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Cyclooxygenase-1 (COX-1) is implicated in ovarian cancer. However, patterns of COX expression and function have been unclear and controversial. In this report, patterns of COX-1 and COX-2 gene expression were obtained from RNA-seq data through The Cancer Genome Atlas. Our analysis revealed markedly higher COX-1 mRNA expression than COX-2 in high-grade serous ovarian cancers (HGSOC) and higher COX-1 expression in HGSOC tumors than 10 other tumor types. High expression of COX-1 in HGSOC tumors was confirmed in an independent tissue microarray. In contrast, lower or similar expression of COX-1 compared to COX-2 was observed in endometrioid, mucinous and clear cell tumors. Stable COX-1 knockdown in HGSOC-representative OVCAR-3 ovarian cancer cells reduced gene expression in multiple pro-tumorigenic pathways. Functional cell viability, clonogenicity, and migration/invasion assays were consistent with transcriptomic changes. These effects were reversed by stable over-expression of COX-1 in SKOV-3 cells. Our results demonstrate a distinct pattern of COX-1 over-expression in HGSOC tumors and strong association of COX-1 with multiple pro-tumorigenic pathways in ovarian cancer cells. These findings provide additional insight into the role of COX-1 in human ovarian cancer and support further development of methods to selectively target COX-1 in the management of HGSOC tumors.
BACKGROUND - PIM1 kinase is coexpressed with c-MYC in human prostate cancers (PCs) and dramatically enhances c-MYC-induced tumorigenicity. Here we examine the effects of a novel oral PIM inhibitor, AZD1208, on prostate tumorigenesis and recurrence.
METHODS - A mouse c-MYC/Pim1-transduced tissue recombination PC model, Myc-CaP allografts, and human PC xenografts were treated with AZD1208 (n = 5-11 per group). Androgen-sensitive and castrate-resistant prostate cancer (CRPC) models were studied as well as the effects of hypoxia and radiation. RNA sequencing was used to analyze drug-induced gene expression changes. Results were analyzed with χ(2) test. Student's t test and nonparametric Mann-Whitney rank sum U Test. All statistical tests were two-sided.
RESULTS - AZD1208 inhibited tumorigenesis in tissue recombinants, Myc-CaP, and human PC xenograft models. PIM inhibition decreased c-MYC/Pim1 graft growth by 54.3 ± 39% (P < .001), decreased cellular proliferation by 46 ± 14% (P = .016), and increased apoptosis by 326 ± 170% (P = .039). AZD1208 suppressed multiple protumorigenic pathways, including the MYC gene program. However, it also downregulated the p53 pathway. Hypoxia and radiation induced PIM1 in prostate cancer cells, and AZD1208 functioned as a radiation sensitizer. Recurrent tumors postcastration responded transiently to either AZD1208 or radiation treatment, and combination treatment resulted in more sustained inhibition of tumor growth. Cell lines established from recurrent, AZD1208-resistant tumors again revealed downregulation of the p53 pathway. Irradiated AZD1208-treated tumors robustly upregulated p53, providing a possible mechanistic explanation for the effectiveness of combination therapy. Finally, an AZD1208-resistant gene signature was found to be associated with biochemical recurrence in PC patients.
CONCLUSIONS - PIM inhibition is a potential treatment for MYC-driven prostate cancers including CRPC, and its effectiveness may be enhanced by activators of the p53 pathway, such as radiation.
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Estrogen receptor-positive (ER(+)) breast cancers adapt to hormone deprivation and become resistant to antiestrogen therapy. Here, we performed deep sequencing on ER(+) tumors that remained highly proliferative after treatment with the aromatase inhibitor letrozole and identified a D189Y mutation in the inhibitory SH2 domain of the SRC family kinase (SFK) LYN. Evaluation of 463 breast tumors in The Cancer Genome Atlas revealed four LYN mutations, two of which affected the SH2 domain. In addition, LYN was upregulated in multiple ER(+) breast cancer lines resistant to long-term estrogen deprivation (LTED). An RNAi-based kinome screen revealed that LYN is required for growth of ER(+) LTED breast cancer cells. Kinase assays and immunoblot analyses of SRC substrates in transfected cells indicated that LYN(D189Y) has higher catalytic activity than WT protein. Further, LYN(D189Y) exhibited reduced phosphorylation at the inhibitory Y507 site compared with LYN(WT). Other SH2 domain LYN mutants, E159K and K209N, also exhibited higher catalytic activity and reduced inhibitory site phosphorylation. LYN(D189Y) overexpression abrogated growth inhibition by fulvestrant and/or the PI3K inhibitor BKM120 in 3 ER(+) breast cancer cell lines. The SFK inhibitor dasatinib enhanced the antitumor effect of BKM120 and fulvestrant against estrogen-deprived ER(+) xenografts but not LYN(D189Y)-expressing xenografts. These results suggest that LYN mutations mediate escape from antiestrogens in a subset of ER(+) breast cancers.
