Inhibitor Of Protein Phosphatase 2A Research Articles

Inhibition of PP2A by LB100 sensitizes bladder cancer cells to chemotherapy by inducing p21 degradation.

Bladder carcinoma (BLCA) is the most common urinary tract malignancy and exhibits a poor response to chemotherapy. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase involved in a wide variety of regulatory cellular processes, including apoptosis and the DNA-damage response (DDR). LB100, a small molecule inhibitor of PP2A, has been shown to act as a chemo-sensitizer in multiple types of cancer. However, the anti-tumor effect and mode of action of LB100 in BLCA have yet to be identified. In vitro and in vivo experiments were performed to assess the anti-tumor effect of LB100 alone or in combination with gemcitabine. Mass spectrometry (MS)-based phosphoproteomics analysis was used to identify the downstream substrates of PP2A and to explore the mechanism underlying LB100-induced DNA damage and apoptosis. In addition, we established a chemo-resistant BLCA cell line (RT-112-R) by prolonged drug exposure and determined the effect of LB100 in enhancing genotoxicity in BLCA cell lines and xenograft mouse models. We found that LB100 is sufficient to induce an anti-tumor response in BLCA cells by inducing DNA damage and apoptosis both in vitro and in vivo. Furthermore, we found that PP2A potentially dephosphorylates p-p21-ser130 to stabilize p21. Inhibition of PP2A by LB100 increased the level of p-p21-ser130, subsequently leading to a reduction in p21 level in a dose-dependent manner. In addition, we found that treatment of LB100 abrogated the G1/S cell cycle checkpoint, resulting in increased phosphorylation of γH2AX in BLCA cells. Moreover, LB100 enhanced genotoxicity in chemo-resistant BLCA cells by inducing DNA damage and apoptosis in vitro and in vivo. Our findings indicate that PP2A may serve as a potential therapeutic target in BLCA through regulating p21 stability.

Reciprocal regulation of CIP2A and AR expression in prostate cancer cells

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein overexpressed in human malignancies, including prostate cancer (PCa). In this study, we aimed to explore the oncogenic function of CIP2A in PCa cells and its underlying mechanism. We showed that 63.3% (38/60 cases) of PCa tissues exhibited a high CIP2A immunostaining, compared to 25% (3/12 cases) of BPH samples (p = 0.023). Furthermore, the protein level of CIP2A was positively correlated with patients’ short survival time and nuclear AR levels in PCa tissues. Compared to PZ-HPV-7, an immortalized prostate cell line, androgen-sensitive LNCaP C-33, androgen-independent LNCaP C-81, or 22Rv1 cells exhibited a high CIP2A level, associated with high protein and phosphorylation levels of AR. While AR expression and activity modulated CIP2A expression, manipulating CIP2A expression in PCa cells regulated their AR protein levels and proliferation. The reduction of CIP2A expression also enhanced the sensitivity of PCa cells toward Enzalutamide treatment. Our data further showed that depletion of polo-kinase 1 (PLK1) expression or activity in C-81 or 22Rv1 cells caused reduced protein levels of c-Myc and AR. Notably, inhibition of PLK1 activity could abolish CIP2A-promoted expressions in c-Myc, AR, and prostate-specific antigen (PSA) in C-33 cells under an androgen-deprived condition, suggesting the role of PLK1 activity in CIP2A-promoted AR expression. In summary, our data showed the existence of a novel regulation between CIP2A and AR protein levels, which is critical for promoting PCa malignancy. Thus, CIP2A could serve as a therapeutic target for PCa.

The Asparaginyl Endopeptidase Legumain: An Emerging Therapeutic Target and Potential Biomarker for Alzheimer's Disease.

