Cell Cycle Regulatory Cyclin D1 Research Articles

Stage-specific requirement for cyclin D1 in glial progenitor cells of the cerebral cortex

Despite the vast abundance of glial progenitor cells in the mouse brain parenchyma, little is known about the molecular mechanisms driving their proliferation in the adult. Here we unravel a critical role of the G1 cell cycle regulator cyclin D1 in controlling cell division of glial cells in the cortical grey matter. We detect cyclin D1 expression in Olig2-immunopositive (Olig2+) oligodendrocyte progenitor cells, as well as in Iba1+ microglia and S100β+ astrocytes in cortices of 3-month-old mice. Analysis of cyclin D1-deficient mice reveals a cell and stage-specific molecular control of cell cycle progression in the various glial lineages. While proliferation of fast dividing Olig2+ cells at early postnatal stages becomes gradually dependent on cyclin D1, this particular G1 regulator is strictly required for the slow divisions of Olig2+/NG2+ oligodendrocyte progenitors in the adult cerebral cortex. Further, we find that the population of mature oligodendrocytes is markedly reduced in the absence of cyclin D1, leading to a significant decrease in the number of myelinated axons in both the prefrontal cortex and the corpus callosum of 8-month-old mutant mice. In contrast, the pool of Iba1+ cells is diminished already at postnatal day 3 in the absence of cyclin D1, while the number of S100β+ astrocytes remains unchanged in the mutant.

CAPER, a novel regulator of human breast cancer progression

CAPER is an estrogen receptor (ER) co-activator that was recently shown to be involved in human breast cancer pathogenesis. Indeed, we reported increased expression of CAPER in human breast cancer specimens. We demonstrated that CAPER was undetectable or expressed at relatively low levels in normal breast tissue and assumed a cytoplasmic distribution. In contrast, CAPER was expressed at higher levels in ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) specimens, where it assumed a predominantly nuclear distribution. However, the functional role of CAPER in human breast cancer initiation and progression remained unknown. Here, we used a lentiviral-mediated gene silencing approach to reduce the expression of CAPER in the ER-positive human breast cancer cell line MCF-7. The proliferation and tumorigenicity of MCF-7 cells stably expressing control or human CAPER shRNAs was then determined via both in vitro and in vivo experiments. Knockdown of CAPER expression significantly reduced the proliferation of MCF-7 cells in vitro. Importantly, nude mice injected with MCF-7 cells harboring CAPER shRNAs developed smaller tumors than mice injected with MCF-7 cells harboring control shRNAs. Mechanistically, tumors derived from mice injected with MCF-7 cells harboring CAPER shRNAs displayed reduced expression of the cell cycle regulators PCNA, MCM7, and cyclin D1, and the protein synthesis marker 4EBP1. In conclusion, knockdown of CAPER expression markedly reduced human breast cancer cell proliferation in both in vitro and in vivo settings. Mechanistically, knockdown of CAPER abrogated the activity of proliferative and protein synthesis pathways.

One Patient, Two Uncommon B-Cell Neoplasms: Solitary Plasmacytoma following Complete Remission from Intravascular Large B-Cell Lymphoma Involving Central Nervous System

Second lymphoid neoplasms are an uncommon but recognized feature of non-Hodgkin's lymphomas, putatively arising secondary to common genetic or environmental risk factors. Previous limited evaluations of clonal relatedness between successive mature B-cell malignancies have yielded mixed results. We describe the case of a man with intravascular large B-cell lymphoma involving the central nervous system who went into clinical remission following immunochemotherapy and brain radiation, only to relapse 2 years later with a plasmacytoma of bone causing cauda equina syndrome. The plasmacytoma stained strongly for the cell cycle regulator cyclin D1 on immunohistochemistry, while the original intravascular large cell lymphoma was negative, a disparity providing no support for clonal identity between the 2 neoplasms. Continued efforts atcataloging and evaluating unique associations of B-cell malignancies are critical to improving understanding of overarching disease biology in B-cell malignancies.

