| 1. |
- Dyczynski, Matheus, et al.
(författare)
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Targeting autophagy by small molecule inhibitors of vacuolar protein sorting 34 (Vps34) improves the sensitivity of breast cancer cells to Sunitinib
- 2018
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Ingår i: Cancer Letters. - : Elsevier. - 0304-3835 .- 1872-7980. ; 435, s. 32-43
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Tidskriftsartikel (refereegranskat)abstract
- Resistance to chemotherapy is a challenging problem for treatment of cancer patients and autophagy has been shown to mediate development of resistance. In this study we systematically screened a library of 306 known anti-cancer drugs for their ability to induce autophagy using a cell-based assay. 114 of the drugs were classified as autophagy inducers; for 16 drugs, the cytotoxicity was potentiated by siRNA-mediated knock-down of Atg7 and Vps34. These drugs were further evaluated in breast cancer cell lines for autophagy induction, and two tyrosine kinase inhibitors, Sunitinib and Erlotinib, were selected for further studies. For the pharmacological inhibition of autophagy, we have characterized here a novel highly potent selective inhibitor of Vps34, SB02024. SB02024 blocked autophagy in vitro and reduced xenograft growth of two breast cancer cell lines, MDA-MB-231 and MCF-7, in vivo. Vps34 inhibitor significantly potentiated cytotoxicity of Sunitinib and Erlotinib in MCF-7 and MDA-MB-231 in vitro in monolayer cultures and when grown as multicellular spheroids. Our data suggests that inhibition of autophagy significantly improves sensitivity to Sunitinib and Erlotinib and that Vps34 is a promising therapeutic target for combination strategies in breast cancer.
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| 2. |
- Dyczynski, Matheus
(författare)
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The role of autophagy in anticancer therapy
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Autophagy is a fundamental catabolic process, which is utilized by nearly every cell and tissue type upon stress exposure and has been shown to contribute to resistance to chemotherapy in a variety of cancers. The subject of this thesis is to shed light on the role of autophagy in chemotherapy and to investigate novel regulators of autophagy. Multiple clinical trials have been started in order to overcome resistance to standard therapy by combining it with lysosomal inhibitor hydroxychloroquine, yet with limited success. This drug has been shown to have poor cell uptake properties in solid tumors due to tumor acidosis. In paper I we found that the compound Salinomycin is a potent autophagy inhibitor in multiple cancer cell lines, especially under acidic conditions. Salinomycin was able to penetrate the acidic core of multicellular spheroids and decrease cell viability and clonogenic survival of colorectal cancer cells. We also show that Salinomycin efficiently blocked autophagic flux in breast cancer cells. In particular, cancer stem cells derived from cell lines or primary breast cancer tumors showed reduced viability and reduced capability to form mammospheres under Salinomycin treatment. Using mass spectrometry, we could confirm pH-dependent intracellular accumulation of Salinomycin. This data proves the potency of Salinomycin as an anti-cancer drug with capacities to modulate autophagy in the acidic tumor microenvironment. Part of the standard treatment regimen of pediatric patients with Acute Lymphoblastic Leukemia (ALL) are glucocorticoids (GC). This metabolic hormone is effective in inducing cell death in ALL cells. GC mediated inhibition of glucose uptake and upregulation of catabolic processes such as autophagy have previously been reported. In paper II we addressed in detail what metabolic changes occur upon GC treatment in ALL cell lines by parallel time-course proteomics, metabolomics and isotope tracing, and by confirming selected findings by cross-referencing with publicly available microarray data and experimentally by qRT-PCR. Our findings confirmed the onset of growth arrest, autophagy and apoptosis. Not only glucose but also glutamine entry into the Citric-Acid-Cycle was inhibited contrasting the upregulation of glutamine-ammonia-ligase (GLUL) expression suggesting the induction of glutamine synthesis. Potentiating the GLUL-mediated reaction rescued cell viability and reduced autophagic flux suggesting that GLUL induction and glutamine synthesis are relevant for the autophagy induction and sensitivity of ALL cells to GCs. This data provides a comprehensive overview of metabolic changes in ALL cells upon GCs' treatment and may shed light on the mechanism of GC-induced cell death in ALL cells. In paper III we used high-content microscopy to screen the FIMM drug library consisting of 306 anticancer drugs and identified 104 autophagy modulators, of which 16 showed cell death potentiation upon siRNA mediated knock-down of ATG7 (autophagy-related protein 7) and VPS34 (vacuolar protein sorting 34), key regulators of autophagy. We validated the hits in 2 breast cancer cell lines, MDA-MB231 and MCF7, and continued to characterize two of the hits, Erlotinib and Sunitinib, in detail. The collaboration with Sprint Bioscience led to the development of SB02024, a specific inhibitor of the VPS34 kinase. We showed that SB02024 could block autophagy in vitro and in in vivo xenograft mouse models. Combination of SB02024 with Sunitinib and Erlotinib increased cytotoxicity by these drugs in either 2D cell culture, colony formation assays, or, in case of Sunitinib, in cells grown in 3D as multicellular spheroids. This data further strengthens the notion that using VPS34 inhibitors in combination with targeted tyrosine kinase inhibitor-based therapy, and particularly Sunitinib, can overcome resistance and emphasizes their value in cancer treatment. RAS protein activator like 2 (RASAL2) is a known tumor-suppressor regulating members of the RAS-family of oncoproteins. In paper IV we describe for the first time a role for RASAL2 in the induction of autophagy. We found that autophagy induction via pharmaceutical mTOR inhibition or amino acid-starvation increased RASAL2 transcription. Furthermore, RASAL2 protein levels were regulated by autophagy-dependent protein degradation. Thus, in the starved cells, RASAL2 mRNA levels were induced while protein levels declined. Also, depletion of autophagy-related protein 7 (ATG7) that impaired autophagy process resulted in a striking increase in RASAL2 protein levels. RNAi-mediated knockdown of RASAL2 inhibited LC3-II accumulation or GFP-LC3 puncta formation. In silico analysis of RASAL2 revealed two potential LC3 interacting region motifs (LIR), which could point to an interaction between these two proteins. These data suggest that RASAL2 is involved in autophagy and is regulated by autophagy in a negative feedback manner.