Alzheimer’s disease (AD) is incurable dementia closely associated with aging. Most cases of AD are sporadic, and very few are inherited; the pathogenesis of sporadic AD is complex and remains to be elucidated. The asparaginyl endopeptidase (AEP) or legumain is the only recognized cysteine protease that specifically hydrolyzes peptide bonds after asparagine residues in mammals. The expression level of AEPs in healthy brains is far lower than that of peripheral organs. Recently, growing evidence has indicated that aging may upregulate and overactivate brain AEPs. The overactivation of AEPs drives the onset of AD through cleaving tau and amyloid precursor proteins (APP), and SET, an inhibitor of protein phosphatase 2A (PP2A). The AEP-mediated cleavage of these peptides enhances amyloidosis, promotes tau hyperphosphorylation, and ultimately induces neurodegeneration and cognitive impairment. Upregulated AEPs and related deleterious reactions constitute upstream events of amyloid/tau toxicity in the brain, and represent early pathological changes in AD. Thus, upregulated AEPs are an emerging drug target for disease modification and a potential biomarker for predicting preclinical AD. However, the presence of the blood–brain barrier greatly hinders establishing body-fluid-based methods to measure brain AEPs. Research on AEP-activity-based imaging probes and our recent work suggest that the live brain imaging of AEPs could be used to evaluate its predictive efficacy as an AD biomarker. To advance translational research in this area, AEP imaging probes applicable to human brain and AEP inhibitors with good druggability are urgently needed.

Paliperidone alleviates MK-801-induced damage to prefrontal cortical neurons via the PP2A/PTEN pathway

BackgroundThe putative mechanisms underlying the efficacy of the US Food and Drug Administration-approved antipsychotic drug paliperidone for the treatment of schizophrenia deserve additional investigation, which is the aim of the present animal study. MethodsThe behavioral activities of mice were recorded in the open field test and light-dark box test. The effects of paliperidone on MK-801-induced neuronal damage in the prefrontal cortex were tested by flow cytometry, TUNEL staining assays, and ROS staining assays. The neuroprotective effects of paliperidone on neural dendrites and synapses were evaluated using Golgi staining and Sholl analysis. An adenovirus vector containing a Ca2+ indicator was used to monitor the calcium ion concentration in the prefrontal cortex. The expression levels of protein phosphatase 2A (PP2A) and phosphatase and tensin homolog (PTEN) were investigated using Western blotting. ResultsThe data showed that MK-801 caused stereotyped behavior in mice and induced synaptic damage and dendritic spine impairment compared with the control, whereas paliperidone ameliorated these changes. Moreover, paliperidone reversed MK-801-induced decreases in PP2A and PTEN levels in prefrontal cortical neurons. Furthermore, in primary cultured cortical neurons and HT-22 cells, paliperidone inhibited cell apoptosis caused by MK-801. In particular, pretreatment with the PP2A inhibitor LB-100 significantly restrained the protective effects of paliperidone on MK-801-treated neurons and on locomotor activity and stereotypical behavior of mice. LimitationsWhether other proteins are involved in this pathway and how the pathway works have not been revealed. ConclusionOur data show that paliperidone alleviates neuronal damage induced by MK-801 via the PP2A/PTEN pathway.

The Mechanism of TNF-α-Mediated Accumulation of Phosphorylated Tau Protein and Its Modulation by Propofol in Primary Mouse Hippocampal Neurons: Role of Mitophagy, NLRP3, and p62/Keap1/Nrf2 Pathway.

Background Neuroinflammation-induced phosphorylated Tau (p-Tau) deposition in central nervous system contributes to neurodegenerative disorders. Propofol possesses neuroprotective properties. We investigated its impacts on tumor necrosis factor-α (TNF-α)-mediated p-Tau deposition in neurons. Methods Mouse hippocampal neurons were exposed to propofol followed by TNF-α. Cell viability, p-Tau, mitophagy, reactive oxygen species (ROS), NOD-like receptor protein 3 (NLRP3), antioxidant enzymes, and p62/Keap1/Nrf2 pathway were investigated. Results TNF-α promoted p-Tau accumulation in a concentration- and time-dependent manner. TNF-α (20 ng/mL, 4 h) inhibited mitophagy while increased ROS accumulation and NLRP3 activation. It also induced glycogen synthase kinase-3β (GSK3β) while inhibited protein phosphatase 2A (PP2A) phosphorylation. All these effects were attenuated by 25 μM propofol. In addition, TNF-α-induced p-Tau accumulation was attenuated by ROS scavenger, NLRP3 inhibitor, GSK3β inhibitor, or PP2A activator. Besides, compared with control neurons, 100 μM propofol decreased p-Tau accumulation. It also decreased ROS and NLRP3 activation, modulated GSK3β/PP2A phosphorylation, leaving mitophagy unchanged. Further, 100 μM propofol induced p62 expression, reduced Keap1 expression, triggered the nuclear translocation of Nrf2, and upregulated superoxide dismutase (SOD) and heme oxygenase-1 (HO-1) expression, which was abolished by p62 knockdown, Keap1 overexpression, or Nrf2 inhibitor. Consistently, the inhibitory effect of 100 μM propofol on ROS and p-Tau accumulation was mitigated by p62 knockdown, Keap1 overexpression, or Nrf2 inhibitor. Conclusions In hippocampal neurons, TNF-α inhibited mitophagy, caused oxidative stress and NLRP3 activation, leading to GSK3β/PP2A-dependent Tau phosphorylation. Propofol may reduce p-Tau accumulation by reversing mitophagy and oxidative stress-related events. Besides, propofol may reduce p-Tau accumulation by modulating SOD and HO-1 expression through p62/Keap1/Nrf2 pathway.

