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| 2. |
- Herdenberg, Carl, 1982-
(författare)
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Molecular and physiological functions of LRIG proteins and netrin-1 in health and disease
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The leucine-rich repeats and immunoglobulin-like domains (LRIG) gene family has three members, LRIG1, LRIG2, and LRIG3, that encode three structurally similar transmembrane proteins. LRIG1 is a receptor tyrosine kinase regulator, tumor suppressor, and stem cell marker in the skin, intestine, and brain. LRIG2 and LRIG3 have been less studied but shown to interact with LRIG1. The different roles and mechanisms of action of LRIG proteins have not yet been fully elucidated. In Caenorhabditis elegans (C. elegans), the LRIG homolog SMA-10 regulates bone morphogenetic protein (BMP) signaling; however, this function has not been demonstrated for mammalian LRIG proteins. In mice, the gene encoding the neurodevelopmental guidance cue netrin-1, Ntn1, interacts with Lrig3 in inner ear development. The physical interactions between LRIG proteins and other proteins are mostly unknown. Here, we describe an LRIG1-centered protein interaction network that regulates growth factor receptor levels. The LRIG1 interactome comprised LRIG2 and LRIG3 as well as many unanticipated proteins. An unbiased pathological examination of female mice with different Lrig3 genotypes (homozygous, heterozygous, or knockout) revealed a reduced incidence of spontaneous fatty liver and lymphocytic hyperplasia of the spleen in Lrig3-null mice. Female Lrig3-null mice also had a lower incidence of microvesicular cytoplasm in the liver after eight weeks on a high-fat diet. To further explore the molecular and physiological functions of LRIG proteins, we generated Lrig-null (Lrig1-/-;Lrig2-/-;Lrig3-/-) mouse embryonic fibroblasts (MEFs), which displayed a deficiency in adipogenesis caused by impaired BMP signaling. LRIG1 and LRIG3, but not LRIG2, sensitized cells to BMP and rescued the adipogenesis deficiency in Lrig-null MEFs. In C. elegans, the LRIG homolog sma-10 was needed for proper lipid accumulation. By analyzing data from the UK Biobank and GENiAL cohort, we found that certain LRIG1 gene variants were associated with a higher body mass index (BMI) yet protected against type 2 diabetes. This effect was probably mediated by altered adipocyte morphology. CRISPR/Cas9-mediated ablation of Ntn1 revealed that the BMP-promoting function of LRIG1 and LRIG3 was opposed by netrin-1, which functioned as an inhibitor of BMP signaling via its receptor neogenin.In summary, the present thesis describes a novel LRIG protein interaction network, the regulation of BMP signaling by LRIG proteins and netrin-1, and an important function of LRIG proteins in regulating fat metabolism with implications for human metabolic health.
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- Hollandsworth, Hannah M., et al.
(författare)
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Fluorophore-conjugated Helicobacter pylori recombinant membrane protein (HopQ) labels primary colon cancer and metastases in orthotopic mouse models by binding CEA-related cell adhesion molecules
- 2020
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Ingår i: Translational Oncology. - : Elsevier. - 1944-7124 .- 1936-5233. ; 13:12
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Tidskriftsartikel (refereegranskat)abstract
- HopQ is an outer-membrane protein of Helicobacter pylori that binds to human carcinoembryonic antigen-related cell-adhesion molecules (CEACAMs) with high specificity. We aimed to investigate fluorescence targeting of CEACAM-expressing colorectal tumors in patient-derived orthotopic xenograft (PDOX) models with fluorescently labeled recombinant HopQ (rHopQ). Western blotting, flow cytometry and ELISA were performed to determine the efficiency of rHopQ binding to CEACAMs. rHopQ was conjugated to IR800DyeCW (rHopQ-IR800). Nude mice received orthotopic implantation of colon cancer tumors. Three weeks later, mice were administered 25 μg or 50 μg HopQ-IR800 and imaged 24 or 48 h later. Intravital images were analyzed for tumor-to-background ratio (TBR). Flow cytometry and ELISA demonstrated binding of HopQ to CEACAM1, 3 and 5. Dose-response intravital imaging in PDOX models demonstrated optimal results 48 h after administration of 50 μg rHopQ-IR800 (TBR = 3.576) in our protocol. Orthotopic models demonstrated clear tumor margins of primary tumors and small regional metastases with a mean TBR = 3.678 (SD ± 1.027). rHopQ showed specific binding to various CEACAMs in PDOX models. rHopQ may be useful for CEACAM-positive tumor and metastasis detection for pre-surgical diagnosis, intra-operative imaging and fluorescence-guided surgery.
