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Sustaining Cyclin D1 Expression Renders Kidney Cancer Cells HIF2-Independent

ABSTRACT: Inactivation of theVHLtumor suppressor gene stabilizes HIF2a, which drives clear cell renal carcinoma (ccRCC). The HIF2ainhibitor belzutifan is approved for ccRCC treatment, butde novoand acquiredresistance are common. HIF2a, bound to ARNT, transcriptionally activates hundreds of genes. We performed genome-wide CRISPRa screens in HIF2a-dependent ccRCC lines treated with the belzutifan tool compound PT2399 to identify HIF2a-responsive genes that confer belzutifan resistance when not downregulated. Sustaining the expression of the HIF2atarget geneCCND1, encoding Cyclin D1, promoted HIF2a-independence/belzutifan resistance. This activity requires Cdk4/6 activation by Cyclin D1, but is not solely due to inhibitory phosphorylation of the canonical Cyclin D1 target, pRB. Indeed, ccRCC lines lacking all three pRB family members remained at least partially HIF2a-dependent. In this context. However, kinase-defective Cyclin D1 variants at least partially overrode belzutifan’s antiproliferative effects, suggesting that ccRCC promotion by Cyclin D1 requires the phosphorylation of pRB paralogs and one or more kinase-independent Cyclin D1 activities.

ORGANISM(S): Homo sapiens

PROVIDER: GSE277044 | GEO | 2025/03/20

REPOSITORIES: GEO

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Sustaining Cyclin D1 Expression Renders Kidney Cancer Cells HIF2-Independent

Project description:Inactivation of theVHLtumor suppressor gene stabilizes HIF2a, which drives clear cell renal carcinoma (ccRCC). The HIF2ainhibitor belzutifan is approved for ccRCC treatment, butde novoand acquiredresistance are common. HIF2a, bound to ARNT, transcriptionally activates hundreds of genes. We performed genome-wide CRISPRa screens in HIF2a-dependent ccRCC lines treated with the belzutifan tool compound PT2399 to identify HIF2a-responsive genes that confer belzutifan resistance when not downregulated. Sustaining the expression of the HIF2atarget geneCCND1, encoding Cyclin D1, promoted HIF2a-independence/belzutifan resistance. This activity requires Cdk4/6 activation by Cyclin D1, but is not solely due to inhibitory phosphorylation of the canonical Cyclin D1 target, pRB. Indeed, ccRCC lines lacking all three pRB family members remained at least partially HIF2a-dependent. In this context. However, kinase-defective Cyclin D1 variants at least partially overrode belzutifan’s antiproliferative effects, suggesting that ccRCC promotion by Cyclin D1 requires the phosphorylation of pRB paralogs and one or more kinase-independent Cyclin D1 activities.

2025-03-20 | GSE277043 | GEO

Sustaining Cyclin D1 Expression Renders Kidney Cancer Cells HIF2-Independent

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To identify genes responsive to treatment with the HIF2a inhibitor PT2399

Project description:HIF2a function is both necessary and sufficient for the growth of VHL-null clear cell Renal Cell Carcinoma (ccRCC). Targeting HIF2a function can therefore be a promising therapeutic strategy. We used microarray analysis to characterize a novel pharmacological inhibitor of HIF2a named PT2399. By comparing genes that are responsive to PT2399 in parental cells vs cells lacking HIF2a, by virtue of CRISPR-mediated genetic editing, we characterized gene signatures that are regulated by PT2399 in a HIF2a dependent manner. Cells were treated with either DMSO (control) or 2uM PT2399 for indicated time periods, total RNA was extracted and analyzed. Please note that the experiments with 786O Parental and HIF2a null cells were conducted independently.

2016-07-03 | E-GEOD-72959 | biostudies-arrayexpress

To identify genes responsive to treatment with the HIF2a inhibitor PT2399

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Project description:As shown by large-scale human genetic data, cancer mutations display strong tissue-selectivity, but how this selectivity arises remains unclear. Here, using experimental models, functional genomics and analysis of patient samples, we demonstrate that the lineage transcription factor paired box 8 (PAX8) is required for oncogenic signalling by two common clear cell renal cell carcinoma (ccRCC)-causing genetic alterations in humans: the germline variant rs7948643 at 11q13.3 and somatic inactivation of the von Hippel-Lindau tumour suppressor (VHL). VHL loss, observed in ~90% of ccRCCs, can lead to hypoxia-inducible factor 2 alpha (HIF2A) stabilization. We show that HIF2A is preferentially recruited to PAX8-bound transcriptional enhancers, including a pro-tumorigenic cyclin D1 (CCND1) enhancer that is controlled by PAX8 and HIF2A. The ccRCC protective allele C at rs7948643 inhibits PAX8 binding at this enhancer and downstream activation of CCND1 expression. Co-option of a PAX8-dependent physiological programme that supports proliferation of normal renal epithelial cells is also required for MYC expression from the ccRCC metastasis-associated amplicons at 8q21.3-q24.3. These results demonstrate that transcriptional lineage factors are essential for the expression of canonical oncogenes and they mediate tissue-specific cancer risk associated with somatic and inherited genetic variants

