Determining the Anticancer Activity of Sphingosine Kinase Inhibitors Containing Heteroatoms in Their Tail Structure

Sphingosine kinase (SK) enzyme, a central player of sphingolipid rheostat, catalyzes the phosphorylation of sphingosine to the bioactive lipid mediator sphingosine 1 phosphate (S1P), which regulates cancer cell proliferation, migration, differentiation, and angiogenesis through its extracellular five G protein-coupled S1P receptors (S1PR1–5). Recently, several research studies on SK inhibitors have taken place in order use them for the development of novel anticancer-targeted therapy. In this study, we designed and synthesized analog derivatives of known SK1 inhibitors, namely RB005 and PF-543, by introducing heteroatoms at their tail structure, as well as investigated their anticancer activities and pharmacokinetic parameters in vitro. Compounds 1–20 of RB005 and PF-543 derivatives containing an aliphatic chain or a tail structure of benzenesulfonyl were synthesized. All compounds of set 1 (1–10) effectively reduced cell viability in both HT29 and HCT116 cells, whereas set 2 derivatives (11–20) showed poor anticancer effect. Compound 10, having the highest cytotoxic effect (48 h, HT29 IC50 = 6.223 µM, HCT116 IC50 = 8.694 µM), induced HT29 and HCT116 cell death in a concentration-dependent manner through the mitochondrial apoptotic pathway, which was demonstrated by increased annexin V-FITC level, and increased apoptotic marker cleaved caspase-3 and cleaved PARP. Compound 10 inhibited SK1 by 20%, and, thus, the S1P level decreased by 42%. Unlike the apoptosis efficacy, the SK1 inhibitory effect and selectivity of the PF-543 derivative were superior to that of the RB005 analog. As a result, compounds with an aliphatic chain tail exhibited stronger apoptotic effects. However, this ability was not proportional to the degree of SK inhibition. Compound 10 increased the protein phosphatase 2A (PP2A) activity (1.73 fold) similar to FTY720 (1.65 fold) and RB005 (1.59 fold), whereas compounds 11 and 13 had no effect on PP2A activation. Since the PP2A activity increased in compounds with an aliphatic chain tail, it can be suggested that PP2A activation has an important effect on anticancer and SK inhibitory activities.

Renal AT 2 Receptors Mediate Natriuresis via Protein Phosphatase PP2A

Background: How signals from activated angiotensin type-2 receptors (AT 2 R) mediate inhibition of sodium ion (Na+) reabsorption in renal proximal tubule cells (RPTCs) is currently unknown. Protein phosphatases including protein phosphatase 2A (PP2A) have been implicated in AT2R signaling in tissues other than kidney. We investigated whether inhibition of protein phosphatase PP2A reduced AT 2 R-mediated natriuresis and evaluated changes in PP2A activity and localization after renal AT 2 R activation in normal 4- and 10-week-old control Wistar-Kyoto rats (WKY) and 4-week-old pre-hypertensive and 10-week-old hypertensive spontaneously hypertensive rats (SHR). Methods and Results: In WKY, direct renal interstitial (RI) administration of selective AT 2 R non-peptide agonist Compound-21 (C-21) increased RI cyclic GMP (cGMP) levels, urine Na + excretion (U Na V), and simultaneously increased PP2A activity ≅ 2-fold in homogenates of renal cortical tubules. The cGMP and natriuretic responses were abolished by concurrent RI administration of protein phosphatase inhibitor calyculin A (CAL). In RPTCs in response to C-21, PP2A subunits A, B55α and C, but not B56γ, were recruited to apical plasma membranes together with AT 2 Rs. CAL treatment abolished C-21-induced translocation of both AT 2 R and PP2A regulatory subunit B55α to apical plasma membranes. Immunoprecipitation of AT 2 R solubilized from renal cortical homogenates demonstrated physical association of AT 2 R with PP2A A, B55α, and C but not B56γ subunits. In contrast, in SHR, administration of C-21 did not alter UNaV or PP2A activity and failed to translocate AT 2 Rs and PP2A subunits to apical plasma membranes. Conclusions: In RPTCs of WKY, PP2A is activated and PP2A subunits AB55αC are recruited to C-21-activated AT 2 Rs during induction of natriuresis. This response is defective in pre-hypertensive and hypertensive SHR, presenting a potential novel therapeutic target for treating renal Na+ retention and hypertension.

