| 1. |
- Pateras, Ioannis S., et al.
(författare)
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Short term starvation potentiates the efficacy of chemotherapy in triple negative breast cancer via metabolic reprogramming
- 2023
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Ingår i: Journal of Translational Medicine. - : BioMed Central (BMC). - 1479-5876. ; 21:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Chemotherapy (CT) is central to the treatment of triple negative breast cancer (TNBC), but drug toxicity and resistance place strong restrictions on treatment regimes. Fasting sensitizes cancer cells to a range of chemotherapeutic agents and also ameliorates CT-associated adverse effects. However, the molecular mechanism(s) by which fasting, or short-term starvation (STS), improves the efficacy of CT is poorly characterized.Methods: The differential responses of breast cancer or near normal cell lines to combined STS and CT were assessed by cellular viability and integrity assays (Hoechst and PI staining, MTT or H2DCFDA staining, immunofluorescence), metabolic profiling (Seahorse analysis, metabolomics), gene expression (quantitative real-time PCR) and iRNA-mediated silencing. The clinical significance of the in vitro data was evaluated by bioinformatical integration of transcriptomic data from patient data bases: The Cancer Genome Atlas (TCGA), European Genome-phenome Archive (EGA), Gene Expression Omnibus (GEO) and a TNBC cohort. We further examined the translatability of our findings in vivo by establishing a murine syngeneic orthotopic mammary tumor-bearing model.Results: We provide mechanistic insights into how preconditioning with STS enhances the susceptibility of breast cancer cells to CT. We showed that combined STS and CT enhanced cell death and increased reactive oxygen species (ROS) levels, in association with higher levels of DNA damage and decreased mRNA levels for the NRF2 targets genes NQO1 and TXNRD1 in TNBC cells compared to near normal cells. ROS enhancement was associated with compromised mitochondrial respiration and changes in the metabolic profile, which have a significant clinical prognostic and predictive value. Furthermore, we validate the safety and efficacy of combined periodic hypocaloric diet and CT in a TNBC mouse model.Conclusions: Our in vitro, in vivo and clinical findings provide a robust rationale for clinical trials on the therapeutic benefit of short-term caloric restriction as an adjuvant to CT in triple breast cancer treatment.
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| 2. |
- Tsioras, Konstantinos, et al.
(författare)
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Analysis of proteome-wide degradation dynamics in ALS SOD1 iPSC-derived patient neurons reveals disrupted VCP homeostasis
- 2023
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Ingår i: Cell Reports. - : Elsevier. - 2211-1247. ; 42:10
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Tidskriftsartikel (refereegranskat)abstract
- Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS) through gain-of-function effects, yet the mechanisms by which misfolded mutant SOD1 (mutSOD1) protein impairs human motor neurons (MNs) remain unclear. Here, we use induced-pluripotent-stem-cell-derived MNs coupled to metabolic stable isotope labeling and mass spectrometry to investigate proteome-wide degradation dynamics. We find several proteins, including the ALS-causal valosin-containing protein (VCP), which predominantly acts in proteasome degradation and autophagy, that degrade slower in mutSOD1 relative to isogenic control MNs. The interactome of VCP is altered in mutSOD1 MNs in vitro, while VCP selectively accumulates in the affected motor cortex of ALS-SOD1 patients. Overexpression of VCP rescues mutSOD1 toxicity in MNs in vitro and in a C. elegans model in vivo, in part due to its ability to modulate the degradation of insoluble mutSOD1. Our results demonstrate that VCP contributes to mutSOD1-dependent degeneration, link two distinct ALS-causal genes, and highlight selective protein degradation impairment in ALS pathophysiology.
