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PURPOSE: The presence of tumor-infiltrating lymphocytes (TILs) in tumors is superior to conventional pathologic staging in predicting patient outcome. However, their presence does not define TIL functionality. Here we developed an assay that tests TIL cytotoxicity in patients with locally advanced rectal cancer before definitive treatment, identifying those who will obtain a pathologic complete response (pCR). We also used the assay to demonstrate the rescue of TIL function after checkpoint inhibition blockade (CIB). PATIENTS AND METHODS: Thirty-four consecutive patients were identified initially, with successful completion of the assay before surgery in those 17 patients who underwent full treatment. An in vitro cytotoxic assay of rectal cancer tumoroids cocultured with patient-matched TILs was established and validated. Newly diagnosed patients were recruited with pretreatment biopsy specimens processed within 1 month. Evaluation of TIL-mediated tumoroid lysis was performed by measuring the mean fluorescence intensity of cell death marker, propidium iodide. CIB (anti-programmed cell death protein 1 [anti-PD-1] antibody) response was also assessed in a subset of patient specimens. RESULTS: Six of the 17 patients achieved an objective pCR on final evaluation of the resected specimen after neoadjuvant chemoradiotherapy. Cytotoxic killing identified the pCR group with a higher mean fluorescence intensity (27,982 [95% CI, 25,340 to 30,625]) compared with the non-pCR cohort (12,428 [95% CI, 9,434 to 15,423]; p < .001). Assessment of the effectiveness of CIB revealed partial restoration of cytotoxicity in TILs with increased PD-1 expression with anti-PD-1 antibody exposure. CONCLUSION: Evaluating TIL function can be undertaken within weeks of the diagnostic biopsy, affording the potential to alter patient management decisions and refine selection for a watch-and-wait protocol. This cytotoxic assay also has the potential to serve as a platform to assist in the additional development of CIB.
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The presence of tumor immune infiltrating cells (TILs), particularly CD8(+) T-cells, is a robust predictor of outcome in patients with colorectal cancer (CRC). We revisited TIL abundance specifically in patients with microsatellite stable (MSS) CRC without evidence of lymph node or metastatic spread. Examination of the density of CD8(+) T-cells in primary tumors in the context of other pro-oncogenic markers was performed to investigate potential regulators of TILs. Two independent cohorts of patients with MSS T2-4N0M0 CRC, enriched for cases with atypical relapse, were investigated. We quantified CD8(+) and CD45RO(+) -TILs, inflammatory markers, NFkBp65, pStat3, Cyclo-oxygenase-2 (COX2) and GRP78 as well as transcription factors (TF), ß-catenin and MYB. High CD8(+) TILs correlated with a better relapse-free survival in both cohorts (p = 0.002) with MYB and its target gene, GRP78 being higher in the relapse group (p = 0.001); no difference in pSTAT3 and p65 was observed. A mouse CRC (CT26) model was employed to evaluate the effect of MYB on GRP78 expression as well as T-cell infiltration. MYB over-expressing in CT26 cells increased GRP78 expression and the analysis of tumor-draining lymph nodes adjacent to tumors showed reduced T-cell activation. Furthermore, MYB over-expression reduced the efficacy of anti-PD-1 to modulate CT26 tumor growth. This high MYB and GRP78 show a reciprocal relationship with CD8(+) TILs which may be useful refining the prediction of patient outcome. These data reveal a new immunomodulatory function for MYB suggesting a basis for further development of anti-GRP78 and/or anti-MYB therapies.
