PPM1F in Dentate Gyrus Modulates Anxiety-Related Behaviors by Regulating BDNF Expression via AKT/JNK/p-H3S10 Pathway.

Anxiety is a serious psychiatric disorder, with a higher incidence rate in women than in men. Protein phosphatase Mg2+/Mn2+-dependent 1F (PPM1F), a serine/threonine phosphatase, has been shown to have multiple biological and cellular functions. However, the effects of PPM1F and its neuronal substrates on anxiety remain largely unclear. In this study, we showed that chronic restraint stress (CRS) induced anxiety-related behaviors only in female mice, while acute restraint stress (ARS) produced anxiety-related behaviors in both male and female mice in light-dark and elevated plus maze tests and induced upregulation of PPM1F and downregulation of brain-derived neurotrophic factor (BDNF) expression in the hippocampus. Adeno-associated virus-mediated overexpression of PPM1F or conditional knockout of BDNF in dentate gyrus (DG) led to a more pronounced anxiety-related behavior in female than in male mice as indicated by the behavioral evaluations. Meanwhile, overexpression of PPM1F in the DG decreased total Bdnf exon-specific messenger RNA expression in the hippocampus with the decreased binding activity of phosphorylated H3S10 to its individual promoters in female mice. Furthermore, we identified that overexpression of PPM1F decreased the phosphorylation levels of AKT and JNK in the hippocampus of female mice. These results may suggest that PPM1F regulates anxiety-related behaviors by modulating BDNF expression and H3S10 phosphorylation-mediated epigenetic modification, which may be served as potentially pathological genes associated with anxiety or other mental diseases.

PPM1F in hippocampal dentate gyrus regulates the depression-related behaviors by modulating neuronal excitability.

Major depressive disorder (MDD) is a common, serious, debilitating mental illness. Protein phosphatase Mg2+/Mn2+-dependent 1F (PPM1F), a serine/threonine phosphatase, has been reported to have multiple biological and cellular functions. However, the effects of PPM1F and its neuronal substrates on depressive behaviors remain largely unknown. Here, we showed that PPM1F is widely distributed in the hippocampus, and chronic unpredictable stress (CUS) can induce increased expression of PPM1F in the hippocampus, which was correlated with depression-associated behaviors. Overexpression of PPM1F mediated by adeno-associated virus (AAV) in the dentate gyrus (DG) produced depression-related behaviors and enhanced susceptibility to subthreshold CUS (SCUS) in both male and female mice, while, knockout of PPM1F in DG produced antidepressant phonotypes under stress conditions. Whole-cell patch-clamp recordings demonstrated that overexpression of PPM1F increased the neuronal excitability of the granule cells in the DG. Consistent with neuronal hyperexcitability, overexpression of PPM1F regulated the expression of certain ion channel genes and induced decreased phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CAMKII) and Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) in hippocampus. These results suggest that PPM1F in the DG regulates depression-related behaviors by modulating neuronal excitability, which might be an important pathological gene for depression or other mental diseases.

Slingshot homolog-1 expression is a poor prognostic factor of pT1 bladder urothelial carcinoma after transurethral resection.