Abstract P1067: IRS Mediated Recruitment Of PI3Kgamma By Insulin Inhibits Beta-adrenergic Receptor Function

Insulin (INS) treatment results in impaired response to β-agonist isoproterenol (ISO) associated with phosphorylated β2-adrenergic receptor (β2AR) and impaired cAMP response. Although INS does not mediate dissociation of G-protein Gβγ subunits, yet surprisingly recruits phosphoinositide 3-kinase γ (PI3Kγ) to the plasma membrane. Correspondingly, knock-down of PI3Kγ (PI3Kγ KD) in HEK 293 or ablation in primary adult cardiomyocytes and fibroblasts (isolated from PI3Kγ knockout mice) abrogated β2AR phosphorylation reflecting a key role for PI3Kγ in retaining β2AR phosphorylation. Also, adult cardiomyocytes isolated from C57Bl6 mice showed significant loss of in vitro ISO-stimulated cardiomyocyte contraction upon INS pre-treatment which was remarkably preserved in the PI3Kγ knockout cardiomyocytes despite INS. As PI3Kγ is traditionally recruited to the βAR complex by the Gβγ subunits, we hypothesized that INS-mediates Gβγ-independent recruitment of PI3Kγ to the βAR complex underlying its dysfunction. INS stimulation is known to recruit insulin receptor substrate (IRS 1 & 2) to the receptor complex, and therefore, we tested whether PI3Kγ interacts with IRS 1/2 leading to Gβγ-independent recruitment of PI3Kγ to the β2AR complex. Immunoprecipitation PI3Kγ following INS showed that PI3Kγ interacts with IRS2 recruiting PI3Kγ to the β2AR complex bypassing the traditional Gβγ-dependent pathway. Since PI3Kγ KD resulted in loss of INS-mediated β2AR phosphorylation, we tested whether PI3Kγ inhibits protein phosphatase 2A (PP2A) function impairing β2AR de-phosphorylation. INS treatment resulted in significant loss of β2AR-associated PP2A activity which was rescued in PI3Kγ KD cells showing that PI3Kγ impairs PP2A function. Furthermore, our studies show that PI3Kγ phosphorylates the endogenous inhibitor of PP2A, I2PP2A which then robustly binds and inhibits PP2A activity impairing β2AR dephosphorylation. Consistently, CRISPR ablation of I2PP2A relieves this inhibition on PP2A leading to increased PP2A activity. This reduces accumulation of phosphorylated βARs despite the presence of PI3Kγ showing an underappreciated regulation of PP2A by insulin through non-canonical recruitment of PI3Kγ that impairs β2AR function.

Coupling to short linear motifs creates versatile PME-1 activities in PP2A holoenzyme demethylation and inhibition.

Protein phosphatase 2A (PP2A) holoenzymes target broad substrates by recognizing short motifs via regulatory subunits. PP2A methylesterase 1 (PME-1) is a cancer-promoting enzyme and undergoes methylesterase activation upon binding to the PP2A core enzyme. Here, we showed that PME-1 readily demethylates different families of PP2A holoenzymes and blocks substrate recognition in vitro. The high-resolution cryoelectron microscopy structure of a PP2A-B56 holoenzyme-PME-1 complex reveals that PME-1 disordered regions, including a substrate-mimicking motif, tether to the B56 regulatory subunit at remote sites. They occupy the holoenzyme substrate-binding groove and allow large structural shifts in both holoenzyme and PME-1 to enable multipartite contacts at structured cores to activate the methylesterase. B56 interface mutations selectively block PME-1 activity toward PP2A-B56 holoenzymes and affect the methylation of a fraction of total cellular PP2A. The B56 interface mutations allow us to uncover B56-specific PME-1 functions in p53 signaling. Our studies reveal multiple mechanisms of PME-1 in suppressing holoenzyme functions and versatile PME-1 activities derived from coupling substrate-mimicking motifs to dynamic structured cores.

PP2A regulates metastasis and vasculogenic mimicry formation via PI3K/AKT/ZEB1 axis in non-small cell lung cancers

Studies have shown that inhibition of PI3K/AKT signaling is a key strategy for the treatment of tyrosine kinase inhibitor resistance in non-small cell lung cancer (NSCLC). Vasculogenic mimicry (VM) not only accelerates tumor progression but also increases drug-induced resistance. As a tumor suppressor, protein phosphatase 2A (PP2A) is a ubiquitous conserved serine/threonine phosphatase. While its effects and mechanisms on VM formation and invasion in NSCLC remain unclear. The present study aimed to investigate the role of PP2A in VM formation and elucidate the underlying mechanisms. Results showed that PP2A could significantly inhibit VM formation and VM-dependent behavior, including invasion and migration both in vitro and in vivo. Activation of PP2A with FTY720 or Ad-PP2A reduced phosphorylated AKT and inhibited ZEB1 transcription, thereby further downregulating the expression of MMP-2, VE-cadherin, and VEGFR-2, whereas inhibition of PP2A with okadaic acid (OA) or Ad-dn-PP2A exerted the opposite effect. Furthermore, PP2A inhibited tumor growth and VM formation in the xenograft tumor model. PI3K inhibitor BENC-511 could potentiate activation of PP2A, leading to inhibition of p-AKT/ZEB1 and VM formation in vitro and in vivo. This study indicated that PP2A could regulate VM formation in NSCLC through the PI3K/AKT/ZEB1 axis. PP2A reactivation or combination with PI3K inhibitor might be a more effective treatment against advanced NSCLC by inhibiting VM formation.

PB1893: THE STUDY OF SET GENE AND PROTEIN PHOSPHATASE 2A IN CHRONIC MYELOID LEUKEMIA AND THEIR ROLE IN DISEASE PROGRESSION

Background: Chronic myeloid leukemia (CML) is characterized by three stages, chronic, accelerated and blastic phases. Tyrosine kinase inhibitors (TKIs) delay the progression of CML into blastic phase. However, resistance to TKIs has emerged due to different factors. Protein phosphatase 2A (PP2A) is a tumor suppressor protein that affect many cellular pathways. Inhibition of PP2A by PP2A inhibitors such as SET is one mechanism responsible for CML progression. Moreover, phosphorylation of C-terminal tyrosine residue (Tyr307) on the PP2A catalytic subunit was associated with decreased protein activity and consequently, loss of its tumor suppression function. Aims: This work studied the role of SET gene expression as well as phosphorylated PP2A expression in CML progression Methods: The study included 50 CML patients under TKI therapy for at least 18 months, and 50 healthy controls. All study participants were tested for SET gene expression level in peripheral blood by RQ-PCR and PP2A (Tyr307) in serum by ELISA. Moreover, patients were analyzed for complete blood count, BCR-ABL1 in peripheral blood by RQ-PCR and bone marrow examination. Results: High SET gene expression in patients was found to be significantly associated with advanced disease phase and worse clinical and laboratory findings while low expression was significantly associated with better molecular response assessed by BCR-ABL1 level. High levels of PP2A (Tyr307) was significantly associated with blastic phase. Moreover, significant positive correlations was found between BCR-ABL1 level and both SET gene expression and PP2A Tyr307 levels. Image:Summary/Conclusion: SET gene and PP2A are important factors in CML progression and can be used as possible targets for treatment in TKI-resistant cases.