In preterm infants, exposure to inflammation increases the risk of bronchopulmonary dysplasia, a chronic, developmental lung disease. Although macrophages are the key cells that initiate lung inflammation, less is known about lung macrophage phenotype and maturation. We hypothesized that fetal lung macrophages mature into distinct subpopulations during mouse development, and that activation could influence macrophage maturation. Expression of the fetal macrophage markers CD68, CD86, CD206, Ym1, fibrinogen-like protein 2, and indolamine-2, 3-dioxygenase was developmentally regulated, with each marker having different temporal patterns. Flow cytometry analysis showed macrophages within the fetal lung were less diverse than the distinctly separate subpopulations in newborn and adult lungs. Similar to adult alveolar macrophages, fetal lung macrophages responded to the TLR4 agonist LPS and the alternative activation cytokines IL-4 and IL-13. Using a macrophage-specific constitutively active IκB Kinase transgenic model (IKFM), we demonstrated that macrophage activation increased proinflammatory gene expression and reduced the response of fetal lung macrophages to IL-4 and IL-13. Activation also increased fetal lung macrophage proliferation. Fetal IKFM lungs contained increased percentages of more mature, CD11b(low)F4/80(high) cells that also expressed higher levels of the alternative activation markers CD204 and CD206. Development of fetal lung macrophages into mature alveolar macrophages may therefore include features of both proinflammatory and alternative activation paradigms.
Copyright © 2014 by The American Association of Immunologists, Inc.
A new mechanism for formation of 7-ketocholesterol was recently described involving cytochrome P-450 (CYP)7A1-catalyzed conversion of 7-dehydrocholesterol into 7-ketocholesterol with cholesterol-7,8-epoxide as a side product. Some patients with cerebrotendinous xanthomatosis (CTX) and all patients with Smith-Lemli-Opitz syndrome (SLO) have markedly increased levels of 7-dehydrocholesterol in plasma and tissues. In addition, the former patients have markedly upregulated CYP7A1. We hypothesized that these patients may produce 7-ketocholesterol from 7-dehydrocholesterol with formation of cholesterol-7,8-epoxide as a side product. In accord with this hypothesis, two patients with CTX were found to have increased levels of 7-ketocholesterol and 7-dehydrocholesterol, as well as a significant level of cholesterol-7,8-epoxide. The latter steroid was not detectable in plasma from healthy volunteers. Downregulation of CYP7A1 activity by treatment with chenodeoxycholic acid reduced the levels of 7-ketocholesterol in parallel with decreased levels of 7-dehydrocholesterol and cholesterol-7,8-epoxide. Three patients with SLO were found to have markedly elevated levels of 7-ketocholesterol as well as high levels of cholesterol-7,8-epoxide. The results support the hypothesis that 7-dehydrocholesterol is a precursor to 7-ketocholesterol in SLO and some patients with CTX.
Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.
During M phase, Endosulfine (Endos) family proteins are phosphorylated by Greatwall kinase (Gwl), and the resultant pEndos inhibits the phosphatase PP2A-B55, which would otherwise prematurely reverse many CDK-driven phosphorylations. We show here that PP2A-B55 is the enzyme responsible for dephosphorylating pEndos during M phase exit. The kinetic parameters for PP2A-B55's action on pEndos are orders of magnitude lower than those for CDK-phosphorylated substrates, suggesting a simple model for PP2A-B55 regulation that we call inhibition by unfair competition. As the name suggests, during M phase PP2A-B55's attention is diverted to pEndos, which binds much more avidly and is dephosphorylated more slowly than other substrates. When Gwl is inactivated during the M phase-to-interphase transition, the dynamic balance changes: pEndos dephosphorylated by PP2A-B55 cannot be replaced, so the phosphatase can refocus its attention on CDK-phosphorylated substrates. This mechanism explains simultaneously how PP2A-B55 and Gwl together regulate pEndos, and how pEndos controls PP2A-B55. DOI: http://dx.doi.org/10.7554/eLife.01695.001.
Cytochrome P450 (P450) 2U1 has been shown to be expressed, at the mRNA level, in human thymus, brain, and several other tissues. Recombinant P450 2U1 was purified and used as a reagent in a metabolomic search for substrates in bovine brain. In addition to fatty acid oxidation reactions, an oxidation of endogenous N-arachidonoylserotonin was characterized. Subsequent NMR and mass spectrometry and chemical synthesis showed that the main product was the result of C-2 oxidation of the indole ring, in contrast to other human P450s that generated different products. N-Arachidonoylserotonin, first synthesized chemically and described as an inhibitor of fatty acid amide hydrolase, had previously been found in porcine and mouse intestine; we demonstrated its presence in bovine and human brain samples. The product (2-oxo) was 4-fold less active than N-arachidonoylserotonin in inhibiting fatty acid amide hydrolase. The rate of oxidation of N-arachidonoylserotonin was similar to that of arachidonic acid, one of the previously identified fatty acid substrates of P450 2U1. The demonstration of the oxidation of N-arachidonoylserotonin by P450 2U1 suggests a possible role in human brain and possibly other sites.
