older adults with poor outcomes. Lack of effective targeted treatment is a major challenge in managing the disease in the clinic. Scaffolding adaptor Gab2 is amplified in a subset of AML. However, the causative role of Gab2 in AML remains to be explored. In this study, it was found that Gab2 was expressed at high levels in AML patient samples and AML cell lines. Experiments by knocking down Gab2 expression using shRNA showed that Gab2 promoted AML cell growth and migration in vitro and in vivo. Further studies using Gab2 mutants and pharmacological inhibitors revealed that Gab2 increased CREB phosphorylation via the SHP-2/Erk signaling pathway. CREB phosphorylation contributed to Gab2-induced cell migration by increasing MMP2 and MMP9 expression. This research indicates that high Gab2 expression promotes AML progression through the SHP2-Erk-CREB signaling pathway. CREB suppression may help treat AML with high Gab2 expression.
Asunto(s)
Leucemia Mieloide Aguda , Metaloproteinasa 2 de la Matriz , Proteínas Adaptadoras Transductoras de Señales , Anciano , Humanos , Leucemia Mieloide Aguda/genética , Metaloproteinasa 9 de la Matriz , ARN Interferente Pequeño/genética , Transducción de SeñalRESUMEN
Mucin 1 (MUC1) has been regarded as an ideal target for cancer treatment, since it is overexpressed in a variety of different cancers including the majority of breast cancer. However, there are still no approved monoclonal antibody drugs targeting MUC1. In this study, we generated a humanized MUC1 (HzMUC1) antibody from our previously developed MUC1 mouse monoclonal antibody that only recognizes MUC1 on the surface of tumor cells. Furthermore, an antibody-drug conjugate (ADC) was generated by conjugating HzMUC1 with monomethyl auristatin (MMAE), and the efficacy of HzMUC1-MMAE on the MUC1-positive HER2+ breast cancer in vitro and in 'Xenograft' model was tested. Results from western blot analysis and immunoprecipitation revealed that the HzMUC1 antibody did not recognize cell-free MUC1-N in sera from breast cancer patients. Confocal microscopy analysis showed that HzMUC1 antibody bound to MUC1 on the surface of breast cancer cells. Results from mapping experiments suggested that HzMUC1 may recognize an epitope present in the interaction region between MUC1-N and MUC1-C. Results from colony formation assay and flow cytometry demonstrated that HzMUC1-MMAE significantly inhibited cell growth by inducing G2/M cell cycle arrest and apoptosis in trastuzumab-resistant HER2-positive breast cancer cells. Meanwhile, HzMUC1-MMAE significantly reduced the growth of HCC1954 xenograft tumors by inhibiting cell proliferation and enhancing cell death. In conclusion, our results indicate that HzMUC1-ADC is a novel therapeutic drug that can overcome trastuzumab resistance of breast cancer. HzMUC1-ADC should also be an effective therapeutic drug for the treatment of different MUC1-positive cancers in clinic.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos Inmunológicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Inmunoconjugados/farmacología , Mucina-1/metabolismo , Trastuzumab/farmacología , Animales , Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos Inmunológicos/metabolismo , Antineoplásicos Inmunológicos/uso terapéutico , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/sangre , Neoplasias de la Mama/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/inmunología , Epítopos , Humanos , Inmunoconjugados/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mucina-1/sangre , Mucina-1/química , Mucina-1/inmunología , Oligopéptidos/química , Oligopéptidos/farmacología , Oligopéptidos/uso terapéutico , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Although endocrine therapies targeting estrogen receptor α (ERα) are effective in managing ER positive (+) breast cancer, many patients have primary resistance or develop resistance to endocrine therapies. In addition, ER+ breast cancer with PIK3CA activating mutations and 11q13-14 amplification have poor survival with unclear mechanism. We uncovered that higher expression of deubiquitinase USP35, located in 11q14.1, was associated with ER+ breast cancer and poor survival. Estrogen enhanced USP35 protein levels by downregulating USP35-targeting miRNA-140-3p and miRNA-26a-5p. USP35 promoted the growth of ER+ breast cancer in vitro and in vivo, and reduced the sensitivity of ER+ breast cancer cells to endocrine therapies such as tamoxifen and fulvestrant. Mechanistically, USP35 enhanced ERα stability by interacting and deubiquitinating ERα, and transcriptional activity of ERα by interacting with ERα in DNA regions containing estrogen response element. In addition, AKT, a key effector of PI3K, phosphorylated USP35 at Serine613, which promoted USP35 nuclear translocation, ERα transcriptional activity, and the growth of ER+ breast cancer cells. Our data indicate that USP35 and ERα form a positive feedback loop in promoting the growth of ER+ breast cancer. USP35 may be a treatment target for ER+ breast cancer with endocrine resistance or with PIK3CA mutations or hyperactivation of the PI3K pathway.
