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The Sonic Hedgehog (SHH) pathway is crucial regulator of embryonic development and stemness. Its alteration leads to medulloblastoma (MB), the most common malignant pediatric brain tumor. The SHH-MB subgroup is the best genetically characterized, however the molecular mechanisms responsible for its pathogenesis are not fully understood and therapeutic benefits are still limited. Here, we show that the pro-oncogenic stemness regulator Spalt-like transcriptional factor 4 (SALL4) is re-expressed in mouse SHH-MB models, and its high levels correlate with worse overall survival in SHH-MB patients. Proteomic analysis revealed that SALL4 interacts with REN/KCTD11 (here REN), a substrate receptor subunit of the Cullin3-RING ubiquitin ligase complex (CRL3REN) and a tumor suppressor lost in ~30% of human SHH-MBs. We demonstrate that CRL3REN induces polyubiquitylation and degradation of wild type SALL4, but not of a SALL4 mutant lacking zinc finger cluster 1 domain (ΔZFC1). Interestingly, SALL4 binds GLI1 and cooperates with HDAC1 to potentiate GLI1 deacetylation and transcriptional activity. Notably, inhibition of SALL4 suppresses SHH-MB growth both in murine and patient-derived xenograft models. Our findings identify SALL4 as a CRL3REN substrate and a promising therapeutic target in SHH-dependent cancers.
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Neoplasias Encefálicas , Neoplasias Cerebelosas , Meduloblastoma , Animales , Humanos , Ratones , Proteínas de Ciclo Celular , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Proteómica , Factores de Transcripción/genética , Transferasas , Proteína con Dedos de Zinc GLI1/genéticaRESUMEN
IMPORTANCE: The novelty of this study lies in the fact that it shows that IRE1 alpha endoribonuclease inhibition by 4µ8C was able to counteract Epstein-Barr virus-driven lymphomagenesis in NOD SCID gamma mice and prevent B-cell immortalization in vitro, unveiling that this drug may be a promising therapeutic approach to reduce the risk of post-transplant lymphoproliferative disorders (PTLD) onset in immune-deficient patients. This hypothesis is further supported by the fact that 4µ8C impaired the survival of PTLD-like cells derived from mice, meaning that it could be helpful also in the case in which there is the possibility that these malignancies have begun to arise.
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Infecciones por Virus de Epstein-Barr , Trastornos Linfoproliferativos , Proteínas Serina-Treonina Quinasas , Animales , Ratones , Endorribonucleasas , Herpesvirus Humano 4 , Trastornos Linfoproliferativos/terapia , Ratones SCID , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína 1 de Unión a la X-Box/antagonistas & inhibidores , Proteína 1 de Unión a la X-Box/metabolismoRESUMEN
Mast cells (MCs) are multifaceted innate immune cells often present in the tumor microenvironment (TME). Several recent findings support their contribution to the transition from chronic inflammation to cancer. However, MC-derived mediators can either favor tumor progression, inducing the spread of the tumor, or exert anti-tumorigenic functions, limiting tumor growth. This apparent controversial role likely depends on the plastic nature of MCs that under different microenvironmental stimuli can rapidly change their phenotype and functions. Thus, the exact effect of unique MC subset(s) during tumor progression is far from being understood. Using a murine model of colitis-associated colorectal cancer, we initially characterized the MC population within the TME and in non-lesional colonic areas, by multicolor flow cytometry and confocal microscopy. Our results demonstrated that tumor-associated MCs harbor a main connective tissue phenotype and release high amounts of Interleukin (IL)-6 and Tumor Necrosis Factor (TNF)-α. This MC phenotype correlates with the presence of high levels of Stem Cell Factor (SCF) and IL-33 inside the tumor. Thus, we investigated the effect of SCF and IL-33 on primary MC cultures and underscored their ability to shape MC phenotype eliciting the production of pro-inflammatory cytokines. Our findings support the conclusion that during colonic transformation a sustained stimulation by SCF and IL-33 promotes the accumulation of a prevalent connective tissue-like MC subset that through the secretion of IL-6 and TNF-α maintains a pro-inflammatory microenvironment.
