microRNA-665 is down-regulated in gastric cancer and inhibits proliferation, invasion, and EMT by targeting PPP2R2A.

Recently, microRNA-665 (miR-665) has been reported to function as both tumour suppressor and oncogene in several cancer types, including gastric cancer, hepatocellular cancer, and lung cancer. However, the biological function of miR-665 and its precise mechanisms in gastric cancer (GC) have not been well clarified. The aim of this study was to study the roles of miR-665/PPP2R2A axis in GC. The levels of PPP2R2A and miR-665 were detected by quantitative PCR assay in GC tissues and cell lines. Moreover, the biological roles of miR-665 and PPP2R2A in GC cells were assessed by cell proliferation, invasion, and epithelial-mesenchymal transition (EMT). The mRNA and protein levels of PPP2R2A were determined by using quantitative PCR and Western blotting assays. Luciferase assays were used to confirm that PPP2R2A was one target of miR-665. In this study, the miR-665 level was dramatically reduced in GC tissues and cell lines, and the PPP2R2A expression was significantly enhanced. What is more, the PPP2R2A expression was negatively related to the miR-665 level in GC tissues. Furthermore, up-regulation of miR-665 obviously restrained GC cells proliferation, invasion, and EMT. We confirmed that miR-665 could directly target PPP2R2A by luciferase reporter assay. Besides, knockdown of PPP2R2A also could markedly inhibit the proliferation, invasion and EMT of GC cells. Finally, overexpression of miR-665 in GC cells partially reversed the promoted effects of PPP2R2A up-regulation. Overexpression of miR-665 restrained GC cells proliferation, invasion and EMT via regulation of PPP2R2A. SIGNIFICANCE OF THE STUDY: miR-665 has been reported to function as oncogene or tumour suppressor in different cancers. However, the precise roles of miR-665 in GC have not been elucidated. Our study for the first time demonstrated that miR-665 level was significantly down-regulated in GC. Additionally, miR-665 overexpression inhibited cell growth, invasion, and EMT of GC. Moreover, our data suggested a significant negative correlation between miR-665 and PPP2R2A expression in GC. MiR-665 suppressed GC cell proliferation, invasion, and EMT by directly targeting PPP2R2A, which suggested important roles for miR-665/PPP2R2A axis in the GC pathogenesis and its potential application in cancer therapy.

Upregulation of miR-614 promotes proliferation and inhibits apoptosis in ovarian cancer by suppressing PPP2R2A expression.

It has previously been demonstrated that microRNAs (miRNAs) have essential roles and participate in various biological processes by regulating their specific target genes. However, the precise role of miRNAs in ovarian cancer (OC) has not yet been elucidated. The present study demonstrated that miR­614 expression levels were significantly upregulated in OC tissues and cell lines, whereas decreased miR­614 demonstrated opposite effects. Furthermore, gain­of­function and loss­of­function experiments indicated that miR­614 overexpression promoted cell proliferation and suppressed cell apoptosis. Protein phosphatase 2 regulatory subunit B α, (PPP2R2A) was identified as a direct target of miR­614 using western blotting and luciferase reporter assays. Notably, silencing of PPP2R2A counter­acted the effect of miR­614 inhibitor in OC cell proliferation and cell apoptosis. Overall, the data suggested that miR­614 promoted cell proliferation and inhibited cell apoptosis of OC cells by targeting PPP2R2A, and may therefore act as a potential target for OC therapy in the future.

Non-genomic mechanisms of protein phosphatase 2A (PP2A) regulation in cancer.

