G-protein Pathway Suppressor Research Articles

GPS2 promotes erythroid differentiation in K562 erythroleukemia cells primarily via NCOR1.

G protein pathway suppressor 2 (GPS2) has been shown to play a pivotal role in human and mouse definitive erythropoiesis in an EKLF-dependent manner. However, whether GPS2 affects human primitive erythropoiesis is still unknown. This study demonstrated that GPS2 positively regulates erythroid differentiation in K562 cells, which have a primitive erythroid phenotype. Overexpression of GPS2 promoted hemin-induced hemoglobin synthesis in K562 cells as assessed by the increased percentage of benzidine-positive cells and the deeper red coloration of the cell pellets. In contrast, knockdown of GPS2 inhibited hemin-induced erythroid differentiation of K562 cells. GPS2 overexpression also enhanced erythroid differentiation of K562 cells induced by cytosine arabinoside (Ara-C). GPS2 induced hemoglobin synthesis by increasing the expression of globin and ALAS2 genes, either under steady state or upon hemin treatment. Promotion of erythroid differentiation of K562 cells by GPS2 mainly relies on NCOR1, as knockdown of NCOR1 or lack of the NCOR1-binding domain of GPS2 potently diminished the promotive effect. Thus, our study revealed a previously unknown role of GPS2 in regulating human primitive erythropoiesis in K562 cells.

Inhibition of K63 ubiquitination by G-Protein pathway suppressor 2 (GPS2) regulates mitochondria-associated translation

G-Protein Pathway Suppressor 2 (GPS2) is an inhibitor of non-proteolytic K63 ubiquitination mediated by the E2 ubiquitin-conjugating enzyme Ubc13. Previous studies have associated GPS2-mediated restriction of ubiquitination with the regulation of insulin signaling, inflammatory responses and mitochondria-nuclear communication across different tissues and cell types. However, a detailed understanding of the targets of GPS2/Ubc13 activity is lacking. Here, we have dissected the GPS2-regulated K63 ubiquitome in mouse embryonic fibroblasts and human breast cancer cells, unexpectedly finding an enrichment for proteins involved in RNA binding and translation on the outer mitochondrial membrane. Validation of selected targets of GPS2-mediated regulation, including the RNA-binding protein PABPC1 and translation factors RPS1, RACK1 and eIF3M, revealed a mitochondrial-specific strategy for regulating the translation of nuclear-encoded mitochondrial proteins via non-proteolytic ubiquitination. Removal of GPS2-mediated inhibition, either via genetic deletion or stress-induced nuclear translocation, promotes the import-coupled translation of selected mRNAs leading to the increased expression of an adaptive antioxidant program. In light of GPS2 role in nuclear-mitochondria communication, these findings reveal an exquisite regulatory network for modulating mitochondrial gene expression through spatially coordinated transcription and translation.

G protein pathway suppressor 2 suppresses aerobic glycolysis through RACK1-mediated HIF-1α degradation in breast cancer

Aerobic glycolysis has been recognized as a hallmark of human cancer. G protein pathway suppressor 2 (GPS2) is a negative regulator of the G protein-MAPK pathway and a core subunit of the NCoR/SMRT transcriptional co-repressor complex. However, how its biological properties intersect with cellular metabolism in breast cancer (BC) development remains poorly elucidated. Here, we report that GPS2 is low expressed in BC tissues and negatively correlated with poor prognosis. Both in vitro and in vivo studies demonstrate that GPS2 suppresses malignant progression of BC. Moreover, GPS2 suppresses aerobic glycolysis in BC cells. Mechanistically, GPS2 destabilizes HIF-1α to reduce the transcription of its downstream glycolytic regulators (PGK1, PGAM1, ENO1, PKM2, LDHA, PDK1, PDK2, and PDK4), and then suppresses cellular aerobic glycolysis. Notably, receptor for activated C kinase 1 (RACK1) is identified as a key ubiquitin ligase for GPS2 to promote HIF-1α degradation. GPS2 stabilizes the binding of HIF-1α to RACK1 by directly binding to RACK1, resulting in polyubiquitination and instability of HIF-1α. Amino acid residues 70–92 aa of the GPS2 N-terminus bind RACK1. A 23-amino-acid-long GPS2-derived peptide was developed based on this N-terminal region, which promotes the interaction of RACK1 with HIF-1α, downregulates HIF-1α expression and significantly suppresses BC tumorigenesis in vitro and in vivo. In conclusion, our findings indicate that GPS2 decreases the stability of HIF-1α, which in turn suppresses aerobic glycolysis and tumorigenesis in BC, suggesting that targeting HIF-1α degradation and treating with peptides may be a promising approach to treat BC.