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Interleucina-33 , Factor de Células Madre , Animales , Ratones , Citocinas , Interleucina-33/genética , Interleucina-6 , Mastocitos , Fenotipo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Colorectal cancer (CRC) is a leading cause of cancer-related mortality and chemoresistance is a major medical issue. The epithelial-to-mesenchymal transition (EMT) is the primary step in the emergence of the invasive phenotype and the Hedgehog-GLI (HH-GLI) and NOTCH signaling pathways are associated with poor prognosis and EMT in CRC. CRC cell lines harboring KRAS or BRAF mutations, grown as monolayers and organoids, were treated with the chemotherapeutic agent 5-Fluorouracil (5-FU) alone or combined with HH-GLI and NOTCH pathway inhibitors GANT61 and DAPT, or arsenic trioxide (ATO) to inhibit both pathways. Treatment with 5-FU led to the activation of HH-GLI and NOTCH pathways in both models. In KRAS mutant CRC, HH-GLI and NOTCH signaling activation co-operate to enhance chemoresistance and cell motility, while in BRAF mutant CRC, the HH-GLI pathway drives the chemoresistant and motile phenotype. We then showed that 5-FU promotes the mesenchymal and thus invasive phenotype in KRAS and BRAF mutant organoids and that chemosensitivity could be restored by targeting the HH-GLI pathway in BRAF mutant CRC or both HH-GLI and NOTCH pathways in KRAS mutant CRC. We suggest that in KRAS-driven CRC, the FDA-approved ATO acts as a chemotherapeutic sensitizer, whereas GANT61 is a promising chemotherapeutic sensitizer in BRAF-driven CRC.
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Development of the cerebellum is characterized by rapid proliferation of cerebellar granule cell precursors (GCPs) induced by paracrine stimulation of Sonic hedgehog (Shh) signaling from Purkinje cells, in the external granular layer (EGL). Then, granule cell precursors differentiate and migrate into the inner granular layer (IGL) of the cerebellum to form a terminally differentiated cell compartment. Aberrant activation of Sonic hedgehog signaling leads to granule cell precursors hyperproliferation and the onset of Sonic hedgehog medulloblastoma (MB), the most common embryonal brain tumor. ß-arrestin1 (ARRB1) protein plays an important role downstream of Smoothened, a component of the Sonic hedgehog pathway. In the medulloblastoma context, ß-arrestin1 is involved in a regulatory axis in association with the acetyltransferase P300, leading to the acetylated form of the transcription factor E2F1 (E2F1-ac) and redirecting its activity toward pro-apoptotic gene targets. This axis in the granule cell precursors physiological context has not been investigated yet. In this study, we demonstrate that ß-arrestin1 has antiproliferative and pro-apoptotic functions in cerebellar development. ß-arrestin1 silencing increases proliferation of Sonic hedgehog treated-cerebellar precursor cells while decreases the transcription of E2F1-ac pro-apoptotic targets genes, thus impairing apoptosis. Indeed, chromatin immunoprecipitation experiments show a direct interaction between ß-arrestin1 and the promoter regions of the pro-apoptotic E2F1 target gene and P27, indicating the double role of ß-arrestin1 in controlling apoptosis and cell cycle exit in a physiological context. Our data elucidate the role of ß-arrestin1 in the early postnatal stages of cerebellar development, in those cell compartments that give rise to medulloblastoma. This series of experiments suggests that the physiological function of ß-arrestin1 in neuronal progenitors is to directly control, cooperating with E2F1 acetylated form, transcription of pro-apoptotic genes.