14-3-3ζ Captures SET in the Cytoplasm, Mediating Tau Pathology and Cognitive Impairments

Background: 14-3-3ζ is overexpressed in brain regions affected by tau pathology in Alzheimer’s disease (AD), and its expression correlates with disease progression. The nuclear protein SET is a specific and efficient endogenous inhibitor of PP2A, which acts as a major protein phosphatase regulating tau phosphorylation and is compromised in AD. However, whether 14-3-3ζ mediates the cytoplasmic retention of SET and its related downstream events in AD remains elusive. Methods: We performed Spearman correlation analysis between 14-3-3 and SET in normal control brains and AD brains in a large-scale proteomic study of AD, and observed the distribution of SET and 14-3-3 in AD brain neurons, thus finding the cytoplasmic retention event of SET. We synthesized the peptides that prevented SET from binding to 14-3-3, and evaluated the effects of these peptides in blocking tau pathology. Results: Here, we reported that there is a significant increase in 14-3-3ζ interaction with SET during aging in 3´Tg AD mice and the human AD brain. 14-3-3ζ captures and sequesters SET in the cytoplasm and subsequently inhibits PP2A activity, induces tau hyperphosphorylation and triggers neurodegeneration, leading to behavioral impairments in mice infected with adeno-associated virus encoding 14-3-3ζ. Interestingly, as a dimer and scaffold protein, 14-3-3ζ was found to bind to SET while simultaneously recruiting CKII, which induced SET phosphorylation and cytoplasmic retention. Conversely, silencing 14-3-3ζ impeded CKII phosphorylation of SET. Moreover, blocking the 14-3-3ζ interaction with SET with the peptide NESGDPSSKST substantially induced SET translocation back to the nucleus, augmented PP2A activity and subsequently led to reduced tau hyperphosphorylation, decreased synapse loss and rescue of cognitive deficits in 14-3-3ζ mice. Furthermore, the peptide NESGDPSSKST but not the scramble peptide attenuated tau pathology and cognitive deficits in a 3´Tg AD mouse model. Conclusions: Our data strongly indicate that 14-3-3ζ captures SET in the cytoplasm and simultaneously recruits CKII, and this interaction plays a critical role in mediating tau-related clinical and neuropathological alterations. Inhibition of the 14-3-3ζ interaction with SET may be therapeutically useful for treating AD.

SET Improved Oocyte Maturation by PP2A and Inhibited Oocyte Apoptosis in Mouse Oocytes

Background: SET is a multifunctional protein involved in a variety of molecular processes such as transcription control, chromatin remodeling, cell apoptosis, and cell-cycle regulation. In ovaries SET is predominantly expressed in theca cells and oocytes. And in PCOS patients the expression of SET was increased than normal people. The current study was designed to determine whether SET play a role in oocyte maturation and apoptosis, which may provide clues for the underlying pathological mechanism of follicular development in PCOS patients.Methods: Oocytes at GV stage were collected from 6-week-old female ICR mice. 20-25 oocytes per group were placed in M16 medium. Then these oocytes were randomly divided into control group or treatment group for further study. Normal distribution was assessed by the Shapiro-Wilk test, and the student t test was used to compare the mRNA and protein levels. Chi-square analysis was used to compare the ratios of oocytes.Results: SET overexpression improved oocyte maturation whereas SET knockdown inhibited oocyte maturation. Moreover, SET negatively regulated PP2A activity in oocytes. Treatment with PP2A inhibitor okadaic acid (OA) promoted oocyte maturation. Furthermore, PP2A knockdown confirmed the key 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 BMP15 and GDF9 and PP2A inhibited their expression. Besides, SET inhibited oocyte apoptosis through decreased the expression of caspase 3 and caspases 8, while PP2A had no effect on oocyte apoptosis. Conclusions: 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 oocyte development disorder in PCOS.

