{"id":{"repo_id":"u-iceland","oai_identifier":"oai:skemman.is:1946/47148"},"canonical_url":"https://search.dev.ndltd.org/etd/u-iceland/oai:skemman.is:1946/47148","repository":{"repo_id":"u-iceland","name":"University of Iceland","base_url":"https://skemman.is/oai/request"},"display":{"title":"The effect of distinct BRCA1 splice forms on drug sensitivity in breast- and ovarian cancer cell lines","abstract":"Germline mutations in BRCA-genes cause higher risk of cancer development, particularly in breast and ovaries. PARP inhibitors are a promising treatment option for carriers of BRCA1/2 mutations diagnosed with these tumor types. Pathogenic spliceogenic variants have been found in BRCA1 that change the expression of BRCA1 transcripts, in which the expression of shorter BRCA1 transcripts (BRCA1 ∆11 and ∆11q) increases, but expression of full-length BRCA1 transcript (BRCA1-FL) decreases. However, additional work is needed to determine how different BRCA1 transcripts can affect the drug sensitivity in breast- and ovarian cancer. The aim of this study was to knock out BRCA1 expression in the T47D breast cancer cell line and the CAOV3 ovarian cancer cell line and overexpress longer and shorter transcripts of BRCA1 in the cells. Subsequently, the aim was to assess their sensitivity to PARP inhibition and thereafter, study how each BRCA1 splice form affects the drug sensitivity of cancer cells. Finally, the aim was to use bioinformatic analysis with RNA sequencing and proteomics data to identify phenotypical changes in T47D cell lines with different expression of BRCA1 variants. CRISPR/Cas9 technique was used to knock out (KO) BRCA1 expression in T47D and CAOV3. The knockout of the gene was confirmed with RT-qPCR and western blot (WB) in T47D cells, but the BRCA1 knockout in CAOV3 was unsuccessful. Therefore, only T47D cell line was used in the project to further explore the drug sensitivity to the PARP inhibitor olaparib. Drug sensitivity was studied by clonogenic assay, and treatment with olaparib demonstrated that cells with loss of BRCA1 expression are more sensitive to PARP inhibitors than cells expressing wildtype (WT) BRCA1. The clonogenic assay also revealed that cells with BRCA1 loss grew slower than control cells. Subsequently, the BRCA1-FL transcript and the shorter transcript BRCA1∆11q were overexpressed in T47D BRCA1-KO cells using lentiviruses. RT-qPCR and WB results showed 3- and 7-fold expression of the BRCA1 splice forms in BRCA1-FL and BRCA1∆11q transduced cells, respectively, compared to control cells. To examine how different BRCA1 splice forms affect the drug sensitivity of breast cancer cells, the cells were treated with olaparib. Results revealed that drug sensitivity was partially reserved when both FL and ∆11q splice forms were overexpressed in T47D cells. RNA sequencing and proteomics data on cells with different splicing in BRCA1 exon 11 showed changed RNA and protein expression with decreased BRCA1-FL expression and increased BRCA1∆11/∆11q transcripts. Moreover, changes in gene regulatory pathways such as DNA repair were also observed. This study demonstrated that loss of BRCA1 expression sensitizes cells to PARP inhibition and that drug sensitivity is partially reserved when different splice variants of BRCA1 are overexpressed in T47D cells. Also, that changed expression of BRCA1 splice variants affects the phenotype of breast cancer cells. Future studies will focus on using the T47D cell model to study further the role of BRCA1 splice forms in breast cancer cells and explore how BRCA1 