RESUMEN
Despite a high clinical need for the treatment of colorectal carcinoma (CRC) as the second leading cause of cancer-related deaths, targeted therapies are still limited. The multifunctional enzyme Transglutaminase 2 (TGM2), which harbors transamidation and GTPase activity, has been implicated in the development and progression of different types of human cancers. However, the mechanism and role of TGM2 in colorectal cancer are poorly understood. Here, we present TGM2 as a promising drug target.In primary patient material of CRC patients, we detected an increased expression and enzymatic activity of TGM2 in colon cancer tissue in comparison to matched normal colon mucosa cells. The genetic ablation of TGM2 in CRC cell lines using shRNAs or CRISPR/Cas9 inhibited cell expansion and tumorsphere formation. In vivo, tumor initiation and growth were reduced upon genetic knockdown of TGM2 in xenotransplantations. TGM2 ablation led to the induction of Caspase-3-driven apoptosis in CRC cells. Functional rescue experiments with TGM2 variants revealed that the transamidation activity is critical for the pro-survival function of TGM2. Transcriptomic and protein-protein interaction analyses applying various methods including super-resolution and time-lapse microscopy showed that TGM2 directly binds to the tumor suppressor p53, leading to its inactivation and escape of apoptosis induction.We demonstrate here that TGM2 is an essential survival factor in CRC, highlighting the therapeutic potential of TGM2 inhibitors in CRC patients with high TGM2 expression. The inactivation of p53 by TGM2 binding indicates a general anti-apoptotic function, which may be relevant in cancers beyond CRC.
Asunto(s)
Carcinogénesis/genética , Neoplasias del Colon/genética , Proteína Glutamina Gamma Glutamiltransferasa 2/genética , Proteína p53 Supresora de Tumor/genética , Animales , Apoptosis/genética , Caspasa 3/genética , Línea Celular Tumoral , Proliferación Celular/genética , Colon/patología , Neoplasias del Colon/patología , Regulación Neoplásica de la Expresión Génica/genética , Células HCT116 , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mapas de Interacción de Proteínas/genética , Transcriptoma/genéticaRESUMEN
Purpose Dysregulated microRNAs are implicated in the pathogenesis and aggressiveness of acute myeloid leukemia (AML). We describe the effect of the hematopoietic stem-cell self-renewal regulating miR-193b on progression and prognosis of AML. Methods We profiled miR-193b-5p/3p expression in cytogenetically and clinically characterized de novo pediatric AML (n = 161) via quantitative real-time polymerase chain reaction and validated our findings in an independent cohort of 187 adult patients. We investigated the tumor suppressive function of miR-193b in human AML blasts, patient-derived xenografts, and miR-193b knockout mice in vitro and in vivo. Results miR-193b exerted important, endogenous, tumor-suppressive functions on the hematopoietic system. miR-193b-3p was downregulated in several cytogenetically defined subgroups of pediatric and adult AML, and low expression served as an independent indicator for poor prognosis in pediatric AML (risk ratio ± standard error, -0.56 ± 0.23; P = .016). miR-193b-3p expression improved the prognostic value of the European LeukemiaNet risk-group stratification or a 17-gene leukemic stemness score. In knockout mice, loss of miR-193b cooperated with Hoxa9/Meis1 during leukemogenesis, whereas restoring miR-193b expression impaired leukemic engraftment. Similarly, expression of miR-193b in AML blasts from patients diminished leukemic growth in vitro and in mouse xenografts. Mechanistically, miR-193b induced apoptosis and a G1/S-phase block in various human AML subgroups by targeting multiple factors of the KIT-RAS-RAF-MEK-ERK (MAPK) signaling cascade and the downstream cell cycle regulator CCND1. Conclusion The tumor-suppressive function is independent of patient age or genetics; therefore, restoring miR-193b would assure high antileukemic efficacy by blocking the entire MAPK signaling cascade while preventing the emergence of resistance mechanisms.