OBJECTIVE: Slingshot homolog-1 (SSH-1) shows an important role in the occurrence and development in various tumors. While, the expression and prognostic implications of SSH-1 in bladder urothelial carcinoma (UC) remain unclear and thus were addressed in this study. METHODS: Immunohistochemistry (IHC) was performed on tissue microarrays composed of 624 bladder UC specimens after transurethral resection of bladder tumor (TUR-BT) to detect SSH-1 expression. The clinic-pathological features were compared between SSH-1( +) and SSH-1(-) subgroups. The Kaplan-Meier curve with log-rank test and univariate/multivariate Cox regression model with stepwise backward elimination methods were performed for survival analyses. RESULTS: In this study, 359 (57.53%) specimens were detected with SSH-1 expression. SSH-1 positivity was significantly associated with higher pathological grade (p = 0.020), lymphovascular invasion (p = 0.006), tumor recurrence (p < 0.001) and progression (p < 0.001) in bladder UC. Besides, SSH-1 positivity predicted a shorter overall survival (OS, p = 0.024), recurrence-free survival (RFS, p < 0.001), progression-free survival (PFS, p = 0.002) and cancer-specific survival (CSS, p = 0.047). Multivariate Cox proportional hazard analysis showed that tumor size (p = 0.007), lymphovascular invasion (p = 0.003), recurrence (p < 0.001), progression (p < 0.001) and SSH-1 expression (p = 0.015) were predictors of poor prognosis in bladder UC patients. CONCLUSIONS: SSH-1 expression was associated with undesirable clinic-pathological characteristics and poor post-operative prognosis in bladder UC patients. SSH-1 might play an important role in bladder UC and serve as a promising predictor of oncological outcomes in patients with bladder UC.

Effect of benzo[a]pyrene on the expression of miR-126, miR-190a and their target genes EGFL7, TP53INP1 and PHLPP1 in primary endometrial cells.

The main topic of this study was to investigate the effect of benzo[a]pyrene (BP) on microRNAs and their target genes expression levels in primary cell cultures from normal and malignant endometrial tissue. MicroRNA-126 (miR-126) and miR-190a were most sensitive to BP treatment. The treatment of both cultures with BP was accompanied by a decrease of miR-126 level and an increase of EGFL7 gene expression level. BP-induced upregulation of miR-190a was detected only in normal cells and it was accompanied with decrease of mRNA levels of TP53INP1 and PHLPP1 genes. Taking into account that BP promoted the proliferation of normal cells and amplified apoptosis of cancer cells, it is possible that miR-190a is involved in general cellular response to BP. The findings of this study indicate that miR-190a and its target genes may be involved in the regulation of cell fate under BP treatment.

LIMK/cofilin pathway and Slingshot are implicated in human colorectal cancer progression and chemoresistance.

Cofilin phospho-regulation is important for actin filament turnover and is implicated in cancer. Phosphorylation of cofilin is mediated by LIM kinases (LIMKs) and dephosphorylation by Slingshot phosphatases (SSH). LIMKs and SSH promote cancer cell invasion and metastasis and represent novel anti-cancer targets. However, little is known regarding LIMK/cofilin and SSH in human colorectal cancer (CRC). In this study, we aimed to address their expression and significance in human CRC. We evaluated expression of non-phosphorylated (active) and phosphorylated cofilin, LIMK1, LIMK2, and SSH1 by immunohistochemistry in 143 human CRC samples in relation to clinicopathologic parameters, response of metastatic disease to chemotherapy, and epithelial-mesenchymal transition (EMT) markers ß-catenin, E-cadherin, and ZEB. We show that active cofilin, LIMK1, LIMK2, and SSH1 are overexpressed in human CRC and are associated with tumor progression parameters. SSH1 is an independent predictor of lymph node metastasis by multivariate analysis. LIMK1 and SSH1 expression is also higher in non-responders to chemotherapy, and SSH1 is shown by multivariate analysis to independently predict response of metastatic disease to chemotherapy. Active cofilin, LIMK1, LIMK2, and SSH1 also correlated with the EMT markers examined. In addition, immunofluorescence analysis showed increased expression of active cofilin, LIMK1, LIMK2, and SSH1 in HT29 colon cancer cells resistant to 5-fluorouracil compared to parental HT29 cells. Our results suggest that F-actin regulators LIMK/cofilin pathway and SSH1 are associated with CRC progression and chemoresistance representing promising tumor biomarkers and therapeutic targets in CRC.

Expression and prognostic roles of magnesium-dependent phosphatase-1 in gastric cancer.