GPS2 ameliorates cigarette smoking-induced pulmonary vascular remodeling by modulating the ras-Raf-ERK axis.

Mitogen-activated protein kinase (MAPK)signaling-mediated smoking-associated pulmonary vascular remodeling (PVR) plays an important role in the pathogenesis of group 3 pulmonary hypertension (PH). And G protein pathway suppressor 2 (GPS2) could suppress G-protein signaling such as Ras and MAPK, but its role in cigarette smoking -induced PVR (CS-PVR) is unclear. An in vivo model of smoke-exposed rats was constructed to assess the role of GPS2 in smoking-induced PH and PVR. In vitro, the effects of GPS2 overexpression and silencing on the function of human pulmonary arterial smooth cells (HPASMCs) and the underlying mechanisms were explored. GPS2 expression was downregulated in rat pulmonary arteries (PAs) and HPASMCs after CS exposure. More importantly, CS-exposed rats with GPS2 overexpression had lower right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and wall thickness (WT%) than those without. And enhanced proliferation and migration of HPASMCs induced by cigarette smoking extract (CSE) can be evidently inhibited by overexpressed GPS2. Besides, GPS2siRNA significantly enhanced the proliferation, and migration of HPASMCs as well as activated Ras and Raf/ERK signaling, while these effects were inhibited by zoledronic acid (ZOL). In addition, GPS2 promoter methylation level in rat PAs and HPASMCs was increased after CS exposure, and 5-aza-2-deoxycytidine (5-aza) inhibited CSE-induced GPS2 hypermethylation and downregulation in vitro. GPS2 overexpression could improve the CS-PVR, suggesting that GPS2 might serve as a novel therapeutic target for PH-COPD in the future.

LINC01305 recruits basonuclin 1 to act on G-protein pathway suppressor 1 to promote esophageal squamous cell carcinoma.

EsophageaL squamous cell carcinoma (ESCC) is one of the most common and lethal tumors, however, its underlying molecular mechanisms are not completely understood and new therapeutic targets are needed. Here, we found that the transcription factor basonuclin 1 (BNC1) was significantly upregulated and closely related to the differentiation and metastasis of ESCC. Furthermore, BNC1, LINC01305, and G-protein pathway suppressor 1 (GPS1) had significant oncogenic roles in ESCC. In addition, in vivo experiments showed that knockdown of BNC1 indeed significantly inhibited the proliferation and metastasis of ESCC. We also revealed the molecular mechanism by which LINC01305 recruits BNC1 to the promoter of GPS1, and then GPS1 could mediate the JNK signaling pathway to promote the proliferation and metastases of ESCC. Taken together, we discovered the novel molecular mechanism by which LINC01305/BNC1 upregulates GPS1 expression to promote the development of ESCC, providing a new therapeutic target for ESCC.

Antagonistic action of GPS2 and KDM1A at enhancers governs alternative macrophage activation by interleukin 4.

The Th2 cytokine interleukin 4 (IL4) promotes macrophage differentiation into alternative subtypes and plays important roles in physiology, in metabolic and inflammatory diseases, in cancer and in tissue regeneration. While the regulatory transcription factor networks governing IL4 signaling are already well-characterized, it is currently less understood which transcriptional coregulators are involved and how they operate mechanistically. In this study, we discover that G protein pathway suppressor 2 (GPS2), a core subunit of the HDAC3 corepressor complex assembled by SMRT and NCOR, represses IL4-dependent enhancer activation in mouse macrophages. Our genome-wide and gene-specific characterization revealed that, instead of directly repressing STAT6, chromatin-bound GPS2 cooperates with SMRT and NCOR to antagonize enhancer activation by lysine demethylase 1A (KDM1A, LSD1). Mechanistically, corepressor depletion increased KDM1A recruitment to enhancers linked to IL4-induced genes, accompanied by demethylation of the repressive histone marks H3K9me2/3 without affecting H3K4me1/2, the classic KDM1A substrates for demethylation in other cellular contexts. This in turn caused enhancer and gene activation already in the absence of IL4/STAT6 and sensitized the STAT6-dependent IL4 responsiveness of macrophages. Thus, our work identified with the antagonistic action of a GPS2-containing corepressor complex and the lysine demethylase KDM1A a hitherto unknown epigenetic corepressor-coactivator switching mechanism that governs alternative macrophage activation.