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Project description:Genomic aberrations of Cyclin D1 (CCND1) and CDK4 in neuroblastoma indicate that dysregulation of the G1 entry checkpoint is an important cell cycle aberration in this pediatric tumor. Here we report that analysis of Affymetrix expression data of primary neuroblastic tumors shows an extensive over-expression of Cyclin D1 and CDK4 which correlates with histological subgroups and prognosis respectively. Immunohistochemical analysis demonstrated an over-expression of Cyclin D1 in neuroblasts and a low Cyclin D1 expression in all cell types in ganglioneuroma. This suggests an involvement of G1 regulating genes in neuronal differentiation processes which we further evaluated using RNA interference against Cyclin D1 and its kinase partner CDK4 in several neuroblastoma cell lines. This resulted in pRb pathway inhibition as shown by an almost complete disappearance of CDK4 specific pRb phosphorylation; reduction of E2F transcriptional activity and a decrease of Cyclin A protein levels. The Cyclin D1 and CDK4 knock-down resulted in a significant reduction in cell proliferation, a G1 specific cell cycle arrest and moreover an extensive neuronal differentiation. Affymetrix microarray profiling of siRNA treated cells revealed a shift in expression profile towards a neuronal phenotype. Several new potential downstream players are identified. We conclude that neuroblastoma functionally depend on over-expression of G1 regulating genes to maintain their undifferentiated phenotype. Keywords: Neuroblastoma, CCND1, Cyclin D1, CDK4

2008-08-20 | GSE8866 | GEO

Stromal HIF2 Regulates Immune Suppression in the Pancreatic Cancer Microenvironment

Project description:Background & Aims. Pancreatic ductal adenocarcinoma (PDAC) has a hypoxic, immunosuppressive stroma, which contributes to its resistance to immune checkpoint blockade therapies. The hypoxia-inducible factors (HIFs) mediate the cellular response to hypoxia, but their role within the PDAC tumor microenvironment remains unknown. Methods. We used a dual recombinase mouse model to delete Hif1a or Hif2a in α-smooth muscle actin (αSMA)-expressing cancer-associated fibroblasts (CAFs) arising within spontaneous pancreatic tumors. The effects of CAF-Hif2a expression on tumor progression and composition of the tumor microenvironment were evaluated by Kaplan-Meier analysis, quantitative real-time polymerase chain reaction, histology, immunostaining, and by both bulk and single-cell RNA sequencing. CAF-macrophage crosstalk was modeled ex vivo using conditioned media from CAFs after treatment with hypoxia and PT2399, a HIF2 inhibitor currently in clinical trials. Syngeneic flank and orthotopic PDAC models were used to assess whether HIF2 inhibition improves response to immune checkpoint blockade. Results. CAF-specific deletion of HIF2, but not HIF1, suppressed PDAC tumor progression and growth, and improved survival of mice by 50% (n = 21-23 mice/group, Log-rank P = 0.0009). Deletion of CAF-HIF2 modestly reduced tumor fibrosis and significantly decreased the intratumoral recruitment of immunosuppressive M2 macrophages and regulatory T cells. Treatment with the clinical HIF2 inhibitor PT2399 significantly reduced in vitro macrophage chemotaxis and M2 polarization, and improved tumor responses to immunotherapy in both syngeneic PDAC mouse models. Conclusions. Together, these data suggest that stromal HIF2 is an essential component of PDAC pathobiology and is a druggable therapeutic target that could relieve tumor microenvironment immunosuppression and enhance immune responses in this disease.

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siRNA profiling of human cell line IMR-32 at time point 0 and transiently transfected with siRNA against GFP, Cyclin D1 and CDK4 at time point 48 hours reveals the undifferentiated phenotype in neuroblastoma depends on Cyclin D1 and CDK4 activity

2008-10-25 | E-GEOD-8866 | biostudies-arrayexpress