OBJECTIVE: Gastric cancer is a leading cause of cancer deaths and has a poor prognosis after diagnosis. Previous studies showed that Magnesium-Dependent Phosphatase-1 (MDP-1) might be a key component for glycosylation in human protein repair, and an alteration of its function has been involved in some aspects of cellular metabolic networks linked to either normal or pathological processe. In this study, we investigate the MDP-1 status in patients with gastric carcinoma, and determine the potential relationship between MDP-1 and clinical outcome. PATIENTS AND METHODS: One hundred and seventy-one consecutive patients with stage I-III gastric carcinoma who had received a D2 gastrectomy were recruited. The MDP-1 expression was determined by immunohistochemistry (IHC). Disease-free survival (DFS) and overall survival (OS) were evaluated. RESULTS: We generate an IHC score on a continuous scale of 0-7. The IHC cutoff point generated by ROC analysis and the threshold IHC score was 2. Low MDP-1 expression was scored for 61 (35.7%) and high MDP-1 expression for 110 (64.3%) patients. We saw a significant down-regulation of MDP-1 expression in G3-4 and stage III tumor tissue compared with G1-2 and stage I-II tumors, p=0.023 and p=0.047. In univariate survival analysis, high expression of MDP-1 predicted a significantly better DFS (56.0 months vs. 25.0 months, p=0.029) and OS (59.0 months vs. 41.0 months, p=0.043) compared with low expression. In a multivariate analysis, the tumor stage was a significant predictor for DFS and OS even after adjustment for all other covariates. The MDP-1 status was a joint predictor for DFS and OS with a multivariate HR 0.728, 95% CI 0.530-0.999, p=0.049 and a multivariate HR 0.745, 95% CI 0.543-1.022, p=0.068, respectively. CONCLUSIONS: We showed that down-regulation of MDP-1 expression was correlated with poorly differentiated carcinoma and later tumor stage, and it predicted a significantly poorer DFS and OS. Down-regulation of MDP-1 expression was a predictor of a poor prognosis for gastric cancer patients, and it may refer to tumor cells that have lost a protective enzymatic system.

Elevated expression of serine/threonine phosphatase type 5 correlates with malignant proliferation in human osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor in adolescents and young adults. However, the involvement of serine/threonine phosphatase type 5 (PP5) in osteosarcoma remains unclear. The aim of this study was to evaluate the functional role of PP5 in osteosarcoma cells. Firstly, we found that PP5 is widely expressed in several human osteosarcoma cell lines. Then we used lentivirus-delivered siRNA to silence PP5 expression in Saos-2 and U2OS cell lines. Knockdown of endogenous PP5 expression by shRNA-expressing lentivirus significantly decreased the viability and proliferation of the osteosarcoma cells. Moreover, FACS analysis showed that knockdown of PP5 expression induced a significant arrest in the G0/G1 phase of the cell cycle, which was associated with the inhibition of cell proliferation. Therefore, knockdown of PP5 is likely to provide a novel alternative to targeted therapy of osteosarcoma and deserves further investigation.

Phosphatase POPX2 Exhibits Dual Regulatory Functions in Cancer Metastasis.

Cancer metastasis is a complex mechanism involving multiple processes. Previously, our integrative proteome, transcriptome, and phosphoproteome study reported that the levels of serine/threonine phosphatase POPX2 were positively correlated with cancer cell motility through modulating MAPK signaling. Surprisingly, here we found that POPX2 knockdown cells induced more numerous and larger tumor nodules in lungs in longer term animal studies. Interestingly, our analysis of DNA microarray data from cancer patient samples that are available in public databases shows that low POPX2 expression is linked to distant metastasis and poor survival rate. These observations suggest that lower levels of POPX2 may favor tumor progression in later stages of metastasis. We hypothesize that POPX2 may do so by modulation of angiogenesis. Secretome analysis of POPX2-knockdown MDA-MB-231 cells using LC-MS/MS-based SILAC quantitative proteomics and cytokine array show that silencing of POPX2 leads to increased secretion of exosomes, which may, in turn, induce multiple pro-angiogenic cytokines. This study, combined with our previous findings, suggests that a single ubiquitously expressed phosphatase POPX2 influences cancer metastasis via modulating multiple biological processes including MAPK signaling and exosome cytokine secretion.

