RESUMO
How cancer-associated chromatin abnormalities shape tumor-immune interaction remains incompletely understood. Recent studies have linked DNA hypomethylation and de-repression of retrotransposons to anti-tumor immunity through the induction of interferon response. Here, we report that inactivation of the histone H3K36 methyltransferase NSD1, which is frequently found in squamous cell carcinomas (SCCs) and induces DNA hypomethylation, unexpectedly results in diminished tumor immune infiltration. In syngeneic and genetically engineered mouse models of head and neck SCCs, NSD1-deficient tumors exhibit immune exclusion and reduced interferon response despite high retrotransposon expression. Mechanistically, NSD1 loss results in silencing of innate immunity genes, including the type III interferon receptor IFNLR1, through depletion of H3K36 di-methylation (H3K36me2) and gain of H3K27 tri-methylation (H3K27me3). Inhibition of EZH2 restores immune infiltration and impairs the growth of Nsd1-mutant tumors. Thus, our work uncovers a druggable chromatin cross talk that regulates the viral mimicry response and enables immune evasion of DNA hypomethylated tumors.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Histona Metiltransferases , Evasão Tumoral , Animais , Camundongos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Cromatina , Metilação de DNA , Neoplasias de Cabeça e Pescoço/genética , Histona Metiltransferases/genética , Histona Metiltransferases/metabolismo , Histonas/genética , Histonas/metabolismo , Interferons/genética , Proteínas Nucleares/metabolismo , Receptores de Interferon/genética , Retroelementos , Evasão Tumoral/genéticaRESUMO
Renal agenesis and hypodysplasia (RHD) are major causes of pediatric chronic kidney disease and are highly genetically heterogeneous. We conducted whole-exome sequencing in 202 case subjects with RHD and identified diagnostic mutations in genes known to be associated with RHD in 7/202 case subjects. In an additional affected individual with RHD and a congenital heart defect, we found a homozygous loss-of-function (LOF) variant in SLIT3, recapitulating phenotypes reported with Slit3 inactivation in the mouse. To identify genes associated with RHD, we performed an exome-wide association study with 195 unresolved case subjects and 6,905 control subjects. The top signal resided in GREB1L, a gene implicated previously in Hoxb1 and Shha signaling in zebrafish. The significance of the association, which was p = 2.0 × 10-5 for novel LOF, increased to p = 4.1 × 10-6 for LOF and deleterious missense variants combined, and augmented further after accounting for segregation and de novo inheritance of rare variants (joint p = 2.3 × 10-7). Finally, CRISPR/Cas9 disruption or knockdown of greb1l in zebrafish caused specific pronephric defects, which were rescued by wild-type human GREB1L mRNA, but not mRNA containing alleles identified in case subjects. Together, our study provides insight into the genetic landscape of kidney malformations in humans, presents multiple candidates, and identifies SLIT3 and GREB1L as genes implicated in the pathogenesis of RHD.
Assuntos
Anormalidades Congênitas/genética , Exoma/genética , Nefropatias/congênito , Rim/anormalidades , Mutação/genética , Proteínas de Neoplasias/genética , Alelos , Animais , Estudos de Casos e Controles , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Feminino , Heterogeneidade Genética , Estudo de Associação Genômica Ampla/métodos , Genótipo , Hereditariedade/genética , Homozigoto , Humanos , Nefropatias/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Fenótipo , RNA Longo não Codificante/genética , Sistema Urinário/anormalidades , Anormalidades Urogenitais/genética , Peixe-ZebraRESUMO
Muscle invasive bladder cancers (BCs) can be divided into 2 major subgroups-basal/squamous (BASQ) tumors and luminal tumors. Since Pparg has low or undetectable expression in BASQ tumors, we tested the effects of rosiglitazone, Pparg agonist, in a mouse model of BASQ BC. We find that rosiglitazone reduces proliferation while treatment with rosiglitazone plus trametinib, a MEK inhibitor, induces apoptosis and reduces tumor volume by 91% after 1 month. Rosiglitazone and trametinib also induce a shift from BASQ to luminal differentiation in tumors, which our analysis suggests is mediated by retinoid signaling, a pathway known to drive the luminal differentiation program. Our data suggest that rosiglitazone, trametinib, and retinoids, which are all FDA approved, may be clinically active in BASQ tumors in patients.
