Inhibition of angiogenesis is an important new modality for cancer treatment. 2-methoxyestradiol (2ME2) is a novel antitumor and antiangiogenic agent, currently in clinical trials, whose molecular mechanism of action remains unclear. Herein, we report that 2ME2 inhibits tumor growth and angiogenesis at concentrations that efficiently disrupt tumor microtubules (MTs) in vivo. Mechanistically, we found that 2ME2 downregulates hypoxia-inducible factor-1 (HIF) at the posttranscriptional level and inhibits HIF-1-induced transcriptional activation of VEGF expression. Inhibition of HIF-1 occurs downstream of the 2ME2/tubulin interaction, as disruption of interphase MTs is required for HIF-alpha downregulation. These data establish 2ME2 as a small molecule inhibitor of HIF-1 and provide a mechanistic link between the disruption of the MT cytoskeleton and inhibition of angiogenesis.
Abstract-Ischemia is a stimulus for production of angiogenic cytokines that activate local vascular cells and mobilize angiogenic cells to the circulation. These responses are impaired in elderly patients with peripheral arterial disease. Hypoxia-inducible factor (HIF)-1 mediates adaptive responses to ischemia, including production of angiogenic cytokines. In this study, we demonstrate that aging and HIF-1 loss-of-function impair the expression of multiple angiogenic cytokines, mobilization of angiogenic cells, maintenance of tissue viability, and recovery of limb perfusion following femoral artery ligation. We show that HIF-1 directly activates transcription of the gene encoding stem cell factor and that mice lacking the cognate receptor C-KIT have impaired recovery from ischemia. Administration of AdCA5, an adenovirus encoding a constitutively active form of HIF-1␣, improved the recovery of perfusion in older mice to levels similar to those in young mice. Injection of AdCA5 into nonischemic limb was sufficient to increase the number of circulating angiogenic cells. These results indicate that HIF-1 activity is necessary and sufficient for the mobilization of angiogenic cells and that HIF-1␣ gene therapy can counteract the pathological effects of aging in a mouse model of limb ischemia.
As the field of cancer immunotherapy continues to advance at a fast pace, treatment approaches and drug development are evolving rapidly to maximize patient benefit. New agents are commonly evaluated for activity in patients who had previously received a programmed death receptor 1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitor as standard of care or in an investigational study. However, because of the kinetics and patterns of response to PD-1/PD-L1 blockade, and the lack of consistency in the clinical definitions of resistance to therapy, the design of clinical trials of new agents and interpretation of results remains an important challenge. To address this unmet need, the Society for Immunotherapy of Cancer convened a multistakeholder taskforce—consisting of experts in cancer immunotherapy from academia, industry, and government—to generate consensus clinical definitions for resistance to PD-(L)1 inhibitors in three distinct scenarios: primary resistance, secondary resistance, and progression after treatment discontinuation. The taskforce generated consensus on several key issues such as the timeframes that delineate each type of resistance, the necessity for confirmatory scans, and identified caveats for each specific resistance classification. The goal of this effort is to provide guidance for clinical trial design and to support analyses of emerging molecular and cellular data surrounding mechanisms of resistance.
We have previously characterized the release of the signal peptide sequence-less fibroblast growth factor (FGF) prototype, FGF-1, in vitro as a stress-induced pathway in which FGF-1 is released as a latent homodimer with the p40 extravesicular domain of p65 synaptotagmin (Syn)-1. To determine the biologic relevance of the FGF-1 release pathway in vivo, we sought to resolve and characterize from ovine brain a purified fraction that contained both FGF-1 and p40 Syn-1 and report that the brain-derived FGF-1:p40 Syn-1 aggregate is associated with the calcium-binding protein, S100A13. Since S100A13 binds the anti-inflammatory compound amlexanox and FGF-1 is involved in inflammation, we examined the effects of amlexanox on the release of FGF-1 and p40 Syn-1 in response to stress in vitro. We report that while amlexanox was able to repress the heat shock-induced release of FGF-1 and p40 Syn-1 in a concentration-dependent manner, it had no effect on the constitutive release of p40 Syn-1 from p40 Syn-1 NIH 3T3 cell transfectants. These data suggest the following: (i) FGF-1 is associated with Syn-1 and S100A13 in vivo; (ii) S100A13 may be involved in the regulation of FGF-1 and p40 Syn-1 release in response to temperature stress in vitro; and (iii) the FGF-1 release pathway may be accessible to pharmacologic regulation.
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