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Approximately half of the world's population is infected with the stomach pathogen Helicobacter pylori. Infection with H. pylori is the main risk factor for distal gastric cancer. Bacterial virulence factors, such as the oncoprotein CagA, augment cancer risk. Yet despite high infection rates, only a fraction of H. pylori-infected individuals develop gastric cancer. This raises the question of defining the specific host and bacterial factors responsible for gastric tumorigenesis. To investigate the tumorigenic determinants, we analyzed gastric tissues from human subjects and animals infected with H. pylori bacteria harboring different CagA status. For laboratory studies, well-defined H. pylori strain B128 and its cancerogenic derivative strain 7.13, as well as various bacterial isogenic mutants were employed. We found that H. pylori compromises key tumor suppressor mechanisms: the host stress and apoptotic responses. Our studies showed that CagA induces phosphorylation of XIAP E3 ubiquitin ligase, which enhances ubiquitination and proteasomal degradation of the host proapoptotic factor Siva1. This process is mediated by the PI3K/Akt pathway. Inhibition of Siva1 by H. pylori increases survival of human cells with damaged DNA. It occurs in a strain-specific manner and is associated with the ability to induce gastric tumor.
Serine threonine kinase 17A (STK17A) is a ubiquitously expressed kinase originally identified as a regulator of apoptosis; however, whether it functionally contributes to colorectal cancer has not been established. Here, we have analyzed STK17A in colorectal cancer and demonstrated decreased expression of STK17A in primary tumors, which is further reduced in metastatic lesions, indicating a potential role in regulating the metastatic cascade. Interestingly, changes in STK17A expression did not modify proliferation, apoptosis, or sensitivity of colorectal cancer cell lines to treatment with the chemotherapeutic 5-fluorouracil. Instead, knockdown induced a robust mesenchymal phenotype consistent with the epithelial-mesenchymal transition, including spindle-like cell morphology, decreased expression of adherens junction proteins, and increased migration and invasion. Additionally, overexpression of decreased cell size and induced widespread membrane blebbing, a phenotype often associated with activation of cell contractility. Indeed, STK17A-overexpressing cells displayed heightened phosphorylation of myosin light chain in a manner dependent on STK17A catalytic activity. Finally, patient-derived tumor organoid cultures were used to more accurately determine STK17A's effect in primary human tumor cells. Loss of STK17A induced morphologic changes, decreased E-cadherin, increased invasion, and augmented organoid attachment on 2D substrates, all together suggesting a more metastatic phenotype. Collectively, these data indicate a novel role for STK17A in the regulation of epithelial phenotypes and indicate its functional contribution to colorectal cancer invasion and metastasis. IMPLICATIONS: Loss of serine threonine kinase 17A occurs in colorectal cancer metastasis, induces mesenchymal morphologies, and contributes to tumor cell invasion and migration in colorectal cancer.
©2019 American Association for Cancer Research.
BACKGROUND - Wilms tumor (WT) is the most common childhood kidney cancer worldwide, yet its incidence and clinical behavior vary according to race and access to adequate healthcare resources. To guide and streamline therapy in the war-torn and resource-constrained city of Baghdad, Iraq, we conducted a first-ever molecular analysis of 20 WT specimens to characterize the biological features of this lethal disease within this challenged population.
METHODS - Next-generation sequencing of ten target genes associated with WT development and treatment resistance (WT1, CTNNB1, WTX, IGF2, CITED1, SIX2, p53, N-MYC, CRABP2, and TOP2A) was completed. Immunohistochemistry was performed for 6 marker proteins of WT (WT1, CTNNB1, NCAM, CITED1, SIX2, and p53). Patient outcomes were compiled.
RESULTS - Mutations were detected in previously described WT "hot spots" (e.g., WT1 and CTNNB1) as well as novel loci that may be unique to the Iraqi population. Immunohistochemistry showed expression domains most typical of blastemal-predominant WT. Remarkably, despite the challenges facing families and care providers, only one child, with combined WT1 and CTNNB1 mutations, was confirmed dead from disease. Median clinical follow-up was 40.5 months (range 6-78 months).
CONCLUSIONS - These data suggest that WT biology within a population of Iraqi children manifests features both similar to and unique from disease variants in other regions of the world. These observations will help to risk stratify WT patients living in this difficult environment to more or less intensive therapies and to focus treatment on cell-specific targets.