The Short Lef-1 Isoform, Lacking The β-Catenin Binding Domain, Is Not Acting As a Dominant Negative Lef-1 Variant, But Is a Hematopoietic Active Protein With Unique DNA Binding Properties

Studies in the hematopoietic system of human and mice have demonstrated that the Wnt signaling pathway is essential for maintenance, activation and proliferation of normal hematopoietic stem cells. Lef-1 is a member of the Lef-1/T-cell-factor (Tcf) family of transcription factors regulated by the canonical Wnt signaling pathway and generally directly interacts with β-catenin in transcriptional complexes to induce expression of target genes, including the cell cycle regulators cyclin D1 and c-myc. We have previously shown that aberrant expression of Lef-1 perturbs normal hematopoietic stem cell (HSC) and progenitor function and induces acute myeloid leukemia in the murine bone marrow transplantation model and that it is a novel prognostic independent factor in patients with normal karyotype AML (Petropoulos et al. JEM, 2008; Metzeler et al., Blood 2012). Furthermore, we could recently demonstrate that shRNA mediated depletion of Lef-1 drastically compromises the function of long-term repopulating stem cells (Edmaier et al., Leukemia, in press), indicating a vital function of Lef-1 at the level of the most primitive stem cell compartment. So far, all these Lef-1 functions are ascribed to the full-length transcript of Lef-1, which acts as transcriptional mediator of Wnt signaling via its β-catenin binding domain, whereas the shorter Lef-1 isoform, deriving from an intronic promoter is thought to function as a dominant negative variant. To dissect the role of the ‘dominant negative' Lef-1 isoform we first retrovirally engineered primary murine bone marrow cells to express the N-terminal deleted Lef-1 (Lef-1d56), lacking the β-catenin binding domain in comparison to the full-length Lef-1 (Lef-1WT) and the empty retroviral EGFP control. Loss of binding to β-catenin was validated by Co-IP for Lef-1d56. Of note, Lef-1d56 induced a 2,05fold and significant increase at the level of clonogenic cells in vitro compared to the control (n=3; p<0,05) and did not differ substantially from the activity of Lef-1 WT. β-catenin binding did not impact Lef-1 activity at the level of the short-term repopulating stem cells as documented in the CFU-S assay with almost comparable CFU-S frequencies between the wild-type and the mutant Lef-1 (d56) (73 CFU-S/1x105 and 64 CFU-S/1x105 , respectively, compared to 38 CFU-S/1x105 for the EGFP control; p<0.05). In clear contrast, loss of β-catenin interaction reduced the CRU frequency dramatically (1:597.197(Lef-1 WT) and 1:1.675.238 (Lef-1 d56), respectively and 1: 1.233.152 for the EGFP control; p=0,066), indicating the necessity of Lef-1 to collaborate with β-catenin at the level of the long-term repopulating stem cell. Retroviral expression of Lef-1 d56 in murine bone marrow stem and progenitor cells for 48h induced a distinct gene expression profile and deregulated more genes than Lef-1WT compared to the EGFP control. Differentially expressed genes between Lef-1d56 and Lef-1WT comprised known hematopoietic factors such as like Gata2, Ets1 and genes associated to the G-protein coupled receptor protein signaling. ChIP-Seq in hematopoietic murine cells revealed unique binding sites for Lef-1d56 compared to Lef-1WT with binding of the Lef-1d56 to promoters of Wnt5a and CD81. Taken together, our data clearly indicate that loss of the β-catenin binding site does not convert Lef-1 into an overall dominant negative variant, but creates a ‘neomorphic' isoform with distinct biological and DNA binding properties. Analyses are ongoing which quantify the expression of LEF-1WT and the β-catenin domain lacking LEF-1 isoform in over 100 patients with normal karyotype AML in comparison to normal human stem and progenitor cells to test potential differences between normal and leukemic cells as well as associations of the two LEF-1 transcripts with the mutational status, gene expression and treatment outcome in this AML patient group. Disclosures:No relevant conflicts of interest to declare.

