324 research outputs found
Ectodomains of the LDL receptor-related proteins LRP1b and LRP4 have anchorage independent functions in vivo.
BACKGROUND: The low-density lipoprotein (LDL) receptor gene family is a highly conserved group of membrane receptors with diverse functions in developmental processes, lipoprotein trafficking, and cell signaling. The low-density lipoprotein (LDL) receptor-related protein 1b (LRP1B) was reported to be deleted in several types of human malignancies, including non-small cell lung cancer. Our group has previously reported that a distal extracellular truncation of murine Lrp1b that is predicted to secrete the entire intact extracellular domain (ECD) is fully viable with no apparent phenotype. METHODS AND PRINCIPAL FINDINGS: Here, we have used a gene targeting approach to create two mouse lines carrying internally rearranged exons of Lrp1b that are predicted to truncate the protein closer to the N-terminus and to prevent normal trafficking through the secretary pathway. Both mutations result in early embryonic lethality, but, as expected from the restricted expression pattern of LRP1b in vivo, loss of Lrp1b does not cause cellular lethality as homozygous Lrp1b-deficient blastocysts can be propagated normally in culture. This is similar to findings for another LDL receptor family member, Lrp4. We provide in vitro evidence that Lrp4 undergoes regulated intramembraneous processing through metalloproteases and gamma-secretase cleavage. We further demonstrate negative regulation of the Wnt signaling pathway by the soluble extracellular domain. CONCLUSIONS AND SIGNIFICANCE: Our results underline a crucial role for Lrp1b in development. The expression in mice of truncated alleles of Lrp1b and Lrp4 with deletions of the transmembrane and intracellular domains leads to release of the extracellular domain into the extracellular space, which is sufficient to confer viability. In contrast, null mutations are embryonically (Lrp1b) or perinatally (Lrp4) lethal. These findings suggest that the extracellular domains of both proteins may function as a scavenger for signaling ligands or signal modulators in the extracellular space, thereby preserving signaling thresholds that are critical for embryonic development, as well as for the clear, but poorly understood role of LRP1b in cancer
Vitamin D-VDR signaling inhibits Wnt/beta-catenin-mediated melanoma progression and promotes anti-tumor immunity
1α,25-dihydroxyvitamin D3 signals via the Vitamin D Receptor (VDR). Higher serum vitamin D is associated with thinner primary melanoma and better outcome, although a causal mechanism has not been established. As melanoma patients commonly avoid sun exposure, and consequent vitamin D deficiency might worsen outcomes, we interrogated 703 primary melanoma transcriptomes to understand the role of vitamin D-VDR signalling and replicated the findings in TCGA metastases. VDR expression was independently protective for melanoma death in both primary and metastatic disease. High tumor VDR expression was associated with upregulation of pathways mediating anti-tumor immunity and correspondingly with higher imputed immune cell scores and histologically detected tumor infiltrating lymphocytes (TILs). High VDR expressing tumors had downregulation of proliferative pathways, notably Wnt/beta-catenin signaling. Deleterious low VDR levels resulted from promoter methylation and gene deletion in metastases. Vitamin D deficiency (< 25 nmol/l ~ 10 ng/ml) shortened survival in primary melanoma in a VDR-dependent manner. In vitro functional validation studies showed that elevated vitamin D-VDR signaling inhibited Wnt/beta-catenin signaling genes. Murine melanoma cells overexpressing VDR produced fewer pulmonary metastases than controls in tail vein metastasis assays. In summary, vitamin D-VDR signaling contributes to controlling pro-proliferative/immunosuppresive Wnt/beta-catenin signaling in melanoma and this is associated with less metastatic disease and stronger host immune responses. This is evidence of the causal relationship between vitamin D-VDR signaling and melanoma survival which should be explored as a therapeutic target in primary resistance to checkpoint blockade
