Inhibitor Of Protein Phosphatase 2A Research Articles

Protein phosphatase 2A inhibitors: a possible pharmacotherapy for benzodiazepine dependence.

Benzodiazepines (BZDs) activate the γ-aminobutyric acid (GABA) subtype A (GABAA) receptors, and thus are widely used medicines for the treatment of anxiety and insomnia. For chronic use, tolerance to BZDs is a major problem. Patients with chronic insomnia that develop tolerance to BZDs lose therapeutic effects but also potentially suffer from BZD dependence resulting in BZD withdrawal. The development of such treatments is important for the appropriate use of BZDs. Research articles regarding investigation of BZD dependencewere searched on PubMed, Embase, and Scopus databases using keywords "benzodiazepine", "dependence", "treatment". When BZDs are taken chronically, continuous GABAA binding results in up-regulation of α-amino-3-hydroxy-5-methyl-4-lisoxazolepropionic acid (AMPA) glutamate receptor function and release of brain-derived neurotrophic factor (BDNF). Released BDNF binds to its specific receptor tropomyosin-related kinase receptor B (TrkB). Enhanced BDNF-TrkB signaling activates protein phosphatase 2A (PP2A). Activated PP2A dephosphorylates GABAA receptors, resulting in the downregulation of the GABAA receptor function. Reduced GABAA receptor function augments long-term potentiation (LTP), AMPA-mediated glutamatergic neuroplasticity, by reducing LTP inhibition by GABAA receptor function. Augmented LTP enhances extreme anxiety, which leads to BZD dependence. Therefore, iInhibiting dephosphorylation of the GABAA receptor by PP2A, PP2A inhibitors could reduce LTP and anxiety, restoring BZD effectiveness and resulting in possible therapeutic effects for BZD dependence.

EXTH-75. IMMUNOVIROTHERAPY WITH PP2A AND PD-1 INHIBITION IN PEDIATRIC HIGH-GRADE GLIOMA

Abstract Pediatric high-grade gliomas (HGGs) represent a significant clinical challenge with poor survival rates. Engineered oncolytic herpes simplex virus (oHSV) has shown potential in several clinical trials at targeting HGGs through their direct lytic effect and induction of an immune response. We have learned from our clinical trial experience (NCT02457845) that a critical barrier to more durable response is augmenting and sustaining the anti-tumor immune response generated by oHSV. LB-100, a protein phosphatase 2A (PP2A) inhibitor, is known to enhance T-cell activity. We hypothesized that LB-100 with anti-PD-1 therapy would overcome the immunosuppressive barriers, enhancing the immunotherapeutic effects of oHSV and improve survival in an immunocompetent murine model. We investigated the therapeutic impact of clinically available, next-generation oHSV, M002, which expresses murine interleukin-12, combined with LB-100 and PD-1 inhibition compared to M002 with LB-100 or anti-PD-1 antibody, each therapy alone, or control by measuring median survival and changes in the tumor immune microenvironment. The triple combination of M002, LB-100, and anti-PD-1 antibody significantly extended median survival compared to control (45 days vs 22.5 days, P=0.001) or each treatment alone, and compared to M002 and LB-100 (44 days vs 28 days, P=0.017) or M002 and anti-PD-1 (45.5 days vs 41 days, P=0.023). This increase in survival was correlated with a robust increase in CD45+ immune cell infiltration and a reduction in T-regulatory cells. We will further investigate the changes in the tumor microenvironment to better understand the specific contributions of different immune cell populations. These data support the hypothesis that LB-100 enhances the efficacy of oHSV by amplifying T-cell-mediated immune responses against tumor cells. This combination therapy demonstrates a promising strategy that warrants further exploration and potential clinical application, emphasizing the critical role of targeting multiple aspects of immune regulation to enhance the efficacy of oncolytic virotherapy in pediatric tumors.

KRAS-mediated upregulation of CIP2A promotes suppression of PP2A-B56α to initiate pancreatic cancer development.

