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.

The luciferase-based in vivo protein-protein interaction assay revealed that CHK1 promotes PP2A and PME-1 interaction.

Protein phosphatase 2A (PP2A) is an essential serine/threonine protein phosphatase, and its dysfunction is involved in the onset of cancer and neurodegenerative disorders. PP2A functions as a trimeric holoenzyme whose composition is regulated by the methyl-esterification (methylation) of the PP2A catalytic subunit (PP2Ac). Protein phosphatase methylesterase-1 (PME-1) is the sole PP2Ac methylesterase, and the higher PME-1 expression is observed in various cancer and neurodegenerative diseases. Apart from serving as a methylesterase, PME-1 acts as a PP2A inhibitory protein, binding directly to PP2Ac and suppressing its activity. The intricate function of PME-1 hinders drug development by targeting the PME-1/PP2Ac axis. This study applied the NanoBiT system, a bioluminescence-based protein interaction assay, to elucidate the molecular mechanism that modulates unknown PME-1/PP2Ac protein-protein interaction (PPI). Compound screening identified that the CHK1 inhibitors inhibited PME-1/PP2Ac association without affecting PP2Ac methylation levels. CHK1 directly phosphorylates PP2Ac to promote PME-1 association. Phospho-mass spectrometry identified multiple phospho-sites on PP2Ac, including the Thr219, that affect PME-1 interaction. An anti-phospho-Thr219 PP2Ac antibody was generated and showed that CHK1 regulates the phosphorylation levels of this site in cells. On the contrary, in vitro phosphatase assay showed that CHK1 is the substrate of PP2A, and PME-1 hindered PP2A-mediated dephosphorylation of CHK1. Our data provides novel insights into the molecular mechanisms governing the PME-1/PP2Ac PPI and the triad relationship between PP2A, PME-1, and CHK1.

Protein phosphatase 6 promotes stemness of colorectal cancer cells.

Colorectal cancer (CRC) remains a significant global health concern, demanding a more profound comprehension of its molecular foundations for the development of improved therapeutic strategies. This study aimed to elucidate the role of protein phosphatase 6 (PP6), a member of the type 2A protein phosphatase family, in CRC. Protein phosphatase 6 functions as a heterotrimer with a catalytic subunit (PP6c), regulatory subunits (PP6Rs; PP6R1, PP6R2, and PP6R3), and scaffold subunits (ANKRD28, ANKRD44, and ANKRD52). Elevated PP6c expression has been identified in CRC tissues compared to normal mucosa, aligning with its potential involvement in CRC pathogenesis. PP6c knockdown resulted in decreased colony-forming ability and in vivo proliferation of various CRC cell lines. Transcriptome analysis revealed that PP6c knockdown resulted in altered expression of genes associated with cancer stemness. Notably, the PP6c-PP6R3 complex is a key player in regulating cancer stem cell (CSC) markers. Additionally, increased PP6c expression was observed in CSC-like cells induced by sphere formation, implicating the role of PP6c in CSC maintenance. This study highlights the role of PP6c in CRC and suggests that it is a potential therapeutic target disrupting a pathway critical for CRC progression and stem cell maintenance.

Transcriptome analysis revealed that PME-1 suppresses inflammatory signaling, activates PI3K/Akt signaling, and promotes epithelial-mesenchymal transition.

Protein phosphatase 2A (PP2A) is an essential serine/threonine protein phosphatase that belongs to the type2A protein phosphatase family with PP4 and PP6. PP2A functions as a trimeric holoenzyme, and the composition of the trimer is regulated by the methyl-esterification (methylation) of PP2A. Demethylation of PP2A is catalyzed by protein phosphatase methyl-esterase-1 (PME-1). Despite the physiological and pathophysiological importance of PME-1, the impact of changes in PME-1 expression on the transcriptome has not been reported. This study provides transcriptome data to gain a comprehensive understanding of the effects of PME-1 knockout on intracellular signaling of mouse embryonic fibroblasts. Our data showed that PME-1 suppresses inflammatory signaling, activates PI3K/Akt signaling, and promotes epithelial-mesenchymal transition.

The impact of SETBP1 mutations in neurological diseases and cancer.

