PBRM1 loss in clear cell renal cell carcinoma leads to a proangiogenic phenotype via the CXCL5/CXCR2 axis that can be targeted by CXCR2 inhibition
PBRM1 loss in clear cell renal cell carcinoma leads to a proangiogenic phenotype via the CXCL5/CXCR2 axis that can be targeted by CXCR2 inhibition
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DissertationDate of Exam
26.05.2025Date of Publication
18.08.2025Advisor
Ritter, ManuelCo-Referee
Junker, KerstinMetadata
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Abstract
Immune checkpoint blockade (ICB) and anti-angiogenic tyrosine kinase inhibitors (TKI) have substantially improved the outcomes of metastatic clear cell renal cell carcinoma (ccRCC). Nevertheless, the anti-angiogenic TKIs targeting the VEGF/ VEGFR axis result only in temporary antitumor response, and most ccRCCs become ultimately TKI-resistant. Mutations of polybromo-1 (PBRM1) occur in about one-third of ccRCC. It is well known that PBRM1-mutated tumors are highly vascularized. This project aims to dissect the molecular mechanism driving the proangiogenic phenotype of PBRM1-mutated ccRCC. In an unbiased approach, pharmacological inhibition of PBRM1 in ccRCC cell line leads to strong upregulation of the CXCR2 ligands CXCL5 and CXCL6. Accordingly, our TCGA in silico analysis revealed that PBRM1-mutant ccRCC exhibited enhanced CXCL1, CXCL2, CXCL3, and CXCL5 expression; furthermore, patients who had high CXCL5 mRNA expression are associated with worse prognosis in TCGA-KIRC. We established CRISPR-Cas9 induced polyclonal PBRM1-knockouts in the ccRCC cell lines 786O and Caki1. We also identified elevated secretion of CXCL1, CXCL2, CXCL5, CXCL6, and CXCL8, all CXCR2-activating chemokines in PBRM1-loss cell lines. Exposing the cells to pro-inflammatory cytokines (TNFα, and IL-17A) leads to hyper-induction of these chemokines. PBRM1-KO conditioned-media (CM) significantly promoted HUVECs viability and culminated in the enhanced phosphorylation of GSK-3β compared to PBRM1-wild-type CM. The addition of CXCR2 blockade to ccRCC not only suppressed the inhibition of GSK3β signaling, but also the proliferation of HUVECs. The association between PBRM1-deficiency and an enhanced tumor angiogenesis was recapitulated using the spheroid sprouting assay, a three-dimensional (3D) in vitro angiogenic model. Augmented angiogenic sprouting was detected in HUVEC-spheroids which were incubated with supernatant from PBRM1-loss cells. CXCL5 was identified as one of the drivers of the pro-angiogenic phenotype of PBRM1-mutated tumors. Of note, enhanced sprouting capacity of the supernatant of PBRM1-KO ccRCC cell lines can be selectively suppressed by CXCR2 inhibitor and CXLC5 blocking antibody. The therapeutic potential of CXCL5/CXCR2 signaling pathway disruption in ccRCC were shown in the CAM assay, underscoring that targeting CXCL5/CXCR2 axis is a promising approach for ccRCC treatment, specifically in PBRM1-defective ccRCC.
Subjects
PBRM1, angiogenesis, renal cell carcinoma, CXCL5/CXCR2
Classification (DDC)
610 Medizin, GesundheitTran, Ngoc Khanh: PBRM1 loss in clear cell renal cell carcinoma leads to a proangiogenic phenotype via the CXCL5/CXCR2 axis that can be targeted by CXCR2 inhibition. - Bonn, 2025. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-84471
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-84471
@phdthesis{handle:20.500.11811/13373,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-84471,
author = {{Ngoc Khanh Tran}},
title = {PBRM1 loss in clear cell renal cell carcinoma leads to a proangiogenic phenotype via the CXCL5/CXCR2 axis that can be targeted by CXCR2 inhibition},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2025,
month = aug,
note = {Immune checkpoint blockade (ICB) and anti-angiogenic tyrosine kinase inhibitors (TKI) have substantially improved the outcomes of metastatic clear cell renal cell carcinoma (ccRCC). Nevertheless, the anti-angiogenic TKIs targeting the VEGF/ VEGFR axis result only in temporary antitumor response, and most ccRCCs become ultimately TKI-resistant. Mutations of polybromo-1 (PBRM1) occur in about one-third of ccRCC. It is well known that PBRM1-mutated tumors are highly vascularized. This project aims to dissect the molecular mechanism driving the proangiogenic phenotype of PBRM1-mutated ccRCC. In an unbiased approach, pharmacological inhibition of PBRM1 in ccRCC cell line leads to strong upregulation of the CXCR2 ligands CXCL5 and CXCL6. Accordingly, our TCGA in silico analysis revealed that PBRM1-mutant