Oncogenic mutations in KRAS are present in ~95% of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) and are considered the initiating event of pancreatic intraepithelial neoplasia (PanIN) precursor lesions. While it is well established that KRAS mutations drive the activation of oncogenic kinase cascades during pancreatic oncogenesis, the effects of oncogenic KRAS signaling on regulation of phosphatases during this process is not fully appreciated. Protein Phosphatase 2A (PP2A) has been implicated in suppressing KRAS-driven cellular transformation and low PP2A activity is observed in PDAC cells compared to non-transformed cells, suggesting that suppression of PP2A activity is an important step in the overall development of PDAC. In the current study, we demonstrate that KRASG12D induces the expression of an endogenous inhibitor of PP2A activity, Cancerous Inhibitor of PP2A (CIP2A), and phosphorylation of the PP2A substrate, c-MYC. Consistent with these findings, KRASG12D sequestered the specific PP2A subunit responsible for c-MYC degradation, B56α, away from the active PP2A holoenzyme in a CIP2A-dependent manner. During PDAC initiation in vivo, knockout of B56α promoted KRASG12D tumorigenesis by accelerating acinar-to-ductal metaplasia (ADM) and the formation of PanIN lesions. The process of ADM was attenuated ex vivo in response to pharmacological re-activation of PP2A utilizing direct small molecule activators of PP2A (SMAPs). Together, our results suggest that suppression of PP2A-B56α through KRAS signaling can promote the MYC-driven initiation of pancreatic tumorigenesis.

A combination of protein phosphatase 2A inhibition and checkpoint immunotherapy: a perfect storm.

Immune checkpoint blockade has emerged as a potent new tool in the war on cancer. However, only a subset of cancer patients benefit from this therapeutic modality, sparking a search for combination therapies to increase the fraction of responding patients. We argue here that inhibition of protein phosphatase 2A (PP2A) is a promising approach to increase responses to immune checkpoint blockade and other therapies that rely on the presence of tumor-reactive Tcells. Inhibition of PP2A increases neoantigen expression on tumor cells, activates the cGAS/STING pathway, suppresses regulatory Tcells, and increases cytotoxic Tcell activation. In preclinical models, inhibition of PP2A synergizes with immune checkpoint blockade and emerging evidence indicates that patients who have tumors with mutations in PP2A respond better to immune checkpoint blockade. Therefore, inhibition of PP2A activity may be an effective way to sensitize cancer cells to immune checkpoint blockade and cell-based therapies using tumor-reactive Tcells.

Penfluridol inhibits melanoma growth and metastasis through enhancing von Hippel‒Lindau tumor suppressor-mediated cancerous inhibitor of protein phosphatase 2A (CIP2A) degradation.

Melanoma's high metastatic potential, especially to the brain, poses significant challenges to patient survival. The blood‒brain barrier (BBB) is a major obstacle to the effective treatment of melanoma brain metastases. We screened antipsychotic drugs capable of crossing the BBB and identified penfluridol (PF) as the most active candidate. PF reduced melanoma cell viability and induced apoptosis. In animal models, PF effectively inhibited melanoma growth and metastasis to the lung and brain. Using immunoprecipitation combined with high-resolution mass spectrometry, and other techniques such as drug affinity responsive target stability, we identified CIP2A as a direct binding protein of PF. CIP2A is highly expressed in melanoma and its metastases, and is linked to poor prognosis. PF can restore Protein Phosphatase 2A activity by promoting CIP2A degradation, thereby inhibiting several key oncogenic pathways, including AKT and c-Myc. Additionally, von Hippel‒Lindau (VHL) is the endogenous E3 ligase for CIP2A, and PF enhances the interaction between VHL and CIP2A, promoting the ubiquitin‒proteasome degradation of CIP2A, thereby inhibiting melanoma growth and metastasis. Overall, this study not only suggests PF's potential in treating melanoma and its brain metastases but also highlights CIP2A degradation as a therapeutic strategy for melanoma.

PP2A activation overcomes leptomeningeal dissemination in group 3 medulloblastoma