SE translocation (SET) is a cancer-promoting factor whose expression is upregulated in many cancers. High SET expression positively correlates with a poor cancer prognosis. SETBP1 (SET-binding protein 1/SEB/MRD29), identified as SET-binding protein, is the causative gene of Schinzel-Giedion syndrome, which is characterized by severe intellectual disability and a distorted facial appearance. Mutations in these genetic regions are also observed in some blood cancers, such as myelodysplastic syndromes, and are associated with a poor prognosis. However, the physiological role of SETBP1 and the molecular mechanisms by which the mutations lead to disease progression have not yet been fully elucidated. In this review, we will describe the current epidemiological data on SETBP1 mutations and shed light on the current knowledge about the SET-dependent and -independent functions of SETBP1.

Characterization of RNF43 frameshift mutations that drive Wnt ligand- and R-spondin-dependent colon cancer.

Loss-of-function mutations in RNF43 induce activation of Wnt ligand-dependent Wnt/ß-catenin signaling through stabilization of the Frizzled receptor, which is often found in microsatellite instability (MSI)-type colorectal cancer (CRC) that develops from sessile serrated adenomas. However, the mechanism underlying how RNF43 mutations promote tumorigenesis remains poorly understood. In this study, we established nine human CRC-derived organoids and found that three organoid lines carried RNF43 frameshift mutations associated with MSI-high and BRAFV600E mutations, suggesting that these CRCs developed through the serrated pathway. RNF43 frameshift mutant organoids required both Wnt ligands and R-spondin for proliferation, indicating that suppression of ZNRF3 and retained RNF43 function by R-spondin are required to achieve an indispensable level of Wnt activation for tumorigenesis. However, active ß-catenin levels in RNF43-mutant organoids were lower than those in APC two-hit mutant CRC, suggesting a lower threshold for Wnt activation in CRC that developed through the serrated pathway. Interestingly, transplantation of RNF43-mutant organoids with intestinal myofibroblasts accelerated the ß-catenin nuclear accumulation and proliferation of xenograft tumors, indicating a key role of stromal cells in the promotion of the malignant phenotype of RNF43-mutant CRC cells. Sequencing of subcloned organoid cell-expressed transcripts revealed that two organoid lines carried monoallelic RNF43 cis-mutations, with two RNF43 frameshift mutations introduced in the same allele and the wild-type RNF43 allele remaining, while the other organoid line carried two-hit biallelic RNF43 trans-mutations. These results suggest that heterozygous RNF43 frameshift mutations contribute to CRC development via the serrated pathway; however, a second-hit RNF43 mutation may be advantageous in tumorigenesis compared with a single-hit mutation through further activation of Wnt signaling. Finally, treatment with the PORCN inhibitor significantly suppressed RNF43-mutant cell-derived PDX tumor development. These results suggest a novel mechanism underlying RNF43 mutation-associated CRC development and the therapeutic potential of Wnt ligand inhibition against RNF43-mutant CRC. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Protein phosphatase 6 dissociates the Beclin 1/Vps34 complex and inhibits autophagy.

Autophagy is an evolutionarily conserved intracellular degradation system and is regulated by various signaling pathways including the Beclin 1/Vacuolar protein sorting 34 (Vps34) complex. Protein phosphatase 6 (PP6) is an essential serine/threonine phosphatase that regulates various biological processes. Recently, we found that PP6 protein is degraded by p62-dependent selective autophagy. In this study, we show that PP6 conversely inhibits autophagy. PP6 associate with the C-terminal region of Beclin 1, which is close to the binding region of Vps34. The protein levels of PP6 affect Beclin 1/Vps34 complex formation and phosphatase activity of PP6 is not involved in this. We also show that chemically induced PP6/Beclin 1 association leads to Vps34 dissociation from Beclin 1. Overall, our data reveal a novel regulatory mechanism for autophagy by PP6.

Autophagy regulates levels of tumor suppressor enzyme protein phosphatase 6.