Asunto(s)
Leucemia Mieloide Aguda/genética , MicroARNs/biosíntesis , Animales , Procesos de Crecimiento Celular/genética , Regulación hacia Abajo , Genes Supresores de Tumor , Xenoinjertos , Proteínas de Homeodominio/genética , Humanos , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/terapia , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , MicroARNs/genética , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/genética , PronósticoRESUMEN
The transcription factor Meis1 drives myeloid leukemogenesis in the context of Hox gene overexpression but is currently considered undruggable. We therefore investigated whether myeloid progenitor cells transformed by Hoxa9 and Meis1 become addicted to targetable signaling pathways. A comprehensive (phospho)proteomic analysis revealed that Meis1 increased Syk protein expression and activity. Syk upregulation occurs through a Meis1-dependent feedback loop. By dissecting this loop, we show that Syk is a direct target of miR-146a, whose expression is indirectly regulated by Meis1 through the transcription factor PU.1. In the context of Hoxa9 overexpression, Syk signaling induces Meis1, recapitulating several leukemogenic features of Hoxa9/Meis1-driven leukemia. Finally, Syk inhibition disrupts the identified regulatory loop, prolonging survival of mice with Hoxa9/Meis1-driven leukemia.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Leucemia Mieloide Aguda/metabolismo , MicroARNs/genética , Proteínas de Neoplasias/metabolismo , Quinasa Syk/metabolismo , Animales , Regulación Leucémica de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Integrina beta3/metabolismo , Estimación de Kaplan-Meier , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Ratones Endogámicos C57BL , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Proteínas de Neoplasias/genética , Transducción de Señal , Quinasa Syk/genéticaRESUMEN
BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide. Growing evidence indicates that tumor-initiating cells (TICs) are responsible for tumor growth and progression. Conventional chemotherapeutics do not sufficiently eliminate TICs, leading to tumor relapse. We aimed to gain insight into TIC biology by comparing the transcriptome of primary TIC cultures and their normal stem cell counterparts to uncover expression differences. METHODS: We established colonosphere cultures derived from the resection of paired specimens of primary tumor and normal mucosa in patients with CRC. These colonospheres, enriched for TICs, were used for differential transcriptome analyses to detect new targets for a TIC-directed therapy. Effects of target inhibition on CRC cells were studied in vitro and in vivo. RESULTS: Pathway analysis of the regulated genes showed enrichment of genes central to PI3K/AKT and Wnt-signaling. We identified CD133 as a marker for a more aggressive CRC subpopulation enriched with TICs in SW480 CRC cells in an in vivo cancer model. Treatment of CRC cells with the selective AKT inhibitor MK-2206 caused a decrease in cell proliferation, particularly in the TIC fraction, resulting in a significant reduction of the stemness capacity to form colonospheres in vitro and to initiate tumor formation in vivo. Consequently, MK-2206 treatment of mice with established xenograft tumors exhibited a significant deceleration of tumor progression. Primary patient-derived tumorsphere growth was significantly inhibited by MK-2206. CONCLUSION: This study reveals that AKT signaling is critical for TIC proliferation and can be efficiently targeted by MK-2206 representing a preclinical therapeutic strategy to repress colorectal TICs.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Compuestos Heterocíclicos con 3 Anillos/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , Antígeno AC133/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colon/citología , Neoplasias Colorrectales/metabolismo , Fluorouracilo/farmacología , Perfilación de la Expresión Génica , Humanos , Mucosa Intestinal/citología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Esferoides Celulares/efectos de los fármacos , Transcriptoma , Proteína p53 Supresora de Tumor/genética , Vía de Señalización Wnt/genéticaRESUMEN
Hematopoietic stem cells (HSCs) maintain blood cell production life-long by their unique abilities of self-renewal and differentiation into all blood cell lineages. Growth arrest and DNA-damage-inducible 45 alpha (GADD45A) is induced by genotoxic stress in HSCs. GADD45A has been implicated in cell cycle control, cell death and senescence, as well as in DNA-damage repair. In general, GADD45A provides cellular stability by either arresting the cell cycle progression until DNA damage is repaired or, in cases of fatal damage, by inducing apoptosis. However, the function of GADD45A in hematopoiesis remains controversial. We revealed the changes in murine HSC fate control orchestrated by the expression of GADD45A at single cell resolution. In contrast to other cellular systems, GADD45A expression did not cause a cell cycle arrest or an alteration in the decision between cell survival and apoptosis in HSCs. Strikingly, GADD45A strongly induced and accelerated the differentiation program in HSCs. Continuous tracking of individual HSCs and their progeny via time-lapse microscopy elucidated that once GADD45A was expressed, HSCs differentiate into committed progenitors within 29 hours. GADD45A-expressing HSCs failed to long-term reconstitute the blood of recipients by inducing multilineage differentiation in vivo. Importantly, γ-irradiation of HSCs induced their differentiation by upregulating endogenous GADD45A. The differentiation induction by GADD45A was transmitted by activating p38 Mitogen-activated protein kinase (MAPK) signaling and allowed the generation of megakaryocytic-erythroid, myeloid, and lymphoid lineages. These data indicate that genotoxic stress-induced GADD45A expression in HSCs prevents their fatal transformation by directing them into differentiation and thereby clearing them from the system.
