Abstract
Prostate cancer is one of the most common cancers in men worldwide. Currently available diagnostic and prognostic tools for this disease, such as prostate specific antigen, suffer from lack ...of specificity and sensitivity, resulting in over- and misdiagnosis. Hence, there is an urgent need for clinically relevant biomarkers capable of distinguishing between aggressive and nonaggressive forms of prostate cancer to aid in stratification, management and therapeutic decisions. To address this unmet need, we investigated the patterns of expression of a panel of 68 plasma-derived microRNAs (miRNAs) in a cohort of African American (AA) and European American (EA) prostate cancer patients (n = 114). miRNA qPCR results were analyzed using in-depth statistical methods, and a bioinformatics analysis was conducted to identify potential targets of the differentially expressed miRNAs. Our data demonstrate that a new previously unreported circulating miRNA signature consisting of a combination of interacting miRNAs (miR-17/miR-192) and an independent miRNA (miR-181a) are capable of segregating aggressive and nonaggressive prostate cancer in both AA and EA patients. The interacting miRNAs outperformed independent miRNAs in identifying aggressiveness. Our results suggest that these circulating miRNAs may constitute novel biomarkers of prostate cancer aggressiveness in both races and warrant further investigation.
We found that a novel combination of interacting plasma miRNAs is capable of distinguishing between aggressive and nonaggressive prostate cancer in AA and EA patients. The interacting miRNAs outperformed single miRNAs in identifying prostate disease aggressiveness and warrant further validation.
Lysine-specific demethylase 5A (KDM5A), an enzyme that removes activating H3K4 di- and trimethylation marks, plays critical roles in controlling transcription and chromatin architecture, yet its ...biological functions largely remain uncharacterized, particularly in the context of human cancer. In the present study, we found that the KDM5A gene was significantly amplified and over-expressed in various human tumors, including breast cancer. Reducing the expression of KDM5A by shRNA knockdown inhibited proliferation of KDM5A-amplified breast cancer cells. More importantly, we demonstrated that KDM5A over-expression was associated with breast cancer drug resistance. Furthermore, knockdown of KDM5A gene expression altered H3K4 methylation and induced upregulation of CDK inhibitors as well as genes mediating apoptotic cell death. Taken together, our study strongly links KDM5A histone demethylase activity to breast cancer proliferation and drug resistance, and suggests KDM5A is a potential target for breast cancer therapy.
Squalene synthase inhibitors, such as squalestatin 1 (SQ1), are cholesterol‐lowering drugs that block the first committed step in sterol biosynthesis. Unlike statins (e.g., pravastatin), inhibition ...of squalene synthase causes accumulation of non‐sterol isoprenoids that can function as signaling molecules that modulate the activities of various nuclear receptors including the constitutive androstane receptor (CAR), farnesoid X receptor (FXR), and peroxisome proliferator‐activated receptor (PPAR). To characterize the effect of isoprenoids on hepatocyte physiology further, we used microarray analysis to compare the gene expression responses in rat and mouse primary cultured hepatocytes that were treated with either SQ1 or pravastatin. In both species, genes involved in cholesterol biosynthesis were significantly up‐regulated by either drug consistent with the depletion of cellular cholesterol. Compared to pravastatin, treatment with SQ1 differentially affected 1796 genes in rat hepatocytes and 3689 in mouse hepatocytes (蠅 ±1.5‐fold change). Among these, we identified 112 conserved genes that were differentially higher and 54 lower in SQ1‐treated hepatocytes. Functional annotation identified that conserved responses were associated with induction of fatty acid metabolism and inhibition of cell cycle processes. The magnitude of gene expression changes was species‐dependent, likely due to differences in isoprenoid metabolism. Our findings indicate in addition to sterol depletion, squalene synthase inhibitors may uniquely influence cellular energy metabolism and cell cycle regulation.
Grant Funding Source: This work was supported by NIH grant HL050710.
