RESUMO
The primary cilium, a signaling organelle projecting from the surface of a cell, controls cellular physiology and behavior. The presence or absence of primary cilia is a distinctive feature of a given tumor type; however, whether and how the primary cilium contributes to tumorigenesis are unknown for most tumors. Medulloblastoma (MB) is a common pediatric brain cancer comprising four groups: SHH, WNT, group 3 (G3), and group 4 (G4). From 111 cases of MB, we show that primary cilia are abundant in SHH and WNT MBs but rare in G3 and G4 MBs. Using WNT and G3 MB mouse models, we show that primary cilia promote WNT MB by facilitating translation of mRNA encoding ß-catenin, a major oncoprotein driving WNT MB, whereas cilium loss promotes G3 MB by disrupting cell cycle control and destabilizing the genome. Our findings reveal tumor type-specific ciliary functions and underlying molecular mechanisms. Moreover, we expand the function of primary cilia to translation control and reveal a molecular mechanism by which cilia regulate cell cycle progression, thereby providing new frameworks for studying cilium function in normal and pathologic conditions.
Assuntos
Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , Animais , Neoplasias Encefálicas/patologia , Ciclo Celular/genética , Neoplasias Cerebelares/genética , Cílios/genética , Humanos , Meduloblastoma/genética , CamundongosRESUMO
Cell-type plasticity within a tumor has recently been suggested to cause a bidirectional conversion between tumor-initiating stem cells and nonstem cells triggered by an inflammatory stroma. NF-κB represents a key transcription factor within the inflammatory tumor microenvironment. However, NF-κB's function in tumor-initiating cells has not been examined yet. Using a genetic model of intestinal epithelial cell (IEC)-restricted constitutive Wnt-activation, which comprises the most common event in the initiation of colon cancer, we demonstrate that NF-κB modulates Wnt signaling and show that IEC-specific ablation of RelA/p65 retards crypt stem cell expansion. In contrast, elevated NF-κB signaling enhances Wnt activation and induces dedifferentiation of nonstem cells that acquire tumor-initiating capacity. Thus, our data support the concept of bidirectional conversion and highlight the importance of inflammatory signaling for dedifferentiation and generation of tumor-initiating cells in vivo.
Assuntos
Desdiferenciação Celular , Transformação Celular Neoplásica , Neoplasias do Colo/patologia , Células-Tronco Neoplásicas/patologia , Animais , Colo/patologia , Células Epiteliais/patologia , Feminino , Humanos , Masculino , Camundongos , NF-kappa B/metabolismo , Via de Sinalização WntRESUMO
The murine kidney and ureter develop in a regionalized fashion from the ureteric bud and its surrounding mesenchyme. Whereas the factors that establish the metanephric cell lineages have been well characterized, much less is known about the molecular cues that specify the ureter. Here, we have identified a crucial patterning function in this process for Tbx18, a T-box transcription factor gene specifically expressed in the mesenchymal primordium of the ureter. Using misexpression and loss-of-function mice combined with molecular profiling approaches, we show that Tbx18 is required and sufficient to repress metanephric mesenchymal gene programs. We identify Wt1 as a functional target of TBX18. Our work suggests that TBX18 acts as a permissive factor in ureter specification by generating a mesenchymal domain around the distal ureteric bud where SHH and BMP4 signaling can occur.
Assuntos
Ureter , Camundongos , Animais , Ureter/metabolismo , Rim/metabolismo , Transdução de Sinais/genética , Linhagem da Célula/genética , Expressão Gênica , Mesoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismoRESUMO
Hematopoietic stem cells (HSCs) reside and self-renew in the bone marrow (BM) niche. Overall, the signaling that regulates stem cell dormancy in the HSC niche remains controversial. Here, we demonstrate that TGF-ß type II receptor-deficient HSCs show low-level Smad activation and impaired long-term repopulating activity, underlining the critical role of TGF-ß/Smad signaling in HSC maintenance. TGF-ß is produced as a latent form by a variety of cells, so we searched for those that express activator molecules for latent TGF-ß. Nonmyelinating Schwann cells in BM proved responsible for activation. These glial cells ensheathed autonomic nerves, expressed HSC niche factor genes, and were in contact with a substantial proportion of HSCs. Autonomic nerve denervation reduced the number of these active TGF-ß-producing cells and led to rapid loss of HSCs from BM. We propose that glial cells are components of a BM niche and maintain HSC hibernation by regulating activation of latent TGF-ß.
