Tomosyn-1 (STXBP5) is a soluble NSF attachment protein receptor complex-binding protein that inhibits vesicle fusion, but the role of tomosyn-2 (STXBP5L) in the mammalian nervous system is still unclear. Here we generated tomosyn-2 null (Tom2(KO/KO)) mice, which showed impaired motor performance. This was accompanied by synaptic changes at the neuromuscular junction, including enhanced spontaneous acetylcholine release frequency and faster depression of muscle motor endplate potentials during repetitive stimulation. The postsynaptic geometric arrangement and function of acetylcholine receptors were normal. We conclude that tomosyn-2 supports motor performance by regulation of transmitter release willingness to sustain synaptic strength during high-frequency transmission, which makes this gene a candidate for involvement in neuromuscular disorders.
Organic anion-transporting polypeptides (OATPs) mediate the hepatic uptake of many drugs, thus co-determining their clearance. Impaired hepatic clearance due to low-activity polymorphisms in human OATP1B1 may increase systemic exposure to SN-38, the active and toxic metabolite of the anticancer pro-drug irinotecan. We investigated the pharmacokinetics and toxicity of irinotecan and SN-38 in Oatp1a/1b-null mice: plasma exposure of irinotecan and SN-38 was increased 2-3 fold after irinotecan dosing (10 mg/kg, i.v.) compared to wild-type mice. Also, liver-to-plasma ratios were significantly reduced, suggesting impaired hepatic uptake of both compounds. After 6 daily doses of irinotecan, Oatp1a/1b-null mice suffered from increased toxicity. However, Oatp1a/1b-null mice had increased levels of carboxylesterase (Ces) enzymes, which caused higher conversion of irinotecan to SN-38 in plasma, potentially complicating pharmacokinetic analyses. Ces inhibitors blocked this increased conversion. Interestingly, liver-specific humanized OATP1B1 and OATP1B3 transgenic mice had normalized hepatic expression of Ces1 genes. Whereas irinotecan liver-to-plasma ratios in these humanized mice were similar to those in Oatp1a/1b-null mice, SN-38 liver-to-plasma ratios returned to wild-type levels, suggesting that human OATP1B proteins mediate SN-38, but not irinotecan uptake in vivo. Upon direct administration of SN-38 (1 mg/kg, i.v.), Oatp1a/1b-null mice had increased SN-38 plasma levels, lower liver concentrations and decreased cumulative biliary excretion of SN-38. Conclusion: Mouse Oatp1a/1b transporters have a role in the plasma clearance of irinotecan and SN-38, while human OATP1B transporters may only affect SN-38 disposition. Oatp1a/1b-null mice have increased expression and activity of Ces1 enzymes, while humanized mice provide a rescue of this phenotype.
Whereas aberrant activation of canonical Wnt/?-catenin signaling underlies the majority of colorectal cancer cases, the contribution of non-canonical Wnt signaling is unclear. As enhanced expression of the most extensively studied non-canonical Wnt ligand WNT5A is observed in various diseases including colon cancer, WNT5A is gaining attention nowadays. Numerous in vitro studies suggest modulating capacities of WNT5A on proliferation, differentiation, migration and invasion, affecting tumor and non-mutant cells. However, a possible contribution of WNT5A to colorectal cancer remains to be elucidated. We have analyzed WNT5A expression in colorectal cancer profiling data sets, altered WNT5A expression in colon cancer cells and used our inducible Wnt5a transgenic mouse model to gain more insight into the role of WNT5A in intestinal cancer. We observed that increased WNT5A expression is associated with poor prognosis of colorectal cancer patients. WNT5A knockdown in human colon cancer cells caused reduced directional migration, deregulated focal adhesion site formation and reduced invasion, whereas Wnt5a administration promoted the directional migration of colon cancer cells. Despite these observed protumorigenic activities of WNT5A, the induction of Wnt5a expression in intestinal tumors of Apc1638N mice was not sufficient to augment malignancy or metastasis by itself. In conclusion, WNT5A promotes adhesion sites to form in a focal fashion and promotes the directional migration and invasion of colon cancer cells. Although these activities appear insufficient by themselves to augment malignancy or metastasis in Apc1638N mice, they might explain the poor colon cancer prognosis associated with enhanced WNT5A expression.
