Cell cycle arrest or survival signaling through αv integrins,activation of PKC and ERK1/2 lead to anoikis resistance of ovarian cancer spheroids

Ovarian cancer is the most lethal gynecologic cancer mainly due to spheroids organization of cancer cells that disseminate within the peritoneal cavity. We have investigated the molecular mechanisms by which ovarian cancer spheroids resist anoikis, choosing as models the 2 well-characterized human ovarian cancer cell lines IGROV1 and SKOV3. These cell lines have the propensity to float as clusters, and were isolated from tumor tissue and ascites, respectively. To form spheroids, IGROV1 and SKOV3 ovarian adenocarcinoma cells were maintained under anchorage-independent culture conditions, in which both lines survive at least a week. A short apoptotic period prior to a survival signaling commitment was observed for IGROV1 cells whereas SKOV3 cells entered G0/G1 phase of the cell cycle. This difference in behavior was due to different signals. With regard to SKOV3 cells, activation of p38 and an increase in p130/Rb occurred once anchorage-independent culture was established. Analyses of the survival signaling pathway switched on by IGROV1 cells showed that activation of ERK1/2 was required to evade apoptosis, an effect partly dependent on PKC activation and αv integrins. αv-integrin expression is essential for survival through activation of ERK1/2 phosphorylation.     

卡铂通过抑制ERK1/2通路诱导人卵巢癌细胞S和G0/G1期阻滞

卡铂(carboplatin, CBP)是一种抗肿瘤活性较强的化疗药物, 通过诱导细胞周期阻滞抑制肿瘤细胞生长, 但其诱导细胞周期阻滞的报告不甚一致. 本研究探索卡铂对卵巢癌HO-8910细胞生长及细胞周期进程的影响. MTS结果显示, 卡铂以浓度和时间依赖方式抑制卵巢癌HO-8910细胞生长, 联合使用ERK1/2通路抑制剂PD98059可使卡铂抗卵巢癌细胞增殖作用增强. 采用Giemsa染色法观察到, 卡铂与PD98059单用或联用均能致卵巢癌细胞发生明显的形态学变化. 流式细胞术检测细胞周期发现, 随卡铂浓度的增高, S期阻滞作用增强; 抑制ERK1/2通路可拮抗卡铂对HO-8910细胞S期阻滞作用, 增加G1期阻滞作用, 而对G2/M期细胞影响不明显. Western印迹结果显示, 随卡铂浓度的增高, p-ERK1/2、Cdc2(Y15)和p Cdc2(T161)的表达逐渐升高, Cyclin E1和Cyclin B1的表达逐渐降低; 抑制ERK1/2通路可将卡铂上调,p-ERK1/2和p-Cdc2(T161)的作用反转为下调作用, 上调Cdc2(Y15)的表达受阻, 抑制Cyclin B1的下调作用, 促进Cyclin E1的下调作用. 本研究结果提示, 卡铂通过抑制ERK1/2激活, 诱导人卵巢癌HO-8910细胞S和G1期阻滞, 抑制卵巢癌细胞生长.     

Suppressed expression of NDRG2 correlates with poor prognosis in pancreatic cancer

Pancreatic cancer is a highly lethal disease with a poor prognosis; the molecular mechanisms of the development of this disease have not yet been fully elucidated. N-myc downstream regulated gene 2 (NDRG2), one of the candidate tumor suppressor genes, is frequently downregulated in pancreatic cancer, but there has been little information regarding its expression in surgically resected pancreatic cancer specimens. We investigated an association between NDRG2 expression and prognosis in 69 primary resected pancreatic cancer specimens by immunohistochemistry and observed a significant association between poor prognosis and NDRG2-negative staining (P = 0.038). Treatment with trichostatin A, a histone deacetylase inhibitor, predominantly up-regulated NDRG2 expression in the NDRG2 low-expressing cell lines (PANC-1, PCI-35, PK-45P, and AsPC-1). In contrast, no increased NDRG2 expression was observed after treatment with 5-aza-2′ deoxycytidine, a DNA demethylating agent, and no hypermethylation was detected in either pancreatic cancer cell lines or surgically resected specimens by methylation specific PCR. Our present results suggest that (1) NDRG2 is functioning as one of the candidate tumor-suppressor genes in pancreatic carcinogenesis, (2) epigenetic mechanisms such as histone modifications play an essential role in NDRG2 silencing, and (3) the expression of NDRG2 is an independent prognostic factor in pancreatic cancer.     