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UNLABELLED: Cyclin E1 is essential for the reentry of quiescent cells into the cell cycle. When hypomorphic mutant Myb mice (Myb(Plt4)) were examined, it was noted that Cyclin E1 (Ccne1) expression was reduced. Furthermore, the induction of Ccne1 in recovering intestinal epithelia following radiation-induced damage was ablated in Myb-mutant mice. These data prompted us to investigate whether Myb directly regulated Ccne1 and to examine whether elevated Myb in colorectal cancer is responsible for Cyclin E1-driven tumor growth. Here, it was found that Myb/MYB and Ccne1/CCNE1 expressions were coupled in both mouse and human adenomas. In addition, the low molecular weight Cyclin E1 was the predominant form in intestinal crypts and adenomatous polyposis coli (Apc)-mutant adenomas. Chromatin immunoprecipitation (ChIP) analysis confirmed that Myb bound directly to the Ccne1 promoter and regulated its endogenous expression. In contrast, Myb(Plt4) served as a dominant-negative factor that inhibited wild-type Myb and this was not apparently compensated for by the transcription factor E2F1 in intestinal epithelial cells. Myb(Plt4/Plt4) mice died prematurely on an Apc(Min/) (+) background associated with hematopoietic defects, including a myelodysplasia; nevertheless, Apc(Min/) (+) mice were protected from intestinal tumorigenesis when crossed to Myb(Plt4/) (+) mice. Knockdown of CCNE1 transcript in murine colorectal cancer cells stabilized chromosome ploidy and decreased tumor formation. These data suggest that Cyclin E1 expression is Myb dependent in normal and transformed intestinal epithelial cells, consistent with a cell-cycle progression and chromosome instability role in cancer. IMPLICATIONS: This study demonstrates that Myb regulates Cyclin E1 expression in normal gastrointestinal tract epithelial cells and is required during intestinal tumorigenesis.
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Carcinogénesis/metabolismo , Neoplasias Colorrectales/metabolismo , Ciclina E/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas Oncogénicas v-myb/genética , Proteínas Oncogénicas/metabolismo , Adenoma/metabolismo , Animales , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Inmunoprecipitación de Cromatina , Inestabilidad Cromosómica , Cromosomas/ultraestructura , Progresión de la Enfermedad , Femenino , Hematopoyesis , Humanos , Inmunoprecipitación , Mucosa Intestinal/metabolismo , Neoplasias Intestinales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Ploidias , Regiones Promotoras GenéticasRESUMEN
The mammalian adult small intestinal epithelium is a rapidly self-renewing tissue that is maintained by a pool of cycling stem cells intermingled with Paneth cells at the base of crypts. These crypt base stem cells exclusively express Lgr5 and require Wnt3 or, in its absence, Wnt2b. However, the Frizzled (Fzd) receptor that transmits these Wnt signals is unknown. We determined the expression profile of Fzd receptors in Lgr5(+) stem cells, their immediate daughter cells, and Paneth cells. Here we show Fzd7 is enriched in Lgr5(+) stem cells and binds Wnt3 and Wnt2b. Conditional deletion of the Fzd7 gene in adult intestinal epithelium leads to stem cell loss in vivo and organoid death in vitro. Crypts of conventional Fzd7 knockout mice show decreased basal Wnt signaling and impaired capacity to regenerate the epithelium following deleterious insult. These observations indicate that Fzd7 is required for robust Wnt-dependent processes in Lgr5(+) intestinal stem cells.
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Receptores Acoplados a Proteínas G/metabolismo , Células Madre/metabolismo , Animales , Células Cultivadas , Receptores Frizzled/metabolismo , Inmunohistoquímica , Inmunoprecipitación , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células de Paneth/citología , Receptores Acoplados a Proteínas G/genética , Células Madre/citología , Vía de Señalización Wnt , Proteína Wnt3/metabolismoRESUMEN
MYB transcriptional elongation is regulated by an attenuator sequence within intron 1 that has been proposed to encode a RNA stem loop (SLR) followed by a polyU tract. We report that NFκBp50 can bind the SLR polyU RNA and promote MYB transcriptional elongation together with NFκBp65. We identified a conserved lysine-rich motif within the Rel homology domain (RHD) of NFκBp50, mutation of which abrogated the interaction of NFκBp50 with the SLR polyU and impaired NFκBp50 mediated MYB elongation. We observed that the TAR RNA-binding region of Tat is homologous to the NFκBp50 RHD lysine-rich motif, a finding consistent with HIV Tat acting as an effector of MYB transcriptional elongation in an SLR dependent manner. Furthermore, we identify the DNA binding activity of NFκBp50 as a key component required for the SLR polyU mediated regulation of MYB. Collectively these results suggest that the MYB SLR polyU provides a platform for proteins to regulate MYB and reveals novel nucleic acid binding properties of NFκBp50 required for MYB regulation.
