Inhibitor Of Protein Phosphatase 2A Research Articles

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. While 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.

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

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.

CIP2A induces PKM2 tetramer formation and oxidative phosphorylation in non-small cell lung cancer

Tumor cells are usually considered defective in mitochondrial respiration, but human non-small cell lung cancer (NSCLC) tumor tissues are shown to have enhanced glucose oxidation relative to adjacent benign lung. Here, we reported that oncoprotein cancerous inhibitor of protein phosphatase 2A (CIP2A) inhibited glycolysis and promoted oxidative metabolism in NSCLC cells. CIP2A bound to pyruvate kinase M2 (PKM2) and induced the formation of PKM2 tetramer, with serine 287 as a novel phosphorylation site essential for PKM2 dimer-tetramer switching. CIP2A redirected PKM2 to mitochondrion, leading to upregulation of Bcl2 via phosphorylating Bcl2 at threonine 69. Clinically, CIP2A level in tumor tissues was positively correlated with the level of phosphorylated PKM2 S287. CIP2A-targeting compounds synergized with glycolysis inhibitor in suppressing cell proliferation in vitro and in vivo. These results indicated that CIP2A facilitates oxidative phosphorylation by promoting tetrameric PKM2 formation, and targeting CIP2A and glycolysis exhibits therapeutic potentials in NSCLC.

PP2A inhibitor SET promotes mTORC1 and Bmi1 signaling through Akt activation and maintains the colony-formation ability of cancer cells

Protein phosphatase 2A (PP2A) is an essential tumor suppressor, with its activity often hindered in cancer cells by endogenous PP2A inhibitory proteins like SE translocation (SET). SET/PP2A axis plays a pivotal role in the colony-formation ability of cancer cells and the stabilization of c-Myc and E2F1 proteins implicated in this process. However, in osteosarcoma cell line HOS, SET knock-down (KD) suppresses the colony-formation ability without affecting c-Myc and E2F1. This study aimed to unravel the molecular mechanism through which SET enhances the colony-formation ability of HOS cells and determine if it is generalized to other cancer cells. Transcriptome analysis unveiled that SET KD suppressed mTORC1 signaling. SET KD inhibited Akt phosphorylation, an upstream kinase for mTORC1. PP2A inhibitor blocked SET KD-mediated decrease in phosphorylation of Akt and a mTORC1 substrate p70S6K. A constitutively active Akt restored decreased colony-formation ability by SET KD, indicating the SET/PP2A/Akt/mTORC1 axis. Additionally, enrichment analysis highlighted that Bmi-1, a polycomb group protein, is affected by SET KD. SET KD decreased Bmi-1 protein by Akt inhibition but not by mTORC1 inhibition, and exogenous Bmi-1 expression rescued the reduced colony formation by SET KD. Four out of eight cancer cell lines exhibited decreased Bmi-1 by SET KD. Further analysis of these cell lines revealed that Myc activity plays a role in SET KD-mediated Bmi-1 degradation. These findings provide new insights into the molecular mechanism of SET-regulated colony-formation ability, which involved Akt-mediated activation of mTORC1/p70S6K and Bmi-1 signaling.

SET protein as an epigenetics target.

The SET gene has four transcripts reported in NCBI, coding two isoforms of SET proteins. The most known function of SET protein is inhibiting protein phosphatase 2A, a tumor suppressor, which has been associated with different biological processes. In this review, our focus was on exploring the other SET functions related to epigenetic mechanisms, which impact cellular migration, cell cycleand apoptosis.

Novel Small‐Molecular Ligands for Prolyl Oligopeptidase to Combat Tauopathies

AbstractBackgroundTauopathies that include e.g. frontotemporal dementia and Alzheimer’s disease, are neurodegenerative diseases characterized by accumulation of hyperphosphorylated Tau in the brain. Tau is a tempting drug target for tauopathies but no Tau‐targeting therapies are in clinical use. We have recently shown that small‐molecular inhibitors of prolyl oligopeptidase (PREP) can activate protein phosphatase 2A (PP2A) by regulating PREP‐PP2A interaction. PP2A is the main phosphatase regulating Tau, and therefore, we decided to test the efficacy of a reference PREP inhibitor, KYP‐2047, and novel PREP ligands on cellular and animal models of tauopathies.MethodThe compounds, KYP‐2047 and HUP‐46, 55 and 59 (1, 5 and 10 μM), were first screened in the GFP‐2N4R‐Tau transfected HEK‐293 cells where the Tau aggregation was induced by small‐dose okadaic acid (OA; 10 nM), a PP2A inhibitor. The results were verified by using 2N4R‐Tau transfected HEK‐293 cells with 10 nM OA. After the treatment the cells were fractioned to RIPA soluble and SDS‐fractions, and total Tau and S262 phosphorylated Tau (pS262 Tau) levels were studied by Western blot (WB). Finally, KYP‐2047 and HUP‐46 were tested in PS19 Tau transgenic mouse. PS19 mice were treated with the ligands for 4 weeks (10 mg/kg/day, Alzet minipump i.p.) starting from 5.5 months old when the behavioral deficits were observed. After the treatment, the mice behavior was tested by Barnes Maze and locomotor activity assay, and total Tau, pS262 Tau and PP2A levels were studied.ResultIn the GFP‐Tau aggregation assay, all compounds decreased the GFP signal as an indicative of Tau aggregation with 10 mM concentration but HUP‐46 was the most effective (Fig. 1). WB that showed that PREP ligands had the most significant effect on total Tau and pS262 Tau in the SDS‐fraction. In the PS19 mice, KYP‐2047 restored normal behavior in Barnes maze and locomotor activity assay. HUP‐46 had similar effect but did not reach significance (Fig. 2). However, both compounds reduced significantly total Tau and pS262 Tau levels in mouse hippocampus and cortex, and activated PP2A (Fig. 3).ConclusionSmall‐molecular PREP ligands, such as KYP‐2047 and HUP‐46 have disease‐modifying effects on cellular and animal models of tauopathies.

An Assessment of Potential Threats to Human Health from Algae Blooms in the Indian River Lagoon (USA) 2018-2021: Unique Patterns of Cytotoxicity Associated with Toxins.

The Indian River Lagoon (IRL), a 156-mile-long estuary located on the eastern coast of Florida, experiences phytoplankton bloom events due to increased seasonal temperatures coupled with anthropogenic impacts. This study aimed to gather data on the toxicity to human cells and to identify secondary metabolites found in water samples collected in the IRL. Water samples from 20 sites of the IRL were collected during the wet and dry seasons over a three-year period. A panel of cell lines was used to test cytotoxicity. Hemagglutination, hemolysis, and inhibition of protein phosphatase 2A (PP2A) were also measured. Cytotoxic blooms were seen both in the south (Microcystis) and the north (Pyrodinium) of the IRL. Each toxin induced a consistent pattern of cytotoxicity in the panel of human cell lines assayed. During blooms, cytotoxicity due to a single type of toxin is obvious from this pattern. In the absence of blooms, the cytotoxicity seen reflected either a mixture of toxins or it was caused by an unidentified toxin. These observations suggest that other toxins with the potential to be harmful to human health may be present in the IRL. Moreover, the presence of toxins in the IRL is not always associated with blooms of known toxin-producing organisms.

A small-molecule inhibitor of TopBP1 exerts anti-MYC activity and synergy with PARP inhibitors.

We have previously identified TopBP1 (topoisomerase IIβ-binding protein 1) as a promising target for cancer therapy, given its role in the convergence of Rb, PI(3)K/Akt, and p53 pathways. Based on this, we conducted a large-scale molecular docking screening to identify a small-molecule inhibitor that specifically targets the BRCT7/8 domains of TopBP1, which we have named 5D4. Our studies show that 5D4 inhibits TopBP1 interactions with E2F1, mutant p53, and Cancerous Inhibitor of Protein Phosphatase 2A. This leads to the activation of E2F1-mediated apoptosis and the inhibition of mutant p53 gain of function. In addition, 5D4 disrupts the interaction of TopBP1 with MIZ1, which in turn allows MIZ1 to bind to its target gene promoters and repress MYC activity. Moreover, 5D4 inhibits the association of the TopBP1-PLK1 complex and prevents the formation of Rad51 foci. When combined with inhibitors of PARP1/2 or PARP14, 5D4 synergizes to effectively block cancer cell proliferation. Our animal studies have demonstrated the antitumor activity of 5D4 in breast and ovarian cancer xenograft models. Moreover, the effectiveness of 5D4 is further enhanced when combined with a PARP1/2 inhibitor talazoparib. Taken together, our findings strongly support the potential use of TopBP1-BRCT7/8 inhibitors as a targeted cancer therapy.

Emerging roles of the CIP2A-TopBP1 complex in genome integrity.

CIP2A is an inhibitor of the tumour suppressor protein phosphatase 2A. Recently, CIP2A was identified as a synthetic lethal interactor of BRCA1 and BRCA2 and a driver of basal-like breast cancers. In addition, a joint role of TopBP1 (topoisomerase IIβ-binding protein 1) and CIP2A for maintaining genome integrity during mitosis was discovered. TopBP1 has multiple functions as it is a scaffold for proteins involved in DNA replication, transcriptional regulation, cell cycle regulation and DNA repair. Here, we briefly review details of the CIP2A-TopBP1 interaction, its role in maintaining genome integrity, its involvement in cancer and its potential as a therapeutic target.

OR01-02 Epigenomic Dysregulation Of PPP1R17 Drives The Cell Cycle In Adenomas Causing Cushing’s Disease

Abstract Disclosure: D. Asuzu: None. D. Mullaney: None. S. Stoica: None. D. Mandal: None. D. Maric: None. A. Elkahloun: None. B. Sisay: None. C. Tatsi: None. L.K. Nieman: None. P. Chittiboina: None. Introduction: Pituitary adenomas causing Cushing’s disease are largely (60%) mutationally bland. Previously, with syngeneic, pairwise transcriptomic analysis, we found a robust upregulation of PPP1R17 in canonically mutated (USP8, USP48 and BRAF) and ‘wildtype’ CD adenomas. PPP1R17 is an endogenous inhibitor of tumor suppressor phosphatase PP2A. Here, we investigated the epigenomic dysregulation of PPP1R17, its interaction with PP2A, and downstream effects on cell cycle regulation. Methods: We performed single nucleus assays for transposase accessible chromatin (snATACseq, 10X Genomics) in adenomas from 4 CD patients, and two syngeneic adenoma-normal pairs along with parallel single nucleus RNAseq (snRNAseq). Transcription factors bound to open chromatin were identified by reverse chromatin immunoprecipitation (rChIP) using CRISPR dCas9-3XFLAG-Biotin (Millipore Sigma) and promoter-specific synthetic guide RNAs (Invitrogen TrueGuide). Bound transcription factors were quantified using mass spectrometry (SimulTOF 300). Fixed human CD adenomas (n=5) were sectioned and stained using multiplexed immunohistochemistry (mIHC). Association between R17 and protein phosphatase 2A (PP2A) was assessed using proximity ligation assays (PLA, Sigma). We overexpressed R17 or GFP controls in murine corticotroph (mCort) cell lines and assessed for cell-cycle changes (Click-IT EdU) and cell proliferation (OneGlo). Results: We mapped the chromatin accessibility landscape of CD adenomas compared to normal corticotrophs. We identified coordinated chromatin accessibility (snATACseq) and transcriptional upregulation (snRNAseq) at the R17 locus exclusively within adenomatous corticotrophs. Using rChIP, we identified enrichment at the R17 promoter of the RNA polymerase II transcriptional coactivator p15 (SUB1) and the histone variant H1.4, indicating active chromatin remodeling. PPP1R17 protein and its target PP2A were overexpressed in the adenoma compartment but not in the adjacent normal gland. PLA confirmed robust interaction between R17 and PP2Ac within the CD adenoma compartment. Mechanistically, mCortR17 cells showed inhibition of PP2A with phosphorylation its known targets - EGFR and ERK. mCortR17 cells showed accelerated cell cycle progression compared to mCortGFP (S-phase 16.4% versus 24.1%, P < 0.001) with increased proliferation (two-way ANOVA P < 0.001). PP2A agonists Fingolimod, DT061 and ABL127 led to proliferation arrest in mCortR17 cells. Conclusions: We identified increased R17 expression as an underlying mechanism of tumor hyperproliferation in CD adenomas. We found chromatin accessibility underlying PPP1R17 overexpression. PP2A agonists reversed the effects of PPP1R17 overexpression in-vitro. Our findings uncover a novel therapeutic phosphatase target for patients with CD. Presentation: Thursday, June 15, 2023

