Cancerous Inhibitor Of Phosphatase 2A Research Articles

Cancerous Inhibitor of Phosphatase 2A (CIP2A): A Sensitive Diagnostic Marker and Therapeutic Agent in Oral Cancer.

Cancerous Inhibitor of Phosphatase 2A (CIP2A): A Sensitive Diagnostic Marker and Therapeutic Agent in Oral Cancer.

Abstract B064: The role of PP2A-B56α in KRAS-mediated pancreatic tumorigenesis

Abstract Protein phosphatase 2A (PP2A) is a major serine-threonine phosphatase that regulates many cellular pathways including KRAS, whose oncogenic mutation is prevalent in 95% of patients with Pancreatic Ductal Adenocarcinoma (PDAC). Previous research has identified a decrease in global PP2A activity and an increase in the expression of PP2A inhibitors in PDAC cell lines, suggesting that suppression of PP2A activity may be pertinent in PDAC maintenance. Importantly, PP2A has low mutation rates in PDAC, making it a viable target for therapeutic reactivation. While PP2A has been shown to have global tumor suppressive capabilities, the regulation of specific pathways by PP2A can be altered based on PP2A holoenzyme composition. Therefore, there is a critical need to understand the mechanisms by which oncogenic KRAS can affect PP2A function and differential substrate targeting in PDAC. The PP2A holoenzyme consists of 3 subunits: the scaffolding subunit (A), the catalytic subunit (C), and the regulatory subunit (B). There are 16 different B subunits that can be incorporated into the PP2A holoenzyme that are responsible for substrate specificity. The B56α subunit of PP2A has been shown to negatively regulate cellular transformation. Our research aims to investigate the mechanisms by which PP2A-B56α is regulated through oncogenic KRAS and how suppression of B56α impacts the initiation and progression of PDAC. To determine how oncogenic KRAS alters the dynamics of PP2A-B56α and overall PP2A activity we utilized tet-inducible KRASG12D cell lines to allow direct manipulation of KRAS mutational activation. Using this system, we have identified time dependent alterations in cancerous inhibitor of PP2A (CIP2A) following induction of KRASG12D expression, indicating that PP2A suppression may be an early event in PDAC initiation. Consistent with this hypothesis, we characterized changes in the acceleration of PDAC formation in vivo using the Ptf1a-Cre; LSL- KRASG12D (KC) genetic mouse model combined with a B56α hypomorph model (KCBhm/hm). Our data show that the loss of B56α accelerates PDAC initiation, with an increase in pancreatic precursor lesion (PanIN) number and a decrease in healthy acinar area. In response to B56α loss, similar acceleration of acinar to ductal metaplasia (ADM) kinetics were observed in a 3D-cultured ADM Assay. Furthermore, when 3D-cultured acinar cells were treated with a small molecule activator of PP2A (SMAP), SMAP treatment resulted in smaller and fewer ductal structures, preventing the ADM process. Collectively, these data suggest that PP2A-B56α plays a regulatory role in cellular plasticity and loss contributes to PDAC initiation. Future studies will investigate how mutant KRAS-mediated CIP2A expression effects overall PP2A phosphatase activity and how subsequent sequestration of B56α contributes to development of PDAC. Together, these studies identify PP2A as a critical regulator of KRAS-induced cellular plasticity and support reactivation of PP2A as a novel therapeutic strategy in PDAC patients. Citation Format: Samantha L Tinsley, Rebecca A. Shelley, Gagan K. Mall, Ella Rose D. Chianis, Mary C. Thoma, Marina Pasca di Magliano, Goutham Narla, Rosalie C. Sears, Brittany L. Allen-Petersen. The role of PP2A-B56α in KRAS-mediated pancreatic tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B064.