Effect of PPM1H on malignant phenotype of human pancreatic cancer cells.

The objective of this study was to investigate the effect of silencing gene protein phosphatase 1H (PPM1H) on malignant phenotype of human pancreatic cancer cell line BxPC-3. In order to explore the function of PPM1H in pancreatic cancer cells, real-time PCR and western blotting were used to detect the expression of PPM1H in different pancreatic cancer cell lines. Human pancreatic cancer cell line BxPC-3 was treated with 10 ng/ml TGF-ß1 and 200 ng/ml BMP2 for 72 h, respectively, and the mRNA and protein expression levels of PPM1H and EMT-related markers (E-cadherin, vimentin) were detected by real-time PCR and western blotting, respectively. Using exogenous RNA interference technology to silence the PPM1H gene, the expression of PPM1H and EMT-related markers at mRNA and protein levels were detected by real-time PCR and western blotting. The cell migration and invasion were measured using Transwell assays. Finally, cell counting kit-8 (CCK-8) and flow cytometry were used to determine the effect of PPM1H on cell proliferation and apoptosis of BxPC-3 cells. The expression levels of PPM1H in all of the examined pancreatic cancer cell lines (BxPC-3, MIA-PACA2, PANC-1, SW1990, PANC-03.27) were lower than that of normal pancreatic ductal epithelial cells (HPDE6-C7) at both mRNA and protein levels. Both TGF-ß1 and BMP2 treatment induced EMT and downregulation of PPM1H in BxPC-3 cells. By using RNA interference to transiently knock down PPM1H expression in BxPC-3 cells, we found that the expression of E-cadherin was downregulated while vimentin was up-regulated. The data suggested that silencing PPM1H gene can induce EMT in BxPC-3 cells. In addition, Transwell migration assays showed that silencing PPM1H gene can promote the invasion and metastasis of BxPC-3 cells. Cell proliferation and apotosis detection demonstrated that silencing PPM1H gene can promote the proliferation and inhibit apoptosis of BxPC-3 cells. In conclusion, PPM1H is aberrantly expressed in human pancreatic cancer cell lines and can be downregulated when EMT is induced by cytokine stimulation. Silencing PPM1H gene can induce EMT in BxPC-3 cells, and promote the invasion and metastasis of BxPC-3 cells. Moreover, silencing PPM1H gene can promote the proliferation and inhibit apoptosis of BxPC-3 cells. PPM1H may be a new tumor-suppressor factor for pancreatic cancer and provides new insight into molecular targets for gene therapy of pancreatic cancer.

PHLPP2 Downregulation Contributes to Lung Carcinogenesis Following B[a]P/B[a]PDE Exposure.

PURPOSE: The carcinogenic capacity of B[a]P/B[a]PDE is supported by epidemiologic studies. However, the molecular mechanisms responsible for B[a]P/B[a]PDE-caused lung cancer have not been well investigated. We evaluated here the role of novel target PHLPP2 in lung inflammation and carcinogenesis upon B[a]P/B[a]PDE exposure. EXPERIMENTAL DESIGN: We used the Western blotting, RT-PCR, [(35)S]methionine pulse and immunohistochemistry staining to determine PHLPP2 downregulation following B[a]P/B[a]PDE exposure. Both B[a]PDE-induced Beas-2B cell transformation model and B[a]P-caused mouse lung cancer model were used to elucidate the mechanisms leading to PHLPP2 downregulation and lung carcinogenesis. The important findings were also extended to in vivo human studies. RESULTS: We found that B[a]P/B[a]PDE exposure downregulated PHLPP2 expression in human lung epithelial cells in vitro and in mouse lung tissues in vivo. The ectopic expression of PHLPP2 dramatically inhibited cell transformation upon B[a]PDE exposure. Mechanistic studies showed that miR-205 induction was crucial for inhibition of PHLPP2 protein translation by targeting PHLPP2-3'-UTR. Interestingly, PHLPP2 expression was inversely associated with tumor necrosis factor alpha (TNFα) expression, with low PHLPP2 and high TNFα expression in lung cancer tissues compared with the paired adjacent normal lung tissues. Additional studies revealed that PHLPP2 exhibited its antitumorigenic effect of B[a]P/B[a]PDE through the repression of inflammatory TNFα transcription. CONCLUSIONS: Our studies not only first time identify PHLPP2 downregulation by lung carcinogen B[a]P/B[a]PDE, but also elucidate a novel molecular mechanisms underlying lung inflammation and carcinogenesis upon B[a]P/B[a]PDE exposure.

