Bisphosphonates suppress insulin‐like growth factor 1‐induced angiogenesis via the HIF‐1α/VEGF signaling pathways in human breast cancer cells

Adjunctive chemotherapy with bisphosphonates has been reported to delay bone metastasis and improve overall survival in breast cancer. Aside from its antiresorptive effect, bisphosphonates exhibit antitumor activities, in vitro and in vivo, via several mechanisms, including antiangiogenesis. In this study, we investigated the potential molecular mechanisms underlying the antiangiogenic effect of non–nitrogen‐containing and nitrogen‐containing bisphosphonates, clodronate and pamidronate, respectively, in insulin‐like growth factor (IGF)‐1 responsive human breast cancer cells. We tested whether bisphosphonates had any effects on hypoxia‐inducible factor (HIF)‐1α/vascular endothelial growth factor (VEGF) axis that plays a pivotal role in tumor angiogenesis, and our results showed that both pamidronate and clodronate significantly suppressed IGF‐1‐induced HIF‐1α protein accumulation and VEGF expression in MCF‐7 cells. Mechanistically, we found that either pamidronate or clodronate did not affect mRNA expression of HIF‐1α, but they apparently promoted the degradation of IGF‐1‐induced HIF‐1α protein. Meanwhile, we found that the presence of pamidronate and clodronate led to a dose‐dependent decease in the newly‐synthesized HIF‐1α protein induced by IGF‐1 in breast cancer cells after proteasomal inhibition, thus, indirectly reflecting the inhibition of protein synthesis. In addition, our results indicated that the inhibitory effects of bisphosphonates on the HIF‐1α/VEGF axis are associated with the inhibition of the phosphoinositide 3‐kinase/AKT/mammalian target of rapamycin signaling pathways. Consistently, we demonstrated that pamidronate and clodronate functionally abrogated both in vitro and in vivo tumor angiogenesis induced by IGF‐1‐stimulated MCF‐7 cells. These findings have highlighted an important mechanism of the pharmacological action of bisphosphonates in the inhibition of tumor angiogenesis in breast cancer cells.     

Norepinephrine induces VEGF expression and angiogenesis by a hypoxia‐inducible factor‐1α protein‐dependent mechanism

A growing number of studies have demonstrated that physiological factors can influence the progression of several cancers via cellular immune function, angiogenesis and metastasis. Recently, stress‐induced catecholamines have been shown to increase the expression of various cancer progressive factors, including vascular endothelial growth factor (VEGF), matrix metalloproteinases and interleukins. However, a detailed mechanism remains to be identified. In this study, we investigated the role of adrenergic receptors and hypoxia‐inducible factor (HIF)‐1α protein in catecholamine‐induced VEGF expression and angiogenesis. Treatment of the cells with norepinephrine (NE) or isoproterenol induced VEGF expression and HIF‐1α protein amount in a dose‐dependent manner. Induction of VEGF expression by NE was abrogated when the cells were transfected with HIF‐1α–specific siRNA. Similarly, adenylate cyclase activator forskolin and cyclic AMP‐dependent protein kinase A inhibitor H‐89 enhanced and decreased HIF‐1α protein amount, respectively. More importantly, conditioned medium of NE‐stimulated cancer cells induced angiogenesis in a HIF‐1α protein–dependent manner. In addition, pretreatment of cells with propranolol, a β‐adrenergic receptor (AR) blocker, completely abolished induction of VEGF expression and HIF‐1α protein amount by NE in all of the tested cancer cells. However, treatment with the α1‐AR blocker prazosin inhibited NE‐induced HIF‐1α protein amount and angiogenesis in SK‐Hep1 and PC‐3 but not MDA‐MB‐231 cells. Collectively, our results suggest that ARs and HIF‐1α protein have critical roles in NE‐induced VEGF expression in cancer cells, leading to stimulation of angiogenesis. These findings will help to understand the mechanism of cancer progression by stress‐induced catecholamines and design therapeutic strategies for cancer angiogenesis.     

