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Mehic, M. S., de Sa, V. K., Hebestreit, S., Heldin, P. & Heldin, C.-H. S. (2018). The role of deubiquitinating enzyme USP17, hyaluronan synthase 2, and hyaluronan in non-small-cell lung cancer oncogenic transformation. Paper presented at International Conference of the American-Association-for-Cancer-Research (AACR), MAY 04-06, 2017, Sao Paulo, BRAZIL. Clinical Cancer Research, 24(1), 96-96
Open this publication in new window or tab >>The role of deubiquitinating enzyme USP17, hyaluronan synthase 2, and hyaluronan in non-small-cell lung cancer oncogenic transformation
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2018 (English)In: Clinical Cancer Research, ISSN 1078-0432, E-ISSN 1557-3265, Vol. 24, no 1, p. 96-96Article in journal, Meeting abstract (Other academic) Published
Abstract [en]

Introduction: Lung cancer is the result of a multistep accumulation of genetic and/or epigenetic alterations; therefore, a better understanding of the molecular mechanism by which these alterations affect lung cancer pathogenesis would provide new diagnostic procedures and prognostic factors for early detection of recurrence. The remarkable qualitative and quantitative modifications of extracellular matrix components as the deubiquitinating enzyme (USP17), hyaluronan (HA), and hyaluronan synthases 2 (HAS 2) may favor invasion, cellular motility, and proliferation in several cancers including lung.

Results: 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. Numerous epithelial cells expressed USP17 and HAS2 in dysplasia compared to squamous cell carcinoma (SqCC) (p=0.001). USP17 and HAS2 were prominently expressed in adenocarcinoma (ADC) (p≤0.005). HA immunostaining indexes were increased in ADC and SqCC compared to normal and dysplasia cells (p=0.05). Consistent with the immunohistochemical analyses, low amounts of hyaluronan and USP17 were observed in SqCC by confocal analysis, coincident with less colocalization as determined by confocal microscopy. In contrast, a high expression of hyaluronan (48% of positive index) and high USP17 expression (78% of positive index) in ADC was consistent with a higher degree of colocalization.

Conclusions: HAS2, hyaluronan and USP17 were expressed at higher levels in particular in preneoplastic lesions and ADC, suggesting a role in NSCLC oncogenic transformation, possibly by promoting cellular division by USP17-mediated. Elucidation of the mechanism of how USP17 and HAS2 cooperate in the regulation of the cell cycle might be of therapeutic importance.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-347110 (URN)10.1158/1557-3265.TCM17-B64 (DOI)000419180000165 ()
Conference
International Conference of the American-Association-for-Cancer-Research (AACR), MAY 04-06, 2017, Sao Paulo, BRAZIL
Available from: 2018-03-26 Created: 2018-03-26 Last updated: 2018-03-26Bibliographically approved
Mehić, M., de Sa, V. K., Hebestreit, S., Heldin, C.-H. & Heldin, P. (2017). The deubiquitinating enzymes USP4 and USP17 target hyaluronan synthase 2 and differentially affect its function. Oncogenesis, 6, Article ID e348.
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.

Keyword
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
Bart, G., Vico, N. O., Hassinen, A., Pujol, F. M., Deen, A. J., Ruusala, A., . . . Tammi, M. I. (2015). Fluorescence Resonance Energy Transfer (FRET) and Proximity Ligation Assays Reveal Functionally Relevant Homo-and Heteromeric Complexes among Hyaluronan Synthases HAS1, HAS2, and HAS3. Journal of Biological Chemistry, 290(18), 11479-11490
Open this publication in new window or tab >>Fluorescence Resonance Energy Transfer (FRET) and Proximity Ligation Assays Reveal Functionally Relevant Homo-and Heteromeric Complexes among Hyaluronan Synthases HAS1, HAS2, and HAS3
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2015 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 18, p. 11479-11490Article in journal (Refereed) Published
Abstract [en]

In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1-3 (HAS1-3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647-23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-255270 (URN)10.1074/jbc.M115.640581 (DOI)000353719400024 ()25795779 (PubMedID)
Available from: 2015-06-22 Created: 2015-06-15 Last updated: 2017-12-04Bibliographically approved
Simpson, M. A. & Heldin, P. (2014). Advances in Cancer Research: Hyaluronan Signaling And Turnover, Volume One Hundred And Twenty Three, Preface. In: Simpson, MA andHeldin, P (Ed.), Hyaluronan Signaling and Turnover: (pp. 15-16). Elsevier
Open this publication in new window or tab >>Advances in Cancer Research: Hyaluronan Signaling And Turnover, Volume One Hundred And Twenty Three, Preface
2014 (English)In: Hyaluronan Signaling and Turnover / [ed] Simpson, MA andHeldin, P, Elsevier, 2014, p. 15-16Chapter in book (Other academic)
Place, publisher, year, edition, pages
Elsevier, 2014
Series
Advances in Cancer Research, ISSN 0065-230X ; 123
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-232762 (URN)000340575400001 ()978-0-12-800092-2 (ISBN)
Available from: 2014-10-01 Created: 2014-09-24 Last updated: 2014-10-01Bibliographically approved
Heldin, P., Basu, K., Kozlova, I. & Porsch, H. (2014). HAS2 and CD44 in Breast Tumorigenesis. In: Simpson, MA and Heldin, P (Ed.), Hyaluronan Signaling and Turnover: (pp. 211-229). Elsevier, 123
Open this publication in new window or tab >>HAS2 and CD44 in Breast Tumorigenesis
2014 (English)In: Hyaluronan Signaling and Turnover / [ed] Simpson, MA and Heldin, P, Elsevier, 2014, Vol. 123, p. 211-229Chapter in book (Refereed)
Abstract [en]

