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Regulation of Platelet-Derived Growth Factor Receptor Signaling and its Targeting in Cancer Therapy
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. (PDGF signal transduction)
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Overactivity of platelet-derived growth factor receptor (PDGFR) is a frequent event in many types of solid tumors. Therefore, it is of great importance to uncover the mechanisms that regulate PDGF/PDGFR signalling, to develop efficient inhibitors targeting this pathway. The first step of downregulation of PDGFR activity upon ligand binding is internalization; thus we investigated how endocytosis pathways affect PDGFR signaling. We showed that in Ras-transformed fibroblasts, the internalization of PDGFR is shifted from the routine clathrin-dependent endocytosis to macropinocytosis, which results in enhanced PDGFR activity and subsequent downstream signalling, promoting anchorage-independent growth.

We were also interested in how intracellular trafficking regulates signalling attenuation of PDGFR. We found that His-domain containing protein tyrosine phosphatase (HD-PTP) positively regulates phosphorylation level of the ubiquitin-ligases c-Cbl and Cbl-b; consistently, silencing of HD-PTP led to a decreased level of PDGFR ubiquitination (paper II). Consequently, internalized PDGFR could not be sorted properly and escaped degradation. This resulted in enhanced activation of phospholipase C γ (PLCγ) and changed kinetics of signal transducer and activator of transcription (STAT) 3 signalling, which further increased colony formation of HD-PTP silenced cells in soft agar, indicating a tumor suppressor role of HD-PTP.

Activation of PDGFR leads to stimulation of downstream pathways. We identified Fer kinase as a critical signal transducer downstream of PDGFR in a proteomic screen. We showed that Fer kinase is essential for PDGF-induced STAT3 activation; as a result (paper III), Fer depletion severely blunted the ability of PDGFR signalling to promote anchorage-independent growth in soft agar and delayed tumor initiation in a mouse model.

The crosstalk between host and tumor plays a critical role in tumor progression. At present most anti-cancer drugs are targeting tumor cells; we were interested in how targeting tumor host cells affects the efficacy of anti-tumor therapy. We found that selective PDGFRβ inhibition in host cells exerted tumor inhibitory effects on growth and vascularization of tumors with autocrine PDGF signaling, whereas tumors lacking such stimulation show only minor response on tumor growth (paper IV). Meanwhile, we demonstrated that PDGF/PDGFRβ signalling promotes expression of NG2, a marker for pericytes.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. , 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1092
Keyword [en]
PDGF, PDGFR, Ras, macropinocytosis, Fer, STAT3, HD-PTP, Cbl, ubiquitination, pericyte, vasculature, tumor, ASKA
National Category
Cell and Molecular Biology
Research subject
Biology with specialization in Molecular Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-248172ISBN: 978-91-554-9220-5 (print)OAI: oai:DiVA.org:uu-248172DiVA: diva2:799179
Public defence
2015-05-20, B42, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2015-04-29 Created: 2015-03-30 Last updated: 2015-11-02
List of papers
1. Macropinocytosis of the PDGF β-receptor promotes fibroblast transformation by H-RasG12V
Open this publication in new window or tab >>Macropinocytosis of the PDGF β-receptor promotes fibroblast transformation by H-RasG12V
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2012 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 23, no 13, 2571-2582 p.Article in journal (Refereed) Published
Abstract [en]

Receptor tyrosine kinase (RTK) signaling is frequently increased in tumor cells, sometimes as a result of decreased receptor down-regulation. To what extent the endocytic trafficking routes can contribute to such RTK hyper-activation is unclear. Here, we show for the first time that fibroblast transformation by H-RasG12V induces the internalization of platelet-derived growth factor β-receptor (PDGFRβ) by macropinocytosis, enhancing its signaling activity and increasing anchorage-independent proliferation. H-RasG12V transformation and PDGFRβ activation synergized in stimulating PI 3-kinase activity, leading to receptor macropinocytosis. PDGFRβ macropinocytosis was both necessary and sufficient for enhanced receptor activation. Blocking macropinocytosis by inhibition of phosphatidylinositol (PI) 3-kinase prevented the increase in receptor activity in transformed cells. Conversely, increasing macropinocytosis by Rabankyrin-5 overexpression was sufficient to enhance PDGFRβ activation in non-transformed cells. Simultaneous stimulation with PDGF-BB and epidermal growth factor (EGF) promoted macropinocytosis of both receptors and increased their activation in non-transformed cells. We propose that H-Ras transformation promotes tumor progression by enhancing growth factor receptor signaling as a result of increased receptor macropinocytosis.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-175983 (URN)10.1091/mbc.E11-04-0317 (DOI)000306287400017 ()22573884 (PubMedID)
Available from: 2012-06-14 Created: 2012-06-14 Last updated: 2017-12-07Bibliographically approved
2. Histidine-domain-containing protein tyrosine phosphatase regulates platelet-derived growth factor receptor intracellular sorting and degradation
Open this publication in new window or tab >>Histidine-domain-containing protein tyrosine phosphatase regulates platelet-derived growth factor receptor intracellular sorting and degradation
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2015 (English)In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 27, no 11, 2209-2219 p.Article in journal (Refereed) Published
Abstract [en]