Asunto(s)
Neoplasias de la Mama/genética , Endopeptidasas/fisiología , Estradiol/farmacología , Receptor alfa de Estrógeno/genética , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Carcinogénesis/metabolismo , Endopeptidasas/genética , Receptor alfa de Estrógeno/efectos de los fármacos , Receptor alfa de Estrógeno/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Células MCF-7 , Ratones , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Metastasis is responsible for the death of most breast cancer patients. Robo1 has been implicated as a tumor suppressor for various cancers including breast cancer. However, it is not well understood how Robo1 expression is regulated during tumorigenesis. In this study, we uncovered that the transmembrane proline rich γ-carboxyglutamic acid protein 4 (PRRG4) promotes breast cancer metastasis by downregulating Robo1. Analysis of mRNA expression data in The Cancer Genome Atlas and immunohistochemistry assay on breast tumor samples showed that PRRG4 expression was higher in breast tumors than in normal breast tissues. Experiments with PRRG4 knockdown and overexpression revealed that PRRG4 promoted migration and invasion of breast cancer cells, and enhanced metastasis in an experimental metastasis model. Mechanistically, we found that PRRG4 via its LPSY and PPPY motifs recruited the E3 ubiquitin ligase NEDD4, which induced ubiquitination and degradation of Robo1, thus contributing to migration and invasion of breast cancer cells. In addition, PRRG4 interacted with and enhanced protein tyrosine kinase Src and FAK activation. Overall, our data support a model that PRRG4 via NEDD4 downregulates the Robo1, resulting in the activation of Src and FAK and promoting breast cancer metastasis. PRRG4 may be a novel target for treating metastatic breast cancer.
Asunto(s)
Neoplasias de la Mama/patología , Regulación hacia Abajo , Proteínas de la Membrana/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Inmunológicos/metabolismo , Secuencias de Aminoácidos , Animales , Línea Celular Tumoral , Movimiento Celular , Transformación Celular Neoplásica , Activación Enzimática , Quinasa 1 de Adhesión Focal/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia , ARN Mensajero/genética , Proteínas RoundaboutRESUMEN
Cholesterol is a risk factor for breast cancer. However, it is still unclear whether the cholesterol biosynthesis pathway plays any significant role in breast carcinogenesis. 24-Dehydrocholesterol reductase (DHCR24) is a key enzyme in the cholesterol synthesis pathway. Although DHCR24 is reported to have different functions in different cancers, it is not clear whether DHCR24 is involved in breast cancer. In this study, we found that DHCR24 expression was higher in breast cancer especially in luminal and HER2 positive breast cancer tissues compared with normal breast. Changes in DHCR24 expression altered cellular cholesterol content without affecting the adherent growth of breast cancer cells. However, DHCR24 knockdown reduced whereas DHCR24 overexpression enhanced breast cancer stem-like cell populations such as mammosphere and aldehyde dehydrogenase positive cell numbers. In addition, DHCR24 overexpression increased the expression of the Hedgehog pathway-regulated genes. Treating DHCR24 overexpressing breast cancer cell lines with the Hedgehog pathway inhibitor GANT61 blocked DHCR24-induced mammosphere growth and increased mRNA levels of the Hedgehog regulated genes. Furthermore, expression of a constitutively activated mutant of Smoothened, a key hedgehog signal transducer, rescued the decreases in mammosphere