Estrogen receptors are involved in polychlorinated biphenyl-induced apoptosis on mouse spermatocyte GC-2 cell line

Polychlorinated biphenyls (PCBs) are widespread persistent environmental contaminants which have been shown to have reproductive toxicity and to disturb spermatogenesis. But the precise mechanism is not clear. A mouse pachytene spermatocyte-derived cell line, GC-2 cells were used in the present study to investigate the toxic effect of PCBs (Aroclor 1254) and explore the underlying molecular mechanism. Results showed that Aroclor 1254 inhibited cell proliferation, caused the arrest of cells in G0/G1 phase and induced apoptosis which might be partly explained by the decreased expression of Bcl-2 and cell cycle regulator cyclin D1 together with the activation of caspase-3. Besides, the treatment of Aroclor 1254 decreased the protein expression of estrogen receptor (ER)-α while increasing that of ERβ. Then the administration of selective ERα agonist PPT partly reversed Aroclor 1254-induced alteration in Bcl-2, caspase-3 and cyclin D1 protein expression while selective ERβ agonist DPN accelerated it. These results suggest that Aroclor 1254, working through ERα and ERβ, interferes with the expression of proteins involved in the balance between cellular apoptosis and proliferation.

Beta-Cell ARNT Is Required for Normal Glucose Tolerance in Murine Pregnancy

AimsInsulin secretion increases in normal pregnancy to meet increasing demands. Inability to increase beta-cell function results in gestational diabetes mellitus (GDM). We have previously shown that the expression of the transcription factor ARNT (Aryl-hydrocarbon Receptor Nuclear Translocator) is reduced in the islets of humans with type 2 diabetes. Mice with a beta-cell specific deletion of ARNT (β-ARNT mice) have impaired glucose tolerance secondary to defective insulin secretion. We hypothesised that ARNT is required to increase beta-cell function during pregnancy, and that β-ARNT mice would be unable to compensate for the beta-cell stress of pregnancy. The aims of this study were to investigate the mechanisms of ARNT regulation of beta-cell function and glucose tolerance in pregnancy.Methodsβ-ARNT females were mated with floxed control (FC) males and FC females with β-ARNT males.ResultsDuring pregnancy, β-ARNT mice had a marked deterioration in glucose tolerance secondary to defective insulin secretion. There was impaired beta-cell proliferation in late pregnancy, associated with decreased protein and mRNA levels of the islet cell-cycle regulator cyclinD2. There was also reduced expression of Irs2 and G6PI. In contrast, in control mice, pregnancy was associated with a 2.1-fold increase in ARNT protein and a 1.6-fold increase in cyclinD2 protein, and with increased beta-cell proliferation.ConclusionsIslet ARNT increases in normal murine pregnancy and beta-cell ARNT is required for cyclinD2 induction and increased beta-cell proliferation in pregnancy.

Brief report: SRF-dependent MiR-210 silences the sonic hedgehog signaling during cardiopoesis

Serum response factor (SRF) wields potent gene silencing activity through its regulation over numerous microRNAs (miRs). Here, SRF directs embryonic stem cell (ESC) progenitor cell lineage specification in part by silencing genes through miR-210. Viral expression of miR-210 in murine ESCs-derived embryoid bodies (EBs) inhibited cell growth and inhibited the appearance of cardiac progenitor markers Nkx2.5 and Gata4 and terminal differentiated contractile proteins Mlc2v and βMHC. Knockdown of miR-210 expression via antisense RNA activated cardiac progenitor gene activity. miR-210 inhibitory activity was attributed to silencing of the Sonic hedgehog (Shh) signaling pathway, which fosters the cardiac progenitor program. miR-210 directly silenced Shh via targeting of the Shh 3'UTR, comparable to the chemical Shh inhibitor, cyclopamine. miR-210 silencing of Shh/Gli1 signaling also blocked expression of the cell cycle regulators Cyclin D1 and Cyclin D2, and EB cell expansion. Absence of SRF expression in SRF null EBs blocked miR-210 expression, coincident with enhanced Shh, and Gli1 gene activity. Thus, SRF-dependent miR-210 expression may operate as a novel silencer of the Shh signaling pathway.

The monoclonal antibody CH12 augments 5-fluorouracil-induced growth suppression of hepatocellular carcinoma xenografts expressing epidermal growth factor receptor variant III