SET Levels Contribute to Cohesion Fatigue

Chromosome instability (CIN) is a major hallmark of cancer cells and believed to drive tumor progression. Several cellular defects including weak centromeric cohesion are proposed to promote CIN, but the molecular mechanisms underlying these defects are poorly understood. In a screening for SET protein levels in various cancer cell lines, we found that most of the cancer cells exhibit higher SET protein levels than non-transformed cells, including RPE-1. Cancer cells with elevated SET often show weak centromeric cohesion, revealed by MG132-induced cohesion fatigue. Partial SET knockdown largely strengthens centromeric cohesion in cancer cells without increasing overall PP2A activity. Pharmacologically increased PP2A activity in these cancer cells barely ameliorates centromeric cohesion. These results suggest that compromised PP2A activity, a common phenomenon in cancer cells, may not be responsible for weak centromeric cohesion. Furthermore, centromeric cohesion in cancer cells can be strengthened by ectopic Sgo1 overexpression and weakened by SET WT, not by Sgo1-binding-deficient mutants. Altogether, these findings demonstrate that SET overexpression contributes to impaired centromeric cohesion in cancer cells and illustrate mis-regulated SET-Sgo1 pathway as an underlying mechanism.

Protein Phosphatase 2A Inhibits Gastric Cancer Cell Glycolysis by Reducing MYC Signaling

Background: The Warburg effect is closely associated malignant phenotypes and poor prognosis in cancer patients. PP2A is a highly conserved eukaryotic serine/threonine protein phosphatase that functions as a tumor suppressor in a variety of human cancers. However, the relationship between PP2A and the Warburg effect has yet to be fully understood. Methods: The expression profile of two endogenous inhibitors of PP2A, SET and CIP2A, are detected by real-time qPCR. Loss-of-function and gain-of-function are performed to demonstrate the roles of PP2A in gastric cancer cell proliferation and glycolysis. Cell biological, molecular, and biochemical approaches are used to uncover the underlying mechanism. Results: In this study, we find that SET and CIP2A are overexpressed in gastric cancer and associates a decreased PP2A activity. Pharmacological activation of PP2A with FTY-720 and DT-061 significantly reduces gastric cancer cell proliferation and glycolytic ability. Importantly, inhibition of PP2A activity by genetic silencing of PPP2R5A induces a growth advantage, which can be largely compromised by addition of the glycolysis inhibitor 2-Deoxy-D-glucose, suggesting a glycolysis-dependent effect of PP2A in gastric cancer. Mechanistically, the well known transcription factor and glycolysis regulator c-Myc is discovered as the functional mediator of PP2A in regulating cell glycolysis. Ectopic expression of a phosphorylation-mutant c-Myc resistant to PP2A (MycT58A) restores the inhibitory effect of FTY-720 and DT-061 on the lactate production and glucose uptake. Furthermore, there is a close association between SET and CIP2A expression and c-Myc gene signatures in gastric cancer samples. Conclusions: This study provides strong evidence of the involvement of PP2A in the Warburg effect and indicates that it could be a novel antitumor strategy to target tumor metabolism in gastric cancer.

Inhibiting protein phosphatase 2A increases the antitumor effect of protein arginine methyltransferase 5 inhibition in models of glioblastoma

Background Despite multi-model therapy of maximal surgical resection, radiation, chemotherapy, and tumor treating fields, the median survival of Glioblastoma (GBM) patients is less than 15 months. Protein Arginine Methyltransferase 5 (PRMT5) catalyzes the symmetric di-methylation of arginine residues and is overexpressed in GBM. Inhibition of PRMT5 causes senescence in stem-like GBM tumor cells. LB100, a first-in-class small molecular inhibitor of Protein Phosphatase 2A (PP2A) can sensitize therapy-resistant tumor cells. Here, we tested the anti-GBM effect of concurrent PRMT5 and PP2A inhibition. Methods Patient-derived primary GBM neurospheres (GBMNS), transfected with PRMT5 target-specific siRNA were treated with LB100 and subjected to in vitro assays including PP2A activity and western blot. The intracranial mouse xenograft model was used to test the in vivo antitumor efficacy of combination treatment. Results We found that PRMT5-depletion increased PP2A activity in GBMNS. LB100 treatment significantly reduced the viability of PRMT5-depleted GBMNS compared to PRMT5 intact GBMNS. LB100 enhanced G1 cell cycle arrest induced by PRMT5-depletion. Combination therapy also increased the expression of phospho-MLKL. Necrostatin-1 rescued PRMT5-depleted cells from the cytotoxic effects of LB100, indicating that necroptosis caused the enhanced cytotoxicity of combination therapy. In the in vivo mouse tumor xenograft model, LB100 treatment combined with transient depletion of PRMT5 significantly decreased tumor size and prolonged survival, while LB100 treatment alone had no survival benefit. Conclusion Overall, combined PRMT5 and PP2A inhibition had significantly greater antitumor effects than PRMT5 inhibition alone.