Leptomeningeal dissemination (LMD) is the primary cause of treatment failure in children with Group 3 medulloblastoma (MB). Building on our previous work on protein phosphatase 2A (PP2A) activation in MB, here we present pre-clinical and molecular data on the effects of two novel classes of PP2A activators on disease processes of LMD in Group 3 MB. The PP2A activators employed in this study are ATUX-6156 and ATUX-6954 (diarylmethylcycloamine sulfonylureas), and ATUX-1215 and ATUX-5800 (diarylmethyl-4-aminotetrahydropyran-sulfonamides). Treatment with these compounds led to suppression of the endogenous PP2A inhibitor, cancerous inhibitor of PP2A (CIP2A), enhanced phosphatase activity (10-60%), and reduced MB viability, migration, and invasion, prerequisites for MB cells to access the cerebrospinal fluid, affecting the initiation stage of LMD. PP2A activator treatment of MB cells led to apoptosis mediated via caspase 9/PARP signaling due to decreased phosphorylation of Bad, impeding the dispersal stage of LMD. Cell proliferation and LMD-driving cellular traits and molecules pertinent to the third stage, colonization, were also affected. Treatment with ATUX-1215 or ATUX-5800 prevented LMD in an intraventricular murine model of MB, possibly mediated by disruption of the CCL2-CCR2 axis by altered NF-kB phosphorylation via disrupted AKT signaling. The present investigation offers proof-of-principle data for PP2A-based reactivation therapy for Group 3 MB and provides the first indications that PP2A reactivation may challenge the current paradigm in targeting the 3-stage process of MB LMD. Further investigations of PP2A activators are warranted as these compounds may prove beneficial as therapeutics for MB.

Protein phosphatase 2A regulates blood cell proliferation and differentiation in Drosophila larval lymph glands.

Protein phosphatase 2A (PP2A), one of the most abundant protein phosphatases, has divergent functions in multiple types of cells. Its inactivation has been closely associated with leukemia diseases. However, the physiological function of PP2A for hematopoiesis has been poorly understood in organisms. Drosophila hematopoiesis parallels the vertebrate counterpart in developmental and functional features but involves a much simpler hematopoietic system. Here, utilizing the Drosophila major larval hematopoietic organ lymph gland, we studied the function of PP2A for hematopoiesis in vivo. By knocking down the expression of Pp2A-29B that encodes the scaffold subunit of the PP2A holoenzyme complex, we found that PP2A silencing in the differentiating hemocytes resulted in their excessive proliferation. Furthermore, this PP2A inhibition downregulated the expression of Smoothened (Smo), a crucial component in the Hedgehog pathway, and smo overexpression was able to rescue the phenotypes of PP2A depletion, indicating that Smo functions as a downstream effector of PP2A to restrict the hemocyte proliferation. PDGF/VEGF-receptor (Pvr) overexpression also restored the Smo expression and lymph gland morphology of PP2A silencing, suggesting a PP2A-Pvr-Smo axis to regulate lymph gland growth and hemocyte proliferation. Moreover, inhibiting PP2A activity in the blood progenitor cells promoted their differentiation, but which was independent with Smo. Together, our data suggested that PP2A plays a dual role in the Drosophila lymph gland by preserving the progenitor population and restraining the hemocyte proliferation, to properly regulate the hematopoietic process.

Paradoxical action of PP2A inhibition and its potential for therapeutic sensitization.

The protein phosphatase 2A (PP2A), a serine/threonine phosphatase, is recognized as a tumor suppressor involved in diverse cellular processes and essential for maintaining cell viability in vivo. However, endogenous inhibitors of PP2A such as cancerous inhibitor of PP2A (CIP2A) and endogenous nuclear protein inhibitor 2 of PP2A (SET) counteract the anticancer function of PP2A, promoting tumorigenesis, development, and drug resistance in tumors. Surprisingly though, contrary to conventional understanding, inhibition of the tumor suppressor gene PP2A with exogenous small molecule compounds can enhance the efficacy of cancer treatment and achieve superior tumor inhibition. Moreover, exogenous PP2A inhibitors resensitize cancers to treatment and provide novel therapeutic strategies for drug-resistant tumors, which warrant further investigation.

Concomitant targeting of FLT3 and SPHK1 exerts synergistic cytotoxicity in FLT3-ITD+ acute myeloid leukemia by inhibiting β-catenin activity via the PP2A-GSK3β axis

