David Z. Qian

Digoxin and other cardiac glycosides inhibit HIF-1α synthesis and block tumor growth

A library of drugs that are in clinical trials or use was screened for inhibitors of hypoxia-inducible factor 1 (HIF-1). Twenty drugs inhibited HIF-1-dependent gene transcription by >88% at a concentration of 0.4 μM. Eleven of these drugs were cardiac glycosides, including digoxin, ouabain, and proscillaridin A, which inhibited HIF-1α protein synthesis and expression of HIF-1 target genes in cancer cells. Digoxin administration increased latency and decreased growth of tumor xenografts, whereas treatment of established tumors resulted in growth arrest within one week. Enforced expression of HIF-1α by transfection was not inhibited by digoxin, and xenografts derived from these cells were resistant to the anti-tumor effects of digoxin, demonstrating that HIF-1 is a critical target of digoxin for cancer therapy.

Targeting Tumor Angiogenesis with Histone Deacetylase Inhibitors: the Hydroxamic Acid Derivative LBH589

Purpose: Angiogenesis is required for tumor progression and represents a rational target for therapeutic intervention. Histone deacetylase (HDAC) inhibitors have been shown to have activity against various tumor cell types by inhibiting proliferation and inducing apoptosis both in vitro and in vivo. HDAC inhibitors have also been reported to inhibit angiogenesis. The goal of this study was to characterize the antiangiogenic and antitumor activity of a recently developed HDAC inhibitor, the hydroxamic derivative LBH589. Materials and Methods: To evaluate the antiangiogenesis activity of LBH589, we did cell cycle analysis, cell proliferation, tube formation, invasion assays in vitro, and Matrigel plug assay in vivo. To determine the antitumor activity of LBH589, we established human prostate carcinoma cell PC-3 xenografts in vivo. To evaluate the effect of LBH589 on endothelial signaling pathways, gene expression, and protein acetylation, we did Western blots and reverse transcription-PCR in human umbilical vein endothelial cells (HUVEC). Immunohistochemical analysis was done to evaluate new blood vessel formation in vivo. Results: LBH589 induced acetylation of histone H3 and α-tubulin protein in HUVECs. Histone and nonhistone protein acetylation correlated with induction of G2-M cell cycle arrest, inhibition of HUVEC proliferation, and viability. Noncytotoxic concentrations of LBH589 inhibited endothelial tube formation, Matrigel invasion, AKT, extracellular signal-regulated kinase 1/2 phosphorylation, and chemokine receptor CXCR4 expression. In vivo dosing of mice with LBH589 (10 mg/kg/d) reduced angiogenesis and PC-3 tumor growth. Conclusion: This study provides evidence that LBH589 induces a wide range of effects on endothelial cells that lead to inhibition of tumor angiogenesis. These results support the role of HDAC inhibitors as a therapeutic strategy to target both the tumor and endothelial compartment and warrant the clinical development of these agents in combination with angiogenesis inhibitors.

Tubulin-Targeting Chemotherapy Impairs Androgen Receptor Activity in Prostate Cancer

Recent insights into the regulation of the androgen receptor (AR) activity led to novel therapeutic targeting of AR function in prostate cancer patients. Docetaxel is an approved chemotherapy for treatment of castration-resistant prostate cancer; however, the mechanism underlying the action of this tubulin-targeting drug is not fully understood. This study investigates the contribution of microtubules and the cytoskeleton to androgen-mediated signaling and the consequences of their inhibition on AR activity in human prostate cancer. Tissue microarrays from docetaxel-treated and untreated prostate cancer patients were comparatively analyzed for prostate-specific antigen (PSA) and AR immunoreactivity. The AR transcriptional activity was determined in prostate cancer cells in vitro, based on PSA mRNA expression and the androgen response element reporter activity. The interaction of AR with tubulin was examined by immunoprecipitation and immunofluorescence. Treatment of prostate cancer patients with docetaxel led to a significant translocation of AR. In untreated specimens, 50% prostate tumor cells exhibited nuclear accumulation of AR, compared with docetaxel-treated tumors that had significantly depleted nuclear AR (38%), paralleled by an increase in cytosolic AR. AR nuclear localization correlated with PSA expression. In vitro, exposure of prostate cancer cells to paclitaxel (1 μmol/L) or nocodazole (5 μg/mL) inhibited androgen-dependent AR nuclear translocation by targeting AR association with tubulin. Introduction of a truncated AR indicated the requirement of the NH(2)-terminal domain for AR-tubulin interaction. Our findings show that in addition to blocking cell division, docetaxel impairs AR signaling, evidence that enables new insights into the therapeutic efficacy of microtubule-targeting drugs in prostate cancer.