ccRCC exhibited enhanced CXCL1, CXCL2, CXCL3, and CXCL5 expression; furthermore, patients who had high CXCL5 mRNA expression are associated with worse prognosis in TCGA-KIRC. We established CRISPR-Cas9 induced polyclonal PBRM1-knockouts in the ccRCC cell lines 786O and Caki1. We also identified elevated secretion of CXCL1, CXCL2, CXCL5, CXCL6, and CXCL8, all CXCR2-activating chemokines in PBRM1-loss cell lines. Exposing the cells to pro-inflammatory cytokines (TNFα, and IL-17A) leads to hyper-induction of these chemokines. PBRM1-KO conditioned-media (CM) significantly promoted HUVECs viability and culminated in the enhanced phosphorylation of GSK-3β compared to PBRM1-wild-type CM. The addition of CXCR2 blockade to ccRCC not only suppressed the inhibition of GSK3β signaling, but also the proliferation of HUVECs. The association between PBRM1-deficiency and an enhanced tumor angiogenesis was recapitulated using the spheroid sprouting assay, a three-dimensional (3D) in vitro angiogenic model. Augmented angiogenic sprouting was detected in HUVEC-spheroids which were incubated with supernatant from PBRM1-loss cells. CXCL5 was identified as one of the drivers of the pro-angiogenic phenotype of PBRM1-mutated tumors. Of note, enhanced sprouting capacity of the supernatant of PBRM1-KO ccRCC cell lines can be selectively suppressed by CXCR2 inhibitor and CXLC5 blocking antibody. The therapeutic potential of CXCL5/CXCR2 signaling pathway disruption in ccRCC were shown in the CAM assay, underscoring that targeting CXCL5/CXCR2 axis is a promising approach for ccRCC treatment, specifically in PBRM1-defective ccRCC.},
url = {https://hdl.handle.net/20.500.11811/13373}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-84471,
author = {{Ngoc Khanh Tran}},
title = {PBRM1 loss in clear cell renal cell carcinoma leads to a proangiogenic phenotype via the CXCL5/CXCR2 axis that can be targeted by CXCR2 inhibition},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2025,
month = aug,
note = {Immune checkpoint blockade (ICB) and anti-angiogenic tyrosine kinase inhibitors (TKI) have substantially improved the outcomes of metastatic clear cell renal cell carcinoma (ccRCC). Nevertheless, the anti-angiogenic TKIs targeting the VEGF/ VEGFR axis result only in temporary antitumor response, and most ccRCCs become ultimately TKI-resistant. Mutations of polybromo-1 (PBRM1) occur in about one-third of ccRCC. It is well known that PBRM1-mutated tumors are highly vascularized. This project aims to dissect the molecular mechanism driving the proangiogenic phenotype of PBRM1-mutated ccRCC. In an unbiased approach, pharmacological inhibition of PBRM1 in ccRCC cell line leads to strong upregulation of the CXCR2 ligands CXCL5 and CXCL6. Accordingly, our TCGA in silico analysis revealed that PBRM1-mutant ccRCC exhibited enhanced CXCL1, CXCL2, CXCL3, and CXCL5 expression; furthermore, patients who had high CXCL5 mRNA expression are associated with worse prognosis in TCGA-KIRC. We established CRISPR-Cas9 induced polyclonal PBRM1-knockouts in the ccRCC cell lines 786O and Caki1. We also identified elevated secretion of CXCL1, CXCL2, CXCL5, CXCL6, and CXCL8, all CXCR2-activating chemokines in PBRM1-loss cell lines. Exposing the cells to pro-inflammatory cytokines (TNFα, and IL-17A) leads to hyper-induction of these chemokines. PBRM1-KO conditioned-media (CM) significantly promoted HUVECs viability and culminated in the enhanced phosphorylation of GSK-3β compared to PBRM1-wild-type CM. The addition of CXCR2 blockade to ccRCC not only suppressed the inhibition of GSK3β signaling, but also the proliferation of HUVECs. The association between PBRM1-deficiency and an enhanced tumor angiogenesis was recapitulated using the spheroid sprouting assay, a three-dimensional (3D) in vitro angiogenic model. Augmented angiogenic sprouting was detected in HUVEC-spheroids which were incubated with supernatant from PBRM1-loss cells. CXCL5 was identified as one of the drivers of the pro-angiogenic phenotype of PBRM1-mutated tumors. Of note, enhanced sprouting capacity of the supernatant of PBRM1-KO ccRCC cell lines can be selectively suppressed by CXCR2 inhibitor and CXLC5 blocking antibody. The therapeutic potential of CXCL5/CXCR2 signaling pathway disruption in ccRCC were shown in the CAM assay, underscoring that targeting CXCL5/CXCR2 axis is a promising approach for ccRCC treatment, specifically in PBRM1-defective ccRCC.},
url = {https://hdl.handle.net/20.500.11811/13373}
}
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