BackgroundApproximately 25–30% of patients with acute myeloid leukemia (AML) have FMS-like receptor tyrosine kinase-3 (FLT3) mutations that contribute to disease progression and poor prognosis. Prolonged exposure to FLT3 tyrosine kinase inhibitors (TKIs) often results in limited clinical responses due to diverse compensatory survival signals. Therefore, there is an urgent need to elucidate the mechanisms underlying FLT3 TKI resistance. Dysregulated sphingolipid metabolism frequently contributes to cancer progression and a poor therapeutic response. However, its relationship with TKI sensitivity in FLT3-mutated AML remains unknown. Thus, we aimed to assess mechanisms of FLT3 TKI resistance in AML.MethodsWe performed lipidomics profiling, RNA-seq, qRT-PCR, and enzyme-linked immunosorbent assays to determine potential drivers of sorafenib resistance. FLT3 signaling was inhibited by sorafenib or quizartinib, and SPHK1 was inhibited by using an antagonist or via knockdown. Cell growth and apoptosis were assessed in FLT3-mutated and wild-type AML cell lines via Cell counting kit-8, PI staining, and Annexin-V/7AAD assays. Western blotting and immunofluorescence assays were employed to explore the underlying molecular mechanisms through rescue experiments using SPHK1 overexpression and exogenous S1P, as well as inhibitors of S1P2, β-catenin, PP2A, and GSK3β. Xenograft murine model, patient samples, and publicly available data were analyzed to corroborate our in vitro results.ResultsWe demonstrate that long-term sorafenib treatment upregulates SPHK1/sphingosine-1-phosphate (S1P) signaling, which in turn positively modulates β-catenin signaling to counteract TKI-mediated suppression of FLT3-mutated AML cells via the S1P2 receptor. Genetic or pharmacological inhibition of SPHK1 potently enhanced the TKI-mediated inhibition of proliferation and apoptosis induction in FLT3-mutated AML cells in vitro. SPHK1 knockdown enhanced sorafenib efficacy and improved survival of AML-xenografted mice. Mechanistically, targeting the SPHK1/S1P/S1P2 signaling synergizes with FLT3 TKIs to inhibit β-catenin activity by activating the protein phosphatase 2 A (PP2A)-glycogen synthase kinase 3β (GSK3β) pathway.ConclusionsThese findings establish the sphingolipid metabolic enzyme SPHK1 as a regulator of TKI sensitivity and suggest that combining SPHK1 inhibition with TKIs could be an effective approach for treating FLT3-mutated AML.

Abstract Tu078: Mechanistic basis of protein phosphatase 2A inhibition by I2PP2A dimerization: A key step in βAR resensitization

Dysfunction of beta-adrenergic receptor (βAR) is a key hallmark of heart failure, wherein mechanisms of phosphorylation-mediated desensitization of βARs are well understood. However, less is known about dephosphorylation-mediated resensitization of βARs that is regulated by protein phosphatase 2A (PP2A) in heart failure. Our previous studies have shown that agonist stimulation of βAR activates phosphoinositide 3-kinase γ (PI3Kγ) which phosphorylates endogenous inhibitor of PP2A (I2PP2A) on serine residues 9 & 93. Phosphorylated I2PP2A (pI2PP2A) binds to PP2A inhibiting its activity thereby, impairing βAR resensitization. Although structural studies have shown I2PP2A to be a dimer, yet little is understood about the mechanistic basis of PP2A inhibition by I2PP2A. We therefore hypothesized that phosphorylation of I2PP2A promotes dimerization driving robust interaction with PP2A resulting in inhibition of PP2A and impairment in βAR resensitization. To test for dimerization, hemagglutinin (HA)-tagged I2PP2A and Myc-tagged I2PP2A were generated and co-expressed in HEK 293 cells. Immunoprecipitation of HA-I2PP2A led to significant co-immunoprecipitation of Myc-I2PP2A reflecting the ability of I2PP2A to dimerize. To determine whether phosphorylation is key for dimerization, phospho-mimetic mutants of I2PP2A (S9,93A (inactive) or S9,93D (active)) were generated. Immunoblotting of cellular lysates with expression of these phospho-mimetics showed that S9,93D exclusively formed dimers, while S9,93A I2PP2A only formed monomers suggesting that phosphorylation is key facilitator of dimerization. The role of I2PP2A phosphorylation in its dimerization and PP2A interaction with consequences on βAR resensitization will be presented. Determining the mechanistic basis of this understudied pathway is critical as human heart failure is associated with increased PI3Kγ activity, elevated expression of I2PP2A and reduced βAR function. Key words: Desensitization, Resensitization, I2PP2A, PP2A, Dimerization

Abstract Tu077: Targeting β-adrenergic receptor resensitization attenuates cardiac dysfunction

