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Investigation of HIF-2α interaction partners and transcriptional activity under hypoxic conditions in diffuse intrinsic pontine glioma cells

Hedberg, Oscar (2023) MOBM02 20231
Degree Projects in Molecular Biology
Abstract
Purpose: The HIF-2α inhibitor PT2385 inhibits complex formation between HIF-2a and ARNT1 and thereby blocks HIF-2a transcriptional activation. It has been proven to extend survival in renal cancer, while its effects in pediatric brain cancer are unknown. This study is to investigate the effect of PT2385 in diffuse intrinsic pontine glioma (DIPG) cells, as well as to investigate HIF-2a interaction partners under hypoxia in these cells. Specifically, the purpose is to find out 1) does HIF-2α form a complex with ARNT and/or alternative interaction partners, and 2) does the possible complex of HIF-2α-alternative interaction partners get affected by PT2385 treatment.

Cells and Methods: Three patient derived cell lines were used in this... (More)
Purpose: The HIF-2α inhibitor PT2385 inhibits complex formation between HIF-2a and ARNT1 and thereby blocks HIF-2a transcriptional activation. It has been proven to extend survival in renal cancer, while its effects in pediatric brain cancer are unknown. This study is to investigate the effect of PT2385 in diffuse intrinsic pontine glioma (DIPG) cells, as well as to investigate HIF-2a interaction partners under hypoxia in these cells. Specifically, the purpose is to find out 1) does HIF-2α form a complex with ARNT and/or alternative interaction partners, and 2) does the possible complex of HIF-2α-alternative interaction partners get affected by PT2385 treatment.

Cells and Methods: Three patient derived cell lines were used in this study. We utilized two DIPG pediatric brain cancer cell lines, the SF8628 DIPG and BCH869, and additionally the U251 glioblastoma cell line as control. Western blotting was used to investigate the expression of ARNT and alternative interaction partners. Co-immunoprecipitation was performed to extract data on HIF-2α complex formation. siRNA-mediated knock down of ARNT1 and alternative interaction partners was used to test the effect of these genes on hypoxia signaling, which was measured using an HRE-luciferase reporter assay.

Results: Western blots confirmed that SF8628 cells expressed detectable levels of ARNT1 and alternative interaction partners. Co-immunoprecipitation showed that PT2385 inhibited the interaction between HIF-2α and its common HIF-2α partner ARNT1, and that an alternative interaction partner can heterodimerize with HIF-2α in SF8628 DIPG cells even in the presence of PT2385. A luciferase reporter driven by the HIF-2α-responsive hypoxia responsive element (HRE) showed that PT2385 treatment did not affect the hypoxia response in SF8628 cells, and siRNA experiments suggested that the alternative interaction partner was unable to activate HREs.

Conclusion: We have data indicating that HIF-2α is able to form a complex with an alternative interaction partner, and that HIF-2α inhibitor treatment does not affect the downstream hypoxia responsive elements when alternative interaction partner is knocked down. (Less)
Popular Abstract (Swedish)
Samverkande proteiner vid syrebrist under inflytande av inhibitorn PT2385 i hjärntumörceller från barn

DIPG är en typ av hjärncancer hos barn som är placerad i övre delen av nacken vilket gör den svår att operera. Ledsamt nog finns ingen behandling för denna sjukdom, och patienter behandlas ofta med strålning för att förlänga livet med några enstaka månader. Hos den typiska patienten så överlever dem ungefär 1 år från diagnos.

Forskare har testat flera olika behandlingar som fungerat i andra cancersjukdomar för att bota DIPG, och en behandling går ut på att motarbeta cancercellernas förmåga att överleva syrebrist. För det är nämligen så att vid snabb tillväxt av en tumör skapas syrebrist i tumören, det gör att cancercellerna skickar... (More)
Samverkande proteiner vid syrebrist under inflytande av inhibitorn PT2385 i hjärntumörceller från barn

DIPG är en typ av hjärncancer hos barn som är placerad i övre delen av nacken vilket gör den svår att operera. Ledsamt nog finns ingen behandling för denna sjukdom, och patienter behandlas ofta med strålning för att förlänga livet med några enstaka månader. Hos den typiska patienten så överlever dem ungefär 1 år från diagnos.