CROSSTALK BETWEEN UBIQUITINATION AND ADP-RIBOSYLATION IN THE MAINTENANCE OF MITOCHONDRIAL HOMEOSTASIS

Abstract Oxidative stress and increased ROS production, as observed in aging and obesity, commonly lead to the accumulation of mitochondrial dysfunctions. This is met by the activation of a robust mitochondria-to-nucleus stress response promoting the rewiring of nuclear gene expression to limit cellular and tissue damage and promote organelle adaptation. Here we will review previous work uncovering the transcriptional cofactor G-Protein Pathway Suppressor 2 (GPS2) as a mediator of mitochondria retrograde signaling and a key nuclear regulator of nuclear-encoded mitochondrial genes, including mitochondrial chaperones/proteases, mitokines, and other protective enzymes such as the ADP-ribosyltransferase NEURL4. We will also discuss unpublished work showing that shuttling of GPS2 between organelles plays a role in coordinating the transcriptional and translational regulation of antioxidant factors and pro-apoptotic genes by promoting the ubiquitination of mitochondria-associated translation factors.

Genome-wide CRISPR knockout screening identified G protein pathway suppressor 2 as a novel tumor suppressor for uveal melanoma metastasis.

Uveal melanoma (UM) is the most common intraocular malignant tumor in adults. Due to the lack of effective treatments for metastatic UM, the survival of UM has not changed over the past 3 decades. Therefore, it is important to identify essential genes regulating the metastasis of UM. In this study, a genome-wide CRISPR knockout screen in an orthotopic mouse model of UM was performed to identify the regulatory genes conferring the metastatic phenotype. Loss-of-function analyses were performed to explore the function of G protein pathway suppressor 2 (GPS2) in UM metastasis in vitro and in vivo. RNA sequencing was performed to investigate the molecular mechanism underlying the function of GPS2 as a tumor suppressor in UM. Among the highest-ranking genes, we found several validated tumor suppressors, such asSHPRH, GPS2, PRPH2, and hsa-mir-1229; GPS2 was chosen as the candidate gene for further studies. GPS2 was lower expressed in the tumor tissues of UM patients. Furthermore, knocking-down GPS2 promoted the proliferation and metastatic abilities of UM cells both in vivo and in vitro. Finally, analysis of the transcriptome data revealed that silencing GPS2 upregulates oncogenic signaling pathways MAPK and PI3K-Akt, and in the meantime downregulates tumor suppressor signaling pathway Slit/Robo in UM cells. Altogether, our study proved that the GPS2 gene functions as a tumor suppressor and might be a novel potential therapeutic target for UM treatment.

G protein pathway suppressor 2 suppresses gastric cancer by destabilizing epidermal growth factor receptor.

G protein pathway suppressor 2 (GPS2) is expressed in most human tissues, including the stomach. However, the biological functions of GPS2 in cancer, as well as the underlying molecular mechanisms, remain poorly understood. Here, we report that GPS2 expression was aberrantly downregulated in gastric cancer (GC) tissues compared with control tissues. Clinicopathologic analysis showed that low GPS2 expression was significantly correlated with pathological grade, lymph node stage, and invasive depth. Kaplan‐Meier analysis indicated that patients with low GPS2 expression showed poorer overall survival rates than those with high GPS2 expression. Moreover, GPS2 overexpression decreased GC cell proliferation, colony formation, tumorigenesis, and invasion. Overexpression of GPS2 reduced the protein expression of epidermal growth factor receptor (EGFR) and inhibited its downstream signaling in GC cells. Interestingly, GPS2 decreased EGFR protein expression, which was reversed by a lysosome inhibitor. Furthermore, GPS2 reduced EGFR protein stability by enhancing the binding of EGFR and an E3 ligase, c‐Cbl, which promoted the ubiquitination of EGFR, ultimately leading to its degradation through the lysosomal pathway. Further analysis indicated that GPS2 activated autophagy and promoted the autophagic flux by destabilizing EGFR. Taken together, these results suggest that low GPS2 expression is associated with GC progression and provide insights into the applicability of the GPS2‐EGFR axis as a potential therapeutic target in GC.