β-adrenergic receptor (βAR) is a key regulator of cardiac function. Agonist activation of βAR leads to phosphorylation-mediated desensitization resulting in βAR trafficking to endosomes. Endosomal βARs undergo resensitization through protein phosphatase 2A (PP2A)-mediated dephosphorylation. We have shown that stimulation of βARs activates phosphoinositide 3-kinase γ (PI3Kγ) that phosphorylates endogenous inhibitor of PP2A (I2PP2A) promoting robust binding to PP2A resulting in PP2A inhibition. βAR dysfunction due to desensitization is considered as a classical hallmark of heart failure, while less is known about resensitization. Plasma membrane and endosomal fractions were isolated from non-failing and failing patient samples to determine whether βAR resensitization is altered in human heart failure. Endosomes from heart failure samples showed significant accumulation of phosphorylated β2ARs which was associated with reduced adenylyl cyclase and βAR-associated PP2A activity compared to non-failing showing that resensitization is inhibited in human heart failure. Since PP2A activity is inhibited by robust binding of I2PP2A to PP2A, we hypothesized that disrupting I2PP2A interaction with PP2A would unlock PP2A inhibition normalizing βAR resensitization. In silico modeling and biochemical studies mapped the PP2A interaction with I2PP2A to the C-terminal region of PP2A (PP2Act). Agonist isoproterenol (ISO) treatment of cells expressing PP2Act showed significant reduction in β2AR phosphorylation along with increased cAMP generation and normalized PP2A activity showing preserved βAR resensitization. While expression of PP2A mutant with deletion in the PP2Act (ΔPP2Act) did not preserve βAR resensitization. Challenging mice with cardiomyocyte-specific expression of PP2Act to ISO showed preserved cardiac function providing insights that targeting resensitization could potentially be a novel therapeutic strategy. Key words: Resensitization, I2PP2A, PP2A, Cardiac hypertrophy

Immunoregulatory Engineering of Semiconducting Charge-Reversal Nanoantioxidant for Ameliorating Cancer Radioimmunotheranostics.

Radiotherapy (RT) is a crucial clinical modality for cancer. However, nonselectivity, toxicity to normal tissues, and radio-resistance severely limit RT applications. This study develops a versatile X-ray theranostic nano-antioxidant (XTN) to prevent normal tissues from oxidative damage and induce systematic and robust anticancer immunity. XTN owns NIR-II photoacoustic (PA) imaging properties for precise discrimination of the tumor margin through, thereby improving the accuracy of RT. Additionally, XTN is a nano-antioxidant to enhance the cell viability of normal cells after irradiation. Most importantly, XTN scavenges reactive oxygen species (ROS) in the TME to preserve the stimulatory activity of released high mobility group protein B1 to dendritic cells (DCs) and recover T cells' immune function. Meanwhile, XTN achieves charge-reversal specifically releasing an immunomodulator (demethylcantharidin, DMC) in the acidic TME. Moreover, the specifically released DMC inhibits protein phosphatase-2A activity and reduces regulatory T cell (Treg) differentiation. In the bilateral 4T1 tumor model, XTN-mediated radioimmunotherapy remarkably boosts a systemic antitumor immune response and induces durable immunological memory against tumor growth.

The PP2A inhibitor LB-100 mitigates lupus nephritis by suppressing tertiary lymphoid structure formation

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multi-organ involvement and autoantibody production. Patients with SLE face a substantial risk of developing lupus nephritis (LN), which imposes a substantial burden on both patients and their families. Protein phosphatase 2A (PP2A) is a widely distributed serine/threonine phosphatase that participates in regulating multiple signaling pathways. Inhibition of PP2A has been implicated in the treatment of various diseases. LB-100, a small molecule inhibitor of PP2A, has demonstrated anti-tumor therapeutic effects and high safety profile in preclinical experiments. However, the role of PP2A and its inhibitor has been insufficiently studied in LN. In this study, we assessed the potential effects of LB-100 in both MRL/lpr mice and R848-induced BALB/c mice. Our findings indicated that LB-100 administration led to reduced spleen enlargement, decreased deposition of immune complexes, ameliorated renal damage, and improved kidney function in both spontaneous and R848-induced lupus mouse models. Importantly, we observed the formation of tertiary lymphoid structures (TLSs) in the kidneys of two distinct lupus mouse models. The levels of signature genes of TLS were elevated in the kidneys of lupus mice, whereas LB-100 mitigated chemokine production and inhibited TLS formation. In addition, we confirmed that inhibition or knockdown of PP2A reduced the production of T cell-related chemokines by renal tubular epithelial cells (RTEC). In summary, our study highlighted the renal protective potential of the PP2A inhibitor LB-100 in two distinct lupus mouse models, suggesting its potential as a novel strategy for treating LN and other autoimmune diseases.