5-Fluorouracil (5-FU) is one of the most common chemotherapeutic agents used for the treatment of hepatocellular carcinoma (HCC). However, chemoresistance has precluded the use of 5-FU alone in clinical regimens. Combination therapies with 5-FU and other anticancer agents are considered to be a therapeutic option for patients with HCC. We previously reported that the expression of epidermal growth factor receptor variant III (EGFRvIII) can decrease the sensitivity of HCC cells to 5-FU. To overcome this problem, in this study, we elucidated the mechanism underlying EGFRvIII-mediated 5-FU resistance. We observed that EGFRvIII expression can induce miR-520d-3p downregulation and the ensuing upregulation of the transcription factor E2F-1 and the enzyme thymidylate synthase (TS), which may lead to drug resistance. Intriguingly, we found that CH12, a monoclonal antibody directed against EGFRvIII, and 5-FU together had an additive antitumor effect on EGFRvIII-positive HCC xenografts and significantly improved survival in all mice with established tumors when compared with either 5-FU or CH12 alone. Mechanistically, compared with 5-FU alone, the combination more noticeably downregulated EGFR phosphorylation and Akt phosphorylation as well as the expression of the apoptotic protector Bcl-xL and the cell cycle regulator cyclin D1. Additionally, the combination upregulated the expression of the cell cycle inhibitor p27 in in vivo treatment. More interestingly, CH12 treatment upregulated miR-520-3p and downregulated E2F-1 and TS at the mRNA and protein levels. Collectively, these observations suggest that the combination of 5-FU with mAb CH12 is a potential means of circumventing EGFRvIII-mediated 5-FU resistance in HCC.

The Cell-Cycle State of Stem Cells Determines Cell Fate Propensity

SummarySelf-renewal and differentiation of stem cells are fundamentally associated with cell-cycle progression to enable tissue specification, organ homeostasis, and potentially tumorigenesis. However, technical challenges have impaired the study of the molecular interactions coordinating cell fate choice and cell-cycle progression. Here, we bypass these limitations by using the FUCCI reporter system in human pluripotent stem cells and show that their capacity of differentiation varies during the progression of their cell cycle. These mechanisms are governed by the cell-cycle regulators cyclin D1–3 that control differentiation signals such as the TGF-β-Smad2/3 pathway. Conversely, cell-cycle manipulation using a small molecule directs differentiation of hPSCs and provides an approach to generate cell types with a clinical interest. Our results demonstrate that cell fate decisions are tightly associated with the cell-cycle machinery and reveal insights in the mechanisms synchronizing differentiation and proliferation in developing tissues.

Cell cycle regulators cyclin D1 and CDK4/6 have estrogen receptor-dependent divergent functions in breast cancer migration and stem cell-like activity

Cyclin D1 and its binding partners CDK4/6 are essential regulators of cell cycle progression and are implicated in cancer progression. Our aim was to investigate a potential regulatory role of these proteins in other essential tumor biological characteristics. Using a panel of breast cancer cell lines and primary human breast cancer samples, we have demonstrated the importance of these cell cycle regulators in both migration and stem-like cell activity. siRNA was used to target cyclin D1 and CDK4/6 expression, having opposing effects on both migration and stem-like cell activity dependent upon estrogen receptor (ER) expression. Inhibition of cyclin D1 or CDK4/6 increases or decreases migration and stem-like cell activity in ER−ve (ER-negative) and ER+ve (ER-positive) breast cancer, respectively. Furthermore, overexpressed cyclin D1 caused decreased migration and stem-like cell activity in ER−ve cells while increasing activity in ER+ve breast cancer cells. Treatment of breast cancer cells with inhibitors of cyclin D1 and CDK4/6 (Flavopiridol/PD0332991), currently in clinical trials, mimicked the effects observed with siRNA treatment. Re-expression of ER in two ER−ve cell lines was sufficient to overcome the effects of either siRNA or clinical inhibitors of cyclin D1 and CDK4/6. In conclusion, cyclin D1 and CDK4/6 have alternate roles in regulation of migration and stem-like cell activity. Furthermore, these effects are highly dependent upon expression of ER. The significance of these results adds to our general understanding of cancer biology but, most importantly, could be used diagnostically to predict treatment response to cell cycle inhibition in breast cancer.