The Small Molecule BC-2059 Inhibits Wingless/Integrated (Wnt)-Dependent Gene Transcription in Cancer through Disruption of the Transducin β-Like 1-β-Catenin Protein Complex.

The central role of β-catenin in the Wnt pathway makes it an attractive therapeutic target for cancers driven by aberrant Wnt signaling. We recently developed a small-molecule inhibitor, BC-2059, that promotes apoptosis by disrupting the β-catenin/transducin β-like 1 (TBL1) complex through an unknown mechanism of action. In this study, we show that BC-2059 directly interacts with high affinity for TBL1 when in complex with β-catenin. We identified two amino acids in a hydrophobic pocket of TBL1 that are required for binding with β-catenin, and computational modeling predicted that BC-2059 interacts at the same hydrophobic pocket. Although this pocket in TBL1 is involved in binding with NCoR/SMRT complex members G Protein Pathway Suppressor 2 (GSP2) and SMRT and p65 NFκB subunit, BC-2059 failed to disrupt the interaction of TBL1 with either NCoR/SMRT or NFκB. Together, our results show that BC-2059 selectively targets TBL1/β-catenin protein complex, suggesting BC-2059 as a therapeutic for tumors with deregulated Wnt signaling pathway. SIGNIFICANCE STATEMENT: This study reports the mechanism of action of a novel Wnt pathway inhibitor, characterizing the selective disruption of the transducin β-like 1/β-catenin protein complex. As Wnt signaling is dysregulated across cancer types, this study suggests BC-2059 has the potential to benefit patients with tumors reliant on this pathway.

Interaction of Nuclear Export Protein with G Protein Pathway Suppressor 2 (GPS2) Facilitates Influenza A Virus Replication by Weakening the Inhibition of GPS2 to RNA Synthesis and Ribonucleoprotein Assembly

The nuclear export protein (NEP) serves multiple functions in the life cycle of influenza A virus (IAV). Identifying novel host proteins that interact with NEP and understanding their functions in IAV replication are of great interest. In this study, we screened and confirmed the direct interaction of G protein pathway suppressor 2 (GPS2) with NEP through a yeast two-hybrid screening assay and glutathione S-transferase-pulldown and co-immunoprecipitation assays. Knockdown or knockout of GPS2 enhanced IAV titers, whereas overexpression of GPS2 impaired IAV replication, demonstrating that GPS2 acted as a negative host factor in IAV replication. Meanwhile, GPS2 inhibited viral RNA synthesis by reducing the assembly of IAV polymerase. Interestingly, IAV NEP interacted with GPS2 and mediated its nuclear export, thereby activated the degradation of GPS2. Thus, NEP-GPS2 interaction weakened the inhibition of GPS2 to viral polymerase activity and benefited virus replication. Overall, this study identified the novel NEP-binding host partner GPS2 as a critical host factor to participate in IAV replication. These findings provided novel insights into the interactions between IAV and host cells, revealing a new function for GPS2 during IAV replication.Importance: NEP is proposed to play multiple biologically important roles in the life cycle of IAV, which largely relies on host factors by interaction. Our study demonstrated that GPS2 could reduce the interaction between PB1 and PB2 and interfere with vRNP assembly. Thus, GPS2 inhibited the RNA synthesis of IAV and negatively regulated its replication. Importantly, IAV NEP interacted with GPS2 and mediated the nuclear export of GPS2, thereby activated the degradation of GPS2. Thus, NEP-GPS2 interaction weakened the inhibition of GPS2 to viral polymerase activity and benefited virus replication.