An energy potential is constructed and trained to succeed in fold recognition for the general population of proteins as well as an important class which has previously been problematic: small, ...disulfide-bearing proteins. The potential is modeled on solvation, with the energy a function of side chain burial and the number of disulfide bonds. An accurate disulfide recognition algorithm identifies cysteine pairs which have the appropriate orientation to form a disulfide bridge. The potential has 22 energy parameters which are optimized so the Protein Data Bank (PDB) structure for each sequence in a training set is the lowest in energy out of thousands of alternative structures. One parameter per amino acid type reflects burial preference and a single parameter is used in an overpacking term. Additionally, one optimized parameter provides a favorable contribution for each disulfide identified in a given protein structure. With little training, the potential is >80% accurate in ungapped threading tests using a variety of proteins. The same level of accuracy is observed in a threading test of small proteins which have disulfide bonds. Importantly, the energy potential is also successful with proteins having uncrosslinked cysteines.
The 8p11-p12 amplicon occurs in approximately 15% of breast cancers in aggressive luminal B-type tumors. Previously, we identified WHSC1L1 as a driving oncogene from this region. Here, we demonstrate ...that over-expression of WHSC1L1 is linked to over-expression of ERα in SUM-44 breast cancer cells and in primary human breast cancers. Knock-down of WHSC1L1, particularly WHSC1L1-short, had a dramatic effect on ESR1 mRNA and ERα protein levels. SUM-44 cells do not require exogenous estrogen for growth in vitro; however, they are dependent on ERα expression, as ESR1 knock-down or exposure to the selective estrogen receptor degrader fulvestrant resulted in growth inhibition. ChIP-Seq experiments utilizing ERα antibodies demonstrated extensive ERα binding to chromatin in SUM-44 cells under estrogen-free conditions. ERα bound to ERE and FOXA1 motifs under estrogen-free conditions and regulated expression of estrogen-responsive genes. Short-term treatment with estradiol enhanced binding of ERα to chromatin and influenced expression of many of the same genes to which ERα was bound under estrogen-free conditions. Finally, knock-down of WHSC1L1 in SUM-44 cells resulted in loss of ERα binding to chromatin under estrogen-free conditions, which was restored upon exposure to estradiol. These results indicate the SUM-44 cells are a good model of a subset of luminal B breast cancers that have the 8p11-p12 amplicon, over-express WHSC1L1, and over-express ERα, but are independent of estrogen for binding to chromatin and regulation of gene expression. Breast cancers such as these, that are dependent on ERα activity but independent of estradiol, are a major cause of breast cancer mortality.
•SUM44 is a model cell line for ERα positive breast cancer with the 8p11 amplicon.•WHSC1L1 is a driving oncogene from the 8p11 amplicon in SUM44 cells.•SUM44 breast cancer cells have high ERα expression, regulated by WHSC1L1 knockdown.•ERα is required for growth and survival of SUM44 cells but is estrogen-independent.•WHSC1L1 knock-down re-sensitizes ERα to estradiol for binding to essential genes.
Network and protein–protein interaction analyses of proteins undergoing Hg2+-induced phosphorylation and dephosphorylation in Hg2+-intoxicated mouse WEHI-231 B cells identified Lyn as the most ...interconnected node. Lyn is a Src family protein tyrosine kinase known to be intimately involved in the B cell receptor (BCR) signaling pathway. Under normal signaling conditions the tyrosine kinase activity of Lyn is controlled by phosphorylation, primarily of two well known canonical regulatory tyrosine sites, Y-397 and Y-508. However, Lyn has several tyrosine residues that have not yet been determined to play a major role under normal signaling conditions, but are potentially important sites for phosphorylation following mercury exposure. In order to determine how Hg2+ exposure modulates the phosphorylation of additional residues in Lyn, a targeted MS assay was developed. Initial mass spectrometric surveys of purified Lyn identified 7 phosphorylated tyrosine residues. A quantitative assay was developed from these results using the multiple reaction monitoring (MRM) strategy. WEHI-231 cells were treated with Hg2+, pervanadate (a phosphatase inhibitor), or anti-Ig antibody (to stimulate the BCR). Results from these studies showed that the phosphoproteomic profile of Lyn after exposure of the WEHI-231 cells to a low concentration of Hg2+ closely resembled that of anti-Ig antibody stimulation, whereas exposure to higher concentrations of Hg2+ led to increases in the phosphorylation of Y-193/Y-194, Y-501 and Y-508 residues. These data indicate that mercury can disrupt a key regulatory signal transduction pathway in B cells and point to phospho-Lyn as a potential biomarker for mercury exposure.