Changes in the Transcriptome of the Human Endometrial Ishikawa Cancer Cell Line Induced by Estrogen, Progesterone, Tamoxifen, and Mifepristone (RU486) as Detected by RNA-Sequencing

BackgroundEstrogen (E2) and progesterone (P4) are key players in the maturation of the human endometrium. The corresponding steroid hormone modulators, tamoxifen (TAM) and mifepristone (RU486) are widely used in breast cancer therapy and for contraception purposes, respectively.Methodology/Principal findingsGene expression profiling of the human endometrial Ishikawa cancer cell line treated with E2 and P4 for 3 h and 12 h, and TAM and RU486 for 12 h, was performed using RNA-sequencing. High levels of mRNA were detected for genes, including PSAP, ATP5G2, ATP5H, and GNB2L1 following E2 or P4 treatment. A total of 82 biomarkers for endometrial biology were identified among E2 induced genes, and 93 among P4 responsive genes. Identified biomarkers included: EZH2, MDK, MUC1, SLIT2, and IL6ST, which are genes previously associated with endometrial receptivity. Moreover, 98.8% and 98.6% of E2 and P4 responsive genes in Ishikawa cells, respectively, were also detected in two human mid-secretory endometrial biopsy samples. TAM treatment exhibited both antagonistic and agonistic effects of E2, and also regulated a subset of genes independently. The cell cycle regulator cyclin D1 (CCND1) showed significant up-regulation following treatment with TAM. RU486 did not appear to act as a pure antagonist of P4 and a functional analysis of RU486 response identified genes related to adhesion and apoptosis, including down-regulated genes associated with cell-cell contacts and adhesion as CTNND1, JUP, CDH2, IQGAP1, and COL2A1. ConclusionsSignificant changes in gene expression by the Ishikawa cell line were detected after treatments with E2, P4, TAM, and RU486. These transcriptome data provide valuable insight into potential biomarkers related to endometrial receptivity, and also facilitate an understanding of the molecular changes that take place in the endometrium in the early stages of breast cancer treatment and contraception usage.

Supersize me—new insights into cortical expansion and gyration of the mammalian brain

During evolution, the mammalian brain massively expanded its size. However, the exact roles of distinct neural precursors, identified in the developing cortex during embryogenesis, for size expansion and surface folding (i.e., gyration) remain largely unknown. New findings by Nonaka-Kinoshita et al advance our understanding of embryonic neural precursor function by identifying cell type-selective functions for size expansion and folding, and challenge previously held concepts of mammalian brain development.

Upregulation of miR-370 contributes to the progression of gastric carcinoma via suppression of FOXO1

FOXO1 is downregulated in a number of cancers. However, the underlying mechanisms are poorly understood. In this study, we report that the expression of miR-370 was upregulated in gastric cancer cell lines and gastric cancer tissues. Overexpression of miR-370 in gastric cancer cells promoted the cell proliferation and anchorage-independent growth, while silencing of miR-370 showed opposite effects. miR-370-induced proliferation was correlated with the downregulation of cyclin-dependent kinase inhibitors, p27Kip1 and p21Cip1, and the upregulation of the cell cycle regulator cyclin D1. Furthermore, we identified that FOXO1 is the functional target of miR-370. Restored expression of FOXO1 together with miR-370 strongly abrogated miR-370-induced cell proliferation. Taken together, our results revealed a novel mechanism of FOXO1 suppression mediated by miR-370 in gastric cancer.

Abstract 1337: The UGT1A6_19_GG genotype is associated with increased breast cancer risk based on a two-stage study.