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A key mechanism driving colorectal cancer (CRC) development is the upregulation of MYC and its targets, including ornithine decarboxylase (ODC), a master regulator of polyamine metabolism. Elevated polyamines promote tumorigenesis in part by activating DHPS-mediated hypusination of the translation factor eIF5A, thereby inducing MYC biosynthesis. Thus, MYC, ODC and eIF5A orchestrate a positive feedback loop that represents an attractive therapeutic target for CRC therapy. Here we show that combined inhibition of ODC and eIF5A induces a synergistic antitumor response in CRC cells, leading to MYC suppression. We found that genes of the polyamine biosynthesis and hypusination pathways are significantly upregulated in colorectal cancer patients and that inhibition of ODC or DHPS alone limits CRC cell proliferation through a cytostatic mechanism, while combined ODC and DHPS/eIF5A blockade induces a synergistic inhibition, accompanied to apoptotic cell death in vitro and in mouse models of CRC and FAP. Mechanistically, we found that this dual treatment causes complete inhibition of MYC biosynthesis in a bimodal fashion, by preventing translational elongation and initiation. Together, these data illustrate a novel strategy for CRC treatment, based on the combined suppression of ODC and eIF5A, which holds promise for the treatment of CRC.
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Neoplasias Colorrectales , Factores de Iniciación de Péptidos , Poliaminas , Proteínas Proto-Oncogénicas c-myc , Animales , Ratones , Apoptosis , Proliferación Celular , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Ornitina Descarboxilasa/genética , Ornitina Descarboxilasa/metabolismo , Ornitina Descarboxilasa/farmacología , Poliaminas/metabolismo , Humanos , Factores de Iniciación de Péptidos/genética , Factores de Iniciación de Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
AIMS: Inherited or somatic mutations in the MRE11, RAD50 and NBN genes increase the incidence of tumours, including medulloblastoma (MB). On the other hand, MRE11, RAD50 and NBS1 protein components of the MRN complex are often overexpressed and sometimes essential in cancer. In order to solve the apparent conundrum about the oncosuppressive or oncopromoting role of the MRN complex, we explored the functions of NBS1 in an MB-prone animal model. MATERIALS AND METHODS: We generated and analysed the monoallelic or biallelic deletion of the Nbn gene in the context of the SmoA1 transgenic mouse, a Sonic Hedgehog (SHH)-dependent MB-prone animal model. We used normal and tumour tissues from these animal models, primary granule cell progenitors (GCPs) from genetically modified animals and NBS1-depleted primary MB cells, to uncover the effects of NBS1 depletion by RNA-Seq, by biochemical characterisation of the SHH pathway and the DNA damage response (DDR) as well as on the growth and clonogenic properties of GCPs. RESULTS: We found that monoallelic Nbn deletion increases SmoA1-dependent MB incidence. In addition to a defective DDR, Nbn+/- GCPs show increased clonogenicity compared to Nbn+/+ GCPs, dependent on an enhanced Notch signalling. In contrast, full NbnKO impairs MB development both in SmoA1 mice and in an SHH-driven tumour allograft. CONCLUSIONS: Our study indicates that Nbn is haploinsufficient for SHH-MB development whereas full NbnKO is epistatic on SHH-driven MB development, thus revealing a gene dosage-dependent effect of Nbn inactivation on SHH-MB development.
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Proteínas de Ciclo Celular , Neoplasias Cerebelosas , Proteínas de Unión al ADN , Meduloblastoma , Animales , Proteínas de Ciclo Celular/genética , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Proteínas de Unión al ADN/genética , Dosificación de Gen , Genes Esenciales , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Meduloblastoma/patología , Ratones , Ratones TransgénicosRESUMEN
Malignant mesothelioma (MM) is a rare orphan aggressive neoplasia with low survival rates. Among the other signaling pathways, ErbB receptors and Hh signaling are deregulated in MM. Thus, molecules involved in these signaling pathways could be used for targeted therapy approaches. The aim of this study was to evaluate the effects of inhibitors of Hh- (GANT-61) and ErbB receptors (Afatinib)-mediated signaling pathways, when used alone or in combination, on growth, cell cycle, cell death and autophagy, modulation of molecules involved in transduction pathways, in three human MM cell lines of different histotypes. The efficacy of the combined treatment was also evaluated in a murine epithelioid MM cell line both in vitro and in vivo. This study demonstrated that combined treatment with two inhibitors counteracting the activation of two different signaling pathways involved in neoplastic transformation and progression, such as those activated by ErbB and Hh signaling, is more effective than the single treatments in reducing MM growth in vitro and in vivo. This study may have clinical implications for the development of targeted therapy approaches for MM.