Assuntos
Medula Óssea/fisiologia , Células-Tronco Hematopoéticas/citologia , Células de Schwann/citologia , Fator de Crescimento Transformador beta3/metabolismo , Animais , Antígenos CD34/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/metabolismo , Células de Schwann/fisiologia , SimpatectomiaRESUMO
Chronic inflammation is accompanied by recurring cycles of tissue destruction and repair and is associated with an increased risk of cancer1-3. However, how such cycles affect the clonal composition of tissues, particularly in terms of cancer development, remains unknown. Here we show that in patients with ulcerative colitis, the inflamed intestine undergoes widespread remodelling by pervasive clones, many of which are positively selected by acquiring mutations that commonly involve the NFKBIZ, TRAF3IP2, ZC3H12A, PIGR and HNRNPF genes and are implicated in the downregulation of IL-17 and other pro-inflammatory signals. Mutational profiles vary substantially between colitis-associated cancer and non-dysplastic tissues in ulcerative colitis, which indicates that there are distinct mechanisms of positive selection in both tissues. In particular, mutations in NFKBIZ are highly prevalent in the epithelium of patients with ulcerative colitis but rarely found in both sporadic and colitis-associated cancer, indicating that NFKBIZ-mutant cells are selected against during colorectal carcinogenesis. In further support of this negative selection, we found that tumour formation was significantly attenuated in Nfkbiz-mutant mice and cell competition was compromised by disruption of NFKBIZ in human colorectal cancer cells. Our results highlight common and discrete mechanisms of clonal selection in inflammatory tissues, which reveal unexpected cancer vulnerabilities that could potentially be exploited for therapeutics in colorectal cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Colite Ulcerativa/genética , Taxa de Mutação , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular Tumoral , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Neoplasias Colorretais/genética , Humanos , Camundongos , Transdução de SinaisRESUMO
Desmoid tumors (DTs), also called desmoid-type fibromatoses, are locally aggressive tumors of mesenchymal origin. In the present study, we developed a novel mouse model of DTs by inducing a local mutation in the Ctnnb1 gene, encoding ß-catenin in PDGFRA-positive stromal cells, by subcutaneous injection of 4-hydroxy-tamoxifen. Tumors in this model resembled histologically clinical samples from DT patients and showed strong phosphorylation of nuclear SMAD2. Knockout of SMAD4 in the model significantly suppressed tumor growth. Proteomic analysis revealed that SMAD4 knockout reduced the level of Cysteine-and-Glycine-Rich Protein 2 (CSRP2) in DTs, and treatment of DT-derived cells with a TGF-ß receptor inhibitor reduced CSRP2 RNA levels. Knockdown of CSRP2 in DT cells significantly suppressed their proliferation. These results indicate that the TGF-ß/CSRP2 axis is a potential therapeutic target for DTs downstream of TGF-ß signaling.
Assuntos
Fibromatose Agressiva , Animais , Humanos , Camundongos , beta Catenina/genética , beta Catenina/metabolismo , Fibromatose Agressiva/genética , Fibromatose Agressiva/patologia , Proteínas com Domínio LIM/genética , Camundongos Knockout , Proteínas Musculares/metabolismo , Proteínas Nucleares/genética , Proteômica , Fator de Crescimento Transformador beta/metabolismo , Regulação para CimaRESUMO
In adult hippocampal neurogenesis, stem/progenitor cells generate dentate granule neurons that contribute to hippocampal plasticity. The establishment of a morphologically defined dendritic arbor is central to the functional integration of adult-born neurons. We investigated the role of canonical Wnt/ß-catenin signaling in dendritogenesis of adult-born neurons. We show that canonical Wnt signaling follows a biphasic pattern, with high activity in stem/progenitor cells, attenuation in immature neurons, and reactivation during maturation, and demonstrate that this activity pattern is required for proper dendrite development. Increasing ß-catenin signaling in maturing neurons of young adult mice transiently accelerated dendritic growth, but eventually produced dendritic defects and excessive spine numbers. In middle-aged mice, in which protracted dendrite and spine development were paralleled by lower canonical Wnt signaling activity, enhancement of ß-catenin signaling restored dendritic growth and spine formation to levels observed in young adult animals. Our data indicate that precise timing and strength of ß-catenin signaling are essential for the correct functional integration of adult-born neurons and suggest Wnt/ß-catenin signaling as a pathway to ameliorate deficits in adult neurogenesis during aging.
