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,¶
From the Biomedical Graduate Program,* the Departments of Medicine and Pathology and Laboratory Medicine, and the Abramson Cancer Center,¶ University of Pennsylvania, Philadelphia, Pennsylvania; and Bayer Pharmaceuticals Corporation, West Haven, Connecticut
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Abstract |
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Additional characterization has shown that sorafenib inhibits other kinases, such as vascular endothelial growth factor (VEGF) receptors (VEGFR)-2 and -3 and platelet-derived growth factor receptor ß, albeit with higher IC50.14 VEGFR2-mediated VEGF activation of endothelial cells (ECs) is critical for angiogenesis. This is attributed to its ability to induce EC signaling pathways that regulate EC proliferation, migration, and survival. The two major pathways that regulate these processes are the PI3K-AKT and Raf-MEK-ERK pathways.17-22 Activation of these pathways in ECs is necessary for angiogenesis.23-27 Given this and the fact that a dominant-negative mutant Raf was shown to inhibit angiogenesis,28 the VEGFR2 and Raf inhibitory activities of sorafenib have led to expectations that this drug is an inhibitor of angiogenesis. Evidence supporting this idea can be found in the observation that microvessel density (MVD) was reduced in tumors treated with sorafenib.14 However, it remains unclear whether the anti-angiogenic effect of sorafenib was a major mechanism for control of tumor growth and if tumor ECs are targeted by this drug in vivo.
We undertook the current study to understand the mechanism of tumor growth control by sorafenib. Our results show that in K1735 murine melanomas treated with this agent, inhibition of tumor angiogenesis contributes significantly to control of tumor growth and, in fact, may be the predominant anti-tumor mechanism. We then examined the effect of therapy on the activity of EC signal transduction pathways in tumors in vivo. Marked inhibition of ERK activation and unaltered AKT activation in tumor vascular ECs was accompanied by inhibition of EC proliferation and induction of EC apoptosis. Together with similar findings in two other tumor models treated with this agent, these data show that sorafenib is a potent inhibitor of ERK activation in tumor ECs and tumor angiogenesis. Finally, our studies suggest methods for assessing the activity of this agent at relevant target sites, which may be useful both for monitoring its effect and understanding its activity against human cancers in clinical trials.
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Materials and Methods |
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Murine K1735 melanoma and RENCA renal carcinoma cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and penicillin/streptomycin.29 Female C3H/HeN and BALB/c mice, 6 to 8 weeks old, were purchased from Charles River (Wilmington, MA) and Taconic Farms (Germantown, NY), respectively, and maintained in microisolate cages. K1735 and RENCA tumors were generated by injecting 2 x 106 viable tumor cells subcutaneously into the lower right flank of C3H/HeN and BALB/c mice, respectively. Tumors were measured by calipers at regular intervals, and volume was calculated using the formula for approximating the volume of a spheroid [0.5 x (diameter)3]. Mice were treated with either vehicle (5% Cremaphore EL/5% ETOH/90% ddH20) or sorafenib (30 mg/kg) by gavage daily. Colo-205 human colon carcinoma tumors were generated in nude mice and treated as previously described.14 Plasma was collected from a subset of animals 3 hours after gavage and analyzed for drug concentration. For histological studies, all animals were perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.4) by intracardiac injection. Tumors were excised and either frozen or processed for paraffin embedding. Size-matched untreated and vehicle-treated tumors were generated for histological analysis. Tumor samples were stained with hematoxylin and eosin (H&E), and the area of necrosis was calculated by Image J software (National Institutes of Health, Bethesda, MD).
Matrigel Assay for Angiogenesis
C3H/HeN mice were injected subcutaneously with 0.5 ml of Matrigel (BD Biosciences, Bedford, MA) mixed with 15 U of heparin (Sigma, St. Louis, MO) with or without 100 ng of basic fibroblast growth factor (bFGF) (R&D Systems, Minneapolis, MN). In treated mice, vehicle or 30 mg/kg of sorafenib was administered daily by gavage for 6 days. Matrigel pellets were harvested on day 6, fixed in 4% paraformaldehyde overnight, and processed for paraffin embedding. Neovascularization was determined as previously described.30 Briefly, sections were stained with Masson-Trichrome and examined for erythrocytes in nuclei-lined luminal structures.