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Receptores ErbB , Mesotelioma Maligno , Animales , Línea Celular Tumoral , Receptores ErbB/metabolismo , Proteínas Hedgehog , Humanos , Ratones , Transducción de Señal , Proteína con Dedos de Zinc GLI1RESUMEN
Contrasting evidence is present regarding the contribution of stem/progenitor cell populations to pancreatic regeneration in diabetes. Interestingly, a cell compartment with stem/progenitor cell features has been identified in the pancreatic duct glands (PDGs). The aims of the present study were to evaluate pancreatic islet injury and regeneration, and the participation of the PDG compartment in type 2 diabetic mellitus (T2DM) and in an experimental model of diabetes. Human pancreata were obtained from normal (N = 5) or T2DM (N = 10) cadaveric organ donors. Experimental diabetes was generated in mice by intraperitoneal injection of 150 mg/kg of streptozotocin (STZ, N = 10); N = 10 STZ mice also received daily intraperitoneal injections of 100 µg of human recombinant PDX1 peptide (STZ + PDX1). Samples were examined by immunohistochemistry/immunofluorescence or RT-qPCR. Serum glucose and c-peptide levels were measured in mice. Islets in T2DM patients showed ß-cell loss, signs of injury and proliferation, and a higher proportion of central islets. PDGs in T2DM patients had a higher percentage of proliferating and insulin+ or glucagon+ cells compared to controls; pancreatic islets could be observed within pancreatic duct walls of T2DM patients. STZ mice were characterized by reduced islet area compared to controls. PDX1 treatment increased islet area and the percentage of central islets compared to untreated STZ mice but did not revert diabetes. In conclusion, T2DM patients show signs of pancreatic islet regeneration and involvement of the PDG niche. PDX1 administration could support increased endocrine pancreatic regeneration in STZ. These findings contribute to defining the role and participation of stem/progenitor cell compartments within the pancreas.
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Target gene silencing is a strategy that can be used to turn off pathways or genes which are difficult to turn off pharmacologically, both because of lack of targeting drugs, or because of the risk of wider off-target effects. Here we describe the design and use of short hairpin RNA (ShRNA) and lentiviral vectors as an efficient technique for silencing NF-kappaB (NF-κB) pathway in cultured cells. This method can be used also in hard to transfect primary cell cultures.
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ARN Interferente Pequeño/genética , Silenciador del Gen , Vectores Genéticos/genética , Lentivirus/genética , Lentivirus/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Interferencia de ARNRESUMEN
The human family of Potassium (K+) Channel Tetramerization Domain (KCTD) proteins counts 25 members, and a significant number of them are still only partially characterized. While some of the KCTDs have been linked to neurological disorders or obesity, a growing tally of KCTDs are being associated with cancer hallmarks or involved in the modulation of specific oncogenic pathways. Indeed, the potential relevance of the variegate KCTD family in cancer warrants an updated picture of the current knowledge and highlights the need for further research on KCTD members as either putative therapeutic targets, or diagnostic/prognostic markers. Homology between family members, capability to participate in ubiquitination and degradation of different protein targets, ability to heterodimerize between members, role played in the main signalling pathways involved in development and cancer, are all factors that need to be considered in the search for new key players in tumorigenesis. In this review we summarize the recent published evidence on KCTD members' involvement in cancer. Furthermore, by integrating this information with data extrapolated from public databases that suggest new potential associations with cancers, we hypothesize that the number of KCTD family members involved in tumorigenesis (either as positive or negative modulator) may be bigger than so far demonstrated. Video abstract.