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Proteínas de Unión al ADN/genética , Genes myb/genética , VIH-1/genética , Subunidad p50 de NF-kappa B/genética , Proteínas de Unión al ADN/metabolismo , VIH-1/patogenicidad , Humanos , Intrones/genética , Secuencias Invertidas Repetidas/genética , Mutación , Subunidad p50 de NF-kappa B/metabolismo , ARN Viral/genéticaRESUMEN
Skin integrity requires an ongoing replacement and repair orchestrated by several cell types. We previously investigated the architecture of the skin of avian myeloblastosis viral oncogene homolog (Myb) knock-out (KO) embryos and wound repair in Myb(+/)(-) mice revealing a need for Myb in the skin, attributed to fibroblast-dependent production of collagen type 1. Here, using targeted Myb deletion in keratin-14 (K14) positive cells we reveal further Myb-specific defects in epidermal cell proliferation, thickness and ultrastructural morphology. This was associated with a severe deficit in collagen type 1 production, reminiscent of that observed in patients with ichthyosis vulgaris and Ehlers-Danlos syndrome. Since collagen type 1 is a product of fibroblasts, the collagen defect observed was unexpected and appears to be directed by the loss of Myb with significantly reduced tumor growth factor beta 1 (Tgfß-1) expression by primary keratinocytes. Our findings support a specific role for Myb in K14+ epithelial cells in the preservation of adult skin integrity and function.
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Colágeno Tipo I/metabolismo , Proteínas Proto-Oncogénicas c-myb/fisiología , Piel/inmunología , Factor de Crecimiento Transformador beta1/fisiología , Animales , Proliferación Celular , Exones , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Eliminación de Gen , Queratina-14/genética , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Rastreo , Piel/metabolismo , TransgenesRESUMEN
BACKGROUND: Conventional laparoscopic surgery uses CO2 that is dry and cold, which can damage peritoneal surfaces. It is speculated that disseminated cancer cells may adhere to such damaged peritoneum and metastasize. We hypothesized that insufflation using humidified-warm CO2, which has been shown to reduce mesothelial damage, will also ameliorate peritoneal inflammation and tumor cell implantation compared to conventional dry-cold CO2. METHODS: Laparoscopic insufflation was modeled in mice along with anesthesia and ventilation. Entry and exit ports were introduced to maintain insufflation using dry-cold or humidified-warm CO2 with a constant flow and pressure for 1 h; then 1000 or 1 million fluorescent-tagged murine colorectal cancer cells (CT26) were delivered into the peritoneal cavity. The peritoneum was collected at intervals up to 10 days after the procedure to measure inflammation, mesothelial damage, and tumor burden using fluorescent detection, immunohistochemistry, and scanning electron microscopy. RESULTS: Rapid temperature control was achieved only in the humidified-warm group. Port-site tumors were present in all mice. At 10 days, significantly fewer tumors on the peritoneum were counted in mice insufflated with humidified-warm compared to dry-cold CO2 (p < 0.03). The inflammatory marker COX-2 was significantly increased in the dry-cold compared to the humidified-warm cohort (p < 0.01), while VEGFA expression was suppressed only in the humidified-warm cohort. Significantly less mesothelial damage and tumor cell implantation was evident from 2 h after the procedure in the humidified-warm cohort. CONCLUSIONS: Mesothelial cell damage and inflammation are reduced by using humidified-warm CO2 for laparoscopic oncologic surgery and may translate to reduce patients' risk of developing peritoneal metastasis.