SET improved oocyte maturation by serine/threonine protein phosphatase 2A and inhibited oocyte apoptosis in mouse oocytes

SET is a multifunctional protein involved in a variety of molecular processes such as cell apoptosis and cell-cycle regulation. In ovaries SET is predominantly expressed in theca cells and oocytes. In polycystic ovary syndrome (PCOS) patients the expression of SET was increased than healthy people. The current study was designed to determine whether SET plays a role in oocyte maturation and apoptosis, which may provide clues for the underlying pathological mechanism of follicular development in PCOS patients. Oocytes at germinal vesicle (GV) stage were collected from 6-week-old female ICR mice ovaries. The expression of SET was manipulated by AdCMV-SET and AdH1-SiRNA/SET adenoviruses. SET overexpression improved oocyte maturation whereas SET knockdown inhibited oocyte maturation. Moreover, SET negatively regulated serine/threonine protein phosphatase 2A (PP2A) activity in oocytes. Treatment with PP2A inhibitor okadaic acid (OA) promoted oocyte maturation. Furthermore, PP2A knockdown confirmed the role of PP2A in oocyte maturation, and OA was able to block the AdH1-SiRNA/SET-mediated inhibition on oocyte maturation. The central role of PP2A in SET-mediated regulation of oocyte maturation was confirmed by the finding that SET increased the expression of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) and PP2A inhibited their expressions. Besides, SET inhibited oocyte apoptosis through decreasing the expression of caspase 3 and caspases 8, while PP2A had no effect on oocyte apoptosis. SET promoted oocyte maturation by inhibiting PP2A activity and inhibited oocyte apoptosis in mouse in-vitro cultured oocytes, which may provide a pathologic pathway leading to impaired oocyte developmental competence in PCOS.

Overexpression of cancerous inhibitor ofPP2A (CIP2A) in acute myeloid leukemia

ABSTRACT Background Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy. Protein phosphatase 2A Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase and tumor suppressor that negatively regulates numerous signal transduction pathways. Cancerous inhibitor of PP2A (CIP2A) is an endogenous inhibitor of PP2A. CIP2A overexpression was shown to be a recurrent event in cytogenetic normal AML patients. The aim of the study is to evaluate the prognostic significance of CIP2A overexpression in patients with AML. Research design and methods The study included 174 newly diagnosed cytogenetic normal AML patients. Detection of CIP2A expression was performed using quantitative real-time PCR. Results CIP2A was overexpressed in 125/174 (71.8%) of patients. Correlation of CIP2A overexpression with other prognostic factors showed significant association with CD34 expression (p = 0.04). CIP2A overexpression was significantly associated with a lower rate of (complete remission) CR (p = 0.019) and shorter disease free survival (DFS) and overall survival (OS) (p < 0.001 and <0.001, respectively). In multivariate analysis, CIP2A overexpression was an independent adverse prognostic factor that negatively affected DFS and OS (p < 0.001, HR:2.8,95%CI:1.7–4.7 and p = 0.002, HR:1.8; 95%CI:1.2–2.65, respectively). Conclusion CIP2A overexpression is a useful prognostic marker in AML.

Oct4 facilitates chondrogenic differentiation of mesenchymal stem cells by mediating CIP2A expression.

Bone development and cartilage formation require strict modulation of gene expression for mesenchymal stem cells (MSCs) to progress through their differentiation stages. Octamer-binding transcription factor 4 (Oct4) expression is generally restricted to developing embryonic pluripotent cells, but its role in chondrogenic differentiation (CD) of MSCs remains unclear. We therefore investigated the role of Oct4 in CD using a microarray, quantitative real-time polymerase chain reaction, and western blotting. The expression of Oct4 was elevated when the CD of cultured MSCs was induced. Silencing Oct4 damaged MSC growth and proliferation and decreased CD, indicated by decreased cartilage matrix formation and the expression of Col2a1, Col10a1, Acan, and Sox9. We found a positive correlation between the expression of CIP2A, a natural inhibitor of protein phosphatase 2A (PP2A) and that of Oct4. Cellular inhibitor of PP2A (CIP2A) expression gradually increased after CD. Overexpression of CIP2A in MSCs with Oct4 depletion promoted cartilage matrix deposition as well as Col2a1, Col10a1, Acan, and Sox9 expression. The chondrogenic induction triggered c-Myc, Akt, ERK, and MEK phosphorylation and upregulated c-Myc and mTOR expression, which was downregulated upon Oct4 knockdown and restored by CIP2A overexpression. These findings indicated that Oct4 functions as an essential chondrogenesis regulator, partly via the CIP2A/PP2A pathway.