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Neoplasias/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Genes Supresores de Tumor , Humanos , Neoplasias/patología , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Oncogenes , Canales de Potasio con Entrada de Voltaje/genéticaRESUMEN
There is a lack of knowledge on the possible influence of systemic conditions on peri-implantitis. The aim of this case-control study is to evaluate the difference in terms of oral pathogens' concentrations in the peri-implant sulcus of a group of patients affected by metabolic syndrome (Mets) compared to healthy subjects. For each patient, peri-implant sulcular biofilm samples were obtained by inserting two sterile endodontic paper points in the deepest aspect of the peri-implant sulcus for 30 s. The quantitative real-time polymerase chain reaction was performed to evaluate total bacterial counts of six pathogens. Patients were screened for peri-implant diseases and clinical and radiographic parameters were recorded. A total of 50 patients was enrolled in the study, 25 affected by Mets and 25 healthy. Significantly higher bacterial counts were discovered for Aggregatibacter actinomycetemcomitans (p = 0.0008), Prevotella intermedia (p = 0.0477) and Staphylococcus aureus (p = 0.034) in MetS patients compared to healthy subjects. Performing a sub-group analysis, considering peri-implant status and dividing patients by MetS diagnosis, no statistically significant (p < 0.05) differences were found. For the first time, a correlation between MetS presence and a greater prevalence of some bacterial species in the peri-implant sulcus was reported, irrespectively from peri-implant status (health vs. disease).
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The Hedgehog (Hh) signaling pathway plays a crucial role in normal embryonic development and adult tissue homeostasis. On the other end, dysregulated Hh signaling triggers a prolonged mitogenic response that may prompt abnormal cell proliferation, favoring tumorigenesis. Indeed, about 30% of medulloblastomas (MBs), the most common malignant childhood cerebellar tumors, exhibit improper activation of the Hh signaling. The oncosuppressor KCASH2 has been described as a suppressor of the Hh signaling pathway, and low KCASH2 expression was observed in Hh-dependent MB tumor. Therefore, the study of the modulation of KCASH2 expression may provide fundamental information for the development of new therapeutic approaches, aimed to restore physiological KCASH2 levels and Hh inhibition. To this end, we have analyzed the TATA-less KCASH2 proximal promoter and identified key transcriptional regulators of this gene: Sp1, a TF frequently overexpressed in tumors, and the tumor suppressor p53. Here, we show that in WT cells, Sp1 binds KCASH2 promoter on several putative binding sites, leading to increase in KCASH2 expression. On the other hand, p53 is involved in negative regulation of KCASH2. In this context, the balance between p53 and Sp1 expression, and the interplay between these two proteins determine whether Sp1 acts as an activator or a repressor of KCASH2 transcription. Indeed, in p53-/- MEF and p53 mutated tumor cells, we hypothesize that Sp1 drives promoter methylation through increased expression of the DNA methyltransferase 1 (DNMT1) and reduces KCASH2 transcription, which can be reversed by Sp1 inhibition or use of demethylating agents. We suggest therefore that downregulation of KCASH2 expression in tumors could be mediated by gain of Sp1 activity and epigenetic silencing events in cells where p53 functionality is lost. This work may open new venues for novel therapeutic multidrug approaches in the treatment of Hh-dependent tumors carrying p53 deficiency.