Assuntos
Hipocampo/metabolismo , Neurônios/metabolismo , Transdução de Sinais/fisiologia , beta Catenina/metabolismo , Envelhecimento/metabolismo , Animais , Proteína Axina/genética , Feminino , Hipocampo/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Via de Sinalização Wnt , beta Catenina/genéticaRESUMO
Experimental techniques for patient-derived cancer stem-cell organoids/spheroids can be powerful diagnostic tools for personalized chemotherapy. However, establishing their cultures from gastric cancer remains challenging due to low culture efficiency and cumbersome methods. To propagate gastric cancer cells as highly proliferative stem-cell spheroids in vitro, we initially used a similar method to that for colorectal cancer stem cells, which, unfortunately, resulted in a low success rate (25%, 18 of 71 cases). We scrutinized the protocol and found that the unsuccessful cases were largely caused by the paucity of cancer stem cells in the sampled tissues as well as insufficient culture media. To overcome these obstacles, we extensively revised our sample collection protocol and culture conditions. We then investigated the following second cohort and, consequently, achieved a significantly higher success rate (88%, 29 of 33 cases). One of the key improvements included new sampling procedures for tumor tissues from wider and deeper areas of gastric cancer specimens, which allowed securing cancer stem cells more reproducibly. Additionally, we embedded tumor epithelial pieces separately in both Matrigel and collagen type-I as their preference to the extracellular matrix was different depending on the tumors. We also added a low concentration of Wnt ligands to the culture, which helped the growth of occasional Wnt-responsive gastric cancer stem-cell spheroids without allowing proliferation of the normal gastric epithelial stem cells. This newly improved spheroid culture method may facilitate further studies, including personalized drug-sensitivity tests prior to drug therapy.
Assuntos
Esferoides Celulares , Neoplasias Gástricas , Humanos , Esferoides Celulares/patologia , Neoplasias Gástricas/patologia , Células-Tronco Neoplásicas/patologiaRESUMO
Members of the Iroquois B (IrxB) homeodomain cluster genes, specifically Irx3 and Irx5, are crucial for heart, limb and bone development. Recently, we reported their importance for oocyte and follicle survival within the developing ovary. Irx3 and Irx5 expression begins after sex determination in the ovary but remains absent in the fetal testis. Mutually antagonistic molecular signals ensure ovary versus testis differentiation with canonical Wnt/ß-catenin signals paramount for promoting the ovary pathway. Notably, few direct downstream targets have been identified. We report that Wnt/ß-catenin signaling directly stimulates Irx3 and Irx5 transcription in the developing ovary. Using in silico analysis of ATAC- and ChIP-Seq databases in conjunction with mouse gonad explant transfection assays, we identified TCF/LEF-binding sequences within two distal enhancers of the IrxB locus that promote ß-catenin-responsive ovary expression. Meanwhile, Irx3 and Irx5 transcription is suppressed within the developing testis by the presence of H3K27me3 on these same sites. Thus, we resolved sexually dimorphic regulation of Irx3 and Irx5 via epigenetic and ß-catenin transcriptional control where their ovarian presence promotes oocyte and follicle survival vital for future ovarian health.