Tumor Immunostaining
Detection of tumor cell hypoxia by EF5 was performed as previously described.29 In brief, EF5 (provided by Dr. Cameron Koch, University of Pennsylvania, Philadelphia, PA) was injected into tumor-bearing mice via tail vein 3 hours before euthanasia. Sections of frozen tumors were stained with Cy3-conjugated anti-EF5 monoclonal antibody (provided by Dr. Cameron Koch). Vasculature in frozen tumor sections was studied by epifluorescence and confocal microscopy as previously described31 using anti-CD31 (PECAM) antibody (Pharmingen, San Jose, CA) to identify ECs followed by Alexa Fluor 488 goat anti-rat IgG (Molecular Probes, Eugene, OR). Pericytes were identified using anti-smooth muscle actin (SMA) (DAKO, Carpinteria, CA) antibody in M.O.M. blocking buffer (Vector Laboratories, Burlingame, CA) followed by Texas Red-conjugated goat anti-mouse IgG antibody (Molecular Probes). MVD measurements were calculated as previously described.32
Cell Signaling
Five-µm paraffin-embedded tumor sections were stained for p-ERK with rabbit anti-phospho-ERK antibody (Cell Signaling, Beverly, MA), for p-AKT with rabbit anti-phospho-AKT antibody (Cell Signaling, MA), and for proliferation using mouse anti-Ki-67 antibody (Vector Laboratories). These were incubated with biotinylated secondary antibodies (Vector Laboratories) and ABC reagent (Vector Laboratories). Immune complexes were detected with 3,3-diaminobenzidine (Vector Laboratories). The same sections were subsequently stained for vessels using CD34 (Abcam, Cambridge, MA) by immunofluorescence. A vessel was counted as positive for p-ERK, p-AKT, or Ki-67 when at least one EC within a vessel stained positively for these antigens. Apoptotic cells were identified by terminal dUTP nick-end labeling (TUNEL) staining using the Apoptag indirect fluorescein isothiocyanate detection kit (Chemicon, Temecula, CA) followed by vessel staining by CD31 immunofluorescence (Pharmingen, La Jolla, CA) on 10-µm frozen tumor sections. IP Lab Software (Scanalitics, Fairfax, VA) was used to quantitate tumor cell staining of Ki-67 and TUNEL.
In Vitro Studies
Cultured mouse brain capillary endothelial cells (MBECs, gift from Dr. Dounan Yu, University of Pennsylvania)33 and human microvascular dermal endothelial cells (HMVEC-d; Clonetics, San Diego, CA) were treated with different concentrations of sorafenib for 2 hours and lysed. Thirty to 60 µg of protein were run on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and probed for p-ERK or p-AKT (Cell Signaling). Blots were subsequently stripped and reprobed for ERK or AKT (Cell Signaling). K1735 tumors were homogenized in lysis buffer. Tumor lysate was loaded at increasing concentrations of protein (50, 100, and 300 µg) along with K1735 tumor cell lysate and run on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. The gel was probed for p-ERK, stripped, and reprobed for ERK. Relative p-ERK and p-AKT expressions were quantitated by calculating (optical density of phosphorylated proteins/optical density of unphosphorylated proteins) and normalized to control levels (Image J).
Statistical Analysis
Assessment of statistical significance for survival analysis was performed by log rank test (R Statistical Software, http://www.r-project.org). All other statistical analyses were performed by Student’s t-test (Excel; Microsoft, Redmond, WA).
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Results |
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C3H/HeN mice bearing subcutaneous K1735 melanoma tumors were treated daily with sorafenib (30 mg/kg) or vehicle by gavage when their tumors reached 2 mm in diameter. Mice were treated for 4 weeks or until their tumors reached euthanizable size (1.2 cm3). After 4 weeks of treatment, all control tumors and eight of nine vehicle-treated tumors were greater than 1.2 cm3, whereas only 3 of 10 sorafenib-treated tumors had reached this size (Figure 1A)
. This difference in growth between vehicle and sorafenib-treated tumors was significant (P < 0.01, log rank test). Similar results were obtained in another experiment in which we tested treatment with sorafenib at 30 and 100 mg/kg/day (data not shown).