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Both CDKN1A (p21 Waf1/Cip1) and Apoptosis signal-regulating kinase 1 (ASK1) play important roles in tumorigenesis. The role of p21 Waf1/Cip1 in attenuating ASK1-induced apoptosis by various stress conditions is well established. However, how ASK1 and p21 Waf1/Cip1 functionally interact during tumorigenesis is still unclear. To address this aspect, we crossed ASK1 knockout (ASK1KO) mice with p21 Waf1/Cip1 knockout (p21KO) mice to compare single and double-mutant mice. We observed that deletion of p21 Waf1/Cip1 leads to increased keratinocyte proliferation but also increased cell death. This is mechanistically linked to the ASK1 axis-induced apoptosis, including p38 and PARP. Indeed, deletion of ASK1 does not alter the proliferation but decreases the apoptosis of p21KO keratinocytes. To analyze as this interaction might affect skin carcinogenesis, we investigated the response of ASK1KO and p21KO mice to DMBA/TPA-induced tumorigenesis. Here we show that while endogenous ASK1 is dispensable for skin homeostasis, ASK1KO mice are resistant to DMBA/TPA-induced tumorigenesis. However, we found that epidermis lacking both p21 and ASK1 reacquires increased sensitivity to DMBA/TPA-induced tumorigenesis. We demonstrate that apoptosis and cell-cycle progression in p21KO keratinocytes are uncoupled in the absence of ASK1. These data support the model that a critical event ensuring the balance between cell death, cell-cycle arrest, and successful divisions in keratinocytes during stress conditions is the p21-dependent ASK1 inactivation.
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Solid tumors often grow in a micro-environment characterized by < 2% O2 tension. This condition, together with the aberrant activation of specific oncogenic patwhays, increases the amount and activity of the hypoxia-inducible factor-1α (HIF-1α), a transcription factor that controls up to 200 genes involved in neoangiogenesis, metabolic rewiring, invasion and drug resistance. Hypoxia also induces endoplasmic reticulum (ER) stress, a condition that triggers cell death, if cells are irreversibly damaged, or cell survival, if the stress is mild.Hypoxia and chronic ER stress both induce chemoresistance. In this review we discuss the multiple and interconnected circuitries that link hypoxic environment, chronic ER stress and chemoresistance. We suggest that hypoxia and ER stress train and select the cells more adapted to survive in unfavorable conditions, by activating pleiotropic mechanisms including apoptosis inhibition, metabolic rewiring, anti-oxidant defences, drugs efflux. This adaptative process unequivocally expands clones that acquire resistance to chemotherapy.We believe that pharmacological inhibitors of HIF-1α and modulators of ER stress, although characterized by low specificty and anti-cancer efficacy when used as single agents, may be repurposed as chemosensitizers against hypoxic and chemorefractory tumors in the next future.
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Hipoxia de la Célula/fisiología , Estrés del Retículo Endoplásmico/fisiología , Respuesta de Proteína Desplegada/fisiología , HumanosRESUMEN
Persistent mortality rates of medulloblastoma (MB) and severe side effects of the current therapies require the definition of the molecular mechanisms that contribute to tumor progression. Using cultured MB cancer stem cells and xenograft tumors generated in mice, we show that low expression of miR-326 and its host gene ß-arrestin1 (ARRB1) promotes tumor growth enhancing the E2F1 pro-survival function. Our models revealed that miR-326 and ARRB1 are controlled by a bivalent domain, since the H3K27me3 repressive mark is found at their regulatory region together with the activation-associated H3K4me3 mark. High levels of EZH2, a feature of MB, are responsible for the presence of H3K27me3. Ectopic expression of miR-326 and ARRB1 provides hints into how their low levels regulate E2F1 activity. MiR-326 targets E2F1 mRNA, thereby reducing its protein levels; ARRB1, triggering E2F1 acetylation, reverses its function into pro-apoptotic activity. Similar to miR-326 and ARRB1 overexpression, we also show that EZH2 inhibition restores miR-326/ARRB1 expression, limiting E2F1 pro-proliferative activity. Our results reveal a new regulatory molecular axis critical for MB progression.