Modeling Partial Monosomy for Human Chromosome 21q11.2-q21.1 Reveals Haploinsufficient Genes Influencing Behavior and Fat Deposition
Haploinsufficiency of part of human chromosome 21 results in a rare condition known as Monosomy 21. This disease displays a variety of clinical phenotypes, including intellectual disability, craniofacial dysmorphology, skeletal and cardiac abnormalities, and respiratory complications. To search for dosage-sensitive genes involved in this disorder, we used chromosome engineering to generate a mouse model carrying a deletion of the Lipi–Usp25 interval, syntenic with 21q11.2-q21.1 in humans. Haploinsufficiency for the 6 genes in this interval resulted in no gross morphological defects and behavioral analysis performed using an open field test, a test of anxiety, and tests for social interaction were normal in monosomic mice. Monosomic mice did, however, display impaired memory retention compared to control animals. Moreover, when fed a high-fat diet (HFD) monosomic mice exhibited a significant increase in fat mass/fat percentage estimate compared with controls, severe fatty changes in their livers, and thickened subcutaneous fat. Thus, genes within the Lipi–Usp25 interval may participate in memory retention and in the regulation of fat deposition
RASSF1A–LATS1 signalling stabilizes replication forks by restricting CDK2-mediated phosphorylation of BRCA2
Genomic instability is a key hallmark of cancer leading to tumour heterogeneity and therapeutic resistance. BRCA2 has a fundamental role in error-free DNA repair but also sustains genome integrity by promoting RAD51 nucleofilament formation at stalled replication forks. CDK2 phosphorylates BRCA2 (pS3291-BRCA2) to limit stabilizing contacts with polymerized RAD51; however, how replication stress modulates CDK2 activity and whether loss of pS3291-BRCA2 regulation results in genomic instability of tumours are not known. Here we demonstrate that the Hippo pathway kinase LATS1 interacts with CDK2 in response to genotoxic stress to constrain pS3291-BRCA2 and support RAD51 nucleofilaments, thereby maintaining genomic fidelity during replication stalling. We also show that LATS1 forms part of an ATR-mediated response to replication stress that requires the tumour suppressor RASSF1A. Importantly, perturbation of the ATR–RASSF1A–LATS1 signalling axis leads to genomic defects associated with loss of BRCA2 function and contributes to genomic instability and ‘BRCA-ness’ in lung cancers
Artificial Intelligence to Predict the BRAF V595E Mutation in Canine Urinary Bladder Urothelial Carcinomas
In dogs, the BRAF mutation (V595E) is common in bladder and prostate cancer and represents a specific diagnostic marker. Recent advantages in artificial intelligence (AI) offer new opportunities in the field of tumour marker detection. While AI histology studies have been conducted in humans to detect BRAF mutation in cancer, comparable studies in animals are lacking. In this study, we used commercially available AI histology software to predict BRAF mutation in whole slide images (WSI) of bladder urothelial carcinomas (UC) stained with haematoxylin and eosin (HE), based on a training (n = 81) and a validation set (n = 96). Among 96 WSI, 57 showed identical PCR and AI-based BRAF predictions, resulting in a sensitivity of 58% and a specificity of 63%. The sensitivity increased substantially to 89% when excluding small or poor-quality tissue sections. Test reliability depended on tumour differentiation (p < 0.01), presence of inflammation (p < 0.01), slide quality (p < 0.02) and sample size (p < 0.02). Based on a small subset of cases with available adjacent non-neoplastic urothelium, AI was able to distinguish malignant from benign epithelium. This is the first study to demonstrate the use of AI histology to predict BRAF mutation status in canine UC. Despite certain limitations, the results highlight the potential of AI in predicting molecular alterations in routine tissue sections
Artificial Intelligence to Predict the BRAF V595E Mutation in Canine Urinary Bladder Urothelial Carcinomas.