Propagation of transient signals requires coordinated suppression of antagonistic phosphatase activity. Protein phosphatase 2A (PP2A) is a broad specificity serine/threonine phosphatase that functions as an antagonist of many signaling pathways associated with growth and proliferation, and endogenous inhibitory mechanisms suppress PP2A activity in response to mitogenic stimuli. These inhibitory mechanisms, including expression and activation of endogenous inhibitor proteins and phosphoregulation of PP2A subunits, are also engaged by aberrant constitutive activation of mitogenic pathways in cancer. Inhibition of PP2A activity has been shown to promote malignant transformation and endogenous inhibitory mechanisms of PP2A have been associated with malignant progression and prognosis in a wide range of cancers. Despite existence of recurrent mutations and other genetic and gene regulatory alterationsin PP2A genes, they collectively appear at relatively low frequency, and in only some cancer types. The non-genomic inhibition of PP2A activity by increased expression of endogenous PP2A inhibitor proteins greatly exceeds the frequency of genetic mutations of PP2A genes in human cancers. This feature makes PP2A an untypical tumor suppressor, and may have influenced its recognition as one of the critical human cell transformation mechanisms. We propose that non-genetic inhibition is the dominant mechanism causing loss of PP2A tumor suppressor function in cancer cells, possibly because these mechanisms do not elicit genomic instability associated with genetic loss of function of specific PP2A subunits.

Determination of dendritic spine morphology by the striatin scaffold protein STRN4 through interaction with the phosphatase PP2A.

Dendritic spines are heterogeneous and exist with various morphologies. Altered spine morphology might underlie the cognitive deficits in neurodevelopmental disorders such as autism, but how different subtypes of dendritic spines are selectively maintained along development is still poorly understood. Spine maturation requires spontaneous activity of N-methyl-d-aspartate (NMDA) receptor and local dendritic protein synthesis. STRN4 (also called zinedin) belongs to the striatin family of scaffold proteins, and some of the potential striatin-interacting proteins are encoded by autism risk genes. Although previous studies have demonstrated their localization in dendritic spines, the function of various striatin family members in the neuron remains unknown. Here, we demonstrate that Strn4 mRNA is present in neuronal dendrites, and the local expression of STRN4 protein depends on NMDA receptor activation. Notably, STRN4 is preferentially expressed in mushroom spines, and STRN4 specifically maintains mushroom spines but not thin spines and filopodia through interaction with the phosphatase PP2A. Our findings have therefore unraveled the local expression of STRN4 as a novel mechanism for the control of dendritic spine morphology.

Self-adjuvanting C18 lipid vinil sulfone-PP2A vaccine: study of the induced immunomodulation against Trichuris muris infection.

Despite the importance of the adjuvant in the immunization process, very few adjuvants merge with the antigens in vaccines. A synthetic self-adjuvant oleic-vinyl sulfone (OVS) linked to the catalytic region of recombinant serine/threonine phosphatase 2A from the nematode Angiostrongylus costaricensis (rPP2A) was used for intranasal immunization in mice previously infected with Trichuris muris The animal intranasal immunization with rPP2A-OVS showed a reduction of 99.01% in the number of the nematode eggs and 97.90% in adult. The immunohistochemical analysis of the intestinal sections showed that in immunized animals with lipopeptide the mucus was significantly higher than in the other experimental groups. Also, these animals presented significantly different chemokine, CCL20 and CCL11, levels. However, although the number and size of Tuft cells did not vary between groups, the intensity of fluorescence per cell was significant in the group immunized with the rPP2A-OVS. The results of the present study suggest that mice immunized with the lipopeptide are capable of activating a combined Th17/Th9 response. This strategy of immunization may be of great applicability not only in immunotherapy and immunoprophylaxis to control diseases caused by nematodes but also in pathologies necessitating action at the level of the Th9 response in the intestinal mucosa.

Promotion of Cell Viability and Histone Gene Expression by the Acetyltransferase Gcn5 and the Protein Phosphatase PP2A in Saccharomyces cerevisiae.

Histone modifications direct chromatin-templated events in the genome and regulate access to DNA sequence information. There are multiple types of modifications, and a common feature is their dynamic nature. An essential step for understanding their regulation, therefore, lies in characterizing the enzymes responsible for adding and removing histone modifications. Starting with a dosage-suppressor screen in Saccharomyces cerevisiae, we have discovered a functional interaction between the acetyltransferase Gcn5 and the protein phosphatase 2A (PP2A) complex, two factors that regulate post-translational modifications. We find that RTS1, one of two genes encoding PP2A regulatory subunits, is a robust and specific high-copy suppressor of temperature sensitivity of gcn5∆ and a subset of other gcn5∆ phenotypes. Conversely, loss of both PP2A(Rts1) and Gcn5 function in the SAGA and SLIK/SALSA complexes is lethal. RTS1 does not restore global transcriptional defects in gcn5∆; however, histone gene expression is restored, suggesting that the mechanism of RTS1 rescue includes restoration of specific cell cycle transcripts. Pointing to new mechanisms of acetylation-phosphorylation cross-talk, RTS1 high-copy rescue of gcn5∆ growth requires two residues of H2B that are phosphorylated in human cells. These data highlight the potential significance of dynamic phosphorylation and dephosphorylation of these deeply conserved histone residues for cell viability.