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- Hui, Zhixuan, et al.
(författare)
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TGFβ-induced EN1 promotes tumor budding of adenoid cystic carcinoma in patient-derived organoid model
- 2024
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Ingår i: International Journal of Cancer. - : John Wiley & Sons. - 0020-7136 .- 1097-0215. ; 154:10, s. 1814-1827
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Tidskriftsartikel (refereegranskat)abstract
- Adenoid cystic carcinoma (ACC) and basal cell adenoma (BCA) share many histological characteristics and often need a differential diagnosis in clinical pathology. Recently, we found homeobox protein engrailed-1 (EN1) was a potential diagnostic marker for ACC in an organoids library of salivary gland tumors (SGTs). Here we aim to confirm EN1 as a differential diagnostic marker for ACC, and further investigate the regulatory mechanism and biological function of EN1 in tumor progression. The transcriptional analysis, quantitative polymerase chain reaction, Western blot and immunohistochemistry staining were performed and revealed that EN1 was specifically and highly expressed in ACC, and accurately differentiated ACC from BCA. Furthermore, TGFβ signaling pathway was found associated with ACC, and the regulation of EN1 through TGFβ was detected in the human ACC cell lines and patient-derived organoids (PDOs). TGFβ-induced EN1 was important in promoting tumor budding in the PDOs model. Interestingly, a high level of EN1 and TGFβ1 in the budding tips was observed in ACC clinical samples, and the expression of EN1 and TGFβ1 in ACC was significantly associated with the clinical stage. In summary, our study verified EN1 is a good diagnostic marker to differentiate ACC from BCA. TGFβ-induced EN1 facilitates the tumor budding of ACC, which might be an important mechanism related to the malignant phenotype of ACC.
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- Kermpatsou, Despoina
(författare)
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Method development for the analysis of protein interactions
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biological processes take place through interactions between macromolecules, such as nucleic acids and proteins. It is, therefore, fundamental to understand the functions of proteins and how they form complexes in order to carry out their role. Importantly, including spatial information in the analyses of protein complexes allows us to account for cell or tissue heterogeneity, highlighting the importance of in situ studies in biologically and clinically relevant material.To that end, methods for the analysis of protein complexes in situ have been developed, such as in situ proximity ligation assay (PLA) and proximity-dependent initiation of hybridisation chain reaction (proxHCR). Both methods depend on antibodies for target recognition and utilise oligonucleotide systems in order to generate reporter signals with fluorescence readout. While in situ PLA employs rolling circle amplification, proxHCR is an enzyme-free method that takes advantage of DNA hybridisation properties. Both methods, however, yield a polymerised reporter signal of protein complex formation.To further the use of proxHCR, we optimised the design of the oligonucleotide system as well as the experimental procedure, so as to increase the robustness and versatility of the assay. In addition, we developed a novel method, MolBoolean, that simultaneously reports the levels of free proteins as well as protein complexes. In this way, we address limitations of earlier methods and provide the opportunity to obtain a more comprehensive picture of biological processes. Our methods provide a means to circumvent the resolution limits of light microscopy by utilising molecular tricks so that protein binding events, that occur below the resolving power of conventional instrumentation, are made visible. The methods presented in the present doctoral thesis provide powerful tools in the analysis of protein interactions and have applications in cell biology studies as well as in diagnostics. Part of this thesis was the examination of the function of 1,25D3-MARRS (membrane-associated, rapid response steroid-binding) receptor, potentially linked to vitamin D3. We investigated the expression and subcellular localisation of 1,25D3-MARRS in an array of cell lines and employed siRNA-mediated depletion to examine effects on cellular processes in androgen-independent prostate cancer cell models. Our data suggest that 1,25D3-MARRS supports cell proliferation and might have a role in cell migration. Additionally, we observe an effect on the regulation of intracellular vitamin D3 levels. With this study, we contribute to the understanding of the role of 1,25D3-MARRS in prostate cancer cells, that could potentially prove of value in the adaptation of therapeutic strategy for prostate carcinoma.
