Phosphorylation of P27 by AKT is required for inhibition of cell cycle progression in cholangiocarcinoma.

BACKGROUND AND OBJECTIVE: P27 is a putative tumor suppressor when located in the nucleus and AKT is an inhibitor of P27 which promotes growth of cholangiocarcinoma. We hypothesized that AKT-dependent phosphorylation at the P27 nuclear localization sequence T157 leads to nuclear export of P27, and thus loss of its tumor suppressive function. This study investigated whether loss of cell cycle regulation in cholangiocarcinoma due to subcellular localization of P27. METHODS: Human cholangiocarcinoma cells were transfected with AKT. P27 was tagged with yellow fluorescence protein. Cell cycle progression was determined by flow cytometry. Migration and invasion of was measured by transwell assay. RESULTS: Overexpression of wildtype P27 or P27-T157A in Mz-ChA-1 cells resulted in G1 arrest; expression of myr-AKT caused translocation of P27-YFP and endogenous P27 from the nucleus to the cytoplasm, leading to inhibition of P27-dependent G1 arrest; the AKT inhibitor and expression of dnAKT increased P27-YFP accumulation in the nucleus and promoted G1 arrest. In contrast, cells expressing YFP-P27-T157A or P27-YFP accumulated only in the nucleus. Co-expression of myr-AKT failed to induce P27-YFP translocation to the cytoplasm or inhibit G1 arrest. Overexpression of P27-T157A significantly increased migration and invasion. CONCLUSIONS: Cholangiocarcinoma growth is associated with nuclear export of P27 that is due to AKT-mediated phosphorylation of P27 at T157.

Growth Arrest Specific 2 (GAS2) is a Critical Mediator of Germ Cell Cyst Breakdown and Folliculogenesis in Mice.

In the mouse ovary, the primordial follicle pool is established through a diverse array of signaling pathways and tissue remodeling events. Growth arrest specific gene two (GAS2) is a highly conserved cytoskeleton-associated protein whose in vivo function remains unclear. In Drosophila, loss of the GAS2 homolog, Pigs, results in infertility. We demonstrate herein that, in the mouse ovary, GAS2 is expressed in the stromal cells surrounding the oocyte cysts on 16.5 dpc, and in stromal cells surrounding growing follicles during juvenile and adult life. We have generated genetically engineered mice with inactivated Gas2. Gas2 homozygous mutant mice are viable but have severely impaired fertility in females, in which oocyte cyst breakdown is disrupted and follicle growth is impaired, with significantly reduced numbers of large antral follicles and corpora lutea. In these mutant mice, the organization of the basal lamina surrounding developing follicles is severely defective at multiple stages of folliculogenesis. We also found that Notch signaling activity was altered in ovaries from Gas2 null mice around the time of birth and during follicular development later in life. These results indicate that GAS2 is a critical and novel regulator of tissue remodeling in the ovary during oocyte cyst breakdown and folliculogenesis.

Recruited metastasis suppressor NM23-H2 attenuates expression and activity of peroxisome proliferator-activated receptor δ (PPARδ) in human cholangiocarcinoma.

BACKGROUND: Peroxisome proliferator-activated receptor δ (PPARδ) is a versatile regulator of distinct biological processes and overexpression of PPARδ in cancer may be partially related to its suppression of its own co-regulators. AIMS: To determine whether recruited suppressor proteins bind to and regulate PPARδ expression, activity and PPARδ-dependent cholangiocarcinoma proliferation. METHODS: Yeast two-hybrid assays were done using murine PPARδ as bait. PPARδ mRNA expression was determined by qPCR. Protein expression was measured by western blot. Immunohistochemistry and fluorescence microscopy were used to determine PPARδ expression and co-localization with NDP Kinase alpha (NM23-H2). Cell proliferation assays were performed to determine cell numbers. RESULTS: Yeast two-hybrid screening identified NM23-H2 as a PPARδ binding protein and their interaction was confirmed. Overexpressed PPARδ or treatment with the agonist GW501516 resulted in increased cell proliferation. NM23-H2 siRNA activated PPARδ luciferase promoter activity, upregulated PPARδ RNA and protein expression and increased GW501516-stimulated CCA growth. Overexpression of NM23-H2 inhibited PPARδ luciferase promoter activity, downregulated PPARδ expression and AKT phosphorylation and reduced GW501516-stimulated CCA growth. CONCLUSIONS: We report the novel association of NM23-H2 with PPARδ and the negative regulation of PPARδ expression by NM23-H2 binding to the C-terminal region of PPARδ. These findings provide evidence that the metastasis suppressor NM23-H2 is involved in the regulation of PPARδ-mediated proliferation.

FXR-induced secretion of FGF15/19 inhibits CYP27 expression in cholangiocytes through p38 kinase pathway.

