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Journal of Translational Medicine May 2023Pancreatic ductal adenocarcinoma (PDAC) has a low survival, its incidence is rising and little therapeutic improvements are expected in the near future. It has been...
Human pancreatic cancer patients with Epithelial-to-Mesenchymal Transition and an aggressive phenotype show a disturbed balance in Protein Phosphatase Type 2A expression and functionality.
BACKGROUND
Pancreatic ductal adenocarcinoma (PDAC) has a low survival, its incidence is rising and little therapeutic improvements are expected in the near future. It has been observed that Epithelial-to-Mesenchymal transition (EMT) contributes (including in PDAC) to a more aggressive cancer phenotype. Additionally, largely unexplored, studies indicate a mechanistic interplay between Protein Phosphatase Type 2A (PP2A) enzymes and EMT that could offer treatment opportunities. The aim was to investigate the relation of a PP2A expression signature (encompassing all PP2A subunits, endogenous inhibitors and activators) with EMT and aggressive pancreatic cancer, and to discuss possible implications.
METHODS
We retrieved different PDAC expression datasets from NCBI to capture the variation in patients, and analyzed these using datamining, survival analysis, differential gene and protein expression. We determined genes highly associated with aggressive PDAC. For in vitro evaluation, Panc-1 cells were treated with the pharmacologic PP2A inhibitor Okadaic Acid (OA). Additionally, two OA-resistant Panc-1 clones were developed and characterized.
RESULTS
In patients, there is a strong correlation between EMT and aggressive PDAC, and between aggressive PDAC and PP2A, with a significant upregulation of PP2A inhibitor genes. Several PP2A genes significantly correlated with decreased survival. In vitro, short-term exposure to OA induced EMT in Panc-1 cells. This shift towards EMT was further pronounced in the OA-resistant Panc-1 clones, morphologically and by pathway analysis. Proteomic analysis and gene sequencing showed that the advanced OA-resistant model most resembles the clinical PDAC presentation (with EMT signature, and with several specific PP2A genes upregulated, and others downregulated).
CONCLUSIONS
We demonstrated a strong association between EMT, altered PP2A expression and aggressive PDAC in patients. Also, in vitro, PP2A inhibition induces EMT. Overall, statistics suggests the mechanistic importance of PP2A dysregulation for PDAC progression. Translationally, our observations indicate that pharmacologic restoration of PP2A activity could be an attractive therapeutic strategy to block or reverse progression.
Topics: Humans; Proteomics; Cell Proliferation; Pancreatic Neoplasms; Carcinoma, Pancreatic Ductal; Epithelial-Mesenchymal Transition; Phosphoprotein Phosphatases; Cell Line, Tumor; Cell Movement; Gene Expression Regulation, Neoplastic
PubMed: 37170215
DOI: 10.1186/s12967-023-04145-z -
Bioscience Reports May 2023Phosphoprotein phosphatases (PPPs) are a ubiquitous class of enzymes which dephosphorylate serine and threonine residues on substrate proteins involved in a wide variety...
Phosphoprotein phosphatases (PPPs) are a ubiquitous class of enzymes which dephosphorylate serine and threonine residues on substrate proteins involved in a wide variety of cellular processes. The active site of PPP enzymes are highly conserved with key residues coordinating the substrate phosphoryl group (the two R-clamp) and two metal ions necessary for catalysis. Because of the diverse number of roles that these enzymes play it is no surprise that they are highly regulated in the cell, often accomplished by binding regulatory subunits. These regulatory subunits are able to dictate substrate specificity, localization, and activity of the bound catalytic subunit. Eukaryotic PPP subtypes have been previously shown to manifest varying degrees of sensitivity to environmental toxins. We present here an evolutionary model which now rationalizes this data. Our re-examination of published structural evidence reveals that Eukaryotic PPP toxin-binding residues also interact with substrate binding residues (the two R-clamp) and ancient regulatory proteins. Such functional interactions could have stabilized PPP sequence early in Eukaryotic evolution, providing a stable target which was co-opted by toxins and their producer organisms.