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Dióxido de Carbono/farmacología , Transformación Celular Neoplásica/efectos de los fármacos , Calor , Inflamación/prevención & control , Insuflación/métodos , Neoplasias Peritoneales/prevención & control , Peritoneo/efectos de los fármacos , Animales , Dióxido de Carbono/administración & dosificación , Transformación Celular Neoplásica/patología , Femenino , Humedad , Inflamación/fisiopatología , Ratones , Ratones Endogámicos BALB C , Neoplasias Peritoneales/fisiopatología , Peritoneo/lesiones , Peritoneo/patología , Células Tumorales CultivadasRESUMEN
Cancers can be addicted to continued and relatively high expression of nuclear oncoproteins. This is evident in colorectal cancer (CRC) where the oncoprotein and transcription factor MYB is over expressed and essential to continued proliferation and tumour cell survival. Historically, targeting transcription factors in the context of cancer has been very challenging. Nevertheless, we formulated a DNA vaccine to generate a MYB-specific immune response in the belief MYB peptides might be aberrantly presented on the cell surface of CRC cells. MYB, like many tumour antigens, is weakly immunogenic as it is a 'self' antigen and is subject to tolerance. To break tolerance, a fusion vaccine was generated comprising a full-length MYB complementary DNA (cDNA) flanked by two potent CD4-epitopes derived from tetanus toxoid. Vaccination was achieved against tumours initiated by two distinct highly aggressive, syngeneic cancer cell lines (CT26 and MC38) that express MYB. This was done in BALB/c and C57BL/6 mouse strains respectively. We introduced multiple inactivating mutations into the oncogene sequence for safety and sub-cloned the cDNA into a Food and Drug Administration (FDA)-compliant vector. We used low dose cyclophosphamide (CY) to overcome T-regulatory cell immune suppression, and anti-program cell death receptor 1 (anti-PD-1) antibodies to block T-cell exhaustion. Anti-PD-1 administered alone slightly delayed tumour growth in MC38 and more effectively in CT26 bearing mice, while CY treatment alone did not. We found that therapeutic vaccination elicits protection when MC38 tumour burden is low, mounts tumour-specific cell killing and affords enhanced protection when MC38 and CT26 tumour burden is higher but only in combination with anti-PD-1 antibody or low dose CY, respectively.
RESUMEN
Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K, are among the most common mutations found in human cancer and have also recently been implicated in a range of overgrowth syndromes in humans. We have used a novel inducible "exon-switch" approach to knock in the constitutively active Pik3ca(H1047R) mutation into the endogenous Pik3ca gene of the mouse. Ubiquitous expression of the Pik3ca(H1047R) mutation throughout the body resulted in a dramatic increase in body weight within 3 weeks of induction (mutant 150 ± 5%; wild-type 117 ± 3%, mean ± sem), which was associated with increased organ size rather than adiposity. Severe metabolic effects, including a reduction in blood glucose levels to 59 ± 4% of baseline (11 days postinduction) and undetectable insulin levels, were also observed. Pik3ca(H1047R) mutant mice died earlier (median survival 46.5 d post-mutation induction) than wild-type control mice (100% survival > 250 days). Although deletion of Akt2 increased median survival by 44%, neither organ overgrowth, nor hypoglycemia were rescued, indicating that both the growth and metabolic functions of constitutive PI3K activity can be Akt2 independent. This mouse model demonstrates the critical role of PI3K in the regulation of both organ size and glucose metabolism at the whole animal level.
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Hipoglucemia/enzimología , Hipoglucemia/genética , Insulina/sangre , Mutación , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Sustitución de Aminoácidos , Animales , Fosfatidilinositol 3-Quinasa Clase I , Femenino , Expresión Génica , Técnicas de Sustitución del Gen , Glucosa/metabolismo , Humanos , Hipoglucemia/metabolismo , Ratones , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Tamaño de los Órganos/genética , Tamaño de los Órganos/fisiología , Proteínas Proto-Oncogénicas c-akt/deficiencia , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Aumento de PesoRESUMEN
Loss of heterozygosity (LOH) of the adenomatous polyposis coli (APC) gene triggers a series of molecular events leading to intestinal adenomagenesis. Haploinsufficiency of the cohesin Rad21 influences multiple initiating events in colorectal cancer (CRC). We identify Rad21 as a gatekeeper of LOH and a ß-catenin target gene and provide evidence that Wnt pathway activation drives RAD21 expression in human CRC. Genome-wide analyses identified Rad21 as a key transcriptional regulator of critical CRC genes and long interspersed element (LINE-1 or L1) retrotransposons. Elevated RAD21 expression tracks with reactivation of L1 expression in human sporadic CRC, implicating cohesin-mediated L1 expression in global genomic instability and gene dysregulation in cancer.