Fanconi anaemia (FA) is a rare recessive disorder marked by developmental abnormalities, bone marrow failure, and a high risk for the development of leukaemia and solid tumours. The inactivation of FA genes, in particular FANCF, has also been documented in sporadic tumours in non-FA patients. To study whether there is a causal relationship between FA pathway defects and tumour development, we have generated a mouse model with a targeted disruption of the FA core complex gene Fancf. Fancf-deficient mouse embryonic fibroblasts displayed a phenotype typical for FA cells: they showed an aberrant response to DNA cross-linking agents as manifested by G(2) arrest, chromosomal aberrations, reduced survival, and an inability to monoubiquitinate FANCD2. Fancf homozygous mice were viable, born following a normal Mendelian distribution, and showed no growth retardation or developmental abnormalities. The gonads of Fancf mutant mice functioned abnormally, showing compromised follicle development and spermatogenesis as has been observed in other FA mouse models and in FA patients. In a cohort of Fancf-deficient mice, we observed decreased overall survival and increased tumour incidence. Notably, in seven female mice, six ovarian tumours developed: five granulosa cell tumours and one luteoma. One mouse had developed tumours in both ovaries. High-resolution array comparative genomic hybridization (aCGH) on these tumours suggests that the increased incidence of ovarian tumours correlates with the infertility in Fancf-deficient mice and the genomic instability characteristic of FA pathway deficiency.
Fanconi anemia (FA) is a heritable disease characterized by bone marrow failure, congenital abnormalities, and cancer predisposition. The 15 identified FA genes operate in a molecular pathway to preserve genomic integrity. Within this pathway the FA core complex operates as an ubiquitin ligase that activates the complex of FANCD2 and FANCI to coordinate DNA repair. The FA core complex is formed by at least 12 proteins. However, only the FANCL subunit displays ubiquitin ligase activity. FANCA and FANCG are members of the FA core complex for which no other functions have been described than to participate in protein interactions. In this study we generated mice with combined null alleles for Fanca and Fancg to identify extended functions for these genes by characterizing the double mutant mice and cells. Double mutant a(-/-)/g(-/-) mice were born at near Mendelian frequencies without apparent developmental abnormalities. Histological analysis of a(-/-)/g(-/-) mice revealed a Leydig cell hyperplasia and frequent vacuolization of Sertoli cells in testes, while ovaries were depleted from developing follicles and displayed an interstitial cell hyperplasia. These gonadal aberrations were associated with a compromised fertility of a(-/-)/g(-/-) males and females. During the first year of life a(-/-)/g(-/-) did not develop malignancies or bone marrow failure. At the cellular level a(-/-)/g(-/-), Fanca(-/-), and Fancg(-/-) cells proved equally compromised in DNA crosslink and homology-directed repair. Overall the phenotype of a(-/-)/g(-/-) double knockout mice and cells appeared highly similar to the phenotype of Fanca or Fancg single knockouts. The lack of an augmented phenotype suggest that null mutations in Fanca or Fancg are fully epistatic, making additional important functions outside of the FA core complex highly unlikely.