Assuntos
Epigênese Genética/fisiologia , Gônadas/embriologia , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Animais , Diferenciação Celular/genética , Células Cultivadas , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Gônadas/metabolismo , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Ovário/embriologia , Ovário/metabolismo , Caracteres Sexuais , Diferenciação Sexual/genética , Testículo/embriologia , Testículo/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Mutations in Adenomatous polyposis coli (APC) underlie familial adenomatous polyposis (FAP), an inherited cancer syndrome characterized by the widespread development of colorectal polyps. APC is best known as a scaffold protein in the ß-catenin destruction complex, whose activity is antagonized by canonical Wnt signaling. Whether other effector pathways mediate APC's tumor suppressor function is less clear. Here we report that activation of YAP, the downstream effector of the Hippo signaling pathway, is a general hallmark of tubular adenomas from FAP patients. We show that APC functions as a scaffold protein that facilitates the Hippo kinase cascade by interacting with Sav1 and Lats1. Consistent with the molecular link between APC and the Hippo signaling pathway, genetic analysis reveals that YAP is absolutely required for the development of APC-deficient adenomas. These findings establish Hippo-YAP signaling as a critical effector pathway downstream from APC, independent from its involvement in the ß-catenin destruction complex.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/fisiopatologia , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Adenoma/enzimologia , Adenoma/fisiopatologia , Polipose Adenomatosa do Colo/enzimologia , Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/patologia , Proteínas de Ciclo Celular , Células Cultivadas , Via de Sinalização Hippo , Humanos , Intestinos/fisiopatologia , Camundongos , Fatores de Transcrição , Proteínas de Sinalização YAP , beta Catenina/metabolismoRESUMO
Fibroblast growth factor receptor inhibitors (FGFRi) were introduced into clinical trials on several cancer types and found to be particularly efficacious on urothelial cancer and cholangiocarcinoma. Although many enrolled patients responded well in clinical trials, there were some patients who did not respond to FGFRi even though their tumors carried the genomic changes that met the enrollment criteria. As already established, fibroblast growth factor receptor (FGFR) and epidermal growth factor receptor (EGFR) share the downstream signaling pathway of MAPK activation. Accordingly, it is conceivable that targeted inhibition of FGFR alone could leave the MAPK signaling unaffected when the signaling through EGFR is relatively strong. To test this hypothesis, we calculated here the FGFR to EGFR mRNA ratio (F/E for short) of biliary tract and urothelial cancer cell lines utilized in preclinical studies. In six biliary tract cancer cell lines, two responsive lines had an F/E of 9.5 and 9.0, whereas the F/E of four nonresponsive lines was 0.1-1.8. In 22 urothelial cancer cell lines, four of the five responsive lines showed an F/E of 2.8-4.9 (median, 3.6), whereas the F/E range of 17 nonresponsive lines was 0.01-2.7 (median, 0.6) (p = 0.004). We further investigated our 47 patient-derived colorectal cancer-stem cell spheroid lines. The 18 responsive lines showed relatively high F/E (median, 16.4), whereas 29 nonresponsive lines had low F/E (median, 9.2) (p = 0.0006). These results suggest that F/E is another strong predictor of responses to FGFRi that is as useful as the current genomic criteria based solely on the FGFR genomic changes.
Assuntos
Neoplasias , Inibidores de Proteínas Quinases , Humanos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Transdução de Sinais , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genéticaRESUMO
BACKGROUND & AIMS: One-third of hepatocellular carcinomas (HCCs) harbor mutations activating the ß-catenin pathway, predominantly via mutations in the CTNNB1 gene itself. Mouse models of Apc loss-of-function are widely used to mimic ß-catenin-dependent tumorigenesis. Given the low prevalence of APC mutations in human HCCs, we aimed to generate liver tumors through CTNNB1 exon 3 deletion (ßcatΔex3). We then compared ßcatΔex3 liver tumors with liver tumors generated via frameshift in exon 15 of Apc (Apcfs-ex15). METHODS: We used hepatocyte-specific and inducible mouse models generated through either a Cre-Lox or a CRISPR/Cas9 approach using adeno-associated virus vectors. Tumors generated by the Cre-Lox models were phenotypically analyzed using immunohistochemistry and were selected for transcriptomic analysis by RNA-sequencing (RNAseq). Mouse RNAseq data were compared to human RNAseq data (8 normal tissues, 48 HCCs, 9 hepatoblastomas) in an integrative analysis. Tumors generated via CRISPR were analyzed using DNA sequencing and immuno-histochemistry. RESULTS: Mice with CTNNB1 exon 3 deletion in hepatocytes developed liver tumors indistinguishable from Apcfs-ex15 liver tumors. Both Apcfs-ex15 and ßcatΔex3 mouse models induced growth of phenotypically distinct tumors (differentiated or undifferentiated). Integrative analysis of human and mouse tumors showed that differentiated mouse tumors cluster with well-differentiated human CTNNB1-mutated tumors. Conversely, undifferentiated mouse tumors cluster with human mesenchymal hepatoblastomas and harbor activated YAP signaling. CONCLUSION: Apcfs-ex15 and ßcatΔex3 mouse models both induce growth of tumors that are transcriptionally similar to either well-differentiated and ß-catenin-activated human HCCs or mesenchymal hepatoblastomas. LAY SUMMARY: New and easy-to-use transgenic mouse models of primary liver cancers have been generated, with mutations in the gene encoding beta-catenin, which are frequent in both adult and pediatric primary liver cancers. The mice develop both types of cancer, constituting a strong preclinical model.