MiR-522 contributes to cell proliferation of human glioblastoma cells by suppressing PHLPP1 expression.

Previous studies have shown that microRNAs play essential roles in cancer growth and progression. Although a number of microRNAs were differentially expressed in glioblastoma (GBM). In this study, we evaluated the miR-522s role in cell proliferation in GBM. Expression of miR-522 is markedly upregulated in GBM tissues and GBM cells compared with the matched non-tumor adjacent brain tissues (TAT) and normal human astrocytes (NHAs). In functional assays, miR-522 promoted GBM cell proliferation, which could be reversed by inhibitor of miR-522. We further identified PH domain leucine-rich repeats protein phosphatase-1 (PHLPP1) as a putative target of miR-522, which is likely a main contributor to the promotion of tumor cell growth observed in our assays. Our results demonstrated that miR-522 promoted tumor cell proliferation and hence may represent a novel therapeutically relevant cellular target to treatment of GBM patients.

Cofilin-phosphatase slingshot-1L (SSH1L) is over-expressed in pancreatic cancer (PC) and contributes to tumor cell migration.

Slingshot-1L (SSH1L), a cofilin-phosphatase, plays a role in actin dynamics and cell migration by reactivating cofilin-1. However, the expression of SSH1L in malignant diseases is poorly understood. The overexpression of SSH1L in cancerous tissue compared to the matched surrounding non-cancerous tissues from patients with late stages (III-IV) of PC was detected in 90% (9/10) of cases by western blotting. The expression of SSH1L was shown to be upregulated in tumor cells from 10.7% (11/102) of patients with pancreatic cancer (PC) by immunohistochemistry (IHC). The positive rate of SSH1L in patients with PC at stage VI (TNM) categorized as grade 3 was of 50% (2/4) and 15% (6/40), respectively. Moreover, SSH1L expression was shown to be up-regulated in the PC cell lines (KLM1, PANC-1 and MIAPaCa-2) with high metastatic potential. Loss of SSH1L expression was associated with an increase in the phosphorylation of cofilin-1 at serine-3 and further inhibited cell migration (but not proliferation) in KLM1, PANC-1 and MIAPaCa-2. Actin polymerization inhibitor cytochalasin-D was sufficient to abrogate cell migration of PC without changing SSH1L expression. These results reveal that SSH1L is upregulated in a subset of PCs and that the SSH1L/cofilin-1 signal pathway is associated positively in PC with cell migration. Our study may thus provide potential targets to prevent and/or treat PC invasion and metastasis in patients with SSH1L-positive PC.

Knockdown of PPP5C inhibits growth of hepatocellular carcinoma cells in vitro.

Ser/Thr protein phosphatase 5 (PPP5C) has been reported to participate in tumor progression. However, its functional role in hepatocellular carcinoma (HCC) remains unknown yet. In this study, we firstly evaluated the expression levels of PPP5C in six HCC cell lines by real-time PCR and found that PPP5C was widely expressed in HCC cells. To explore the role of PPP5C in HCC cell growth, lentivirus-mediated short hairpin RNA (shRNA) was employed to silence PPP5C expression in HepG2 and Bel-7404 cells. The expression of PPP5C was significantly downregulated in PPP5C knockdown cells. Knockdown of PPP5C markedly suppressed the proliferation and colony formation ability of HCC cells. Moreover, cell cycle analysis showed that PPP5C depletion in HepG2 cells led to G0/G1 phase and G2/M phase arrest. We demonstrate for the first time that PPP5C is essential for growth of HCC cells, which suggests that inhibition of PPP5C by RNAi may be a potential therapeutic strategy for the treatment of HCC.

Low expression of PHLPP1 in sacral chordoma and its association with poor prognosis.

Sacral chordoma is a rare spine tumor with a high recurrence rate even after optimal therapy. Previous studies have demonstrated that the PI3K/AKT pathway plays a pivotal role in chordoma, and high expression of pAKT is associated with poor prognosis. Recently, PHLPP was recognized to be a tumor suppressor that targets AKT. We analyzed the expression of PHLPP1 and AKT2 in 37 chordoma samples and 11 fetal nucleus pulposus samples by immunohistochemical staining. Of the chordoma cases, 40.5% (15/37) showed strong cytoplasmic staining (score ≥3) for PHLPP1, which was significantly lower than the 90.9% (10/11) of fetal nucleus pulposus samples (P = 0.004). Conversely, strong immunohistochemical staining for AKT2 was observed in 75.7% (28/37) of chordoma samples, which was significantly higher than 36.4% (4/11) of fetal nucleus pulposus (P = 0.021). Kaplan-Meier survival curves and log-rank test showed that patients with high expression of PHLPP1 experienced longer progression free survival time than those with low PHLPP1 expression (P = 0.011). Further multivariate Cox regression analysis indicated that PHLPP1 expression level and surgical approaches were independent risk factors for chordoma recurrence (P = 0.023 and P = 0.022). However, PHLPP1 expression was not statistically related to patients' total survival time. Conclusively, our results suggest that PHLPP1 plays a crucial role in sacral chordoma, and may be a promising biomarker for prognosis. Meanwhile, manipulation of PHLPP1 expression is also a potential therapeutic approach for the treatment of sacral chordoma.

Resolvin D1 reverses reactivity and Ca2+ sensitivity induced by ET-1, TNF-α, and IL-6 in the human pulmonary artery.

Pulmonary hypertension (PH) is a rare and progressive disease characterized by an inflammatory status and vessel wall remodeling, resulting in increased pulmonary artery resistance. During the last decade, treatments have been proposed; most of them target the endothelial pathways that stimulate smooth muscle cell relaxation. However, PH remains associated with significant morbidity. We hypothesized that inflammation plays a crucial role in the severity of the abnormal vasoconstriction in PH. The goal of this study was to assess the effects of resolvin D1 (RvD1), a potent anti-inflammatory agent, on the pharmacological reactivity of human pulmonary arteries (HPAs) via an in vitro model of induced hyperreactivity. The effects of RvD1 and monoacylglyceride compounds were measured on contractile activity and Ca(2+) sensitivity developed by HPAs that had been pretreated (or not) under proinflammatory conditions with either 10 ng/ml TNF-α or 10 ng/ml IL-6 or under hyperreactive conditions with 5 nM endothelin-1. The results demonstrated that, compared with controls, 24-h pretreatment with TNF-α, IL-6, or endothelin-1 increased reactivity and Ca(2+) sensitivity of HPAs as revealed by agonist challenges with 80 mM KCl, 1 µM serotonin (5-hydroxytryptamine), 30 nM U-46619, and 1 µM phorbol 12,13-dibutyrate. However, 300 nM RvD1 as well as 1 µM monoacylglyceride-docosapentaenoic acid monoglyceride strongly reversed the overresponsiveness induced by both proinflammatory and hyperreactive treatments. In pretreated pulmonary artery smooth muscle cells, Western blot analyses revealed that RvD1 treatment decreased the phosphorylation level of CPI-17 and expression of transmembrane protein member 16A while increasing the detection of G protein-coupled receptor 32. The present data demonstrate that RvD1, a trihydroxylated docosahexaenoic acid derivative, decreases induced overreactivity in HPAs via a reduction in CPI-17 phosphorylation and transmembrane protein member 16A expression.

Overexpression of wip1 is associated with biologic behavior in human clear cell renal cell carcinoma.

Wild-type p53-induced phosphatase (Wip1 or PPM1D) has been reported to be aberrantly expressed in various cancers and correlated with the malignant behavior of cancer cells. However, the function of Wip1 in RCC remains unclear. The present study investigated its abnormal expression and dysfunctions in clear cell renal cell carcinoma (ccRCC) in vitro. With the combination of immunohistochemistry, western blotting, immunofluorescence, qRT-PCR, and cell proliferation, migration and invasion assays, we found that levels of Wip1 mRNA and protein were dramatically increased in human ccRCC tissues (P<0.001 for both), and upregulation of Wip1 was significantly associated with depth of invasion (P<0.001), Distant metastasis (P = 0.001), lymph node status (P<0.001) and Fuhrman grade (P<0.001). Wip1 knockdown inhibited the proliferation, migration and invasion of 786-O and RLC-310 cells, whereas Wip1 overexpression promoted the growth and aggressive phenotype of 786-O and RLC-310 cells in vitro. The uni- and multivariate analyses indicated that expression of Wip1 was an independent predictor for survival of ccRCC patients (P = 0.003, P = 0.027 respectively). Wip1- negative patients had a higher tumor-free/overall survival rate than patients with high Wip1 expression (P = 0.001, P = 0.002 respectively). Overexpression of Wip1 is useful in the prediction of survival in ccRCC patients.

Loss of PPM1A expression enhances invasion and the epithelial-to-mesenchymal transition in bladder cancer by activating the TGF-ß/Smad signaling pathway.

The transforming growth factor-ß (TGF-ß) signaling pathway is believed to contribute to carcinoma development by increasing cell invasiveness and metastasis and inducing the epithelial-to-mesenchymal transition (EMT). Protein phosphatase PPM1A has been reported to dephosphorylate TGF-ß-activated Smad2/3, thus inhibiting the TGF-ß signaling pathway. In this study, we investigated the role of PPM1A in bladder cancer. PPM1A protein expression was analyzed in 145 bladder cancer specimens. The loss of PPM1A expression was predictive of poor survival and high muscle-invasiveness. PPM1A was more commonly deficient among muscle-invasive relapse samples compared to primary tumors in twenty paired bladder cancer tissues. Functional studies indicated that blockade of PPM1A through lentivirus-mediated RNA interference significantly promoted urinary bladder cancer (BCa) cell motility, the EMT in vitro and metastasis in vivo, and these effects were dependent on the TGF-ß/Smad signaling pathway. The increase in p-Smad2/3 induced by TGF-ß1 correlated with the degree of PPM1A depletion in BCa cells, which resulted in an altered expression profile of TGF-ß-inducible genes. The correlations between PPM1A and biomarkers related to the TGF-ß signaling pathway and tumor invasion were also detected in BCa samples. These results demonstrate that loss of PPM1A is associated with the development of tumor invasion in bladder cancer patients.

Loss expression of PHLPP1 correlates with lymph node metastasis and exhibits a poor prognosis in patients with gastric cancer.

BACKGROUND: PHLPP1 functions as an antitumor factor in several human cancers, but the expression pattern and clinical significance of PHLPP1 in gastric cancer have yet to be determined. The aim of this study is to assess the expression of PHLPP1 in gastric cancer and its impact on the prognosis of patients with gastric cancer. METHODS: A total of 202 consecutive patients with gastric cancer who had undergone gastrectomy were enrolled in this study. The expressions of PHLPP1 were assessed by immunohistochemistry method. Survival analysis according to PHLPP1 expression was calculated. RESULTS: The positive rates of PHLPP1 protein in primary gastric cancer tissues and metastatic lymph nodes were significantly lower than in normal stomach mucosa tissues (56.9% vs. 96.7%, 38.8% vs. 96.7%, both P < 0.001). The overall survival (OS) time and relapse-free survival (RFS) time in PHLPP1-positive patients were significantly longer than in PHLPP1-negative patients (both P < 0.001). Moreover, PHLPP1 was an independent prognostic factor for OS and RFS of gastric cancer patients (HR 0.55, 95% CI 0.36-0.85, P = 0.007; HR 0.52, 95% CI 0.31-0.87, P = 0.013; respectively). CONCLUSIONS: The loss expression of PHLPP1 was observed in gastric cancer and PHLPP1 is an independent prognostic factor for patients with gastric cancer.