KRAS-mediated upregulation of CIP2A promotes suppression of PP2A-B56α to initiate pancreatic cancer development.

Oncogenic mutations in KRAS are present in approximately 95% of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) and are considered the initiating event of pancreatic intraepithelial neoplasia (PanIN) precursor lesions. While it is well established that KRAS mutations drive the activation of oncogenic kinase cascades during pancreatic oncogenesis, the effects of oncogenic KRAS signaling on regulation of phosphatases during this process is not fully appreciated. Protein Phosphatase 2A (PP2A) has been implicated in suppressing KRAS-driven cellular transformation. However, low PP2A activity is observed in PDAC cells compared to non-transformed cells, suggesting that suppression of PP2A activity is an important step in the overall development of PDAC. In the current study, we demonstrate that KRASG12D induces the expression of both an endogenous inhibitor of PP2A activity, Cancerous Inhibitor of PP2A (CIP2A), and the PP2A substrate, c-MYC. Consistent with these findings, KRASG12D sequestered the specific PP2A subunit responsible for c-MYC degradation, B56α, away from the active PP2A holoenzyme in a CIP2A-dependent manner. During PDAC initiation in vivo, knockout of B56α promoted KRASG12D tumorigenesis by accelerating acinar-to-ductal metaplasia (ADM) and the formation of PanIN lesions. The process of ADM was attenuated ex vivo in response to pharmacological re-activation of PP2A utilizing direct small molecule activators of PP2A (SMAPs). Together, our results suggest that suppression of PP2A-B56α through KRAS signaling can promote the MYC-driven initiation of pancreatic tumorigenesis.

RETRACTION: "Okadaic acid promotes epithelial-mesenchymal transition of hepatocellular carcinoma cells by inhibiting protein phosphatase 2A".

J. Kong, D. Li, S. Zhang, H. Zhang, Y. Fu, B. Qian, C. Bei, S. Tan, X. Zhu, "Okadaic acid promotes epithelial-mesenchymal transition of hepatocellular carcinoma cells by inhibiting protein phosphatase 2A," Journal of Cellular Biochemistry 122, no. 9 (2021): 993-1002, https://doi.org/10.1002/jcb.29629 The above article, published online on 6 January 2020 in Wiley Online Library (wileyonlinelibrary.com) has been retracted by agreement between the authors; the journal's Editor in Chief, Christian Behl; and Wiley Periodicals LLC. The retraction has been agreed following an investigation based on allegations raised by third parties. During the investigation, several flaws and inconsistencies were found within the provided data. The authors admitted substantial mistakes during figure compilation for this manuscript. Thus, the conclusions of this article are considered invalid by the editors.

The protein phosphatase-2A subunit PR130 is involved in the formation of cytotoxic protein aggregates in pancreatic ductal adenocarcinoma cells

As a major source of cellular serine and threonine phosphatase activity, protein phosphatase-2A (PP2A) modulates signaling pathways in health and disease. PP2A complexes consist of catalytic, scaffolding, and B-type subunits. Seventeen PP2A B-type subunits direct PP2A complexes to selected substrates. It is ill-defined how PP2A B-type subunits determine the growth and drug responsiveness of tumor cells. Pancreatic ductal adenocarcinoma (PDAC) is a disease with poor prognosis. We analyzed the responses of murine and human mesenchymal and epithelial PDAC cells to the specific PP2A inhibitor phendione. We assessed protein levels by immunoblot and proteomics and cell fate by flow cytometry, confocal microscopy, and genetic manipulation. We show that murine mesenchymal PDAC cells express significantly higher levels of the PP2A B-type subunit PR130 than epithelial PDAC cells. This overexpression of PR130 is associated with a dependency of such metastasis-prone cells on the catalytic activity of PP2A. Phendione induces apoptosis and an accumulation of cytotoxic protein aggregates in murine mesenchymal and human PDAC cells. These processes occur independently of the frequently mutated tumor suppressor p53. Proteomic analyses reveal that phendione upregulates the chaperone HSP70 in mesenchymal PDAC cells. Inhibition of HSP70 promotes phendione-induced apoptosis and phendione promotes a proteasomal degradation of PR130. Genetic elimination of PR130 sensitizes murine and human PDAC cells to phendione-induced apoptosis and protein aggregate formation. These data suggest that the PP2A-PR130 complex dephosphorylates and thereby prevents the aggregation of proteins in tumor cells.Graphical