PubMed: 37144562
DOI: 10.1042/BSR20230378 -
Frontiers in Physiology 2023The actin regulatory protein, cofilin plays a key signaling role in many cells for numerous cellular responses including in proliferation, development, motility,...
The actin regulatory protein, cofilin plays a key signaling role in many cells for numerous cellular responses including in proliferation, development, motility, migration, secretion and growth. In the pancreas it is important in islet insulin secretion, growth of pancreatic cancer cells and in pancreatitis. However, there are no studies on its role or activation in pancreatic acinar cells. To address this question, we studied the ability of CCK to activate cofilin in pancreatic acinar cells, AR42J cells and CCK-R transfected Panc-1 cells, the signaling cascades involved and its effect on enzyme secretion and MAPK activation, a key mediator of pancreatic growth. CCK (0.3 and 100 nM), TPA, carbachol, Bombesin, secretin and VIP decreased phospho-cofilin (i.e., activate cofilin) and both phospho-kinetic and inhibitor studies of cofilin, LIM kinase (LIMK) and Slingshot Protein Phosphatase (SSH1) demonstrated these conventional activators of cofilin were not involved. Serine phosphatases inhibitors (calyculin A and okadaic acid), however inhibited CCK/TPA-cofilin activation. Studies of various CCK-activated signaling cascades showed activation of PKC/PKD, Src, PAK4, JNK, ROCK mediated cofilin activation, but not PI3K, p38, or MEK. Furthermore, using both siRNA and cofilin inhibitors, cofilin activation was shown to be essential for CCK-mediated enzyme secretion and MAPK activation. These results support the conclusion that cofilin activation plays a pivotal convergent role for various cell signaling cascades in CCK mediated growth/enzyme secretion in pancreatic acini.
PubMed: 37138671
DOI: 10.3389/fphys.2023.1147572 -
Marine Drugs Apr 2023Palytoxin is considered one of the most potent biotoxins. As palytoxin-induced cancer cell death mechanisms remain to be elucidated, we investigated this effect on...
Palytoxin is considered one of the most potent biotoxins. As palytoxin-induced cancer cell death mechanisms remain to be elucidated, we investigated this effect on various leukemia and solid tumor cell lines at low picomolar concentrations. As palytoxin did not affect the viability of peripheral blood mononuclear cells (PBMC) from healthy donors and did not create systemic toxicity in zebrafish, we confirmed excellent differential toxicity. Cell death was characterized by a multi-parametric approach involving the detection of nuclear condensation and caspase activation assays. zVAD-sensitive apoptotic cell death was concomitant with a dose-dependent downregulation of antiapoptotic Bcl-2 family proteins Mcl-1 and Bcl-xL. Proteasome inhibitor MG-132 prevented the proteolysis of Mcl-1, whereas the three major proteasomal enzymatic activities were upregulated by palytoxin. Palytoxin-induced dephosphorylation of Bcl-2 further exacerbated the proapoptotic effect of Mcl-1 and Bcl-xL degradation in a range of leukemia cell lines. As okadaic acid rescued cell death triggered by palytoxin, protein phosphatase (PP)2A was involved in Bcl-2 dephosphorylation and induction of apoptosis by palytoxin. At a translational level, palytoxin abrogated the colony formation capacity of leukemia cell types. Moreover, palytoxin abrogated tumor formation in a zebrafish xenograft assay at concentrations between 10 and 30 pM. Altogether, we provide evidence of the role of palytoxin as a very potent and promising anti-leukemic agent, acting at low picomolar concentrations in cellulo and in vivo.
Topics: Animals; Humans; Leukocytes, Mononuclear; Zebrafish; Down-Regulation; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins c-bcl-2; Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Leukemia; bcl-X Protein
PubMed: 37103372
DOI: 10.3390/md21040233 -
Comparative Biochemistry and... Sep 2023Globally around 24 million elderly population are dealing with dementia, and this pathological characteristic is commonly seen in people suffering from Alzheimer's...