Protein phosphatase 6 (PP6) is an essential serine/threonine protein phosphatase that acts as an important tumor suppressor. However, increased protein levels of PP6 have been observed in some cancer types, and they correlate with poor prognosis in glioblastoma. This raises a question about how PP6 protein levels are regulated in normal and transformed cells. In this study, we show that PP6 protein levels increase in response to pharmacologic and genetic inhibition of autophagy. PP6 associates with autophagic adaptor protein p62/SQSTM1 and is degraded in a p62-dependent manner. Accordingly, protein levels of PP6 and p62 fluctuate in concert under different physiological and pathophysiological conditions. Our data reveal that PP6 is regulated by p62-dependent autophagy and suggest that accumulation of PP6 protein in tumor tissues is caused at least partially by deficiency in autophagy.

Established fibrous peritoneal metastasis in an immunocompetent mouse model similar to clinical immune microenvironment of gastric cancer.

BACKGROUND: Peritoneal metastasis (PM) in gastric cancer (GC) is characterized by diffusely infiltrating and proliferating cancer cells accompanied by extensive stromal fibrosis in the peritoneal space. The prognosis of GC with PM is still poor regardless of the various current treatments. In order to elucidate the cause of difficulties in PM treatment, we compared the tumor immune microenvironment (TME) in primary and PM lesions in GC. In addition, a PM model with fibrous stroma was constructed using immunocompetent mice to determine whether its TME was similar to that in patients. METHODS: Immuno-histochemical analyses of infiltrating immune cells were performed in paired primary and PM lesions from 28 patients with GC. A C57BL/6 J mouse model with PM was established using the mouse GC cell line YTN16 either with or without co-inoculation of mouse myofibroblast cell line LmcMF with α-SMA expression. The resected PM from each mouse model was analyzed the immunocompetent cells using immunohistochemistry. RESULTS: The number of CD8+ cells was significantly lower in PM lesions than in primary lesions (P < 0.01). Conversely, the number of CD163+ cells (M2 macrophages) was significantly higher in PM lesions than in primary lesions (P = 0.016). Azan staining revealed that YTN16 and LmcMF co-inoculated tumors were more fibrous than tumor with YTN16 alone (P < 0.05). Co-inoculated fibrous tumor also showed an invasive growth pattern and higher progression than tumor with YTN16 alone (P = 0.045). Additionally, YTN16 and LmcMF co-inoculated tumors showed lower infiltration of CD8+ cells and higher infiltration of M2 macrophages than tumors with YTN16 alone (P < 0.05, P < 0.05). These results indicate that LmcMF plays as cancer-associated fibroblasts (CAFs) by crosstalk with YTN16 and CAFs contribute tumor progression, invasion, fibrosis, and immune suppression. CONCLUSIONS: This model is the first immunocompetent mouse model similar to TME of human clinical PM with fibrosis. By using this model, new treatment strategies for PM, such as anti-CAFs therapies, may be developed.

Hedgehog Signals Mediate Anti-Cancer Drug Resistance in Three-Dimensional Primary Colorectal Cancer Organoid Culture.

Colorectal cancer is one of the most common causes of cancer death worldwide. In patients with metastatic colorectal cancer, combination treatment with several anti-cancer drugs is employed and improves overall survival in some patients. Nevertheless, most patients with metastatic disease are not cured owing to the drug resistance. Cancer stem cells are known to regulate resistance to chemotherapy. In the previous study, we established a novel three-dimensional organoid culture model from tumor colorectal tissues of human patients using an air-liquid interface (ALI) method, which contained numerous cancer stem cells and showed resistance to 5-fluorouracil (5-FU) and Irinotecan. Here, we investigate which inhibitor for stem cell-related signal improves the sensitivity for anti-cancer drug treatment in tumor ALI organoids. Treatment with Hedgehog signal inhibitors (AY9944, GANT61) decreases the cell viability of organoids compared with Notch (YO-01027, DAPT) and Wnt (WAV939, Wnt-C59) signal inhibitors. Combination treatment of AY9944 or GANT61 with 5-FU, Irinotecan or Oxaliplatin decreases the cell viability of tumor organoids compared with each anti-cancer drug alone treatment. Treatment with AY9944 or GANT61 inhibits expression of stem cell markers c-Myc, CD44 and Nanog, likely through the decrease of their transcription factor, GLI-1 expression. Combination treatment of AY9944 or GANT61 with 5-FU or Irinotecan also prevents colony formation of colorectal cancer cell lines HCT116 and SW480. These findings suggest that Hedgehog signals mediate anti-cancer drug resistance in colorectal tumor patient-derived ALI organoids and that the inhibitors are useful as a combinational therapeutic strategy against colorectal cancer.