Paradoxical Activation of Oncogenic Signaling as a Cancer Treatment Strategy.

Cancer homeostasis depends on a balance between activated oncogenic pathways driving tumorigenesis and engagement of stress response programs that counteract the inherent toxicity of such aberrant signaling. Although inhibition of oncogenic signaling pathways has been explored extensively, there is increasing evidence that overactivation of the same pathways can also disrupt cancer homeostasis and cause lethality. We show here that inhibition of protein phosphatase 2A (PP2A) hyperactivates multiple oncogenic pathways and engages stress responses in colon cancer cells. Genetic and compound screens identify combined inhibition of PP2A and WEE1 as synergistic in multiple cancer models by collapsing DNA replication and triggering premature mitosis followed by cell death. This combination also suppressed the growth of patient-derived tumors in vivo. Remarkably, acquired resistance to this drug combination suppressed the ability of colon cancer cells to form tumors in vivo. Our data suggest that paradoxical activation of oncogenic signaling can result in tumor-suppressive resistance. Significance: A therapy consisting of deliberate hyperactivation of oncogenic signaling combined with perturbation of the stress responses that result from this is very effective in animal models of colon cancer. Resistance to this therapy is associated with loss of oncogenic signaling and reduced oncogenic capacity, indicative of tumor-suppressive drug resistance.

Abstract 7062: CIP2A enhances mitochondrial respiratory electron transport chain complexes and induces lung cancer in vivo

Abstract Identification of oncogenes that can induce cancer in vivo is critical to elucidation of tumorigenesis and development of targeted therapies. Here, we reported for the first time that forced ectopic expression of CIP2A, the cancerous inhibitor of protein phosphatase 2A (PP2A), in lung tissues induced lung adenocarcinoma in mice. CIP2A upregulated the transcription and translation of mitochondrial DNA (mtDNA), and increases the enzymatic activity of mitochondrial electron transport chain (ETC) complex IV and V, thus enhancing the function of mitochondria and promoting the level of oxidative phosphorylation in tumors. CIP2A inhibited the binding of PP2A-B56α to FOXO3a, leading to the enhancement of the phosphorylation level of FOXO3a. The phosphorylated FOXO3a then translocated into the mitochondrion, and acted as a transcription factor to regulate CIP2A-mediated metabolic reprogramming in cancer cells. We found that oxidative phosphorylation inhibitors effectively inhibited CIP2A-induced cell proliferation. In non-small cell lung cancer, CIP2A level in tumor tissues was positively associated with the level of mtDNA. Our results indicated that CIP2A is able to induce lung adenocarcinoma in mice and has a critical role in mitochondrial oxidative phosphorylation, and represents a promising therapeutic target for non-small cell lung cancer. Competing interests: The authors declare no competing interests. Citation Format: Fu-Ying Yang, Di Wang, Li-Jun liang, Gui-Zhen Wang, Guang-Biao Zhou. CIP2A enhances mitochondrial respiratory electron transport chain complexes and induces lung cancer in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7062.

