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Clinical Cancer Research 14, 3589, June 1, 2008. doi: 10.1158/1078-0432.CCR-07-4306
© 2008 American Association for Cancer Research
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Cancer Therapy: Preclinical

Combination Strategy Targeting the Hypoxia Inducible Factor-1{alpha} with Mammalian Target of Rapamycin and Histone Deacetylase Inhibitors

Henk M.W. Verheul1,2, Brenda Salumbides1, Karen Van Erp1, Hans Hammers1, David Z. Qian1, Tolib Sanni1, Peter Atadja3 and Roberto Pili2

Authors' Affiliations: 1 Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore Maryland; 2 The University Medical Center Utrecht, Utrecht, The Netherlands; and 3 Novartis Institute for Biomedical Research, East Hanover, New Jersey

Requests for reprints: Roberto Pili, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, 1650 Orleans Street, Cancer Research Building I, 1M52, Baltimore, MD 21231. Phone: 410-502-7482; Fax: 410-614-8160; E-mail: piliro{at}jhimi.edu.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Purpose: The hypoxia-inducible factor-1{alpha} (HIF-{alpha}) is a key regulator of tumor angiogenesis. Mammalian target of rapamycin (mTOR) and histone deacetylase (HDAC) inhibitors suppress tumor-induced angiogenesis by reducing tumor HIF-1{alpha} protein expression. Thus, we hypothesized that combination treatment of rapamycin and the HDAC inhibitor LBH589 has greater antiangiogenic and antitumor activity compared with single agents.

Experimental Design: To evaluate the effect of LBH589 and rapamycin on HIF-1{alpha} in human prostate PC3, renal C2 carcinoma cell lines, and endothelial cells (human umbilical vein endothelial cells), we did Western blot analysis. To determine the antitumor activity of LBH589 and rapamycin, cell proliferation assays and xenograft experiments were conducted.

Results: Western blotting showed that combination treatment of human umbilical vein endothelial cells, C2 and PC3, significantly reduced HIF-1{alpha} protein expression compared with single agents. Treatment with rapamycin resulted in inhibition of the downstream signals of the mTOR pathway and increased phosphorylation of Akt in C2 cells, whereas the constitutively activated Akt in PC3 cells was not modulated. LBH589 decreased both constitutively expressed and rapamycin-induced phosphorylated Akt levels in PC3 and C2 cell lines. In clonogenic assays, the combination treatment had a greater inhibitory effect in PC3 cells (93 ± 1.4%) compared with single agents (66 ± 9% rapamycin and 43 ± 4% LBH589). Combination of rapamycin and LBH589 significantly inhibited PC3 and C2 in vivo tumor growth and angiogenesis as measured by tumor weight and microvessel density.

Conclusions: Combination treatment of mTOR and HDAC inhibitors represents a rational therapeutic strategy targeting HIF-1{alpha} that warrants clinical testing.

Angiogenesis is required for tumor progression and represents a rational target for therapeutic intervention as proposed by Judah Folkman since 1971 (1). The hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) plays a key role in tumor angiogenic phenotype (2, 3). In normoxic conditions, HIF-1{alpha} is hydroxylated at the proline residue and degraded by interaction with the von Hipple-Lindau protein complex and proteosome machinery. Under low oxygen tension or in the presence of oncogenic transformation, HIF-1{alpha} protein is stabilized and translocated into the nucleus for specific gene expression regulation including the key angiogenic growth factor vascular endothelial growth factor (VEGF). In certain tumors (that is, renal cell carcinoma), genetic and epigenetic silencing of the von Hipple-Lindau tumor suppressor gene may also induce overexpression of HIF-1{alpha}. Therefore, HIF-1{alpha} has become an attractive target for the development of anticancer drugs (4). Not only VEGF is up-regulated by HIF-1{alpha} but also other growth factors such as platelet-derived growth factor-β polypeptide and transforming growth factor-{alpha}. In addition, HIF-1{alpha} regulates intracellular pH, metabolism, cell invasion, and autophagy and prevents cell death (5).