Specific PP2A Catalytic Subunits Are a Prerequisite for Positive Growth Effects in Arabidopsis Co-Cultivated with Azospirillum brasilense and Pseudomonas simiae

Plant growth-promoting rhizobacteria (PGPR) stimulate plant growth, but the underlying mechanism is poorly understood. In this study, we asked whether PROTEIN PHOSPHATASE 2A (PP2A), a regulatory molecular component of stress, growth, and developmental signaling networks in plants, contributes to the plant growth responses induced by the PGPR Azospirillum brasilense (wild type strain Sp245 and auxin deficient strain FAJ0009) and Pseudomonas simiae (WCS417r). The PGPR were co-cultivated with Arabidopsis wild type (WT) and PP2A (related) mutants. These plants had mutations in the PP2A catalytic subunits (C), and the PP2A activity-modulating genes LEUCINE CARBOXYL METHYL TRANSFERASE 1 (LCMT1) and PHOSPHOTYROSYL PHOSPHATASE ACTIVATOR (PTPA). When exposed to the three PGPR, WT and all mutant Arabidopsis revealed the typical phenotype of PGPR-treated plants with shortened primary root and increased lateral root density. Fresh weight of plants generally increased when the seedlings were exposed to the bacteria strains, with the exception of catalytic subunit double mutant c2c5. The positive effect on root and shoot fresh weight was especially pronounced in Arabidopsis mutants with low PP2A activity. Comparison of different mutants indicated a significant role of the PP2A catalytic subunits C2 and C5 for a positive response to PGPR.

STEM-23. INHIBITING PROTEIN PHOSPHATASE 2A INCREASES THE ANTITUMOR EFFECT OF PROTEIN ARGININE METHYLTRANSFERASE 5 INHIBITION IN MODELS OF GLIOBLASTOMA

BACKGROUND: Despite multi-model therapy of maximal surgical resection, radiation, chemotherapy, and tumor treating fields, median survival of Glioblastoma (GBM) patients is less than two years. Protein Arginine Methyltransferase 5 (PRMT5) catalyzes the symmetric di-methylation of arginine residues and is overexpressed in GBM. Inhibition of PRMT5 causes senescence of immature GBM tumor cells. LB100, first-in-class small molecule inhibitor of Protein Phosphatase 2A (PP2A) can sensitize therapy-resistant tumor cells. Here, we tested the anti-GBM effect of concurrent PRMT5 and PP2A inhibition. Methods: Patient-derived primary GBM neurospheres (GBMNS), transfected with PRMT5 target-specific siRNA were treated with LB100 and subjected to in vitro assays including PP2A activity and western blot. The intracranial mouse xenograft model was used to test the in vivo antitumor efficacy of combination treatment. Results: We found that PRMT5-depletion increased PP2A activity in GBMNS. LB100 treatment significantly reduced the viability of PRMT5-depleted GBMNS compared to PRMT5 intact GBMNS. LB100 enhanced the G1 cell cycle arrest induced by PRMT5-depletion. Combination therapy also increased the expression of phospho-MLKL. Necrostatin-1 rescued PRMT5-depleted cells from the cytotoxic effects of LB100, indicating that necroptosis caused the enhanced cytotoxicity of combination therapy. In the in vivo mouse tumor xenograft model, LB100 treatment combined with transient depletion of PRMT5 significantly decreased tumor size and prolonged mice survival, while LB100 treatment alone had no survival benefit. Conclusion: Overall, combined PRMT5 and PP2A inhibition had significantly greater antitumor effects than PRMT5 inhibition alone.