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Neoplasias Cerebelosas/metabolismo , Regulación hacia Abajo , Factor de Transcripción E2F1/biosíntesis , Regulación Neoplásica de la Expresión Génica , Meduloblastoma/metabolismo , MicroARNs/biosíntesis , Proteínas de Neoplasias/biosíntesis , ARN Neoplásico/biosíntesis , beta-Arrestina 1/biosíntesis , Animales , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/mortalidad , Neoplasias Cerebelosas/patología , Factor de Transcripción E2F1/genética , Femenino , Células HEK293 , Humanos , Masculino , Meduloblastoma/genética , Meduloblastoma/mortalidad , Meduloblastoma/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , MicroARNs/genética , Proteínas de Neoplasias/genética , ARN Neoplásico/genética , beta-Arrestina 1/genéticaRESUMEN
Eukaryotic Translation Initiation Factor 5A (EIF5A) is a translation factor regulated by hypusination, a unique posttranslational modification catalyzed by deoxyhypusine synthetase (DHPS) and deoxyhypusine hydroxylase (DOHH) starting from the polyamine spermidine. Emerging data are showing that hypusinated EIF5A regulates key cellular processes such as autophagy, senescence, polyamine homeostasis, energy metabolism, and plays a role in cancer. However, the effects of EIF5A inhibition in preclinical cancer models, the mechanism of action, and specific translational targets are still poorly understood. We show here that hypusinated EIF5A promotes growth of colorectal cancer (CRC) cells by directly regulating MYC biosynthesis at specific pausing motifs. Inhibition of EIF5A hypusination with the DHPS inhibitor GC7 or through lentiviral-mediated knockdown of DHPS or EIF5A reduces the growth of various CRC cells. Multiplex gene expression analysis reveals that inhibition of hypusination impairs the expression of transcripts regulated by MYC, suggesting the involvement of this oncogene in the observed effect. Indeed, we demonstrate that EIF5A regulates MYC elongation without affecting its mRNA content or protein stability, by alleviating ribosome stalling at five distinct pausing motifs in MYC CDS. Of note, we show that blockade of the hypusination axis elicits a remarkable growth inhibitory effect in preclinical models of CRC and significantly reduces the size of polyps in APCMin/+ mice, a model of human familial adenomatous polyposis (FAP). Together, these data illustrate an unprecedented mechanism, whereby the tumor-promoting properties of hypusinated EIF5A are linked to its ability to regulate MYC elongation and provide a rationale for the use of DHPS/EIF5A inhibitors in CRC therapy.
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Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Lisina/análogos & derivados , Factores de Iniciación de Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas de Unión al ARN/metabolismo , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/patología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Neoplasias Colorrectales/genética , Regulación hacia Abajo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Lisina/metabolismo , Ratones Desnudos , Sistemas de Lectura Abierta/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidores , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Factores de Iniciación de Péptidos/química , Péptidos/metabolismo , Poliaminas/metabolismo , Biosíntesis de Proteínas , Proteínas de Unión al ARN/química , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
Hypoxia is a condition commonly observed in the core of solid tumors. The hypoxia-inducible factors (HIF) act as hypoxia sensors that orchestrate a coordinated response increasing the pro-survival and pro-invasive phenotype of cancer cells, and determine a broad metabolic rewiring. These events favor tumor progression and chemoresistance. The increase in glucose and amino acid uptake, glycolytic flux, and lactate production; the alterations in glutamine metabolism, tricarboxylic acid cycle, and oxidative phosphorylation; the high levels of mitochondrial reactive oxygen species; the modulation of both fatty acid synthesis and oxidation are hallmarks of the metabolic rewiring induced by hypoxia. This review discusses how metabolic-dependent factors (e.g., increased acidification of tumor microenvironment coupled with intracellular alkalinization, and reduced mitochondrial metabolism), and metabolic-independent factors (e.g., increased expression of drug efflux transporters, stemness maintenance, and epithelial-mesenchymal transition) cooperate in determining chemoresistance in hypoxia. Specific metabolic modifiers, however, can reverse the metabolic phenotype of hypoxic tumor areas that are more chemoresistant into the phenotype typical of chemosensitive cells. We propose these metabolic modifiers, able to reverse the hypoxia-induced metabolic rewiring, as potential chemosensitizer agents against hypoxic and refractory tumor cells.