eEF-2 kinase is a critical regulator of Warburg effect through controlling PP2A-A synthesis.

Cancer cells predominantly metabolize glucose by glycolysis to produce energy in order to meet their metabolic requirement, a phenomenon known as Warburg effect. Although Warburg effect is considered a peculiarity critical for survival and proliferation of cancer cells, the regulatory mechanisms behind this phenomenon remain incompletely understood. We report here that eukaryotic elongation factor-2 kinase (eEF-2K), a negative regulator of protein synthesis, has a critical role in promoting glycolysis in cancer cells. We showed that deficiency in eEF-2K significantly reduced the uptake of glucose and decreased the productions of lactate and adenosine triphosphate in tumor cells and in the Ras-transformed mouse embryonic fibroblasts. We further demonstrated that the promotive effect of eEF-2K on glycolysis resulted from the kinase-mediated restriction of synthesis of the protein phosphatase 2A-A (PP2A-A), a key factor that facilitates the ubiquitin-proteasomal degradation of c-Myc protein, as knockdown of eEF-2K expression led to a significant increase in PP2A-A protein synthesis and remarkable downregulation of c-Myc and pyruvate kinase M2 isoform, the key glycolytic enzyme transcriptionally activated by c-Myc. In addition, depletion of eEF-2K reduced the ability of the transformed cells to proliferate and enhanced the sensitivity of tumor cells to chemotherapy both in vitro and in vivo. These results, which uncover a role of the eEF-2K-mediated control of PP2A-A in tumor cell glycolysis, provide new insights into the regulation of the Warburg effect.

Blocking protein phosphatase 2A signaling prevents endothelial-to-mesenchymal transition and renal fibrosis: a peptide-based drug therapy.

Endothelial-to-mesenchymal transition (EndMT) contributes to the emergence of fibroblasts and plays a significant role in renal interstitial fibrosis. Protein phosphatase 2A (PP2A) is a major serine/threonine protein phosphatase in eukaryotic cells and regulates many signaling pathways. However, the significance of PP2A in EndMT is poorly understood. In present study, the role of PP2A in EndMT was evaluated. We demonstrated that PP2A activated in endothelial cells (EC) during their EndMT phenotype acquisition and in the mouse model of obstructive nephropathy (i.e., UUO). Inhibition of PP2A activity by its specific inhibitor prevented EC undergoing EndMT. Importantly, PP2A activation was dependent on tyrosine nitration at 127 in the catalytic subunit of PP2A (PP2Ac). Our renal-protective strategy was to block tyrosine127 nitration to inhibit PP2A activation by using a mimic peptide derived from PP2Ac conjugating a cell penetrating peptide (CPP: TAT), termed TAT-Y127WT. Pretreatment with TAT-Y127WT was able to prevent TGF-ß1-induced EndMT. Administration of the peptide to UUO mice significantly ameliorated renal EndMT level, with preserved density of peritubular capillaries and reduction in extracellular matrix deposition. Taken together, these results suggest that inhibiting PP2Ac nitration using a mimic peptide is a potential preventive strategy for EndMT in renal fibrosis.