A yeast mitotic activator sensitises the shoot apical meristem to become floral in day-neutral tobacco.

True day-neutral (DN) plants flower regardless of day-length and yet they flower at characteristic stages. DN Nicotiana tabacum cv. Samsun, makes about forty nodes before flowering. The question still persists whether flowering starts because leaves become physiologically able to export sufficient floral stimulus or the shoot apical meristem (SAM) acquires developmental competence to interpret its arrival. This question was addressed using tobacco expressing the Schizosaccharomyces pombe cell cycle gene, Spcdc25, as a tool. Spcdc25 expression induces early flowering and we tested a hypothesis that this phenotype arises because of premature floral competence of the SAM. Scions of vegetative Spcdc25 plants were grafted onto stocks of vegetative WT together with converse grafts and flowering onset followed (as the time since sowing and number of leaves formed till flowering). Spcdc25 plants flowered significantly earlier with fewer leaves, and, unlike WT, also formed flowers from axillary buds. Scions from vegetative Spcdc25 plants also flowered precociously when grafted to vegetative WT stocks. However, in a WT scion to Spcdc25 stock, the plants flowered at the same time as WT. SAMs from young vegetative Spcdc25 plants were elongated (increase in SAM convexity determined by tracing a circumference of SAM sections) with a pronounced meristem surface cell layers compared with WT. Presumably, Spcdc25 SAMs were competent for flowering earlier than WT and responded to florigenic signal produced even in young vegetative WT plants. Precocious reproductive competence in Spcdc25 SAMs comprised a pronounced mantle, a trait of prefloral SAMs. Hence, we propose that true DN plants export florigenic signal since early developmental stages but the SAM has to acquire competence to respond to the floral stimulus.

microRNA-224 promotes cell proliferation and tumor growth in human colorectal cancer by repressing PHLPP1 and PHLPP2.

PURPOSE: To investigate the clinicopathologic significance, role, and mechanism of action of microRNA-224 (miR-224) in colorectal cancer. EXPERIMENTAL DESIGN: Real-time PCR was used to quantify miR-224 expression. The association of miR-224 with the clinicopathologic features and survival was evaluated in 110 colorectal cancer patients. The role of miR-224 in colorectal cancer was investigated using in vitro and in vivo assays. Luciferase reporter assays were conducted to confirm target gene associations. RESULTS: miR-224 was overexpressed in colorectal cancer. High-level expression of miR-224 was significantly associated with an aggressive phenotype and poor prognosis. Overexpression of miR-224 promoted colorectal cancer cell proliferation in vitro and tumor growth in vivo. Specifically, miR-224 accelerated the G1-S phase transition through activation of AKT/FOXO3a signaling, downregulation of p21Cip1 and p27Kip1, and upregulation of cyclin D1. Moreover, both PH domain leucine-rich-repeats protein phosphatase 1 (PHLPP1) and PHLPP2, antagonists of PI3K/AKT signaling, were confirmed as bona fide targets of miR-224. miR-224 directly targeted the 3'-untranslated regions of the PHLPP1 and PHLPP2 mRNAs and repressed their expression. CONCLUSION: This study reveals functional and mechanistic links between miRNA-224 and the tumor suppressors PHLPP1 and PHLPP2 in the pathogenesis of colorectal cancer. miR-224 not only plays important roles in the regulation of cell proliferation and tumor growth in colorectal cancer, but also has potential as a prognostic marker or therapeutic target for colorectal cancer.