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| 6. |
- Mallikarjuna, Pramod, et al.
(författare)
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The Synergistic Cooperation between TGF-Cancer and Fibrosis
- 2022
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Ingår i: Biomolecules. - : MDPI. - 2218-273X. ; 12:5
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Forskningsöversikt (refereegranskat)abstract
- Transforming growth factor β (TGF-β) is a multifunctional cytokine regulating homeostasis and immune responses in adult animals and humans. Aberrant and overactive TGF-β signaling promotes cancer initiation and fibrosis through epithelial–mesenchymal transition (EMT), as well as the invasion and metastatic growth of cancer cells. TGF-β is a key factor that is active during hypoxic conditions in cancer and is thereby capable of contributing to angiogenesis in various types of cancer. Another potent role of TGF-β is suppressing immune responses in cancer patients. The strong tumor-promoting effects of TGF-β and its profibrotic effects make it a focus for the development of novel therapeutic strategies against cancer and fibrosis as well as an attractive drug target in combination with immune regulatory checkpoint inhibitors. TGF-β belongs to a family of cytokines that exert their function through signaling via serine/threonine kinase transmembrane receptors to intracellular Smad proteins via the canonical pathway and in combination with co-regulators such as the adaptor protein and E3 ubiquitin ligases TRAF4 and TRAF6 to promote non-canonical pathways. Finally, the outcome of gene transcription initiated by TGF-β is context-dependent and controlled by signals exerted by other growth factors such as EGF and Wnt. Here, we discuss the synergistic cooperation between TGF-β and hypoxia in development, fibrosis and cancer.
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| 7. |
- Rakhimova, Olena, et al.
(författare)
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Cytokine Secretion, Viability, and Real-Time Proliferation of Apical-Papilla Stem Cells Upon Exposure to Oral Bacteria
- 2020
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Ingår i: Frontiers in Cellular and Infection Microbiology. - : Frontiers Media S.A.. - 2235-2988. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- The use of stem cells from the apical papilla (SCAPs) has been proposed as a means of promoting root maturation in permanent immature teeth, and plays a significant role in regenerative dental procedures. However, the role of SCAPs may be compromised by microenvironmental factors, such as hypoxic conditions and the presence of bacteria from infected dental root canals. We aim to investigate oral bacterial modulation of SCAP in terms of binding capacity using flow cytometry and imaging, real-time cell proliferation monitoring, and cytokine secretion (IL-6, IL-8, and TGF-β isoforms) under anaerobic conditions. SCAPs were exposed to key species in dental root canal infection, namely Actinomyces gerensceriae, Slackia exigua, Fusobacterium nucleatum, and Enterococcus faecalis, as well as two probiotic strains, Lactobacillus gasseri strain B6 and Lactobacillus reuteri (DSM 17938). We found that A. gerensceriae, S. exigua, F. nucleatum, and E. faecalis, but not the Lactobacillus probiotic strains bind to SCAPs on anaerobic conditions. Enterococcus faecalis and F. nucleatum exhibited the strongest binding capacity, resulting in significantly reduced SCAP proliferation. Notably, F. nucleatum, but not E. faecalis, induce production of the proinflammatory chemokine IL-8 and IL-10 from SCAPs. Production of TGF-β1 and TGF-β2 by SCAPs was dependent on species, cell line, and time, but secretion of TGF-β3 did not vary significantly over time. In conclusion, SCAP response is compromised when exposed to bacterial stimuli from infected dental root canals in anaerobic conditions. Thus, stem cell-mediated endodontic regenerative studies need to include microenvironmental conditions, such as the presence of microorganisms to promote further advantage in the field.
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| 8. |
- Song, Jie, 1984-, et al.
