Integrated genomic analysis of 456 pancreatic ductal adenocarcinomas identified 32 recurrently mutated genes that aggregate into 10 pathways: KRAS, TGF-β, WNT, NOTCH, ROBO/SLIT signalling, G1/S transition, SWI-SNF, chromatin modification, DNA repair and RNA processing. Expression analysis defined 4 subtypes: (1) squamous; (2) pancreatic progenitor; (3) immunogenic; and (4) aberrantly differentiated endocrine exocrine (ADEX) that correlate with histopathological characteristics. Squamous tumours are enriched for TP53 and KDM6A mutations, upregulation of the TP63∆N transcriptional network, hypermethylation of pancreatic endodermal cell-fate determining genes and have a poor prognosis. Pancreatic progenitor tumours preferentially express genes involved in early pancreatic development (FOXA2/3, PDX1 and MNX1). ADEX tumours displayed upregulation of genes that regulate networks involved in KRAS activation, exocrine (NR5A2 and RBPJL), and endocrine differentiation (NEUROD1 and NKX2-2). Immunogenic tumours contained upregulated immune networks including pathways involved in acquired immune suppression. These data infer differences in the molecular evolution of pancreatic cancer subtypes and identify opportunities for therapeutic development.

The cis-acting response element, A2RE, which is sufficient for cytoplasmic mRNA trafficking in oligodendrocytes, binds a small group of rat brain proteins. Predominant among these is heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor for cytoplasmic trafficking of RNAs bearing A2RE-like sequences. We have now identified the other A2RE-binding proteins as hnRNP A1/A1 B , hnRNP B1, and four isoforms of hnRNP A3. The rat and human hnRNP A3 cDNAs have been sequenced, revealing the existence of alternatively spliced mRNAs. In Western blotting, 38-, 39-, 41-, and 41.5-kDa components were all recognized by antibodies against a peptide in the glycine-rich region of hnRNP A3, but only the 41-and 41.5-kDa bands bound antibodies to a 15-residue N-terminal peptide encoded by an alternatively spliced part of exon 1. The identities of these four proteins were verified by Edman sequencing and mass spectral analysis of tryptic fragments generated from electrophoretically separated bands. Sequence-specific binding of bacterially expressed hnRNP A3 to A2RE has been demonstrated by biosensor and UV cross-linking electrophoretic mobility shift assays. Mutational analysis and confocal microscopy data support the hypothesis that the hnRNP A3 isoforms have a role in cytoplasmic trafficking of RNA.Establishment of asymmetry in cells requires selective localization of proteins. This may be accomplished by directed protein transport, a well established pathway for plasma membrane and secreted proteins, or by trafficking and subsequent localization of mRNA. Localization of RNA has been intensively studied in Drosophila and Xenopus oocytes (for reviews see Refs. 1-6) and more recently in mammalian somatic cells (7-12).In 1982 Subsequent experiments demonstrated that MBP mRNA is translated close to myelin and the protein rapidly incorporated into the nascent membrane (14 -16) and lead to a model in which MBP mRNA is recruited into RNA transport granules in the perikaryon and then transported, by indirect attachment to the microtubule-bound motor protein kinesin, to the myelin compartment at the cell periphery (10, 17-21). The granules are localized in the myelin compartment, and the RNA cargo is translated, with the MBP being incorporated into the myelin membrane. Deletion studies led to the conclusion that a small element in the 3Ј-untranslated region of the MBP mRNA, the RNA transport sequence (RTS), is sufficient and necessary for this cytoplasmic RNA transport in oligodendrocytes (17). Cytoplasmic trafficking of RNA encoding ␤-actin is also dependent on inclusion in transport granules that are attached to the cytoskeleton. In fibroblasts ␤-actin mRNA transport is microfilament-dependent (9, 22), whereas microtubules are implicated in transport of this mRNA in neurons (23,24).trans-Acting factors have been isolated in pull-down experiments with RTS-labeled magnetic particles. The predominant RTS-binding protein from a number of rat tissues is heterogeneous nuclear ribonucleoprotein (hnRNP) A2 (25), a constituent of...

The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multi-tasking protein that acts in the cytoplasm and nucleus. We have explored the possibility that this protein is associated with telomeres and participates in their maintenance. