Biomarkers for Early Cancer Detection: A Landscape View of Recent Advancements, Spotlighting Pancreatic and Liver Cancers.

Cancer is one of the leading causes of death worldwide. Early cancer detection is critical because it can significantly improve treatment outcomes, thus saving lives, reducing suffering, and lessening psychological and economic burdens. Cancer biomarkers provide varied information about cancer, from early detection of malignancy to decisions on treatment and subsequent monitoring. A large variety of molecular, histologic, radiographic, or physiological entities or features are among the common types of cancer biomarkers. Sizeable recent methodological progress and insights have promoted significant developments in the field of early cancer detection biomarkers. Here we provide an overview of recent advances in the knowledge related to biomolecules and cellular entities used for early cancer detection. We examine data from the CAS Content Collection, the largest human-curated collection of published scientific information, as well as from the biomarker datasets at Excelra, and analyze the publication landscape of recent research. We also discuss the evolution of key concepts and cancer biomarkers development pipelines, with a particular focus on pancreatic and liver cancers, which are known to be remarkably difficult to detect early and to have particularly high morbidity and mortality. The objective of the paper is to provide a broad overview of the evolving landscape of current knowledge on cancer biomarkers and to outline challenges and evaluate growth opportunities, in order to further efforts in solving the problems that remain. The merit of this review stems from the extensive, wide-ranging coverage of the most up-to-date scientific information, allowing unique, unmatched breadth of landscape analysis and in-depth insights.

Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase.

Many microorganisms use flavin-dependent thymidylate synthase (FDTS) to synthesize the essential nucleotide 2'-deoxythymidine 5'-monophosphate (dTMP) from 2'-deoxyuridine 5'-monophosphate (dUMP), 5,10-methylenetetrahydrofolate (CH2THF), and NADPH. FDTSs have a structure that is unrelated to the thymidylate synthase used by humans and a very different mechanism. Here we report nuclear magnetic resonance evidence that FDTS ionizes N3 of dUMP using an active-site arginine. The ionized form of dUMP is largely responsible for the changes in the flavin absorbance spectrum of FDTS upon dUMP binding. dUMP analogues also suggest that the phosphate of dUMP acts as the base that removes the proton from C5 of the dUMP-methylene intermediate in the FDTS-catalyzed reaction. These findings establish additional differences between the mechanisms of FDTS and human thymidylate synthase.

Multivalency in the inhibition of oxidative protein folding by arsenic(III) species.

The renewed use of arsenicals as chemotherapeutics has rekindled interest in the biochemistry of As(III) species. In this work, simple bis- and tris-arsenical derivatives were synthesized with the aim of exploiting the chelate effect in the inhibition of thiol-disulfide oxidoreductases (here, Quiescin sulfhydryl oxidase, QSOX, and protein disulfide isomerase, PDI) that utilize two or more CxxC motifs in the catalysis of oxidative protein folding. Coupling 4-aminophenylarsenoxide (APAO) to acid chloride or anhydride derivatives yielded two bis-arsenical prototypes, BA-1 and BA-2, and a tris-arsenical, TA-1. Unlike the monoarsenical, APAO, these new reagents proved to be strong inhibitors of oxidative protein folding in the presence of a realistic intracellular concentration of competing monothiol (here, 5 mM reduced glutathione, GSH). However, this inhibition does not reflect direct inactivation of QSOX or PDI, but avid binding of MVAs to the reduced unfolded protein substrates themselves. Titrations of reduced riboflavin-binding protein with MVAs show that all 18 protein -SH groups can be captured by these arsenicals. With reduced RNase, addition of substoichiometric levels of MVAs is accompanied by the formation of Congo Red- and Thioflavin T-positive fibrillar aggregates. Even with Kd values of ∼50 nM, MVAs are ineffective inhibitors of PDI in the presence of millimolar levels of competing GSH. These results underscore the difficulties of designing effective and specific arsenical inhibitors for folded enzymes and proteins. Some of the cellular effects of arsenicals likely reflect their propensity to associate very tightly and nonspecifically to conformationally mobile cysteine-rich regions of proteins, thereby interfering with folding and/or function.

An arsenical-maleimide for the generation of new targeted biochemical reagents.