Histone deacetylase (HDAC) inhibitors represent an emergent class of therapeutic agents that induce tumor cell cytostasis, differentiation, and apoptosis in various hematologic and solid malignancies (6). HDAC inhibitors have also been reported to inhibit tumor angiogenesis (7, 8). Angiogenesis inhibition by HDAC inhibitors may be in part mediated by HIF-1{alpha} down-regulation in both tumor and endothelial cells and consequently down-regulation of VEGF and other HIF-1{alpha}-regulated angiogenesis-related genes (7). Class II HDAC associate with HIF-{alpha} and are important modifiers of HIF-1{alpha} protein stability via a von Hipple-Lindau–independent but proteosome-dependent pathway (8). We have recently reported that the HDAC inhibitor LBH589 reduced tumor growth and angiogenesis in a preclinical prostate cancer model (9).

Another pathway involved in tumor growth and angiogenesis is the mammalian target of rapamycin (mTOR) signaling pathway especially when the tumor suppressor gene PTEN is mutated (10). The mTOR signaling cascade downstream of the phosphatidylinositol 3-kinase/Akt pathway plays a central role in cell survival and proliferation. Dysregulation of the mTOR pathway has been implicated in cancer cell growth and survival. Inhibition of the mTOR pathway reduces angiogenesis and tumor growth in a variety of tumor types (11). VEGF production and signaling is partly dependent on mTOR-induced expression of HIF-1{alpha} (12). The mTOR inhibitor rapamycin was initially developed as an immunosuppressant and has been in clinical development as an anticancer agent and HIF-1{alpha} inhibitor (13). The preclinical and clinical development of other mTOR inhibitors, such as CCI-779 and RAD001, suggests an improved bioavailability of these agents compared with rapamycin (14, 15). Treatment with CCI-779 has been reported to induce survival benefit in patients with advanced real cell cancer and poor prognosis and has been recently approved by the Food and Drug Administration for the treatment of advanced kidney cancer (16). Phase II studies with RAD001 have shown clinical activity in patients with kidney cancer, and the results from the phase III trial are eagerly awaited (17).

Drug resistance represents a major hurdle in cancer therapeutics. Inhibition of the mTOR pathway has been shown to sensitize tumor cells for chemotherapeutic agents such as doxorubicin and radiotherapy (18, 19). On the other hand, it has been reported both in vitro and in vivo that inhibition of the mTOR pathway can induce Akt phosphorylation as well as tyrosine kinase receptor activation that regulates alternative pathways involved in potential mechanisms of resistance (20). In this study, we hypothesized that inhibition of the HIF pathway by different mechanisms can increase antitumor activity of targeted therapies. We assessed the biological activity of combination treatment of a HDAC inhibitor (LBH589) and the mTOR inhibitor rapamycin both in vitro and in vivo. Not only HIF suppression was increased by the combination, but we also observed that either constitutive or rapamycin-induced phosphorylation of Akt was abrogated by concomitant treatment with LBH589. In vitro and in vivo studies in a s.c. prostate cancer model and an orthotopic renal cell cancer model revealed that combination treatment of the mTOR inhibitor rapamycin and the HDAC inhibitor LBH589 had a greater antitumor activity compared with either agent alone.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Reagents and cell culture. von Hipple-Lindau–deficient renal cell carcinoma cell line UMRC2 (C2) cells were kindly provided Drs. Jennifer Isaacs and Len Neckers (National Cancer Institute, NIH). Cells were cultured in DMEM with 10% fetal bovine serum. Human umbilical vein endothelial cells (HUVEC) were purchased from Clonetics and cultured in complete EGM containing EGM bullet kit (Cambrex). HDAC inhibitor LBH589 was provided by Novartis. Rapamycin was purchased from Sigma. Before each assay, cells were starved overnight with 0.5% fetal bovine serum. Assays were done in 2% fetal bovine serum or indicated otherwise.