MiR-338-5p sensitizes glioblastoma cells to radiation through regulation of genes involved in DNA damage response.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor. Despite radical surgery and radiotherapy supported by chemotherapy, the disease still remains incurable with an extremely low median survival rate of 12-15 months from the time of initial diagnosis. The main cause of treatment failure is considered to be the presence of cells that are resistant to the treatment. MicroRNAs (miRNAs) as regulators of gene expression are involved in the tumor pathogenesis, including GBM. MiR-338 is a brain-specific miRNA which has been described to target pathways involved in proliferation and differentiation. In our study, miR-338-3p and miR-338-5p were differentially expressed in GBM tissue in comparison to non-tumor brain tissue. Overexpression of miR-338-3p with miRNA mimic did not show any changes in proliferation rates in GBM cell lines (A172, T98G, U87MG). On the other hand, pre-miR-338-5p notably decreased proliferation and caused cell cycle arrest. Since radiation is currently the main treatment modality in GBM, we combined overexpression of pre-miR-338-5p with radiation, which led to significantly decreased cell proliferation, increased cell cycle arrest, and apoptosis in comparison to irradiation-only cells. To better elucidate the mechanism of action, we performed gene expression profiling analysis that revealed targets of miR-338-5p being Ndfip1, Rheb, and ppp2R5a. These genes have been described to be involved in DNA damage response, proliferation, and cell cycle regulation. To our knowledge, this is the first study to describe the role of miR-338-5p in GBM and its potential to improve the sensitivity of GBM to radiation.

Upregulation of PP2Ac predicts poor prognosis and contributes to aggressiveness in hepatocellular carcinoma.

Protein phosphatase 2A (PP2A) is a heterotrimeric protein phosphatase consisting of a 36-kD catalytic C subunit (PP2Ac). This study aimed to explore the prognostic and biological significance of PP2Ac in human hepatocellular carcinoma (HCC). High PP2Ac expression was significantly (P < 0.01) associated with serum hepatitis B surface antigen positivity, serum hepatitis B e antigen positivity, liver cirrhosis, moderate to poor differentiation grade, advanced disease stage, intrahepatic metastasis, and early recurrence in HCC. Multivariate analysis revealed PP2Ac as an independent prognostic factor for overall survival. Enforced expression of hepatitis B virus X protein (HBx) and its carboxyl-terminal truncated isoform induced PP2Ac expression in HCC cells. Co-immunoprecipitation assay revealed a direct interaction between PP2Ac and HBx. Small interfering RNA-mediated knockdown of PP2Ac significantly inhibited in vitro cell proliferation, colony formation, migration, and invasion and reduced tumor growth in an xenograft mouse model. In contrast, overexpression of PP2Ac promoted HCC cell proliferation, colony formation, and tumorigenesis. Additionally, silencing of PP2Ac impaired the growth-promoting effects on HepG2 HCC cells elicited by overexpression of carboxyl-terminal truncated HBx. Gene expression profiling analysis showed that PP2Ac downregulation modulated the expression of numerous genes involved in cell cycle and apoptosis regulation. Collectively, PP2Ac upregulation has a poor prognostic impact on the overall survival of HCC patients and contributes to the aggressiveness of HCC. PP2Ac may represent a potential therapeutic target for HCC.

RhoB loss induces Rac1-dependent mesenchymal cell invasion in lung cells through PP2A inhibition.

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, which is mainly due to its high risk of metastatic dissemination. One critical point of this process is the ability of cancer cells to detach from the primary tumor and migrate through the extracellular matrix; however, the underlying molecular mechanisms are not yet fully understood. In the present study, we identified the small GTPase RhoB as a key regulator of bronchial cell morphology in a three-dimensional (3D) matrix. RhoB loss, which is frequently observed during lung cancer progression, induced an epithelial-mesenchymal transition (EMT) characterized by an increased proportion of invasive elongated cells in 3D. The process was mediated by Slug induction and E-cadherin repression. In addition, downregulation of RhoB induced Akt1 activation, which in turn activated Rac1 through the guanine-exchange factor Trio to control cell shape rearrangement. Further, we provide evidence that RhoB interacted with and positively regulates phosphatase PP2A through the recruitment of its regulatory subunit B55, which was found to be crucial for Akt dephosphorylation. B55 inhibition completely suppressed RhoB-mediated PP2A regulation. Finally, we show that PP2A inactivation, by targeting either its catalytic or its regulatory B55 subunit, completely reversed RhoB-dependent morphological changes and also fully prevented the ability of RhoB to decrease the invasiveness of bronchial cells. Altogether, these results highlight a novel signaling axis and describe new molecular mechanisms that could explain the tumor suppressor role of RhoB in lung cancer. Therefore, we propose that RhoB could be responsible for early metastatic prevention by inhibiting the EMT-derived invasiveness of lung cells through the control of PP2A activity.