Regulation of Beclin 1 Protein Phosphorylation and Autophagy by Protein Phosphatase 2A (PP2A) and Death-associated Protein Kinase 3 (DAPK3).

Autophagy is an evolutionarily conserved intracellular degradation system that is involved in cell survival and activated in various diseases, including cancer. Beclin 1 is a central scaffold protein that assembles components for promoting or inhibiting autophagy. Association of Beclin 1 with its interacting proteins is regulated by the phosphorylation of Beclin 1 by various Ser/Thr kinases, but the Ser/Thr phosphatases that regulate these phosphorylation events remain unknown. Here we identify Ser-90 in Beclin 1 as a regulatory site whose phosphorylation is markedly enhanced in cells treated with okadaic acid, an inhibitor of protein phosphatase 2A (PP2A). Beclin 1 Ser-90 phosphorylation is induced in skeletal muscle tissues isolated from starved mice. The Beclin 1 S90A mutant blocked starvation-induced autophagy. We found association of PP2A B55α with Beclin 1, which dissociate by starvation. We also found that death-associated protein kinase 3 directly phosphorylates Beclin 1 Ser-90. We propose that physiological regulation of Beclin 1 Ser-90 phosphorylation by PP2A and death-associated protein kinase 3 controls autophagy.

Establishment of a dog primary prostate cancer organoid using the urine cancer stem cells.

Dog spontaneously develop prostate cancer (PC) like humans. Because most dogs with PC have a poor prognosis, they could be used as a translational model for advanced PC in humans. Stem cell-derived 3-D organoid culture could recapitulate organ structures and physiology. Using patient tissues, a human PC organoid culture system was established. Recent study has shown that urine cells also possess the characteristic of stem cells. However, urine cell-derived PC organoids have never been produced. Therefore, we generated PC organoids using the dog urine samples. Urine organoids were successfully generated from each dog with PC. Each organoid showed cystic structures and resembled the epithelial structures of original tissues. Expression of an epithelial cell marker, E-cadherin, and a myofibloblast marker, α-SMA, was observed in the urine organoids. The organoids also expressed a basal cell marker, CK5, and a luminal cell marker, CK8. CD49f-sorted basal cell organoids rapidly grew compared with CD24-sorted luminal cell organoids. The population of CD44-positive cells was the highest in both organoids and the original urine cells. Tumors were successfully formed with the injection of the organoids into immunodeficient mice. Treatment with a microtubule inhibitor, docetaxel, but not a cyclooxygenase inhibitor, piroxicam, and an mTOR inhibitor, rapamycin, decreased the cell viability of organoids. Treatment with a Hedgehog signal inhibitor, GANT61, increased the radiosensitivity in the organoids. These findings revealed that PC organoids using urine might become a useful tool for investigating the mechanisms of the pathogenesis and treatment of PC in dogs.

Protein Ser/Thr phosphatase-6 is required for maintenance of E-cadherin at adherens junctions.

BACKGROUND: Epithelial tissues depend on intercellular homodimerization of E-cadherin and loss of E-cadherin is central to the epithelial to mesenchymal transition seen in multiple human diseases. Signaling pathways regulate E-cadherin function and cellular distribution via phosphorylation of the cytoplasmic region by kinases such as casein kinases but the protein phosphatases involved have not been identified. RESULTS: This study shows protein Ser/Thr phosphatase-6 catalytic subunit (PP6c) is expressed in epithelial tissue and its mRNA and protein are robustly up-regulated in epithelial cell lines at high vs. low density. PP6c accumulates at adherens junctions, not tight junctions, co-immunoprecipitates with E-cadherin-catenin complexes without a canonical SAPS subunit, and associates directly with the E-cadherin cytoplasmic tail. Inducible shRNA knockdown of PP6c dispersed E-cadherin from the cell surface and this response was reversed by chemical inhibition of casein kinase-1 and prevented by alanine substitution of Ser846 in murine E-cadherin. CONCLUSIONS: PP6c associates with E-cadherin in adherens junctions and is required to oppose casein kinase-1 to maintain cell surface localization of E-cadherin. There is feedback signaling to enhance PP6c transcription and boost protein levels in high density epithelial cells.

