Erratum: Specific targeting of PDGFRß in the stroma inhibits growth and angiogenesis in tumors with high PDGF-BB expression: Erratum.

[This corrects the article DOI: 10.7150/thno.37851.].

Specific targeting of PDGFRß in the stroma inhibits growth and angiogenesis in tumors with high PDGF-BB expression.

PDGF-BB/PDGFRß signaling plays an important role during vascularization by mediating pericyte recruitment to the vasculature, promoting the integrity and function of vessels. Until now it has not been possible to assess the specific role of PDGFRß signaling in tumor progression and angiogenesis due to lack of appropriate animal models and molecular tools. Methods: In the present study, we used a transgenic knock-in mouse strain carrying a silent mutation in the PDGFRß ATP binding site that allows specific targeting of PDGFRß using the compound 1-NaPP1. To evaluate the impact of selective PDGFRß inhibition of stromal cells on tumor growth we investigated four tumor cell lines with no or low PDGFRß expression, i.e. Lewis lung carcinoma (LLC), EO771 breast carcinoma, B16 melanoma and a version of B16 that had been engineered to overexpress PDGF-BB (B16/PDGF-BB). Results: We found that specific impairment of PDGFRß kinase activity by 1-NaPP1 treatment efficiently suppressed growth in tumors with high expression of PDGF-BB, i.e. LLC and B16/PDGF-BB, while the clinically used PDGFRß kinase inhibitor imatinib did not suppress tumor growth. Notably, tumors with low levels of PDGF-BB, i.e. EO771 and B16, neither responded to 1-NaPP1 nor to imatinib treatment. Inhibition of PDGFRß by either drug impaired tumor vascularization and also affected pericyte coverage; however, specific targeting of PDGFRß by 1-NaPP1 resulted in a more pronounced decrease in vessel function with increased vessel apoptosis in high PDGF-BB expressing tumors, compared to treatment with imatinib. In vitro analysis of PDGFRß ASKA mouse embryo fibroblasts and the mesenchymal progenitor cell line 10T1/2 revealed that PDGF-BB induced NG2 expression, consistent with the in vivo data. Conclusion: Specific targeting of PDGFRß signaling significantly inhibits tumor progression and angiogenesis depending on PDGF-BB expression. Our data suggest that targeting PDGFRß in the tumor stroma could have therapeutic value in patients with high tumor PDGF-BB expression.

Imatinib increases oxygen delivery in extracellular matrix-rich but not in matrix-poor experimental carcinoma.

BACKGROUND: Imatinib causes increased turnover of stromal collagen, reduces collagen fibril diameter, enhances extracellular fluid turnover and lowers interstitial fluid pressure (IFP) in the human colonic carcinoma KAT-4/HT-29 (KAT-4) xenograft model. METHODS: We compared the effects of imatinib on oxygen levels, vascular morphology and IFP in three experimental tumor models differing in their content of a collagenous extracellular matrix. RESULTS: Neither the KAT4 and CT-26 colonic carcinoma models, nor B16BB melanoma expressed PDGF ß-receptors in the malignant cells. KAT-4 tumors exhibited a well-developed ECM in contrast to the other two model systems. The collagen content was substantially higher in KAT-4 than in CT-26, while collagen was not detectable in B16BB tumors. The pO2 was on average 5.4, 13.9 and 19.3 mmHg in KAT-4, CT-26 and B16BB tumors, respectively. Treatment with imatinib resulted in similar pO2-levels in all three tumor models but only in KAT-4 tumors did the increase reach statistical significance. It is likely that after imatinib treatment the increase in pO2 in KAT-4 tumors is caused by increased blood flow due to reduced vascular resistance. This notion is supported by the significant reduction observed in IFP in KAT-4 tumors after imatinib treatment. Vessel area varied between 4.5 and 7% in the three tumor models and was not affected by imatinib treatment. Imatinib had no effect on the fraction of proliferating cells, whereas the fraction of apoptotic cells increased to a similar degree in all three tumor models. CONCLUSION: Our data suggest that the effects of imatinib on pO2-levels depend on a well-developed ECM and provide further support to the suggestion that imatinib acts by causing interstitial stroma cells to produce a less dense ECM, which would in turn allow for an increased blood flow. The potential of imatinib treatment to render solid tumors more accessible to conventional treatments would therefore depend on the degree of tumor desmoplasia.