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Resistencia a Antineoplásicos , Hipoxia/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Aminoácidos/metabolismo , Animales , Ciclo del Ácido Cítrico , Dimerización , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal , Glucosa/metabolismo , Glutamina/metabolismo , Humanos , Ácido Láctico/metabolismo , Ratones , Mitocondrias/metabolismo , Neoplasias/metabolismo , Oxidación-Reducción , Fosforilación Oxidativa , Oxígeno/metabolismo , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Microambiente TumoralRESUMEN
Colorectal cancer (CRC) is a leading cause of cancer death. Chemoresistance is a pivotal feature of cancer cells leading to treatment failure and ATP-binding cassette (ABC) transporters are responsible for the efflux of several molecules, including anticancer drugs. The Hedgehog-GLI (HH-GLI) pathway is a major signalling in CRC, however its role in chemoresistance has not been fully elucidated. Here we show that the HH-GLI pathway favours resistance to 5-fluorouracil and Oxaliplatin in CRC cells. We identified potential GLI1 binding sites in the promoter region of six ABC transporters, namely ABCA2, ABCB1, ABCB4, ABCB7, ABCC2 and ABCG1. Next, we investigated the binding of GLI1 using chromatin immunoprecipitation experiments and we demonstrate that GLI1 transcriptionally regulates the identified ABC transporters. We show that chemoresistant cells express high levels of GLI1 and of the ABC transporters and that GLI1 inhibition disrupts the transporters up-regulation. Moreover, we report that human CRC tumours express high levels of the ABCG1 transporter and that its expression correlates with worse patients' prognosis. This study identifies a new mechanism where HH-GLI signalling regulates CRC chemoresistance features. Our results indicate that the inhibition of Gli1 regulates the ABC transporters expression and therefore should be considered as a therapeutic option in chemoresistant patients.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Neoplasias Colorrectales/metabolismo , Proteínas Hedgehog/metabolismo , Proteína con Dedos de Zinc GLI1/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Fluorouracilo/farmacología , Regulación Neoplásica de la Expresión Génica , Proteínas Hedgehog/genética , Humanos , Estimación de Kaplan-Meier , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Oxaliplatino/farmacología , Regiones Promotoras Genéticas/genética , Unión Proteica , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína con Dedos de Zinc GLI1/genéticaRESUMEN
Originally identified as an RNA polymerase II interactor, Che-1/AATF (Che-1) has now been recognized as a multifunctional protein involved in cell-cycle regulation and cancer progression, as well as apoptosis inhibition and response to stress. This protein displays a peculiar nucleolar localization and it has recently been implicated in pre-rRNA processing and ribosome biogenesis. Here, we report the identification of a novel function of Che-1 in the regulation of ribosomal RNA (rRNA) synthesis, in both cancer and normal cells. We demonstrate that Che-1 interacts with RNA polymerase I and nucleolar upstream binding factor (UBF) and promotes RNA polymerase I-dependent transcription. Furthermore, this protein binds to the rRNA gene (rDNA) promoter and modulates its epigenetic state by contrasting the recruitment of HDAC1. Che-1 downregulation affects RNA polymerase I and UBF recruitment on rDNA and leads to reducing rDNA promoter activity and 47S pre-rRNA production. Interestingly, Che-1 depletion induces abnormal nucleolar morphology associated with re-distribution of nucleolar proteins. Finally, we show that upon DNA damage Che-1 re-localizes from rDNA to TP53 gene promoter to induce cell-cycle arrest. This previously uncharacterized function of Che-1 confirms the important role of this protein in the regulation of ribosome biogenesis, cellular proliferation and response to stress.