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Regulation of Hyaluronan Synthesis and Signaling via CD44 in Cancer
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Hyaluronan is a ubiquitous glycosaminoglycan which is an important constituent of the extracellular matrix (ECM). In addition to organizing the extracellular matrix and regulating tissue homeostasis, hyaluronan, by binding to its main cell surface receptor CD44, is involved in intracellular signaling pathways regulating major cellular processes during development, wound healing, inflammation and cancer. Accumulation of hyaluronan in cancer promotes progression of the disease and correlates with poor prognosis. This thesis focuses on the regulation of hyaluronan synthesis and its signaling in normal and cancer cells.

Cancer cells in solid tumors are surrounded by stroma, which has an essential role in the growth and metastasis of tumors. Prominent members of the tumor stroma are fibroblasts, which synthesize ECM components, such as hyaluronan, and secrete growth factors, and activate intracellular signaling pathways. We demonstrate a cross-talk between the receptors for platelet-derived growth factor BB (PDGF-BB), transforming growth factor β (TGFβ) and CD44 in dermal fibroblasts. We found that PDGF-BB can activate the Smad signaling pathway downstream of the TGFβ receptor I (TβRI), and that PDGF-BB-induced migration depends on TβRI. CD44 forms a ternary complex with the receptors for PDGF-BB and TGFβ, and negatively regulates their signaling. Furthermore, we demonstrate that TGFβ stimulation of mammary epithelial cells transcriptionally upregulates hyaluronan synthase 2 (HAS2), which is essential for TGFβ-induced epithelial-mesenchymal transition (EMT); in this process, polarized epithelial cells adapt a mesenchymal phenotype which facilitates migration and invasion.

HAS2 protein activity and stability is regulated by posttranslational modifications, including ubiquitination. We investigated the ubiquitination of HAS2 in aggressive breast cancer cells, whose metastasizing capability depends on HAS2-synthesized hyaluronan. We identified two deubiquitinating enzymes, USP4 and USP17, which target HAS2 and affect its activity and stability.

In summary, these studies increase the knowledge about the regulation of hyaluronan production and its role in cancer progression.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , p. 54
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1291
Keywords [en]
Hyaluronan, CD44, TGFβ, PDGF-BB, cancer, signaling, hyaluronan synthase, epithelial-mesenchymal transition
National Category
Cell and Molecular Biology
Research subject
Biology with specialization in Molecular Biology; Medical Science
Identifiers
URN: urn:nbn:se:uu:diva-312485ISBN: 978-91-554-9797-2 (print)OAI: oai:DiVA.org:uu-312485DiVA, id: diva2:1065110
Public defence
2017-03-03, B/B42, Biomedical Centre (BMC), Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2017-02-10 Created: 2017-01-10 Last updated: 2018-01-13
List of papers
1. Platelet-derived Growth Factor beta-Receptor, Transforming Growth Factor beta Type I Receptor, and CD44 Protein Modulate Each Other's Signaling and Stability
Open this publication in new window or tab >>Platelet-derived Growth Factor beta-Receptor, Transforming Growth Factor beta Type I Receptor, and CD44 Protein Modulate Each Other's Signaling and Stability
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2014 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 28, p. 19747-19757Article in journal (Refereed) Published
Abstract [en]

Growth factors, such as platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta(TGF beta), are key regulators of cellular functions, including proliferation, migration, and differentiation. Growth factor signaling is modulated by context-dependent cross-talk between different signaling pathways. We demonstrate in this study that PDGF-BB induces phosphorylation of Smad2, a downstream mediator of the canonical TGF beta pathway, in primary dermal fibroblasts. The PDGF-BB-mediated Smad2 phosphorylation was dependent on the kinase activities of both TGF beta type I receptor (T beta RI) and PDGF beta-receptor (PDGFR beta), and it was prevented by inhibitory antibodies against TGF beta. Inhibition of the activity of the T beta RI kinase greatly reduced the PDGF-BB-dependent migration in dermal fibroblasts. Moreover, we demonstrate that the receptors for PDGF-BB and TGF beta interact physically in primary dermal fibroblasts and that stimulation with PDGF-BB induces internalization not only of PDGFR beta but also of T beta RI. In addition, silencing of PDGFR beta by siRNA decreased the stability of T beta RI and delayed TGF beta-induced signaling. We further show that the hyaluronan receptor CD44 interacts with both PDGFR beta and T beta RI. Depletion of CD44 by siRNA increased signaling via PDGFR beta and T beta RI by stabilizing the receptor proteins. Our data suggest that cross-talk between PDGFR beta and T beta RI occurs in dermal fibroblasts and that CD44 negatively modulates signaling via these receptors.