ARHGAP9 knockdown promotes lung adenocarcinoma metastasis by activating Wnt/β-catenin signaling pathway via suppressing DKK2

This study aims to elucidate the effect of ARHGAP9 on lung adenocarcinoma (LUAD) metastasis, and preliminarily explore its molecular mechanism. As a result, we found that ARHGAP9 was downregulated and correlated with poor prognosis of LUAD. ARHGAP9 knockdown promoted LUAD cell proliferation, migration and invasion, inhibited cell apoptosis and reduced G0G1 cell cycle arrest, in contrast to the results of ARHGAP9 overexpression. Further RNA sequencing analysis demonstrated that ARHGAP9 knockdown in H1299 cells significantly reduced DKK2 (dickkopf related protein 2) expression. Silencing ARHGAP9 in H1299 cells while overexpressing DKK2, DKK2 reversed the promoted effects of ARHGAP9 knockdown on LUAD cell proliferation, migration and invasion. Meanwhile, the activity of Wnt/β-catenin signaling pathway was also reduced. Taken together, these data indicated that ARHGAP9 knockdown promoted LUAD metastasis by activating Wnt/β-catenin signaling pathway via suppressing DKK2. This may provide a new strategy for LUAD treatment.     

HIF-1 and NDRG2 contribute to hypoxia-induced radioresistance of cervical cancer Hela cells

Hypoxia inducible factor 1 (HIF-1), the key mediator of hypoxia signaling pathways, has been shown involved in hypoxia-induced radioresistance. However, the underlying mechanisms are unclear. The present study demonstrated that both hypoxia and hypoxia mimetic cobalt chloride could increase the radioresistance of human cervical cancer Hela cells. Meanwhile, ectopic expression of HIF-1 could enhance the resistance of Hela cells to radiation, whereas knocking-down of HIF-1 could increase the sensitivity of Hela cells to radiation in the presence of hypoxia. N-Myc downstream-regulated gene 2 (NDRG2), a new HIF-1 target gene identified in our lab, was found to be upregulated by hypoxia and radiation in a HIF-1-dependent manner. Overexpression of NDRG2 resulted in decreased sensitivity of Hela cells to radiation while silencing NDRG2 led to radiosensitization. Moreover, NDRG2 was proved to protect Hela cells from radiation-induced apoptosis and abolish radiation-induced upregulation of Bax. Taken together, these data suggest that both HIF-1 and NDRG2 contribute to hypoxia-induced tumor radioresistance and that NDRG2 acts downstream of HIF-1 to promote radioresistance through suppressing radiation-induced Bax expression. It would be meaningful to further explore the clinical application potential of HIF-1 and NDRG2 blockade as radiosensitizer for tumor therapy.     

The regulation of NDRG2 expression during ATLL development after HTLV-1 infection

N-myc downstream-regulated gene 2 (NDRG2) is a candidate tumor suppressor that is frequently downregulated in adult T-cell leukemia/lymphoma (ATLL) and functions to negatively regulate several cellular signaling pathways as PP2A recruiter. To clarify the molecular mechanisms of suppression of NDRG2 expression, we initially determined the expression pattern of NDRG2 in various types of T-cells and ATLL cells. NDRG2 expression was significantly upregulated in HTLV-1/Tax-immortalized T-cells, which was mediated by NF-κB activation through Tax expression. On the other hand, NDRG2 expression was suppressed in HTLV-1-infected cell lines and various types of ATLL cells, which was dependent on the DNA methylation of the NDRG2 promoter. We found that the expression of enhancer of zeste homolog 2 (EZH2), a member of the polycomb family, is increased in ATLL, and that EZH2 directly binds to the NDRG2 promoter and induces DNA methylation of the NDRG2 promoter. Since the expression of EZH2 were anti-parallelly regulated with the NDRG2 expression, EZH2 might be one of the most important regulators of the downregulation of NDRG2, contributing to enhanced activation of signaling pathways during ATLL development.     