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; necrotic areas accounted for 44 ± 28% of the cut surface of sorafenib-treated tumors versus 1 ± 1% of the cut surface of vehicle-treated tumors, P < 0.05). These results show that sorafenib treatment controls K1735 tumor growth, causing massive regional tumor cell death consistent with ischemic necrosis. Sorafenib Inhibits bFGF-Induced Matrigel Neovascularization
To demonstrate that sorafenib can inhibit angiogenesis in vivo, Matrigel pellets containing 100 ng/ml bFGF were implanted subcutaneously in C3H/HeN mice and the hosts subjected to daily treatment with either 30 mg/kg sorafenib or vehicle. After 6 days, plugs were removed and analyzed by Masson-Trichrome staining.30
Untreated and vehicle-treated Matrigel showed large numbers of vascular structures that reached the center of the pellet (Figure 2, B and C)
. In contrast, Matrigel treated with sorafenib had fewer vascular structures, and these were located predominantly at the periphery of the pellets (Figure 2D)
. Sorafenib treatment, however, did not reduce the number of vessels to levels seen in Matrigel without bFGF (Figure 2A)
. These results show that sorafenib treatment inhibits but does not completely abrogate bFGF-induced angiogenesis in vivo.
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To determine whether sorafenib treatment inhibits angiogenesis and vascular development in tumors, we examined development of hypoxia in treated tumors. Because necrosis interferes with detection of hypoxia, and extensive necrosis was seen in K1735 tumors treated with sorafenib for 3 to 4 weeks, we studied K1735 tumors treated with sorafenib for only 7 days. We started treatment after tumors reached 5 mm in diameter to ensure that they would be sufficiently large for analysis at the end of the week of therapy. Hypoxic tumor cells were labeled in vivo with EF5, which was subsequently detected by immunostaining with Cy3-conjugated anti-EF5 antibody.34
As previously seen,29
untreated K1735 tumors had few, if any, regions of EF5 labeling (data not shown). Vehicle-treated tumors had limited areas of EF5 staining, which were not intense (Figure 2, E and F)
. In contrast, tumors treated with sorafenib had widespread areas of intense EF5 staining (Figure 2, G and H)
. As in K1735 tumors treated successfully with other anti-angiogenic agents,29
EF5 staining in sorafenib-treated tumors tended to be in tumor regions most distant from perfused vessels.
To investigate the effect of treatment on MVD, tumors treated for 7 days with vehicle or sorafenib were immunostained with anti-CD31 (PECAM) antibody. MVD was significantly reduced in sorafenib-treated tumors compared to vehicle-treated tumors (P < 0.01) (Figure 3A)
. To assess vessel pericyte coverage, tumor sections were immunostained with anti-smooth muscle actin (SMA) and anti-CD31 antibodies. Pericyte coverage of tumor vessels increased significantly after sorafenib treatment but not after vehicle treatment (Figure 3B)
. Thick (50 µm) tumor sections were stained with anti-CD31 and examined by confocal microscopy to assess vessel morphology. Vessels in vehicle-treated tumors appeared only slightly larger in caliber and resembled those in untreated tumors in arborization (Figure 3, C and D)
. In contrast, vessels in sorafenib-treated tumors were obviously larger in caliber and were much less arborized (Figure 3E)
. The marked increase in tumor cell hypoxia, the decrease in tumor MVD, the increase in the percentage of pericyte-covered vessels, and the changes in vessel morphology are hallmarks of angiogenesis inhibition in K1735 tumors.29,32,35,36
These led us to conclude that sorafenib is a potent inhibitor of angiogenesis and that this activity contributes significantly to its control of tumor growth.