Objective Deregulation of the Wnt signalling pathway by mutations in the Apc or ?-catenin genes underlies colorectal carcinogenesis. As a result, ?-catenin stabilises, translocates to the nucleus, and activates gene transcription. Intestinal tumours show a heterogeneous pattern of nuclear ?-catenin, with the highest levels observed at the invasion front. Activation of receptor tyrosine kinases in these tumour areas by growth factors expressed by surrounding stromal cells phosphorylate ?-catenin at tyrosine residues, which is thought to increase ?-catenin nuclear translocation and tumour invasiveness. This study investigates the relevance of ?-catenin tyrosine phosphorylation for Wnt signalling and intestinal tumorigenesis in vivo. Design A conditional knock-in mouse model was generated into which the phospho-mimicking Y654E modification in the endogenous ?-catenin gene was introduced. Results This study provided in vivo evidence that ?-catenin(E654) is characterised by reduced affinity for cadherins, increased signalling and strongly increased phosphorylation at serine 675 by protein kinase A (PKA). In addition, homozygosity for the ?-catenin(E654) targeted allele caused embryonic lethality, whereas heterozygosity predisposed to intestinal tumour development, and strongly enhanced Apc-driven intestinal tumour initiation associated with increased nuclear accumulation of ?catenin. Surprisingly, the expression of ?-catenin(E654) did not affect histological grade or induce tumour invasiveness. Conclusions A thus far unknown mechanism was uncovered in which Y654 phosphorylation of ?-catenin facilitates additional phosphorylation at serine 675 by PKA. In addition, in contrast to the current belief that ?-catenin Y654 phosphorylation increases tumour progression to a more invasive phenotype, these results show that it rather increases tumour initiation by enhancing Wnt signalling.
For amphiphilic anticancer drugs, such as the anthracyclin doxorubicin (Dox), uptake by tumor cells involves slow diffusion across the plasma membrane, a limiting factor in clinical oncology. Previously, we discovered that preinsertion of short-chain sphingolipids such as N-octanoyl-glucosylceramide (GC) in the tumor cell membrane enhances cellular Dox uptake. In the present study, we apply this strategy in vitro and in vivo by coadministering GC and Dox in a lipid nanovesicle (LNV). GC enrichment of Dox-LNVs strongly enhanced in vitro cytotoxicity toward B16 melanoma and A431 carcinoma, as evidenced by 6-fold decreased IC(50) values compared with Dox-LNVs. This correlated with enhanced cellular Dox uptake observed by confocal microscopy. Intravital optical imaging in window chamber-bearing mice with orthotopically implanted B16 melanoma demonstrated enhanced GC-mediated Dox delivery to tumor cells. Treatment of nude mice bearing human A431 xenografts with 6 mg/kg GC-Dox-LNVs almost doubled the tumor growth delay compared with Dox-LNVs. A second administration of 5 mg/kg after 3 d induced even 3-fold delay in tumor growth, while no systemic toxicity was found. GC-enriched Dox-LNVs displayed superior in vitro and in vivo antitumor activity, without systemic toxicity. This new drug delivery concept, aiming at increased membrane permeability for amphiphilic drugs, provides an opportunity to improve cancer chemotherapy.
The induction of skin cancer involves both mutagenic and proliferative responses of the epidermis to ultraviolet (UV) light. It is believed that tumor initiation requires the mutagenic replication of damaged DNA by translesion synthesis (TLS) pathways. The mechanistic basis for the induction of proliferation, providing tumor promotion, is poorly understood. Here, we have investigated the role of TLS in the initiation and promotion of skin carcinogenesis, using a sensitive nucleotide excision repair-deficient mouse model that carries a hypomorphic allele of the error-prone TLS gene Rev1. Despite a defect in UV-induced mutagenesis, skin carcinogenesis was accelerated in these mice. This paradoxical phenotype was caused by the induction of inflammatory hyperplasia of the mutant skin that provides strong tumor promotion. The induction of hyperplasia was associated with mild and transient replicational stress of the UV-damaged genome, triggering DNA damage signaling and senescence. The concomitant expression of Interleukin-6 (IL-6) is in agreement with an executive role for IL-6 and possibly other cytokines in the autocrine induction of senescence and the paracrine induction of inflammatory hyperplasia. In conclusion, error-prone TLS suppresses tumor-promoting activities of UV light, thereby controlling skin carcinogenesis.