Low-dose endothelial monocyte-activating polypeptide-II increases permeability of blood-tumor barrier via a PKC-ζ/PP2A-dependent signaling mechanism.

Our previous study demonstrated that low-dose endothelial monocyte-activating polypeptide-II (EMAP-II) induces blood-tumor barrier (BTB) opening via the RhoA/Rho kinase/protein kinase C (PKC)-α/ß signaling pathway and that PKC-ζ is involved in this process via other mechanisms. In the present study, using an in vitro BTB model, we detected the exact signaling mechanisms by which PKC-ζ activation affects EMAP-II-induced BTB hyperpermeability. Our results showed that three types of serine/threonine (Ser/Thr) protein phosphatases (PPs), namely PP1, PP2A, and PP2B, were expressed by rat brain microvascular endothelial cells (RBMECs). There was an interaction between PKC-ζ and PP2A in RBMECs. In addition, EMAP-II induced a significant increase in both the expression and the activity of PP2A in RBMECs. Inhibition of PKC-ζ with PKC-ζ pseudosubstrate inhibitor (PKC-ζ-PI) completely blocked EMAP-II-induced PP2A activation. Conversely, inhibition of PP2A with okadaic acid (OA) had no effect on EMAP-II-induced PKC-ζ activation. Like PKC-ζ-PI, OA partially prevented EMAP-II-induced BTB hyperpermeability and occludin redistribution in RBMECs. Neither PKC-ζ-PI nor OA affected EMAP-II-induced phosphorylation of myosin light chain and redistribution of actin cytoskeleton in RBMECs. Taken together, our present study demonstrated that low-dose EMAP-II increases BTB permeability by activating the PKC-ζ/PP2A signaling pathway, which consequently leads to the disruption of TJs and impairment of endothelial barrier function.

MicroRNA-136 Promotes Vascular Muscle Cell Proliferation Through the ERK1/2 Pathway by Targeting PPP2R2A in Atherosclerosis.

Aberrant proliferation of vascular smooth muscle cells [VSMCs] is implicated in the pathogenesis of vascular pathologies such as atherosclerosis and restenosis. Accumulating evidences have revealed that microRNAs are involved in cell proliferation in various pathological conditions. In the present study, we showed that miR-136 was up regulated in human coronary atherosclerotic plaques when compared with normal coronary artery tissues. Moreover, miR-136 levels were up regulated in proliferative vascular smooth muscle cells induced by platelet-derived growth factor [PDGF] or serum. In cultured VSMCs, over expression of miR-136 stimulated cell proliferation. PPP2R2A was proved to be the direct target gene of miR-136 and knockdown of PPP2R2A had a proliferative effect on VSMCs. miR-136-induced PPP2R2A down-regulation was accompanied by increased expression of ERK1/2 phosphorylation. Inhibition of ERK1/2 abolished the effect of miR-136 and knockdown of PPP2R2A on VSMCs proliferation. In summary, aberrant miR-136 up regulation in atherosclerosis contributes to abnormal VSMC proliferation through suppressing the ERK1/2 pathway by targeting PPP2R2A. Our study also suggested that specific modulation of miR-136 in human VSMCs may provide a potential approach for the treatment of atherosclerosis.

The protein phosphatase Siw14 controls caffeine-induced nuclear localization and phosphorylation of Gln3 via the type 2A protein phosphatases Pph21 and Pph22 in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae Siw14, a tyrosine phosphatase involved in the response to caffeine, participates in regulation of the phosphorylation and intracellular localization of Gln3, a GATA transcriptional activator of nitrogen catabolite repression-sensitive genes. In Δsiw14 cells, the phosphorylation level of Gln3 is decreased and the nuclear localization of Gln3 is stimulated by caffeine. However, the mechanism by which Siw14 controls the localization and function of Gln3 remains unclear, although the nuclear localization of Gln3 is known to be induced by activation of the type 2A phosphatases (PP2As) Pph21 and Pph22, and the type 2A-related phosphatase Sit4. In this study, we show that the increased nuclear localization of Gln3 in response to caffeine caused by disruption of the SIW14 gene is dependent on the Sit4 and PP2A phosphatases. We also show that decreased phosphorylation of Gln3 caused by disruption of the SIW14 gene is completely suppressed by deletion of both PPH21 and PPH22, but only partially suppressed by deletion of SIT4. Taking these results together, we conclude that Siw14 functions upstream of Pph21 and Pph22 as an inhibitor of the phosphorylation and localization of Gln3, and that Sit4 acts independently of Siw14.

