Carina Hellberg

Protein-tyrosine phosphatases and cancer

Tyrosine phosphorylation is an important signalling mechanism in eukaryotic cells. In cancer, oncogenic activation of tyrosine kinases is a common feature, and novel anticancer drugs have been introduced that target these enzymes. Tyrosine phosphorylation is also controlled by protein-tyrosine phosphatases (PTPs). Recent evidence has shown that PTPs can function as tumour suppressors. In addition, some PTPs, including SHP2, positively regulate the signalling of growth-factor receptors, and can be oncogenic. An improved understanding of how these enzymes function and how they are regulated might aid the development of new anticancer agents.

PDGF and Vessel Maturation

Pericytes are smooth muscle-like cells found in close contact with the endothelium in capillaries, where they regulate the morphology and function of the vessels. During vessel formation, platelet-derived growth factor-BB (PDGF-BB) is required for the recruitment and differentiation of pericytes. Tumor vessels display abnormal morphology and increased endothelial proliferation, resulting in leaky, tortuous vessels that are often poorly perfused. These vessels typically display decreased pericyte density, and the tumor-associated pericytes often express abnormal markers and show abnormal morphology. Anti-angiogenic therapy targeting pro-angiogenic growth factor pathways has been applied to a broad range of solid tumors with varying results. Studies utilizing mouse models indicate that the presence of pericytes protect endothelial cells against inhibition of vascular endothelial growth factor (VEGF) signaling. Simultaneous inhibition of PDGF receptors on pericytes therefore improves the effect of VEGF inhibitors on endothelial cells and enhances anti-angiogenic therapy.

Site-selective regulation of platelet-derived growth factor beta receptor tyrosine phosphorylation by T-cell protein tyrosine phosphatase.

ABSTRACT The platelet-derived growth factor (PDGF) β receptor mediates mitogenic and chemotactic signals. Like other tyrosine kinase receptors, the PDGF β receptor is negatively regulated by protein tyrosine phosphatases (PTPs). To explore whether T-cell PTP (TC-PTP) negatively regulates the PDGF β receptor, we compared PDGF β receptor tyrosine phosphorylation in wild-type and TC-PTP knockout (ko) mouse embryos. PDGF β receptors were hyperphosphorylated in TC-PTP ko embryos. Fivefold-higher ligand-induced receptor phosphorylation was observed in TC-PTP ko mouse embryo fibroblasts (MEFs) as well. Reexpression of TC-PTP partly abolished this difference. As determined with site-specific phosphotyrosine antibodies, the extent of hyperphosphorylation varied among different autophosphorylation sites. The phospholipase Cγ1 binding site Y1021, previously implicated in chemotaxis, displayed the largest increase in phosphorylation. The increase in Y1021 phosphorylation was accompanied by increased phospholipase Cγ1 activity and migratory hyperresponsiveness to PDGF. PDGF β receptor tyrosine phosphorylation in PTP-1B ko MEFs but not in PTPε ko MEFs was also higher than that in control cells. This increase occurred with a site distribution different from that seen after TC-PTP depletion. PDGF-induced migration was not increased in PTP-1B ko cells. In summary, our findings identify TC-PTP as a previously unrecognized negative regulator of PDGF β receptor signaling and support the general notion that PTPs display site selectivity in their action on tyrosine kinase receptors.

VE-PTP regulates VEGFR2 activity in stalk cells to establish endothelial cell polarity and lumen formation

Vascular endothelial growth factor (VEGF) guides the path of new vessel sprouts by inducing VEGF receptor-2 activity in the sprout tip. In the stalk cells of the sprout, VEGF receptor-2 activity is downregulated. Here, we show that VEGF receptor-2 in stalk cells is dephosphorylated by the endothelium-specific vascular endothelial-phosphotyrosine phosphatase (VE-PTP). VE-PTP acts on VEGF receptor-2 located in endothelial junctions indirectly, via the Angiopoietin-1 receptor Tie2. VE-PTP inactivation in mouse embryoid bodies leads to excess VEGF receptor-2 activity in stalk cells, increased tyrosine phosphorylation of VE-cadherin and loss of cell polarity and lumen formation. Vessels in ve-ptp−/− teratomas also show increased VEGF receptor-2 activity and loss of endothelial polarization. Moreover, the zebrafish VE-PTP orthologue ptp-rb is essential for polarization and lumen formation in intersomitic vessels. We conclude that the role of Tie2 in maintenance of vascular quiescence involves VE-PTP-dependent dephosphorylation of VEGF receptor-2, and that VEGF receptor-2 activity regulates VE-cadherin tyrosine phosphorylation, endothelial cell polarity and lumen formation.