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| 3. |
- Achour, Cyrinne, 1991-
(författare)
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Canonical and non-canonical functions of METTL3 in breast cancer
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Gene expression is spatially and temporally regulated at multiple levels. N6-methyladenosine (m6A) is the most prevalent internal modification in messenger RNA (mRNA) and long noncoding RNA (lncRNAs). m6A plays important roles in multiple cellular processes including stem cell pluripotency, adipogenesis, spermatogenesis, neurogenesis, circadian rhythm and development by modulating RNA splicing, export, stability, degradation and translation. Although aberrant m6A methylation has been reported in various types of cancer, the underlying molecular functions of METTL3, the solely catalytic subunit of the m6A-methylase complex, has yet to be defined.m6A has been recently identified in nascent pre-mRNA, and more specifically intronic m6A has been linked to exon skipping events. The occurrence of impaired alternative splicing (AS) is frequently found during the development of cancer. We performed transcriptome wide analysis in breast cancer cell lines and explored AS events. Our results define an AS signature for breast tumorigenesis. We found that METTL3 modulates AS directly through m6A deposition at the intron-exon junctions or indirectly by the m6A deposition in transcripts encoding for splicing factors and transcription factors. In particular, we show that MYC mRNA harbours the m6A mark, suggesting that METTL3 regulates AS indirectly via the regulation of MYC expression. Indeed, the targets of MYC overlapped with METTL3-associated AS events. Importantly, five of the AS events identified and validated in vitro, are linked to a worse prognosis in breast cancer patients. Additionally, we show that METTL3 enhances the breast cancer phenotype through a dual mechanism depending on its sub-cellular localization. We find that the canonical nuclear function of METTL3 decorates transcripts that are involved in cell proliferation and migration. We observe that METTL3 is highly expressed in the cytoplasmic compartment of breast cancer cells from patients. Remarkably, we uncover that the cytoplasmic METTL3 interacts with subunits of the exocyst, whose subunit EXOC7 has been linked to cell adhesion, migration and invasion. Notably, we show that breast cancer cell lines depleted of METTL3 display less gelatinase activity and invadopodia formation, supporting the role of METTL3 in cell invasion via exocytosis.m6A is a reversible modification, which can be demethylated by the erasers FTO and ALKBH5. Depletion of FTO has been shown to increase the level of m6A in mRNA, however recent studies have reported that FTO could demethylate N6,2´-O-dimethyladenosine (m6Am), adjacent to the 7-methylguanosine cap on mRNA. In the cellular model of colorectal cancer CRC1, depletion of FTO leads to a cancer stem cell phenotype and confers chemotherapy resistance. By performing m6A-RNA immunoprecipitation followed by sequencing (MeRIP), we show that knockdown of FTO in CRC1 cells does not affect the global level of m6A in mRNA but of m6Am level.
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| 4. |
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| 5. |
- Alhouayek, Mireille, et al.
(författare)
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Role of pannexin-1 in the cellular uptake, release and hydrolysis of anandamide by T84 colon cancer cells
- 2019
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- The large pore ion channel pannexin-1 (Panx1) has been reported to play a role in the cellular uptake and release of anandamide (AEA) in the hippocampus. It is not known whether this is a general mechanism or limited to the hippocampus. We have investigated this pharmacologically using T84 colon cancer cells. The cells expressed Panx1 at the mRNA level, and released ATP in a manner that could be reduced by treatment with the Panx1 inhibitors carbenoxolone and mefloquine and the Panxl substrate SR101. However, no significant effects of these compounds upon the uptake or hydrolysis of exogenously applied AEA was seen. Uptake by T84 cells of the other main endocannabinoid 2-arachidonoylglycerol and the AEA homologue palmitoylethanolamide was similarly not affected by carbenoxolone or mefloquine. Total release of tritium from [H-3]AEA-prelabelled T84 cells over 10 min was increased, rather than inhibited by carbenoxolone and mefloquine. Finally, AEA uptake by PC3 prostate cancer and SH-SY5Y neuroblastoma cells, which express functional Panx1 channels, was not inhibited by carbenoxolone. Thus, in contrast to the hippocampus, Panx1 does not appear to play a role in AEA uptake and release from the cells studied under the conditions used.
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| 6. |
- Andreae, Laura C., et al.