Western blotting of HIF-1{alpha}, tissue factor, and cell signaling cascade. Western blot analyses were carried out on either 4% to 15% gradient gel or 7.5% gel according to methods described previously (8). The monoclonal or polyclonal antibodies for p21, p70S6kinase (Thr389), pAkt (Ser473), and acetylated histone 3 were purchased from Cell Signaling, HIF-1{alpha} was from R&D Systems, and tissue factor was from American Diagnostics. Cells were seeded, and after attaching, they were starved overnight at low serum. Subsequently, cells were treated for 24 h, and for each condition, 10 to 50 µg total protein lysates were used for Western blot analysis to detect protein levels. HUVEC were treated with 25 ng/mL VEGF and the accumulation of HIF-1 was induced by an hypoxia mimetic (100 µmol/L CoCl2; Sigma) in HUVEC. The same blots were probed with β-actin or vinculin (Sigma) as equal loading control.

Tumor cell VEGF production. VEGF concentrations were determined in supernatant of tumor cells by ELISA according to standard procedures (R&D Systems) and corrected for cell number. DMEM with 2% did not contain detectable VEGF.

Tissue factor activity assay. Tissue factor activity assay was done as described previously (21). Briefly, after starvation and subsequent treatment of HUVEC for 6 h and C2 cells for 24 h in 96-well plates, cells were washed and subsequently incubated with HEPES (0.2% glucose, pH 7.4) containing activated factor VIIa (500 ng/mL; American Diagnostica) and factor X (1 unit/mL; American Diagnostica) and incubated for 45 min at 37°C, allowing the formation of factor Xa by the tissue factor-factor VIIa complex. Next, the reaction was stopped with assay buffer containing 75 mmol/L EDTA. The supernatant containing factor Xa was transferred to a new well and incubated with the chromogenic substrate S-2765 (DiaPharma; 2 mmol/L diluted in H2O). This substrate is converted by activated factor Xa as an indirect measure for tissue factor activity (in arbitrary units). The 96-well plates were read in an automated plate reader at 405 nm for 10 min.

Colony formation assay. Exponentially growing tumor cells were seeded at 2,000 per well in six-well plates and allowed to attach for 24 h and subsequently starved in serum-free medium overnight. Cells were treated with either rapamycin or LBH589 or combination at different concentrations in complete medium containing DMSO less than 0.001% or 0.1%, respectively. After 72 h, cells were rinsed, and fresh medium was added. Cells were cultured for 7 days in 10% fetal bovine serum–containing medium and then fixed and stained with crystal violet. Each condition was counted in duplicate (10 fields per well) on an inverted microscope. The experiments were repeated two times with similar results. One well is represented from each treatment set (n = 3 per single agent or combination). Images of stained colonies were captured using Kodak Image Station 440cF and area of interest was digitally analyzed by Image-Pro Plus (version 3.0, Media Cybernetics). Percentage inhibition by area of interest was calculated using the following formula with untreated control as baseline:

Formula

Additivism and synergism were calculated according to the Bliss model (22). Bliss additivism model predicts the combined response C for two single compounds with effects A and B is

Formula
where each effect is expressed as fractional inhibition between 0 and 1 (23).

Tumor cell proliferation assay. Cell proliferation assays were done in 96-well plates and proliferation was determined measured by XTT according standard procedures. Proliferation was measured in 72-h incubation experiments. Experiments were repeated twice.

In vivo tumor experiments. Animal protocols were approved by the Institutional Care and Use Committee at the Johns Hopkins Medical Institutions. Orthotopic C2 tumor model: 6-week-old male athymic nude mice were housed under pathogen-free conditions. Luciferase-transfected C2 cells (2 x 106) were placed orthotopically into the subcapsular space of the left kidney. Tumor imaging was done by i.p. administration of luciferin using bioluminescence technology (Xenogen system). For the s.c. prostate cancer model, 2 x 106 PC3 prostate cancer cells were s.c. injected on both flanks of 6-week-old male athymic nude mice. Tumor growth was assessed twice weekly by using caliper, and the size was expressed in mm3 using the standard formula: length x (width)2 x 0.52. Before starting the treatment, mice were divided in homogenous groups (5-7 per group) according to tumor burden determined by either luciferase expression (C2) or size (PC3). Treatment-related toxicity was determined by mouse weights weekly. Tumor weights were determined when mice were sacrificed at the end of the study. Mice were treated with rapamycin at 2 or 3.75 mg/kg/d by oral gavage and i.p. injection of LBH589 at 5 or 10 mg/kg/d.