Assuntos
Carcinoma Hepatocelular , Hepatoblastoma , Neoplasias Hepáticas , beta Catenina , Animais , Carcinoma Hepatocelular/patologia , Hepatoblastoma/metabolismo , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Transgênicos , Mutação , beta Catenina/genéticaRESUMO
The Wnt signaling antagonist, sclerostin, is a potent suppressor of bone acquisition that also mediates endocrine communication between bone and adipose. As a result, Sost-/- mice exhibit dramatic increases in bone formation but marked decreases in visceral and subcutaneous adipose that are secondary to alterations in lipid synthesis and utilization. While interrogating the mechanism by which sclerostin influences adipocyte metabolism, we observed paradoxical increases in the adipogenic potential and numbers of CD45- :Sca1+ :PDGFRα+ adipoprogenitors in the stromal vascular compartment of fat pads isolated from male Sost-/- mice. Lineage tracing studies indicated that sclerostin deficiency blocks the differentiation of PDGFRα+ adipoprogenitors to mature adipocytes in association with increased Wnt/ß-catenin signaling. Importantly, osteoblast/osteocyte-specific Sost gene deletion mirrors the accumulation of PDGFRα+ adipoprogenitors, reduction in fat mass, and improved glucose metabolism evident in Sost-/- mice. These data indicate that bone-derived sclerostin regulates multiple facets of adipocyte physiology ranging from progenitor cell commitment to anabolic metabolism.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adipócitos/metabolismo , Adipogenia/genética , Osso e Ossos/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Células-Tronco/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Tecido Adiposo/metabolismo , Animais , Glicemia/metabolismo , Células Cultivadas , Técnicas de Inativação de Genes/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/metabolismo , Osteócitos/metabolismo , Osteogênese/genéticaRESUMO
Endometrioid endometrial carcinomas (EECs) carry multiple driver mutations even when they are low grade. However, the biological significance of these concurrent mutations is unknown. We explored the interactions among three signature EEC mutations: loss-of-function (LOF) mutations in PTEN, gain-of-function (GOF) mutations of phosphoinositide 3-kinase (PI3K), and CTNNB1 exon 3 mutations, utilizing in vivo mutagenesis of the mouse uterine epithelium. While epithelial cells with a monoallelic mutation in any one of three genes failed to propagate in the endometrium, any combination of two or more mutant alleles promoted the growth of epithelium, causing simple hyperplasia, in a dose-dependent manner. Notably, Ctnnb1 exon 3 deletion significantly increased the size of hyperplastic lesions by promoting the growth of PTEN LOF and/or PI3K GOF mutant cells through the activation of neoadenogenesis pathways. Although these three mutations were insufficient to cause EEC in intact female mice, castration triggered malignant transformation, leading to myometrial invasion and serosal metastasis. Treatment of castrated mice with progesterone or estradiol attenuated the neoplastic transformation. This study demonstrates that multiple driver mutations are required for premalignant cells to break the growth-repressing field effect of normal endometrium maintained by ovarian steroids and that CTNNB1 exon 3 mutations play critical roles in the growth of preneoplastic cells within the endometrium of premenopausal women and in the myometrial invasion of EECs in menopausal women.