Propofol protects against angiotensin II-induced mouse hippocampal HT22 cells apoptosis via inhibition of p66Shc mitochondrial translocation.

Hippocampal neuronal oxidative stress and apoptosis have been reported to be involved in cognitive impairment, and angiotensin II could induce hippocampal oxidative stress and apoptosis. Propofol is a widely used intravenous anesthetic agent in clinical practice, and it demonstrates significant neuroprotective activities. In this study, we investigated the mechanism how propofol protected mouse hippocampal HT22 cells against angiotensin II-induced oxidative stress and apoptosis. Cell viability was evaluated with CCK8 kit. Protein expressions of active caspase 3, cytochrome c, p66(Shc), p-p66(shc)-Ser(36), protein kinase C ßII (PKCßII), Pin-1 and phosphatase A2 (PP2A) were measured by Western blot. Superoxide anion (O2(.-)) accumulation was measured with the reduction of ferricytochrome c. Compared with the control group, angiotensin II up-regulated expression of PKCßII, Pin-1 and PP2A, induced p66(Shc)-Ser(36) phosphorylation, and facilitated p66(Shc) mitochondrial translocation, resulting in O2(.-) accumulation, mitochondrial cytochrome c release, caspase 3 activation, and the inhibition of cell viability. Importantly, we found propofol inhibited angiotensin II-induced PKCßII and PP2A expression and improved p66(Shc) mitochondrial translocation, O2(.-) accumulation, mitochondrial cytochrome c release, caspase 3 activation, inhibition of cell viability. On the other hand, propofol had no effects on angiotensin II-induced Pin-1 expression and p66(Shc)-Ser(36) phosphorylation. Moreover, the protective effects of propofol on angiotensin II-induced HT22 apoptosis were similar with calyculin A, an inhibitor of PP2A and CGP53353, an inhibitor of PKCßII. However, the protective effect of propofol could be reversed by FTY720, an activator of PP2A, rather than PMA, an activator of PKCßII. Our data indicated that propofol down-regulated PP2A expression, inhibiting dephosphorylation of p66(Shc)-Ser(36) and p66(Shc) mitochondrial translocation, decreasing O2(.-) accumulation, reducing mitochondrial cytochrome c release, inhibiting caspase 3 activation. By these mechanisms, it protects mouse hippocampal HT22 cells against angiotensin II-induced apoptosis.

Intermittent hypoxia-induced protein phosphatase 2A activation reduces PC12 cell proliferation and differentiation.

BACKGROUND: Intermittent hypoxia (IH) plays a critical role in sleep breathing disorder-associated hippocampus impairments, including neurocognitive deficits, irreversible memory and learning impairments. IH-induced neuronal injury in the hippocampus may result from reduced precursor cell proliferation and the relative numbers of postmitotic differentiated neurons. However, the mechanisms underlying IH-induced reactive oxygen species (ROS) generation effects on cell proliferation and neuronal differentiation remain largely unknown. RESULTS: ROS generation significantly increased after 1-4 days of IH without increased pheochromocytoma-12 (PC12) cell death, which resulted in increased protein phosphatase 2A (PP2A) mRNA and protein levels. After 3-4 days of IH, extracellular signal-regulated kinases 1/2 (ERK1/2) protein phosphorylation decreased, which could be reversed by superoxide dismutase (SOD), 1,10-phenanthroline (Phe), the PP2A phosphorylation inhibitors, okadaic acid (OKA) and cantharidin, and the ERK phosphorylation activator nicotine (p < 0.05). In particular, the significantly reduced cell proliferation and increased proportions of cells in the G0/G1 phase after 1-4 days of IH (p < 0.05), which resulted in decreased numbers of PC12 cells, could be reversed by treatment with SOD, Phe, PP2A inhibitors and an ERK activator. In addition, the numbers of nerve growth factor (NGF)-induced PC12 cells with neurite outgrowths after 3-4 days of IH were less than those after 4 days of RA, which was also reversed by SOD, Phe, PP2A inhibitors and an ERK activator. CONCLUSIONS: Our results suggest that IH-induced ROS generation increases PP2A activation and subsequently downregulates ERK1/2 activation, which results in inhibition of PC12 cell proliferation through G0/G1 phase arrest and NGF-induced neuronal differentiation.

