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BACKGROUND: Non-toxic approaches to enhance radiotherapy outcomes are beneficial, particularly in ageing populations. Based on preclinical findings showing that high-fibre diets sensitised bladder tumours to irradiation by modifying the gut microbiota, along with clinical evidence of prebiotics enhancing anti-cancer immunity, we hypothesised that dietary fibre and its gut microbiota modification can radiosensitise tumours via secretion of metabolites and/or immunomodulation. We investigated the efficacy of high-fibre diets combined with irradiation in immunoproficient C57BL/6 mice bearing bladder cancer flank allografts. RESULT: Psyllium plus inulin significantly decreased tumour size and delayed tumour growth following irradiation compared to 0.2% cellulose and raised intratumoural CD8+ cells. Post-irradiation, tumour control positively correlated with Lachnospiraceae family abundance. Psyllium plus resistant starch radiosensitised the tumours, positively correlating with Bacteroides genus abundance and increased caecal isoferulic acid levels, associated with a favourable response in terms of tumour control. Psyllium plus inulin mitigated the acute radiation injury caused by 14 Gy. Psyllium plus inulin increased caecal acetate, butyrate and propionate levels, and psyllium alone and psyllium plus resistant starch increased acetate levels. Human gut microbiota profiles at the phylum level were generally more like mouse 0.2% cellulose profiles than high fibre profiles. CONCLUSION: These supplements may be useful in combination with radiotherapy in patients with pelvic malignancy. Video Abstract.
Assuntos
Fibras na Dieta , Suplementos Nutricionais , Microbioma Gastrointestinal , Inulina , Camundongos Endogâmicos C57BL , Psyllium , Neoplasias da Bexiga Urinária , Animais , Camundongos , Microbioma Gastrointestinal/efeitos dos fármacos , Inulina/administração & dosagem , Neoplasias da Bexiga Urinária/radioterapia , Neoplasias da Bexiga Urinária/patologia , Humanos , Feminino , Lesões por Radiação/prevenção & controle , Intestinos/microbiologia , Intestinos/efeitos da radiação , Linfócitos T CD8-PositivosRESUMO
BACKGROUND: Genomic regions identified by genome-wide association studies (GWAS) for bladder cancer risk provide new insights into etiology. OBJECTIVE: To identify new susceptibility variants for bladder cancer in a meta-analysis of new and existing genome-wide genotype data. DESIGN, SETTING, AND PARTICIPANTS: Data from 32 studies that includes 13,790 bladder cancer cases and 343,502 controls of European ancestry were used for meta-analysis. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSES: Log-additive associations of genetic variants were assessed using logistic regression models. A fixed-effects model was used for meta-analysis of the results. Stratified analyses were conducted to evaluate effect modification by sex and smoking status. A polygenic risk score (PRS) was generated on the basis of known and novel susceptibility variants and tested for interaction with smoking. RESULTS AND LIMITATIONS: Multiple novel bladder cancer susceptibility loci (6p.22.3, 7q36.3, 8q21.13, 9p21.3, 10q22.1, 19q13.33) as well as improved signals in three known regions (4p16.3, 5p15.33, 11p15.5) were identified, bringing the number of independent markers at genome-wide significance (p < 5 × 10-8) to 24. The 4p16.3 (FGFR3/TACC3) locus was associated with a stronger risk for women than for men (p-interaction = 0.002). Bladder cancer risk was increased by interactions between smoking status and genetic variants at 8p22 (NAT2; multiplicative p value for interaction [pM-I] = 0.004), 8q21.13 (PAG1; pM-I = 0.01), and 9p21.3 (LOC107987026/MTAP/CDKN2A; pM-I = 0.02). The PRS based on the 24 independent GWAS markers (odds ratio per standard deviation increase 1.49, 95% confidence interval 1.44-1.53), which also showed comparable results in two prospective cohorts (UK Biobank, PLCO trial), revealed an approximately fourfold difference in the lifetime risk of bladder cancer according to the PRS (e.g., 1st vs 10th decile) for both smokers and nonsmokers. CONCLUSIONS: We report novel loci associated with risk of bladder cancer that provide clues to its biological underpinnings. Using 24 independent markers, we constructed a PRS to stratify lifetime risk. The PRS combined with smoking history, and other established risk factors, has the potential to inform future screening efforts for bladder cancer. PATIENT SUMMARY: We identified new genetic markers that provide biological insights into the genetic causes of bladder cancer. These genetic risk factors combined with lifestyle risk factors, such as smoking, may inform future preventive and screening strategies for bladder cancer.