Immunohistochemistry of microvessel density. Zinc-fixed, paraffin-embedded tissue was generated from each tumor to quantify differences in microvessel density (MVD) between control and experimental groups. Sections were incubated (18 h at 4°C) with anti-CD31 antibody (PharMingen), a specific marker for endothelial cells after deparaffinization. Sections were incubated with a secondary biotin-conjugated rabbit anti-goat IgG antibody (1:100) for 30 min at room temperature. Sections were incubated with avidin-biotin peroxidase complex (Vector Laboratories) as per manufacturer's instructions. Sections were then incubated with 3,3'-diaminobenzidine solution, washed, and counterstained with methyl green. MVD was determined with software program Image Pro [at least 10 fields (x20) per tumor tissue section were analyzed].

Statistical analysis. Statistical analysis was done using Student's t test and ANOVA to compare multiple groups. P values < 0.05 were considered statistically significant. All experiments were done twice. Error bars reflect SE.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Effect of rapamycin and LBH589 on HIF-1{alpha} expression in tumor cells and endothelial cells. To test whether down-regulation of HIF-1{alpha} by mTOR and HADC inhibition is enhanced by combination treatment, we incubated two tumor cell lines with rapamycin and LBH589. In parallel, HUVEC were treated with this combination treatment under VEGF stimulation (25 ng/mL, 24 hours) and hypoxic-mimetic (100 µmol/L CoCl2, 6 hours) conditions. Combination of rapamycin and LBH589 abrogated HIF-1{alpha} protein expression compared with single agents in both C2 renal cell cancer cells and HUVEC (Fig. 1 ). In PC3 cells, HIF-1{alpha} inhibition was also induced by combination therapy but to a lesser extent (data not shown).


Figure 1
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  		Fig. 1. HIF-1{alpha} inhibition by rapamycin and LBH589. C2 cells and HUVEC were starved overnight and subsequently treated with either 50 nmol/L LBH589, 100 nmol/L rapamycin, or combination. HUVEC were stimulated with VEGF to induce tissue factor expression. To mimic hypoxia, 100 µmol/L CoCl2 was added to the culture medium for the last 6 h. Cells were treated for 24 h, and for each condition, 50 µg total protein lysates were used for Western blot analysis to detect HIF-1{alpha} protein levels. The same blot was probed for β-actin as equal loading control.

 
Effects of rapamycin and LBH589 on Akt and downstream intracellular signaling proteins. As expected, treatment with LBH589 resulted in both C2 and PC3 cells increased acetylation of histone 3 and p21 expression (Fig. 2A ). In C2 cells, but not in PC3 cells, combination treatment of LBH589 and rapamycin reduced p21 expression. As reported, rapamycin may induce pAkt in tumor cells (20). Western blotting analysis revealed that treatment with rapamycin in C2 tumor cells induced pAkt, whereas, in PC3 cells, pAkt was constitutively phosphorylated due to PTEN deletion as reported previously (Fig. 2A). In both C2 and PC3 cells, pAkt expression was reduced by combination treatment with LBH589. Downstream in the mTOR pathway, phosphorylation of kinase protein (pS6kinase) was reduced by rapamycin but not affected by LBH589 (Fig. 2B). In addition, tissue factor expression, the main initiator of the coagulation cascade, known to be induced by rapamycin in endothelial cells, was inhibited by treatment with rapamycin and further reduced by treatment with LBH589 in the C2 tumor cell line (Fig. 2B). Functional inhibition of tissue factor activity of C2 cells by combination treatment was confirmed in a tissue factor activity assay in vitro (Fig. 2C). In addition, rapamycin-induced tissue factor activity in HUVEC was also decreased by LBH589 back to baseline. VEGF production by both C2 and PC3 cells was reduced by treatment with LBH589 and combination treatment with only minor reduction by rapamycin treatment (Fig. 2D; ref. 9).