(författare)
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Chick Lrrn2, a novel downstream effector of Hoxb1 and Shh, functions in the selective targeting of rhombomere 4 motor neurons
- 2009
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Ingår i: Neural development. - : Springer Science and Business Media LLC. - 1749-8104. ; 4, s. 27-
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Tidskriftsartikel (refereegranskat)abstract
- Background; Capricious is a Drosophila adhesion molecule that regulates specific targeting of a subset of motor neurons to their muscle target. We set out to identify whether one of its vertebrate homologues, Lrrn2, might play an analogous role in the chick.Results; We have shown that Lrrn2 is expressed from early development in the prospective rhombomere 4 (r4) of the chick hindbrain. Subsequently, its expression in the hindbrain becomes restricted to a specific group of motor neurons, the branchiomotor neurons of r4, and their pre-muscle target, the second branchial arch (BA2), along with other sites outside the hindbrain. Misexpression of the signalling molecule Sonic hedgehog (Shh) via in ovo electroporation results in upregulation of Lrrn2 exclusively in r4, while the combined expression of Hoxb1 and Shh is sufficient to induce ectopic Lrrn2 in r1/2. Misexpression of Lrrn2 in r2/3 results in axonal rerouting from the r2 exit point to the r4 exit point and BA2, suggesting a direct role in motor axon guidance.Conclusion; Lrrn2 acts downstream of Hoxb1 and plays a role in the selective targeting of r4 motor neurons to BA2.
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| 7. |
- Bergman, Joakim, et al.
(författare)
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Intrathecal treatment trial of rituximab in progressive MS: An open-label phase 1b study
- 2018
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Ingår i: Neurology. - : Wolters Kluwer. - 1526-632X .- 0028-3878. ; 91:20
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVES: To perform a phase 1b assessment of the safety and feasibility of intrathecally delivered rituximab as a treatment for progressive multiple sclerosis (PMS) and to evaluate the effect of treatment on disability and CSF biomarkers during a 1-year follow-up period. METHODS: Three doses of rituximab (25 mg with a 1-week interval) were administered in 23 patients with PMS via a ventricular catheter inserted into the right frontal horn and connected to a subcutaneous Ommaya reservoir. Follow-ups were performed at 1, 3, 6, 9, and 12 months. RESULTS: Mild to moderate vertigo and nausea were common but temporary adverse events associated with intrathecal rituximab infusion, which was otherwise well tolerated. The only severe adverse event was a case of low-virulent bacterial meningitis that was treated effectively. Of 7 clinical assessments, only 1 showed statistically significant improvement 1 year after treatment. No treatment effect was observed during the follow-up period among 6 CSF biomarkers. CONCLUSIONS: Intrathecal administration of rituximab was well tolerated. However, it may involve a risk for injection-related infections. The lack of a control group precludes conclusions being drawn regarding treatment efficacy. CLINICALTRIALSGOV IDENTIFIER: NCT01719159. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that intrathecal rituximab treatment is well tolerated and feasible in PMS but involves a risk of severe infections. © 2018 American Academy of Neurology.
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| 8. |
- Bergman, Joakim, 1989-
(författare)
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Studies of the Biology of Intrathecal Treatment in Progressive MS
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: Multiple Sclerosis (MS) is a chronic, inflammatory, autoimmune disease, affecting the central nervous system (CNS). About 85% of afflicted present with a relapsing-remitting form of the disease (RRMS), for which a breakthrough in treatment was made in 2008 with rituximab, an antibody directed towards CD20, a surface antigen on B-cells. These findings also contributed to cementing the importance of the B-cell’s role in MS pathophysiology. However, MS also exist as a progressive phenotype, affecting most MS patients either from onset or after a transition from RRMS, and for progressive MS the same treatment effect of anti-CD20 has not been observed. Still, studies have found follicle-like structures containing B-cells in meninges and subarachnoid space of the cortex in progressive MS brains, supporting the involvement of B-cells. Evidence also support the existence of a chronic, low-grade inflammatory process compartmentalised within the CNS that correlates with the progressive phase of MS, which may present a treatment barrier towards anti-CD20. Peripherally administrated therapeutic antibodies cross the intact blood-brain barrier with low efficiency with only 0.1-0.5% of the plasma concentration occurring in the cerebrospinal fluid (CSF). Intrathecal (IT) administration circumvents the blood-brain barrier, presenting an opportunity to better target the CNS B-cells.Aims: To evaluate the safety and feasibility of intrathecal anti-CD20 therapy with rituximab in progressive MS, its effect on disease progression through clinical parameters, and impact on biomarkers in CSF. Furthermore, this thesis aimed to evaluate the effect on biomarkers representative of cell injury related to insertion of a ventricular catheter for drug administration and to examine the interstitial milieu in the brain through microdialysis (MD).Methods: The thesis is based on the open-label, phase IIb, multicentre clinical trial Intrathecal Treatment Trial in Progressive Multiple Sclerosis (ITT-PMS; EudraCT 2008-002626-11), in which 23 participants received IT treatment with rituximab, and the extended follow-up study, ITT-PMS extension (EudraCT 2012-000721-53). All participants received a ventricular catheter and an Ommaya reservoir for drug administration through a neurosurgical procedure, and 10 participants received a MD catheter in parallel to the ventricular catheter for 10 days. The treatment effect was evaluated by regular clinical evaluations and analyses of CSF. The clinical outcome was evaluated through walking and upper-limb function tests, and by clinical evaluation scales. Levels of selected CSF biomarkers were analysed from the same time-points as the clinical evaluations.Results: After the completion of the extension trial, one clinical parameter (cognitive performance) showed improvement but could most likely be explained by a learning effect. Worsening of walking speed was observed, while the remaining clinical parameters showed no change. Two severe adverse events occurred in the form of low-virulent bacterial meningitis caused by Propionibacterium, but both were treated effectively with antibiotics without residual symptoms. A ‘spike’ was noticed in the level of lumbar CSF neurofilament light (NFL) following surgery but returned to pre-surgery baseline within 6-12 months. No change was observed for any of the other lumbar CSF biomarkers. No meaningful correlation of protein levels was observed when comparing MD samples, ventricular CSF, and lumbar CSF.Conclusions: Intrathecal treatment through intraventricular administration was well tolerated but not without risks. A continued progression was observed in gait impairment. The insertion of the ventricular catheter caused white matter injury, measured through an increase in NFL in lumbar CSF, in direct association with the surgical procedure. No impact was observed on other CSF biomarkers. There was a poor correlation between different CNS compartments regarding protein levels, arguing for caution in drawing conclusions about brain pathophysiology from lumbar CSF samples.
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| 9. |
- Cunningham, Thomas J., et al.
(författare)
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DNA Editing for Amyotrophic Lateral Sclerosis : Leading Off First Base
- 2020
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Ingår i: The CRISPR Journal. - : Mary Ann Liebert. - 2573-1599 .- 2573-1602. ; 3:2, s. 75-77
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Gene therapy in a mouse model for amyotrophic lateral sclerosis (ALS) illustrates the rapid deployment of base editing in therapeutic modeling of neurodegenerative disease.
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| 10. |
- Devoy, Anny, et al.
(författare)
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Generation and analysis of innovative genomically humanized knockin SOD1, TARDBP (TDP-43), and FUS mouse models
- 2021
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Ingår i: iScience. - : Elsevier. - 2589-0042. ; 24:12
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Tidskriftsartikel (refereegranskat)abstract
- Amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) is a fatal neurodegenerative disorder, and continued innovation is needed for improved understanding and for developing therapeutics. We have created next-generation genomically humanized knockin mouse models, by replacing the mouse genomic region of Sod1, Tardbp (TDP-43), and Fus, with their human orthologs, preserving human protein biochemistry and splicing with exons and introns intact. We establish a new standard of large knockin allele quality control, demonstrating the utility of indirect capture for enrichment of a genomic region of interest followed by Oxford Nanopore sequencing. Extensive analysis shows that homozygous humanized animals only express human protein at endogenous levels. Characterization of humanized FUS animals showed that they are phenotypically normal throughout their lifespan. These humanized strains are vital for preclinical assessment of interventions and serve as templates for the addition of coding or non-coding human ALS/FTD mutations to dissect disease pathomechanisms, in a physiological context.
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