Abstract PO049: Compartment-specific profiling for evaluation of disrupted signaling in early stage, low-grade endometrial cancer

Abstract Understanding tumor microenvironment is critical to improving conservative management (non-surgical) strategies for early stage, low grade endometrial cancer (EC). This remains an unmet clinical need for women who wish to maintain fertility and those who cannot undergo surgery due to medical co-morbidities. Current regimens rely on progestin treatment, but resistance and early relapse require improved therapeutic strategies. We sought to evaluate alterations in gene expression in the epithelial and stromal compartment of EC compared to normal endometrium to identify targets for combination therapies. RNA was extracted from microdissected epithelial and stromal compartments of formalin fixed paraffin embedded early stage, low grade EC and normal endometrium (NE). Comprehensive transcriptome profiling was performed with Affymetrix Clariom D assay and pathway analysis was performed. Immunohistochemistry (IHC) was used for validation. Functional studies of differentially expressed genes were performed in Hec1A EC cell lines. Among differentially expressed genes in EC vs NE, multiple altered signaling pathways were identified. EIF2 signaling, eIF4 and p70S6K signaling, and PI3K/AKT/mTOR were the most significant pathways activated in both epithelial and stromal compartments. In the epithelia compartment, the most significant gene expression changes included reduced expression of RBPMS (RNA Binding Protein with Multiple Splicing), SORD (Sorbitol Dehydrogenase), DDX17 (DEAD-Box Helicase 17), and TPT1 (Translationally-Controlled Tumor Protein) in EC, while MTDH (Metadherin) was significantly increased in EC. Similar changes were observed in the stroma compartment, but with lower fold changes than that observed in gland. Together with decreased RBPMS expression, decreased RBPMS-interacting molecules IGF2, TCF7L2 (Transcription Factor 7 Like 2), FOS (Proto-Oncogene C-Fos) and increased GPS2 (G protein Pathway Suppressor 2) were also observed. Differential expression of proteins in gland and stroma was also confirmed by IHC. An inverse correlation of RBPMS expression and EC proliferation was also observed in EC at the protein level. Enhanced AKT and S6 protein phosphorylation were observed by RBPMS silencing in Hec1A endometrial cells. Reverse Phase Protein Array demonstrated activation of Caveolin 1, FOXO3 (Forkhead Box O3), MT-CO1 (Mitochondrially Encoded Cytochrome C Oxidase I), L1CAM (L1 Cell Adhesion Molecule) with RBPMS silencing in Hec1A cell. These molecules are involved in pathways such as molecular mechanisms of cancer, estrogen receptor signaling, and senescence pathway. While estrogen-regulated changes were prominent in early stage low-grade EC, the identification of additional signaling changes provide support for an ongoing trial of levonorgestrel IUD + everolimus (mTOR inhibitor) and can be probed further to identify new targets for combination conservative management strategies. Additionally, biomarkers from these pathways, such as RBPMS, can be studied to guide the selection of progestin-only therapy versus combination therapy. Citation Format: Qian Zhang, Xiaoping Su, Joseph Celestino, Ying Yuan, Samuel C. Mok, Karen H. Lu, Melinda S. Yates. Compartment-specific profiling for evaluation of disrupted signaling in early stage, low-grade endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO049.

Loss of G-Protein Pathway Suppressor 2 Promotes Tumor Growth Through Activation of AKT Signaling.