Combination therapy using imatinib and vatalanib improves the therapeutic efficiency of paclitaxel towards a mouse melanoma tumor.

Melanomas respond poorly to chemotherapy. In this study, we investigated the sensitization of B16 mouse melanoma tumors to paclitaxel by a combination of two tyrosine kinase inhibitors: vatalanib, targeting vascular endothelial growth factor receptors, and imatinib, an inhibitor targeting for example, Abl/BCR-ABL, the platelet-derived growth factor receptor, and stem cell factor receptor c-Kit. C57Bl6/J mice carrying B16/PDGF-BB mouse melanoma tumors were treated daily with vatalanib (25 mg/kg), imatinib (100 mg/kg), or a combination of these drugs. Paclitaxel was given subcutaneously twice during the study. The effects of the drugs on tumor cell proliferation in vitro were determined by counting cells. B16/PDGF-BB mouse melanoma tumors were not sensitive to paclitaxel at doses of either 5 or 20 mg/kg. However, the tumor growth was significantly reduced by 58%, in response to paclitaxel (5 mg/kg) when administered with daily doses of both vatalanib and imatinib. Paclitaxel only inhibited the in-vitro growth of B16/PDGF-BB tumor cells when given in combination with imatinib. Imatinib presumably targets c-Kit, as the cells do not express platelet-derived growth factor receptor and as another c-Abl inhibitor was without effect. This was supported by data from three c-Kit-expressing human melanoma cell lines showing varying sensitization to paclitaxel by the kinase inhibitors. In addition, small interfering RNA knockdown of c-Kit sensitized the cells to paclitaxel. These data show that combination of two tyrosine kinase inhibitors, imatinib and vatalanib, increases the effects of paclitaxel on B16/PDGF-BB tumors, thus suggesting a novel strategy for the treatment of melanomas expressing c-Kit.

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.

Activation of protein kinase C alpha is necessary for sorting the PDGF beta-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.

An activating mutation in the PDGF receptor-beta causes abnormal morphology in the mouse placenta.

An oncogenic D842V mutation in the platelet-derived growth factor (PDGF) alpha-receptor (Pdgfra) has recently been described in patients with gastrointestinal stromal tumors. In order to test if the same mutation would confer oncogenic properties to the homologous PDGF beta-receptor (Pdgfrb), the corresponding aspartic acid residue at position 849 of Pdgfrb was changed into valine (D849V) using a knock-in strategy. This mutation turned out to be dominantly lethal and caused death even in chimeras (from 345 transferred chimeric blastocysts, no living coat chimeras were detected). Experiments employing mouse embryonic fibroblasts (MEFs) indicated hyperactivity of the mutant receptor. The mutant receptor was phosphorylated in a ligand-independent manner and, in contrast to wild-type MEFs, mutant cells proliferated even in the absence of ligand. Knockout experiments have previously indicated a role for Pdgfrb in placental development. We therefore analyzed wild-type and Pdgfrb D849V chimeric placentas from different gestational stages. No differences were detected at embryonic days 11.5 and 13.5 (n=4). At embryonic day 17.5, however, chimeric placentas (n=3/4) displayed abnormalities both in the labyrinth and in the chorionic plate. The changes included hyper-proliferation of alpha-smooth muscle actin and platelet/endothelial cell adhesion molecule-1 positive cells in the labyrinth and cells in the chorionic plate. In addition, the fetal blood vessel compartment of the labyrinth was completely disorganized.

Platelet-derived growth factor receptor-beta constitutive activity promotes angiogenesis in vivo and in vitro.