growth and Hedgehog regulated gene expression induced by knockdown of DHCR24. These results indicate that DHCR24 promotes the growth of breast cancer stem-like cells in part through enhancing the Hedgehog signaling pathway. Our data suggest that cholesterol contribute to breast carcinogenesis by enhancing Hedgehog signaling and cancer stem-like cell populations. Enzymes including DHCR24 involved in cholesterol biosynthesis should be considered as potential treatment targets for breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas Hedgehog/genética , Células Madre Neoplásicas/patología , Proteínas del Tejido Nervioso/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Transducción de Señal/genética , Antineoplásicos/farmacología , Mama/efectos de los fármacos , Mama/patología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Colesterol/genética , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Células HEK293 , Humanos , Células MCF-7 , Células Madre Neoplásicas/efectos de los fármacos , Piridinas/farmacología , Pirimidinas/farmacología , ARN Mensajero/genética , Transducción de Señal/efectos de los fármacosRESUMEN
As a subtype of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) is characterized by a chromosomal translocation, most of which result in the production of a PML-RAR alpha fusion protein. Although the overall survival rate of APL patients has improved dramatically due to all-trans retinoic acid (ATRA) treatment, ATRA-resistance remains a clinical challenge in the management of APL. Therefore, alternative agents should be considered for ATRA-resistant APL patients. Here, we report that antimalaria drug primaquine phosphate (PRQ) exhibits an anti-leukemia effect on both ATRA-sensitive cell line NB4 and ATRA-resistant APL cell lines, NB4-LR2, NB4-LR1, and NB4-MR2. Moreover, PRQ significantly inhibited primary colony formation of untreated or relapsed APL patients. Further study showed that PRQ could induce the apoptosis of APL cells by inhibiting NF-κB signaling pathway. The in vivo study showed that PRQ significantly inhibited NB4-LR2 xenograft tumors growth. These results suggest that PRQ is a potential therapeutic agent for ATRA-resistant APL patients.
Asunto(s)
Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Leucemia Promielocítica Aguda/patología , FN-kappa B/metabolismo , Primaquina/farmacología , Transducción de Señal , Tretinoina/farmacología , Adulto , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Primaquina/química , Transducción de Señal/efectos de los fármacos , Ensayo de Tumor de Célula Madre , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
We recently demonstrated that leukocyte Ig-like receptor 4 (LILRB4) expressed by monocytic acute myeloid leukemia (AML) cells mediates T-cell inhibition and leukemia cell infiltration via its intracellular domain. The cytoplasmic domain of LILRB4 contains three immunoreceptor tyrosine-based inhibitory motifs (ITIMs); the tyrosines at positions 360, 412, and 442 are phosphorylation sites. Here, we analyzed how the ITIMs of LILRB4 in AML cells mediate its function. Our in vitro and in vivo data show that Y412 and Y442, but not Y360, of LILRB4 are required for T-cell inhibition, and all three ITIMs are needed for leukemia cell infiltration. We constructed chimeric proteins containing the extracellular domain of LILRB4 and the intracellular domain of LILRB1 and vice versa. The intracellular domain of LILRB4, but not that of LILRB1, mediates T-cell suppression and AML cell migration. Our studies thus defined the unique signaling roles of LILRB4 ITIMs in AML cells.