X-linked inhibitor of apoptosis (XIAP) plays an important role in preventing apoptotic cell death. XIAP has been shown to participate in signaling pathways, including Wnt signaling. XIAP regulates Wnt signaling by promoting the monoubiquitylation of the co-repressor Groucho/TLE family proteins, decreasing its affinity for the TCF/Lef family of transcription factors and allowing assembly of transcriptionally active β-catenin-TCF/Lef complexes. We now demonstrate that XIAP is phosphorylated by GSK3 at threonine 180, and that an alanine mutant (XIAP) exhibits decreased Wnt activity compared to wild-type XIAP in cultured human cells and in embryos. Although XIAP ubiquitylates TLE3 at wild-type levels , it exhibits a reduced capacity to ubiquitylate and bind TLE3 in human cells. XIAP binds Smac (also known as DIABLO) and inhibits Fas-induced apoptosis to a similar degree to wild-type XIAP. Our studies uncover a new mechanism by which XIAP is specifically directed towards a Wnt signaling function versus its anti-apoptotic function. These findings have implications for development of anti-XIAP therapeutics for human cancers.
© 2018. Published by The Company of Biologists Ltd.
The histologic differential diagnosis between intramuscular myxoma and low-grade myxofibrosarcoma can be quite difficult in some cases. To identify a diagnostic immunohistochemical marker, we compared the staining profiles of 19 different antigens, including cell cycle proteins, apoptosis proteins, and proliferative markers, and selected other signaling and structural proteins in these two tumors. Ten cases each of intramuscular myxoma and low-grade myxofibrosarcoma were stained with antibodies directed against apoptosis regulatory proteins (Bcl2, activated caspase-3, phospho-H2A.X, and cleaved PARP), cell cycle regulatory proteins (Rb1, Cyclin-A, CDKN1B, and Cdt1), proliferative markers (KI67, MCM2, phospho-histone H3, and geminin), cell signalling molecules (c-Myc, EGF, EGFR, PLA2G4A, and HSP90), a dendritic cell marker (CD209), and the extracellular matrix proteoglycan decorin. Staining patterns of myxoma and myxofibrosarcoma were compared using Fisher's exact test and the Mann-Whitney test. For each potential diagnostic marker studied, the proportions of cases scored as positive on both dichotomous or ordinal scales were not significantly different between myxoma and myxofibrosarcoma. Myxoma and myxofibrosarcoma share a common immunophenotype for each of the markers studied. Distinction between these tumors is still predominantly based on morphologic criteria.
Herein we report the discovery and SAR of an indole-based protease activated receptor-4 (PAR-4) antagonist scaffold derived from a similarity search of the Vanderbilt HTS collection, leading to MLPCN probe ML354 (VU0099704). Using a novel PAC-1 fluorescent αIIbβ3 activation assay this probe molecule antagonist was found to have an IC50 of 140nM for PAR-4 with 71-fold selectivity versus PAR-1 (PAR-1IC50=10μM).
Copyright © 2014 Elsevier Ltd. All rights reserved.
Anti-apoptosis engineering is an established technique to prolong the viability of mammalian cell cultures used for industrial production of recombinant proteins. However, the effect of overexpressing anti-apoptotic proteins on central carbon metabolism has not been systematically studied. We transfected CHO-S cells to express Bcl-2∆, an engineered anti-apoptotic gene, and selected clones that differed in their Bcl-2∆ expression and caspase activity. (13)C metabolic flux analysis (MFA) was then applied to elucidate the metabolic alterations induced by Bcl-2∆. Expression of Bcl-2Δ reduced lactate accumulation by redirecting the fate of intracellular pyruvate toward mitochondrial oxidation during the lactate-producing phase, and it significantly increased lactate re-uptake during the lactate-consuming phase. This flux redistribution was associated with significant increases in biomass yield, peak viable cell density (VCD), and integrated VCD. Additionally, Bcl-2∆ expression was associated with significant increases in isocitrate dehydrogenase and NADH oxidase activities, both rate-controlling mitochondrial enzymes. This is the first comprehensive (13)C MFA study to demonstrate that expression of anti-apoptotic genes has a significant impact on intracellular metabolic fluxes, especially in controlling the fate of pyruvate carbon, which has important biotechnology applications for reducing lactate accumulation and enhancing productivity in mammalian cell cultures.
Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
Aurora kinases play a key role in mitosis and are frequently overexpressed in a variety of tumor cells. Inhibition of aurora kinases results in mitotic arrest and death of cancer cells, and has been explored as an anticancer strategy. However, how aurora inhibition kills cancer cells is poorly understood. In this study, we found that inhibition of aurora kinases by siRNA or small-molecule inhibitors led to induction of p53 upregulated modulator of apoptosis (PUMA), a BH3-only Bcl-2 family protein, in colorectal cancer cells irrespective of p53 status. Deficiency in PUMA increased polyploidy, improved cell survival, and abrogated mitochondria-mediated apoptosis induced by aurora kinase inhibitors. In response to aurora kinase inhibition, PUMA was directly activated by p65 through the canonical NF-κB pathway following AKT inhibition. Furthermore, PUMA was necessary for the chemosensitization and in vivo antitumor effects of aurora kinase inhibitors in colon cancer cells. These results suggest that PUMA induction mediates the apoptotic response to mitotic arrest imposed by aurora kinase inhibition, and may be a useful indicator for the anticancer activity of aurora kinase inhibitors.
Wilms tumor (WT) is the most common childhood kidney cancer and retains gene expression profiles reminiscent of the embryonic kidney. We have shown previously that CITED1, a transcriptional regulator that labels the self-renewing, multipotent nephron progenitor population of the developing kidney, is robustly expressed across all major WT disease and patient characteristics. In this malignant context, CITED1 becomes enriched in the nucleus, which deviates from its cytosolic predominance in embryonic nephron progenitors. We designed the current studies to test the functional and mechanistic effects of differential CITED1 subcellular localization on WT behavior. To mimic its subcellular distribution observed in clinical WT specimens, CITED1 was misexpressed ectopically in the human WT cell line, WiT49, as either a wild-type (predominantly cytosolic) or a mutant, but transcriptionally active, protein (two point mutations in its nuclear export signal, CITED1ΔNES; nuclear-enriched). In vitro analyses showed that CITED1ΔNES enhanced WiT49 proliferation and colony formation in soft agar relative to wild-type CITED1 and empty vector controls. The nuclear-enriched CITED1ΔNES cell line showed the greatest tumor volumes after xenotransplantation into immunodeficient mice (n=15 animals per cell line). To elucidate CITED1 gene targets in this model, microarray profiling showed that wild-type CITED1 foremost upregulated LGR5 (stem cell marker), repressed CDH6 (early marker of epithelial commitment of nephron progenitors), and altered expression of specific WNT pathway participants. In summary, forced nuclear enrichment of CITED1 in a human WT cell line appears to enhance tumorigenicity, whereas ectopic cytosolic expression confers stem-like properties and an embryonic phenotype, analogous to the developmental context.
BACKGROUND - Higher frequency of Smad4 inactivation or loss of expression is observed in metastasis of colorectal cancer (CRC) leading to unfavourable survival and contributes to chemoresistance. However, the molecular mechanism of how Smad4 regulates chemosensitivity of CRC is unknown.
METHODS - We evaluated how the loss of Smad4 in CRC enhanced chemoresistance to 5-fluorouracil (5-FU) using two CRC cell lines in vitro and in vivo. Immunoblotting with cell and tumour lysates and immunohistochemical analyses with tissue microarray were performed.
RESULTS - Knockdown or loss of Smad4 induced tumorigenicity, migration, invasion, angiogenesis, metastasis, and 5-FU resistance. Smad4 expression in mouse tumours regulated cell-cycle regulatory proteins leading to Rb phosphorylation. Loss of Smad4 activated Akt pathway that resulted in upregulation of anti-apoptotic proteins, Bcl-2 and Bcl-w, and Survivin. Suppression of phosphatidylinositol-3-kinase (PI3K)/Akt pathway by LY294002 restored chemosensitivity of Smad4-deficient cells to 5-FU. Vascular endothelial growth factor-induced angiogenesis in Smad4-deficient cells might also lead to chemoresistance. Low levels of Smad4 expression in CRC tissues correlated with higher levels of Bcl-2 and Bcl-w and with poor overall survival as observed in immunohistochemical staining of tissue microarrays.
CONCLUSION - Loss of Smad4 in CRC patients induces resistance to 5-FU-based therapy through activation of Akt pathway and inhibitors of this pathway may sensitise these patients to 5-FU.