PI3K–mTORC2 but not PI3K–mTORC1 Regulates Transcription of HIF2A/EPAS1 and Vascularization in Neuroblastoma

Abstract

Hypoxia-inducible factor (HIF) is a master regulator of cellular responses to oxygen deprival with a critical role in mediating the angiogenic switch in solid tumors. Differential expression of the HIF subunits HIF1a and HIF2a occurs in many human tumor types, suggesting selective implications to biologic context. For example, high expression of HIF2a that occurs in neuroblastoma is associated with stem cell-like features, disseminated disease, and poor clinical outcomes, suggesting pivotal significance for HIF2 control in neuroblastoma biology. In this study, we provide novel insights into how HIF2a expression is transcriptionally controlled by hypoxia and how this control is abrogated by inhibition of insulin-like growth factor-1R/INSR-driven phosphoinositide 3-kinase (PI3K) signaling. Reducing PI3K activity was sufficient to decrease HIF2a mRNA and protein expression in a manner with smaller and less vascularized tumors in vivo. PI3K-regulated HIF2A mRNA expression was independent of Akt or mTORC1 signaling but relied upon mTORC2 signaling. HIF2A mRNA was induced by hypoxia in neuroblastoma cells isolated from metastatic patient-derived tumor xenografts, where HIF2A levels could be reduced by treatment with PI3K and mTORC2 inhibitors. Our results suggest that targeting PI3K and mTORC2 in aggressive neuroblastomas with an immature phenotype may improve therapeutic efficacy.

Anti‐tumor effects of PIM / PI 3K/ mTOR triple kinase inhibitor IBL ‐302 in neuroblastoma

“…Cells were lysed in RIPA buffer supplemented with Complete Protease Inhibitor (Roche, Basel, Switzerland) and phosSTOP (Roche) as previously described (Mohlin et al , ). Proteins were separated on SDS–PAGE gels and transferred to PVDF‐ or Hybond‐C extra nitrocellulose membranes (Bio‐Rad, Hercules, CA).…”

“…Current treatment strategies include high-dose chemotherapy, surgery, radiotherapy, and anti-GD2 therapy. We and others have shown that targeting the PI3K/mTOR pathway could be a viable treatment for aggressive neuroblastoma (Chesler et al, 2006;Johnsen et al, 2008;Segerström et al, 2011;Chanthery et al, 2012;Mohlin et al, 2013Mohlin et al, , 2015Cage et al, 2015;Stewart et al, 2015;Vaughan et al, 2016). Several studies reported anti-tumor effects and improved survival rates in preclinical neuroblastoma models using PI3K inhibitors LY294002 (Chesler et al, 2006), PI-103 (Segerström et al, 2011), dactolisib/NVP-BEZ235 (Chanthery et al, 2012;Stewart et al, 2015;Vaughan et al, 2016), ZSTK474 (Vaughan et al, 2016), PIK-75 (Cage et al, 2015), PW-12 (Cage et al, 2015), BKM120 (Stewart et al, 2015), and/or SF1126 (Erdreich-Epstein et al, 2016).…”

“…In addition, AKT inhibitor perifosine (Li et al, 2010(Li et al, , 2011 and mTOR inhibitors rapamycin (Johnsen et al, 2008), CCI-779 (Johnsen et al, 2008), and Torin (Vaughan et al, 2016) were demonstrated to decrease proliferation in vitro and consequent tumor growth in vivo. One explanation to the promising preclinical results from inhibiting PI3K/AKT/mTOR could be the anti-angiogenic downstream effects (Johnsen et al, 2008;Chanthery et al, 2012;Mohlin et al, 2015). The majority of reports also showed that neuroblastoma cells with high expression of MYCN were more sensitive to PI3K inhibition and/or that treatment resulted in downregulated N-Myc protein levels in vitro and in vivo (Cage et al, 2015;Chanthery et al, 2012;Chesler et al, 2006, Erdreich-Epstein et al, 2016, Johnsen et al, 2008Vaughan et al, 2016).…”

A HIF – LIMD 1 negative feedback mechanism mitigates the pro‐tumorigenic effects of hypoxia

“…We propose that inhibition of hypoxia-driven LIMD1 expression, resulting in enhanced HIF-a protein stability, drives HIF2A mRNA expression. Stabilisation of HIF-a protein and hypoxia has previously been described as inducers of HIF2A mRNA expression Mohlin et al, 2015). Many HIF-activated genes have been identified as prognostic and diagnostic markers.…”

The role of hypoxia‐inducible factor‐2 alpha in angiogenesis

“…In breast cancer, methylated CpG binding protein 3 binds to the EPAS1 promoter and enhances its transcription by demethylating CpG located around transcriptional start site, causing the activation of HIF‐2α‐mediated angiogenesis (Cui et al, ). In addition, it has been reported that EPAS1 transcription is regulated by the IGF‐induced PI3K‐mTORC2 system promoting vascularization in neuroblastoma (Mohlin et al, ). The deubiquitylase Cezanne (also known as OTUD7B) is also involved in the regulation of HIF‐2α mRNA synthesis via E2F1, which directly binds to the EPAS1 promoter (Moniz et al, ).…”