Acriflavine inhibits HIF-1 dimerization, tumor growth, and vascularization

HIF-1 is a heterodimeric transcription factor that mediates adaptive responses to hypoxia and plays critical roles in cancer progression. Using a cell-based screening assay we have identified acriflavine as a drug that binds directly to HIF-1α and HIF-2α and inhibits HIF-1 dimerization and transcriptional activity. Pretreatment of mice bearing prostate cancer xenografts with acriflavine prevented tumor growth and treatment of mice bearing established tumors resulted in growth arrest. Acriflavine treatment inhibited intratumoral expression of angiogenic cytokines, mobilization of angiogenic cells into peripheral blood, and tumor vascularization. These results provide proof of principle that small molecules can inhibit dimerization of HIF-1 and have potent inhibitory effects on tumor growth and vascularization.

Class II Histone Deacetylases Are Associated with VHL-Independent Regulation of Hypoxia-Inducible Factor 1α

Hypoxia-inducible factor 1 alpha (HIF-1 alpha) plays a critical role in transcriptional gene activation involved in tumor angiogenesis. A novel class of agents, the histone deacetylase (HDAC) inhibitors, has been shown to inhibit tumor angiogenesis and HIF-1 alpha protein expression. However, the molecular mechanism responsible for this inhibition remains to be elucidated. In the current study, we investigated the molecular link between HIF-1 alpha inhibition and HDAC inhibition. Treatment of the VHL-deficient human renal cell carcinoma cell line UMRC2 with the hydroxamic HDAC inhibitor LAQ824 resulted in a dose-dependent inhibition of HIF-1 alpha protein via a VHL-independent mechanism and reduction of HIF-1 alpha transcriptional activity. HIF-1 alpha inhibition by LAQ824 was associated with HIF-1 alpha acetylation and polyubiquitination. HIF-1 alpha immunoprecipitates contained HDAC activity. Then, we tested different classes of HDAC inhibitors with diverse inhibitory activity of class I versus class II HDACs and assessed their capability of targeting HIF-1 alpha. Hydroxamic acid derivatives with known activity against both class I and class II HDACs were effective in inhibiting HIF-1 alpha at low nanomolar concentrations. In contrast, valproic acid and trapoxin were able to inhibit HIF-1 alpha only at concentrations that are effective against class II HDACs. Coimmunoprecipitation studies showed that class II HDAC4 and HDAC6 were associated with HIF-1 alpha protein. Inhibition by small interfering RNA of HDAC4 and HDAC6 reduced HIF-1 alpha protein expression and transcriptional activity. Taken together, these results suggest that class II HDACs are associated with HIF-1 alpha stability and provide a rationale for targeting HIF-1 alpha with HDAC inhibitors against class II isozymes.

Anthracycline chemotherapy inhibits HIF-1 transcriptional activity and tumor-induced mobilization of circulating angiogenic cells

Using a cell-based reporter gene assay, we screened a library of drugs in clinical use and identified the anthracycline chemotherapeutic agents doxorubicin and daunorubicin as potent inhibitors of hypoxia-inducible factor 1 (HIF-1)-mediated gene transcription. These drugs inhibited HIF-1 by blocking its binding to DNA. Daily administration of doxorubicin or daunorubicin potently inhibited the transcription of a HIF-1-dependent reporter gene as well as endogenous HIF-1 target genes encoding vascular endothelial growth factor, stromal-derived factor 1, and stem cell factor in tumor xenografts. CXCR4+/sca1+, VEGFR2+/CD34+, and VEGFR2+/CD117+ bone-marrow derived cells were increased in the peripheral blood of SCID mice bearing prostate cancer xenografts but not in tumor-bearing mice treated for 5 days with doxorubicin or daunorubicin, which dramatically reduced tumor vascularization. These results provide a molecular basis for the antiangiogenic effect of anthracycline therapy and have important implications for refining the use of these drugs to treat human cancer more effectively.

The histone deacetylase inhibitor NVP-LAQ824 Inhibits angiogenesis and has a greater antitumor effect in combination with the vascular endothelial growth factor receptor tyrosine kinase inhibitor PTK787/ZK222584

Chromatin remodeling agents such as histone deacetylase inhibitors have been shown to modulate gene expression in tumor cells and inhibit tumor growth and angiogenesis. Vascular endothelial growth factor (VEGF) and VEGF receptors represent critical molecular targets for antiangiogenesis therapy. In this study, we investigated the biological effect of the histone deacetylase inhibitor NVP-LAQ824 in combination with the VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 on tumor growth and angiogenesis. We report that treatment with NVP-LAQ824 affected tumor and endothelial cells and was associated with increased histone acetylation, p21 up-regulation, and growth inhibition. In addition, NVP-LAQ824 treatment inhibited the expression of angiogenesis-related genes such as angiopoietin-2, Tie-2, and survivin in endothelial cells and down-regulated hypoxia-inducible factor 1-α and VEGF expression in tumor cells. Combination treatment with NVP-LAQ824 and PTK787/ZK222584 was more effective than single agents in inhibiting in vitro and in vivo VEGF-induced angiogenesis. Endothelial cell proliferation, tube formation, and invasion into the Matrigel plugs were reduced. In mouse models with established subcutaneous prostate (PC3) and orthotopic breast tumors (MDA-MB321), this combination treatment induced 80 to 85% inhibition of tumor growth without overt toxicity. These results suggest that the combination of histone deacetylase inhibitors and VEGF receptor inhibitors may target multiple pathways in tumor progression and angiogenesis and represents a novel therapeutic approach in cancer treatment.