CIP2A interacts with AKT1 to promote the malignant biological behaviors of oral squamous cell carcinoma by upregulating the GSK‑3β/β‑catenin pathway.

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies worldwide, which is associated with a poor prognosis. The present study aimed to investigate the role of cancerous inhibitor of protein phosphatase 2A (CIP2A) in OSCC and its regulatory effect on AKT1. Firstly, CIP2A and AKT1 expression in OSCC cells was detected by western blotting. After silencing CIP2A, cell viability and cell proliferation were assessed using the Cell Counting Kit-8 assay and 5-ethynyl-2'-deoxyuridine staining. Cell apoptosis was evaluated by TUNEL staining and the expression of apoptosis-related proteins was assessed using western blotting. Wound healing, Transwell and tube formation assays were performed to evaluate CAL-27 cell migration, invasion and human umbilical vein endothelial cell (HUVEC) tube formation. The interaction between CIP2A and AKT1 was identified by co-immunoprecipitation (co-IP). In addition, AKT1 was overexpressed in CIP2A-silenced CAL-27 cells to perform rescue experiments to analyze the malignant biological functions of CAL-27 cells. Finally, the expression of proteins in the glycogen synthase kinase (GSK)-3β/β-catenin pathway was determined by western blot analysis. Markedly elevated CIP2A and AKT1 expression was observed in OSCC cells. CIP2A knockdown inhibited the viability, proliferation, migration and invasion, and promoted the apoptosis of CAL-27 cells. Concurrently, CIP2A loss-of-function attenuated tube formation. Results of Co-IP confirmed there was an interaction between CIP2A and AKT1. Rescue experiments suggested that AKT1 overexpression alleviated the inhibitory effects of CIP2A knockdown on the viability, proliferation, migration and invasion of CAL-27 cells, as well as tube formation in HUVECs . Additionally, CIP2A silencing significantly downregulated phosphorylated-GSK-3β and β-catenin expression, which was reversed by AKT1 overexpression. In conclusion, CIP2A could interact with AKT1 to promote the malignant biological behaviors of OSCC cells by upregulating the GSK-3β/β-catenin pathway. These findings may provide a targeted therapy for OSCC treatment.

Inhibition of PP2A ameliorates intervertebral disc degeneration by reducing annulus fibrosus cells apoptosis via p38/MAPK signal pathway

BackgroundIntervertebral disc degeneration (IVDD) is considered one of the main reasons for low back pain (LBP). To date, the specific pathology of IVDD remains unclear. The annulus fibrosus (AF) is an important part of the intervertebral disc, and AF cell oxidative stress, apoptosis plays a vital role in disc degeneration. Protein phosphatase 2 A (PP2A), a serine/threonine phosphatase, has regulatory functions in various processes, including apoptosis and autophagy. However, thus far, the effect of PP2A on IVDD is not clear. MethodsAF cells derived from caudal intervertebral discs in SD rats were used to analyze the levels of oxidative stress, apoptosis and degeneration as well as PP2A expression. A PP2A agonist (FTY720), inhibitor (microcystin-LR) and siRNA (si-PPP2CA) were employed in IVDD induced by H2O2 to investigate the levels of apoptosis and degeneration. The p38/MAPK signal pathways were evaluated, and a p38 inhibitor (SB203580) and ERK inhibitor (U0126) were added for verification. Finally, FTY720 and microcystin-LR were administered to IVDD rats to assess the effects on levels of apoptosis and degeneration and the relief of IVDD. ResultsThe expression of PP2A was increased in rat AF cells after H2O2 intervention.The levels of apoptosis and degeneration were higher with upregulation of PP2A but were significantly reduced after inhibition of PP2A. The PP2A inhibitor relieved cell apoptosis and degeneration by downregulating the p38/MAPK pathway. In vivo, the knockdown of PP2A resulted in a more complete morphology of discs and less apoptotic and degenerative expression. ConclusionsThis study suggests that the downregulation of PP2 A could reduce AF cell apoptosis and degeneration via the p38/MAPK pathway. It also revealed that the inhibition of PP2 A is expected to be a therapeutic target for IVDD.