Assuntos
Arilamina N-Acetiltransferase , Neoplasias da Bexiga Urinária , Masculino , Humanos , Feminino , Estudo de Associação Genômica Ampla , Estudos Prospectivos , Fatores de Risco , Genótipo , Neoplasias da Bexiga Urinária/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Proteínas Associadas aos Microtúbulos , Proteínas de Membrana , Proteínas Adaptadoras de Transdução de SinalRESUMO
With an ageing population, there is an urgent need to find alternatives to current standard-of-care chemoradiation schedules in the treatment of pelvic malignancies. The gut microbiota may be exploitable, having shown a valuable role in improving patient outcomes in anticancer immunotherapy. These bacteria feed on dietary fibres, which reach the large intestine intact, resulting in the production of beneficial metabolites, including short-chain fatty acids. The gut microbiota can impact radiotherapy (RT) treatment responses and itself be altered by the radiation. Evidence is emerging that manipulation of the gut microbiota by dietary fibre supplementation can improve tumour responses and reduce normal tissue side effects following RT, although data on tumour response are limited to date. Both may be mediated by immune and non-immune effects of gut microbiota and their metabolites. Alternative approaches include use of probiotics and faecal microbiota transplantation (FMT). Current evidence will be reviewed regarding the use of dietary fibre interventions and gut microbiota modification in improving outcomes for pelvic RT patients. However, data regarding baseline (pre-RT) gut microbiota of RT patients and timing of dietary fibre manipulation (before or during RT) is limited, heterogenous and inconclusive, thus more robust clinical studies are required before these strategies can be applied clinically.
Assuntos
Fibras na Dieta , Neoplasias , HumanosRESUMO
DNA-protein crosslinks (DPCs) are a specific type of DNA lesion in which proteins are covalently attached to DNA. Unrepaired DPCs lead to genomic instability, cancer, neurodegeneration, and accelerated aging. DPC proteolysis was recently identified as a specialized pathway for DPC repair. The DNA-dependent protease SPRTN and the 26S proteasome emerged as two independent proteolytic systems. DPCs are also repaired by homologous recombination (HR), a canonical DNA repair pathway. While studying the cellular response to DPC formation, we identify ubiquitylation and SUMOylation as two major signaling events in DNA replication-coupled DPC repair. DPC ubiquitylation recruits SPRTN to repair sites, promoting DPC removal. DPC SUMOylation prevents DNA double-strand break formation, HR activation, and potentially deleterious genomic rearrangements. In this way, SUMOylation channels DPC repair toward SPRTN proteolysis, which is a safer pathway choice for DPC repair and prevention of genomic instability.
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Dano ao DNA , Reparo do DNA , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Sumoilação , Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA de Neoplasias/biossíntese , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/genética , Feminino , Células HEK293 , Células HeLa , Recombinação Homóloga , Humanos , Masculino , Proteólise , Mutações Sintéticas LetaisRESUMO
DNA end resection converts broken ends of double-stranded DNA (dsDNA) to 3'-single-stranded DNA (3'-ssDNA). The extent of resection regulates DNA double-strand break (DSB) repair pathway choice and thereby genomic stability. Here, we characterize an optimized immunofluorescence (IF) microscopy-based protocol for measuring ssDNA in mammalian cells by labeling genomic DNA with 5-bromo-2'-deoxyuridine (BrdU). BrdU foci can be detected under non-denaturing conditions by anti-BrdU antibody, providing an accurate and reliable readout of DNA end resection in most mammalian cell lines. For complete details on the use and execution of this protocol, please refer to Kilgas et al. (2021).