Cholangiocytes, bile duct lining cells, actively adjust the amount of cholesterol and bile acids in bile through expression of enzymes and channels involved in transportation and metabolism of the cholesterol and bile acids. Herein, we report molecular mechanisms regulating bile acid biosynthesis in cholangiocytes. Among the cytochrome p450 (Cyp) enzymes involved in bile acid biosynthesis, sterol 27-hydroxylase (Cyp27) that is the rate-limiting enzyme for the acidic pathway of bile acid biosynthesis expressed in cholangiocytes. Expression of other Cyp enzymes for the basic bile acid biosynthesis was hardly detected. The Cyp27 expression was negatively regulated by a hydrophobic bile acid through farnesoid X receptor (FXR), a nuclear receptor activated by bile acid ligands. Activated FXR exerted the negative effects by inducing an expression of fibroblast growth factor 15/19 (FGF15/19). Similar to its repressive function against cholesterol 7α-hydroxylase (Cyp7a1) expression in hepatocytes, secreted FGF15/19 triggered Cyp27 repression in cholangiocytes through interaction with its cognate receptor fibroblast growth factor receptor 4 (FGFR4). The involvements of FXR and FGFR4 for the bile acid-induced Cyp27 repression were confirmed in vivo using knockout mouse models. Different from the signaling in hepatocytes, wherein the FGF15/19-induced repression signaling is mediated by c-Jun N-terminal kinase (JNK), FGF15/19-induced Cyp27 repression in cholangiocytes was mediated by p38 kinase. Thus, the results collectively suggest that cholangiocytes may be able to actively regulate bile acid biosynthesis in cholangiocytes and even hepatocyte by secreting FGF15/19. We suggest the presence of cholangiocyte-mediated intrahepatic feedback loop in addition to the enterohepatic feedback loop against bile acid biosynthesis in the liver.

Antisense oligonucleotide suppression of human IGF-1R inhibits the growth and survival of in vitro cultured epithelial ovarian cancer cells.

BACKGROUND: Preclinical evaluation of the anti-neoplastic activity of antisense oligonucleotide (AS) suppression of human insulin-like growth factor I receptor (IGF-IR) in human epithelial ovarian cancer (EOC). METHODS: Ovarian cancer cells from 36 patients with EOC were investigated under serum-free tissue culture conditions. IGF-I production was evaluated by standard ELISA. IGF-IR and phosphorylated IRS-1, AKT, and MAP kinase expression and protein levels were evaluated by immunohistochemistry and Western blotting. Cancer cell growth and proliferation assays were performed in triplicates using MTT assay. Apoptosis was evaluated by TUNNEL assay. RESULTS: All ovarian cancer tissue samples tested produced IGF-I and expressed IGF-IR, supporting the existence of an autocrine loop. Treatment of primary ovarian cancer cell lines with an IGF-1R AS inhibited growth and proliferation and decreased clonogenicity in soft agar assay. AS treatment was demonstrated to inhibit the expression of IGF-1R and decrease the concentration of phosphorylated IRS-1, AKT, and MAP kinase signaling protein downstream of the IGF-IR. We also observed that the IGF-1R AS sensitized cancer cell lines to cisplatin in vitro through the PI3K pathway. CONCLUSIONS: IGF-IR enhances the proliferation and tumorigenicity of human ovarian cancer cells and inhibition of IGF-IR by AS oligonucleotide treatment potentiates the activity of cisplatin in vitro. Therefore, IGF-1R is a potential molecular target in ovarian cancer.

Production of a mouse line with a conditional Crim1 mutant allele.

Crim1 is a developmentally expressed, transmembrane protein essential for normal embryonic development. We generated mice engineered to contain a Crim1 conditional null allele by flanking exons three and four of Crim1 with unidirectional LoxP sites. After crossing Crim1+/FLOX mice with a CMV-Cre line, a Crim1+/Δflox colony was established after germline transmission of the deleted allele. We then analyzed genomic DNA, mRNA transcripts, and protein expression from Crim1Δflox/Δflox null mice to confirm the nature of the genomic lesion. Crim1Δflox/Δflox mice displayed phenotypes similar to those previously described for a Crim1 gene-trap mutant, Crim1KST264/KST264, including perinatal lethality, digit syndactyly, eye, and kidney abnormalities, with varying penetrance and severity. The production of a conditional mutant allele represents a valuable resource for the study of the tissue-specific roles for Crim1, and for understanding the pleimorphic phenotypes associated with Crim1 mutation.

Loss of spindle assembly checkpoint-mediated inhibition of Cdc20 promotes tumorigenesis in mice.

Genomic instability is a hallmark of human cancers. Spindle assembly checkpoint (SAC) is a critical cellular mechanism that prevents chromosome missegregation and therefore aneuploidy by blocking premature separation of sister chromatids. Thus, SAC, much like the DNA damage checkpoint, is essential for genome stability. In this study, we report the generation and analysis of mice carrying a Cdc20 allele in which three residues critical for the interaction with Mad2 were mutated to alanine. The mutant Cdc20 protein (AAA-Cdc20) is no longer inhibited by Mad2 in response to SAC activation, leading to the dysfunction of SAC and aneuploidy. The dysfunction could not be rescued by the additional expression of another Cdc20 inhibitor, BubR1. Furthermore, we found that Cdc20(AAA/AAA) mice died at late gestation, but Cdc20(+/AAA) mice were viable. Importantly, Cdc20(+/AAA) mice developed spontaneous tumors at highly accelerated rates, indicating that the SAC-mediated inhibition of Cdc20 is an important tumor-suppressing mechanism.