Protein phosphatase-2A is down-regulated in patients within clear cell renal cell carcinoma.

BACKGROUND: Protein phosphatase-2A (PP2A) is one of the major cellular serine-threonine phosphatases. It positively regulates apoptosis and negatively regulates the mitogenic pathway, suggesting that loss of it might be involved in cancer development. Recent studies found its association with breast, lung and colorectal cancer; however, its expression profile and its prognostic value in clear cell renal cell carcinoma (ccRCC) have not been investigated. METHODS: Real-time quantitative PCR (qRT-PCR) and Western blot were used to explore PP2A expression in ccRCC and normal renal tissues. Moreover immunohistochemistry (ICH) was used to detect the expression of PP2A in ccRCC. Spearman's rank correlation, Kaplan-Meier plots and Cox proportional hazards regression model were used to analyze the data. RESULTS: Down-regulated expression of PP2A mRNA and protein was observed in the majority of ccRCC by qRT-PCR and Western blot when compared with their paired normal renal tissues. Clinic pathological analysis was showed a significant correlation existed between the lower expression of PP2A protein with the histological grade, lymph node metastasis and tumor distant metastasis (P<0.05); Survival analysis by Kaplan-Meier survival curve and log-rank test demonstrated that reduced PP2A expression in cancer tissue predicted poorer overall survival (OS) compared with group in higher expression. Notably, multivariate analyses by Cox's proportional hazard model revealed that expression of PP2A was an independent prognostic factor in ccRCC. CONCLUSIONS: These results suggest that the aberrant expression of PP2A in human ccRCC is possibly involved with tumorigenesis and development, and the PP2A protein could act as a potential biomarker for prognosis assessment of renal cancer. Further studies on the cellular functions of PP2A need to address these issues.

Restoration of PPP2CA expression reverses epithelial-to-mesenchymal transition and suppresses prostate tumour growth and metastasis in an orthotopic mouse model.

BACKGROUND: Emergence of castration-resistance in prostate cancer (PCa) is invariably associated with aggressive and metastatic disease. Previously, we reported promotion of castration-resistance upon downregulation of PPP2CA (encoding catalytic subunit of protein phosphatase 2A (PP2A), α-isoform); however, its role in PCa growth and metastasis remained undetermined. METHODS: PPP2CA was overexpressed/silenced in PCa cells by stable transfection. Gene expression was examined by reverse transcription polymerase chain reaction, immunoblot and immunofluorescence analyses, and transcriptional activity measured by luciferase-based promoter-reporter assay. Effect on PCa phenotype was studied in vitro and in orthotopic mouse model, and immunohistochemical/histological analyses performed to assess proliferation/apoptosis and confirm metastatic lesions. RESULTS: An inverse association of PPP2CA expression was observed with epithelial-to-mesenchymal transition (EMT) and aggressive PCa phenotype. PPP2CA restoration resulted in decreased nuclear accumulation and transcriptional activity of ß-catenin/NF-κB, and restitution of their activity abrogated PPP2CA-induced EMT reversal and suppression of PCa invasiveness. Akt mediated PPP2CA loss-induced nuclear accumulation of ß-catenin/NF-κB through inactivation of Gsk3-ß and IκB-α, respectively. Animal studies revealed a suppressive effect of PPP2CA expression on PCa growth and metastasis. CONCLUSIONS: Our findings suggest that PPP2CA downregulation serves as a molecular link between gain of castration-resistance and aggressive PCa phenotype, and its restoration could be an effective preventive/therapeutic approach against the advanced disease.

cAMP signaling inhibits radiation-induced ATM phosphorylation leading to the augmentation of apoptosis in human lung cancer cells.