FAT1 modulates EMT and stemness genes expression in hypoxic glioblastoma

Glioblastoma (GBM) is characterized by the presence of hypoxia, stemness and local invasiveness. We have earlier demonstrated that FAT1 promotes invasiveness, inflammation and upregulates HIF‐1α expression and its signaling in hypoxic GBM. Here, we have identified the role of FAT1 in regulating EMT (epithelial‐mesenchymal transition) and stemness characteristics in GBM. The expression of FAT1, EMT (Snail/LOX/Vimentin/N‐cad), stemness (SOX2/OCT4/Nestin/REST) and hypoxia markers (HIF‐1α/VEGF/PGK1/CA9) was upregulated in ≥39% of GBM tumors (n = 31) with significant positive correlation (p ≤ 0.05) of the expression of FAT1 with LOX/Vimentin/SOX2/HIF‐1α/PGK1/VEGF/CA9. Furthermore, positive correlation (p ≤ 0.01) of FAT1 with Vimentin/N‐cad/SOX2/REST/HIF‐1α has been observed in TCGA GBM‐dataset (n = 430). Analysis of cells (U87MG/A172) exposed to severe hypoxia (0.2%O₂) revealed elevated mRNA expression of FAT1, EMT (Snail/LOX/Vimentin/N‐cad), stemness (SOX2/OCT4/Nestin/REST) and hypoxia markers (HIF‐1α/PGK1/VEGF/CA9) as compared to their normoxic (20%O₂) counterparts. FAT1 knockdown in U87MG/A172 maintained in severe hypoxia and in normoxic primary glioma cultures led to significant reduction of EMT/stemness markers as compared to controls. HIF‐1α knockdown in U87MG cells markedly reduced the expression of all the EMT/stemness markers studied except for Nestin and SOX2 which were more under the influence of FAT1. This indicates FAT1 has a novel regulatory effect on EMT/stemness markers both via or independent of HIF‐1α. The functional relevance of our study was corroborated by significant reduction in the number of soft‐agar colonies formed in hypoxic‐siFAT1 treated U87MG cells. Hence, our study for the first time reveals FAT1 as a novel regulator of EMT/stemness in hypoxic GBM and suggests FAT1 as a potential therapeutic candidate.     

Clinicopathological significance of nuclear factor‐kappa B,HIF‐1 alpha,and vascular endothelial growth factor expression in stage III colorectal cancer

Nuclear factor‐κB (NF‐κB), hypoxia‐inducible factor 1α (HIF‐1α), and vascular endothelial growth factor (VEGF) are involved in cell proliferation, invasion, angiogenesis, and metastases. The principal objective of this study was to assess the prognostic significance of NF‐κB, HIF‐1α, and VEGF expression in stage III colorectal cancer. Tumor tissues from 148 patients with stage III colorectal carcinoma, all of whom underwent potentially curative resection, were immunohistochemically evaluated using monoclonal antibodies against NF‐κB, HIF‐1α, and VEGF. Positivity rates of NF‐κB, HIF‐1α, and VEGF were 47.3%, 42.6%, and 61.5%, respectively. NF‐κB expression in tumor tissues was correlated significantly with HIF‐1α expression (P < 0.001), VEGF expression (P = 0.044), and the presence of vascular invasion (P = 0.013). Univariate analysis demonstrated that NF‐κB expression was associated with poor 5‐year overall survival (55.8 months vs 76.9 months, P = 0.012). Multivariate analysis verified that NF‐κB was independently associated with adverse outcomes (relative risk: 1.92, P = 0.049). However, HIF‐1α and VEGF did not appear to be related to clinical outcomes. NF‐κB expression in tumor tissue is associated with angiogenesis and poor 5‐year overall survival in stage III colorectal cancer patients. (Cancer Sci 2010; 00: 000–000)     

Major vault protein forms complexes with hypoxia‐inducible factor (HIF)‐1α and reduces HIF‐1α level in ACHN human renal adenocarcinoma cells

Vaults are evolutionarily highly conserved ribonucleoprotein (RNP) particles with a hollow barrel‐like structure. Although roles in multidrug resistance and innate immunity have been suggested, the physiological function of vaults remains unclear. Major vault protein (MVP), the main component of the vault particle, has been reported to be induced by hypoxia. However, there are no reports about the effect of vaults on cellular responses to hypoxia. We thus examined whether vaults are implicated in cellular responses to hypoxia. In this study, we focused on hypoxia‐inducible factor‐1α (HIF‐1α), which is a master regulator of hypoxic responses, and found that: (i) MVP knockdown by RNA interference increases HIF‐1α protein levels induced by hypoxia and hypoxia mimetics; (ii) MVP knockdown does not affect HIF‐1α mRNA levels, but decreases the ubiquitination and degradation of HIF‐1α protein; and (iii) vaults form complexes with HIF‐1α, PHD2, and pVHL. Taken together, these results suggest that vaults function as scaffolds in HIF‐1α degradation pathway and promote the ubiquitination and degradation of HIF‐1α. (Cancer Sci 2010; 101: 920–926)     

Knockdown of prolyl‐4‐hydroxylase domain 2 inhibits tumor growth of human breast cancer MDA‐MB‐231 cells by affecting TGF‐β1 processing

