Class I HDAC inhibitors enhance YB-1 acetylation and oxidative stress to block sarcoma metastasis.

Outcomes for metastatic Ewing sarcoma and osteosarcoma are dismal and have not changed for decades. Oxidative stress attenuates melanoma metastasis, and melanoma cells must reduce oxidative stress to metastasize. We explored this in sarcomas by screening for oxidative stress sensitizers, which identified the class I HDAC inhibitor MS-275 as enhancing vulnerability to reactive oxygen species (ROS) in sarcoma cells. Mechanistically, MS-275 inhibits YB-1 deacetylation, decreasing its binding to 5'-UTRs of NFE2L2 encoding the antioxidant factor NRF2, thereby reducing NFE2L2 translation and synthesis of NRF2 to increase cellular ROS. By global acetylomics, MS-275 promotes rapid acetylation of the YB-1 RNA-binding protein at lysine-81, blocking binding and translational activation of NFE2L2, as well as known YB-1 mRNA targets, HIF1A, and the stress granule nucleator, G3BP1. MS-275 dramatically reduces sarcoma metastasis in vivo, but an MS-275-resistant YB-1K81-to-alanine mutant restores metastatic capacity and NRF2, HIF1α, and G3BP1 synthesis in MS-275-treated mice. These studies describe a novel function for MS-275 through enhanced YB-1 acetylation, thus inhibiting YB-1 translational control of key cytoprotective factors and its pro-metastatic activity.

Heterogeneous distribution of trastuzumab in HER2-positive xenografts and metastases: role of the tumor microenvironment.

Most HER2-positive metastatic breast cancer patients continue to relapse. Incomplete access to all target HER2-positive cells in metastases and tumor tissues is a potential mechanism of resistance to trastuzumab. The location of locally bound trastuzumab was evaluated in HER2-positive tissues in vivo and as in vivo xenografts or metastases models in mice. Microenvironmental elements of tumors were related to bound trastuzumab using immunohistochemical staining and include tight junctions, vasculature, vascular maturity, vessel patency, hypoxia and HER2 to look for correlations. Trastuzumab was evaluated alone and in combination with bevacizumab. Dynamic contrast-enhanced magnetic resonance imaging parameters of overall vascular function, perfusion and apparent permeability were compared with matched histological images of trastuzumab distribution and vascular patency. Trastuzumab distribution is highly heterogeneous in all models examined, including avascular micrometastases of the brain and lung. Trastuzumab distributes well through the extravascular compartment even in conditions of high HER2 expression and poor convective flow in vivo. Microregional patterns of trastuzumab distribution in vivo do not consistently correlate with vascular density, patency, function or maturity; areas of poor trastuzumab access are not necessarily those with poor vascular supply. The number of vessels with perivascular trastuzumab increases with time and higher doses and dramatically decreases when pre-treated with bevacizumab. Areas of HER2-positive tissue without bound trastuzumab persist in all conditions. These data directly demonstrate tissue- and vessel-level barriers to trastuzumab distribution in vivo that can effectively limit access of the drug to target cells in brain metastases and elsewhere.

Targeting the tumour vasculature: exploitation of low oxygenation and sensitivity to NOS inhibition by treatment with a hypoxic cytotoxin.

Many cancer research efforts focus on exploiting genetic-level features that may be targeted for therapy. Tissue-level features of the tumour microenvironment also represent useful therapeutic targets. Here we investigate the presence of low oxygen tension and sensitivity to NOS inhibition of tumour vasculature as potential tumour-specific features that may be targeted by hypoxic cytotoxins, a class of therapeutics currently under investigation. We have previously demonstrated that tirapazamine (TPZ) mediates central vascular dysfunction in tumours. TPZ is a hypoxic cytotoxin that is also a competitive inhibitor of NOS. Here we further investigated the vascular-targeting activity of TPZ by combining it with NOS inhibitor L-NNA, or with low oxygen content gas breathing. Tumours were analyzed via multiplex immunohistochemical staining that revealed irreversible loss of perfusion and enhanced tumour cell death when TPZ was combined with either low oxygen or a NOS inhibitor. Tumour growth rate was reduced by TPZ + NOS inhibition, and tumours previously resistant to TPZ-mediated vascular dysfunction were sensitized by low oxygen breathing. Additional mapping analysis suggests that tumours with reduced vascular-associated stroma may have greater sensitivity to these effects. These results indicate that poorly oxygenated tumour vessels, also being abnormally organized and with inadequate smooth muscle, may be successfully targeted for significant anti-cancer effects by inhibition of NOS and hypoxia-activated prodrug toxicity. This strategy illustrates a novel use of hypoxia-activated cytotoxic prodrugs as vascular targeting agents, and also represents a novel mechanism for targeting tumour vessels.