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| 3. |
- Pellegrini, Paola, et al.
(författare)
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Tumor acidosis enhances cytotoxic effects and autophagy inhibition by salinomycin on cancer cell lines and cancer stem cells
- 2016
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Ingår i: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 7:24, s. 35703-35723
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Tidskriftsartikel (refereegranskat)abstract
- Sustained autophagy contributes to the metabolic adaptation of cancer cells to hypoxic and acidic microenvironments. Since cells in such environments are resistant to conventional cytotoxic drugs, inhibition of autophagy represents a promising therapeutic strategy in clinical oncology. We previously reported that the efficacy of hydroxychloroquine (HCQ), an autophagy inhibitor under clinical investigation is strongly impaired in acidic tumor environments, due to poor uptake of the drug, a phenomenon widely associated with drug resistance towards many weak bases. In this study we identified salinomycin (SAL) as a potent inhibitor of autophagy and cytotoxic agent effective on several cancer cell lines under conditions of transient and chronic acidosis. Since SAL has been reported to specifically target cancer-stem cells (CSC), we used an established model of breast CSC and CSC derived from breast cancer patients to examine whether this specificity may be associated with autophagy inhibition. We indeed found that CSC-like cells are more sensitive to autophagy inhibition compared to cells not expressing CSC markers. We also report that the ability of SAL to inhibit mammosphere formation from CSC-like cells was dramatically enhanced in acidic conditions. We propose that the development and use of clinically suitable SAL derivatives may result in improved autophagy inhibition in cancer cells and CSC in the acidic tumor microenvironment and lead to clinical benefits.
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| 4. |
- Ramachandran, Mohanraj, 1988-, et al.
(författare)
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Safe and effective treatment of experimental neuroblastoma and glioblastoma using systemically administered triple microRNA-detargeted oncolytic Semliki Forest virus
- 2017
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Ingår i: Clinical Cancer Research. - : American Association for Cancer Research. - 1078-0432 .- 1557-3265. ; 23:6, s. 1519-1530
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE:Glioblastoma multiforme (GBM) and high-risk neuroblastoma are cancers with poor outcome. Immunotherapy in the form of neurotropic oncolytic viruses is a promising therapeutic strategy for these malignancies. Here we evaluate the oncolytic potential of the neurovirulent and partly interferon (IFN)-β-resistant Semliki Forest virus (SFV)-4 in GBMs and neuroblastomas. To reduce neurovirulence we constructed SFV4miRT, which is attenuated in normal CNS cells through insertion of microRNA target sequences for miR124, miR125, miR134 Experimental Design:Oncolytic activity of SFV4miRT was examined in mouse neuroblastoma and GBM cell lines and in patient-derived human glioblastoma cell cultures (HGCC). In vivo neurovirulence and therapeutic efficacy was evaluated in two syngeneic orthotopic glioma models (CT-2A, GL261) and syngeneic subcutaneous neuroblastoma model (NXS2). The role of IFN-β in inhibiting therapeutic efficacy was investigated.RESULTS:The introduction of microRNA target sequences reduced neurovirulence of SFV4 in terms of attenuated replication in mouse CNS cells and ability to cause encephalitis when administered intravenously. A single intravenous injection of SFV4miRT prolonged survival and cured 4 of 8 mice (50%) with NXS2 and 3 of 11 mice (27%) with CT-2A, but not for GL261 tumor bearing mice. In vivo therapeutic efficacy in different tumor models inversely correlated to secretion of IFN-β by respective cells upon SFV4 infection in vitro Similarly, killing efficacy of HGCC lines inversely correlated to IFN-β response and interferon-α⁄β receptor (IFNAR)-1 expression.CONCLUSIONS:SFV4miRT has reduced neurovirulence, while retaining its oncolytic potential. SFV4miRT is an excellent candidate for treatment of GBMs and neuroblastomas with low IFN-β secretion.
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