Abstract PR-008: The role of PP2A-B56α in acinar-to-ductal metaplasia and initiation of pancreatic ductal adenocarcinoma

Abstract Protein phosphatase 2A (PP2A) is a major serine-threonine phosphatase that regulates many cellular pathways including KRAS, whose oncogenic mutation is prevalent in 95% of patients with Pancreatic Ductal Adenocarcinoma (PDAC). Previous research has identified a decrease in global PP2A activity and an increase in the expression PP2A inhibitors in PDAC cell lines, suggesting that suppression of PP2A activity may be pertinent in PDAC maintenance. Importantly, PP2A has low mutation rates in PDAC, making it a viable target for therapeutic reactivation. While PP2A has been shown to have global tumor suppressive capabilities, the regulation of specific pathways by PP2A can be altered based on PP2A holoenzyme composition. Therefore, there is a critical need to understand the mechanisms by which oncogenic KRAS can affect PP2A function and differential substrate targeting in PDAC. The PP2A holoenzyme consists of 3 subunits: the scaffolding subunit (A), the catalytic subunit (C), and the regulatory subunit (B). There are 16 different B subunits that can be incorporated into the PP2A holoenzyme that are responsible for substrate specificity. The B56α subunit of PP2A has been shown to negatively regulate cellular transformation and has decreased expression in aggressive subtypes of PDAC, indicating that suppression of this subunit may aid in PDAC tumorgenicity. Our research aims to investigate the mechanisms by which PP2A-B56α is regulated downstream of oncogenic KRAS and how suppression of B56α impacts the initiation and progression of PDAC. In order to determine how oncogenic KRAS alters the dynamics of PP2A-B56α and overall PP2A activity we utilized tet-inducible KRASG12D cell lines to allow direct manipulation of KRAS mutational activation. Using this system, we have identified time dependent alterations in cancerous inhibitor of PP2A (CIP2A) following induction of KRASG12D expression, indicating that PP2A suppression may be an early event in PDAC initiation. Consistent with this hypothesis, we characterized changes in the acceleration of PDAC formation in vivo using the Ptf1a-Cre; LSL-KRASG12D (KC) genetic mouse model combined with a B56α hypomorph model (KCBhm/hm). Our data show that the loss of B56α accelerates PDAC initiation, with an increase in pancreatic precursor lesion (PanIN) number and a decrease in healthy acinar area. Similar results were seen in an ex vivo acinar-to-ductal metaplasia assay, suggesting that B56α alters pancreatic cellular plasticity in a cell-autonomous manner. Future studies will investigate how mutant KRAS expression effects overall PP2A phosphatase activity, holoenzyme formation, and sequestration of B56α by endogenous inhibitors to further understand how suppression of PP2A-B56α contributes to development of PDAC. Together, these studies identify PP2A as a critical regulator of KRAS-induced cellular plasticity and support therapeutic reactivation of PP2A as a novel therapeutic strategy in PDAC patients. Citation Format: Samantha L. Tinsley, Rebecca A. Shelley, Mary C. Thoma, Goutham Narla, Rosalie C. Sears, Brittany A. Allen-Petersen. The role of PP2A-B56α in acinar-to-ductal metaplasia and initiation of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PR-008.

CIP2A Causes Tau/APP Phosphorylation, Synaptopathy, and Memory Deficits in Alzheimer’s Disease

SUMMARYProtein phosphatase 2A (PP2A) inhibition causes hy-perphosphorylation of tau and APP in Alzheimer’s disease (AD). However, the mechanisms underlying the downregulation of PP2A activity in AD brain remain unclear. We demonstrate that Cancerous Inhibitor of PP2A (CIP2A), an endogenous PP2A inhibitor, is overexpressed in AD brain. CIP2A-mediated PP2A inhibition drives tau/APP hyperphosphorylation and increases APP β-cleavage and Aβ production. Increase in CIP2A expression also leads to tau mislocalization to dendrites and spines and synaptic degeneration. In mice, injection of AAV-CIP2A to hippocampus induced AD-like cognitive deficits and impairments in long-term potentiation (LTP) and exacerbated AD pathologies in neurons. Indicative of disease exacerbating the feedback loop, we found that increased CIP2A expression and PP2A inhibition in AD brains result from increased Aβ production. In summary, we show that CIP2A overexpression causes PP2A inhibition and AD-related cellular pathology and cognitive deficits, pointing to CIP2A as a potential target for AD therapy.