Assuntos
Hiperplasia Endometrial/patologia , Neoplasias do Endométrio/fisiopatologia , Ovário/fisiopatologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , beta Catenina/genética , Alelos , Transformação Celular Neoplásica , Progressão da Doença , Hiperplasia Endometrial/enzimologia , Hiperplasia Endometrial/metabolismo , Neoplasias do Endométrio/enzimologia , Feminino , Humanos , MutaçãoRESUMO
Cancer stem cell (CSC)-specific markers may be potential therapeutic targets. We previously identified that Dclk1, a tuft cell marker, marks tumor stem cells (TSCs) in mouse intestinal adenomas. Based on the analysis of mouse Dclk1+ tumor cells, we aimed to identify a CSC-specific cell surface marker in human colorectal cancers (hCRCs) and validate the therapeutic effect of targeting it. IL17RB was distinctively expressed by Dclk1+ mouse intestinal tumor cells. Using Il17rb-CreERT2-IRES-EGFP mice, we show that IL17RB marked intestinal TSCs in an IL13-dependent manner. Tuft cell-like cancer cells were detected in a subset of hCRCs. In these hCRCs, lineage-tracing experiments in CRISPR-Cas9-mediated IL17RB-CreERT2 knockin organoids and xenograft tumors revealed that IL17RB marks CSCs that expand independently of IL-13. We observed up-regulation of POU2F3, a master regulator of tuft cell differentiation, and autonomous tuft cell-like cancer cell differentiation in the hCRCs. Furthermore, long-term ablation of IL17RB-expressing CSCs strongly suppressed the tumor growth in vivo. These findings reveal insights into a CSC-specific marker IL17RB in a subset of hCRCs, and preclinically validate IL17RB+ CSCs as a cancer therapeutic target.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Células-Tronco Neoplásicas/patologia , Receptores de Interleucina-17/metabolismo , Animais , Biomarcadores Tumorais/genética , Sistemas CRISPR-Cas/genética , Carcinogênese , Diferenciação Celular , Linhagem da Célula , Quinases Semelhantes a Duplacortina , Técnicas de Introdução de Genes , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Transgênicos , Fatores de Transcrição de Octâmero/metabolismo , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/metabolismo , Receptores de Interleucina-17/genética , Esferoides Celulares , Imagem com Lapso de Tempo , Células Tumorais Cultivadas , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Inactivating mutations of Arid1a, a subunit of the Switch/sucrose nonfermentable chromatin remodeling complex, have been reported in multiple human cancers. Intestinal deletion of Arid1a has been reported to induce colorectal cancer in mice; however, its functional role in intestinal homeostasis remains unclear. We investigated the functional role of Arid1a in intestinal homeostasis in mice. We found that intestinal deletion of Arid1a results in loss of intestinal stem cells (ISCs), decreased Paneth and goblet cells, disorganized crypt-villous structures, and increased apoptosis in adult mice. Spheroids did not develop from intestinal epithelial cells deficient for Arid1a Lineage-tracing experiments revealed that Arid1a deletion in Lgr5+ ISCs leads to impaired self-renewal of Lgr5+ ISCs but does not perturb intestinal homeostasis. The Wnt signaling pathway, including Wnt agonists, receptors, and target genes, was strikingly down-regulated in Arid1a-deficient intestines. We found that Arid1a directly binds to the Sox9 promoter to support its expression. Remarkably, overexpression of Sox9 in intestinal epithelial cells abrogated the above phenotypes, although Sox9 overexpression in intestinal epithelial cells did not restore the expression levels of Wnt agonist and receptor genes. Furthermore, Sox9 overexpression permitted development of spheroids from Arid1a-deficient intestinal epithelial cells. In addition, deletion of Arid1a concomitant with Sox9 overexpression in Lgr5+ ISCs restores self-renewal in Arid1a-deleted Lgr5+ ISCs. These results indicate that Arid1a is indispensable for the maintenance of ISCs and intestinal homeostasis in mice. Mechanistically, this is mainly mediated by Sox9. Our data provide insights into the molecular mechanisms underlying maintenance of ISCs and intestinal homeostasis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Mucosa Intestinal/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição SOX9/metabolismo , Células-Tronco/metabolismo , Animais , Células Epiteliais/metabolismo , Homeostase/fisiologia , Intestinos/fisiologia , Camundongos , Regiões Promotoras Genéticas/fisiologia , Fatores de Transcrição , Via de Sinalização Wnt/fisiologiaRESUMO
The circadian clock system exists in most organs and regulates diverse physiological processes, including growth. Here, we used a prostate-specific Bmal1-knockout mouse model (pBmal1 KO: PbsnCre+; Bmal1fx/fx) and immortalized human prostate cells (RWPE-1 and WPMY-1) to elucidate the role of the peripheral prostate clock on prostate growth. Bmal1 KO resulted in significantly decreased ventral and dorsolateral lobes with less Ki-67-positive epithelial cells than the controls. Next, the cap analysis of gene expression revealed that genes associated with cell cycles were differentially expressed in the pBmal1 KO prostate. Cdkn1a (coding p21) was diurnally expressed in the control mouse prostate, a rhythm which was disturbed in pBmal1 KO. Meanwhile, the knockdown of BMAL1 in epithelial RWPE-1 and stromal WPMY-1 cell lines decreased proliferation. Furthermore, RWPE-1 BMAL1 knockdown increased G0/G1-phase cell numbers but reduced S-phase numbers. These findings indicate that core clock gene Bmal1 is involved in prostate growth via the modulation of the cell cycle and provide a rationale for further research to link the pathogenesis of benign prostatic hyperplasia or cancer with the circadian clock.