Identification of a subset of pericytes that respond to combination therapy targeting PDGF and VEGF signaling

The aim of our study was to further explore the use of anti‐angiogenic therapy targeting the vascular endothelial growth factor receptor (VEGFR) on endothelial cells while simultaneously targeting platelet‐derived growth factor receptors (PDGFRs) on adjacent pericytes. B16 mouse melanoma tumors exogenously expressing PDGF‐BB (B16/PDGF‐BB) display higher pericyte coverage on the vasculature compared to the parental B16 tumors (B16/mock). These models were used to investigate the effects of combination therapy targeting VEGFR and PDGFR signaling on size‐matched tumors. Combination therapy using 25 mg/kg/day of the VEGFR inhibitor PTK787 and 100 mg/kg/day of the PDGFR inhibitor STI571 decreased the tumor growth rate of both tumor types, but the inhibition was only significant in the B16/PDGF‐BB tumors. Combination therapy induced vessel remodeling, primarily by reducing the vessel density in B16/mock tumors, and by reducing the vessel size in B16/PDGF‐BB tumors. When analyzing the effects of combination therapy on tumor vessel pericytes, it was found to primarily reduce the subpopulation of α‐smooth muscle actin and PDGFRβ‐positive pericytes partly detached from the tumor vessels, without affecting the number of pericytes closely attached to the endothelium, which also express desmin. Taken together, these data demonstrate an increased benefit of targeting both VEGFR and PDGFR pathways in B16/PDGF‐BB tumors, and demonstrates that the increased tumor growth inhibition in this model is accompanied by a reduction in a specific subset of pericytes, characterized by being loosely attached to endothelial cells and negative for the pericyte marker desmin.

Selective activation of oxidized PTP1B by the thioredoxin system modulates PDGF-β receptor tyrosine kinase signaling

The inhibitory reversible oxidation of protein tyrosine phosphatases (PTPs) is an important regulatory mechanism in growth factor signaling. Studies on PTP oxidation have focused on pathways that increase or decrease reactive oxygen species levels and thereby affect PTP oxidation. The processes involved in reactivation of oxidized PTPs remain largely unknown. Here the role of the thioredoxin (Trx) system in reactivation of oxidized PTPs was analyzed using a combination of in vitro and cell-based assays. Cells lacking the major Trx reductase TrxR1 (Txnrd1−/−) displayed increased oxidation of PTP1B, whereas SHP2 oxidation was unchanged. Furthermore, in vivo-oxidized PTP1B was reduced by exogenously added Trx system components, whereas SHP2 oxidation remained unchanged. Trx1 reduced oxidized PTP1B in vitro but failed to reactivate oxidized SHP2. Interestingly, the alternative TrxR1 substrate TRP14 also reactivated oxidized PTP1B, but not SHP2. Txnrd1-depleted cells displayed increased phosphorylation of PDGF-β receptor, and an enhanced mitogenic response, after PDGF-BB stimulation. The TrxR inhibitor auranofin also increased PDGF-β receptor phosphorylation. This effect was not observed in cells specifically lacking PTP1B. Together these results demonstrate that the Trx system, including both Trx1 and TRP14, impacts differentially on the oxidation of individual PTPs, with a preference of PTP1B over SHP2 activation. The studies demonstrate a previously unrecognized pathway for selective redox-regulated control of receptor tyrosine kinase signaling.

Dynamic changes in the expression of DEP-1 and other PDGF receptor-antagonizing PTPs during onset and termination of neointima formation

Growth factor‐dependent tissue remodeling, such as restenosis, is believed to be predominantly regulated by changes in expression of receptor‐tyrosine‐kinases (RTKs) and their ligands. As endogenous antagonists of RTKs, protein‐tyrosine‐phosphatases (PTPs) are additional candidate regulators of these processes. Using laser‐capture‐microdissection and quantitative RT‐polymerase chain reaction (qRT‐PCR), we investigated the layer‐specific expression of the four platelet‐derived growth factor (PDGF) isoforms, the PDGF‐ and receptors, and five PTPs implied in control of PDGF‐receptor signaling 8 and 14 days after balloon injury of the rat carotid. Results were correlated with analyses of PDGF‐ receptor phosphorylation and vascular smooth muscle cell (VSMC) proliferation in vivo. The expression levels of all components, as well as receptor activation and VSMC proliferation, showed specific changes, which varied between media and neointima. Interestingly, PTP expression—particularly, DEP‐1 μlevels—appeared to be the dominating factor determining receptor‐phosphorylation and VSMC proliferation. In support of these findings, cultured DEP‐1–/–cells displayed increased PDGF‐dependent cell signaling. Hyperactivation of PDGF‐induced signaling was also observed after siRNA‐down‐regulation of DEP‐1 in VSMCs. The results indicate a previously unrecognized role of PDGF‐receptor‐targeting PTPs in controlling neointima formation. In more general terms, the observations indicate transcriptional regulation of PTPs as an important mechanism for controlling onset and termination of RTK‐dependent tissue remodeling.—FASEB J. 21, 523–534 (2007)