We previously identified a Drosophila maternal effect-lethal mutant named 'no poles' (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the rapid cycles of syncytial embryogenesis because of activation of a Chk2-mediated DNA checkpoint. NOPO is the Drosophila homolog of human TNF receptor associated factor (TRAF)-interacting protein (TRIP), which has been implicated in TNF signaling. NOPO and TRIP contain RING domains closely resembling those of known E3 ubiquitin ligases. We herein sought to elucidate the mechanism by which TRIP/NOPO promotes genomic stability by performing a yeast two-hybrid screen to identify potential substrates/interactors. We identified members of the Y-family of DNA polymerases that facilitate replicative bypass of damaged DNA (translesion synthesis) as TRIP interactors. We show that TRIP and NOPO co-immunoprecipitate with human and Drosophila Polη, respectively, from cultured cells. We generated a null mutation in Drosophila Polη (dPolη) and found that dPolη-derived embryos have increased sensitivity to ultraviolet irradiation and exhibit nopo-like mitotic spindle defects. dPolη and nopo interact genetically in that overexpression of dPolη in hypomorphic nopo-derived embryos suppresses nopo phenotypes. We observed enhanced ubiquitylation of Polη by TRIP and NOPO E3 ligases in human cells and Drosophila embryos, respectively, and show that TRIP promotes hPolη localization to nuclear foci in human cells. We present a model in which TRIP/NOPO ubiquitylates Polη to positively regulate its activity in translesion synthesis.
Acute Helicobacter pylori infection of gastric epithelial cells induces CagA oncoprotein- and peptidoglycan (SLT)-dependent mobilization of NF-κB p50 homodimers that bind to H-K-ATPase α-subunit (HKα) promoter and repress HKα gene transcription. This process may facilitate gastric H. pylori colonization by induction of transient hypochlorhydria. We hypothesized that H. pylori also regulates HKα expression posttranscriptionally by miRNA interaction with HKα mRNA. In silico analysis of the HKα 3' untranslated region (UTR) identified miR-1289 as a highly conserved putative HKα-regulatory miRNA. H. pylori infection of AGS cells transfected with HKα 3' UTR-Luc reporter construct repressed luciferase activity by 70%, whereas ΔcagA or Δslt H. pylori infections partially abrogated repression. Transfection of AGS cells expressing HKα 3' UTR-Luc construct with an oligoribonucleotide mimetic of miR-1289 induced maximal repression (54%) of UTR activity within 30 min; UTR activity was unchanged by nontargeting siRNA transfection. Gastric biopsies from patients infected with cagA(+) H. pylori showed a significant increase in miR-1289 expression compared with uninfected patients or those infected with cagA(-) H. pylori. Finally, miR-1289 expression was necessary and sufficient to attenuate biopsy HKα protein expression in the absence of infection. Taken together, these data indicate that miR-1289 is upregulated by H. pylori in a CagA- and SLT-dependent manner and targets HKα 3' UTR, affecting HKα mRNA translation. The sensitivity of HKα mRNA 3' UTR to binding of miR-1289 identifies a novel regulatory mechanism of gastric acid secretion and offers new insights into mechanisms underlying transient H. pylori-induced hypochlorhydria.
Collagens constitute nearly 30% of all proteins in our body. Type IV collagen is a major and crucial component of basement membranes. Collagen chains undergo several posttranslational modifications that are indispensable for proper collagen function. One of these modifications, prolyl 3-hydroxylation, is accomplished by a family of prolyl 3-hydroxylases (P3H1, P3H2, and P3H3). The present study shows that P3H2-null mice are embryonic-lethal by embryonic day 8.5. The mechanism of the unexpectedly early lethality involves the interaction of non-3-hydroxylated embryonic type IV collagen with the maternal platelet-specific glycoprotein VI (GPVI). This interaction results in maternal platelet aggregation, thrombosis of the maternal blood, and death of the embryo. The phenotype is completely rescued by producing double KOs of P3H2 and GPVI. Double nulls are viable and fertile. Under normal conditions, subendothelial collagens bear the GPVI-binding sites that initiate platelet aggregation upon blood exposure during injuries. In type IV collagen, these sites are normally 3-hydroxylated. Thus, prolyl 3-hydroxylation of type IV collagen has an important function preventing maternal platelet aggregation in response to the early developing embryo. A unique link between blood coagulation and the ECM is established. The newly described mechanism may elucidate some unexplained fetal losses in humans, where thrombosis is often observed at the maternal/fetal interface. Moreover, epigenetic silencing of P3H2 in breast cancers implies that the interaction between GPVI and non-3-hydroxylated type IV collagen might also play a role in the progression of malignant tumors and metastasis.