High fructose induces dysfunctional vasodilatation via PP2A-mediated eNOS Ser1177 dephosphorylation

BackgroundProcessed foods are popular and contain large amounts of industrial fructose, which changes people’s diet and exacerbates the negative health effects of high fructose. Several studies have shown that excessive intake of fructose has a major impact on vascular disease. However, the mechanism of the effect of high fructose on blood vessels is currently unclear.MethodsThe effect of fructose on the vasodilatation of isolated thoracic aortic rings was observed by using wire myography in wild-type (WT) mice. Cell viability and nitric oxide (NO) production were assessed by the corresponding kits in mouse vascular endothelial cells. The effect of fructose on endothelial nitric oxide synthase (eNOS) and protein phosphatase 2A (PP2A) and their changes in phosphorylation were detected by using Western blots. Moreover, a PP2A inhibitor (okadaic acid, OA) was used to evaluate the relationship between fructose and PP2A. Furthermore, PP2ACα endothelial-specific knockout (PP2A cKO) mice were used to detect the vasodilatation of in vitro fructose-incubated thoracic aortic rings by using wire myography.ResultsHigh fructose induced endothelium-dependent dysfunctional vasodilatation. High fructose reduced acetylcholine (Ach)-induced vasodilation but did not affect sodium nitroprusside (SNP)-induced vasodilation. Accordingly, NO production and the phosphorylation level of eNOS at serine (Ser) 1177 (P-eNOS) in vascular endothelial cells were remarkably reduced without changes in cell viability. The expression of protein phosphatase 2A catalytic subunit (PP2AC) was increased and the expression of phosphorylated PP2AC (P-PP2A, tyrosine [Tyr] 307) was significantly decreased. Nevertheless, these effects were reversed by OA. Moreover, knockout of the PP2A gene could recover the response of vessels to Ach under high fructose stimulation.ConclusionsOur observations demonstrate an underlying mechanism of fructose-induced dysfunctional vasodilatation. Fructose could activate PP2A, which leads to decrease in the phosphorylation of eNOS at Ser1177 and the reduction of NO release, thus leading to the occurrence of endothelium-dependent dysfunctional vasodilatation.

Valproic acid decreases vascular smooth muscle cell proliferation via protein phosphatase 2A-mediated p70 S6 kinase inhibition

Valproic acid (VPA) has been used to treat epilepsy and bipolar disorder. Although the abnormal proliferation of vascular smooth muscle cells (VSMCs) is a well-established contributor to the development of various vascular diseases including atherosclerosis, the effect of VPA on VSMC proliferation and its mechanism of action have not been fully revealed. Herein, we investigated the molecular mechanism by which VPA inhibits rat VSMC proliferation. VPA dose-dependently decreased VSMC proliferation, which was accompanied by the dose-dependent decrease in phosphorylation of p70 S6 kinase (p70S6K) at Thr389 (p-p70S6K-Thr389), and overexpression of the p70S6K-T389E mutant gene significantly reversed VPA-inhibited VSMC proliferation. Co-treatment with okadaic acid, a specific protein phosphatase 2A (PP2A) inhibitor, significantly restored p-p70S6K-Thr389. Furthermore, knockdown of PP2Ac gene expression by siRNA significantly reversed VPA-inhibited p-p70S6K-Thr389 and VSMC proliferation. Confocal microscopic analyses and co-immunoprecipitation results clearly showed that the physical binding of p70S6K and PP2Ac was promoted by VPA. Valpromide, a VPA's structural derivative with no histone deacetylase (HDAC) inhibition activity, as well as VPA and sodium butyrate, an HDAC inhibitor similar to VPA, decreased VSMC proliferation and p-p70S6K-Thr389, indicating that HDAC is not involved in VPA-inhibited VSMC proliferation. Finally, the inhibitory effects of VPA on p-p70S6K-Thr389 and VSMC proliferation were reiterated in a platelet-derived growth factor (PDGF)-induced in vitro atherosclerosis model. In conclusion, our results demonstrate that VPA decreased cell proliferation via PP2A-mediated inhibition of p-p70S6K-Thr389 in basal and PDGF-stimulated VSMCs. The results suggest that VPA could be used in the treatment and prevention of atherosclerosis and in-stent restenosis.