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Poliposis Adenomatosa del Colon/genética , Haploinsuficiencia , Proteínas Nucleares/fisiología , Fosfoproteínas/fisiología , Poliposis Adenomatosa del Colon/metabolismo , Células Madre Adultas/fisiología , Animales , Proteínas de Ciclo Celular , Proliferación Celular , Inestabilidad Cromosómica , Colon/patología , Daño del ADN , Proteínas de Unión al ADN , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Ratones , Ratones Transgénicos , Retroelementos/genética , Regulación hacia ArribaRESUMEN
Most colon cancers arise from somatic mutations in the tumor suppressor gene APC (adenomatous polyposis coli), and these mutations cause constitutive activation of the Wnt-to-ß-catenin pathway in the intestinal epithelium. Because Wnt-ß-catenin signaling is required for homeostasis and regeneration of the adult intestinal epithelium, therapeutic targeting of this pathway is challenging. We found that genetic activation of the cytokine-stimulated pathway mediated by the receptor gp130, the associated Jak (Janus kinase) kinases, and the transcription factor Stat3 (signal transducer and activator of transcription 3) was required for intestinal regeneration in response to irradiation-induced damage in wild-type mice and for tumorigenesis in Apc-mutant mice. Systemic pharmacological or partial genetic inhibition of gp130-Jak-Stat3 signaling suppressed intestinal regeneration, the growth of tumors in Apc-mutant mice, and the growth of colon cancer xenografts. The growth of Apc-mutant tumors depended on gp130-Jak-Stat3 signaling for induction of the polycomb repressor Bmi-1, and the associated repression of genes encoding the cell cycle inhibitors p16 and p21. However, suppression of gp130-Jak-Stat3 signaling did not affect Wnt-ß-catenin signaling or homeostasis in the intestine. Thus, these data not only suggest a molecular mechanism for how the gp130-Jak-Stat3 pathway can promote cancer but also provide a rationale for therapeutic inhibition of Jak in colon cancer.
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Neoplasias del Colon/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Genes APC/fisiología , Mucosa Intestinal/fisiología , Regeneración/fisiología , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismo , Animales , Neoplasias del Colon/genética , Receptor gp130 de Citocinas/metabolismo , Cartilla de ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Técnicas Histológicas , Inmunohistoquímica , Janus Quinasa 1/metabolismo , Luciferasas , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Complejo Represivo Polycomb 1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/metabolismo , Vía de Señalización Wnt/genéticaRESUMEN
Deletion studies confirm Wnt, Notch and Myb transcriptional pathway engagement in intestinal tumorigenesis. Nevertheless, their contrasting and combined roles when activated have not been elucidated. This is important as these pathways are not ablated but rather are aberrantly activated during carcinogenesis. Using ApcMin/+ mice as a source of organoids we documented their transition, on a clone-by-clone basis, to cyst-like spheres with constitutively activated Wnt pathway, increased self-renewal and growth and reduced differentiation. We then looked at this transition when Myb and/or Notch1 are activated. Activated Notch promoted cyst-like organoids. Conversely growth and propagation of cyst-like, but not normal organoids were Notch-independent. Activated Myb promoted normal, but not cyst-like organoids. Interestingly the Wnt, Notch and Myb pathways were all involved in regulating the expression of the intestinal stem cell (ISC) gene Lgr5 in organoids, while ISC gene and Notch target Olfm4 was dominantly repressed by Wnt. These findings parallel mouse intestinal adenoma formation where Notch promoted the initiation, but not growth, of Wnt-driven Olfm4-repressed colon tumors. Also Myb was essential for colon tumor initiation and collateral mouse pathologies. These data reveal the complex interplay and hierarchy of transcriptional networks that operate in ISCs and uncover a shift in pathway-dependencies during tumor initiation.
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Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-myb/metabolismo , Receptores Notch/metabolismo , Células Madre/metabolismo , Proteínas Wnt/metabolismo , Adenoma/metabolismo , Adenoma/mortalidad , Adenoma/patología , Animales , Carcinogénesis , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Mucosa Intestinal/metabolismo , Intestinos/citología , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Organoides/citología , Organoides/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Notch/genética , Transducción de Señal , Células Madre/citologíaRESUMEN
Adult stem cells reside in most parts of the body where high tissue turn-over is evident. However there are vastly different demands on the number of cells that might be produced and no better examples of each extreme are the neurogenic zones of the brain, and the crypt compartments of the intestines. From a perspective of understanding the function of the transcription factor Myb, we have explored the biology of stem cell niches in both these radically different tissues. Each tissue has remarkable features, provide different in vivo and in vitro options for manipulation and open up novel insights into damage responses and diseases like cancer. A variety of studies using mouse models, conditional and hypomorphic Myb mutants, radiation induced damage and primary in vitro assays have advanced our understanding of both stem cell niches and has revealed a previously unrecognised role for Myb in the regulation of stem cells.