splice forms affect the phenotype and drug sensitivity of ovarian cancer by using similar methods.","abstract_html":"Germline mutations in BRCA-genes cause higher risk of cancer development, particularly in breast and ovaries. PARP inhibitors are a promising treatment option for carriers of BRCA1/2 mutations diagnosed with these tumor types. Pathogenic spliceogenic variants have been found in BRCA1 that change the expression of BRCA1 transcripts, in which the expression of shorter BRCA1 transcripts (BRCA1 ∆11 and ∆11q) increases, but expression of full-length BRCA1 transcript (BRCA1-FL) decreases. However, additional work is needed to determine how different BRCA1 transcripts can affect the drug sensitivity in breast- and ovarian cancer. The aim of this study was to knock out BRCA1 expression in the T47D breast cancer cell line and the CAOV3 ovarian cancer cell line and overexpress longer and shorter transcripts of BRCA1 in the cells. Subsequently, the aim was to assess their sensitivity to PARP inhibition and thereafter, study how each BRCA1 splice form affects the drug sensitivity of cancer cells. Finally, the aim was to use bioinformatic analysis with RNA sequencing and proteomics data to identify phenotypical changes in T47D cell lines with different expression of BRCA1 variants. CRISPR/Cas9 technique was used to knock out (KO) BRCA1 expression in T47D and CAOV3. The knockout of the gene was confirmed with RT-qPCR and western blot (WB) in T47D cells, but the BRCA1 knockout in CAOV3 was unsuccessful. Therefore, only T47D cell line was used in the project to further explore the drug sensitivity to the PARP inhibitor olaparib. Drug sensitivity was studied by clonogenic assay, and treatment with olaparib demonstrated that cells with loss of BRCA1 expression are more sensitive to PARP inhibitors than cells expressing wildtype (WT) BRCA1. The clonogenic assay also revealed that cells with BRCA1 loss grew slower than control cells. Subsequently, the BRCA1-FL transcript and the shorter transcript BRCA1∆11q were overexpressed in T47D BRCA1-KO cells using lentiviruses. RT-qPCR and WB results showed 3- and 7-fold expression of the BRCA1 splice forms in BRCA1-FL and BRCA1∆11q transduced cells, respectively, compared to control cells. To examine how different BRCA1 splice forms affect the drug sensitivity of breast cancer cells, the cells were treated with olaparib. Results revealed that drug sensitivity was partially reserved when both FL and ∆11q splice forms were overexpressed in T47D cells. RNA sequencing and proteomics data on cells with different splicing in BRCA1 exon 11 showed changed RNA and protein expression with decreased BRCA1-FL expression and increased BRCA1∆11/∆11q transcripts. Moreover, changes in gene regulatory pathways such as DNA repair were also observed. This study demonstrated that loss of BRCA1 expression sensitizes cells to PARP inhibition and that drug sensitivity is partially reserved when different splice variants of BRCA1 are overexpressed in T47D cells. Also, that changed expression of BRCA1 splice variants affects the phenotype of breast cancer cells. Future studies will focus on using the T47D cell model to study further the role of BRCA1 splice forms in breast cancer cells and explore how BRCA1 splice forms affect the phenotype and drug sensitivity of ovarian cancer by using similar methods.","abstract_has_math":false,"creators":["Sif Heiðarsdóttir 1998-"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Háskóli