Abstract Over 20 breast cancer susceptibility loci have been identified mainly by genome-wide association studies (GWAS). These include Fibroblast Growth Factor Receptor (FGFR2) and loci close to the cell cycle regulator Cyclin D1 (CCND1) at 11q13. Phase I and II enzymes are anticipated to also contribute to breast cancer risk due to their roles in the inactivation, detoxification and elimination of endogenous and exogenous carcinogenic compounds. However, a GWAS approach is inappropriate for many genes due to their high degree of sequence homologies. For the UDP glucuronosyltransferase 1 family, polypeptide A6 (UGT1A6), a key player in Phase II steroid metabolism, we pursued a candidate approach using a specific assay design for gene amplification. We focused on the UGT1A6_19_T&amp;gt;G polymorphism (rs6759892) because this variant has been suggested to decrease enzyme activity. Moreover, our prior efforts showed an association between the UGT1A6_19_GG genotype and increased breast cancer risk (OR 1.17; 95% CI: 1.03-1.34; p = 0.014) in two population-based breast cancer case-control studies from Germany (3,139 cases and 5,466 controls) (MARIE-GENICA, 2010, Breast Cancer Res Treat 119:463-74). To validate these findings we extended the analysis to four independent study collections from Finland (KBCP, OBCS), Germany (BBCC) and Sweden (SASBAC) comprising 3,635 cases and 2,648 controls. Moreover, we performed a pooled analysis of all study subjects (7,418 cases and 8,720 controls) all being of European descent. UGT1A6_19_T&amp;gt;G genotyping was done by Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and statistical analysis by logistic regression adjusted for age and study using SPSS. Power calculation was done with nQuery Advisor ® which showed that the pooled study had an 80% power to detect a minimum OR of 1.06 (α = 0.05, two-sided test). The analysis of the BBCC, KBCP, OBCS and SASBAC collections confirmed our previous findings showing an increased overall breast cancer risk among women with the UGT1A6_19_GG genotype (OR 1.13; 95% CI: 1.05-1.22; p = 0.001). The pooled study showed a similar effect (OR 1.09; 95% CI: 1.04-1.14; p = 0.0004). We conclude based on the significant association between the UGT1A6_19_GG variant and breast cancer that a decreased conjugation activity of UGT1A6 might elevate the exposure to carcinogenic compounds thereby increasing the risk of breast cancer. Citation Format: Ofure M. Obazee, Christina Justenhoven, Stefan Winter, Sylvia Rabstein, Anne Lotz, Volker Harth, Beate Pesch, Thomas Brüning, Christian Baisch, Jaana M. Hartikainen, Arto Mannermaa, Robert Winqvist, Peter Fasching, Per Hall, Jenny Chang-Claude, Ute Hamann, Yon-Dschun Ko, Hiltrud Brauch. The UGT1A6_19_GG genotype is associated with increased breast cancer risk based on a two-stage study. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1337. doi:10.1158/1538-7445.AM2013-1337

Inhibition of STAT3 with orally active JAK inhibitor, AZD1480, decreases tumor growth in Neuroblastoma and Pediatric SarcomasIn vitroandIn vivo

The IL-6/JAK/STAT pathway is a key signal transduction pathway implicated in the pathogenesis of many human cancers, suggesting that kinase inhibitors targeting JAK/STAT3 may have a broad spectrum of antitumor activity. AZD1480, a pharmacological JAK1/2 inhibitor, exhibits anti-tumor potency in multiple adult malignancies. To evaluate the efficacy of inhibition of JAK/STAT3 signal transduction pathway we assessed the activity of AZD1480 in pediatric malignancies using preclinical models of three highly malignant pediatric solid tumors: neuroblastoma (NB), rhabdomyosarcoma (RMS) and the Ewing Sarcoma Family Tumors (ESFT). In this study, we employed panels of biomedical and biological experiments to evaluate the in vitro and in vivo activity of AZD1480 in NB, RMS and ESFT. Our data indicate that AZD1480 blocks endogenous as well as IL-6 induced STAT3 activation. AZD1480 decreases cell viability in 7/7NB, 7/7RMS and 2/2 ESFT cell lines (median EC50 is 1.5 μM, ranging from 0.36-5.37 μM). AZD1480 induces cell growth inhibition and caspase-dependent apoptosis in vitro and decreases expression of STAT3 target genes, including cell cycle regulators CyclinD1, 3 and CDC25A, anti-apoptotic genes Bcl-2 and survivin, the metastasis-related factor TIMP-1 and c-Myc. In vivo studies showed AZD1480 significantly decreased tumor growth and prolonged overall survival in tumor-bearing mice. Tumors from AZD1480-treated mice showed inhibition of activated STAT3 as well as decreased expression of STAT3 downstream targets. Our study provides strong evidence of the anti-tumor growth potency of JAK inhibitor AZD1480 in pediatric solid tumors, providing proof-of principle that inhibition of the JAK/STAT3 signal transduction could be a promising therapeutic target for high-risk pediatric solid tumors.