Assuntos
Bromodesoxiuridina/química , DNA de Cadeia Simples , Microscopia de Fluorescência/métodos , Bromodesoxiuridina/metabolismo , Linhagem Celular Tumoral , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Instabilidade Genômica/genética , HumanosRESUMO
PURPOSE: Preclinical studies using ultra-high dose rate (FLASH) irradiation have demonstrated reduced normal tissue toxicity compared with conventional dose rate (CONV) irradiation, although this finding is not universal. We investigated the effect of temporal pulse structure and average dose rate of FLASH compared with CONV irradiation on acute intestinal toxicity. MATERIALS AND METHODS: Whole abdomens of C3H mice were irradiated with a single fraction to various doses, using a 6 MeV electron linear accelerator with single pulse FLASH (dose rate = 2-6 × 106 Gy/s) or conventional (CONV; 0.25 Gy/s) irradiation. At 3.75 days postirradiation, fresh feces were collected for 16S rRNA sequencing to assess changes in the gut microbiota. A Swiss roll-based crypt assay was used to quantify acute damage to the intestinal crypts to determine how tissue toxicity was affected by the different temporal pulse structures of FLASH delivery. RESULTS: We found statistically significant improvements in crypt survival for mice irradiated with FLASH at doses between 7.5 and 12.5 Gy, with a dose modifying factor of 1.1 for FLASH (7.5 Gy, P < .01; 10 Gy, P < .05; 12.5 Gy, P < .01). This sparing effect was lost when the delivery time was increased, either by increasing the number of irradiation pulses or by prolonging the time between 2 successive pulses. Sparing was observed for average dose rates of ≥280 Gy/s. Fecal microbiome analysis showed that FLASH irradiation caused fewer changes to the microbiota than CONV irradiation. CONCLUSIONS: This study demonstrates that FLASH irradiation can spare mouse small intestinal crypts and reduce changes in gut microbiome composition compared with CONV irradiation. The higher the average dose rate, the larger the FLASH effect, which is also influenced by temporal pulse structure of the delivery.
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Trato Gastrointestinal , Aceleradores de Partículas , Animais , Camundongos , Camundongos Endogâmicos C3H , RNA Ribossômico 16S , Dosagem RadioterapêuticaRESUMO
BACKGROUND AND PURPOSE: Pelvic radiotherapy (RT) often results in gastrointestinal toxicity and clinical trials have demonstrated a potential benefit of dietary supplements in alleviating acute effects. However, no prophylactic agents have been approved to date for relief of gastrointestinal side-effects caused by pelvic radiation. In this systematic review, we evaluated the efficacy of dietary supplements in preventing or alleviating symptoms of gastrointestinal toxicity in patients undergoing pelvic RT. MATERIALS AND METHODS: CENTRAL, MEDLINE, EMBASE, and ClinicalTrials.gov were searched up to June 2020 for randomised controlled trials. Interventions included four supplement categories: biotics, glutamine, poly-unsaturated fatty acids and polyphenols. Efficacy was determined with reference to outcomes based on symptoms of acute gastrointestinal toxicity, including diarrhoea, nausea, vomiting, flatulence/bloating, bowel movement frequency, tenesmus and rectal bleeding. RESULTS: Twenty-three randomised controlled trials (1919 patients) were identified in this review. Compared with placebo, probiotics (RR = 0.71; 95% CI: 0.52 to 0.99), synbiotics (RR = 0.45; 95% CI: 0.28 to 0.73) and polyphenols (RR = 0.30; 95% CI: 0.13 to 0.70) were significantly associated with a lower risk of diarrhoea. Biotic supplements also reduced the risk of moderate to severe diarrhoea (RR = 0.49; 95% CI: 0.36 to 0.67) and the need for anti-diarrhoeal medication (RR = 0.64; 95%CI: 0.44 to 0.92). In contrast, glutamine had no effect on acute symptoms (RR = 1.05; 95% CI: 0.86 to 1.29). There was a non-significant trend for reduction in nausea and mean bowel movements per day using dietary supplements. CONCLUSIONS: Biotic supplements, especially probiotics and synbiotics, reduce acute symptoms of gastrointestinal toxicity in patients undergoing pelvic radiotherapy.