Abstract 371: Hypoxia-induced centrosome loss as a driver of chromosomal instability in prostate cancer

Abstract PURPOSE: This study aims to identify a causal mechanism of centrosome loss previously observed in primary prostate cancer. Prostate cancer progression is accompanied by bursts of chromosomal instability (CIN), including chromosomal translocations, oncogene amplifications, and chromothripsis. While high CIN correlates with most metrics of aggressive disease, we lack a mechanistic understanding of how CIN originates in prostate cancer. Recently, we discovered that cells in primary prostate tumors lack centrosomes, cytoplasmic organelles that ensure the fidelity of chromosome segregation by organizing the shape of the mitotic spindle. Experimental elimination of centrosomes in immortalized, non-tumorigenic prostate cell lines was sufficient to generate extensive CIN, resulting in oncogenic transformation of these lines when sub-cutaneously injected into NSG mice. Because we identified centrosome loss as a potential driver of CIN, we sought to understand mechanisms that can trigger centrosome loss in the prostate. Although centrosome loss naturally occurs during the development of some tissues, for example centrosome elimination during oogenesis, the mechanisms that trigger loss are not known. Therefore, we first investigated microenvironmental changes in early prostate cancer as a potential cause. Hypoxia, pathologically low oxygen concentration, is common in the aging prostate due to loss of vasculature and is associated with poor prostate cancer prognosis and high CIN. Therefore, we hypothesized that hypoxic exposure induces centrosome loss in prostate cells. Indeed, we found that exposure to 1% oxygen concentrations leads to progressive centrosome loss in non-tumorigenic, immortalized prostate epithelial cell lines. Using immunofluorescence of centrosomes in cultured cells, we found that hypoxia-induced centrosome loss is independent of HIF transcription but requires activation of the confluence-dependent Hippo signaling pathway. Mechanistically, we found that centrosome disassembly begins with the removal of the pericentriolar material (PCM), the outer shell of the centrosome that nucleates microtubules. We were able to block centrosome disassembly by over-expressing a constitutively-active mutant of Polo-like Kinase 1, which normally strengthens the PCM during mitosis to promote centrosome maturation. Furthermore, centrosome loss was prevented by treatment with the Protein Phosphatase 2A (PP2A) inhibitor Okadaic Acid. Using RNAseq, we have identified potential PP2A regulatory subunits that are up-regulated in cells undergoing hypoxia-induced centrosome loss. Our model is that hypoxia induces dephosphorylation of the PCM, mechanically weakening the centrosome and leaving it vulnerable to disassembly. Together with previous findings, we conclude that hypoxia-induced centrosome disassembly is a plausible driver of CIN in early prostate cancer. Citation Format: John M. Ryniawec, Gregory C. Rogers, Anne E. Cress. Hypoxia-induced centrosome loss as a driver of chromosomal instability in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 371.

Protein phosphatase 4 mediates palmitic acid-induced endothelial dysfunction by decreasing eNOS phosphorylation at serine 633 in HUVECs

Plasma saturated free fatty acid (FFA)-induced endothelial dysfunction (ED) contributes to the pathogenesis of atherosclerosis and cardiovascular diseases. However, the mechanism underlying saturated FFA-induced ED remains unclear. This study demonstrated that palmitic acid (PA) induced ED by activating the NADPH oxidase (NOX)/ROS signaling pathway to activate protein phosphatase 4 (PP4) and protein phosphatase 2A (PP2A), thereby reducing endothelial nitric oxide synthase (eNOS) phosphorylation at Ser633 and Ser1177, respectively. Okadaic acid (OA) and fostriecin (FST), which are inhibitors of PP2A, inhibited the PA-induced decreases in eNOS phosphorylation at Ser633 and Ser1177. The antioxidants N-acetylcysteine (NAC) and apocynin (APO) or knockdown of gp91phox or p67phox (NOX subunits) restored PA-mediated downregulation of PP4R2 protein expression and eNOS Ser633 phosphorylation. Knockdown of the PP4 catalytic subunit (PP4c) specifically increased eNOS Ser633 phosphorylation, while silencing the PP2A catalytic subunit (PP2Ac) restored only eNOS Ser1177 phosphorylation. Furthermore, PA dramatically decreased the protein expression of the PP4 regulatory subunit R2 (PP4R2) but not the other regulatory subunits. PP4R2 overexpression increased eNOS Ser633 phosphorylation, nitric oxide (NO) production, cell migration and tube formation but did not change eNOS Ser1177 phosphorylation levels. Coimmunoprecipitation (Co-IP) suggested that PP4R2 and PP4c interacted with the PP4R3α and eNOS proteins. In summary, PA decreases PP4R2 protein expression through the Nox/ROS pathway to activate PP4, which contributes to ED by dephosphorylating eNOS at Ser633. The results of this study suggest that PP4 is a novel therapeutic target for ED and ED-associated vascular diseases.