In vivo profiling of hypoxic gene expression in gliomas using the hypoxia marker EF5 and laser-capture microdissection.

Hypoxia is a key determinant of tumor aggressiveness, yet little is known regarding hypoxic global gene regulation in vivo. We used the hypoxia marker EF5 coupled with laser-capture microdissection to isolate RNA from viable hypoxic and normoxic regions of 9L experimental gliomas. Through microarray analysis, we identified several mRNAs (including the HIF targets Vegf, Glut-1, and Hsp27) with increased levels under hypoxia compared with normoxia both in vitro and in vivo. However, we also found striking differences between the global in vitro and in vivo hypoxic mRNA profiles. Intriguingly, the mRNA levels of a substantial number of immunomodulatory and DNA repair proteins including CXCL9, CD3D, and RAD51 were found to be downregulated in hypoxic areas in vivo, consistent with a protumorigenic role of hypoxia in solid tumors. Immunohistochemical staining verified increased HSP27 and decreased RAD51 protein levels in hypoxic versus normoxic tumor regions. Moreover, CD8(+) T cells, which are recruited to tumors upon stimulation by CXCL9 and CXCL10, were largely excluded from viable hypoxic areas in vivo. This is the first study to analyze the influence of hypoxia on mRNA levels in vivo and can be readily adapted to obtain a comprehensive picture of hypoxic regulation of gene expression and its influence on biological functions in solid tumors.

The GCN2-ATF4 pathway is critical for tumour cell survival and proliferation in response to nutrient deprivation.

The transcription factor ATF4 regulates the expression of genes involved in amino acid metabolism, redox homeostasis and ER stress responses, and it is overexpressed in human solid tumours, suggesting that it has an important function in tumour progression. Here, we report that inhibition of ATF4 expression blocked proliferation and survival of transformed cells, despite an initial activation of cytoprotective macroautophagy. Knockdown of ATF4 significantly reduced the levels of asparagine synthetase (ASNS) and overexpression of ASNS or supplementation of asparagine in trans, reversed the proliferation block and increased survival in ATF4 knockdown cells. Both amino acid and glucose deprivation, stresses found in solid tumours, activated the upstream eukaryotic initiation factor 2alpha (eIF2alpha) kinase GCN2 to upregulate ATF4 target genes involved in amino acid synthesis and transport. GCN2 activation/overexpression and increased phospho-eIF2alpha were observed in human and mouse tumours compared with normal tissues and abrogation of ATF4 or GCN2 expression significantly inhibited tumour growth in vivo. We conclude that the GCN2-eIF2alpha-ATF4 pathway is critical for maintaining metabolic homeostasis in tumour cells, making it a novel and attractive target for anti-tumour approaches.

Foxd3 is required in the trophoblast progenitor cell lineage of the mouse embryo.

The murine blastocyst contains two nonoverlapping pools of progenitor cells: the embryonic component contributes to the fetus and generates embryonic stem cells in vitro, whereas the extraembryonic pool contributes to the placenta and generates trophoblast stem cells in vitro. The transcriptional repressor Foxd3 is required for maintenance of the epiblast and the in vitro establishment of embryonic stem cell lines. Here, we demonstrate that Foxd3 is also required in the trophoblast lineage. Trophoblast progenitors in Foxd3-/- embryos do not self-renew and are not multipotent, but instead give rise to an excess of trophoblast giant cells. Injection of Foxd3-/- blastocysts with wild type ES cells fails to rescue Foxd3-/- placentas and such chimeras die around 10 days of embryogenesis. These results indicate an essential role for Foxd3 in two nonoverlapping progenitor cell populations that require different secreted factors to maintain their multipotent properties in vitro and give rise to divergent tissues in vivo. Moreover, this provides support for the hypothesis that there are conserved molecular mechanisms for maintaining the self-renewing properties of diverse progenitor cell types.