Serine/Threonine Protein Phosphatase 2A Regulates the Transport of Axonal Mitochondria.

Microtubule-based transport provides mitochondria to distant regions of neurons and is essential for neuronal health. To identify compounds that increase mitochondrial motility, we screened 1,641 small-molecules in a high-throughput screening platform. Indirubin and cantharidin increased mitochondrial motility in rat cortical neurons. Cantharidin is known to inhibit protein phosphatase 2A (PP2A). We therefore tested two other inhibitors of PP2A: LB-100 and okadaic acid. LB-100 increased mitochondrial motility, but okadaic acid did not. To resolve this discrepancy, we knocked down expression of the catalytic subunit of PP2A (PP2CA). This long-term inhibition of PP2A more than doubled retrograde transport of axonal mitochondria, confirming the importance of PP2A as a regulator of mitochondrial motility and as the likely mediator of cantharidin’s effect.

SP1-induced lncRNA DUBR promotes stemness and oxaliplatin resistance of hepatocellular carcinoma via E2F1-CIP2A feedback.

Oxaliplatin-based chemotherapy is widely used to treat advanced hepatocellular carcinoma (HCC), but many patients develop drug resistance that leads to tumor recurrence. Cancer stem cells (CSCs) are known to contribute to chemoresistance, the underlying mechanism, however, remains largely unknown. In this study, we discovered a specificity protein 1 (SP1)-induced long noncoding RNA--DPPA2 upstream binding RNA (DUBR) and its high expression in HCC tissues and liver CSCs. DUBR was associated with HCC progression and poor chemotherapy response. Moreover, DUBR facilitated the stemness and oxaliplatin resistance of HCC in vitro and in vivo. Mechanistically, DUBR upregulated cancerous inhibitor of protein phosphatase 2A (CIP2A) expression through E2F1-mediated transcription regulation. DUBR also exerted function by binding microRNA (miR)-520d-5p as a competing endogenous RNA to upregulate CIP2A at mRNA level. CIP2A, in turn, stabilized E2F1 protein and activated the Notch1 signaling pathway, thereby increasing the stemness feature of HCC and leading to chemoresistance. In conclusion, we identified SP1/DUBR/E2F1-CIP2A as a critical axis to activate the Notch1 signaling pathway and promote stemness and chemoresistance of HCC. Therefore, DUBR could be a potential target in HCC treatment.

Chk1 Inhibition Ameliorates Alzheimer’s Disease Pathogenesis and Cognitive Dysfunction Through CIP2A/PP2A Signaling

Alzheimer’s disease (AD) is the most common neurodegenerative disease with limited therapeutic strategies. Cell cycle checkpoint protein kinase 1 (Chk1) is a Ser/Thr protein kinase which is activated in response to DNA damage, the latter which is an early event in AD. However, whether DNA damage-induced Chk1 activation participates in the development of AD and Chk1 inhibition ameliorates AD-like pathogenesis remain unclarified. Here, we demonstrate that Chk1 activity and the levels of protein phosphatase 2A (PP2A) inhibitory protein CIP2A are elevated in AD human brains, APP/PS1 transgenic mice, and primary neurons with Aβ treatment. Chk1 overexpression induces CIP2A upregulation, PP2A inhibition, tau and APP hyperphosphorylation, synaptic impairments, and cognitive memory deficit in mice. Moreover, Chk1 inhibitor (GDC0575) effectively increases PP2A activity, decreases tau phosphorylation, and inhibits Aβ overproduction in AD cell models. GDC0575 also reverses AD-like cognitive deficits and prevents neuron loss and synaptic impairments in APP/PS1 mice. In conclusion, our study uncovers a mechanism by which DNA damage-induced Chk1 activation promotes CIP2A-mediated tau and APP hyperphosphorylation and cognitive dysfunction in Alzheimer’s disease and highlights the therapeutic potential of Chk1 inhibitors in AD.