Assuntos
Fatores de Transcrição ARNTL , Relógios Circadianos , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Proteínas CLOCK/genética , Ritmo Circadiano/fisiologia , Humanos , Antígeno Ki-67 , Masculino , Camundongos , Camundongos Knockout , Próstata/metabolismoRESUMO
Several studies have demonstrated a multiphasic role for Wnt signaling during embryonic cardiogenesis and developed protocols that enrich for cardiac derivatives during in vitro differentiation of human pluripotent stem cells (hPSCs). However, few studies have investigated the role of Wnt signaling in the specification of cardiac progenitor cells (CPCs) toward downstream fates. Using transgenic mice and hPSCs, we tracked endothelial cells (ECs) that originated from CPCs expressing NKX2.5. Analysis of EC-fated CPCs at discrete phenotypic milestones during hPSC differentiation identified reduced Wnt activity as a hallmark of EC specification, and the enforced activation or inhibition of Wnt reduced or increased, respectively, the degree of vascular commitment within the CPC population during both hPSC differentiation and mouse embryogenesis. Wnt5a, which has been shown to exert an inhibitory influence on Wnt signaling during cardiac development, was dynamically expressed during vascular commitment of hPSC-derived CPCs, and ectopic Wnt5a promoted vascular specification of hPSC-derived and mouse embryonic CPCs.
Assuntos
Embrião de Mamíferos/metabolismo , Células Endoteliais/metabolismo , Coração/embriologia , Células-Tronco Pluripotentes/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Embrião de Mamíferos/citologia , Células Endoteliais/citologia , Humanos , Camundongos , Camundongos Transgênicos , Células-Tronco Pluripotentes/citologia , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismoRESUMO
The organized array of smooth muscle cells (SMCs) and fibroblasts in the walls of visceral tubular organs arises by patterning and differentiation of mesenchymal progenitors surrounding the epithelial lumen. Here, we show that the TBX2 and TBX3 transcription factors have novel and required roles in regulating these processes in the murine ureter. Co-expression of TBX2 and TBX3 in the inner mesenchymal region of the developing ureter requires canonical WNT signaling. Loss of TBX2/TBX3 in this region disrupts activity of two crucial drivers of the SMC program, Foxf1 and BMP4 signaling, resulting in decreased SMC differentiation and increased extracellular matrix. Transcriptional profiling and chromatin immunoprecipitation experiments revealed that TBX2/TBX3 directly repress expression of the WNT antagonists Dkk2 and Shisa2, the BMP antagonist Bmper and the chemokine Cxcl12 These findings suggest that TBX2/TBX3 are effectors of canonical WNT signaling in the ureteric mesenchyme that promote SMC differentiation by maintaining BMP4 and WNT signaling in the inner region, while restricting CXCL12 signaling to the outer layer of fibroblast-fated mesenchyme.
Assuntos
Padronização Corporal , Diferenciação Celular , Mesoderma/embriologia , Proteínas com Domínio T/metabolismo , Ureter/embriologia , Via de Sinalização Wnt , Animais , Proteína Morfogenética Óssea 4/metabolismo , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Camundongos , Modelos Biológicos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Peristaltismo , Proteínas com Domínio T/genética , Transcriptoma/genética , Ureter/metabolismo , Ureter/patologiaRESUMO
Cancer cells alter their metabolism for the production of precursors of macromolecules. However, the control mechanisms underlying this reprogramming are poorly understood. Here we show that metabolic reprogramming of colorectal cancer is caused chiefly by aberrant MYC expression. Multiomics-based analyses of paired normal and tumor tissues from 275 patients with colorectal cancer revealed that metabolic alterations occur at the adenoma stage of carcinogenesis, in a manner not associated with specific gene mutations involved in colorectal carcinogenesis. MYC expression induced at least 215 metabolic reactions by changing the expression levels of 121 metabolic genes and 39 transporter genes. Further, MYC negatively regulated the expression of genes involved in mitochondrial biogenesis and maintenance but positively regulated genes involved in DNA and histone methylation. Knockdown of MYC in colorectal cancer cells reset the altered metabolism and suppressed cell growth. Moreover, inhibition of MYC target pyrimidine synthesis genes such as CAD, UMPS, and CTPS blocked cell growth, and thus are potential targets for colorectal cancer therapy.