The finding that arsenic trioxide is an effective treatment for acute promyelocytic leukemia has renewed interest in the pharmacological uses of inorganic and organic arsenicals. Here we synthesized and characterized the reactivity of an arsenical-maleimide (As-Mal) that can be efficiently conjugated to exposed cysteine residues in peptides and proteins with the ultimate goal of directing these As(III) species to vicinal thiols in susceptible targets within cells and tissues. As-Mal conjugated to a surface cysteine in thioredoxin provides a more potent inhibitor for Escherichia coli thioredoxin reductase than comparable simple inorganic or organic arsenicals. As-Mal can be coupled to all of the eight cysteine residues of reduced unfolded ribonuclease A or to site-specific locations using appropriate cysteine mutations. We observed particularly strong binding to the two CxxC motifs of protein disulfide isomerase using a mutant RNase in which As-Mal was specifically incorporated at residues 26 and 110. As-Mal will serve as a facile reagent for the incorporation of As(III) species into a wide range of thiol-containing proteins, biomaterials, and surfaces.

SR protein family members display diverse activities in the formation of nascent and mature mRNPs in vivo.

The SR proteins are a family of pre-mRNA splicing factors with additional roles in gene regulation. To investigate individual family members in vivo, we generated a comprehensive panel of stable cell lines expressing GFP-tagged SR proteins under endogenous promoter control. Recruitment of SR proteins to nascent FOS RNA was transcription dependent and RNase sensitive, with unique patterns of accumulation along the gene specified by the RNA recognition motifs (RRMs). In addition, all SR protein interactions with Pol II were RNA dependent, indicating that SR proteins are not preassembled with Pol II. SR protein interactions with RNA were confirmed in situ by FRET/FLIM. Interestingly, SC35-GFP also exhibited FRET with DNA and failed to associate with cytoplasmic mRNAs, whereas all other SR proteins underwent nucleocytoplasmic shuttling and associated with specific nuclear and cytoplasmic mRNAs. Because different constellations of SR proteins bound nascent, nuclear, and cytoplasmic mRNAs, mRNP remodeling must occur throughout an mRNA's lifetime.

The splicing factor Prp17 interacts with the U2, U5 and U6 snRNPs and associates with the spliceosome pre- and post-catalysis.

Saccharomyces cerevisiae PRP17-null mutants are temperature-sensitive for growth. In vitro splicing with extracts lacking Prp17 are kinetically slow for the first step of splicing and are arrested for the second step at temperatures greater than 34 degrees C. In the present study we show that these stalled spliceosomes are compromised for an essential conformational switch that is triggered by Prp16 helicase. These results suggest a plausible mechanistic basis for the second-step arrest in prp17Delta extracts and support a role for Prp17 in conjunction with Prp16. To understand the association of Prp17 with spliceosomes we used a functional epitope-tagged protein in co-immunoprecipitation experiments. Examination of co-precipitated snRNAs (small nuclear RNAs) show that Prp17 interacts with U2, U5 and U6 snRNPs (small nuclear ribonucleoproteins) but it is not a core component of any one snRNP. Prp17 association with in-vitro-assembled spliceosome complexes on actin pre-mRNAs was also investigated. Although the U5 snRNP proteins Prp8 and Snu114 are found in early pre-spliceosomes that contain all five snRNPs, Prp17 is not detectable at this step; however, Prp17 is present in the subsequent pre-catalytic A1 complex, containing unspliced pre-mRNA, formed after the dissociation of U4 snRNP. Thus Prp17 joins the spliceosome prior to both catalytic reactions. Our results indicate continued interactions in catalytic spliceosomes that contain reaction intermediates and in post-splicing complexes containing the lariat intron. These Prp17-spliceosome association analyses provide a biochemical basis for the delayed first step in prp17Delta and explain the previously known multiple genetic interactions between Prp17, factors of the Prp19-complex [NTC (nineteen complex)], functional elements in U2 and U5 snRNAs and other second-step splicing factors.

Spliceosomal small nuclear ribonucleoprotein particles repeatedly cycle through Cajal bodies.

The Cajal body (CB) is a nuclear structure closely associated with import and biogenesis of small nuclear ribonucleoprotein particles (snRNPs). Here, we tested whether CBs also contain mature snRNPs and whether CB integrity depends on the ongoing snRNP splicing cycle. Sm proteins tagged with photoactivatable and color-maturing variants of fluorescent proteins were used to monitor snRNP behavior in living cells over time; mature snRNPs accumulated in CBs, traveled from one CB to another, and they were not preferentially replaced by newly imported snRNPs. To test whether CB integrity depends on the snRNP splicing cycle, two human orthologues of yeast proteins involved in distinct steps in spliceosome disassembly after splicing, hPrp22 and hNtr1, were depleted by small interfering RNA treatment. Surprisingly, depletion of either protein led to the accumulation of U4/U6 snRNPs in CBs, suggesting that reassembly of the U4/U6.U5 tri-snRNP was delayed. Accordingly, a relative decrease in U5 snRNPs compared with U4/U6 snRNPs was observed in CBs, as well as in nuclear extracts of treated cells. Together, the data show that particular phases of the spliceosome cycle are compartmentalized in living cells, with reassembly of the tri-snRNP occurring in CBs.

Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells.

Coupling between transcription and RNA processing is a key gene regulatory mechanism. Here we use chromatin immunoprecipitation to detect transcription-dependent accumulation of the precursor mRNA (pre-mRNA) splicing factors hnRNP A1, U2AF65 and U1 and U5 snRNPs on the intron-containing human FOS gene. These factors were poorly detected on intronless heat-shock and histone genes, a result that opposes direct recruitment by RNA polymerase II (Pol II) or the cap-binding complex in vivo. However, an observed RNA-dependent interaction between U2AF65 and active forms of Pol II may stabilize U2AF65 binding to intron-containing nascent RNA. We establish chromatin-RNA immunoprecipitation and show that FOS pre-mRNA is cotranscriptionally spliced. Notably, the topoisomerase I inhibitor camptothecin, which stalls elongating Pol II, increased cotranscriptional splicing factor accumulation and splicing in parallel. This provides direct evidence for a kinetic link between transcription, splicing factor recruitment and splicing catalysis.

Genome-wide analysis of pre-mRNA splicing: intron features govern the requirement for the second-step factor, Prp17 in Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Removal of pre-mRNA introns is an essential step in eukaryotic genome interpretation. The spliceosome, a ribonucleoprotein performs this critical function; however, precise roles for many of its proteins remain unknown. Genome-wide consequences triggered by the loss of a specific factor can elucidate its function in splicing and its impact on other cellular processes. We have employed splicing-sensitive DNA microarrays, with yeast open reading frames and intron sequences, to detect changes in splicing efficiency and global expression. Comparison of expression profiles, for intron-containing transcripts, among mutants of two second-step factors, Prp17 and Prp22, reveals their unique and shared effects on global splicing. This analysis enabled the identification of substrates dependent on Prp17. We find a significant Prp17 role in splicing of introns which are longer than 200nts and note its dispensability when introns have a < or =13-nucleotide spacing between their branch point nucleotide and 3 ' splice site. In vitro splicing of substrates with varying branch nucleotide to 3 ' splice site distances supports the differential Prp17 dependencies inferred from the in vivo analysis. Furthermore, we tested the predicted dispensability of Prp17 for splicing short introns in the evolutionarily distant yeast, Schizosaccharomyces pombe, where the genome contains predominantly short introns. SpPrp17 was non-essential at all growth temperatures implying that functional evolution of splicing factors is integrated with genome evolution. Together our studies point to a role for budding yeast Prp17 in splicing of subsets of introns and have predictive value for deciphering the functions of splicing factors in gene expression and regulation in other eukaryotes.

Dependence of pre-mRNA introns on PRP17, a non-essential splicing factor: implications for efficient progression through cell cycle transitions.

Saccharomyces cerevisiae PRP17 (CDC40) encodes a second-step pre-mRNA splicing factor with a role in cell division. The functions of Prp17 in specific cell cycle transitions were examined using temperature-sensitive alleles in arrest/release experiments. We find that G(1)/S and G(2)/M transitions depend on Prp17. G(1)-synchronized prp17::LEU2 cells arrest at non-permissive temperatures as unbudded haploid cells with low levels of CLN1, CLB5 and RNR1 transcripts. This indicates a Prp17 execution point at or prior to Start. Reduced levels of alpha-tubulin protein, a mitotic spindle component, underlie the benomyl sensitivity of prp17 mutants and possibly their G(2)/M arrest. Splicing of TUB1 and TUB3 transcripts, which encode alpha-tubulin, was analyzed in prp17 and other second-step factor mutants. TUB1 splicing is inefficient in prp17, prp16 and prp22, and marginally affected in prp18, slu7-1 and psf1-1. TUB3 splicing is similarly affected. In vitro splicing with TUB3 pre-mRNA demonstrates a compromised second step in prp17::LEU2 extracts, implicating a direct role for Prp17 in its efficient splicing. Genomic replacement of an intronless TUB1 gene relieves the benomyl sensitivity of prp17 mutants; however, they remain temperature sensitive, implying multiple limiting factors for mitosis. The data suggest that integration of splicing with the cell cycle is important for G(1)/S and G(2)/M transitions.