Assuntos
Apoptose , Proliferação de Células , Modelos Animais de Doenças , Piridonas , Pirimidinonas , Rosiglitazona , Neoplasias da Bexiga Urinária , Animais , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/genética , Piridonas/farmacologia , Piridonas/uso terapêutico , Pirimidinonas/farmacologia , Pirimidinonas/uso terapêutico , Rosiglitazona/farmacologia , Rosiglitazona/uso terapêutico , Camundongos , Apoptose/efeitos dos fármacos , Humanos , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Invasividade Neoplásica , Feminino , PPAR gama/metabolismo , PPAR gama/agonistas , Tiazolidinedionas/farmacologia , Tiazolidinedionas/uso terapêutico , Diferenciação Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Retinoides/farmacologia , Retinoides/uso terapêuticoRESUMO
Bladder cancers (BCs) can be divided into 2 major subgroups displaying distinct clinical behaviors and mutational profiles: basal/squamous (BASQ) tumors that tend to be muscle invasive, and luminal/papillary (LP) tumors that are exophytic and tend to be non-invasive. Pparg is a likely driver of LP BC and has been suggested to act as a tumor suppressor in BASQ tumors, where it is likely suppressed by MEK-dependent phosphorylation. Here we tested the effects of rosiglitazone, a Pparg agonist, in a mouse model of BBN-induced muscle invasive BC. Rosiglitazone activated Pparg signaling in suprabasal epithelial layers of tumors but not in basal-most layers containing highly proliferative invasive cells, reducing proliferation but not affecting tumor survival. Addition of trametinib, a MEK inhibitor, induced Pparg signaling throughout all tumor layers, and eradicated 91% of tumors within 7-days of treatment. The 2-drug combination also activated a luminal differentiation program, reversing squamous metaplasia in the urothelium of tumor-bearing mice. Paired ATAC-RNA-seq analysis revealed that tumor apoptosis was most likely linked to down-regulation of Bcl-2 and other pro-survival genes, while the shift from BASQ to luminal differentiation was associated with activation of the retinoic acid pathway and upregulation of Kdm6a, a lysine demethylase that facilitates retinoid-signaling. Our data suggest that rosiglitazone, trametinib, and retinoids, which are all FDA approved, may be clinically active in BASQ tumors in patients. That muscle invasive tumors are populated by basal and suprabasal cell types with different responsiveness to PPARG agonists will be an important consideration when designing new treatments.
RESUMO
The anaerobic actinobacterium Gardnerella was first isolated from the bladder by suprapubic aspiration more than 50 years ago. Since then, Gardnerella has been increasingly recognized as a common and often abundant member of the female urinary microbiome (urobiome). Some studies even suggest that the presence of Gardnerella is associated with urological disorders in women. We recently reported that inoculation of Gardnerella into the bladders of mice results in urothelial exfoliation. Here, we performed whole bladder RNA-seq in our mouse model to identify additional host pathways involved in the response to Gardnerella bladder exposure. The transcriptional response to Gardnerella reflected the urothelial turnover that is a consequence of exfoliation while also illustrating the activation of pathways involved in inflammation and immunity. Additional timed exposure experiments in mice provided further evidence of a potentially clinically relevant consequence of bladder exposure to Gardnerella-increased susceptibility to subsequent UTI caused by uropathogenic Escherichia coli. Together, these data provide a broader picture of the bladder's response to Gardnerella and lay the groundwork for future studies examining the impact of Gardnerella on bladder health.