Figure 2
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  		Fig. 2. Differential effect of LBH589 and rapamycin on p21 expression in C2 and PC3 cells. C2 and PC3 cells were treated with either 50 nmol/L LBH589 or 100 nmol/L rapamycin, or combination for 24 h. A, p21 and histone 3 acetylation expressions were analyzed by Western blotting. The same blot was probed with β-actin as equal loading control. p21 expression was reduced by rapamycin in C2 cells but not in the PTEN mutated PC3 cells. Western blotting of pAkt expression shows an increased expression in the C2 cells on treatment with rapamycin, which is reduced by LBH589. In the PC3 cells, pAkt is constitutively phosphorylated, and no inhibition of LBH589 was observed. B, in C2 cells, Western blotting shows inhibition of the downstream of mTOR phosphorylation of p70S6kinase. In addition, tissue factor is reduced in C2 cells by rapamycin and LBH589. The same blot was probed for vinculin as equal loading control. C, tissue factor activity in a chromogenic assay with C2 cells and HUVEC revealed that LBH589 reduces rapamycin-induced tissue factor activity in HUVEC, whereas, in addition to LBH589, rapamycin reduced constitutive tissue factor activity in C2 cells. Columns, mean of three independent experiments; bars, SD. *, P < 0.05 versus positive control (VEGF stimulation of HUVEC/untreated C2 cells). D, inhibition of VEGF production by rapamycin and LBH589. C2 and PC3 cells were treated with rapamycin and LBH589 as single agent or in combination as described above. After 18 h, medium was repleted with fresh medium containing the same treatment. Six hours after repletion, supernatant was obtained, cells were counted, and VEGF concentrations were determined (all conditions in duplicate). Combination treatment significantly inhibited VEGF production in both cell lines as tested by ANOVA (P < 0.022).

 
Effect of rapamycin and LBH589 on PC3 tumor cell proliferation. To determine the antiproliferative activity of rapamycin and LBH589, a standard colony formation assay was conducted (24). Treatment of PC3 cells with rapamycin and LBH589 resulted in a 66 ± 9% and 43 ± 4% growth inhibition, respectively (Fig. 3A and B ). Combination treatment resulted in a greater inhibition of PC3 cell growth up to 93 ± 1.4% at the highest tested concentration of rapamycin (100 nmol/L) and LBH (10 nmol/L) in combination. The antitumor effect of combination treatment revealed a greater effect that reached synergism at lower concentrations.


Figure 3
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  		Fig. 3. Clonogenic assay with PC3 cells; treatment with combination of rapamycin and LBH589. Tumor cells were allowed to attach for 24 h and subsequently starved in serum-free medium overnight. Subsequently, cells were treated with either rapamycin or LBH589 for 72 h. After washing, cells were culture for 7 d in 10% fetal bovine serum–containing medium and then were fixed and stained with crystal violet. Experiments were repeated twice. A, colony formation assay showed that tumor cells (PC3) were treated concomitantly with rapamycin plus LBH589 in increasing doses of 0 to 100 and 0 to 10 nmol/L, respectively. One well is represented from each treatment set (n = 3 per single agent or combination). Representative picture. B, each condition was tested in triplicate, quantitated (10 fields per well) by imaging software, and reported as percentage of area occupied by cells. Results are expressed as percentage of inhibition. The maximal inhibitory activity of monotherapy rapamycin resulted in a 66 ± 9% growth inhibition and with LBH589 in 43 ± 4% growth inhibition, whereas combination treatment resulted in a greater inhibition of the outgrowth of PC3 cells up to 93 ± 1.4% at the highest tested dose of rapamycin (100 nmol/L) and LBH589 (10 nmol/L) in combination. Additivism and synergism were calculated according to the Bliss model (see Materials and Methods).