Protein phosphatase 6 promotes transforming growth factor-ß signaling in mouse embryonic fibroblasts.

Transforming growth factor-beta (TGF-ß) is a multifunctional cytokine that controls various cellular processes. Protein phosphatase 6 (PP6) is an evolutionarily conserved serine/threonine protein phosphatase with diverse functions in cell signaling. However, it has not been linked to TGF-ß signaling. We found that TGF-ß treatment increased PP6 protein levels via transcriptional and post-translational regulation. Loss of the Ppp6c gene suppressed TGF-ß-induced canonical Smad3 phosphorylation and its transcriptional activity. PP6 knockout also inhibited non-canonical p38 mitogen-activated protein kinase (MAPK) pathway. Moreover, PP6 depletion suppressed cell migration induced by TGF-ß. These findings uncovered the role of PP6 as a positive regulator for TGF-ß signaling.

Age-related alterations in protein phosphatase 2A methylation levels in brains of cynomolgus monkeys: a pilot study.

The abnormal activity of PP2A, a dominant member of type 2A serine/threonine protein phosphatase, has been implicated in the development of Alzheimer's disease (AD) and dementia with Lewy bodies (DLB). PP2A is a holoenzyme, and protein methylation of the catalytic subunit, PP2Ac, alters the complex composition. A decrease in PP2Ac methylation levels has been reported in AD and DLB. Aging is the most common risk factor for AD and DLB, but the relationship between aging and PP2A has not been studied in detail. Cynomolgus monkey show increased phosphorylation levels of tau and α-synuclein with aging. In this study, we investigated the alterations in the PP2A activity regulation with aging in monkey brains from 2 to 43 years of age using fractionated proteins. We found that type 2A protein phosphatase activity decreased with aging in cytoplasmic and nuclear-soluble fractions. PP2Ac methylation level was decreased in cytoplasmic and sarkosyl-insoluble fractions. A principal component analysis using PP2Ac, demethylated PP2Ac and PP2A methylesterase PME-1 levels in cytoplasmic and nuclear-soluble fractions as attributes showed that aged monkeys were in the same cluster. Our results show that brain aging in cynomolgus monkeys is closely related to changes in PP2A methylation.

Cancer-specific apoptosis induction in canine lymphoma cell lines by the endocytosis inhibitor dynasore.

Canine lymphoma is the most common cancer in dogs and has a poor prognosis. We recently found that the endocytosis inhibitor dynasore suppresses the viability of human cancer cell lines, especially hematopoietic cancers, by inducing apoptosis. In the present study, we examined the anticancer effects of dynasore on five previously established canine lymphoma cell lines (CLBL-1, Ema, Nody-1, CLC, and GL-1). Dynasore suppressed cell viability in these canine lymphoma cell lines more effectively than in human cancer cell lines. It also induced apoptosis in CLBL-1 and Ema cells but not in peripheral blood mononuclear cells in healthy dogs or in Madin-Darby canine kidney (MDCK) cells, suggesting that the ability of dynasore to induce apoptosis is cancer-specific. Furthermore, dynasore induced a DNA damage response in CLBL-1 and Ema cells, suggesting that it acts as a genotoxic agent in canine lymphoma cell lines. These findings suggest that endocytosis inhibitors may provide a new anticancer treatment for canine lymphoma.

The protein level of the tumour-promoting factor SET is regulated by cell density.

SET/I2PP2A is a multifunctional protein that acts as an intrinsic inhibitor of the tumour suppressor protein phosphatase 2A and as a histone chaperone. Increased SET levels have been observed in various cancers; however, the underlying molecular mechanisms remain unclear. In this study, we found that SET protein accumulates with the increasing density of cultured cells. This phenomenon was observed not only in cancer cell lines but also in non-cancer cell lines. The mRNA levels of SET were not affected by the cell density. Proteasome inhibition decreased SET levels, whereas autophagy inhibition led to SET accumulation, indicating the involvement of autophagy. The mRNA and protein expression of SETBP1, which stabilizes the SET protein, increased with cell density. The decrease in SET level due to the loss of SETBP1 was more pronounced in wild-type cells than that in autophagy-deficient cells. These results have revealed a mechanism underlying the regulation of SET level, wherein increased cell density induces SETBP1 expression and protects SET from autophagy.