Íslands"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024-05-21T09:53:55Z","date_published":"2024-05-21T09:53:55Z","updated_at":"2026-07-27T21:42:56Z","subjects":["Lífeindafræði","Stökkbreytingar","Krabbamein"],"languages":["en"],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/1946/47148","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Háskóli Íslands"]},{"key":"dc:creator","label":"Author","values":["Sif Heiðarsdóttir 1998-"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2024-05-21T09:53:54Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2024-05-21T09:53:54Z"]},{"key":"dc:date.issued","label":"Date","values":["2024-05-21T09:53:55Z"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Lífeindafræði","Stökkbreytingar","Krabbamein"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/1946/47148"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Germline mutations in BRCA-genes cause higher risk of cancer development, particularly in breast and ovaries. PARP inhibitors are a promising treatment option for carriers of BRCA1/2 mutations diagnosed with these tumor types. Pathogenic spliceogenic variants have been found in BRCA1 that change the expression of BRCA1 transcripts, in which the expression of shorter BRCA1 transcripts (BRCA1 ∆11 and ∆11q) increases, but expression of full-length BRCA1 transcript (BRCA1-FL) decreases. However, additional work is needed to determine how different BRCA1 transcripts can affect the drug sensitivity in breast- and ovarian cancer. The aim of this study was to knock out BRCA1 expression in the T47D breast cancer cell line and the CAOV3 ovarian cancer cell line and overexpress longer and shorter transcripts of BRCA1 in the cells. Subsequently, the aim was to assess their sensitivity to PARP inhibition and thereafter, study how each BRCA1 splice form affects the drug sensitivity of cancer cells. Finally, the aim was to use bioinformatic analysis with RNA sequencing and proteomics data to identify phenotypical changes in T47D cell lines with different expression of BRCA1 variants. CRISPR/Cas9 technique was used to knock out (KO) BRCA1 expression in T47D and CAOV3. The knockout of the gene was confirmed with RT-qPCR and western blot (WB) in T47D cells, but the BRCA1 knockout in CAOV3 was unsuccessful. Therefore, only T47D cell line was used in the project to further explore the drug sensitivity to the PARP inhibitor olaparib. Drug sensitivity was studied by clonogenic assay, and treatment with olaparib demonstrated that cells with loss of BRCA1 expression are more sensitive to PARP inhibitors than cells expressing wildtype (WT) BRCA1. The clonogenic assay also revealed that cells with BRCA1 loss grew slower than control cells. Subsequently, the BRCA1-FL transcript and the shorter transcript BRCA1∆11q were overexpressed in T47D BRCA1-KO cells using lentiviruses. RT-qPCR and WB results showed 3- and 7-fold expression of the BRCA1 splice forms in BRCA1-FL and BRCA1∆11q transduced cells, respectively, compared to control cells. To examine how different BRCA1 splice forms affect the drug sensitivity of breast cancer cells, the cells were treated with olaparib. Results revealed that drug sensitivity was partially reserved when both FL and ∆11q splice forms were overexpressed in T47D cells. RNA sequencing and proteomics data on cells with different splicing in BRCA1 exon 11 showed changed RNA and protein expression with decreased BRCA1-FL expression and increased BRCA1∆11/∆11q transcripts. Moreover, changes in gene regulatory pathways such as DNA repair were also observed. This study demonstrated that loss of BRCA1 