OBJECTIVE: Knockout studies have demonstrated crucial roles for the platelet-derived growth factor-B and its cognate receptor, platelet-derived growth factor receptor-beta (PDGFR-beta), in blood vessel maturation, that is, the coverage of newly formed vessels with mural cells/pericytes. This study describes the consequences of a constitutively activating mutation of the PDGFR-beta (Pdgfrb(D849V)) introduced into embryonic stem cells with respect to vasculogenesis/angiogenesis in vitro and in vivo. METHODS AND RESULTS: Embryonic stem cells were induced to either form teratomas in vivo or embryoid bodies, an in vitro model for mouse embryogenesis. Western blotting studies on embryoid bodies showed that expression of a single allele of the mutant Pdgfrb led to increased levels of PDGFR-beta tyrosine phosphorylation and augmented downstream signal transduction. This was accompanied by enhanced vascular development, followed by exaggerated angiogenic sprouting with abundant pericyte coating as shown by immunohistochemistry/immunofluorescence. Pdgfrb(D849V/+) embryoid bodies were characterized by increased expression of vascular endothelial growth factor (VEGF)-A and VEGF receptor-2; neutralizing antibodies against VEGF-A/VEGF receptor-2 blocked vasculogenesis and angiogenesis in mutant embryoid bodies. Moreover, Pdgfrb(D849V/+) embryonic stem cell-derived teratomas in nude mice were more densely vascularized than wild-type teratomas. CONCLUSIONS: Increased PDGFR-beta kinase activity is associated with elevated expression of VEGF-A and VEGF receptor-2, acting directly on endothelial cells and resulting in increased vessel formation.

Deletion of exon I of SMAD7 in mice results in altered B cell responses.

The members of the TGF-beta superfamily, i.e., TGF-beta isoforms, activins, and bone morphogenetic proteins, regulate growth, differentiation, and apoptosis, both during embryonic development and during postnatal life. Smad7 is induced by the TGF-beta superfamily members and negatively modulates their signaling, thus acting in a negative, autocrine feedback manner. In addition, Smad7 is induced by other stimuli. Thus, it can fine-tune and integrate TGF-beta signaling with other signaling pathways. To investigate the functional role(s) of Smad7 in vivo, we generated mice deficient in exon I of Smad7, leading to a partial loss of Smad7 function. Mutant animals are viable, but significantly smaller on the outbred CD-1 mouse strain background. Mutant B cells showed an overactive TGF-beta signaling measured as increase of phosphorylated Smad2-positive B cells compared with B cells from wild-type mice. In agreement with this expected increase in TGF-beta signaling, several changes in B cell responses were observed. Mutant B cells exhibited increased Ig class switch recombination to IgA, significantly enhanced spontaneous apoptosis in B cells, and a markedly reduced proliferative response to LPS stimulation. Interestingly, LPS treatment reverted the apoptotic phenotype in the mutant cells. Taken together, the observed phenotype highlights a prominent role for Smad7 in development and in regulating the immune system's response to TGF-beta.

A gain of function mutation in the activation loop of platelet-derived growth factor beta-receptor deregulates its kinase activity.

The platelet-derived growth factor receptors (PDGFRs) are receptor tyrosine kinases implicated in multiple aspects of cell growth, differentiation, and survival. Recently, a gain of function mutation in the activation loop of the human PDGFRalpha has been found in patients with gastrointestinal stromal tumors. Here we show that a mutation in the corresponding codon in the activation loop of the murine PDGFRbeta, namely an exchange of asparagine for aspartic acid at amino acid position 849 (D849N), confers transforming characteristics to embryonic fibroblasts from mutant mice, generated by a knock-in strategy. By comparing the enzymatic properties of the wild-type versus the mutant receptor protein, we demonstrate that the D849N mutation lowers the threshold for kinase activation, causes a dramatic alteration in the pattern of tyrosine phosphorylation kinetics following ligand stimulation, and induces a ligand-independent phosphorylation of several tyrosine residues. These changes result in deregulated recruitment of specific signal transducers. The GTPase-activating protein for Ras (RasGAP), a negative regulator of the Ras mitogenic pathway, displayed a delayed binding to the mutant receptor. Moreover, we have observed enhanced ligand-independent ERK1/2 activation and an increased proliferation of mutant cells. The p85 regulatory subunit of the phosphatidylinositol 3 '-kinase was constitutively associated with the mutant receptor, and this ligand-independent activation of the phosphatidylinositol 3'-kinase pathway may explain the observed strong protection against apoptosis and increased motility in cellular wounding assays. Our findings support a model whereby an activating point mutation results in a deregulated PDGFRbeta with oncogenic predisposition.