Globally around 24 million elderly population are dealing with dementia, and this pathological characteristic is commonly seen in people suffering from Alzheimer's disease (AD). Despite having multiple treatment options that can mitigate AD symptoms, there is an imperative call to advance our understanding of the disease pathogenesis to unfold disease-modifying treatments/therapies. To explore the driving mechanisms of AD development, we stretch out further to study time-dependant changes after Okadaic acid (OKA)-induced AD-like conditions in zebrafish. We evaluated the pharmacodynamics of OKA at two-time points, i.e., after 4-days and 10-days exposure to zebrafish. T-Maze was utilized to observe the learning and cognitive behaviour, and inflammatory gene expressions such as 5-Lox, Gfap, Actin, APP, and Mapt were performed in zebrafish brains. To scoop everything out from the brain tissue, protein profiling was performed using LCMS/MS. Both time course OKA-induced AD models have shown significant memory impairment, as evident from T-Maze. Gene expression studies of both groups have reported an overexpression of 5-Lox, GFAP, Actin, APP, and OKA 10D group has shown remarkable upregulation of Mapt in zebrafish brains. In the case of protein expression, the heatmap suggested an important role of some common proteins identified in both groups, which can be explored further to investigate their mechanism in OKA-induced AD pathology. Presently, the preclinical models available to understand AD-like conditions are not completely understood. Hence, utilizing OKA in the zebrafish model can be of great importance in understanding the pathology of AD progression and as a screening tool for drug discovery.
Topics: Aged; Animals; Humans; Alzheimer Disease; Zebrafish; Proteomics; Actins; Brain; Okadaic Acid; Genomics; Disease Models, Animal
PubMed: 37100105
DOI: 10.1016/j.cbpc.2023.109636 -
Journal of Cancer Research and Clinical... Sep 2023Okadaic acid class of tumor promoters are transformed into endogenous protein inhibitors of PP2A, SET, and CIP2A in human cancers. This indicates that inhibition of PP2A... (Review)
Review
PURPOSE
Okadaic acid class of tumor promoters are transformed into endogenous protein inhibitors of PP2A, SET, and CIP2A in human cancers. This indicates that inhibition of PP2A activity is a common mechanism of cancer progression in humans. It is important to study the roles of SET and CIP2A vis-à-vis their clinical significance on the basis of new information gathered from a search of PubMed.
RESULTS AND DISCUSSION
The first part of this review introduces the carcinogenic roles of TNF-α and IL-1, which are induced by the okadaic acid class of compounds. The second part describes unique features of SET and CIP2A in cancer progression for several types of human cancer: (1) SET-expressing circulating tumor cells (SET-CTCs) in breast cancer, (2) knockdown of CIP2A and increased PP2A activity in chronic myeloid leukemia, (3) CIP2A and epidermal growth factor receptor (EGFR) activity in erlotinib sensitive- and resistant-non-small cell lung cancer, (4) SET antagonist EMQA plus radiation therapy against hepatocellular carcinoma, (5) PP2A inactivation as a common event in colorectal cancer, (6) prostate cancer susceptibility variants, homeobox transcription factor (HOXB13 T) and CIP2A T, and (7) SET inhibitor OP449 for pre-clinical investigation of pancreatic cancer. In the Discussion, the binding complex of SET is briefly introduced, and overexpression of SET and CIP2A proteins is discussed in relation to age-associated chronic inflammation (inflammaging).
CONCLUSION
This review establishes the concept that inhibition of PP2A activity is a common mechanism of human cancer progression and activation of PP2A activity leads to effective anticancer therapy.
Topics: Male; Humans; Okadaic Acid; Carcinoma, Non-Small-Cell Lung; Carcinogens; Lung Neoplasms; Membrane Proteins; Cell Line, Tumor; Intracellular Signaling Peptides and Proteins; Liver Neoplasms; Autoantigens
PubMed: 37097392
DOI: 10.1007/s00432-023-04800-4 -
BioRxiv : the Preprint Server For... Mar 2023Protein phosphorylation is an essential regulatory mechanism that controls most cellular processes, including cell cycle progression, cell division, and response to...