G Protein Suppressor 2 (GPS2) is a multifunctional protein that exerts important roles in inflammation and metabolism in adipose, liver, and immune cells. GPS2 has recently been identified as a significantly mutated gene in breast cancer and other malignancies and proposed to work as a putative tumor suppressor. However, molecular mechanisms by which GPS2 prevents cancer development and/or progression are largely unknown. Here, we have profiled the phenotypic changes induced by GPS2 depletion in MDA-MB-231 triple negative breast cancer cells and investigated the underlying molecular mechanisms. We found that GPS2-deleted MDA-MB-231 cells exhibited increased proliferative, migratory, and invasive properties in vitro, and conferred greater tumor burden in vivo in an orthotopic xenograft mouse model. Transcriptomic, proteomic and phospho-proteomic profiling of GPS2-deleted MBA-MB-231 revealed a network of altered signals that relate to cell growth and PI3K/AKT signaling. Overlay of GPS2-regulated gene expression with MDA-MB-231 cells modified to express constitutively active AKT showed significant overlap, suggesting that sustained AKT activation is associated with loss of GPS2. Accordingly, we demonstrate that the pro-oncogenic phenotypes associated with GPS2 deletion are rescued by pharmacological inhibition of AKT with MK2206. Collectively, these observations confirm a tumor suppressor role for GPS2 and reveal that loss of GPS2 promotes breast cancer cell proliferation and tumor growth through uncontrolled activation of AKT signaling. Moreover, our study points to GPS2 as a potential biomarker for a subclass of breast cancers that would be responsive to PI3K-class inhibitor drugs.

Loss of G protein pathway suppressor 2 in human adipocytes triggers lipid remodeling by upregulating ATP binding cassette subfamily G member 1

ObjectiveAdipogenesis is critical for adipose tissue remodeling during the development of obesity. While the role of transcription factors in the orchestration of adipogenic pathways is already established, the involvement of coregulators that transduce regulatory signals into epigenome alterations and transcriptional responses remains poorly understood. The aim of our study was to investigate which pathways are controlled by G protein pathway suppressor 2 (GPS2) during the differentiation of human adipocytes. MethodsWe generated a unique loss-of-function model by RNAi depletion of GPS2 in human multipotent adipose-derived stem (hMADS) cells. We thoroughly characterized the coregulator depletion-dependent pathway alterations during adipocyte differentiation at the level of transcriptome (RNA-seq), epigenome (ChIP-seq H3K27ac), cistrome (ChIP-seq GPS2), and lipidome. We validated the in vivo relevance of the identified pathways in non-diabetic and diabetic obese patients. ResultsThe loss of GPS2 triggers the reprogramming of cellular processes related to adipocyte differentiation by increasing the responses to the adipogenic cocktail. In particular, GPS2 depletion increases the expression of BMP4, an important trigger for the commitment of fibroblast-like progenitors toward the adipogenic lineage and increases the expression of inflammatory and metabolic genes. GPS2-depleted human adipocytes are characterized by hypertrophy, triglyceride and phospholipid accumulation, and sphingomyelin depletion. These changes are likely a consequence of the increased expression of ATP-binding cassette subfamily G member 1 (ABCG1) that mediates sphingomyelin efflux from adipocytes and modulates lipoprotein lipase (LPL) activity. We identify ABCG1 as a direct transcriptional target, as GPS2 depletion leads to coordinated changes of transcription and H3K27 acetylation at promoters and enhancers that are occupied by GPS2 in wild-type adipocytes. We find that in omental adipose tissue of obese humans, GPS2 levels correlate with ABCG1 levels, type 2 diabetic status, and lipid metabolic status, supporting the in vivo relevance of the hMADS cell-derived in vitro data. ConclusionOur study reveals a dual regulatory role of GPS2 in epigenetically modulating the chromatin landscape and gene expression during human adipocyte differentiation and identifies a hitherto unknown GPS2-ABCG1 pathway potentially linked to adipocyte hypertrophy in humans.

GPS2 promotes erythroid differentiation by control of the stability of EKLF protein

AbstractErythropoiesis is a complex multistage process that involves differentiation of early erythroid progenitors to enucleated mature red blood cells, in which lineage-specific transcription factors play essential roles. Erythroid Krüppel-like factor (EKLF/KLF1) is a pleiotropic erythroid transcription factor that is required for the proper maturation of the erythroid cells, whose expression and activation are tightly controlled in a temporal and differentiation stage-specific manner. Here, we uncover a novel role of G-protein pathway suppressor 2 (GPS2), a subunit of the nuclear receptor corepressor/silencing mediator of retinoic acid and thyroid hormone receptor corepressor complex, in erythrocyte differentiation. Our study demonstrates that knockdown of GPS2 significantly suppresses erythroid differentiation of human CD34+ cells cultured in vitro and xenotransplanted in nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor γ-chain null mice. Moreover, global deletion of GPS2 in mice causes impaired erythropoiesis in the fetal liver and leads to severe anemia. Flow cytometric analysis and Wright-Giemsa staining show a defective differentiation at late stages of erythropoiesis in Gps2−/− embryos. Mechanistically, GPS2 interacts with EKLF and prevents proteasome-mediated degradation of EKLF, thereby increasing EKLF stability and transcriptional activity. Moreover, we identify the amino acids 191-230 region in EKLF protein, responsible for GPS2 binding, that is highly conserved in mammals and essential for EKLF protein stability. Collectively, our study uncovers a previously unknown role of GPS2 as a posttranslational regulator that enhances the stability of EKLF protein and thereby promotes erythroid differentiation.