GPR158, an Orphan Member of G Protein-Coupled Receptor Family C: Glucocorticoid-Stimulated Expression and Novel Nuclear Role

Members of the large G protein-coupled receptor (GPCR) clan are implicated in many physiological and disease processes, making them important therapeutic drug targets. In the present study, we follow up on results of a pilot study suggesting a functional relationship between glucocorticoid (GC)-induced ocular hypertension and GPR158, one of three orphan members of the GPCR Family C. GC treatment increases levels of GPR158 mRNA and protein through transcriptional mechanisms, in cultured trabecular meshwork (TBM) cells derived from the eye's aqueous outflow pathway. Like treatment with GCs, transient overexpression of GPR158 stimulates cell proliferation, while siRNA knockdown of endogenous GPR158 has the opposite effect. Both endogenous and overexpressed GPR158 show an unusual subcellular localization pattern, being found almost entirely in the nucleus. However, when cells are treated with inhibitors of clathrin-mediated endocytosis, GPR158 is shifted to the plasma membrane. Mutation of a bipartite nuclear localization signal (NLS) in the 8th helix also shifts GPR158 out of the nucleus, but in this case the protein is found in vesicles localized in the cytoplasm. These results suggest that newly synthesized GPR158 first traffics to the plasma membrane, where it rapidly undergoes endocytosis and translocation to the nucleus. Significantly, mutation of the NLS abrogates GPR158-mediated enhancement of cell proliferation, indicating a functional requirement for nuclear localization. GPR158 overexpression upregulates levels of the cell cycle regulator cyclin D1, but mutation of the NLS reverses this. Overexpression of GPR158 enhances the barrier function of a TBM cell monolayer, which is associated with an increase in the levels of tight junction proteins ZO-1 and occludin, similar to reported studies on GC treatment. Regulated paracellular permeability controls aqueous outflow facility in vivo. Since GCs stimulate GPR158 expression, the result is consistent with a role for elevation of GPR158 expression in GC-induced ocular hypertension.

Clinical implications of deregulated CDK4 and Cyclin D1 expression in patients with human hepatocellular carcinoma

Deregulated cell cycle can contribute to the unscheduled proliferation in cancer cells. Overexpression of cell cycle regulators CDK4 and Cyclin D1 has been reported in many cancers. The aim of this study is to determine the clinical implications of CDK4 and Cyclin D1 in hepatocellular carcinoma (HCC). The levels of mRNA and protein were analyzed by quantitative real-time RT-PCR and immunohistochemistry, respectively, in 59 paired HCC and the neighboring noncancer tissues. The relationship between CDK4 and Cyclin D1 expression, clinicopathological parameters, and prognosis was investigated. Our data demonstrated that the mRNA level of CDK4 was up-regulated (p = 0.019), while that of Cyclin D1 was down-regulated (p = 0.002), in HCC. Immunohistochemical data confirmed that CDK4 protein was increased in 73 % and Cyclin D1 protein was decreased in 66 % of HCC samples. Overexpression of CDK4 was correlated with HBV (p = 0.054, borderline significant), tumor size (p = 0.014), and stage (p = 0.010). The Kaplan-Meier survival curves showed that high CDK4 was correlated with a poor survival rate (I vs. II, p < 0.001; I vs. III, p < 0.001). Univariate analysis showed that tumor size (p = 0.002), stage (p = 0.021), and high CDK4 score (I vs. II-III, p < 0.001) were significant prognostic factors. Multivariate analysis showed that tumor size (p = 0.007) and high CDK4 score (I vs. II-III, p < 0.001) were independent factors for overall survival of HCC. The expression of Cyclin D1 was not correlated with CDK4 expression, tumor grades, survival rate, and any clinicopathological parameters. CDK4 could provide a clinical prognostic marker for HCC progression.

Convergence of oncogenic and hormone receptor pathways promotes metastatic phenotypes

Cyclin D1b is a splice variant of the cell cycle regulator cyclin D1 and is known to harbor divergent and highly oncogenic functions in human cancer. While cyclin D1b is induced during disease progression in many cancer types, the mechanisms underlying cyclin D1b function remain poorly understood. Herein, cell and human tumor xenograft models of prostate cancer were utilized to resolve the downstream pathways that are required for the protumorigenic functions of cyclin D1b. Specifically, cyclin D1b was found to modulate the expression of a large transcriptional network that cooperates with androgen receptor (AR) signaling to enhance tumor cell growth and invasive potential. Notably, cyclin D1b promoted AR-dependent activation of genes associated with metastatic phenotypes. Further exploration determined that transcriptional induction of SNAI2 (Slug) was essential for cyclin D1b-mediated proliferative and invasive properties, implicating Slug as a critical driver of disease progression. Importantly, cyclin D1b expression highly correlated with that of Slug in clinical samples of advanced disease. In vivo analyses provided strong evidence that Slug enhances both tumor growth and metastatic phenotypes. Collectively, these findings reveal the underpinning mechanisms behind the protumorigenic functions of cyclin D1b and demonstrate that the convergence of the cyclin D1b/AR and Slug pathways results in the activation of processes critical for the promotion of lethal tumor phenotypes.