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Células Madre Adultas/metabolismo , Encéfalo/metabolismo , Epigénesis Genética , Mucosa Intestinal/metabolismo , Proteínas Proto-Oncogénicas c-myb/genética , Nicho de Células Madre/genética , Células Madre Adultas/citología , Animales , Encéfalo/citología , Ciclo Celular , Regulación del Desarrollo de la Expresión Génica , Intestinos/citología , Ratones , Ratones Transgénicos , MicroARNs/genética , MicroARNs/metabolismo , Especificidad de Órganos , Proteínas Proto-Oncogénicas c-myb/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
The colony stimulating factor-1 (CSF-1) receptor (CSF-1R) directly regulates the development of Paneth cells (PC) and influences proliferation and cell fate in the small intestine (SI). In the present study, we have examined the role of CSF-1 and the CSF-1R in the large intestine, which lacks PC, in the steady state and in response to acute inflammation induced by dextran sulfate sodium (DSS). As previously shown in mouse, immunohistochemical (IHC) analysis of CSF-1R expression showed that the receptor is baso-laterally expressed on epithelial cells of human colonic crypts, indicating that this expression pattern is shared between species. Colons from Csf1r null and Csf1(op/op) mice were isolated and sectioned for IHC identification of enterocytes, enteroendocrine cells, goblet cells and proliferating cells. Both Csf1r(-/-) and Csf1(op/op) mice were found to have colon defects in enterocytes and enteroendocrine cell fate, with excessive goblet cell staining and reduced cell proliferation. In addition, the gene expression profiles of the cell cycle genes, cyclinD1, c-myc, c-fos, and c-myb were suppressed in Csf1r(-/-) colonic crypt, compared with those of WT mice and the expression of the stem cell marker gene Lgr5 was markedly reduced. However, analysis of the proliferative responses of immortalized mouse colon epithelial cells (lines; Immorto-5 and YAMC) indicated that CSF-1R is not a major regulator of colonocyte proliferation and that its effects on proliferation are indirect. In an examination of the acute inflammatory response, Csf1r(+/-) male mice were protected from the adverse affects of DSS-induced colitis compared with WT mice, while Csf1r(+/-) female mice were significantly less protected. These data indicate that CSF-1R signaling plays an important role in colon homeostasis and stem cell gene expression but that the receptor exacerbates the response to inflammatory challenge in male mice.
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Colon/inmunología , Colon/metabolismo , Inflamación/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Animales , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Homeostasis/genética , Homeostasis/inmunología , Humanos , Inmunohistoquímica , Técnicas In Vitro , Inflamación/genética , Masculino , Ratones , Ratones Mutantes , Receptor de Factor Estimulante de Colonias de Macrófagos/genéticaRESUMEN
Gastrointestinal (GI) homeostasis requires the action of multiple pathways. There is some controversy regarding whether small intestine (SI) Paneth cells (PCs) play a central role in orchestrating crypt architecture and their relationship with Lgr5+ve stem cells. Nevertheless, we previously showed that germline CSF-1 receptor (Csf1r) knock out (KO) or Csf1 mutation is associated with an absence of mature PC, reduced crypt proliferation and lowered stem cell gene, Lgr5 expression. Here we show the additional loss of CD24, Bmi1 and Olfm4 expression in the KO crypts and a high resolution 3D localization of CSF-1R mainly to PC. The induction of GI-specific Csf1r deletion in young adult mice also led to PC loss over a period of weeks, in accord with the anticipated long life span of PC, changed distribution of proliferating cells and this was with a commensurate loss of Lgr5 and other stem cell marker gene expression. By culturing SI organoids, we further show that the Csf1r(-/-) defect in PC production is intrinsic to epithelial cells as well as definitively affecting stem cell activity. These results show that CSF-1R directly supports PC maturation and that in turn PCs fashion the intestinal stem cell niche.