The prolyl‐4‐hydroxylase domain 1–3 (PHD1–3) enzymes are regulating the protein stability of the α‐subunit of the hypoxia‐inducible factor‐1 (HIF‐1), which mediates oxygen‐dependent gene expression. PHD2 is the main isoform regulating HIF‐1α hydroxylation and thus stability in normoxia. In human cancers, HIF‐1α is overexpressed as a result of intratumoral hypoxia which in turn promotes tumor progression. The role of PHD2 for tumor progression is in contrast far from being thoroughly understood. Therefore, we established PHD2 knockdown clones of MDA‐MB‐231 breast cancer cells and analyzed their tumor‐forming potential in a SCID mouse model. Tumor progression was significantly impaired in the PHD2 knockdown MDA‐MB‐231 cells, which could be partially rescued by re‐establishing PHD2 expression. In a RNA profile screen, we identified the secreted phosphoprotein 1 (SPP1) as one target, which is differentially regulated as a consequence of the PHD2 knockdown. Knockdown of PHD2 drastically reduced the SPP1 expression in MDA‐MB‐231 cells. A correlation of SPP1 and PHD2 expression was additionally verified in 294 invasive breast cancer biopsies. In subsequent analyses, we identified that PHD2 alters the processing of transforming growth factor (TGF)‐β1, which is highly involved in SPP1 expression. The altered processing capacity was associated with a dislocation of the pro‐protein convertase furin. Thus, our data demonstrate that in MDA‐MB‐231 cells PHD2 might affect tumor‐relevant TGF‐β1 target gene expression by altering the TGF‐β1 processing capacity.     

Overcoming evasive resistance from vascular endothelial growth factor a inhibition in sarcomas by genetic or pharmacologic targeting of hypoxia‐inducible factor 1α

Increased levels of hypoxia and hypoxia‐inducible factor 1α (HIF‐1α) in human sarcomas correlate with tumor progression and radiation resistance. Prolonged antiangiogenic therapy of tumors not only delays tumor growth but may also increase hypoxia and HIF‐1α activity. In our recent clinical trial, treatment with the vascular endothelial growth factor A (VEGF‐A) antibody, bevacizumab, followed by a combination of bevacizumab and radiation led to near complete necrosis in nearly half of sarcomas. Gene Set Enrichment Analysis of microarrays from pretreatment biopsies found that the Gene Ontology category “Response to hypoxia” was upregulated in poor responders and that the hierarchical clustering based on 140 hypoxia‐responsive genes reliably separated poor responders from good responders. The most commonly used chemotherapeutic drug for sarcomas, doxorubicin (Dox), was recently found to block HIF‐1α binding to DNA at low metronomic doses. In four sarcoma cell lines, HIF‐1α shRNA or Dox at low concentrations blocked HIF‐1α induction of VEGF‐A by 84–97% and carbonic anhydrase 9 by 83–93%. HT1080 sarcoma xenografts had increased hypoxia and/or HIF‐1α activity with increasing tumor size and with anti‐VEGF receptor antibody (DC101) treatment. Combining DC101 with HIF‐1α shRNA or metronomic Dox had a synergistic effect in suppressing growth of HT1080 xenografts, at least in part via induction of tumor endothelial cell apoptosis. In conclusion, sarcomas respond to increased hypoxia by expressing HIF‐1α target genes that may promote resistance to antiangiogenic and other therapies. HIF‐1α inhibition blocks this evasive resistance and augments destruction of the tumor vasculature.     

Hypoxia‐inducible factor‐1α and nuclear factor‐κB play important roles in regulating programmed cell death ligand 1 expression by epidermal growth factor receptor mutants in non‐small‐cell lung cancer cells