Complete Inhibition of STAT5 Phosphorylation Is Achieved By Combination of JAK1/2 and PI3K/mTOR Inhibitors in in Vitro and In Vivo MPN Models

Autonomous activation of JAK/STAT pathway by JAK2 V617F (VF) and similar mutations in myeloproliferative neoplasms (MPN) is associated with enhanced signaling of other downstream pathways including the PI3K/mTOR. Targeting the PI3K pathway resulted in inhibition of JAK2 VF cells (J Cell Mol Med 2013;17:1385), and the Torc1 inhibitor Everolimus (RAD001) showed clinical benefits in a phase I/II trial (Blood 2011;118:2069). In this study we evaluated the effects of BKM120, a pan-PI3K specific inhibitor, alone and in combination with the JAK1/JAK2 inhibitor Ruxolitinib (Ruxo) and Everolimus or PP242 (Torc1/2 inhibitor), in different MPN models and correlated efficacy with the extent of STAT5 phosphorylation inhibition.In vitro, we used BaF3 and BaF3-EPOR cells expressing wild type (WT) or VF mutated JAK2, the human VF mutated HEL and SET2 cell lines, and primary CD34+ cells from MPN patients. The combination index (CI) according to Chou and Talalay was used to evaluate drug combination; a CI <1 indicates synergistic activity. For in vivo studies we used two mouse models: (1) SCID mice receiving iv BaF3-EPOR VF-luciferase (luc) cells (Mol Cancer Ther 2010;9:1945) were randomized on day 6 to different treatment groups based on baseline luminescence and animals were sacrificed when became moribund; (2) C57Bl6/J JAK2VF knock-in (KI) mice constitutively expressing JAK2VF protein in heterozygous status (Blood 2013; 122:1464) were treated for 15 days before analysis.BKM120 preferentially inhibited BaF3VF and BaF3-EpoR-VF cells compared to the WT counterpart (IC50: 364±200nM and 1100±207nM vs 5300±800nM and 3122±1000nM respectively; P<0.05) and was similarly effective against JAK2VF HEL and SET2 cells. Western blot analysis showed marked reduction of phospho (p)-mTOR and its target p-4EBP1 as well as downregulation of p-STAT5. BKM120 reduced colony formation from MF and PV CD34+ cells at concentrations 2- to 8-fold lower than controls (P<0.01). EEC colony growth was inhibited at very low nM concentrations (IC50: 9±4nM). In vivo, BKM120 alone at a dose of 60mpk increased survival of BaF3VF-luc injected mice from 21d (controls) to 28d (P<0.05). Then we evaluated triple combinations of BKM120 and Ruxo plus either Everolimus or PP242 and found strong synergism against BaF3-EpoR-VF cells (CI=0.27 and 0.52). Furthermore, triple combinations of low doses (30nM each) BKM120, Ruxo and Everolimus reduced colony formation from BM cells of JAK2VF KI mice preferentially as compared to WT mice (-93.7% vs -44.7%). In vivo, triple combination treatment of KI mice with low drug doses (30mpk BKM120 + 30mpk Ruxo + 1.5mpk Everolimus) resulted in impressive improvement of splenomegaly: median spleen reduction compared to controls was -44.5%, -36.2%, -8%, for 60mpk BKM120, 60mpk Ruxo, 60mpk Everolimus as single agents and -69.5%, for triple treatment. Similarly greater activity was seen concerning reduction of leukocytosis and reticulocyte count. Compatible with findings in vitro, the level of p-4eBP1 and p-STAT5 in the spleen were significantly reduced in mice receiving combined treatment versus single agents. We explored the mechanisms by which PI3K/mTOR inhibitors resulted in reduced p-STAT5. We found that Ruxo treatment reduced tyrosine (Tyr) phosphorylation of the STAT5a isoform while leaving serines (Ser) unaffected, while BKM120 and Everolimus specifically resulted in dephosphorylation of STAT5b serine residues, in particular Ser193 and Ser731; accordingly, combined treatment resulted in complete STAT5 inactivation by dephosphorylation of both Ser and Tyr residues. Interestingly, the mRNA and protein levels of Cip2a, Cancerous Inhibitor of Phosphatase 2a (PP2a, that is normally involved in dephosphorylation of p-STAT residues), were strongly reduced by PI3K pathway inhibitors both in cell lines and in samples from patients responsive to Everolimus treatment. PP2a inhibition by Calyculin A resulted in enhanced STAT5b Ser193 and Ser731 phosphorylation while slightly altered STAT5a phosphoresidues suggesting a role of PP2a and its repressor Cip2a in PI3K/STAT5-dependent activation of JAK/STAT signaling.Concurrent inhibition of the JAK2 and PI3K pathways by Ruxo plus BKM120 and Everolimus results in enhanced activity in preclinical models of MPN and underlines the importance of complete STAT5 inactivation in order to magnify treatment efficacy, thereby providing a rationale for clinical trials. DisclosuresVannucchi:Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shire: Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees.