Asunto(s)
Puntos de Control del Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Diferenciación Celular/genética , Células Madre Hematopoyéticas , Proteínas Nucleares/genética , Animales , Apoptosis/genética , Proteínas de Ciclo Celular/biosíntesis , Proliferación Celular/genética , Supervivencia Celular/genética , Daño del ADN/genética , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis/genética , Humanos , Ratones , Proteínas Nucleares/biosíntesis , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Haematopoietic stem cells (HSCs) require the right composition of microRNAs (miR) for proper life-long balanced blood regeneration. Here we show a regulatory circuit that prevents excessive HSC self-renewal by upregulation of miR-193b upon self-renewal promoting thrombopoietin (TPO)-MPL-STAT5 signalling. In turn, miR-193b restricts cytokine signalling, by targeting the receptor tyrosine kinase c-KIT. We generated a miR-193b knockout mouse model to unravel the physiological function of miR-193b in haematopoiesis. MiR-193b(-/-) mice show a selective gradual enrichment of functional HSCs, which are fully competent in multilineage blood reconstitution upon transplantation. The absence of miR-193b causes an accelerated expansion of HSCs, without altering cell cycle or survival, but by decelerating differentiation. Conversely, ectopic miR-193b expression restricts long-term repopulating HSC expansion and blood reconstitution. MiR-193b-deficient haematopoietic stem and progenitor cells exhibit increased basal and cytokine-induced STAT5 and AKT signalling. This STAT5-induced microRNA provides a negative feedback for excessive signalling to restrict uncontrolled HSC expansion.
Asunto(s)
Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , MicroARNs/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptores de Trombopoyetina/metabolismo , Factor de Transcripción STAT5/metabolismo , Trombopoyetina/metabolismo , Animales , Proliferación Celular/genética , Citometría de Flujo , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Receptores de Citocinas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Imagen de Lapso de TiempoRESUMEN
The balance of self-renewal and differentiation in long-term repopulating hematopoietic stem cells (LT-HSC) must be strictly controlled to maintain blood homeostasis and to prevent leukemogenesis. Hematopoietic cytokines can induce differentiation in LT-HSCs; however, the molecular mechanism orchestrating this delicate balance requires further elucidation. We identified the tumor suppressor GADD45G as an instructor of LT-HSC differentiation under the control of differentiation-promoting cytokine receptor signaling. GADD45G immediately induces and accelerates differentiation in LT-HSCs and overrides the self-renewal program by specifically activating MAP3K4-mediated MAPK p38. Conversely, the absence of GADD45G enhances the self-renewal potential of LT-HSCs. Videomicroscopy-based tracking of single LT-HSCs revealed that, once GADD45G is expressed, the development of LT-HSCs into lineage-committed progeny occurred within 36 hr and uncovered a selective lineage choice with a severe reduction in megakaryocytic-erythroid cells. Here, we report an unrecognized role of GADD45G as a central molecular linker of extrinsic cytokine differentiation and lineage choice control in hematopoiesis.
Asunto(s)
Citocinas/farmacología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Animales , Diferenciación Celular/fisiología , Citometría de Flujo , Péptidos y Proteínas de Señalización Intracelular/genética , MAP Quinasa Quinasa Quinasa 4/genética , MAP Quinasa Quinasa Quinasa 4/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía por Video , Proteinas GADD45RESUMEN
Generating heat and maintaining body temperature is the primary function of brown adipose tissue (BAT). Previous studies have implicated microRNAs, including miR-193b and miR-365-1, in BAT differentiation. We used mouse genetics to further understand the specific contributions of these two miRs. BAT function in mice with an inactivated miR-193b-365-1 locus, as determined by their response to the selective ß3 adrenergic receptor agonist CL316.243 and their tolerance to cold exposure, was normal and expression of genes associated with functional BAT, including Prdm16 and Ucp1, was unaffected. In addition, genome-wide expression profiles of miRNAs and mRNAs in BAT in the presence and absence of miR-193b-365-1 were determined. In summary, these data demonstrate, in contrast to earlier work, that the development, differentiation, and function of BAT do not require the presence of miR-193b and miR-365-1.