Unbiased data mining identifies cell cycle transcripts that predict non-indolent Gleason score 7 prostate cancer.

BACKGROUND: Patients with newly diagnosed non-metastatic prostate adenocarcinoma are typically classified as at low, intermediate, or high risk of disease progression using blood prostate-specific antigen concentration, tumour T category, and tumour pathological Gleason score. Classification is used to both predict clinical outcome and to inform initial management. However, significant heterogeneity is observed in outcome, particularly within the intermediate risk group, and there is an urgent need for additional markers to more accurately hone risk prediction. Recently developed web-based visualization and analysis tools have facilitated rapid interrogation of large transcriptome datasets, and querying broadly across multiple large datasets should identify predictors that are widely applicable. METHODS: We used camcAPP, cBioPortal, CRN, and NIH NCI GDC Data Portal to data mine publicly available large prostate cancer datasets. A test set of biomarkers was developed by identifying transcripts that had: 1) altered abundance in prostate cancer, 2) altered expression in patients with Gleason score 7 tumours and biochemical recurrence, 3) correlation of expression with time until biochemical recurrence across three datasets (Cambridge, Stockholm, MSKCC). Transcripts that met these criteria were then examined in a validation dataset (TCGA-PRAD) using univariate and multivariable models to predict biochemical recurrence in patients with Gleason score 7 tumours. RESULTS: Twenty transcripts met the test criteria, and 12 were validated in TCGA-PRAD Gleason score 7 patients. Ten of these transcripts remained prognostic in Gleason score 3 + 4 = 7, a sub-group of Gleason score 7 patients typically considered at a lower risk for poor outcome and often not targeted for aggressive management. All transcripts positively associated with recurrence encode or regulate mitosis and cell cycle-related proteins. The top performer was BUB1, one of four key MIR145-3P microRNA targets upregulated in hormone-sensitive as well as castration-resistant PCa. SRD5A2 converts testosterone to its more active form and was negatively associated with biochemical recurrence. CONCLUSIONS: Unbiased mining of large patient datasets identified 12 transcripts that independently predicted disease recurrence risk in Gleason score 7 prostate cancer. The mitosis and cell cycle proteins identified are also implicated in progression to castration-resistant prostate cancer, revealing a pivotal role for loss of cell cycle control in the latter.

C. elegans SUP-46, an HNRNPM family RNA-binding protein that prevents paternally-mediated epigenetic sterility.

BACKGROUND: In addition to DNA, gametes contribute epigenetic information in the form of histones and non-coding RNA. Epigenetic programs often respond to stressful environmental conditions and provide a heritable history of ancestral stress that allows for adaptation and propagation of the species. In the nematode C. elegans, defective epigenetic transmission often manifests as progressive germline mortality. We previously isolated sup-46 in a screen for suppressors of the hexosamine pathway gene mutant, gna-2(qa705). In this study, we examine the role of SUP-46 in stress resistance and progressive germline mortality. RESULTS: We identified SUP-46 as an HNRNPM family RNA-binding protein, and uncovered a highly novel role for SUP-46 in preventing paternally-mediated progressive germline mortality following mating. Proximity biotinylation profiling of human homologs (HNRNPM, MYEF2) identified proteins of ribonucleoprotein complexes previously shown to contain non-coding RNA. Like HNRNPM and MYEF2, SUP-46 was associated with multiple RNA granules, including stress granules, and also formed granules on active chromatin. SUP-46 depletion disrupted germ RNA granules and caused ectopic sperm, increased sperm transcripts, and chronic heat stress sensitivity. SUP-46 was also required for resistance to acute heat stress, and a conserved "MYEF2" motif was identified that was needed for stress resistance. CONCLUSIONS: In mammals, non-coding RNA from the sperm of stressed males has been shown to recapitulate paternal stress phenotypes in the offspring. Our results suggest that HNRNPM family proteins enable stress resistance and paternally-mediated epigenetic transmission that may be conserved across species.

The eggshell in the C. elegans oocyte-to-embryo transition.

In egg-laying animals, embryonic development takes place within the highly specialized environment provided by the eggshell and its underlying extracellular matrix. Far from being simply a passive physical support, the eggshell is a key player in many early developmental events. Herein, we review current understanding of eggshell structure, biosynthesis, and function in zygotic development of the nematode, C. elegans. Beginning at sperm contact or entry, eggshell layers are produced sequentially. The earlier outer layers are required for secretion or organization of inner layers, and layers differ in composition and function. Developmental events that depend on the eggshell include polyspermy barrier generation, high fidelity meiotic chromosome segregation, osmotic barrier synthesis, polar body extrusion, anterior-posterior polarization, and organization of membrane and cortical proteins. The C. elegans eggshell is proving to be an excellent, tractable system to study the molecular cues of the extracellular matrix that instruct cell polarity and early development.

The eggshell is required for meiotic fidelity, polar-body extrusion and polarization of the C. elegans embryo.