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Activation of the Raf-MEK-ERK kinase pathway in ECs has been shown to be necessary for neovascularization,25-27
so we asked whether sorafenib treatment inhibited ERK phosphorylation in K1735 tumor vasculature. Tumors treated for 1 and 4 weeks were stained for phospho-ERK (p-ERK) using anti-p-ERK antibody by immunohistochemistry and for vessels using anti-CD34 antibody by immunofluorescence. In untreated and vehicle-treated K1735 tumors, p-ERK staining was frequently seen in ECs and was usually nuclear (Figure 4A)
. The specificity of this stain was confirmed by immunostaining these tumors with a different (monoclonal) anti-p-ERK antibody and through use of a blocking peptide for the polyclonal anti-p-ERK antibody (data not shown). In addition, p-MEK staining co-localized with p-ERK staining in tumor ECs, indicating that p-ERK activation resulted from Raf-MEK-ERK signaling in these EC (Supplemental Figure S1 at http://ajp.amjpathol.org). Interestingly, p-ERK staining was rarely seen in the tumor cells themselves in most K1735 tumors. The dearth of detectable p-ERK in tumor cells was confirmed by Western blot analysis of K1735 tumor lysates. This was a surprising finding, because K1735 cells cultured in vitro contain abundant p-ERK (Figure 5A)
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22% of vessels in untreated K1735 tumors stained for p-ERK and that 28 and 18% of vessels stained in tumors treated with vehicle for 1 and 4 weeks, respectively (Figure 4, A and B
; P > 0.05). In contrast, vascular p-ERK expression was significantly reduced in sorafenib-treated tumors with 14% of vessels stained after 1 week of treatment (P < 0.05) (Figure 4, A and B)
. p-ERK expression further decreased with continued treatment, such that only 3% of tumor vessels stained with p-ERK after 4 weeks of treatment (P < 0.01). In treated tumors with reduced vascular p-ERK staining, staining for total ERK was not decreased (data not shown). Another EC signaling pathway known to be activated during angiogenesis is the PI3K-AKT pathway. We examined activation of this pathway and the effect of sorafenib treatment by staining tumor sections for phospho-AKT (p-AKT). In contrast to p-ERK staining, anti-p-AKT antibody stained tumor cells as well as ECs in K1735 tumors (Figure 4A)
. Treatment with sorafenib for 1 or 4 weeks did not significantly change the percentage of vessels staining for p-AKT (Figure 4, A and C)
.
The decrease in vascular p-ERK staining but not p-AKT staining in tumors treated with sorafenib was also seen in the two other tumor types examined, Colo-205 human colon carcinoma xenografts and RENCA renal cell carcinoma tumors (Figure 4D
, Table 1
). Quantitation of p-ERK and p-AKT expression in tumor vasculature and changes induced by therapy for all tumors examined are summarized in Table 1
.
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. Sorafenib Inhibits Proliferation and Promotes Apoptosis in Tumor Vasculature
Angiogenesis requires EC proliferation, and anti-angiogenic agents inhibit EC proliferation and/or induce EC apoptosis. Inhibition of K1735 tumor angiogenesis by sorafenib suggests that one, the other, or both processes are affected by treatment. To examine proliferation in K1735 vasculature, tumor sections were stained for the proliferation-associated antigen, Ki-67, by immunohistochemistry followed by CD34 vessel staining by immunofluorescence. Quantification revealed that a similar percentage of vessels (14%) in untreated and vehicle-treated tumors stained for Ki-67 (data not shown). In contrast, a much lower percentage of vessels (2%) stained for Ki-67 after 7 days of sorafenib treatment (Table 1
, P < 0.01). A significant decrease in Ki-67 staining was also seen in the vascular ECs of treated Colo-205 and RENCA tumors (Table 1)
. Cell death in K1735 tumors was studied by TUNEL staining and CD31 immunostaining. There was significantly more EC death in sorafenib-treated tumor vasculature compared to untreated or vehicle-treated tumor vasculature (7 ± 3 versus 3 ± 2%, P < 0.01; Table 2
). These data show that both decreased formation of new tumor vessels and regression of existing tumor vessels contribute to sorafenib’s inhibition of tumor vasculature development. Because a decrease in tumor vasculature could be attributed to primary effects on tumor cells, we investigated tumor cell proliferation and apoptosis in K1735 tumors after sorafenib treatment. There was no decrease in tumor cell staining for Ki-67 after sorafenib treatment, but treatment increased tumor cell TUNEL staining (1.4 ± 0.5 versus 0.8 ± 0.2%, P < 0.01; Table 2
).