Targeting Vascular Endothelial Growth Factor for Relapsed and Refractory Adult Acute Myelogenous Leukemias: Therapy with Sequential 1-β-d-Arabinofuranosylcytosine, Mitoxantrone, and Bevacizumab

Purpose: Vascular endothelial growth factor (VEGF) promotes acute myelogenous leukemia (AML) cell growth and survival and may contribute to drug resistance. bevacizumab, an anti-VEGF monoclonal antibody, exhibits clinical activity against diverse malignancies when administered with cytotoxic chemotherapy. We conducted a Phase II clinical trial of bevacizumab administered after chemotherapy to adults with refractory or relapsed AML, using a timed sequential therapy (TST) approach. Experimental Design: bevacizumab 10 mg/kg was administered on day 8 after 1-β-d-arabinofuranosylcytosine 2 g/m2/72 h beginning day 1 and mitoxantrone 40 mg/m2 beginning day 4. In vivo laboratory correlates included AML cell VEGF receptor-1 (FLT-1) expression, marrow microvessel density, and free serum VEGF before and during TST with bevacizumab. Results: Forty-eight adults received induction therapy. Myelosuppression occurred in all of the patients similar to other TST regimens. Toxicities were decreased ejection fraction (6%), cerebrovascular bleed (4%), and mortality of 15%. Overall response was 23 of 48 (48%), with complete response (CR) in 16 (33%). Eighteen (14 CR and 4 partial response) underwent one consolidation cycle and 5 (3 CR and 2 partial response) underwent allogeneic transplant. Median overall and disease-free survivals for CR patients were 16.2 months (64%, 1 year) and 7 months (35%, 1 year). Marrow blasts demonstrated FLT-1 staining before bevacizumab and marked decrease in microvessel density after bevacizumab. VEGF was detected in pretreatment serum in 67% of patients tested, increased by day 8 in 52%, and decreased in 93% (67% undetectable) 2 h after bevacizumab. Conclusions: In this single arm study, cytotoxic chemotherapy followed by bevacizumab yields a favorable CR rate and duration in adults with AML that is resistant to traditional treatment approaches. The clearance of marrow blasts in some patients after bevacizumab suggests that VEGF neutralization might result directly in leukemic cell death. The potential biological and clinical activity of bevacizumab in AML warrants additional clinical and laboratory study.

Vascular Endothelial Growth Factor Trap Blocks Tumor Growth, Metastasis Formation, and Vascular Leakage in an Orthotopic Murine Renal Cell Cancer Model

Purpose: Angiogenesis inhibitors have shown clinical benefit in patients with advanced renal cell cancer, but further therapeutic improvement is needed. Vascular endothelial growth factor (VEGF) Trap is a newly developed VEGF-blocking agent with stronger affinity and broader activity than the anti-VEGF antibody bevacizumab. In this study, we tested the activity of VEGF Trap in an orthotopic murine model of renal cancer with spontaneous lung metastases. Experimental Design: Murine syngeneic renal cell carcinoma cells (RENCA) transfected with a luciferase-expressing vector were injected into the renal capsule of BALB/c mice. I.p. treatment with VEGF Trap or control protein (10 or 25 mg/kg twice weekly) was started shortly after tumor injection to prevent tumor development (prevention model) or after established tumors were formed to inhibit tumor growth and metastasis formation (intervention model). Results: In the prevention model, VEGF Trap inhibited tumor growth by 87 ± 14% compared with control (P = 0.007) and significantly prolonged survival. In the intervention model, VEGF Trap inhibited tumor growth by 74 ± 9% (P < 0.001) and the formation of lung metastases was inhibited by 98% (P < 0.004). Microvascular density was reduced by 66% due to VEGF Trap treatment (P < 0.001). In addition, VEGF Trap prevented fibrinogen leakage into the tumor microenvironment representative for reduced vascular leaking as shown by immunohistochemical staining. Conclusions: VEGF Trap is a potent inhibitor of RENCA tumor growth and metastasis formation and blocks the biological function of VEGF in vivo. These results support further clinical development of VEGF Trap for renal cell cancer and other cancer types.

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

Purpose: The hypoxia-inducible factor-1α (HIF-α) 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-1α 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-1α 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-1α 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-1α that warrants clinical testing.