Endotoxin conditioning induces VCP/p97-mediated and inducible nitric-oxide synthase-dependent Tyr284 nitration in protein phosphatase 2A.

Endotoxins activate Toll-like receptors and reprogram cells to be refractory to secondary exposure. Here we found that activation of different Toll-like receptors elicited a time- and dose-dependent increase in the levels of the protein phosphatase 2A catalytic subunit (PP2Ac) but not its partner A subunit. We purified the lipopolysaccharide-induced form of PP2A by chromatography plus immunoprecipitation and used mass spectrometry to identify VCP/p97 as a novel partner for PP2Ac. Endogenous VCP/p97 and PP2Ac were co-immunoprecipitated from primary murine macrophages and human lymphocytes. GST-VCP/p97 bound purified PP2A in pulldown assays, showing direct protein-protein interaction. Endotoxin conditioning of macrophages induced formation of 3-nitrotyrosine in the PP2Ac associated with VCP/p97, a response severely reduced in macrophages from iNOS knock-out mice. The reaction of purified PP2A with peroxynitrite dissociated the A subunit, and 3-nitro-Tyr(284) was identified in PP2Ac by mass spectrometry. Myc-PP2Ac (Y284F) expressed in cells was resistant to peroxynitrite-induced nitration and reduction of A subunit binding. Transient expression of either VCP/p97 or PP2Ac was sufficient to elevate levels of the dual specificity phosphatase DUSP1, reduce p38 MAPK activation, and suppress tumor necrosis factor-alpha release. We propose that VCP/p97-mediated Tyr nitration of PP2A increases the levels of phosphatases PP2A and DUSP1 to contribute to the refractory response of conditioned cells.

Protein phosphatase 6 promotes neurite outgrowth by promoting mTORC2 activity in N2a cells.

Understanding the molecular mechanism of neuronal differentiation is important to overcome the incurable diseases caused by nervous system damage. Neurite outgrowth is prerequisite for neuronal differentiation and regeneration, and cAMP response element-binding protein (CREB) is one of the major transcriptional factors positively regulating this process. Neuronal differentiation stimuli activate mammalian target of rapamycin (mTOR) complex 2 (mTORC2)/Akt signalling to phosphorylate CREB; however, the precise molecular mechanism of this event has not been fully understood. In this manuscript, we show that neuronal differentiation stimuli increased a protein level of protein phosphatase 6 (PP6), a member of type 2A Ser/Thr protein phosphatases. PP6 knockdown suppressed mTORC2/Akt/CREB signalling and results in failure of neurite outgrowth. SIN1 is a unique component of mTORC2 that enhances mTORC2 activity towards Akt when it is in dephosphorylated form. We found PP6 knockdown increased SIN1 phosphorylation. These data suggest that PP6 may positively regulate neurite outgrowth by dephosphorylating SIN1 to activate mTORC2/Akt/CREB signalling.

Perphenazine exerts antitumor effects on HUT78 cells through Akt dephosphorylation by protein phosphatase 2A.

Sezary syndrome is a rare type of non-Hodgkin lymphoma. Protein phosphatase 2A (PP2A) is an important tumor suppressor whose activity is widely inhibited in a variety of tumors. Recently, reactivation of PP2A has attracted increasing attention as a promising approach for cancer therapy. Phenothiazine anti-psychotic perphenazine (PPZ) exerts antitumor effects by reactivating PP2A. The present study investigated the molecular mechanism underling the antitumor effects of PPZ in the neuroblastoma rat sarcoma oncogene (NRAS)-mutated Sezary syndrome cell line, HUT78. The results of the present study demonstrated that PPZ induced the dephosphorylation of Akt and ERK1/2, and triggered apoptosis in HUT78 cells. In addition, a PP2A inhibitor blocked the PPZ-mediated dephosphorylation of Akt but did not affect that of ERK1/2. The pharmacological inhibition of Akt and ERK1/2 signaling revealed that Akt activity serves an important role in the survival of HUT78 cells. The present data suggested that suppressing Akt activity by PP2A activation may be an attractive antitumor strategy for NRAS-mutated Sezary syndrome.