Emodin inhibits the growth of hepatoma cells: Finding the common anti-cancer pathway using Huh7, Hep3B, and HepG2 cells

Emodin—a major component of Rheum palmatum L.—exerts antiproliferative effects in cancer cells that are regulated by different signaling pathways. Hepatocellular carcinoma has high-incidence rates and is associated with poor prognosis and high mortality rates. This study was designed to evaluate the effects of emodin on human hepatocarcinoma cell viability and investigate its mechanisms of action in Huh7, Hep3B, and HepG2 cells. To define the molecular changes associated with this process, expression profiles were compared in emodin-treated hepatoma cells by cDNA microarray hybridization, quantitative RT-PCRs, and Western blot analysis. G2/M phase arrest was observed in all 3 cell lines. Cell cycle regulatory gene analysis showed increased protein levels of cyclin A, cyclin B, Chk2, Cdk2, and P27 in hepatoma cells after time courses of emodin treatment, and Western blot analysis showed decreased protein levels of Cdc25c and P21. Microarray expression profile data and quantitative PCR revealed that 15 representative genes were associated with emodin treatment response in hepatoma cell lines. The RNA expression levels of CYP1A1, CYP1B1, GDF15, SERPINE1, SOS1, RASD1, and MRAS were upregulated and those of NR1H4, PALMD, and TXNIP were downregulated in all three hepatoma cells. Moreover, at 6 h after emodin treatment, the levels of GDF15, CYP1A1, CYP1B1, and CYR61 were upregulated. Here, we show that emodin treatment caused G2/M arrest in liver cancer cells and increased the expression levels of various genes both in mRNA and protein level. It is likely that these genes act as biomarkers for hepatocellular carcinoma therapy.     

2-Amino-1,3,4-thiadiazole derivative (FABT) inhibits the extracellular signal-regulated kinase pathway and induces cell cycle arrest in human non-small lung carcinoma cells

The anticancer potential of 2-amino-1,3,4-thiadiazole compounds has been documented by in vitro and in vivo studies. In our previous research, we described the synthesis as well as the antiproliferative and neuroprotective activities of 2-(4-fluorophenyloamino)-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole (FABT). The aim of the present study was to investigate the molecular mechanisms involved in FABT-induced growth inhibition in A549 lung carcinoma cells. Western blotting analysis revealed that FABT inhibited the activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, and Real-time PCR analysis showed no changes in the expression of P44ERK1 and CREB1 genes. Furthermore, FABT induced cell cycle arrest in the GO/G1 phase and enhanced p27/Kip1 expression. Our results suggest that FABT acts by inhibiting ERK1/2 pathway and cell cycle progression through G1 into S phase in A549 cells. Further studies are needed to completely explain the molecular mechanisms of anticancer action of this 2-aminothiadiazole derivative.     

Interaction between abscisic acid receptor PYL3 and protein phosphatase type 2C in response to ABA signaling in maize

Abscisic acid (ABA) is a ubiquitous hormone that regulates plant growth, development and responses to environmental stresses. In recent researches, pyrabactin resistance 1-like protein (PYL) and protein phosphatase type 2C (PP2C) were identified as the direct receptor and the second component of ABA signaling pathway, respectively. However, a lot of PYL and PP2C members were found in Arabidopsis and several other plants. Some of them were found not to be involved in ABA signaling. Because of the complex diversity of the genome, few documents have been available on the molecular details of the ABA signal perception system in maize. In the present study, we conducted bioinformatics analysis to find out the candidates (ZmPYL3 and ZmPP2C16) of the PYL and PP2C members most probably involved in ABA signaling in maize, cloned their encoding genes (ZmPYL3 and ZmPP2C16), verified the interaction between these two proteins in response to exogenous ABA induction by yeast two-hybrid assay and bimolecular fluorescence complementation, and investigated the expression patterns of these two genes under the induction of exogenous ABA by real-time fluorescence quantitative PCR. The results indicated that the ZmPYL3 and ZmPP2C16 proteins interacted in vitro and in vivo in response to the induction of exogenous ABA. The downregulated expression of the ZmPYL3 gene and the upregulated expression of the ZmPP2C16 gene are responsive to the induction of exogenous ABA. The ZmPYL3 and ZmPP2C16 proteins are the most probable members of the receptors and the second components of ABA signaling pathway, respectively.     

Identification of NDRG1-regulated genes associated with invasive potential in cervical and ovarian cancer cells

N-myc downstream regulated gene 1 (NDRG1) is an important gene regulating tumor invasion. In this study, shRNA technology was used to suppress NDRG1 expression in CaSki (a cervical cancer cell line) and HO-8910PM (an ovarian cancer cell line). In vitro assays showed that NDRG1 knockdown enhanced tumor cell adhesion, migration and invasion activities without affecting cell proliferation. cDNA microarray analysis revealed 96 deregulated genes with more than 2-fold changes in both cell lines after NDRG1 knockdown. Ten common upregulated genes (LPXN, DDR2, COL6A1, IL6, IL8, FYN, PTP4A3, PAPPA, ETV5 and CYGB) and one common downregulated gene (CLCA2) were considered to enhance tumor cell invasive activity. BisoGenet network analysis indicated that NDRG1 regulated these invasion effector genes/proteins in an indirect manner. Moreover, NDRG1 knockdown also reduced pro-invasion genes expression such as MMP7, TMPRSS4 and CTSK. These results suggest that regulation of invasion and metastasis by NDRG1 is a highly complicated process.     