In dogs, the BRAF mutation (V595E) is common in bladder and prostate cancer and represents a specific diagnostic marker. Recent advantages in artificial intelligence (AI) offer new opportunities in the field of tumour marker detection. While AI histology studies have been conducted in humans to detect BRAF mutation in cancer, comparable studies in animals are lacking. In this study, we used commercially available AI histology software to predict BRAF mutation in whole slide images (WSI) of bladder urothelial carcinomas (UC) stained with haematoxylin and eosin (HE), based on a training (n = 81) and a validation set (n = 96). Among 96 WSI, 57 showed identical PCR and AI-based BRAF predictions, resulting in a sensitivity of 58% and a specificity of 63%. The sensitivity increased substantially to 89% when excluding small or poor-quality tissue sections. Test reliability depended on tumour differentiation (p < 0.01), presence of inflammation (p < 0.01), slide quality (p < 0.02) and sample size (p < 0.02). Based on a small subset of cases with available adjacent non-neoplastic urothelium, AI was able to distinguish malignant from benign epithelium. This is the first study to demonstrate the use of AI histology to predict BRAF mutation status in canine UC. Despite certain limitations, the results highlight the potential of AI in predicting molecular alterations in routine tissue sections
Urinary ATP as an indicator of infection and inflammation of the urinary tract in patients with lower urinary tract symptoms
BACKGROUND:
Adenosine-5'-triphosphate (ATP) is a neurotransmitter and inflammatory cytokine implicated in the pathophysiology of lower urinary tract disease. ATP additionally reflects microbial biomass thus has potential as a surrogate marker of urinary tract infection (UTI). The optimum clinical sampling method for ATP urinalysis has not been established. We tested the potential of urinary ATP in the assessment of lower urinary tract symptoms, infection and inflammation, and validated sampling methods for clinical practice.
METHODS:
A prospective, blinded, cross-sectional observational study of adult patients presenting with lower urinary tract symptoms (LUTS) and asymptomatic controls, was conducted between October 2009 and October 2012. Urinary ATP was assayed by a luciferin-luciferase method, pyuria counted by microscopy of fresh unspun urine and symptoms assessed using validated questionnaires. The sample collection, storage and processing methods were also validated.
RESULTS:
75 controls and 340 patients with LUTS were grouped as without pyuria (n = 100), pyuria 1-9 wbc ?l(-1) (n = 120) and pyuria ?10 wbc ?l(-1) (n = 120). Urinary ATP was higher in association with female gender, voiding symptoms, pyuria greater than 10 wbc ?l(-1) and negative MSU culture. ROC curve analysis showed no evidence of diagnostic test potential. The urinary ATP signal decayed with storage at 23°C but was prevented by immediate freezing at ??-20°C, without boric acid preservative and without the need to centrifuge urine prior to freezing.
CONCLUSIONS:
Urinary ATP may have a role as a research tool but is unconvincing as a surrogate, clinical diagnostic marker
Loss of Rassf1a cooperates with Apc(Min) to accelerate intestinal tumourigenesis
Promoter methylation of the RAS-association domain family 1, isoform A gene (RASSF1A) is one of the most frequent events found in human tumours. In this study we set out to test the hypothesis that loss of Rassf1a can co-operate with inactivation of the adenomatous polyposis coli (Apc) gene to accelerate intestinal tumourigenesis using the Apc-Min (Apc(Min/+)) mouse model, as mutational or deletional inactivation of APC is a frequent early event in the genesis of intestinal cancer. Further, loss of RASSF1A has also been reported to occur in premalignant adenomas of the bowel. RASSF1A has been implicated in an array of pivotal cellular processes, including regulation of the cell cycle, apoptosis, microtubule stability and most recently in the β-catenin signalling pathway. By interbreeding isoform specific Rassf1a knockout mice with Apc(+/Min) mice we showed that loss of Rassf1a results in a significant increase in adenomas of the small intestine and accelerated intestinal tumourigenesis leading to the earlier death of adenocarcinoma-bearing mice and decreased overall survival. Comparative genomic hybridization of adenomas from Rassf1a(−/−); Apc(+/Min) mice revealed no evidence of aneuploidy or gross chromosomal instability (no difference to adenomas from Rassf1a(+/+); Apc(+/Min) mice). Immunohistochemical analysis of adenomas revealed increased nuclear β-catenin accumulation in adenomas from Rassf1a(−/−); Apc(+/Min) mice, compared to those from Rassf1a(+/+); Apc(+/Min) mice, but no differences in proliferation marker (Ki67) staining patterns. Collectively these data demonstrate co-operation between inactivation of Rassf1a and Apc resulting in accelerated intestinal tumourigenesis, with adenomas showing increased nuclear accumulation of β-catenin, supporting a mechanistic link via loss of the known interaction of Rassf1 with β-TrCP that usually mediates degradation of β-catenin
A high-throughput in vivo screening method in the mouse for identifying regulators of metastatic colonization
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