Influenza virus infection can be accompanied by life-threatening immune pathology most likely due to excessive antiviral responses. Inhibitory immune receptors may restrain such overactive immune responses. To study the role of the inhibitory immune receptor CD200R and its ligand CD200 during influenza infection, we challenged wild-type and CD200(-/-) mice with influenza virus. We found that CD200(-/-) mice in comparison to wild-type controls when inoculated with influenza virus developed more severe disease, associated with increased lung infiltration and lung endothelium damage. CD200(-/-) mice did develop adequate adaptive immune responses and were able to control viral load, suggesting that the severe disease was caused by a lack of control of the immune response. Interestingly, development of disease was completely prevented by depletion of T cells before infection, despite dramatically increased viral load, indicating that T cells are essential for the development of disease symptoms. Our data show that lack of CD200-CD200R signaling increases immune pathology during influenza infection, which can be reduced by T cell depletion.
The Fanconi anemia (FA) core complex member FANCM remodels synthetic replication forks and recombination intermediates. Thus far, only one FA patient with FANCM mutations has been described, but the relevance of these mutations for the FA phenotype is uncertain. To provide further experimental access to the FA-M complementation group we have generated Fancm-deficient mice by deleting exon 2. FANCM deficiency caused hypogonadism in mice and hypersensitivity to cross-linking agents in mouse embryonic fibroblasts (MEFs), thus phenocopying other FA mouse models. However, Fancm(Delta2/Delta2) mice also showed unique features atypical for FA mice, including underrepresentation of female Fancm(Delta2/Delta2) mice and decreased overall and tumor-free survival. This increased cancer incidence may be correlated to the role of FANCM in the suppression of spontaneous sister chromatid exchanges as observed in MEFs. In addition, FANCM appeared to have a stimulatory rather than essential role in FANCD2 monoubiquitination. The FA-M mouse model presented here suggests that FANCM functions both inside and outside the FA core complex to maintain genome stability and to prevent tumorigenesis.
During skeletogenesis, protein levels of beta-catenin in the canonical Wnt signaling pathway determine lineage commitment of skeletal precursor cells to osteoblasts and chondrocytes. Adenomatous polyposis coli (Apc) is a key controller of beta-catenin turnover by down-regulating intracellular levels of beta-catenin.
Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant hereditary predisposition to the development of multiple colorectal adenomas and of a broad spectrum of extra-intestinal tumors. Moreover, somatic APC mutations play a rate-limiting and initiating role in the majority of sporadic colorectal cancers. Notwithstanding its multifunctional nature, the main tumor suppressing activity of the APC gene resides in its ability to regulate Wnt/beta-catenin signaling. Notably, genotype-phenotype correlations have been established at the APC gene between the length and stability of the truncated proteins encoded by different mutant alleles, the corresponding levels of Wnt/beta-catenin signaling activity they encode for, and the incidence and distribution of intestinal and extra-intestinal tumors. Here, we report a novel mouse model, Apc1572T, obtained by targeting a truncated mutation at codon 1572 in the endogenous Apc gene. This hypomorphic mutant allele results in intermediate levels of Wnt/beta-catenin signaling activation when compared with other Apc mutations associated with multifocal intestinal tumors. Notwithstanding the constitutive nature of the mutation, Apc(+/1572T) mice have no predisposition to intestinal cancer but develop multifocal mammary adenocarcinomas and subsequent pulmonary metastases in both genders. The histology of the Apc1572T primary mammary tumours is highly heterogeneous with luminal, myoepithelial, and squamous lineages and is reminiscent of metaplastic carcinoma of the breast in humans. The striking phenotype of Apc(+/1572T) mice suggests that specific dosages of Wnt/beta-catenin signaling activity differentially affect tissue homeostasis and initiate tumorigenesis in an organ-specific fashion.