Protein Phosphatase 2A as a Therapeutic Target in Pulmonary Diseases.

New disease targets and medicinal chemistry approaches are urgently needed to develop novel therapeutic strategies for treating pulmonary diseases. Emerging evidence suggests that reduced activity of protein phosphatase 2A (PP2A), a complex heterotrimeric enzyme that regulates dephosphorylation of serine and threonine residues from many proteins, is observed in multiple pulmonary diseases, including lung cancer, smoke-induced chronic obstructive pulmonary disease, alpha-1 antitrypsin deficiency, asthma, and idiopathic pulmonary fibrosis. Loss of PP2A responses is linked to many mechanisms associated with disease progressions, such as senescence, proliferation, inflammation, corticosteroid resistance, enhanced protease responses, and mRNA stability. Therefore, chemical restoration of PP2A may represent a novel treatment for these diseases. This review outlines the potential impact of reduced PP2A activity in pulmonary diseases, endogenous and exogenous inhibitors of PP2A, details the possible PP2A-dependent mechanisms observed in these conditions, and outlines potential therapeutic strategies for treatment. Substantial medicinal chemistry efforts are underway to develop therapeutics targeting PP2A activity. The development of specific activators of PP2A that selectively target PP2A holoenzymes could improve our understanding of the function of PP2A in pulmonary diseases. This may lead to the development of therapeutics for restoring normal PP2A responses within the lung.

Dual Synergistic Tumor-Specific Polymeric Nanoparticles for Efficient Chemo-Immunotherapy.

Chemo-immunotherapy has made significant progress in cancer treatment. However, the cancer cell self-defense mechanisms, including cell cycle checkpoint and programmed cell death-ligand 1 (PD-L1) upregulation, have greatly hindered the therapeutic efficacy. Herein, norcantharidin (NCTD)-platinum (Pt) codelivery nanoparticles (NC-NP) with tumor-sensitive release profiles are designed to overcome the self-defense mechanisms via synergistic chemo-immunotherapy. NC-NP remains stable under normal physiological conditions but quickly releases 1,2-diaminocyclohexane-platinum(II) (DACHPt, a parent drug of oxaliplatin) and NCTD in response to the tumor acidity. NCTD inhibits protein phosphatase 2A (PP2A) activity to relieve cell cycle arrest and downregulates the tumor PD-L1 expression to disrupt the programmed cell death-1 (PD-1)/PD-L1 interaction, synergistically enhancing Pt-based chemotherapy and immunogenic cell death-induced immunotherapy. As a result, NC-NP exhibits potent synergistic cytotoxicity and promotes T cell recruitment to generate robust antitumor immune responses. The dual synergism exhibits potent antitumor activity against orthotopic 4T1 tumors, providing a promising chemo-immunotherapy paradigm for cancer treatment.

Celastrol impairs tumor growth by modulating the CIP2A-GSK3β-MCL-1 axis in gastric cancer cells.