BACKGROUND: Fertilization restores the diploid state and begins the process by which the single-cell oocyte is converted into a polarized, multicellular organism. In the nematode, Caenorhabditis elegans, two of the earliest events following fertilization are secretion of the chitinous eggshell and completion of meiosis, and in this report we demonstrate that the eggshell is essential for multiple developmental events at the one-cell stage. RESULTS: We show that the GLD (Germline differentiation abnormal)-1-regulated hexosamine pathway enzyme, glucosamine-6-phosphate N-acetyltransferase (GNA)-2, is required for synthesis of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), the substrate for eggshell chitin synthesis by chitin synthase-1 (CHS-1). Furthermore, while chs-1(RNAi) or combined RNAi with the chitin-binding proteins, CEJ-1 and B0280.5, does not interfere with normal meiotic timing, lagging chromosomes are observed at meiosis, and polar-body extrusion fails. We also demonstrate that chitin, and either CEJ-1 or B0280.5, are essential for the osmotic/permeability barrier and for movement of the sperm pronucleus/centrosome complex to the cortex, which is associated with the initiation of polarization. CONCLUSION: Our results indicate that the eggshell is required in single-cell C. elegans development, playing an essential role in multiple actin-dependent early events. Furthermore, the earliest meiotic roles precede osmotic barrier formation, indicating that the role of the eggshell is not limited to generation of the osmotic barrier.

The directed migration of gonadal distal tip cells in Caenorhabditis elegans requires NGAT-1, a ß1,4-N-acetylgalactosaminyltransferase enzyme.

Glycoproteins such as growth factor receptors and extracellular matrix have well-known functions in development and cancer progression, however, the glycans at sites of modification are often heterogeneous molecular populations which makes their functional characterization challenging. Here we provide evidence for a specific, discrete, well-defined glycan modification and regulation of a stage-specific cell migration in Caenorhabditis elegans. We show that a chain-terminating, putative null mutation in the gene encoding a predicted ß1,4-N-acetylgalactosaminyltransferase, named ngat-1, causes a maternally rescued temperature sensitive (ts) defect in the second phase of the three phase migration pattern of the posterior, but not the anterior, hermaphrodite Distal Tip Cell (DTC). An amino-terminal partial deletion of ngat-1 causes a similar but lower penetrance ts phenotype. The existence of multiple ts alleles with distinctly different molecular DNA lesions, neither of which is likely to encode a ts protein, indicates that NGAT-1 normally prevents innate temperature sensitivity for phase 2 DTC pathfinding. Temperature shift analyses indicate that the ts period for the ngat-1 mutant defect ends by the beginning of post-embryonic development-nearly 3 full larval stages prior to the defective phase 2 migration affected by ngat-1 mutations. NGAT-1 homologs generate glycan-terminal GalNAc-ß1-4GlcNAc, referred to as LacdiNAc modifications, on glycoproteins and glycolipids. We also found that the absence of the GnT1/Mgat1 activity [UDP-N-acetyl-D-glucosamine:α-3-D-mannoside ß-1,2-N-acetylglucosaminyltransferase 1 (encoded by C. elegans gly-12, gly-13, and gly-14 and homologous to vertebrate GnT1/Mgat1)], causes a similar spectrum of DTC phenotypes as ngat-1 mutations-primarily affecting posterior DTC phase 2 migration and preventing manifestation of the same innate ts period as ngat-1. GnT1/Mgat1 is a medial Golgi enzyme known to modify mannose residues and initiate N-glycan branching, an essential step in the biosynthesis of hybrid, paucimannose and complex-type N-glycans. Quadruple mutant animals bearing putative null mutations in ngat-1 and the three GnT genes (gly-12, gly-13, gly-14) were not enhanced for DTC migration defects, suggesting NGAT-1 and GnT1 act in the same pathway. These findings suggest that GnTI generates an N-glycan substrate for NGAT-1 modification, which is required at restrictive temperature (25°C) to prevent, stabilize, reverse or compensate a perinatal thermo-labile process (or structure) causing late larval stage DTC phase 2 migration errors.

Eggshell chitin and chitin-interacting proteins prevent polyspermy in C. elegans.

Development requires fertilization by a single sperm. In Caenorhabditis elegans, fertilization occurs in a sperm-filled spermatheca, implying the barrier to polyspermy is generated in this compartment. Eggshell chitin synthesis is initiated at fertilization, and chitin is deposited before the zygote exits the spermatheca. Whereas polyspermy is very rare in wild-type, here we report an incidence of 14%-51% in zygotes made chitin deficient by loss of chitin synthase-1 (CHS-1), the CHS-1 substrate UDP-N-acetylglucosamine, the CHS-1-interacting protein EGG-3, or the sperm-provided protein SPE-11. The spe-11(hc90) mutant deposits chitin at the male end but fails to complete a continuous layer. The polyspermy barrier is also compromised by loss of the chitin-binding protein CBD-1 or the GLD-1-regulated LDL receptor-like EGG-1, together with its homolog, EGG-2. Loss of CBD-1 or EGG-1/2 disrupts oocyte cortical distribution of CHS-1, as well as MBK-2 and EGG-3. In CBD-1 or EGG-1/2 deficiency, chitin is synthesized but the eggshell is fractured, suggesting aberrantly clustered CHS-1/MBK-2/EGG-3 may fail to support construction of a continuous eggshell. Together, our results show that eggshell chitin is required to prevent polyspermy in C. elegans, in addition to its previously reported requirement in polar body extrusion and polarization of the zygote.