Asunto(s)
Movimiento Celular/inmunología , Tolerancia Inmunológica , Glicoproteínas de Membrana/inmunología , Proteínas de Neoplasias/inmunología , Receptores Inmunológicos/inmunología , Linfocitos T/inmunología , Secuencias de Aminoácidos , Animales , Movimiento Celular/genética , Humanos , Leucemia Mieloide Aguda , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Proteínas de Neoplasias/genética , Receptores Inmunológicos/genética , Linfocitos T/patología , Células THP-1RESUMEN
Therapeutic strategies are urgently needed for patients with acute myeloid leukemia (AML). Leukocyte immunoglobulin-like receptor B4 (LILRB4), which suppresses T-cell activation and supports tissue infiltration of AML cells, represents an attractive drug target for anti-AML therapeutics. Here, we report the identification and development of an LILRB4-specific humanized mAb that blocks LILRB4 activation. This mAb, h128-3, showed potent activity in blocking the development of monocytic AML in various models including patient-derived xenograft mice and syngeneic immunocompetent AML mice. MAb h128-3 enhanced the anti-AML efficacy of chemotherapy treatment by stimulating mobilization of leukemia cells. Mechanistic studies revealed four concordant modes of action for the anti-AML activity of h128-3: (i) reversal of T-cell suppression, (ii) inhibition of monocytic AML cell tissue infiltration, (iii) antibody-dependent cellular cytotoxicity, and (iv) antibody-dependent cellular phagocytosis. Therefore, targeting LILRB4 with antibody represents an effective therapeutic strategy for treating monocytic AML.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Apolipoproteínas E/metabolismo , Transformación Celular Neoplásica/inmunología , Transformación Celular Neoplásica/metabolismo , Leucemia Mieloide Aguda/etiología , Leucemia Mieloide Aguda/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Anticuerpos Bloqueadores , Anticuerpos Monoclonales , Citotoxicidad Celular Dependiente de Anticuerpos , Antineoplásicos Inmunológicos/uso terapéutico , Apolipoproteínas E/química , Apoptosis , Línea Celular , Transformación Celular Neoplásica/efectos de los fármacos , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Humanos , Leucemia Mieloide Aguda/patología , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/química , Ratones , Ratones Noqueados , Modelos Biológicos , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Conejos , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/química , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Scaffolding adaptor Gab2 is overexpressed in a subset of high-grade ovarian cancer. Our published work shows that Gab2 via PI3K enhances migratory behaviors and epithelial to mesenchymal transition (EMT) features of ovarian cancer cells in vitro. However, it is still unclear how Gab2/PI3K pathway reuglates EMT characteristics and whether Gab2 promotes the growth of ovarian cancer stem cell (CSC)-like population and metastatic growth. In this study, we examined the effects of Gab2 expression on CSC-like cell growth using Aldefluor and tumorshpere assays commonly used for assessing ovarian cancer cells with CSC properties. Gab2 overexpression increased the number of ALDH+ cells and tumorsphere formation in two different ovarian cancer cell lines OVCAR5 and OVCAR8, whereas knockdown of Gab2 decreased the number of ALDH+ cells and tumorsphere formation in Caov-3â¯cells. Furthermore, Gab2 promoted metastatic tumor growth of OVCAR5 in nude mice. Mechanistically, we uncovered that Gab2 via PI3K specifically inhibited miR-200c expression. miR-200c downregulation contributed to the Gab2-enhanced cell migratory behaviors, EMT properties, and the expansion of ALDH+ cells and tumorspheres. Furthermore, Gab2 promoted CD44 expression and cell migration/invasion through miR-200c downregulation. Our findings support a model that Gab2-PI3K pathway via miR-200c downregulation promotes CD44 expression, EMT characteristics, and CSC-like cell growth. Therapies involving miR-200c or targeting CD44 should help treat ovarian cancer with high Gab2 expression.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Regulación Neoplásica de la Expresión Génica , MicroARNs/fisiología , Proteínas de Neoplasias/fisiología , Neoplasias Ováricas/genética , ARN Neoplásico/fisiología , Animales , Movimiento Celular , Regulación hacia Abajo , Femenino , Xenoinjertos , Humanos , Receptores de Hialuranos/antagonistas & inhibidores , Receptores de Hialuranos/biosíntesis , Receptores de Hialuranos/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/genética , Invasividad Neoplásica , Metástasis de la Neoplasia , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Fosfatidilinositol 3-Quinasas/fisiología , Interferencia de ARN , ARN Neoplásico/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacologíaRESUMEN
