ARNT-dependent HIF-2 transcriptional activity is not sufficient to regulate downstream target genes in neuroblastoma
(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.
(Less)
- author
- Persson, Camilla U. LU ; von Stedingk, Kristoffer LU ; Fredlund, Elina LU ; Bexell, Daniel LU ; Påhlman, Sven LU ; Wigerup, Caroline LU and Mohlin, Sofie LU
- organization
- publishing date
- 2020-03-15
- 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
-
- pmid:31945318
- scopus:85077917535
- 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
- 2024-07-10 09:29:18
@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}}, }