RESUMO
The ATPase p97 is a central component of the ubiquitin-proteasome degradation system. p97 uses its ATPase activity and co-factors to extract ubiquitinated substrates from different cellular locations, including DNA lesions, thereby regulating DNA repair pathway choice. Here, we find that p97 physically and functionally interacts with the MRE11-RAD50-NBS1 (MRN) complex on chromatin and that inactivation of p97 blocks the disassembly of the MRN complex from the sites of DNA damage upon ionizing radiation (IR). The inhibition of p97 function results in excessive 5'-DNA end resection mediated by MRE11 that leads to defective DNA repair and radiosensitivity. In addition, p97 inhibition by the specific small-molecule inhibitor CB-5083 increases tumor cell killing following IR both in vitro and in vivo. Mechanistically, this is mediated via increased MRE11 nuclease accumulation. This suggests that p97 inhibitors might be exploited to improve outcomes for radiotherapy patients.
Assuntos
Adenosina Trifosfatases/metabolismo , Morte Celular/genética , DNA/genética , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/metabolismo , Radiação Ionizante , HumanosRESUMO
PURPOSE: Chemoradiation therapy is the standard of care in muscle-invasive bladder cancer (MIBC). Although agents such as gemcitabine can enhance tumor radiosensitivity, their side effects can limit patient eligibility and treatment efficacy. This study investigates ultrasound and microbubbles for targeting gemcitabine delivery to reduce normal-tissue toxicity in a murine orthotopic MIBC model. MATERIALS AND METHODS: CD1-nude mice were injected orthotopically with RT112 bladder tumor cells. Conventional chemoradiation involved injecting gemcitabine (10 mg/kg) before 6 Gy targeted irradiation of the bladder area using the Small Animal Radiation Research Platform (SARRP). Ultrasound-mediated gemcitabine delivery (10 mg/kg gemcitabine) involved either coadministration of microbubbles with gemcitabine or conjugating gemcitabine onto microbubbles followed by exposure to ultrasound (1.1 MHz center frequency, 1 MPa peak negative pressure, 1% duty cycle, and 0.5 Hz pulse repetition frequency) before SARRP irradiation. The effect of ultrasound and microbubbles alone was also tested. Tumor volumes were measured by 3D ultrasound imaging. Acute normal-tissue toxicity from 12 Gy to the lower bowel area was assessed using an intestinal crypt assay in mice culled 3.75 days posttreatment. RESULTS: A significant delay in tumor growth was observed with conventional chemoradiation therapy and both microbubble groups (P < .05 compared with the radiation-only group). Transient weight loss was seen in the microbubble groups, which resolved within 10 days posttreatment. A positive correlation was found between weight loss on day 3 posttreatment and tumor growth delay (P < .05; R2 = 0.76). In contrast with conventional chemoradiation therapy, ultrasound-mediated drug delivery methods did not exacerbate the acute intestinal toxicity using the crypt assay. CONCLUSIONS: Ultrasound and microbubbles offer a promising new approach for improving chemoradiation therapy for muscle-invasive bladder cancer, maintaining a delay in tumor growth but with reduced acute intestinal toxicity compared with conventional chemoradiation therapy.