Some driver gene mutations, including epidermal growth factor receptor (EGFR), have been reported to be involved in expression regulation of the immunosuppressive checkpoint protein programmed cell death ligand 1 (PD‐L1), but the underlying mechanism remains obscure. We investigated the potential role and precise mechanism of EGFR mutants in PD‐L1 expression regulation in non‐small‐cell lung cancer (NSCLC) cells. Examination of pivotal EGFR signaling effectors in 8 NSCLC cell lines indicated apparent associations between PD‐L1 overexpression and phosphorylation of AKT and ERK, especially with increased protein levels of phospho‐IκBα (p‐IκBα) and hypoxia‐inducible factor‐1α (HIF‐1α). Flow cytometry results showed stronger membrane co‐expression of EGFR and PD‐L1 in NSCLC cells with EGFR mutants compared with cells carrying WT EGFR. Additionally, ectopic expression or depletion of EGFR mutants and treatment with EGFR pathway inhibitors targeting MEK/ERK, PI3K/AKT, mTOR/S6, IκBα, and HIF‐1α indicated strong accordance among protein levels of PD‐L1, p‐IκBα, and HIF‐1α in NSCLC cells. Further treatment with pathway inhibitors significantly inhibited xenograft tumor growth and p‐IκBα, HIF‐1α, and PD‐L1 expression of NSCLC cells carrying EGFR mutant in nude mice. Moreover, immunohistochemical analysis revealed obviously increased protein levels of p‐IκBα, HIF‐1α, and PD‐L1 in NSCLC tissues with EGFR mutants compared with tissues carrying WT EGFR. Non‐small‐cell lung cancer tissues with either p‐IκBα or HIF‐1α positive staining were more likely to possess elevated PD‐L1 expression compared with tissues scored negative for both p‐IκBα and HIF‐1α. Our findings showed important roles of phosphorylation activation of AKT and ERK and potential interplay and cooperation between NF‐κB and HIF‐1α in PD‐L1 expression regulation by EGFR mutants in NSCLC.     

Repressive role of stabilized hypoxia inducible factor 1α expression on transforming growth factor β‐induced extracellular matrix production in lung cancer cells

Activation of transforming growth factor β (TGF‐β) combined with persistent hypoxia often affects the tumor microenvironment. Disruption of cadherin/catenin complexes induced by these stimulations yields aberrant extracellular matrix (ECM) production, characteristics of epithelial‐mesenchymal transition (EMT). Hypoxia‐inducible factors (HIF), the hallmark of the response to hypoxia, play differential roles during development of diseases. Recent studies show that localization of cadherin/catenin complexes at the cell membrane might be tightly regulated by protein phosphatase activity. We aimed to investigate the role of stabilized HIF‐1α expression by protein phosphatase activity on dissociation of the E‐cadherin/β‐catenin complex and aberrant ECM expression in lung cancer cells under stimulation by TGF‐β. By using lung cancer cells treated with HIF‐1α stabilizers or carrying doxycycline‐dependent HIF‐1α deletion or point mutants, we investigated the role of stabilized HIF‐1α expression on TGF‐β‐induced EMT in lung cancer cells. Furthermore, the underlying mechanisms were determined by inhibition of protein phosphatase activity. Persistent stimulation by TGF‐β and hypoxia induced EMT phenotypes in H358 cells in which stabilized HIF‐1α expression was inhibited. Stabilized HIF‐1α protein expression inhibited the TGF‐β‐stimulated appearance of EMT phenotypes across cell types and species, independent of de novo vascular endothelial growth factor A (VEGFA) expression. Inhibition of protein phosphatase 2A activity abrogated the HIF‐1α‐induced repression of the TGF‐β‐stimulated appearance of EMT phenotypes. This is the first study to show a direct role of stabilized HIF‐1α expression on inhibition of TGF‐β‐induced EMT phenotypes in lung cancer cells, in part, through protein phosphatase activity.     

The critical role of mast cell‐derived hypoxia‐inducible factor‐1α in human and mice melanoma growth

Mast cells play an important role in tumorigenesis. Histamine released from mast cells stimulates new vessel formation by acting through the histamine1 (H1) receptor. Despite the evidence of the role of mast cells in tumor growth and angiogenesis, the potential mechanism remains to be elucidated. Therefore, we investigated the role of mast cell‐derived HIF‐1α in melanoma growth. Here, we identify that the most positive cells for HIF‐1α staining are seen in mast cells of human and animal melanoma tissue. The number of the stromal cell types (fibroblasts, macrophages and endothelial cells) was also increased in melanoma tissues. In activated bone marrow‐derived mast cells (BMMCs), expressions of HIF‐1α and VEGF were increased. Histamine also induced the expressions of HIF‐1α and VEGF in BMMCs. H1 receptor antagonists significantly improved overall survival rates and substantially suppressed tumor growth as well as the infiltration of mast cells and levels of VEGF through the inhibition of HIF‐1α expression in B16F10 melanoma‐bearing mice. Furthermore, the injection of HIF‐1α depleted BMMCs markedly inhibited the growth of tumors and migration of mast cells and increased the survival rate of the mice. These findings emphasize that the growth of melanoma can actually be exacerbated by mast cell‐derived HIF‐1α. In aggregate, our results reveal a novel role for mast cell‐derived HIF‐1α in the melanoma microenvironment and have important implications for the design of therapeutic strategies.