Inhibitory phosphorylation of separase is essential for genome stability and viability of murine embryonic germ cells.

Activity of separase, a cysteine protease that cleaves sister chromatid cohesin at the onset of anaphase, is tightly regulated to ensure faithful chromosome segregation and genome stability. Two mechanisms negatively regulate separase: inhibition by securin and phosphorylation on serine 1121. To gauge the physiological significance of the inhibitory phosphorylation, we created a mouse strain in which Ser1121 was mutated to Ala (S1121A). Here we report that this S1121A point mutation causes infertility in mice. We show that germ cells in the mutants are depleted during development. We further demonstrate that S1121A causes chromosome misalignment during proliferation of the postmigratory primordial germ cells, resulting in mitotic arrest, aneuploidy, and eventual cell death. Our results indicate that inhibitory phosphorylation of separase plays a critical role in the maintenance of sister chromatid cohesion and genome stability in proliferating postmigratory primordial germ cells.

The Notch signaling pathway controls the size of the ocular lens by directly suppressing p57Kip2 expression.

The size of an organ must be tightly controlled so that it fits within an organism. The mammalian lens is a relatively simple organ composed of terminally differentiated, amitotic lens fiber cells capped on the anterior surface by a layer of immature, mitotic epithelial cells. The proliferation of lens epithelial cells fuels the growth of the lens, thus controling the size of the lens. We report that the Notch signaling pathway defines the boundary between proliferation and differentiation in the developing lens. The loss of Notch signaling results in the loss of epithelial cells to differentiation and a much smaller lens. We found that the Notch effector Herp2 is expressed in lens epithelium and directly suppresses p57Kip2 expression, providing a molecular link between Notch signaling and the cell cycle control machinery during lens development.

Loss of Cdc20 causes a securin-dependent metaphase arrest in two-cell mouse embryos.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase mediating targeted proteolysis through ubiquitination of protein substrates to control the progression of mitosis. The APC/C recognizes its substrates through two adapter proteins, Cdc20 and Cdh1, which contain similar C-terminal domains composed of seven WD-40 repeats believed to be involved in interacting with their substrates. During the transition from metaphase to anaphase, APC/C-Cdc20 mediates the ubiquitination of securin and cyclin B1, allowing the activation of separase and the onset of anaphase and mitotic exit. APC/C-Cdc20 and APC/C-Cdh1 have overlapping substrates. It is unclear whether they are redundant for mitosis. Using a gene-trapping approach, we have obtained mice which lack Cdc20 function. These mice show failed embryogenesis. The embryos were arrested in metaphase at the two-cell stage with high levels of cyclin B1, indicating an essential role of Cdc20 in mitosis that is not redundant with that of Cdh1. Interestingly, Cdc20 and securin double mutant embryos could not maintain the metaphase arrest, suggesting a role of securin in preventing mitotic exit.

A transgenic approach for RNA interference-based genetic screening in mice.

Genetic screening is the most powerful method through which to uncover gene function. It has been applied very successfully in lower organisms but seldom attempted in mammalian species because of their long generation time. In this study, we exploit RNA interference (RNAi) for its potential use in genetic screening in mice. We show that RNAi-induced gene knockdown can be generated through introducing small hairpin RNA-expressing constructs into the mouse as transgenes via conventional pronuclear injection. The knockdown effect can be transmitted for many generations in these transgenic animals. In a small-scale screening for developmental defects in the kidney, we uncovered a potential role of Id4 in the formation of the renal medulla. Our results demonstrate the feasibility of using RNAi for genetic screening in mice.

Securin and separase phosphorylation act redundantly to maintain sister chromatid cohesion in mammalian cells.

The spindle assembly checkpoint monitors the integrity of the spindle microtubules, which attach to sister chromatids at kinetochores and play a vital role in preserving genome stability by preventing missegregation. A key target of the spindle assembly checkpoint is securin, the separase inhibitor. In budding yeast, loss of securin results in precocious sister chromatid separation when the microtubule spindle is disrupted. However, in contrast to budding yeast, mammalian securin is not required for spindle checkpoint, suggesting that there are redundant mechanisms controlling the dissolution of sister chromatid cohesion in the absence of securin. One candidate mechanism is the inhibitory phosphorylation of separase. We generated a nonphosphorylable point mutant (S1121A) separase allele in securin-/- mouse embryonic stem cells. Securin(-/-)separase(+/S1121A) cells are viable but fail to maintain sister chromatid cohesion in response to the disruption of spindle microtubules, show enhanced sensitivity to nocodazole, and cannot recover from prometaphase arrest.