expression sensitizes cells to PARP inhibition and that drug sensitivity is partially reserved when different splice variants of BRCA1 are overexpressed in T47D cells. Also, that changed expression of BRCA1 splice variants affects the phenotype of breast cancer cells. Future studies will focus on using the T47D cell model to study further the role of BRCA1 splice forms in breast cancer cells and explore how BRCA1 splice forms affect the phenotype and drug sensitivity of ovarian cancer by using similar methods.","Meðfæddar stökkbreytingar í BRCA-genum valda aukinni áhættu á að fá krabbamein, einkum í brjóstum og eggjastokkum. Þekkt er að meinvaldandi breytingar geti valdið splæsiröskun umrita BRCA1 gensins þar sem tjáning á styttri BRCA1 umritum (BRCA1 ∆11 og ∆11q) eykst, en tjáning á fulllengdar BRCA1 umriti (BRCA1 full length-FL) minnkar. Einnig er þekkt að algjört tap á BRCA1 tjáningu auki næmni æxla við PARP hindrun, en frekari rannsókna er þörf á hvaða áhrif ólík BRCA1 umrit hafa á lyfjanæmi brjósta- og eggjastokkakrabbameinsfrumna. Markmið þessa verkefnis var að slá út tjáningu BRCA1 í brjóstakrabbameinsfrumulínunni T47D og eggjastokkakrabbameinsfrumulínunni CAOV3 og yfirtjá lengri og styttri umrit BRCA1 í frumunum. Í framhaldi að mæla næmni frumnanna við PARP hindrum og með þeim hætti rannsaka áhrif einstakra BRCA1 splæsiafbrigða á lyfjanæmi krabbameinsfrumna. Enn fremur var markmiðið að nota lífgagnatæknigreiningu með RNA raðgreiningar- og próteinmengjagögnum til að greina svipgerðarbreytingar í T47D frumulínum með mismunandi tjáningu af BRCA1 umritum. CRISPR/Cas9 aðferð var notuð til að slá út tjáningu BRCA1 í T47D og CAOV3. Útsláttur gensins var staðfestur með RT-qPCR og western blot (WB) í T47D frumum, en ekki tókst að slá út tjáningu BRCA1 í CAOV3. Í framhaldi var einungis unnið með T47D í verkefninu og frumurnar skoðaðar m.t.t. lyfjanæmis fyrir PARP hindranum olaparib. Lyfjanæmisprófanir með clonogenic assay, þar sem frumur voru meðhöndlaðar með olaparib, sýndu að frumur með tap á BRCA1 eru næmari fyrir PARP hindrum en frumur með óbreytta BRCA1 tjáningu. Clonogenic assay sýndi einnig fram á að frumur með tap á BRCA1 uxu hægar en viðmiðunarfrumur. Í framhaldi voru fulllengdar BRCA1 (BRCA1-FL) og stutt BRCA1 umrit (BRCA1∆11q) yfirtjáð í T47D BRCA1-KO frumum með lentivírus. RT-qPCR og WB niðurstöður sýndu að yfirtjáning tókst, og var hún 3-7 föld miðað við viðmiðunarfrumur. Til að skoða hvaða áhrif mismunandi BRCA1 splæsiafbrigði hafa á lyfjanæmi brjóstakrabbameinsfrumna voru frumur meðhöndlaðar með PARP hindranum olaparib. Niðurstöður sýndu að lyfjanæmi gekk til baka að hluta, bæði þegar FL og ∆11q splæsiform voru yfirtjáð í T47D frumunum. RNA raðgreining og próteinmengjagagnagreining á T47D frumum með splæsibreytingar í útröð 11 í BRCA1 sýndi breytta RNA og próteintjáningu í frumum með minni BRCA1-FL tjáningu og aukningu á BRCA1∆11/∆11q umritum. Auk þess var munur á ýmsum genastjórnunarferlum t.d. DNA viðgerðarferlum. Í rannsókninni var sýnt fram á að tap á BRCA1 tjáningu gerir frumur næmar fyrir PARP hindrun, og að lyfjanæmi gengur til baka að hluta til þegar ólík splæsiafbrigði BRCA1 eru yfirtjáð í T47D frumum. Einnig að breytt tjáning BRCA1 splæsiafbrigða hefur áhrif á svipgerð brjóstakrabbameinsfrumna. Framtíðar rannsóknir munu snúa að því að nota T47D frumumódelið til að rannsaka frekari hlutverk BRCA1 splæsiafbrigða í brjóstakrabbameinsfrumum og einnig að kanna áhrif BRCA1 splæsiafbrigða á svipgerð og lyfjanæmi eggjastokkakrabbameins með samskonar aðferðum."]