494 Enhanced Senescence Phenotype In Human Medulloblastoma via Protein Phosphatase 2A Inhibition and Radiation Therapy

INTRODUCTION: Medulloblastoma is the most common malignant pediatric brain tumor. Radiation(RT) is a part of standard care but causes significant neurocognitive impairment. Strategies to sensitize RT is critical to improving the quality of life and long-term survival of patients. Previous studies show that inhibition of protein-phosphatase 2A(PP2A)–a ubiquitous serine/threonine phosphatase–enhanced sensitivity to RT in multiple brain tumor xenografts. However, the mechanism behind enhanced radio-cytotoxicity is poorly understood. We hypothesize that PP2A inhibition combined with RT will increase cytoplasmic double-stranded DNA (dsDNA) and induce cGAS-cGAMP-STING signaling, promoting cellular senescence and thereby decreasing tumor growth. METHODS: D425-WT, a human group-3 medulloblastoma cell line, was used to generate a CRISPR sgPP2A knock out, D425-KO. We radiated WT and KO cells in vitro, quantified dsDNA in cytoplasmic extract using dsDNA Assay kit, and measured cGAMP levels in whole cell lysate using ELISA. Through FACS, we quantified the proportion of b-gal-expressing senescent cells. We measured protein expression of senescence markers (p21, p16, g-h2aX) by Western blot(WB). Finally, we injected D425 WT or KO cells orthotopically into cerebellum of nude NU/J mice followed by RT (10Gy) 3 days post-injection and monitored survival. RESULTS: In response to RT, D425-KO compared to WT showed significant increase in cytoplasmic dsDNA and intracellular cGAMP--a cyclic di-nucleotide STING agonist. There was also an increase in b-gal+ senescent cells and senescence markers expression in WB. In vivo, PP2AKO alone extended longevity, but PP2AKO with RT further prolonged survival compared to KO or RT alone. CONCLUSION: PP2A inhibition enhances RT induced cytosolic dsDNA damage and STING activation, promoting cellular senescence in vitro and sensitivity to RT in vivo.

DNA-Damage-Response-Targeting Mitochondria-Activated Multifunctional Prodrug Strategy for Self-Defensive Tumor Therapy.

We report a novel multifunctional construct, M1, designed explicitly to target the DNA damage response in cancer cells. M1 contains both a floxuridine (FUDR) and protein phosphatase 2A (PP2A) inhibitor combined with a GSH-sensitive linker. Further conjugation of the triphenylphosphonium moiety allows M1 to undergo specific activation in the mitochondria, where mitochondria-mediated apoptosis is observed. Moreover, M1 has enormous effects on genomic DNA ascribed to FUDR's primary function of impeding DNA/RNA synthesis combined with diminishing PP2A-activated DNA repair pathways. Importantly, mechanistic studies highlight the PP2A obtrusion in FUDR/5-fluorouracil (5-FU) therapy and underscore the importance of its inhibition to harbor therapeutic potential. HCT116 cell xenograft-bearing mice that have a low response rate to 5-FU show a prominent effect with M1, emphasizing the importance of DNA damage response targeting strategies using tumor-specific microenvironment-activatable systems.

DNA‐Damage‐Response‐Targeting Mitochondria‐Activated Multifunctional Prodrug Strategy for Self‐Defensive Tumor Therapy

AbstractWe report a novel multifunctional construct, M1, designed explicitly to target the DNA damage response in cancer cells. M1 contains both a floxuridine (FUDR) and protein phosphatase 2A (PP2A) inhibitor combined with a GSH‐sensitive linker. Further conjugation of the triphenylphosphonium moiety allows M1 to undergo specific activation in the mitochondria, where mitochondria‐mediated apoptosis is observed. Moreover, M1 has enormous effects on genomic DNA ascribed to FUDR's primary function of impeding DNA/RNA synthesis combined with diminishing PP2A‐activated DNA repair pathways. Importantly, mechanistic studies highlight the PP2A obtrusion in FUDR/5‐fluorouracil (5‐FU) therapy and underscore the importance of its inhibition to harbor therapeutic potential. HCT116 cell xenograft‐bearing mice that have a low response rate to 5‐FU show a prominent effect with M1, emphasizing the importance of DNA damage response targeting strategies using tumor‐specific microenvironment‐activatable systems.