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ARNT-dependent HIF-2 transcriptional activity is not sufficient to regulate downstream target genes in neuroblastoma

Persson, Camilla U. LU ; von Stedingk, Kristoffer LU ; Fredlund, Elina LU ; Bexell, Daniel LU ; Påhlman, Sven LU ; Wigerup, Caroline LU and Mohlin, Sofie LU orcid (2020) In Experimental Cell Research 388(2).
Abstract

Background: Hypoxia-inducible factor (HIF)-2α associates with poor outcome in neuroblastoma and glioblastoma, and gain-of-function mutations in the EPAS1 gene (encoding HIF-2α) have been reported in paragangliomas and pheochromocytomas. Specific targeting of a druggable hydrophobic pocket in the HIF-2α PAS-B domain with PT2385 have demonstrated promising clinical results for clear cell renal cell carcinoma (ccRCC). Here, we investigated the effect of PT2385-mediated inhibition of ARNT dependent HIF-2 activity. Methods: Neuroblastoma patient-derived xenograft (PDX) cells were treated with PT2385 and analyzed for HIF-2-dependent gene expression, HIF activity, HIF-2α protein localization, response to chemotherapy and orthotopic tumor... (More)

Background: Hypoxia-inducible factor (HIF)-2α associates with poor outcome in neuroblastoma and glioblastoma, and gain-of-function mutations in the EPAS1 gene (encoding HIF-2α) have been reported in paragangliomas and pheochromocytomas. Specific targeting of a druggable hydrophobic pocket in the HIF-2α PAS-B domain with PT2385 have demonstrated promising clinical results for clear cell renal cell carcinoma (ccRCC). Here, we investigated the effect of PT2385-mediated inhibition of ARNT dependent HIF-2 activity. Methods: Neuroblastoma patient-derived xenograft (PDX) cells were treated with PT2385 and analyzed for HIF-2-dependent gene expression, HIF activity, HIF-2α protein localization, response to chemotherapy and orthotopic tumor growth in vivo. Two-sided student t-test was used. Results: We detected high levels of HIF-2α protein in perivascular niches in neuroblastoma PDXs in vivo and at oxygenated conditions in PDX-derived cell cultures in vitro, particularly in the cytoplasmic fraction. Nuclear HIF-2α expression was reduced following PT2385 treatment, but surprisingly, virtually no effects on tumor growth in vivo or expression of canonical HIF downstream target genes in vitro were observed. In coherence, RNA sequencing of PT2385-treated PDX cells revealed a virtually unaffected transcriptome. Treatment with PT2385 did not affect cellular response to chemotherapy. In contrast, HIF-2α protein knockdown resulted in profound downregulation of target genes. Conclusions: The lack of effect from PT2385 treatment in combination with high cytoplasmic HIF-2α expression at normoxia suggest that HIF-2α have additional roles than acting as an ARNT dependent transcription factor. It is important to further unravel the conditions at which HIF-2α has transcriptional and non-transcriptional roles in neuroblastoma.

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author
; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Cancer, Cancer therapy, HIF-2α, Hypoxia, Hypoxia inducible factor (HIF), Neuroblastoma, Patient-derived xenograft cells, Pseudohypoxia, PT2385
in
Experimental Cell Research
volume
388
issue
2
article number
111845
publisher
Academic Press
external identifiers
  • scopus:85077917535
  • pmid:31945318
ISSN
0014-4827
DOI
10.1016/j.yexcr.2020.111845
language
English
LU publication?
yes
id
34f738bf-1056-4bd1-9e08-87f6c01c95dd
date added to LUP
2020-01-30 14:47:55
date last changed
2024-06-13 10:26:23
@article{34f738bf-1056-4bd1-9e08-87f6c01c95dd,
  abstract     = {{<p>Background: Hypoxia-inducible factor (HIF)-2α associates with poor outcome in neuroblastoma and glioblastoma, and gain-of-function mutations in the EPAS1 gene (encoding HIF-2α) have been reported in paragangliomas and pheochromocytomas. Specific targeting of a druggable hydrophobic pocket in the HIF-2α PAS-B domain with PT2385 have demonstrated promising clinical results for clear cell renal cell carcinoma (ccRCC). Here, we investigated the effect of PT2385-mediated inhibition of ARNT dependent HIF-2 activity. Methods: Neuroblastoma patient-derived xenograft (PDX) cells were treated with PT2385 and analyzed for HIF-2-dependent gene expression, HIF activity, HIF-2α protein localization, response to chemotherapy and orthotopic tumor growth in vivo. Two-sided student t-test was used. Results: We detected high levels of HIF-2α protein in perivascular niches in neuroblastoma PDXs in vivo and at oxygenated conditions in PDX-derived cell cultures in vitro, particularly in the cytoplasmic fraction. Nuclear HIF-2α expression was reduced following PT2385 treatment, but surprisingly, virtually no effects on tumor growth in vivo or expression of canonical HIF downstream target genes in vitro were observed. In coherence, RNA sequencing of PT2385-treated PDX cells revealed a virtually unaffected transcriptome. Treatment with PT2385 did not affect cellular response to chemotherapy. In contrast, HIF-2α protein knockdown resulted in profound downregulation of target genes. Conclusions: The lack of effect from PT2385 treatment in combination with high cytoplasmic HIF-2α expression at normoxia suggest that HIF-2α have additional roles than acting as an ARNT dependent transcription factor. It is important to further unravel the conditions at which HIF-2α has transcriptional and non-transcriptional roles in neuroblastoma.</p>}},
  author       = {{Persson, Camilla U. and von Stedingk, Kristoffer and Fredlund, Elina and Bexell, Daniel and Påhlman, Sven and Wigerup, Caroline and Mohlin, Sofie}},
  issn         = {{0014-4827}},
  keywords     = {{Cancer; Cancer therapy; HIF-2α; Hypoxia; Hypoxia inducible factor (HIF); Neuroblastoma; Patient-derived xenograft cells; Pseudohypoxia; PT2385}},
  language     = {{eng}},
  month        = {{03}},
  number       = {{2}},
  publisher    = {{Academic Press}},
  series       = {{Experimental Cell Research}},
  title        = {{ARNT-dependent HIF-2 transcriptional activity is not sufficient to regulate downstream target genes in neuroblastoma}},
  url          = {{http://dx.doi.org/10.1016/j.yexcr.2020.111845}},
  doi          = {{10.1016/j.yexcr.2020.111845}},
  volume       = {{388}},
  year         = {{2020}},
}