Assuntos
Infecções por Escherichia coli , Infecções Urinárias , Escherichia coli Uropatogênica , Animais , Infecções por Escherichia coli/microbiologia , Feminino , Gardnerella , Expressão Gênica , Humanos , Camundongos , Bexiga Urinária/microbiologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/genéticaRESUMO
Pparg, a nuclear receptor, is downregulated in basal subtype bladder cancers that tend to be muscle invasive and amplified in luminal subtype bladder cancers that tend to be non-muscle invasive. Bladder cancers derive from the urothelium, one of the most quiescent epithelia in the body, which is composed of basal, intermediate, and superficial cells. We find that expression of an activated form of Pparg (VP16;Pparg) in basal progenitors induces formation of superficial cells in situ, that exit the cell cycle, and do not form tumors. Expression in basal progenitors that have been activated by mild injury however, results in luminal tumor formation. We find that these tumors are immune deserted, which may be linked to down-regulation of Nf-kb, a Pparg target. Interestingly, some luminal tumors begin to shift to basal subtype tumors with time, down-regulating Pparg and other luminal markers. Our findings have important implications for treatment and diagnosis of bladder cancer.
Assuntos
PPAR gama/metabolismo , Transdução de Sinais , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/patologia , Animais , Biomarcadores Tumorais/metabolismo , Carcinogênese , Carcinógenos/toxicidade , Diferenciação Celular , Proliferação de Células , Proteína Vmw65 do Vírus do Herpes Simples/genética , Proteína Vmw65 do Vírus do Herpes Simples/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , PPAR gama/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Neoplasias da Bexiga Urinária/induzido quimicamente , Neoplasias da Bexiga Urinária/metabolismo , Urotélio/efeitos dos fármacos , Urotélio/imunologia , Urotélio/patologiaRESUMO
The urothelium is an epithelial barrier lining the bladder that protects against infection, fluid exchange and damage from toxins. The nuclear receptor Pparg promotes urothelial differentiation in vitro, and Pparg mutations are associated with bladder cancer. However, the function of Pparg in the healthy urothelium is unknown. Here we show that Pparg is critical in urothelial cells for mitochondrial biogenesis, cellular differentiation and regulation of inflammation in response to urinary tract infection (UTI). Superficial cells, which are critical for maintaining the urothelial barrier, fail to mature in Pparg mutants and basal cells undergo squamous-like differentiation. Pparg mutants display persistent inflammation after UTI, and Nf-KB, which is transiently activated in response to infection in the wild type urothelium, persists for months. Our observations suggest that in addition to its known roles in adipogegnesis and macrophage differentiation, that Pparg-dependent transcription plays a role in the urothelium controlling mitochondrial function development and regeneration.
Assuntos
Diferenciação Celular , Células Epiteliais/metabolismo , Expressão Gênica , Genes Mitocondriais/genética , PPAR gama/metabolismo , Urotélio/metabolismo , Animais , Humanos , Inflamação/complicações , Inflamação/genética , Camundongos Knockout , Camundongos Transgênicos , Mutação , PPAR gama/genética , Bexiga Urinária/citologia , Neoplasias da Bexiga Urinária/genética , Infecções Urinárias/complicações , Urotélio/citologiaRESUMO
The urothelium is an epithelia barrier lined by a luminal layer of binucleated, octoploid, superficial cells. Superficial cells are critical for production and transport of uroplakins, a family of proteins that assemble into a waterproof crystalline plaque that helps protect against infection and toxic substances. Adult urothelium is nearly quiescent, but rapidly regenerates in response to injury. Yet the mechanism by which binucleated, polyploid, superficial cells are produced remains unclear. Here, we show that superficial cells are likely to be derived from a population of binucleated intermediate cells, which are produced from mononucleated intermediate cells via incomplete cytokinesis. We show that binucleated intermediate and superficial cells increase DNA content via endoreplication, passing through S phase without entering mitosis. The urothelium can be permanently damaged by repetitive or chronic injury or disease. Identification of the mechanism by which superficial cells are produced may be important for developing strategies for urothelial repair.