Histidine domain-containing protein tyrosine phosphatase (HD-PTP) is a putative phosphatase that has been shown to affect the signaling and downregulation of certain receptor tyrosine kinases. To investigate if HD-PTP affects platelet-derived growth factor receptor beta (PDGFR beta) signaling, we employed the overexpression of HA-tagged HD-PTP, as well as siRNA-mediated and lentivirus shRNA-mediated silencing of HD-PTP in NIH3T3 cells. We found that HD-PTP was recruited to the PDGFR beta in a ligand-dependent manner. Depletion of HD-PTP resulted in an inability of PDGF-BB to promote tyrosine phosphorylation of the ubiquitin ligases c-Cbl and Cbl-b, with a concomitant missorting and reduction of the degradation of activated PDGFRS. In contrast, ligand-induced internalization of PDGFR beta was unaffected by HD-FTP silencing. Furthermore, the levels of STAM and Hrs of the ESCRT0 machinery were decreased, and immunofluorescence staining showed that in HD-PTP-depleted cells, PDGFR beta accumulated in large aberrant intracellular structures. After the reduction of HD-PTP expression, an NIH3T3-derived cell line that has autocrine PDGF-BB signaling (sis-3 T3) showed increased ability of anchorage-independent growth. However, exogenously added PDGF-BB promoted efficient additional colony formation in control cells, but was not able to do so in HD-PTP-depleted cells. Furthermore, cells depleted of HD-PTP migrated faster than control cells. In summary, HD-PTP affects the intracellular sorting of activated PDGFRS and the migration, proliferation and tumorigenicity of cells stimulated by PDGF.

Keyword
HD-PTP, PDGFR beta, STAT3, c-Cbl, Cbl-b, PLC gamma, ESCRT, Ubiquitination
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-264805 (URN)10.1016/j.cellsig.2015.07.020 (DOI)000361777500008 ()26232618 (PubMedID)
Funder
Swedish Cancer Society, 130519
Available from: 2015-11-02 Created: 2015-10-19 Last updated: 2017-12-01Bibliographically approved
3. The Fer Tyrosine Kinase Is Important for Platelet-derived Growth Factor-BB-induced Signal Transducer and Activator of Transcription 3 (STAT3) Protein Phosphorylation, Colony Formation in Soft Agar, and Tumor Growth in Vivo.
Open this publication in new window or tab >>The Fer Tyrosine Kinase Is Important for Platelet-derived Growth Factor-BB-induced Signal Transducer and Activator of Transcription 3 (STAT3) Protein Phosphorylation, Colony Formation in Soft Agar, and Tumor Growth in Vivo.
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2013 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 22, 15736-15744 p.Article in journal (Refereed) Published
Abstract [en]

Fer is a cytoplasmic tyrosine kinase that is activated in response to platelet-derived growth factor (PDGF) stimulation. In the present report, we show that Fer associates with the activated PDGF β-receptor (PDGFRβ) through multiple autophosphorylation sites, i.e. Tyr-579, Tyr-581, Tyr-740, and Tyr-1021. Using low molecular weight inhibitors, we found that PDGF-BB-induced Fer activation is dependent on PDGFRβ kinase activity, but not on the enzymatic activity of Src or Jak kinases. In cells in which Fer was down-regulated using siRNA, PDGF-BB was unable to induce phosphorylation of STAT3, whereas phosphorylations of STAT5, ERK1/2, and Akt were unaffected. PDGF-BB-induced activation of STAT3 occurred also in cells expressing kinase-dead Fer, suggesting a kinase-independent adaptor role of Fer. Expression of Fer was dispensable for PDGF-BB-induced proliferation and migration but essential for colony formation in soft agar. Tumor growth in vivo was delayed in cells depleted of Fer expression. Our data suggest a critical role of Fer in PDGF-BB-induced STAT3 activation and cell transformation.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-201966 (URN)10.1074/jbc.M113.476424 (DOI)000319822300029 ()23589302 (PubMedID)
Available from: 2013-06-18 Created: 2013-06-18 Last updated: 2017-12-06Bibliographically approved
4. Specific targeting of PDGFRβ kinase activity in host cells inhibits growth and angiogenesis of tumors with high PDGF-BB expression
Open this publication in new window or tab >>Specific targeting of PDGFRβ kinase activity in host cells inhibits growth and angiogenesis of tumors with high PDGF-BB expression
(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-248167 (URN)
Available from: 2015-03-30 Created: 2015-03-30 Last updated: 2015-07-07

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