NDRG2 is a new HIF-1 target gene necessary for hypoxia-induced apoptosis in A549 cells.

The NDRG2 gene belongs to a family of N-Myc downstream-regulated genes (NDRGs) and is expressed in many normal tissues. NDRG2 gene expression has been shown to be regulated in the stress response of certain cells. However, its function is not yet fully understood. Many studies have demonstrated that hypoxia, one of the stress responses, induced apoptosis in several cell types. In the current study, we investigated NDRG2 involvement in hypoxia response and found that NDRG2 expression was markedly up-regulated in several tumor cell lines exposed to hypoxic conditions or similar stresses at the mRNA and protein level. We also observed that the expression of NDRG2 was regulated by Hypoxia-inducible factor 1 (HIF-1) in tumor cells under hypoxia. Three hypoxia-responsive elements (HREs) in the NDRG2 promoter were identified. HRE1 could directly bind Hif-1 in vivo. Importantly, we found that silencing or enforcing the expression of NDRG2 could strongly inhibit or increase apoptosis. In addition, our data also showed that Ndrg2 was able to be translocated from the cytoplasm to the nucleus, and the segment from 101 to 178 amino acids of Ndrg2 is responsible for its translocation. Taken together, this study suggests that NDRG2 is a Hif-1 target gene and closely related with hypoxia-induced apoptosis in A549 cells.     

Elevated cyclin G2 expression intersects with DNA damage checkpoint signaling and is required for a potent G2/M checkpoint arrest response to doxorubicin

To maintain genomic integrity DNA damage response (DDR), signaling pathways have evolved that restrict cellular replication and allow time for DNA repair. CCNG2 encodes an unconventional cyclin homolog, cyclin G2 (CycG2), linked to growth inhibition. Its expression is repressed by mitogens but up-regulated during cell cycle arrest responses to anti-proliferative signals. Here we investigate the potential link between elevated CycG2 expression and DDR signaling pathways. Expanding our previous finding that CycG2 overexpression induces a p53-dependent G(1)/S phase cell cycle arrest in HCT116 cells, we now demonstrate that this arrest response also requires the DDR checkpoint protein kinase Chk2. In accord with this finding we establish that ectopic CycG2 expression increases phosphorylation of Chk2 on threonine 68. We show that DNA double strand break-inducing chemotherapeutics stimulate CycG2 expression and correlate its up-regulation with checkpoint-induced cell cycle arrest and phospho-modification of proteins in the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) signaling pathways. Using pharmacological inhibitors and ATM-deficient cell lines, we delineate the DDR kinase pathway promoting CycG2 up-regulation in response to doxorubicin. Importantly, RNAi-mediated blunting of CycG2 attenuates doxorubicin-induced cell cycle checkpoint responses in multiple cell lines. Employing stable clones, we test the effect that CycG2 depletion has on DDR proteins and signals that enforce cell cycle checkpoint arrest. Our results suggest that CycG2 contributes to DNA damage-induced G(2)/M checkpoint by enforcing checkpoint inhibition of CycB1-Cdc2 complexes.     

N-myc downstream-regulated gene 2 controls astrocyte morphology via Rho-GTPase signaling

