Angiogenesis, the summation of multiple cellular and biological processes culminating in the propagation of blood vessels, has been the subject of extensive examination in the context of tumor biology over the past four decades since first proposed by Judah Folkman in 1971 (1
). Solid tumor growth and progression is dependent on tumor-associated angiogenesis. Tumor expression and circulating levels of angiogenic factors have been correlated with aggressive tumor growth, predilection for metastasis, and prognosis in a wide array of solid tumors, including lung cancer (2
). Although many putative regulators of angiogenesis have been identified, two secreted factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been particularly strongly implicated in tumor-associated angiogenesis (5
). VEGF and bFGF interact with distinct families of tyrosine kinase receptors (RTK) on the surface of endothelial cells, and activate multiple downstream signaling pathways. Together these pathways promote endothelial cell survival, proliferation, migration, invasion, and tube formation, resulting in formation of new vascular networks (6
Lung cancer is the leading cause of cancer mortality in both men and women in the US (8
). The only anti-angiogenic therapy currently approved for use in lung cancer is the α-VEGF monoclonal antibody bevacizumab. A landmark phase III clinical study, ECOG 4599, randomized patients with advanced non-small cell lung cancer (NSCLC) to a standard chemotherapy doublet with or without bevacizumab (9
). This study demonstrated a statistically significant improvement in both progression-free and overall survival in favor of the bevacizumab-containing arm. There are, however, major limitations to the clinical utility of bevacizumab. The absolute improvements in progression-free and overall survival in this study were modest (1.7 and 2 months, respectively). Due to episodes of fatal hemoptysis in a prior study (10
), enrollment was restricted to patients with non-squamous tumors, with no history of hemoptysis, with no brain metastases, with no indication for use of anti-coagulants, and with good performance status. Even in this carefully selected subpopulation bevacizumab use was associated with increased treatment-related deaths (p = 0.001) including 5 episodes of fatal hemoptysis (vs. 0 on the control arm), and with significantly increased rates of hypertension, proteinuria, bleeding, neutropenia, febrile neutropenia, thrombocytopenia, hyponatremia, rash, and headache (p < 0.05 for each of these). Across disease types, bevacizumab use has been associated with increased treatment-related mortality (11
). The financial cost of bevacizumab, given the limited efficacy and significant toxicity, was seen by many as excessive, at upwards of $500,000 per year of life gained (12
). A final concern comes from what was intended as a “confirmatory” trial, the AVAiL study, which randomized a similar patient population to a different standard chemotherapy doublet with or without bevacizumab (14
). This study failed to demonstrate a statistically significant difference in survival. In summary, while evidence is strong that angiogenesis is critical to tumor growth and progression, less toxic, less cost-prohibitive, and more effective therapies are needed. These observations strongly support further development of novel anti-angiogenic strategies for patients with lung cancer.
Itraconazole is an orally bioavailable, FDA-approved agent belonging to the family of azole antifungal drugs, which inhibit the enzyme lanosterol 14α-demethylase (14-DM) responsible for the conversions of lanosterol to ergosterol in fungi and lanosterol to cholesterol in humans, respectively. Initially reported by Chong et al.
, itraconazole has been identified as a potent inhibitor of endothelial cell proliferation and matrigel stimulated angiogenesis, with inhibition of 14-DM and sterol biosynthesis only partially explaining this novel anti-proliferative activity (15
). Further efforts to characterize the mechanism of inhibition of endothelial cell proliferation are ongoing, with recent reports suggesting perturbation of cholesterol trafficking pathways imparted by itraconazole as a possible mechanism contributing to this activity (16
). Of note, itraconazole has also recently been implicated as an antagonist of the hedgehog signaling pathway in models of hedgehog pathway deregulation (17
). Pre-clinical evaluation of the anti-angiogenic capacity of itraconazole in relevant in vitro
models of angiogenesis and in vivo
models of cancer are clearly required in order to determine the viability of pursuing further clinical development of itraconazole as an anti-angiogenic agent.
Tumor cell lines implanted into immunodeficient mice comprise the most commonly used platform for in vivo
preclinical cancer therapeutic testing. However, ex vivo
derivation of stable cell lines in tissue culture is associated with profound changes in cellular morphology, growth characteristics, chromosome structure, gene copy number, and gene expression (18
), changes which are not reversed by reintroduction of cell lines into mice (21
). In sharp contrast to the harsh biological conditions in which tumors naturally arise, typical tissue culture conditions include relatively high oxygen tension, high glucose concentration, and low hydrostatic and oncotic pressures. These are precisely conditions in which maintenance of angiogenic drive, in particular, is not relevant. To evaluate the in vivo
effects of itraconazole, here we employ an alternative approach based on primary lung cancer xenografts. The primary xenograft model depends on immediate transfer of human cancers from patients into recipient mice, without intervening tissue culture or cell line derivation ex vivo
. We have previously reported that gene expression profiles of lung cancer primary xenografts more closely reflects those of the human cancers than do profiles of cell lines derived from the same parental tumor when re-implanted as standard (secondary) xenografts (21
). These observations are supported by data from other investigators exploring primary xenografts (22
Here we describe the results of a series of in vitro and in vivo analyses evaluating the putative anti-angiogenic activities of itraconazole. We employ several in vitro assays using human umbilical vein endothelial cells (HUVEC) to separately probe specific hallmarks of endothelial cell function as they relate to angiogenic processes. These functional competencies include proliferative capacity, migration, chemotactic potential, and the ability to spontaneously form an extracellular matrix (ECM) supported tube network. The capacity of itraconazole to modulate these functions was explored in the presence of multiple angiogenic stimuli including VEGF and bFGF. We further investigate the in vivo activity of itraconazole as an inhibitor of tumor-associated angiogenesis and of tumor growth, both as a single agent and in combination with standard cytotoxic chemotherapy. These studies offer the first assessment of the efficacy of itraconazole as an anti-angiogenic agent and as an anti-cancer therapeutic in a primary disease model.