Protein phosphorylation is an essential regulatory mechanism that controls most cellular processes, including cell cycle progression, cell division, and response to extracellular stimuli, among many others, and is deregulated in many diseases. Protein phosphorylation is coordinated by the opposing activities of protein kinases and protein phosphatases. In eukaryotic cells, most serine/threonine phosphorylation sites are dephosphorylated by members of the Phosphoprotein Phosphatase (PPP) family. However, we only know for a few phosphorylation sites which specific PPP dephosphorylates them. Although natural compounds such as calyculin A and okadaic acid inhibit PPPs at low nanomolar concentrations, no selective chemical PPP inhibitors exist. Here, we demonstrate the utility of endogenous tagging of genomic loci with an auxin-inducible degron (AID) as a strategy to investigate specific PPP signaling. Using Protein Phosphatase 6 (PP6) as an example, we demonstrate how rapidly inducible protein degradation can be employed to identify dephosphorylation SITES and elucidate PP6 biology. Using genome editing, we introduce AID-tags into each allele of the PP6 catalytic subunit (PP6c) in DLD-1 cells expressing the auxin receptor Tir1. Upon rapid auxin-induced degradation of PP6c, we perform quantitative mass spectrometry-based proteomics and phosphoproteomics to identify PP6 substrates in mitosis. PP6 is an essential enzyme with conserved roles in mitosis and growth signaling. Consistently, we identify candidate PP6c-dependent phosphorylation sites on proteins implicated in coordinating the mitotic cell cycle, cytoskeleton, gene expression, and mitogen-activated protein kinase (MAPK) and Hippo signaling. Finally, we demonstrate that PP6c opposes the activation of large tumor suppressor 1 (LATS1) by dephosphorylating Threonine 35 (T35) on Mps One Binder (MOB1), thereby blocking the interaction of MOB1 and LATS1. Our analyses highlight the utility of combining genome engineering, inducible degradation, and multiplexed phosphoproteomics to investigate signaling by individual PPPs on a global level, which is currently limited by the lack of tools for specific interrogation.
PubMed: 36993243
DOI: 10.1101/2023.03.25.534211 -
Marine Drugs Feb 2023Marine toxins (MTs) are a group of structurally complex natural products with unique toxicological and pharmacological activities. In the present study, two common...
Marine toxins (MTs) are a group of structurally complex natural products with unique toxicological and pharmacological activities. In the present study, two common shellfish toxins, okadaic acid (OA) () and OA methyl ester (), were isolated from the cultured microalgae strain PL11. OA can significantly activate the latent HIV but has severe toxicity. To obtain more tolerable and potent latency reversing agents (LRAs), we conducted the structural modification of OA by esterification, yielding one known compound () and four new derivatives (-). Flow cytometry-based HIV latency reversal activity screening showed that compound possessed a stronger activity (EC = 46 ± 13.5 nM) but was less cytotoxic than OA. The preliminary structure-activity relationships (SARs) indicated that the carboxyl group in OA was essential for activity, while the esterification of carboxyl or free hydroxyls were beneficial for reducing cytotoxicity. A mechanistic study revealed that compound promotes the dissociation of P-TEFb from the 7SK snRNP complex to reactivate latent HIV-1. Our study provides significant clues for OA-based HIV LRA discovery.
Topics: Humans; Okadaic Acid; HIV Infections; Virus Latency; HIV-1; Marine Toxins; Dinoflagellida
PubMed: 36976207
DOI: 10.3390/md21030158 -
Circulation Research Apr 2023Small-conductance Ca-activated K (SK)-channel inhibitors have antiarrhythmic effects in animal models of atrial fibrillation (AF), presenting a potential novel...
BACKGROUND
Small-conductance Ca-activated K (SK)-channel inhibitors have antiarrhythmic effects in animal models of atrial fibrillation (AF), presenting a potential novel antiarrhythmic option. However, the regulation of SK-channels in human atrial cardiomyocytes and its modification in patients with AF are poorly understood and were the object of this study.
METHODS
Apamin-sensitive SK-channel current (I) and action potentials were recorded in human right-atrial cardiomyocytes from sinus rhythm control (Ctl) patients or patients with (long-standing persistent) chronic AF (cAF).