Contribution of HDAC3 to transcriptional repression by the human papillomavirus 31 E8^E2 protein.

Human papillomaviruses (HPV) such as HPV16 and HPV31 encode an E8^E2 protein that acts as a repressor of viral replication and transcription. E8^E2's repression activities are mediated via the interaction with host-cell NCoR (nuclear receptor corepressor)/SMRT (silencing mediator of retinoid and thyroid receptors) corepressor complexes, which consist of NCoR, its homologue SMRT, GPS2 (G-protein pathway suppressor 2), HDAC3 (histone deacetylase 3), TBL1 (transducin b-like protein 1) and its homologue TBLR1 (TBL1-related protein 1). We now provide evidence that transcriptional repression by HPV31 E8^E2 is NCoR/SMRT-dependent but surprisingly always HDAC3-independent when analysing different HPV promoters. This is in contrast to the majority of several cellular transcription factors using NCoR/SMRT complexes whose transcriptional repression activities are both NCoR/SMRT- and HDAC3-dependent. However, NCoR/SMRT-dependent but HDAC3-independent repression has been described for specific cellular genes, suggesting that this may not be specific for HPV promoters but could be a feature of a subset of NCoR/SMRT-HDAC3 regulated genes.

Abstract P4-08-05: Novel tumor suppressor regulating the PI3K/AKT pathway in triple negative breast cancer (TNBC)

Abstract The PI3K/AKT signaling pathway is a known oncogenic pathway in breast cancer, involved in essential cellular processes necessary for cancer development and progression, including altered proliferation, metabolism, and cell survival. Mutations in this pathway occur in 20% of all TNBC cases, making it the most deregulated pathway in the only subclass of breast cancer with no targeted therapy. Few clinical trials testing PI3K/AKT pathway inhibition in these breast cancers have shown limited long term efficacy; the heterogeneity of tumors and variable response rates in clinical studies underscore the necessity to better understand the mechanisms governing TNBC and the PI3K/AKT pathway in order to predict patient response to targeted therapies. In particular, appropriate biomarkers are required to identify the tumor subtypes that may be more responsive to PI3K pathway inhibition. While regulation of AKT activation through a classic phosphorylation signaling cascade is well understood, recent studies have revealed a non-canonical pathway contributing to regulate AKT activation and possibly providing an unexpected mechanism for resistance to PI3K inhibition. Non-canonical regulation of AKT is provided by non-proteolytic ubiquitination of AKT favoring its recruitment to the plasma membrane and leading to hyperactivation, but a full understanding of the mechanism and players involved in this process is missing. Our lab has recently identified G-Protein Pathway Suppressor 2 (GPS2) as a novel regulator of the insulin signaling pathway through the inhibition of AKT ubiquitination in the adipose tissue. Here, we explore the role of GPS2 as a tumor suppressor in the context of breast cancer. Deletion of GPS2 in aggressive MDA-MB231 TNBC cells results in increased AKT phosphorylation associated with greater proliferation, migration and invasion in vitro, with all phenotypes mitigated in presence of the allosteric AKT inhibitor MK2206.Transcriptomic profiling of wild-type MB231, MB231-GPS2-KO, and myristoylated AKT (MB231-myrAKT) revealed a significant overlap between the MB231-GPS2-KO and myrAKT signatures. Conversely, deletion of GPS2 in MDA-MB468, a TNBC cell line with an activating mutation in the PI3K/AKT pathway, do not show any increase in proliferation, migration or invasion, but remain sensitive to AKT inhibition. Together these results indicate that loss of GPS2-mediated inhibition of AKT ubiquitination is sufficient to drive activation of the PI3K/AKT pathway in absence of other activating mutations. Furthermore, preliminary analysis of an orthotopic model of MB231-GPS2-KO cells in NOD scid gamma (NSG) mice resulted in greater breast tumor sizes when compared to the parental cell line, confirming GPS2 putative tumor suppressor role in vivo. Further studies are required to investigate GPS2 expression levels and mutational status in tumor samples, and to confirm GPS2 relevance as a predictive biomarker for treatment outcome, thus exposing an additional category of tumors that could be responsive to PI3K/AKT class inhibition and/or would benefit from alternative strategies to prevent resistance. Citation Format: Stefanie Chan, Emma Smith, Stefano Monti, M Dafne Cardamone, Valentina Perissi. Novel tumor suppressor regulating the PI3K/AKT pathway in triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-08-05.