Transformation of human ovarian surface epithelial cells by Krüppel-like factor 8

Previously we demonstrated that Krüppel-like factor 8 (KLF8) participates in oncogenic transformation of mouse fibroblasts and is highly overexpressed in human ovarian cancer. In this work, we first correlated KLF8 overexpression with the aggressiveness of ovarian patient tumors and then tested if KLF8 could transform human ovarian epithelial cells. Using the immortalized non-tumorigenic human ovarian surface epithelial cell line T80 and retroviral infection, we generated cell lines that constitutively overexpress KLF8 alone or its combination with the known ovarian oncogenes c-Myc, Stat3c and/or Akt and examined the cell lines for anchorage-independent growth and tumorigenesis. The soft agar clonogenic assay showed that T80/KLF8 cells formed significantly more colonies than the mock cells. Interestingly, the cells expressing both KLF8 and c-Myc formed the largest amounts of colonies greater than the sum of colonies formed by the cells expressing KLF8 and c-Myc alone. These results suggested that KLF8 might be a weak oncogene that works cooperatively with c-Myc to transform ovarian cells. Surprisingly, overexpression of KLF8 alone was sufficient to induce tumorigenesis in nude mice resulting in short life span whether the T80/KLF8 cells were injected subcutaneously, intraperitoneally or orthotopically into the ovarian bursa. Histopathological studies confirmed that the T80/KLF8 tumors were characteristic of human serous ovarian carcinomas. Comparative expression profiling and functional studies identified the cell cycle regulators cyclin D1 and USP44 as primary KLF8 targets and effectors for the T80 transformation. Overall, we identified KLF8 overexpression as an important factor in human ovarian carcinoma pathogenesis.

CITED2 is a novel direct effector of peroxisome proliferator‐activated receptor γ in suppressing hepatocellular carcinoma cell growth

Previous reports from these authors found that activation of peroxisome proliferator-activated receptor gamma (PPARγ) suppressed hepatocellular carcinoma (HCC). This study sought to identify the molecular target of PPARγ and characterize its antitumor effect in HCC. Optimal PPARγ binding activity was obtained using the PPARγ agonist rosiglitazone (100 μM) as determined by enzyme-linked immunosorbent assay. Under PPARγ activation, 114 PPARγ downstream targets associated with cancer development were identified by oligonucleotide microarray and Gene Ontology analysis. Among them, Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2 (CITED2) was the most prominent PPARγ-bound target, as determined by chromatin immunoprecipitation-polymerase chain reaction. CITED2 messenger RNA and protein was significantly down-regulated in primary HCCs compared with their adjacent nontumor tissues. PPARγ induced expression of CITED2 in HCC cell lines after adenovirus-PPARγ transduction. The biological function of CITED2 was evaluated by loss- and gain-of-function assays. CITED2 knockdown in the hepatocyte cell line LO2 and HCC cell line Hep3B significantly increased cell viability and clonogenicity, and promoted G1 -S phase transition in both cell lines. In contrast, ectopic expression of CITED2 in HepG2 and BEL7404 HCC cell lines significantly suppressed cell growth. The tumor suppressive effect of CITED2 was associated with up-regulation of cyclin-dependent kinase inhibitors p15(INK4B) , p21(Wat1/Cip1) , p27(Kip1) , antiproliferative regulator interferon alpha 1, proapoptotic mediators including tumor necrosis factor receptor superfamily member 1A (TNFRSF1A), TNFRSF25, caspase-8, granzyme A, and the tumor suppressor gene maspin. CITED2 was also associated with the down-regulation of cell cycle regulator cyclin D1, oncogene telomerase reverse transcriptase, and proinvasion/metastasis gene matrix metallopeptidase 2. CITED2 is a direct effector of PPARγ for tumor suppression. Cancer 2013. © 2012 American Cancer Society.