(författare)
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The ubiquitin-ligase TRAF6 and TGFβ type I receptor form a complex with Aurora kinase B contributing to mitotic progression and cytokinesis in cancer cells
- 2022
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Ingår i: EBioMedicine. - : Elsevier. - 2352-3964. ; 82
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundTransforming growth factor β (TGFβ) is overexpressed in several advanced cancer types and promotes tumor progression. We have reported that the intracellular domain (ICD) of TGFβ receptor (TβR) I is cleaved by proteolytic enzymes in cancer cells, and then translocated to the nucleus in a manner dependent on the endosomal adaptor proteins APPL1/2, driving an invasiveness program. How cancer cells evade TGFβ-induced growth inhibition is unclear.MethodsWe performed microarray analysis to search for genes regulated by APPL1/2 proteins in castration-resistant prostate cancer (CRPC) cells. We investigated the role of TβRI and TRAF6 in mitosis in cancer cell lines cultured in 10% FBS in the absence of exogenous TGFβ. The molecular mechanism of the ubiquitination of AURKB by TRAF6 in mitosis and the formation of AURKB–TβRI complex in cancer cell lines and tissue microarrays was also studied.FindingsDuring mitosis and cytokinesis, AURKB–TβRI complexes formed in midbodies in CRPC and KELLY neuroblastoma cells. TRAF6 induced polyubiquitination of AURKB on K85 and K87, protruding on the surface of AURKB to facilitate its activation. AURKB–TβRI complexes in patient's tumor tissue sections correlated with the malignancy of prostate cancer.InterpretationThe AURKB–TβRI complex may become a prognostic biomarker for patients with risk of developing aggressive PC.
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| 9. |
- Thakur, Noopur, et al.
(författare)
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Smad7 Enhances TGF-β-Induced Transcription of c-Jun and HDAC6 Promoting Invasion of Prostate Cancer Cells
- 2020
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Ingår i: iScience. - : Cell Press. - 2589-0042. ; 23:9
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Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor β (TGF-β) enhances migration and invasion of cancer cells, causing life-threatening metastasis. Smad7 expression is induced by TGF-β to control TGF-β signaling in a negative feedback manner. Here we report an additional function of Smad7, i.e., to enhance TGF-β induction of c-Jun and HDAC6 via binding to their regulatory regions, promoting migration and invasion of prostate cancer cells. Lysine 102 in Smad7 is crucial for binding to specific consensus sites in c-Jun and HDAC6, even when endogenous Smad2, 3, and 4 were silenced by siRNA. A correlation between the mRNA expression of Smad7 and HDAC6, Smad7 and c-Jun, and c-Jun and HDAC6 was found in public databases from analyses of prostate cancer tissues. High expression of Smad7, HDAC6, and c-Jun correlated with poor prognosis for patients with prostate cancer. The knowledge that Smad7 can activate transcription of proinvasive genes leading to prostate cancer progression provides clinically relevant information.
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| 10. |
- Tumkur Sitaram, Raviprakash, 1974-, et al.
(författare)
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Role of Wnt Signaling Pathways in Clear Cell Renal Cell Carcinoma Pathogenesis in Relation to VHL and HIF Status
- 2020
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Ingår i: Clinical Oncology and Research. - 2613-4942. ; 3:3
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Forskningsöversikt (refereegranskat)abstract
- Renal cell carcinoma (RCC) encompasses various tumor types characterized by a variety of genetic abnormalities. The genetic changes, like mutations, deletions, and epigenetic alterations, can affect the signaling components and signaling networks, causing the modification of tumor pathogenesis and prognosis of RCC. The most prevalent RCC, clear cell RCC (ccRCC), is asymptomatic in the early stages, refractory to chemotherapy and radiation therapy, and has a poorer prognosis compared with the papillary and chromophobe ccRCC types. Loss of the VHL gene and upregulation of oxygen sensors, hypoxia-inducible factor alphas (HIF-α), which promote different growth factors, is a signature of sporadic ccRCC. The VHL-HIF-α and Wnt/β-catenin pathways are closely connected and contribute to the ontogeny of ccRCC. This review confines to ccRCC and the role of the Wnt/β-catenin signaling pathways and its crosstalk with VHL/HIF.
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