},{"key":"dc:title","label":"Title","values":["The effect of distinct BRCA1 splice forms on drug sensitivity in breast- and ovarian cancer cell lines","Áhrif BRCA1 splæsiforma á lyfjanæmi brjósta- og eggjastokkakrabbameinslína"]}]}],"canonical_facts":{"dc:contributor":["Háskóli Íslands"],"dc:creator":["Sif Heiðarsdóttir 1998-"],"dc:date.accessioned":["2024-05-21T09:53:54Z"],"dc:date.available":["2024-05-21T09:53:54Z"],"dc:date.issued":["2024-05-21T09:53:55Z"],"dc:description.abstract":["Germline mutations in BRCA-genes cause higher risk of cancer development, particularly in breast and ovaries. PARP inhibitors are a promising treatment option for carriers of BRCA1/2 mutations diagnosed with these tumor types. Pathogenic spliceogenic variants have been found in BRCA1 that change the expression of BRCA1 transcripts, in which the expression of shorter BRCA1 transcripts (BRCA1 ∆11 and ∆11q) increases, but expression of full-length BRCA1 transcript (BRCA1-FL) decreases. However, additional work is needed to determine how different BRCA1 transcripts can affect the drug sensitivity in breast- and ovarian cancer. The aim of this study was to knock out BRCA1 expression in the T47D breast cancer cell line and the CAOV3 ovarian cancer cell line and overexpress longer and shorter transcripts of BRCA1 in the cells. Subsequently, the aim was to assess their sensitivity to PARP inhibition and thereafter, study how each BRCA1 splice form affects the drug sensitivity of cancer cells. Finally, the aim was to use bioinformatic analysis with RNA sequencing and proteomics data to identify phenotypical changes in T47D cell lines with different expression of BRCA1 variants. CRISPR/Cas9 technique was used to knock out (KO) BRCA1 expression in T47D and CAOV3. The knockout of the gene was confirmed with RT-qPCR and western blot (WB) in T47D cells, but the BRCA1 knockout in CAOV3 was unsuccessful. Therefore, only T47D cell line was used in the project to further explore the drug sensitivity to the PARP inhibitor olaparib. Drug sensitivity was studied by clonogenic assay, and treatment with olaparib demonstrated that cells with loss of BRCA1 expression are more sensitive to PARP inhibitors than cells expressing wildtype (WT) BRCA1. The clonogenic assay also revealed that cells with BRCA1 loss grew slower than control cells. Subsequently, the BRCA1-FL transcript and the shorter transcript BRCA1∆11q were overexpressed in T47D BRCA1-KO cells using lentiviruses. RT-qPCR and WB results showed 3- and 7-fold expression of the BRCA1 splice forms in BRCA1-FL and BRCA1∆11q transduced cells, respectively, compared to control cells. To examine how different BRCA1 splice forms affect the drug sensitivity of breast cancer cells, the cells were treated with olaparib. Results revealed that drug sensitivity was partially reserved when both FL and ∆11q splice forms were overexpressed in T47D cells. RNA sequencing and proteomics data on cells with different splicing in BRCA1 exon 11 showed changed RNA and protein expression with decreased BRCA1-FL expression and increased BRCA1∆11/∆11q transcripts. Moreover, changes in gene regulatory pathways such as DNA repair were also observed. This study demonstrated that loss of BRCA1 expression sensitizes cells to PARP inhibition and that drug sensitivity is partially reserved when different splice variants of BRCA1 are overexpressed in T47D cells. Also, that changed expression of BRCA1 splice variants affects the phenotype of breast cancer cells. Future studies will focus on using the T47D cell model to study further the role of BRCA1 splice