Copy number variation profiling in pharmacogenes using panel-based exome resequencing and correlation to human liver expression.

Structural variants including copy number variations (CNV) have gained widespread attention, especially in pharmacogenomics but for several genes functional relevance and clinical evidence are still lacking. Detection of CNVs in next-generation sequencing data is challenging but offers widespread applications. We developed a cohort-based CNV detection workflow to extract CNVs from read counts of targeted NGS of 340 genes involved in absorption, distribution, metabolism and excretion (ADME) of drugs. We applied our method to 150 human liver tissue samples and correlated identified CNVs to mRNA expression levels. In total, we identified 445 deletions (73%) and 167 duplications (27%) in 36 pharmacogenes including all well-known CNVs of CYPs, GSTs, SULTs, UGTs, numerous described rare CNVs of CYP2E1, SLC16A3 or UGT2B15 as well as novel observations, e.g., for SLC22A12, SLC22A17 and GPS2 (G Protein Pathway Suppressor 2). We were able to fine-map complex CNVs of CYP2A6 and CYP2D6 with exon resolution. Correlation analysis confirmed known expression patterns for common CNVs and suggested an influence on expression variability for some rare CNVs. Our straightforward CNV detection workflow can be easily applied to any NGS coverage data and helped to analyze CNVs in an ADME-NGS panel of 340 pharmacogenes to improve genotype-phenotype correlations.

SUN-133 G-protein Pathway Suppressor 2 (GPS2) Enhanced the Large Conductance Ca2+ Activated Potassium (BK) Channels Activity through modulating WNK4 Ubiquitination

BK channel plays an important role in renal potassium secretion. G-protein pathway suppressor 2 (GPS2) is a multifunctional protein and transcriptional regulation factor that is involved in the G-protein mitogen-activated protein kinase (MAPK) signaling pathway. We have previously reported that WNK4 inhibits BK channel activity by stimulating ERK1/2 and p38 signaling pathways. We have recently shown that GPS2 enhances BK channel activity and its protein expression by reducing ERK1/2 signaling-mediated degradation of the channel.

Hepatocyte-specific loss of GPS2 in mice reduces non-alcoholic steatohepatitis via activation of PPAR\u03b1

Obesity triggers the development of non-alcoholic fatty liver disease (NAFLD), which involves alterations of regulatory transcription networks and epigenomes in hepatocytes. Here we demonstrate that G protein pathway suppressor 2 (GPS2), a subunit of the nuclear receptor corepressor (NCOR) and histone deacetylase 3 (HDAC3) complex, has a central role in these alterations and accelerates the progression of NAFLD towards non-alcoholic steatohepatitis (NASH). Hepatocyte-specific Gps2 knockout in mice alleviates the development of diet-induced steatosis and fibrosis and causes activation of lipid catabolic genes. Integrative cistrome, epigenome and transcriptome analysis identifies the lipid-sensing peroxisome proliferator-activated receptor α (PPARα, NR1C1) as a direct GPS2 target. Liver gene expression data from human patients reveal that Gps2 expression positively correlates with a NASH/fibrosis gene signature. Collectively, our data suggest that the GPS2-PPARα partnership in hepatocytes coordinates the progression of NAFLD in mice and in humans and thus might be of therapeutic interest.