Immune checkpoint blockade therapy has been successful in treating some types of cancer but has not shown clinical benefits for treating leukaemia1. This result suggests that leukaemia uses unique mechanisms to evade this therapy. Certain immune inhibitory receptors that are expressed by normal immune cells are also present on leukaemia cells. Whether these receptors can initiate immune-related primary signalling in tumour cells remains unknown. Here we use mouse models and human cells to show that LILRB4, an immunoreceptor tyrosine-based inhibition motif-containing receptor and a marker of monocytic leukaemia, supports tumour cell infiltration into tissues and suppresses T cell activity via a signalling pathway that involves APOE, LILRB4, SHP-2, uPAR and ARG1 in acute myeloid leukaemia (AML) cells. Deletion of LILRB4 or the use of antibodies to block LILRB4 signalling impeded AML development. Thus, LILRB4 orchestrates tumour invasion pathways in monocytic leukaemia cells by creating an immunosuppressive microenvironment. LILRB4 represents a compelling target for the treatment of monocytic AML.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/patología , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Escape del Tumor/inmunología , Animales , Apolipoproteínas E/metabolismo , Arginasa/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Tolerancia Inmunológica/inmunología , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Masculino , Glicoproteínas de Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Receptores Inmunológicos , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Escape del Tumor/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Acute myeloid leukemia (AML) is one of the most common types of acute leukemia in adults with the lowest survival rate of all leukemia. Resistance to cytarabine and anthracycline-based chemotherapy is a major cause of treatment failure. Thus, finding new drugs with anti-leukemia activities and minimal side effect is urgently needed. Here through screening more than 1000 drugs approved by the Food and Drug Administration (FDA) of United States, the anthelmintic drug mebendazole (MBZ) was found to inhibit the growth of AML cell lines (THP-1, U937, NB4 and K562) and bone marrow mononuclear cells (BM-MNCs) from AML patients at pharmacologically achievable concentrations. In contrast, similar concentration of MBZ had little inhibitory effect on the growth of normal peripheral blood mononuclear cells (PBMC) or human umbilical vein endothelial cells (HUVEC). In addition, MBZ induced mitotic arrest and mitotic catastrophe in AML cells based on nuclear morphology, cell cycle distribution, mitotic marker analyses and the number of multinucleated cells and apoptotic cells. Furthermore, MBZ treatment inhibited activation of Akt and Erk in AML leukemic cells. Finally, MBZ repressed the progression of leukemic cells in vivo and prolonged survival in AML xenograft mouse model. Taken together, our results suggest that MBZ could be a potential new therapeutic agent for the treatment of AML patients.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Leucemia Mieloide Aguda/patología , Mebendazol/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Células K562 , Leucocitos Mononucleares/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células U937 , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous group of hematopoietic malignancies due to sophisticated genetic mutations and epigenetic dysregulation. MicroRNAs (miRNAs), a class of small non-coding RNAs, are important regulators of gene expression in all biological processes, including leukemogenesis. Recently, miR-375 has been reported to be a suppressive miRNA in multiple types of cancers, but its underlying anti-leukemia activity in AML is largely unknown. METHODS: Quantitative reverse transcriptase PCR (qRT-PCR) was used to measure the expression of miR-375 and HOXB3 in leukemic cells and normal controls. Targets of miR-375 were confirmed by western blot and luciferase assay. Phenotypic effects of miR-375 overexpression and HOXB3 knockdown were assessed using viability (trypan blue exclusion assay), colony formation/replating, as well as tumor xenograft assays in vivo. RESULTS: The expression of miR-375 was substantially decreased in leukemic cell lines and primary AML blasts compared with normal controls, because DNA hypermethylation of precursor-miR-375 (pre-miR-375) promoter was discovered in leukemic cells but not in normal controls. Lower expression of miR-375 predicted poor outcome in AML patients. Furthermore, forced expression of miR-375 not only decreased proliferation and colony