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Discussion |
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In response to growth factor stimulation, cells activate the conserved Raf-MEK-ERK pathway to transduce signals that regulate differentiation, growth, and proliferation.37-40
In vitro, ECs activate this pathway on stimulation of multiple proangiogenic EC receptors including, VEGFR2, FGFR2, Tie2, integrins (
vß3 and vß5), and EDG1,17,18,21,41-44
and EC activation of this pathway is necessary for angiogenesis.22,25-27
Inhibition of EC signaling through Raf-MEK-ERK was shown in tumors treated with sorafenib by decreased immunohistochemical staining for p-ERK. In control K1735 tumors, 18 to 28% of vessels stained for p-ERK (Figure 4B)
. Because quiescent vessels in most normal mouse organs do not stain (data not shown), this is a sign of activated vasculature in tumors. Sorafenib treatment decreased the number of tumor vessels staining for p-ERK by 85 to 90%. This was accompanied by a significant decrease in Ki-67 staining within treated tumor endothelium. We believe this decrease reflects sorafenib inhibition of the Raf-MEK-ERK pathway in ECs and is not merely a manifestation of angiogenesis inhibition, because EC p-ERK expression was not reduced in K1735 tumors treated with the angiogenesis inhibitor rIL-12 (22 ± 4% untreated versus 19 ± 6% treated; P > 0.05). Residual staining for p-ERK in tumor ECs could be attributable to incomplete kinase inhibition by sorafenib or attributable to the activity of alternative pathways of ERK activation.45
Interestingly, ECs seemed to either stain or not stain for p-ERK, and gradations of p-ERK staining (and, presumably, of p-ERK levels) were not evident. In contrast to its consistent and marked inhibition of EC ERK activation, AKT activation in tumor ECs, revealed by p-AKT staining, is not significantly diminished by sorafenib treatment.
The virtually undetectable levels of p-ERK in K1735 tumor cells in vivo was unexpected. If p-ERK levels reflect activity of the Raf-MEK-ERK pathway, it implies that activity of this pathway is low. Perhaps this should not be so surprising, because K1735 tumor cells do not contain a mutant Ras or B-Raf46
to constitutively activate this pathway. Relative inactivity of the targeted signaling pathway in tumor cells highlights the therapeutic significance of sorafenib inhibitory effects on ECs. From this, it may be argued that angiogenesis inhibition played a prominent role in sorafenib efficacy against K1735 tumors only because tumor cell cytotoxic/cytostatic effects were relatively weak in these tumors. Indeed, it is difficult to know whether deleterious effects seen among K1735 cells in treated tumors were a direct effect of sorafenib or a consequence of angiogenesis inhibition. Although effects on tumor cells may play a larger role when the malignant cells depend more on Raf-MEK-ERK activation, angiogenesis dependency on activation of this pathway in ECs will probably not vary, so that angiogenesis inhibition should be a consistently significant feature of sorafenib therapy. This thesis is supported by the results of our Colo-205 tumor studies. These tumor cells have an activating B-Raf mutation14
and express p-ERK in vivo (Figure 4D)
. Sorafenib treatment inhibited p-ERK in EC (Table 1)
much more than in tumor cells,14
indicating that, even when tumor cells have activated Raf-MEK-ERK signaling, sorafenib inhibits signaling in ECs and produces anti-angiogenic effects.
Candidate targets of sorafenib inhibition in EC include VEGFR2 and Raf. Inhibition of either VEGFR2 or Raf can produce angiogenesis inhibition,28,47,48 so both are reasonable therapeutic targets in sorafenib-treated tumors. Unfortunately, the pattern of EC signaling inhibition does not allow identification of the kinase targeted. Down-modulation of the p-ERK would be expected if either or both kinases were inhibited by sorafenib. Absence of p-AKT down-modulation does not argue for Raf or against VEGFR2 being the target, because receptors other than VEGFR2 (eg, Tie2) may be responsible for activation of the PI3K-AKT pathway in tumor ECs. Distinguishing whether Raf or VEGFR2 (or both) is the kinase targeted by sorafenib in ECs in vivo may be therapeutically significant, because many angiogenic factors besides VEGF signal through the Raf-MEK-ERK pathway, and inhibiting Raf may attenuate the action of all these factors.
The approaches used here to study EC dynamics and treatment response in mouse tumors can be adapted to study similar parameters and events in human tumors. Phospho-ERK in microvessels reflects EC stimulation and activation and is present in a significant fraction of vascular ECs in different types of human cancers. Examples of human melanoma and renal cell carcinoma stained for CD34 and p-ERK are shown in Figure 6
. Sorafenib is being tested in patients with these cancers in clinical trials,15,16
and the presence of p-ERK in vascular ECs in these cancers indicates that their vessels are likely targets of therapy. If tumor tissue obtained during treatment were also available for study, this type of histological analysis could also report the effects of targeted anti-angiogenic agents and provide a definitive assessment of therapeutic response.
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Acknowledgements |
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Footnotes |
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Supported by the National Institutes of Health (grants RO1 CA99519 and RO1 CA77851 to W.M.F.L.).
Supplemental material for this article can be found on http://ajp.amjpathol.org.
Accepted for publication August 10, 2006.
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