 
Antitumor effect of rapamycin and LBH589 in vivo. In mice with s.c. injected PC3 cells and orthotopically implanted C2 renal cell cancer, combination treatment with rapamycin and LBH589 significantly resulted in a greater antitumor effect compared with either agent alone (Fig. 4A-C ). Growth of PC3 tumors were significantly inhibited by 78 ± 7% with low-dose combination treatment of rapamycin (2 mg/kg/d) and LBH589 (5 mg/kg/d) compared with single-agent treatment (P = 0.00018, ANOVA, n = 6). This low dose of rapamycin (2 mg/kg) resulted in plasma achievable concentrations (4.12 ± 3.22 ng/mL). Single-agent treatment at these doses resulted in growth inhibition of 54 ± 19% and 42 ± 12% with rapamycin and LBH589, respectively. At the maximal tolerated dose of the combination treatment, growth inhibition of PC3 tumors in vivo resulted in a significant regression of tumors compared with start of treatment. Tumor volume after 49 days after injection was compared with start of treatment (P = 0.01, t test; Fig. 4B). In these experiments, rapamycin was administered at a dose of 3.75 mg/kg/d, whereas LBH589 was administered at a dose of 10 mg/kg/d. Single-agent treatment at this dose resulted in growth inhibition of 88 ± 1.9% and 74 ± 0.7%. Combination treatment was significantly more active than single-agent treatment as tested by ANOVA (P = 0.00023). Significant inhibition of tumor volume by combination treatment was confirmed by tumor weights as shown in Fig. 4C. In the C2 tumor model, tumor growth by combination treatment was almost completely blocked in a 36-day treatment experiment (Fig. 4D). A greater growth inhibition (86 ± 10%) was observed with the combination treatment compared with either agent alone (3.75 mg/kg rapamycin, 69 ± 4% inhibition; 10 mg/kg LBH589, 78 ± 5% inhibition). Combination treatment was significantly more active than single-agent treatment as tested by ANOVA (P = 0.007).


Figure 4
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  		Fig. 4. Tumor growth inhibition in vivo by combination treatment of rapamycin plus LBH589. PC3 prostate cancer cells (2 x 106) were s.c. injected on both flanks of 6-week-old male athymic nude mice and growth was measured twice weekly by using caliper and the size was expressed in mm3 using the standard formula: length x (width)2 x 0.52. Before start of treatment, mice were divided in 5 to 7 mice per group according to tumor volume. A, daily treatment with low-dose rapamycin (2 mg/kg by oral gavage) and LBH589 (5 mg/kg i.p.) of PC3 tumor-bearing mice. PC3 tumor growth was significantly greater inhibited by 78 ± 7% with combination treatment compared with single-agent treatment (P = 0.00018, ANOVA, n = 6). Single-agent treatment at these doses resulted in growth inhibition of 54 ± 19% and 42 ± 12% with rapamycin and LBH589, respectively. B, at the maximal tolerated dose, rapamycin was administered at a dose of 3.75 mg/kg/d, whereas LBH589 was administered at a dose of 10 mg/kg/d. Combination treatment resulted in a significant regression of PC3 tumors compared with start of treatment. Tumor volume after 49 days after injection was compared with start of treatment (P = 0.01, t test). Single-agent treatment at this dose resulted in growth inhibition of 88 ± 1.9% and 74 ± 0.7% by rapamycin and LBH589, respectively. Combination treatment was significantly more active than single-agent treatment as tested by ANOVA (P = 0.00023). C, experiment was terminated because controls had to be euthanized. Significant inhibition of tumor growth by combination treatment was confirmed by tumor weights. D, in the C2 tumor model, 2 x 106 luciferase-transfected C2 cells were placed orthotopically into the subcapsular space of the left kidney. Tumor growth by combination treatment was almost completely blocked in a 36-day treatment experiment. A significantly additive growth inhibition of 86 ± 10% of the combination treatment was observed compared with 69 ± 4% and 78 ± 5% inhibition of either agent alone with rapamycin (3.75 mg/kg) and LBH589 (10 mg/kg), respectively. Combination treatment was significantly more active than single-agent treatment as tested by ANOVA (P = 0.007).

 
Effect of rapamycin and LBH589 on MVD. Despite previous reports in pancreatic cancer models, we were not able to detect increased intratumoral/intravascular thrombosis by treatment with rapamycin in both C2 and PC3 tumors (H&E; data not shown). In contrast, in both in vivo models, angiogenesis as reflected by MVD was significantly inhibited by combination treatment compared with either agent alone (see Fig. 5A and B ). Combination treatment of the C2 tumors reduced the area of MVD to 0.8 ± 0.1% compared with 2.2 ± 0.4% of controls, 1.25 ± 0.2% with LBH589, and 1 ± 0.1% with rapamycin treatment (P = 0.003, ANOVA). Combination treatment of the PC3 tumors reduced the area of MVD to 1.3 ± 0.2% compared with 7.4 ± 0.6% in controls, 3.7 ± 0.4% in LBH589-treated tumors, and 2.9 ± 0.3% in rapamycin-treated tumors (P = 0.0005, ANOVA).