RESULTS
I was significantly higher, and apamin caused larger action potential prolongation in cAF- versus Ctl-cardiomyocytes. Sensitivity analyses in an in silico human atrial cardiomyocyte model identified I and I as major regulators of repolarization. Increased I in cAF was not associated with increases in mRNA/protein levels of SK-channel subunits in either right- or left-atrial tissue homogenates or right-atrial cardiomyocytes, but the abundance of SK2 at the sarcolemma was larger in cAF versus Ctl in both tissue-slices and cardiomyocytes. Latrunculin-A and primaquine (anterograde and retrograde protein-trafficking inhibitors) eliminated the differences in SK2 membrane levels and I between Ctl- and cAF-cardiomyocytes. In addition, the phosphatase-inhibitor okadaic acid reduced I amplitude and abolished the difference between Ctl- and cAF-cardiomyocytes, indicating that reduced calmodulin-Thr80 phosphorylation due to increased protein phosphatase-2A levels in the SK-channel complex likely contribute to the greater I in cAF-cardiomyocytes. Finally, rapid electrical activation (5 Hz, 10 minutes) of Ctl-cardiomyocytes promoted SK2 membrane-localization, increased I and reduced action potential duration, effects greatly attenuated by apamin. Latrunculin-A or primaquine prevented the 5-Hz-induced I-upregulation.
CONCLUSIONS
I is upregulated in patients with cAF due to enhanced channel function, mediated by phosphatase-2A-dependent calmodulin-Thr80 dephosphorylation and tachycardia-dependent enhanced trafficking and targeting of SK-channel subunits to the sarcolemma. The observed AF-associated increases in I, which promote reentry-stabilizing action potential duration shortening, suggest an important role for SK-channels in AF auto-promotion and provide a rationale for pursuing the antiarrhythmic effects of SK-channel inhibition in humans.
Topics: Animals; Humans; Atrial Fibrillation; Apamin; Primaquine; Calmodulin; Heart Atria; Myocytes, Cardiac; Anti-Arrhythmia Agents; Action Potentials; Small-Conductance Calcium-Activated Potassium Channels
PubMed: 36927079
DOI: 10.1161/CIRCRESAHA.122.321858 -
European Journal of Medicinal Chemistry May 2023Protein phosphatase 2A (PP2A) is an important Ser/Thr phosphatase that participates in the regulation of multiple cellular processes. This implies that any deficient...
C-glycosides analogues of the okadaic acid central fragment exert neuroprotection via restoration of PP2A-phosphatase activity: A rational design of potential drugs for Alzheimer's disease targeting tauopathies.
Protein phosphatase 2A (PP2A) is an important Ser/Thr phosphatase that participates in the regulation of multiple cellular processes. This implies that any deficient activity of PP2A is the responsible of severe pathologies. For instance, one of the main histopathological features of Alzheimer's disease is neurofibrillary tangles, which are mainly comprised by hyperphosphorylated forms of tau protein. This altered rate of tau phosphorylation has been correlated with PP2A depression AD patients. With the goal of preventing PP2A inactivation in neurodegeneration scenarios, we have aimed to design, synthesize and evaluate new ligands of PP2A capable of preventing its inhibition. To achieve this goal, the new PP2A ligands present structural similarities with the central fragment C19-C27 of the well-established PP2A inhibitor okadaic acid (OA). Indeed, this central moiety of OA does not exert inhibitory actions. Hence, these compounds lack PP2A-inhibiting structural motifs but, in contrast, compete with PP2A inhibitors, thus recovering phosphatase activity. Proving this hypothesis, most compounds showed a good neuroprotective profile in neurodegeneration models related to PP2A impairment, highlighting derivative 10, named ITH12711, as the most promising one. This compound (1) restored in vitro and cellular PP2A catalytic activity, measured on a phospho-peptide substrate and by western-blot analyses, (2) proved good brain penetration measured by PAMPA, and (3) prevented LPS-induced memory impairment of mice in the object recognition test. Thus, the promising outcomes of the compound 10 validate our rational approach to design new PP2A-activating drugs based on OA central fragment.
Topics: Mice; Animals; Alzheimer Disease; Okadaic Acid; Neuroprotection; Tauopathies; tau Proteins; Protein Phosphatase 2; Phosphorylation
PubMed: 36905916
DOI: 10.1016/j.ejmech.2023.115245