formation in leukemic cells but also reduced xenograft tumor size and prolonged the survival time in a leukemia xenograft mouse model. Mechanistically, overexpression of miR-375 reduced HOXB3 expression and repressed the activity of a luciferase reporter through binding 3'-untranslated regions (3'-UTR) of HOXB3 mRNA. Overexpression of HOXB3 partially blocked miR-375-induced arrest of proliferation and reduction of colony number, suggesting that HOXB3 plays an important role in miR-375-induced anti-leukemia activity. Knockdown of HOXB3 by short hairpin RNAs reduced the expression of cell division cycle associated 3 (CDCA3), which decreased cell proliferation. Furthermore, HOXB3 induced DNA methyltransferase 3B (DNMT3B) expression to bind in the pre-miR-375 promoter and enhanced DNA hypermethylation of pre-miR-375, leading to the lower expression of miR-375. CONCLUSIONS: Collectively, we have identified a miR-375-HOXB3-CDCA3/DNMT3B regulatory circuitry which contributes to leukemogenesis and suggests a therapeutic strategy of restoring miR-375 expression in AML.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/genética , Regulación Leucémica de la Expresión Génica , Proteínas de Homeodominio/genética , Leucemia Mieloide/genética , MicroARNs/genética , Regiones no Traducidas 3'/genética , Enfermedad Aguda , Adulto , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Femenino , Células HL-60 , Proteínas de Homeodominio/metabolismo , Humanos , Células K562 , Estimación de Kaplan-Meier , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Masculino , Ratones Endogámicos NOD , Ratones Noqueados , Ratones Desnudos , Ratones SCID , Persona de Mediana Edad , Trasplante Heterólogo , Adulto Joven , ADN Metiltransferasa 3BRESUMEN
Androgen receptor (AR) is widely expressed in different subtypes of breast cancer (BC). However, it is unclear how AR functions in HER2 positive (+) BC. Knockdown of AR with shRNAs and a new generation anti-androgen drug, Enzalutamide, were used to explore the involvement of AR on the growth of HER2 + BC cells (HCC1954 and SKBR3). AR shRNA or Enzalutamide inhibited the growth of SKBR3 cells at a similar extend compared to trastuzumab, an approved HER2 targeted drug. Combining Enzalutamide with trastuzumab further decreased the growth of HCC1954 and SKBR3 cells compared with single agent alone in vitro. Biochemical analysis revealed that inhibiting AR resulted in decreased HER2 phosphorylation and activation of Erk and Akt, without affecting the HER2 and HER3 expression. The in vivo efficacy of Enzalutamide was further tested using the HCC1954 xenograft model. Enzalutamide impaired the growth of HCC1954 tumor at a level comparable to that by trastuzumab. Enzalutamide decreased Ki67 staining and increased activated caspase3 staining compared with vehicle control in HCC1954 tumors. Our results indicate AR plays an important role in promoting the growth of HER2 + BC by cross-talking with the HER2 signaling. AR drug may be used as an alternative second line therapy for treating HER2 + BC.
Asunto(s)
Antagonistas de Receptores Androgénicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Feniltiohidantoína/análogos & derivados , Receptor ErbB-2/biosíntesis , Receptores Androgénicos/metabolismo , Trastuzumab/farmacología , Animales , Benzamidas , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Nitrilos , Feniltiohidantoína/farmacología , Receptor ErbB-2/genética , Receptor ErbB-3/biosíntesis , Receptor ErbB-3/genética , Receptores Androgénicos/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
SH2-domain containing protein tyrosine phosphatase 1 (Shp1/PTPN6) is mainly expressed in hematopoietic cells and acts a negative signaling regulator. Although Shp1 is also expressed in epithelial cells, the function of shp1 in normal epithelial is still less well understood, especially in regulating the growth of epithelial cells. In this study, different shRNAs and siRNAs against Shp1 were used to knockdown Shp1 expression in MCF10A, an immortalized mammary epithelial cell line. Shp1 knockdown resulted in inhibited cell growth in part due to lower percentage of MCF10A cells entering into S phase and reduced cyclin D1 expression. Accordingly, EGF-induced tyrosyl phosphorylation of EGFR and Stat5 was significantly inhibited in cells with Shp1 knockdown compared with control whereas EGF-induced Akt and Erk phosphorylation was not affected by Shp1 knockdown. Further analysis revealed that Shp1 knockdown lead to decreased EGFR protein expression without affecting EGFR mRNA expression or increasing EGFR protein degradation. Our data indicate that Shp1 functions as a positive regulator and acts in a novel mechanism through promoting EGFR protein expression in mammary epithelial cells.