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Intestino Delgado/citología , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Nicho de Células Madre , Células Madre/citología , Células Madre/metabolismo , Animales , Antígeno CD24/metabolismo , Diferenciación Celular/genética , Proliferación Celular , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos/genética , Organoides/citología , Organoides/metabolismo , Células de Paneth/citología , Células de Paneth/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/deficiencia , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptores Notch/metabolismo , Nicho de Células Madre/genética , Proteínas Wnt/metabolismoRESUMEN
Radiation-induced brain injury occurs in many patients receiving cranial radiation therapy, and these deleterious effects are most profound in younger patients. Impaired neurocognitive functions in both humans and rodents are associated with inflammation, demyelination, and neural stem cell dysfunction. Here we evaluated the utility of lithium and a synthetic retinoid receptor agonist in reducing damage in a model of brain-focused irradiation in juvenile mice. We found that lithium stimulated brain progenitor cell proliferation and differentiation following cranial irradiation while also preventing oligodendrocyte loss in the dentate gyrus of juvenile mice. In response to inflammation induced by radiation, which may have encumbered the optimal reparative action of lithium, we used the anti-inflammatory synthetic retinoid Am80 that is in clinical use in the treatment of acute promyelocytic leukemia. Although Am80 reduced the number of cyclooxygenase-2-positive microglial cells following radiation treatment, it did not enhance lithium-induced neurogenesis recovery, and this alone was not significantly different from the effect of lithium on this proinflammatory response. Similarly, lithium was superior to Am80 in supporting the restoration of new doublecortin-positive neurons following irradiation. These data suggest that lithium is superior in its restorative effects to blocking inflammation alone, at least in the case of Am80. Because lithium has been in routine clinical practice for 60 years, these preclinical studies indicate that this drug might be beneficial in reducing post-therapy late effects in patients receiving cranial radiotherapy and that blocking inflammation in this context may not be as advantageous as previously suggested.
Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/efectos de la radiación , Irradiación Craneana/efectos adversos , Inflamación/patología , Litio/farmacología , Animales , Conducta Animal/efectos de los fármacos , Benzoatos/farmacología , Encéfalo/patología , Diferenciación Celular , Proliferación Celular , Trastornos del Conocimiento/patología , Ciclina D1/metabolismo , Ciclooxigenasa 2/metabolismo , Giro Dentado/patología , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neurogénesis , Neuronas/citología , Neuronas/metabolismo , Oligodendroglía/patología , Traumatismos Experimentales por Radiación/tratamiento farmacológico , Traumatismos Experimentales por Radiación/patología , Células Madre/citología , Células Madre/metabolismo , Tetrahidronaftalenos/farmacologíaRESUMEN
Rapid advances have been made in the understanding of how the highly proliferative gastrointestinal tract epithelium is regulated under homeostasis and disease. The identification of putative intestinal stem cell (ISC) genes and the ability to culture ISC capable of generating all four lineages plus the architecture of small intestinal (SI) crypts has been transformative. Here, we show that transcription factor Myb governs ISC gene expression, particularly Lgr5. Lgr5 is associated with cells that have the capacity to generate all cell lineages in SI organoid cultures and colorectal cancer cells, which overexpress Myb. Furthermore, Wnt signaling and Myb cooperate in maximal Lgr5 promoter activation while hypomorphic Myb (plt4/plt4) mice have decreased Lgr5 expression. After ionizing radiation (IR), ISC genes are elevated; but in plt4/plt4 mice, this response is substantially subdued. ISC genes bmi-1 and olfm4 are expressed at subnormal levels in plt4/plt4 mice, and bmi-1 is induced with IR to half the level in mutant mice. dcamkl-1 and olfm4 failed to recover after IR in both wild-type (wt) and mutant mice. Although not considered as an ISC gene, cyclinE1 is nevertheless used to assist cells in the emergence from a quiescent state (an expectation of ISC following IR) and is overexpressed after IR in wt mice but does not change from a very low base in plt4/plt4 mice. Self-renewal assays using organoid cultures and inducible Myb knockout studies further highlighted the dependence of ISC on Myb consistent with role in other stem cell-containing tissues.