Forskare har testat flera olika behandlingar som fungerat i andra cancersjukdomar för att bota DIPG, och en behandling går ut på att motarbeta cancercellernas förmåga att överleva syrebrist. För det är nämligen så att vid snabb tillväxt av en tumör skapas syrebrist i tumören, det gör att cancercellerna skickar ut signaler för att skapa fler blodkärl för att få mer syre till att växa sig större.

Ifall vi kan lyckas motarbeta DIPG cellernas förmåga att överleva syrebristen så kan det mycket möjligen förhindra att tumören blir större så att patienten kan överleva. En medicin vi undersökt är PT2385 och har visat lovande resultat vid andra cancertyper, som njurcancer där 66% av patienterna hade positiva effekter.

Dock när vi undersökte PT2385 så såg vi att gener ansvariga för anpassningen till låga syrenivåer inte påverkades av medicinen i DIPG celler odlade i laboratoriet. Under experimenten upptäcktes även en ny proteininteraktion vilket vi misstänkte var anledning att PT2385 inte hade en effekt, men de nya proteinerna påverkade tyvärr inte de syrebristgener vi riktat in oss på med eller.


Dessa fynd belyser de utmaningar som forskare står inför när de försöker hitta effektiva behandlingar för DIPG. Medan PT2385 visade lovande resultat vid andra cancersorter var dess påverkan på DIPG-celler inte som förväntat. Denna kunskap är dock värdefull då den ger nya perspektiv för att bättre förstå de underliggande mekanismerna hos DIPG för att identifiera nya möjliga behandlingsmetoder.

Masterexamensprojekt i Molekylärbiologi 30 hp 2023
Biologiska institutionen, Lunds universitet
Handledare: Alexander Pietras
Lund university | Laboratory medicine | Translational Cancer Research (Less)
Please use this url to cite or link to this publication:
author
Hedberg, Oscar
supervisor
organization
course
MOBM02 20231
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
9136010
date added to LUP
2023-09-01 14:21:21
date last changed
2023-09-01 14:21:21
@misc{9136010,
  abstract     = {{Purpose: The HIF-2α inhibitor PT2385 inhibits complex formation between HIF-2a and ARNT1 and thereby blocks HIF-2a transcriptional activation. It has been proven to extend survival in renal cancer, while its effects in pediatric brain cancer are unknown. This study is to investigate the effect of PT2385 in diffuse intrinsic pontine glioma (DIPG) cells, as well as to investigate HIF-2a interaction partners under hypoxia in these cells. Specifically, the purpose is to find out 1) does HIF-2α form a complex with ARNT and/or alternative interaction partners, and 2) does the possible complex of HIF-2α-alternative interaction partners get affected by PT2385 treatment.

Cells and Methods: Three patient derived cell lines were used in this study. We utilized two DIPG pediatric brain cancer cell lines, the SF8628 DIPG and BCH869, and additionally the U251 glioblastoma cell line as control. Western blotting was used to investigate the expression of ARNT and alternative interaction partners. Co-immunoprecipitation was performed to extract data on HIF-2α complex formation. siRNA-mediated knock down of ARNT1 and alternative interaction partners was used to test the effect of these genes on hypoxia signaling, which was measured using an HRE-luciferase reporter assay.

Results: Western blots confirmed that SF8628 cells expressed detectable levels of ARNT1 and alternative interaction partners. Co-immunoprecipitation showed that PT2385 inhibited the interaction between HIF-2α and its common HIF-2α partner ARNT1, and that an alternative interaction partner can heterodimerize with HIF-2α in SF8628 DIPG cells even in the presence of PT2385. A luciferase reporter driven by the HIF-2α-responsive hypoxia responsive element (HRE) showed that PT2385 treatment did not affect the hypoxia response in SF8628 cells, and siRNA experiments suggested that the alternative interaction partner was unable to activate HREs.

Conclusion: We have data indicating that HIF-2α is able to form a complex with an alternative interaction partner, and that HIF-2α inhibitor treatment does not affect the downstream hypoxia responsive elements when alternative interaction partner is knocked down.}},
  author       = {{Hedberg, Oscar}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Investigation of HIF-2α interaction partners and transcriptional activity under hypoxic conditions in diffuse intrinsic pontine glioma cells}},
  year         = {{2023}},
}