•Inorganic mercury (Hg2+) induces changes in the WEHI-231 B cell phosphoproteome.•The B cell receptor (BCR) signaling pathway was the pathway most affected by Hg2+.•The Src family phosphoprotein kinase Lyn was the most interconnected node.•Lyn is likely central to the immunotoxic potential of Hg2+.•Lyn phosphorylation profiles may be biomarkers for Hg2+ intoxication of B cells.
Acute myeloid leukemia (AML) in Down syndrome (DS) children has several unique features including a predominance of the acute megakaryocytic leukemia (AMkL) phenotype, higher event-free survivals ...compared to non-DS children using cytosine arabinoside (ara-C)/anthracycline-based protocols and a uniform presence of somatic mutations in the X-linked transcription factor gene, GATA1. Several chromosome 21-localized transcription factor oncogenes including ETS2 may contribute to the unique features of DS AMkL. ETS2 transcripts measured by real-time RT-PCR were 1.8- and 4.1-fold, respectively, higher in DS and non-DS megakaryoblasts than those in non-DS myeloblasts. In a doxycycline-inducible erythroleukemia cell line, K562pTet-on/ETS2, induction of ETS2 resulted in an erythroid to megakaryocytic phenotypic switch independent of GATA1 levels. Microarray analysis of doxycycline-induced and doxycycline-uninduced cells revealed an upregulation by ETS2 of cytokines (for example, interleukin 1 and CSF2) and transcription factors (for example, TAL1), which are key regulators of megakaryocytic differentiation. In the K562pTet-on/ETS2 cells, ETS2 induction conferred differences in sensitivities to ara-C and daunorubicin, depending on GATA1 levels. These results suggest that ETS2 expression is linked to the biology of AMkL in both DS and non-DS children, and that ETS2 acts by regulating expression of hematopoietic lineage and transcription factor genes involved in erythropoiesis and megakaryopoiesis, and in chemotherapy sensitivities.
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SULT1C2 is a cytosolic sulfotransferase that catalyzes the bioactivation of arylamines. However, little is known about the mechanisms regulating expression of this enzyme. Treatment of ...primary cultured rodent hepatocytes with the squalene synthase inhibitor, squalestatin 1, increases CYP2B expression by causing accumulation of an endogenous isoprenoid that activates the constitutive androstane receptor (CAR). Treatment of primary cultured rat hepatocytes with 0.1 μM squalestatin 1 for 48 hr increased SULT1C2 mRNA content by ~3‐fold, as indicated by microarray and real‐time RT‐PCR analyses. Real‐time RT‐PCR also demonstrated that treatment of primary cultured rat hepatocytes with the HMG‐CoA reductase inhibitor, pravastatin (30 μM), decreased SULT1C2 mRNA content by ~50% while treatment with the HMG‐CoA reductase product, mevalonate (10 mM), increased the SULT1C2 mRNA level by ~3‐fold. Also, pravastatin treatment abolished squalestatin 1‐mediated SULT1C2 induction, while mevalonate supplementation restored induction. Treatment of primary cultured rat hepatocytes with the prototypic CAR activator, phenobarbital (100 μM), had no effect on SULT1C2 expression. These results indicate that SULT1C2 expression is regulated by one or more endogenous isoprenoids and suggest that this regulation is not mediated through CAR. Supported by NIH grants HL050710 and ES005823.
Environmental exposure to mercury is suggested to contribute to human immune dysfunction. To shed light on the mechanism, we identified changes in the phosphoproteomic profile of the WEHI-231 B cell ...line after intoxication with Hg2+. These changes were compared to changes in the phosphoproteome that were induced by pervanadate or okadaic acid exposure. Both 250 μM HgCl2 and pervanadate, a known phosphotyrosine phosphatase inhibitor, caused an increase in the number of proteins identified after TiO2 affinity selection and LC-MS/MS analysis. Pervanadate treatment had a larger effect than Hg2+ on the number of Scansite motifs that were tyrosine-phosphorylated, 17, and Ingenuity canonical signaling pathways activated, 4, with score >5.0. However, Hg2+ had a more focused effect, primarily causing tyrosine-phosphorylation in src homology 2 domains in proteins that are in the B cell receptor signaling pathway. The finding that many of the changes induced by Hg2+ overlap with those of pervanadate, indicates that at high concentrations Hg2+ inhibits protein tyrosine phosphatases.
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