Erratum to: CIP2A is overexpressed and involved in the pathogenesis of chronic myelocytic leukemia by interacting with breakpoint cluster region-Abelson leukemia virus

To detect the expression of cancerous inhibitor of phosphatase 2A (CIP2A) in chronic myelocytic leukemia (CML) and investigate the mechanism underlying CIP2A knockdown-mediated cell proliferation and apoptosis as well as the interaction of CIP2A with breakpoint cluster region- Abelson leukemia virus (BCR-ABL). CIP2A mRNA and protein expression in chronic myelocytic leukemia-chronic (CML-CP) patients and healthy controls were determined by RT-PCR and Western blot. In vivo, c-Myc expression, PP2A activity, cell proliferation, and apoptosis of CML cells were detected with CIP2A depletion. In addition, the relationship among CIP2A, BCR-ABL, and tyrosine phosphatase SHP-1 was explored by depleting/overexpressing CIP2A or inhib- iting BCR-ABL. The level of CIP2A mRNA was higher in CML-CP patients than healthy controls (56/74, 75.7 % vs. 1/35, 2.9 %, P 0.001), and CIP2A protein was overex- pressed in corresponding specimens. CIP2A knockdown by siRNA reduced the level of c-Myc protein and clonogenic formation, inhibited the activity of PP2A, K562 cell prolif- eration, and promoted cell apoptosis. Suppressing BCR- ABL by imatinib mesylate (IM) significantly decreased CIP2A expression. CIP2A knockdown decreased BCR-ABL but increased SHP-1 expression, and CIP2A overexpression had the reverse effect. CIP2A is overexpressed in CML-CP patients, and its expression may promote CML pathogenesis. CIP2A and BCR-ABL can regulate each other in a positive feedback loop. CIP2A may be a useful therapeutic target in CML-CP, particularly in patients with IM resistance. How- ever, further studies are needed to validate the interaction between CIP2A and BCR-ABL using other tyrosine kinase inhibitors than IM.

CIP2A is overexpressed and involved in the pathogenesis of chronic myelocytic leukemia by interacting with breakpoint cluster region-Abelson leukemia virus.

To detect the expression of cancerous inhibitor of phosphatase 2A (CIP2A) in chronic myelocytic leukemia (CML) and investigate the mechanism underlying CIP2A knockdown-mediated cell proliferation and apoptosis as well as the interaction of CIP2A with breakpoint cluster region-Abelson leukemia virus (BCR-ABL). CIP2A mRNA and protein expression in chronic myelocytic leukemia-chronic (CML-CP) patients and healthy controls were determined by RT-PCR and Western blot. In vivo, c-Myc expression, PP2A activity, cell proliferation, and apoptosis of CML cells were detected with CIP2A depletion. In addition, the relationship among CIP2A, BCR-ABL, and tyrosine phosphatase SHP-1 was explored by depleting/overexpressing CIP2A or inhibiting BCR-ABL. The level of CIP2A mRNA was higher in CML-CP patients than healthy controls (56/74, 75.7 % vs. 1/35, 2.9 %, P < 0.001), and CIP2A protein was overexpressed in corresponding specimens. CIP2A knockdown by siRNA reduced the level of c-Myc protein and clonogenic formation, inhibited the activity of PP2A, K562 cell proliferation, and promoted cell apoptosis. Suppressing BCR-ABL by imatinib mesylate (IM) significantly decreased CIP2A expression. CIP2A knockdown decreased BCR-ABL but increased SHP-1 expression, and CIP2A overexpression had the reverse effect. CIP2A is overexpressed in CML-CP patients, and its expression may promote CML pathogenesis. CIP2A and BCR-ABL can regulate each other in a positive feedback loop. CIP2A may be a useful therapeutic target in CML-CP, particularly in patients with IM resistance. However, further studies are needed to validate the interaction between CIP2A and BCR-ABL using other tyrosine kinase inhibitors than IM.