Astrocyte undergoes morphology changes that are closely associated with the signaling communications at synapses. N-myc downstream-regulated gene 2 (NDRG2) is specifically expressed in astrocytes and is associated with several important astrocyte functions, but its potential role(s) relating to astrocyte morphological changes remain unknown. Here, primary astrocytes were prepared from neonatal Ndrg2^+/+ and Ndrg2^−/− pups, and the drug Y27632 was used to induce stellation. We then used a variety of methods to measure the levels of NDRG2, α-Actinin4, and glial fibrillary acidic protein (GFAP), and the activity of RhoA, Rac1, and Cdc42 in Y27632-treated astrocytes as well as in Ndrg2^+/+, Ndrg2^−/−, or Ndrg2^−/− + lentivirus (restore NDRG2 expression) astrocytes. We also conducted live-imaging and proteomics studies of the cultured astrocytes. We found that induction of astrocytes stellation (characterized by cytoplasmic retraction and process outgrowth) resulted in increased NDRG2 protein expression and Rac1 activity and in reduced α-Actinin4 protein expression and RhoA activity. Ndrg2 deletion induced astrocyte flattening, whereas the restoration of NDRG2 expression induced stellation. Ndrg2 deletion also significantly increased α-Actinin4 protein expression and RhoA activity yet reduced GFAP protein expression and Rac1 activity, and these trends were reversed by restoration of NDRG2 expression. Collectively, our results showed that Ndrg2 deletion promoted cell proliferation, interrupted stellation capability, and extensively altered the protein expression profiles of proteins that function in Rho-GTPase signaling. These findings suggest that NDRG2 functions to regulate astrocytes morphology via altering the accumulation of the Rho-GTPase signaling pathway components, thereby supporting that NDRG2 should be understood as a regulator of synaptic plasticity and thus neuronal communications.     

Identification and characterization of an alternative splice variant of Mpl with a high affinity for TPO and its activation of ERK1/2 signaling

The thrombopoietin receptor is a crucial element in thrombopoietin-initiated signaling pathways, which stimulates the differentiation of normal hematopoietic progenitor cells, the maturation of megakaryocytes, and the generation of platelets. In this study, we identified a novel activating variant of thrombopoietin receptor, termed Mpl-D, in human megakaryoblastic leukemia Dami cells and demonstrated that the binding affinity of the Mpl-D receptor for thrombopoietin is enhanced. Cell cycle analysis revealed that in the presence of thrombopoietin, most Mpl-D expressing NIH3T3 (NIH3T3/Mpl-D) cells were prevalent in G1 phase while the S and G2/M populations were less frequently observed. Unexpectedly, thrombopoietin induced strong and prolonged ERK1/2 signaling in NIH3T3/Mpl-D cells compared with its receptor wild-type expressing NIH3T3 (NIH3T3/Mpl-F) cells. Further analysis of the mRNA levels of cyclin D1/D2 in NIH3T3/Mpl-D cells demonstrated markedly down-regulated expression compared to NIH3T3/Mpl-F cells in the presence of thrombopoietin. Thus, the prolonged activation of ERK1/2 by Mpl-D might lead to G1 cell cycle arrest through a profound reduction of cyclin D1/D2 in order to support cell survival without proliferation. We also provided tertiary structural basis for the Mpl-D and thrombopoietin interaction, which might provide insights into how Mpl-D effectively increases binding to thrombopoietin and significantly contributes to its specific signaling pathway. These results suggest a new paradigm for the regulation of cytokine receptor expression and function through the alternative splicing variant of Mpl in Dami cells, which may play a role in the pathogenesis of megakaryoblastic leukemia.     

Regulations of Reversal of Senescence by PKC Isozymes in Response to 12-O-Tetradecanoylphorbol-13-Acetate via Nuclear Translocation of pErk1/2

The mechanism by which 12-O-tetradecanoylphorbol-13-acetate (TPA) bypasses cellular senescence was investigated using human diploid fibroblast (HDF) cell replicative senescence as a model. Upon TPA treatment, protein kinase C (PKC) α and PKCβ1 exerted differential effects on the nuclear translocation of cytoplasmic pErk1/2, a protein which maintains senescence. PKCα accompanied pErk1/2 to the nucleus after freeing it from PEA-15pS¹⁰⁴ via PKCβ1 and then was rapidly ubiquitinated and degraded within the nucleus. Mitogen-activated protein kinase docking motif and kinase activity of PKCα were both required for pErk1/2 transport to the nucleus. Repetitive exposure of mouse skin to TPA downregulated PKCα expression and increased epidermal and hair follicle cell proliferation. Thus, PKCα downregulation is accompanied by in vivo cell proliferation, as evidenced in 7, 12-dimethylbenz(a)anthracene (DMBA)-TPA-mediated carcinogenesis. The ability of TPA to reverse senescence was further demonstrated in old HDF cells using RNA-sequencing analyses in which TPA-induced nuclear PKCα degradation freed nuclear pErk1/2 to induce cell proliferation and facilitated the recovery of mitochondrial energy metabolism. Our data indicate that TPA-induced senescence reversal and carcinogenesis promotion share the same molecular pathway. Loss of PKCα expression following TPA treatment reduces pErk1/2-activated SP1 biding to the p21^WAF1 gene promoter, thus preventing senescence onset and overcoming G1/S cell cycle arrest in senescent cells.