Asunto(s)
Genes myb , Yeyuno/citología , Proteínas Proto-Oncogénicas c-myb/metabolismo , Células Madre/metabolismo , Animales , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Ciclina E/genética , Ciclina E/metabolismo , Rayos gamma , Regulación Neoplásica de la Expresión Génica , Humanos , Yeyuno/metabolismo , Yeyuno/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIH , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myb/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células Madre/citología , Activación Transcripcional , Vía de Señalización WntRESUMEN
MYB oncogene upregulation is associated with estrogen receptor (ER)-positive breast cancer, but disease requirements for MYB function in vivo have not been explored. In this study, we provide evidence of a critical requirement for MYB functions in models of human and murine breast cancer. In human breast cancer, we found that MYB expression was critical for tumor cell growth both in vitro and in vivo in xenograft settings. In transgenic knockout mice, tissue-specific deletion of the murine MYB gene caused a transient defect in mammary gland development that was reflected in delayed ductal branching and defective apical bud formation. In mouse mammary tumor virus (MMTV)-NEU mice where tumors are initiated by activation of HER2, MYB deletion was sufficient to abolish tumor formation. In the more aggressive MMTV-PyMT model system, MYB deletion delayed tumorigenesis significantly. Together, the findings in these transgenic knockout models implied that MYB was critical during an early window in mammary development when it was essential for tumor initiation, even though MYB loss did not exert a lasting impact upon normal mammary function. Two important MYB-target genes that promote cell survival, BCL2 and GRP78/BIP, were each elevated compared with nontransformed mammary epithelial cells, thereby promoting survival as confirmed in colony formation assays in vitro. Taken together, our findings establish a role for MYB at the hub of ER- and HER2-dependent pathways in mammary carcinogenesis.
Asunto(s)
Neoplasias Mamarias Experimentales/etiología , Proteínas Oncogénicas v-myb/fisiología , Animales , Antígenos Virales de Tumores/genética , Línea Celular Tumoral , Chaperón BiP del Retículo Endoplásmico , Receptor alfa de Estrógeno/análisis , Etilnitrosourea , Femenino , Neoplasias Mamarias Experimentales/química , Virus del Tumor Mamario del Ratón/genética , Ratones , Receptor ErbB-2/análisisRESUMEN
Studies employing mouse models have identified crypt base and position +4 cells as strong candidates for intestinal epithelial stem cells. Equivalent cell populations are thought to exist in the human intestine; however robust and specific protein markers are lacking. Here, we show that in the human small and large intestine, PHLDA1 is expressed in discrete crypt base and some position +4 cells. In small adenomas, PHLDA1 was expressed in a subset of undifferentiated and predominantly Ki-67-negative neoplastic cells, suggesting that a basic hierarchy of differentiation is retained in early tumorigenesis. In large adenomas, carcinomas, and metastases PHLDA1 expression became widespread, with increased expression and nuclear localization at invasive margins. siRNA-mediated suppression of PHLDA1 in colon cancer cells inhibited migration and anchorage-independent growth in vitro and tumor growth in vivo. The integrins ITGA2 and ITGA6 were downregulated in response to PHLDA1 suppression, and accordingly cell adhesion to laminin and collagen was significantly reduced. We conclude that PHLDA1 is a putative epithelial stem cell marker in the human small and large intestine and contributes to migration and proliferation in colon cancer cells.
Asunto(s)
Células Epiteliales/citología , Regulación Neoplásica de la Expresión Génica , Mucosa Intestinal/metabolismo , Células Madre/metabolismo , Factores de Transcripción/genética , Animales , Diferenciación Celular , Línea Celular Tumoral , Movimiento Celular , Neoplasias del Colon/metabolismo , Células HCT116 , Humanos , Integrina alfa2/metabolismo , Integrina alfa6/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Células Madre/citologíaRESUMEN
Adoptive immunotherapy involving genetic modification of T cells with antigen-specific, chimeric, single-chain receptors is a promising approach for the treatment of cancer. To determine whether gene-modified T cells could induce antitumor effects without associated autoimmune pathology, we assessed the ability of T cells expressing an anti-Her-2 chimeric receptor to eradicate tumor in Her-2 transgenic mice that express human Her-2 as a self-antigen in brain and mammary tissues. In adoptive transfer studies, we demonstrated significant improvement in the survival of mice bearing Her-2(+) 24JK tumor following administration of anti-Her-2 T cells compared with control T cells. The incorporation of a lymphoablative step prior to adoptive transfer of anti-Her-2 T cells and administration of IL-2 were both found to further enhance survival. The reduction in tumor growth was also correlated with localization of transferred T cells at the tumor site. Furthermore, an antigen-specific recall response could be induced in long-term surviving mice following rechallenge with Her-2(+) tumor. Importantly, antitumor effects were not associated with any autoimmune pathology in normal tissue expressing Her-2 antigen. This study highlights the therapeutic potential of using gene-engineered T cells as a safe and effective treatment of cancer.