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Quimiorradioterapia/efeitos adversos , Desoxicitidina/análogos & derivados , Órgãos em Risco/efeitos da radiação , Lesões por Radiação/prevenção & controle , Neoplasias da Bexiga Urinária/terapia , Animais , Antimetabólitos Antineoplásicos/efeitos adversos , Biotinilação , Linhagem Celular Tumoral , Quimiorradioterapia/métodos , Meios de Contraste/administração & dosagem , Desoxicitidina/administração & dosagem , Desoxicitidina/efeitos adversos , Desoxicitidina/síntese química , Feminino , Humanos , Intestinos/efeitos da radiação , Camundongos , Camundongos Nus , Microbolhas , Invasividade Neoplásica , Carga Tumoral , Ultrassonografia , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/diagnóstico por imagem , Neoplasias da Bexiga Urinária/patologia , GencitabinaRESUMO
In this study we compared three different microbubble-based approaches to the delivery of a widely used chemotherapy drug, gemcitabine: (i) co-administration of gemcitabine and microbubbles (Gem+MB); (ii) conjugates of microbubbles and gemcitabine-loaded liposomes (GemlipoMB); and (iii) microbubbles with gemcitabine directly bound to their surfaces (GembioMB). Both in vitro and in vivo investigations were carried out, respectively, in the RT112 bladder cancer cell line and in a murine orthotopic muscle-invasive bladder cancer model. The in vitro (in vivo) ultrasound exposure conditions were a 1 (1.1) MHz centre frequency, 0.07 (1.0) MPa peak negative pressure, 3000 (20,000) cycles and 100 (0.5) Hz pulse repetition frequency. Ultrasound exposure produced no significant increase in drug uptake either in vitro or in vivo compared with the drug-only control for co-administered gemcitabine and microbubbles. In vivo, GemlipoMB prolonged the plasma circulation time of gemcitabine, but only GembioMB produced a statistically significant increase in cleaved caspase 3 expression in the tumor, indicative of gemcitabine-induced apoptosis.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/farmacocinética , Desoxicitidina/análogos & derivados , Sistemas de Liberação de Medicamentos/métodos , Microbolhas , Terapia por Ultrassom , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/terapia , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Desoxicitidina/administração & dosagem , Desoxicitidina/farmacocinética , Desoxicitidina/uso terapêutico , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Nus , Células Tumorais Cultivadas , GencitabinaRESUMO
The human MRE11/RAD50/NBS1 (MRN) complex plays a crucial role in sensing and repairing DNA DSB. MRE11 possesses dual 3'-5' exonuclease and endonuclease activity and forms the core of the multifunctional MRN complex. We previously identified a C-terminally truncated form of MRE11 (TR-MRE11) associated with post-translational MRE11 degradation. Here we identified SPRTN as the essential protease for the formation of TR-MRE11 and characterised the role of this MRE11 form in its DNA damage response (DDR). Using tandem mass spectrometry and site-directed mutagenesis, the SPRTN-dependent cleavage site for MRE11 was identified between 559 and 580 amino acids. Despite the intact interaction of TR-MRE11 with its constitutive core complex proteins RAD50 and NBS1, both nuclease activities of truncated MRE11 were dramatically reduced due to its deficient binding to DNA. Furthermore, lack of the MRE11 C-terminal decreased HR repair efficiency, very likely due to abolished recruitment of TR-MRE11 to the sites of DNA damage, which consequently led to increased cellular radiosensitivity. The presence of this DNA repair-defective TR-MRE11 could explain our previous finding that the high MRE11 protein expression by immunohistochemistry correlates with improved survival following radical radiotherapy in bladder cancer patients.
Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteína Homóloga a MRE11/metabolismo , Tolerância a Radiação , Neoplasias da Bexiga Urinária/radioterapia , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Proteínas de Ligação a DNA/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Células HEK293 , Humanos , Proteína Homóloga a MRE11/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteólise , Especificidade por Substrato , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologiaRESUMO
BACKGROUND: Patients with pelvic malignancies often receive radiosensitising chemotherapy with radiotherapy to improve survival; however, this is at the expense of increased normal tissue toxicity, particularly in elderly patients. Here, we explore if an alternative, low-cost, and non-toxic approach can achieve radiosensitisation in mice transplanted with human bladder cancer cells. Other investigators have shown slower growth of transplanted tumours in mice fed high-fibre diets. We hypothesised that mice fed a high-fibre diet would have improved tumour control following ionising radiation (IR) and that this would be mediated through the gut microbiota. RESULTS: We investigated the effects of four different diets (low-fibre, soluble high-fibre, insoluble high-fibre, and mixed soluble/insoluble high-fibre diets) on tumour growth in immunodeficient mice implanted with human bladder cancer flank xenografts and treated with ionising radiation, simultaneously investigating the composition of their gut microbiomes by 16S rRNA sequencing. A significantly higher relative abundance of Bacteroides acidifaciens was seen in the gut (faecal) microbiome of the soluble high-fibre group, and the soluble high-fibre diet resulted in delayed tumour growth after irradiation compared to the other groups. Within the soluble high-fibre group, responders to irradiation had significantly higher abundance of B. acidifaciens than non-responders. When all mice fed with different diets were pooled, an association was found between the survival time of mice and relative abundance of B. acidifaciens. The gut microbiome in responders was predicted to be enriched for carbohydrate metabolism pathways, and in vitro experiments on the transplanted human bladder cancer cell line suggested a role for microbial-generated short-chain fatty acids and/or other metabolites in the enhanced radiosensitivity of the tumour cells. CONCLUSIONS: Soluble high-fibre diets sensitised tumour xenografts to irradiation, and this phenotype was associated with modification of the microbiome and positively correlated with B. acidifaciens abundance. Our findings might be exploitable for improving radiotherapy response in human patients.
Assuntos
Bacteroides/fisiologia , Carcinoma/radioterapia , Fibras na Dieta/administração & dosagem , Microbioma Gastrointestinal/efeitos da radiação , Neoplasias da Bexiga Urinária/radioterapia , Animais , Bacteroides/efeitos da radiação , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos NusRESUMO
Approximately 75% of bladder cancers are non-muscle invasive (NMIBC). Of these, up to 53% of cases progress to life-threatening muscle-invasive bladder cancer (MIBC). Patients with high-grade stage T1 (HGT1) NMIBC frequently undergo radical cystectomy (RC), although this represents overtreatment for many. Identification of progressors versus non-progressors could spare unnecessary treatment. Recent studies have confirmed that urothelial carcinoma is composed of two main molecular subtypes, basal and luminal, with 12% of basal tumours exhibiting epithelial-to-mesenchymal transition (EMT). Levels of immune cell infiltration have been shown to be subtype-specific. Here, we performed immunohistochemistry (IHC) for 11 antibodies relating to molecular subtypes or EMT in 26 cases of HGT1 urothelial carcinoma cases with 6 matched samples subsequently obtained at cystectomy (n = 6; 1 muscle-invasive, 5 non-muscle-invasive; 3 = pTis, 1 = pT1, 1 = pTa) and at recurrence (n = 2, pT2). RNAScope was also conducted in a subset. Expression patterns in HGT1 specimens versus MIBC (pT2+) were examined, and correlated with disease-specific survival (DSS). Levels of stromal tumour-infiltrating lymphocytes (sTILs) were assessed manually to determine whether lymphocyte infiltration was associated with DSS and whether differences existed between HGT1 and MIBC. Molecular subtype markers demonstrated increased prognostic potential compared to the EMT markers assessed. Increased expression of the luminal markers FOXA1 and SCUBE2, were found to be significantly associated with better DFS. No EMT markers were significantly associated with DFS. In areas of non-invasive papillary urothelial carcinoma, but not invasive carcinoma, sTIL levels were found to be significantly associated with DFS. While differences were observed between HGT1 cases that progressed versus those that did not, a larger cohort study is required for validation of these findings. Taken together, an emphasis on molecular subtype markers, rather than EMT markers, may be preferable when studying biomarkers of HGT1 urothelial carcinoma in the future.