forms in breast cancer cells and explore how BRCA1 splice forms affect the phenotype and drug sensitivity of ovarian cancer by using similar methods.","Meðfæddar stökkbreytingar í BRCA-genum valda aukinni áhættu á að fá krabbamein, einkum í brjóstum og eggjastokkum. Þekkt er að meinvaldandi breytingar geti valdið splæsiröskun umrita BRCA1 gensins þar sem tjáning á styttri BRCA1 umritum (BRCA1 ∆11 og ∆11q) eykst, en tjáning á fulllengdar BRCA1 umriti (BRCA1 full length-FL) minnkar. Einnig er þekkt að algjört tap á BRCA1 tjáningu auki næmni æxla við PARP hindrun, en frekari rannsókna er þörf á hvaða áhrif ólík BRCA1 umrit hafa á lyfjanæmi brjósta- og eggjastokkakrabbameinsfrumna. Markmið þessa verkefnis var að slá út tjáningu BRCA1 í brjóstakrabbameinsfrumulínunni T47D og eggjastokkakrabbameinsfrumulínunni CAOV3 og yfirtjá lengri og styttri umrit BRCA1 í frumunum. Í framhaldi að mæla næmni frumnanna við PARP hindrum og með þeim hætti rannsaka áhrif einstakra BRCA1 splæsiafbrigða á lyfjanæmi krabbameinsfrumna. Enn fremur var markmiðið að nota lífgagnatæknigreiningu með RNA raðgreiningar- og próteinmengjagögnum til að greina svipgerðarbreytingar í T47D frumulínum með mismunandi tjáningu af BRCA1 umritum. CRISPR/Cas9 aðferð var notuð til að slá út tjáningu BRCA1 í T47D og CAOV3. Útsláttur gensins var staðfestur með RT-qPCR og western blot (WB) í T47D frumum, en ekki tókst að slá út tjáningu BRCA1 í CAOV3. Í framhaldi var einungis unnið með T47D í verkefninu og frumurnar skoðaðar m.t.t. lyfjanæmis fyrir PARP hindranum olaparib. Lyfjanæmisprófanir með clonogenic assay, þar sem frumur voru meðhöndlaðar með olaparib, sýndu að frumur með tap á BRCA1 eru næmari fyrir PARP hindrum en frumur með óbreytta BRCA1 tjáningu. Clonogenic assay sýndi einnig fram á að frumur með tap á BRCA1 uxu hægar en viðmiðunarfrumur. Í framhaldi voru fulllengdar BRCA1 (BRCA1-FL) og stutt BRCA1 umrit (BRCA1∆11q) yfirtjáð í T47D BRCA1-KO frumum með lentivírus. RT-qPCR og WB niðurstöður sýndu að yfirtjáning tókst, og var hún 3-7 föld miðað við viðmiðunarfrumur. Til að skoða hvaða áhrif mismunandi BRCA1 splæsiafbrigði hafa á lyfjanæmi brjóstakrabbameinsfrumna voru frumur meðhöndlaðar með PARP hindranum olaparib. Niðurstöður sýndu að lyfjanæmi gekk til baka að hluta, bæði þegar FL og ∆11q splæsiform voru yfirtjáð í T47D frumunum. RNA raðgreining og próteinmengjagagnagreining á T47D frumum með splæsibreytingar í útröð 11 í BRCA1 sýndi breytta RNA og próteintjáningu í frumum með minni BRCA1-FL tjáningu og aukningu á BRCA1∆11/∆11q umritum. Auk þess var munur á ýmsum genastjórnunarferlum t.d. DNA viðgerðarferlum. Í rannsókninni var sýnt fram á að tap á BRCA1 tjáningu gerir frumur næmar fyrir PARP hindrun, og að lyfjanæmi gengur til baka að hluta til þegar ólík splæsiafbrigði BRCA1 eru yfirtjáð í T47D frumum. Einnig að breytt tjáning BRCA1 splæsiafbrigða hefur áhrif á svipgerð brjóstakrabbameinsfrumna. Framtíðar rannsóknir munu snúa að því að nota T47D frumumódelið til að rannsaka frekari hlutverk BRCA1 splæsiafbrigða í brjóstakrabbameinsfrumum og einnig að kanna áhrif BRCA1 splæsiafbrigða á svipgerð og lyfjanæmi eggjastokkakrabbameins með samskonar aðferðum."],"dc:identifier.uri":["http://hdl.handle.net/1946/47148"],"dc:language.iso":["en"],"dc:subject":["Lífeindafræði","Stökkbreytingar","Krabbamein"],"dc:title":["The effect of distinct BRCA1 splice forms on drug sensitivity in breast- and ovarian cancer cell lines","Áhrif BRCA1 splæsiforma á lyfjanæmi brjósta- og eggjastokkakrabbameinslína"],"dc:type":["Thesis"]},"updated_at":"2026-07-27T21:42:56Z"}