Metastatic spread of breast cancer cells, facilitated by the epithelial-mesenchymal transition (EMT) process, is responsible for the majority of breast cancer mortality. Increased levels of hyaluronan due to deregulation of hyaluronan-synthesizing enzymes, like HAS2, and expression of CD44, the key receptor for hyaluronan, are correlated to poor outcome of patients with basal-like breast cancer. TGFβ induces HAS2 and CD44, both of which are required in the course of efficient TGFβ-induced EMT processes by mammary epithelial cells. Elucidation of the molecular mechanisms underlying tumor-stroma interactions in breast cancer including the regulation of HAS2 and CD44 expression may contribute to the development of better strategies to treat breast cancer patients.

Place, publisher, year, edition, pages
Elsevier, 2014
Series
Advances in Cancer Research, ISSN 0065-230X ; 123
Keyword
Epithelial-Mesenchymal Transition, Cancer Cell-Lines, Hyaluronan Synthase 2, Growth Factor-Bb, Gene-Expression, Tgf-Beta, Stem-Cells, Tumor Microenvironment, Adhesion Molecules, Mesothelial Cells
National Category
Basic Medicine
Identifiers
urn:nbn:se:uu:diva-231936 (URN)10.1016/B978-0-12-800092-2.00008-3 (DOI)000340575400016 ()25081531 (PubMedID)978-0-12-800092-2 (ISBN)
Available from: 2014-09-11 Created: 2014-09-11 Last updated: 2018-01-11Bibliographically approved
Porsch, H., Mehic, M., Olofsson, B., Heldin, P. & Heldin, C.-H. (2014). Platelet-derived Growth Factor beta-Receptor, Transforming Growth Factor beta Type I Receptor, and CD44 Protein Modulate Each Other's Signaling and Stability. Journal of Biological Chemistry, 289(28), 19747-19757
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
Simpson, M. A. & Heldin, P. (2014). Preface. Advances in Cancer Research, 123, xv-xvi
Open this publication in new window or tab >>Preface
2014 (English)In: Advances in Cancer Research, ISSN 0065-230X, E-ISSN 2162-5557, Vol. 123, p. xv-xviArticle in journal, Editorial material (Refereed) Published
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-231940 (URN)10.1016/B978-0-12-800092-2.09986-X (DOI)25081537 (PubMedID)
Available from: 2014-09-11 Created: 2014-09-11 Last updated: 2017-12-05Bibliographically approved
Tzanakakis, G., Kovalszky, I., Heldin, P. & Nikitovic, D. (2014). Proteoglycans/Glycosaminoglycans: from basic research to clinical practice. BioMed Research International, 2014, Article ID 295254.
Open this publication in new window or tab >>Proteoglycans/Glycosaminoglycans: from basic research to clinical practice
2014 (English)In: BioMed Research International, ISSN 2314-6133, E-ISSN 2314-6141, Vol. 2014, article id 295254Article in journal, Editorial material (Refereed) Published
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-242711 (URN)10.1155/2014/295254 (DOI)000347809000001 ()25587531 (PubMedID)
Available from: 2015-01-30 Created: 2015-01-30 Last updated: 2017-12-05Bibliographically approved
Heldin, P., Basu, K., Olofsson, B., Porsch, H., Kozlova, I. & Kahata, K. (2013). Deregulation of hyaluronan synthesis, degradation and binding promotes breast cancer. Journal of Biochemistry (Tokyo), 154(5), 395-408
Open this publication in new window or tab >>Deregulation of hyaluronan synthesis, degradation and binding promotes breast cancer
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2013 (English)In: Journal of Biochemistry (Tokyo), ISSN 0021-924X, E-ISSN 1756-2651, Vol. 154, no 5, p. 395-408Article in journal (Refereed) Published
Abstract [en]

Clinical and experimental data indicate that hyaluronan accumulates in breast cancer compared with normal breast epithelium, which correlates to poor prognosis. In this review, we discuss the expression of genes encoding enzymes that synthesize or degrade hyaluronan, i.e. hyaluronan synthases and hyaluronidases or bind hyaluronan, i.e. CD44 and receptor for hyaluronan-mediated motility (RHAMM, also designated as HMMR or CD168), in relation to breast cancer progression. Hyaluronan and hyaluronan receptors have multi-faceted roles in signalling events in breast cancer. A better understanding of the molecular mechanisms underlying these signalling pathways is highly warranted and may lead to improvement of cancer treatment.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-211067 (URN)10.1093/jb/mvt085 (DOI)000326652500001 ()24092768 (PubMedID)
Available from: 2013-11-19 Created: 2013-11-19 Last updated: 2017-12-06Bibliographically approved
Porsch, H., Bernert, B., Mehić, M., Theocharis, A. D., Heldin, C.-H. & Heldin, P. (2013). Efficient TGF beta-induced epithelial-mesenchymal transition depends on hyaluronan synthase HAS2. Oncogene, 32(37), 4355-4365
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
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ORCID iD: ORCID iD iconorcid.org/0000-0001-9818-4052

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