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma/química , Carcinoma/patologia , Transição Epitelial-Mesenquimal , Neoplasias da Bexiga Urinária/química , Neoplasias da Bexiga Urinária/patologia , Biomarcadores Tumorais/genética , Carcinoma/genética , Carcinoma/imunologia , Cistectomia , Progressão da Doença , Humanos , Imuno-Histoquímica , Linfócitos do Interstício Tumoral/imunologia , Invasividade Neoplásica , Fenótipo , Projetos Piloto , Medição de Risco , Fatores de Risco , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/imunologiaRESUMO
PURPOSE: Muscle-invasive bladder cancer has a 40% to 60% 5-year survival rate with radical treatment by surgical removal of the bladder or radiation therapy-based bladder preservation techniques, including concurrent chemoradiation. Elderly patients cannot tolerate current chemoradiation therapy regimens and often receive only radiation therapy, which is less effective. We urgently need effective chemotherapy agents for use with radiation therapy combinations that are nontoxic to normal tissues and tolerated by elderly patients. METHODS AND MATERIALS: We have identified histone deacetylase (HDAC) inhibitors as promising agents to study. Pan-HDAC inhibition, using panobinostat, is a good strategy for radiosensitization, but more selective agents may be more useful radiosensitizers in a clinical setting, resulting in fewer systemic side effects. Herein, we study the HDAC class I-selective agent romidepsin, which we predict to have fewer off-target effects than panobinostat while maintaining an effective level of tumor radiosensitization. RESULTS: In vitro effects of romidepsin were assessed by clonogenic assay and showed that romidepsin was effective in the nanomolar range in different bladder cancer cells and radiosensitized these cells. The radiosensitizing effect of romidepsin was confirmed in vivo using superficial xenografts. The drug/irradiation combination treatment resulted in significant tumor growth delay but did not increase the severity of acute (3.75 days) intestinal normal tissue toxicity or late toxicity at 29 weeks. Moreover, we showed that romidepsin treatment impaired both homologous recombination and nonhomologous end joining DNA repair pathways, suggesting that the disruption of DNA repair pathways caused by romidepsin is a key mechanism for its radiosensitizing effect in bladder cancer cells. CONCLUSIONS: This study demonstrates that romidepsin is an effective radiosensitizer in vitro and in vivo and does not increase the acute and late toxicity after ionizing radiation. Romidepsin is already in clinical use for the cutaneous T-cell lymphoma, but a phase 1 clinical trial of romidepsin as a radiosensitizer could be considered in muscle-invasive bladder cancer.
Assuntos
Depsipeptídeos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Radiossensibilizantes/farmacologia , Neoplasias da Bexiga Urinária/patologia , Acetilação/efeitos dos fármacos , Acetilação/efeitos da radiação , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Depsipeptídeos/efeitos adversos , Inibidores de Histona Desacetilases/efeitos adversos , Histonas/metabolismo , Humanos , Órgãos em Risco/efeitos da radiação , Radiossensibilizantes/efeitos adversos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The E3 ubiquitin ligase RNF8 (RING finger protein 8) is a pivotal enzyme for DNA repair. However, RNF8 hyper-accumulation is tumour-promoting and positively correlates with genome instability, cancer cell invasion, metastasis and poor patient prognosis. Very little is known about the mechanisms regulating RNF8 homeostasis to preserve genome stability. Here, we identify the cellular machinery, composed of the p97/VCP ubiquitin-dependent unfoldase/segregase and the Ataxin 3 (ATX3) deubiquitinase, which together form a physical and functional complex with RNF8 to regulate its proteasome-dependent homeostasis under physiological conditions. Under genotoxic stress, when RNF8 is rapidly recruited to sites of DNA lesions, the p97-ATX3 machinery stimulates the extraction of RNF8 from chromatin to balance DNA repair pathway choice and promote cell survival after ionising radiation (IR). Inactivation of the p97-ATX3 complex affects the non-homologous end joining DNA repair pathway and hypersensitises human cancer cells to IR. We propose that the p97-ATX3 complex is the essential machinery for regulation of RNF8 homeostasis under both physiological and genotoxic conditions and that targeting ATX3 may be a promising strategy to radio-sensitise BRCA-deficient cancers.