Figure 5
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  		Fig. 5. Immunohistochemistry of C2 and PC3 harvested tumors after treatment with rapamycin and LBH589. Zinc-fixed, paraffin-embedded tissue was generated from each tumor for further analysis by immunohistochemistry to quantify differences in MVD between control and experimental groups according to standard protocols as described in Materials and Methods. A and B, left, an assembly of pictures shows a representative area of the vascular density of the tumor from the different treatment groups. Brown, endothelial cells. Right, quantification of the brown staining revealed that the percent area occupied by vasculature in the combination group was significantly reduced compared with monotherapy or control tumors. A, combination treatment of the C2 tumors reduced the area of MVD to 0.8 ± 0.1% compared with 2.2 ± 0.4% of controls, 1.25 ± 0.2% of LBH589, and 1 ± 0.1% of rapamycin treatment (P = 0.003, ANOVA). B, combination treatment of the PC3 tumors reduced the area of MVD to 1.3 ± 0.2% compared with 7.4 ± 0.6% of controls, 3.7 ± 0.4% of LBH589, and 2.9 ± 0.3% of rapamycin treatment (P = 0.0005, ANOVA).

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
Targeting the mTOR pathway or inhibiting HDAC activity are rational therapeutic strategies currently in clinical development. Despite the antitumor activity, the role of mTOR and HDAC inhibitors as single agents for cancer treatment appears to be limited. In solid tumors (that is, prostate and renal cell cancer), both the mTOR pathway and HDAC modifications are important for tumor progression (13, 25). The results of our studies indicate that the rationally designed combination treatment with HDAC and mTOR pathway inhibition targeting HIF-1{alpha} has clinical potential as an anticancer treatment strategy.

HDAC and mTOR inhibitors are known to inhibit tumor angiogenesis (7, 26). Post-transcriptional modulation of HIF-{alpha} has been reported by both classes of agents. Targeting this transcriptional factor represents an intense field of research in cancer therapeutics (24–26). In the present study, we observed that concomitant exposure of tumor and endothelial cells to HDAC and mTOR inhibitors led to a greater inhibition of HIF-1{alpha} protein expression. It is conceivable that inhibition of HIF-{alpha} by the proposed combination strategy may occur at different levels including translation, transcriptional activity, and protein stability. Reduction of HIF-{alpha} protein levels was associated with decreased tumor angiogenesis. The block of the HIF-1{alpha} pathway has clinical implications especially in the treatment of advanced renal cell carcinoma. Molecular targeted therapies with VEGFR tyrosine kinase inhibitors have been reported to have clinical activity in metastatic renal cell carcinoma patients and have been recently approved by the Food and Drug Administration (27). However, there are preclinical and clinical evidences that tumor "escape" to anti-VEGF therapy occurs due to hypoxia and HIF-1{alpha} up-regulation (28). A rational combination strategy blocking HIF-{alpha} with HDAC and mTOR inhibitors may affect tumor cell and endothelial cell adaptation to anti-VEGF therapies.

The combination treatment with plasma achievable concentrations of rapamycin and LBH589 had also a direct antitumor effect as shown by the in vitro clonogenic assay. Interestingly, exposure to LBH589 inhibited rapamycin-induced pAkt in C2 renal cells. It has been reported extensively that phosphorylation of Akt not only leads to the activation of the mTOR pathway but also activates alternative cell growth signals (29). Recently, Akt phosphorylation induced by treatment with rapamycin has been suggested to play a role in the resistance to mTOR inhibitors (20). Activated Akt may attenuate rapamycin growth-inhibitory effects serving as a negative feedback mechanism. Our findings indicate that concomitant treatment with LBH589 may eventually overcome resistance by decreasing rapamycin-induced Akt phosphorylation. In support of this model, rapamycin combined with the phosphatidylinositol 3-kinase inhibitor LY294002 has been reported to a have a greater antitumor effect (30). Based on our results, we hypothesized that this combination may have greater and prolonged antitumor activity. In a clonogenic assay with PC3 cells, we observed a greater antitumor activity of the combination treatment compared with either agent alone. In our in vivo models of renal cell cancer and prostate cancer, the combination treatment of rapamycin and LBH589 had a significant antitumor activity compared with either agent alone. In the PC3 model at low doses, an additive tumor inhibition was observed, whereas at the maximal tolerated dose a significant reduction of tumor size was obtained after 3 weeks of treatment. In both in vivo models of prostate and renal carcinoma, combination treatment reduced the angiogenic potential of these tumors as reflected by a significantly decreased MVD.