The MAP kinase and PI3 kinase pathways have been identified as the most common pathways that mediate oncogenic transformation in melanoma, and the majority of compounds developed for melanoma treatment target one or the other of these pathways. In addition to such targeted therapies, immunotherapeutic approaches have shown promising results. A combination of these two treatment modalities could potentially result in further improvement of treatment outcome. To preclinically identify efficient treatment combinations and to optimize therapy protocols in terms of sequence and timing, mouse models will be required. We have crossed and characterized the Tyr::CreER(T2);PTEN(F-/-);BRAF(F-V600E/+) inducible melanoma model on a C57BL/6J background. Tumors from this model harbor the BRAF(V600E) mutation and are PTEN-deficient, making them highly suitable for the testing of targeted therapies. Furthermore, we crossed the model onto this specific background for use in immunotherapy studies, because most experiments in this field have been performed in C57BL/6J mice. Selective inhibition of BRAF(V600E) by PLX4720 treatment of melanoma-bearing mice resulted in a strong decrease of tumor outgrowth. Furthermore, the inducible melanomas had immune cell infiltrates similar to those found in human melanoma, and tumor-infiltrating lymphocytes could be cultured from these tumors. Our data indicate that the C57BL/6J Tyr::CreER(T2);PTEN(F-/-);BRAF(F-V600E/+) melanoma model could be used as a standard model in which targeted and immunotherapy combinations can be tested in a high-throughput manner.
Wnt5a is essential during embryonic development, as indicated by mouse Wnt5a knockout embryos displaying outgrowth defects of multiple structures including the gut. The dynamics of Wnt5a involvement in these processes is unclear, and perinatal lethality of Wnt5a knockout embryos has hampered investigation of Wnt5a during postnatal stages in vivo. Although in vitro studies have suggested a relevant role for Wnt5a postnatally, solid evidence for a significant impact of Wnt5a within the complexity of an adult organism is lacking. We generated a tightly-regulated inducible Wnt5a transgenic mouse model and investigated the effects of Wnt5a induction during different time-frames of embryonic development and in adult mice, focusing on the gastrointestinal tract. When induced in embryos from 10.5 dpc onwards, Wnt5a expression led to severe outgrowth defects affecting the gastrointestinal tracts, limbs, facial structures and tails, closely resembling the defects observed in Wnt5a knockout mice. However, Wnt5a induction from 13.5 dpc onwards did not cause this phenotype, indicating that the most critical period for Wnt5a in embryonic development is prior to 13.5 dpc. In adult mice, induced Wnt5a expression did not reveal abnormalities, providing the first in vivo evidence that Wnt5a has no major impact on mouse intestinal homeostasis postnatally. Protein expression of Wnt5a receptor Ror2 was strongly reduced in adult intestine compared to embryonic stages. Moreover, we uncovered a regulatory process where induction of Wnt5a causes downregulation of its receptor Ror2. Taken together, our results indicate a role for Wnt5a during a restricted time-frame of embryonic development, but suggest no impact during homeostatic postnatal stages.
Bmi1 is a member of the polycomb repressive complex 1 and plays different roles during embryonic development, depending on the developmental context. Bmi1 over expression is observed in many types of cancer, including tumors of astroglial and neural origin. Although genetic depletion of Bmi1 has been described to result in tumor inhibitory effects partly through INK4A/Arf mediated senescence and apoptosis and also through INK4A/Arf independent effects, it has not been proven that Bmi1 can be causally involved in the formation of these tumors. To see whether this is the case, we developed two conditional Bmi1 transgenic models that were crossed with GFAP-Cre mice to activate transgenic expression in neural and glial lineages. We show here that these mice generate intermediate and anterior lobe pituitary tumors that are positive for ACTH and beta-endorphin. Combined transgenic expression of Bmi1 together with conditional loss of Rb resulted in pituitary tumors but was insufficient to induce medulloblastoma therefore indicating that the oncogenic function of Bmi1 depends on regulation of p16(INK4A)/Rb rather than on regulation of p19(ARF)/p53. Human pituitary adenomas show Bmi1 overexpression in over 50% of the cases, which indicates that Bmi1 could be causally involved in formation of these tumors similarly as in our mouse model.
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