Assuntos
Adenosina Trifosfatases/metabolismo , Ataxina-3/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Homeostase , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Adenosina Trifosfatases/genética , Ataxina-3/genética , Sobrevivência Celular , Cromatina/genética , Proteínas de Ligação a DNA/genética , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
CONTEXT: In the past few years, research has suggested that molecular subtypes in muscle-invasive bladder cancer (MIBC) may be exploited to accelerate developments in clinical disease management and novel therapeutics. OBJECTIVE: To review MIBC mouse models from a molecular subtype perspective, their advantages and limitations, and their applications in translational medicine, based on a PubMed search for publications from January 2000 to February 2018. EVIDENCE ACQUISITION: Publications relevant to MIBC mouse models and their molecular subtypes were identified in a literature review. EVIDENCE SYNTHESIS: We classified the models according to the technique used for their establishment. For xenotransplant and allograft models, the inoculated cells and inoculated locations are the major determinants of molecular subtypes. Although the cell lines used in xenotransplant models can cover most of the basal-squamous and luminal subtypes, allograft models offer a more realistic environment in which to reconstruct aspects of the associated stromal and immune features. Autochthonous models, using genetic and/or chemical stimuli to induce disease progression, can also generate models with basal-squamous and luminal subtypes, but further molecular characterisation is needed since other mutational variants may be introduced in these models. CONCLUSIONS: We identified preclinical MIBC models with different subtype specifications and assessed their promise and current limitations. These models are versatile tools that can reproduce the molecular complexity of MIBC and support novel therapeutic development. PATIENT SUMMARY: Understanding which models of muscle-invasive bladder cancer most accurately represent the clinical situation is important for the development of novel drugs and disease management strategies. We review the different models currently available and their relevance to different clinical subtypes.
Assuntos
Modelos Animais de Doenças , Neoplasias Musculares/genética , Neoplasias Musculares/patologia , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Humanos , Camundongos , Neoplasias Musculares/terapia , Transplante de Neoplasias , Células Tumorais Cultivadas/transplante , Microambiente Tumoral , Neoplasias da Bexiga Urinária/terapiaRESUMO
PURPOSE: Organ-confined muscle-invasive bladder cancer is treated with cystectomy or bladder preservation techniques, including radiation therapy. There are currently no biomarkers to inform management decisions and aid patient choice. Previously we showed high levels of MRE11 protein, assessed by immunohistochemistry (IHC), predicted outcome after radiation therapy, but not cystectomy. Therefore, we sought to develop the MRE11 IHC assay for clinical use and define its relationship to clinical outcome in samples from 2 major clinical trials. METHODS AND MATERIALS: Samples from the BCON and BC2001 randomized controlled trials and a cystectomy cohort were stained using automated IHC methods and scored for MRE11 in 3 centers in the United Kingdom. RESULTS: Despite step-wise creation of scoring cards and standard operating procedures for staining and interpretation, there was poor intercenter scoring agreement (kappa, 0.32; 95% confidence interval, 0.17-0.47). No significant associations between MRE11 scores and cause-specific survival were identified in BCON (n = 132) and BC2001 (n = 221) samples. Reoptimized staining improved agreement between scores from BCON tissue microarrays (n = 116), but MRE11 expression was not prognostic for cause-specific survival. CONCLUSIONS: Manual IHC scoring of MRE11 was not validated as a reproducible biomarker of radiation-based bladder preservation success. There is a need for automated quantitative methods or a reassessment of how DNA-damage response relates to clinical outcomes.