The role of p21 induction by HDAC inhibition is still controversial and its contribution to induction of apoptosis is unclear (31). In contrast, rapamycin is known to reduce p21 expression (32). Rapamycin has been reported to induce primarily G1 cell cycle arrest, whereas for HDAC inhibition G2 arrest may be responsible for the induction of apoptosis. Under our experimental conditions, rapamycin blocked p21 induction by LBH589. Further studies are needed to get a better understanding how these agents in combination affect cell cycle checkpoints. Interestingly, the in vivo experiments were indicative for a cytostatic effect as suggested by the delayed tumor growth inhibition rather than induction of tumor cell apoptosis measured by immunohistochemistry.

A previous report has shown that the in vivo antitumor activity of rapamycin might be due in part to the induction of intratumoral thrombosis in a pancreatic cancer model (33). In the prostate and renal cell cancer models used, no intratumoral thrombosis was observed. Our in vitro results revealed that rapamycin reduced tissue factor expression and activity in PC3 and C2 tumor cells in contrast to its effect on endothelial cells (34). Reduction of lipopolysaccharide-induced tissue factor activity by rapamycin has been reported previously in mononuclear cells and smooth muscle cells (35). Rapamycin is known to induce tissue factor expression and activity on endothelial cells (34). For the first time, our results suggest that treatment with a HDACI reduced tissue factor expression and activity in both tumor cells and VEGF-stimulated endothelial cells. This reduction in endothelial cells might be mediated by a reduction in nuclear factor-{kappa}B binding to chromatin (36). In addition, we observed that rapamycin-induced tissue factor expression and activity was decreased by HDAC inhibition. Taken together, these data support the potential of this combination treatment, in view also that several studies have shown the role of tissue factor in promoting tumor angiogenesis and metastasis (37).

In our studies, the maximum tolerated dose of rapamycin and LBH589 was relatively well tolerated in the combination group, although some weight loss and bone marrow suppression were observed (9, 38). Blood cell counts in mice treated with full-dose LBH589 as single agent or in combination with rapamycin revealed hematologic toxicity with >50% decrease in leukocyte and platelet counts. Single-agent rapamycin had no effect on blood counts. The hematologic toxicity of HDAC inhibitors is well known in the clinic; therefore, intermittent dosing is mostly used with these agents (39). Fifty-percent reduction of maximal tolerated doses of rapamycin and LBH589 in combination studies achieved an in vivo additive effect without overt toxicity, suggesting that combination of lower doses of HADC and mTOR inhibitors may allow continuous effective treatment with decreased toxicity.

In conclusion, combination treatment with the mTOR inhibitor rapamycin and the HDAC inhibitor LBH589 is an attractive combination strategy for the treatment of prostate and renal cell cancer. As the clinical development of both mTOR and HDAC inhibitors proceeds rapidly, it will be important to test this novel combination strategy in rationally designed clinical trials.


   Disclosure of Potential Conflicts of Interest
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No potential conflicts of interest were disclosed.


   Acknowledgments
 
We want to thank Drs. Ming Zhao and Michelle Rudek for assessing rapamycin plasma concentrations in mice and the Cell Imaging Facility at the Sidney Kimmel Comprehensive Cancer Center for the assistance.


   Footnotes
 
Grant support: Drug Development Fellowship Program of the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins and Adriane van Coevorden Stichting grant (H.M.W. Verheul), Commonwealth Foundation (H.M.W. Verheul and R. Pili), and a donation from Marsha and John Fausti (R. Pili).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: H.M.W. Verheul is a recipient of the American Society of Clinical Oncology Young Investigator's Award 2006.

Received 8/27/07; revised 2/ 8/08; accepted 2/11/08.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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