National Category
Basic Medicine
Identifiers
urn:nbn:se:uu:diva-230095 (URN)10.1074/jbc.M114.547273 (DOI)000339326800046 ()
Available from: 2014-09-02 Created: 2014-08-19 Last updated: 2018-01-11Bibliographically approved
2. Efficient TGF beta-induced epithelial-mesenchymal transition depends on hyaluronan synthase HAS2
Open this publication in new window or tab >>Efficient TGF beta-induced epithelial-mesenchymal transition depends on hyaluronan synthase HAS2
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2013 (English)In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 32, no 37, p. 4355-4365Article in journal (Refereed) Published
Abstract [en]

Epithelial-mesenchymal transition (EMT) is a developmental program, which can be adopted by cancer cells to increase their migration and ability to form metastases. Transforming growth factor β (TGFβ) is a well-studied inducer of EMT. We demonstrate that TGFβ potently stimulates hyaluronan synthesis via upregulation of hyaluronan synthase 2 (HAS2) in NMuMG mammary epithelial cells. This stimulatory effect requires the kinase active type I TGFβ receptor and is dependent on Smad signaling and activation of the p38 mitogen-activated protein kinase. Knockdown of HAS2 inhibited the TGFβ-induced EMT by about 50%, as determined by the phase contrast microscopy and immunostaining using the EMT marker ZO-1. Furthermore, real-time PCR analysis of the EMT markers fibronectin, Snail1 and Zeb1 revealed decreased expressions upon HAS2 suppression, using specific small interfering RNA (siRNA) for HAS2. Removal of the extracellular hyaluronan by Streptomyces hyaluronidase or inhibiting the binding to its cell surface receptor CD44 by blocking antibodies, did not inhibit TGFβ-induced EMT. Interestingly, HAS2 suppression completely abolished the TGFβ-induced cell migration, whereas CD44 knockdown did not. These observations suggest that TGFβ-dependent HAS2 expression, but not extracellular hyaluronan, has an important regulatory role in TGFβ-induced EMT.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-190691 (URN)10.1038/onc.2012.475 (DOI)000324404200004 ()23108409 (PubMedID)
Available from: 2013-01-08 Created: 2013-01-08 Last updated: 2017-12-06Bibliographically approved
3. The deubiquitinating enzymes USP4 and USP17 target hyaluronan synthase 2 and differentially affect its function
Open this publication in new window or tab >>The deubiquitinating enzymes USP4 and USP17 target hyaluronan synthase 2 and differentially affect its function
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2017 (English)In: Oncogenesis, E-ISSN 2157-9024, Vol. 6, article id e348Article in journal (Refereed) Published
Abstract [en]

The levels of hyaluronan, a ubiquitous glycosaminoglycan prominent in the extracellular matrix, is balanced through the actions of hyaluronan-synthesizing enzymes (HAS1, 2 and 3) and degrading hyaluronidases (Hyal 1, 2, 3 and PH20). Hyaluronan accumulates in rapidly remodeling tissues, such as breast cancer, due to deregulated expression of the HAS2 gene and/or alterations of HAS2 activity. The activity of HAS2 is regulated by post-translational modifications, including ubiquitination. In order to identify deubiquitinating enzymes (DUBs) that are involved in de-ubiquitination of HAS2, a complementary (cDNA) library of 69 Flag-HA-tagged human DUBs cloned into retroviral vectors was screened in human embryonic kidney (HEK) 293T cells for their ability to de-ubiquitinate myc-tagged HAS2. Several DUBs were found to decrease the ubiquitination of 6myc-HAS2, among which, the most effective were USP17 and USP4. USP17 efficiently removed polyubiquitination, whereas USP4 preferentially removed monoubiquitination of 6myc-HAS2. Co-immunoprecipitation studies revealed interactions between HAS2 and USP17, as well as between HAS2 and USP4, in membrane preparations of HEK293T cells. USP17 significantly stabilized 6myc-HAS2 protein levels, whereas USP4 did not. The silencing of USP17 led to decreased hyaluronan production, whereas the suppression of USP4 increased hyaluronan synthesis. Importantly, high levels of USP17 and HAS2 were detected in a panel of cancer cell lines compared to normal cells, and immunohistochemical stainings revealed higher expression of USP17 and HAS2 in tissues of lung cancer patients compared to normal tissue. In conclusion, USP17 and USP4 differently affect HAS2 ubiquitination, and the stability and function of HAS2.

Keywords
cell cycle, ubiquitination, DUBs, growth, hyaluronan, cancer
National Category
Cell and Molecular Biology
Research subject
Biology with specialization in Molecular Biology; Biochemistry
Identifiers
urn:nbn:se:uu:diva-312483 (URN)10.1038/oncsis.2017.45 (DOI)000406047300003 ()28604766 (PubMedID)
Funder
Swedish Cancer Society
Available from: 2017-01-12 Created: 2017-01-10 Last updated: 2018-01-13Bibliographically approved

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