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Efficient TGF beta-induced epithelial-mesenchymal transition depends on hyaluronan synthase HAS2
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab.
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2013 (English)In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 32, no 37, 4355-4365 p.Article 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.

Place, publisher, year, edition, pages
2013. Vol. 32, no 37, 4355-4365 p.
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-190691DOI: 10.1038/onc.2012.475ISI: 000324404200004PubMedID: 23108409OAI: oai:DiVA.org:uu-190691DiVA: diva2:584035
Available from: 2013-01-08 Created: 2013-01-08 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Importance of Hyaluronan Metabolism and Signalling in Tumour Progression
Open this publication in new window or tab >>Importance of Hyaluronan Metabolism and Signalling in Tumour Progression
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Hyaluronan, an unbranched glycosaminoglycan of the extracellular matrix, has an amazingly simple structure. Initially thought to fulfil only hydrating and space-filling functions in tissues, evidence generated during the past decades shows that hyaluronan is involved in intriguingly complex signalling events in health and disease. In cancer, increased hyaluronan levels have been correlated with poor patient survival.

The research underlying this thesis sheds light on the interplay between hyaluronan, its producing and degrading enzymes as well as the triggered intracellular signalling in the metastatic cascade. Utilising breast cancer and normal mammary cells, paper I and II investigate the initial steps of tumour progression: proliferation, invasion and epithelial-mesenchymal transition. Hyaluronan synthase 2 plays a central role in all these processes. In paper III, the focus is shifted toward growth factor-induced hyaluronan production. Stimulation with PDGF-BB, which can be secreted by tumour cells, increased hyaluronan production via upregulation of HAS2 in fibroblast cultures. Finally, paper IV discusses the involvement of hyaluronidases and CD44 in angiogenesis and intravasation – events that are associated with advanced cancer stages.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 59 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 855
Keyword
Hyaluronan, CD44, cancer, growth factors
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-190715 (URN)978-91-554-8574-0 (ISBN)
Public defence
2013-02-27, B42, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2013-02-04 Created: 2013-01-08 Last updated: 2013-04-02Bibliographically approved
2. Importance of Hyaluronan-CD44 Signaling in Tumor Progression: Crosstalk with TGFβ and PDGF-BB Signaling
Open this publication in new window or tab >>Importance of Hyaluronan-CD44 Signaling in Tumor Progression: Crosstalk with TGFβ and PDGF-BB Signaling
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In order for solid tumors to metastasize, tumor cells must acquire the ability to invade the surrounding tissue and intravasate into blood- or lymph vessels, survive in the circulation and then extravasate at a distant site to form a new tumor. Overexpression of the glycosaminoglycan hyaluronan, and its adhesion receptor CD44, correlate with breast cancer progression. This thesis focuses on the role of hyaluronan in tumor invasion and metastasis.

In paper I, we demonstrated that upregulation of the hyaluronan synthesizing enzyme hyaluronan synthase 2 (HAS2) was crucial for transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transition (EMT) in mammary epithelial cells. In paper II, we further demonstrated that silencing of HAS2 decreased the invasive behavior of bone-metastasizing breast cancer cells, via upregulation of tissue inhibitor for metalloproteinase 1 (TIMP1), and dephosphorylation of focal adhesion kinase (FAK).

During tumorigenesis, stromal cells, such as fibroblasts, play important roles and several growth factors are synthesized, promoting crosstalk between different cell surface receptors. In paper III, we investigated the crosstalk between the hyaluronan receptor CD44 and the receptors for TGFβ and platelet-derived growth factor BB (PDGF-BB) in dermal fibroblasts. We found that the receptors for the three molecules form a ternary complex, and that PDGF-BB can activate the Smad pathway downstream of TGFβRI. Importantly, CD44 negatively modulated the signaling of both PDGF-BB and TGFβ.

In paper IV, we studied the process by which breast cancer cells invade blood-vessels and the role of hyaluronan and CD44 in angiogenesis. Importantly, CD44, or the hyaluronan degrading enzyme hyaluronidase 2 (HYAL2), decreased the capacity of endothelial cells to form tubes in a 3D in vivo-like assay.  Collectively, our studies add to the understanding of the role of hyaluronan in tumor progression.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 62 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 894
Keyword
extracellular matrix, growth factor, hyaluronan synthase, hyaluronidase, epithelial-mesenchymal transition
National Category
Medical and Health Sciences
Research subject
Molecular Cellbiology
Identifiers
urn:nbn:se:uu:diva-198165 (URN)978-91-554-8649-5 (ISBN)
Public defence
2013-05-24, B42, Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2013-05-03 Created: 2013-04-10 Last updated: 2015-09-11Bibliographically approved
3. Regulation of Hyaluronan Synthesis and Signaling via CD44 in Cancer
Open this publication in new window or tab >>Regulation of Hyaluronan Synthesis and Signaling via CD44 in Cancer
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. 54 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1291
Keyword
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:nbn:se:uu:diva-312485 (URN)978-91-554-9797-2 (ISBN)
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: 2017-02-15

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Porsch, HelenaBernert, BeritMehić, MerimaHeldin, Carl-HenrikHeldin, Paraskevi

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