High Cancerous Inhibitor of PP2A (CIP2A) expression has been reported in solid and hematologic malignancies and is inversely associated with prognosis in Gastric Cancer, the non-small cell lung cancer, et al. CIP2A can be a drug target for the development of novel anti-gastric cancer agent. Our study was designed to explore the anti-cancer effect of celastrol, a small natural compound, and whether it has an anti-proliferative effect through inducing CIP2A degradation against gastric cancer cells. Employing human gastric cancer cells AGS and BCG-823 cells, the effects of celastrol on cell proliferation, apoptosis and cell cycle was specifically investigated via Annexin V-FITC/PI staining and CCK8 assay. The functional association between celastrol and CIP2A was evaluated by using CIP2A knockdown and overexpression technique. The mechanism of underlying celastrol-triggering anti-gastric cancer effect was detected by real-time PCR and western blot analysis. Celastrol concentration- and time-dependently induced CIP2A degradation and led to gastric cancer cell apoptosis. More in depth studies revealed specific activation of Protein phosphatase 2A (PP2A)-GSK3β-MCL-1 signaling pathway was involved in pro-apoptosis effect of celastrol, due to celastrol-triggering degradation of CIP2A, which mainly suppressed PP2A activity. Our findings highlight that celastrol has therapeutic potential via inducing apoptosis of gastric cancer cells.

67-kDa laminin receptor mediates oolonghomobisflavan B-induced cell growth inhibition in melanoma

BackgroundOolonghomobisflavans are unique polyphenols found in oolong teas. Oolonghomobisflavan B (OHBFB), a dimer of (–)-epigallocatechin-3-O-gallate (EGCG), is an active compound found in green tea. PurposeOHBFB has been reported to exert an inhibitory effect on lipase enzyme activity. However, little is known regarding its intercellular signaling induction effect. Further, there are no reports describing the anti-cancer effects of OHBFB. MethodsThe effect of OFBFB on B16 melanoma cells was evaluated by cell counting, and its mechanisms were determined by western blot analysis with or without protein phosphatase 2A (PP2A) inhibitor treatment. Intracellular cyclic adenosine monophosphate (cAMP) levels were evaluated by time-resolved fluorescence resonance energy transfer analysis. Quartz crystal microbalance (QCM) analysis was performed to assess the binding of OHBFB to 67LR. ResultsCell growth assay and western blot analyses showed that OHBFB inhibited melanoma cell growth, followed by myosin phosphatase target subunit 1 (MYPT1) and myosin regulatory light chain (MRLC) dephosphorylation via protein phosphatase 2A (PP2A)-dependent mechanisms. These effects are mediated by intracellular cAMP- and protein kinase A (PKA) A-dependent mechanisms. QCM analysis identified the 67-kDa laminin receptor (67LR) as an OHBFB receptor with a Kd of 3.7 µM. We also demonstrated for the first time that OHBFB intake suppresses tumor growth in vivo. ConclusionsTaken together, these results indicate that the cAMP/PKA/PP2A/MYPT1/MRLC pathway is a key mediator of melanoma cell growth inhibition following OHBFB binding to 67LR and that OHBFB suppresses tumor growth in vivo.

Targeting PP2A-dependent autophagy enhances sensitivity to ruxolitinib in JAK2V617F myeloproliferative neoplasms

The Janus kinase 2 (JAK2)-driven myeloproliferative neoplasms (MPNs) are chronic malignancies associated with high-risk complications and suboptimal responses to JAK inhibitors such as ruxolitinib. A better understanding of cellular changes induced by ruxolitinib is required to develop new combinatory therapies to improve treatment efficacy. Here, we demonstrate that ruxolitinib induced autophagy in JAK2V617F cell lines and primary MPN patient cells through the activation of protein phosphatase 2A (PP2A). Inhibition of autophagy or PP2A activity along with ruxolitinib treatment reduced proliferation and increased the death of JAK2V617F cells. Accordingly, proliferation and clonogenic potential of JAK2V617F-driven primary MPN patient cells, but not of normal hematopoietic cells, were markedly impaired by ruxolitinib treatment with autophagy or PP2A inhibitor. Finally, preventing ruxolitinib-induced autophagy with a novel potent autophagy inhibitor Lys05 improved leukemia burden reduction and significantly prolonged the mice’s overall survival compared with ruxolitinib alone. This study demonstrates that PP2A-dependent autophagy mediated by JAK2 activity inhibition contributes to resistance to ruxolitinib. Altogether, our data support that targeting autophagy or its identified regulator PP2A could enhance sensitivity to ruxolitinib of JAK2V617F MPN cells and improve MPN patient care.