BACKGROUND: The ataxia-telangiectasia mutated (ATM) protein kinase plays a central role in coordinating the cellular response to radiation-induced DNA damage. cAMP signaling regulates various cellular responses including metabolism and gene expression. This study aimed to investigate the mechanism through which cAMP signaling regulates ATM activation and cellular responses to ionizing radiation in lung cancer cells. METHODS: Lung cancer cells were transfected with constitutively active stimulatory G protein (GαsQL), and irradiated with γ-rays. The phosphorylation of ATM and protein phosphatase 2A was analyzed by western blotting, and apoptosis was assessed by western blotting, flow cytometry, and TUNNEL staining. The promoter activity of NF-κB was determined by dual luciferase reporter assay. BALB/c mice were treated with forskolin to assess the effect in the lung tissue. RESULTS: Transient expression of GαsQL significantly inhibited radiation-induced ATM phosphorylation in H1299 human lung cancer cells. Treatment with okadaic acid or knock down of PP2A B56δ subunit abolished the inhibitory effect of Gαs on radiation-induced ATM phosphorylation. Expression of GαsQL increased phosphorylation of the B56δ and PP2A activity, and inhibition of PKA blocked Gαs-induced PP2A activation. GαsQL enhanced radiation-induced cleavage of caspase-3 and PARP and increased the number of early apoptotic cells. The radiation-induced apoptosis was increased by inhibition of NF-κB using PDTC or inhibition of ATM using KU55933 or siRNA against ATM. Pretreatment of BALB/c mice with forskolin stimulated phosphorylation of PP2A B56δ, inhibited the activation of ATM and NF-κB, and augmented radiation-induced apoptosis in the lung tissue. GαsQL expression decreased the nuclear levels of the p50 and p65 subunits and NF-κB-dependent activity after γ-ray irradiation in H1299 cells. Pretreatment with prostaglandin E2 or isoproterenol increased B56δ phosphorylation, decreased radiation-induced ATM phosphorylation and increased apoptosis. CONCLUSIONS: cAMP signaling inhibits radiation-induced ATM activation by PKA-dependent activation of PP2A, and this signaling mechanism augments radiation-induced apoptosis by reducing ATM-dependent activation of NF-κB in lung cancer cells.

Expression and mechanism of regulation of PP2A/Pr65 in ameloblastoma.

OBJECTIVE: This study aimed to investigate the expression of PP2A/PR65 protein in ameloblastoma and the molecular mechanisms underlying the regulation of PP2A/PR65. The association between PP2A/PR65 and the clinicopathological characteristics of tumor specimens in ameloblastoma were to provide a theoretical basis for the diagnosis, therapy and prognosis of ameloblastoma. STUDY DESIGN: Streptavidin-peroxidase (S-P) immunohistochemical staining was used to detect PP2A/Pr65 expression changes in a total of 68 cases of ameloblastoma, six ameloblastic carcinomas, 21 squamous cell carcinomas and seven normal oral mucosas. Western blot was used to analyze PP2A/PR65 protein expression in 15 cases of ameloblastoma and three cases of normal oral mucosa. RESULTS: Of the 68 cases analyzed, four cases were negative, 25 cases were weakly positive, 20 cases were moderately positive and 19 cases were strongly positive. In six cases of ameloblastic carcinoma, three cases were weak positive, one case was positive, two cases were strongly positive and none were negative. In 21 cases of squamous cell carcinomas, three cases were negative, 17 cases were weakly positive, one case was moderately positive and none were strongly positive. Western blot analysis showed that, PP2A/Pr65 protein expression was lower in ameloblastoma tissue compared with normal oral mucosa. CONCLUSIONS: Reduced expression of PP2A/PR65 in ameloblastoma compared with normal oral mucosa indicates that PP2A/PR65 is involved in the occurrence and development of ameloblastoma.