Activation of Protein Kinase C α Is Necessary for Sorting the PDGF β-Receptor to Rab4a-dependent Recycling

Previous studies showed that loss of the T-cell protein tyrosine phosphatase (TC-PTP) induces Rab4a-dependent recycling of the platelet-derived growth factor (PDGF) beta-receptor in mouse embryonic fibroblasts (MEFs). Here we identify protein kinase C (PKC) alpha as the critical signaling component that regulates the sorting of the PDGF beta-receptor at the early endosomes. Down-regulation of PKC abrogated receptor recycling by preventing the sorting of the activated receptor into EGFP-Rab4a positive domains on the early endosomes. This effect was mimicked by inhibition of PKCalpha, using myristoylated inhibitory peptides or by knockdown of PKCalpha with shRNAi. In wt MEFs, short-term preactivation of PKC by PMA caused a ligand-induced PDGF beta-receptor recycling that was dependent on Rab4a function. Together, these observations demonstrate that PKC activity is necessary for recycling of ligand-stimulated PDGF beta-receptor to occur. The sorting also required Rab4a function as it was prevented by expression of EGFP-Rab4aS22N. Preventing receptor sorting into recycling endosomes increased the rate of receptor degradation, indicating that the sorting of activated receptors at early endosomes directly regulates the duration of receptor signaling. Activation of PKC through the LPA receptor also induced PDGF beta-receptor recycling and potentiated the chemotactic response to PDGF-BB. Taken together, our present findings indicate that sorting of PDGF beta-receptors on early endosomes is regulated by sequential activation of PKCalpha and Rab4a and that this sorting step could constitute a point of cross-talk with other receptors.

Combined Anti-Angiogenic Therapy Targeting PDGF and VEGF Receptors Lowers the Interstitial Fluid Pressure in a Murine Experimental Carcinoma

Elevation of the interstitial fluid pressure (IFP) of carcinoma is an obstacle in treatment of tumors by chemotherapy and correlates with poor drug uptake. Previous studies have shown that treatment with inhibitors of platelet-derived growth factor (PDGF) or vascular endothelial growth factor (VEGF) signaling lowers the IFP of tumors and improve chemotherapy. In this study, we investigated whether the combination of PDGFR and VEGFR inhibitors could further reduce the IFP of KAT-4 human carcinoma tumors. The tumor IFP was measured using the wick-in-needle technique. The combination of STI571 and PTK/ZK gave an additive effect on the lowering of the IFP of KAT-4 tumors, but the timing of the treatment was crucial. The lowering of IFP following combination therapy was accompanied by vascular remodeling and decreased vascular leakiness. The effects of the inhibitors on the therapeutic efficiency of Taxol were investigated. Whereas the anti-PDGF and anti-VEGF treatment did not significantly inhibit tumor growth, the inhibitors enhanced the effect of chemotherapy. Despite having an additive effect in decreasing tumor IFP, the combination therapy did not further enhance the effect of chemotherapy. Simultaneous targeting of VEGFR and PDGFR kinase activity may be a useful strategy to decrease tumor IFP, but the timing of the inhibitors should be carefully determined.

Differential tyrosine phosphorylation of fibroblast growth factor (FGF) receptor-1 and receptor proximal signal transduction in response to FGF-2 and heparin

The sulfated regions in heparan sulfate and heparin are known to affect fibroblast growth factor (FGF) function. We have studied the mechanism whereby heparin directs FGF-2-induced FGF receptor-1 (FGFR-1) signal transduction. FGF-2 alone stimulated maximal phosphorylation of Src homology domain 2 tyrosine phosphatase (SHP-2) and the adaptor molecule Crk, in heparan sulfate-deficient Chinese hamster ovary (CHO) 677 cells expressing FGFR-1. In contrast, for phospholipase Cgamma(1) (PLCgamma(1)) and the adaptor molecule Shb to be maximally tyrosine-phosphorylated, cells had to be stimulated with both FGF-2 and heparin (100 ng/ml). Tyrosine residues 463 in the juxtamembrane domain and 766 in the C-terminal tail in FGFR-1 are known to bind Crk and PLCgamma(1), respectively. Analysis of tryptic phosphopeptide maps of FGFR-1 from cells stimulated with FGF-2 alone and FGF-2 together with heparin showed that FGF-2 alone stimulated a several-fold increase in tyrosine 463 in the juxtamembrane domain. In contrast, heparin had to be included in order for tyrosine 766 to be phosphorylated to the same fold level. Our data imply that tyrosine 463 is phosphorylated and able to transduce signals in response to FGF-2 treatment alone; furthermore, we suggest that FGFR-1 dimerization/kinase activation is stabilized by heparin.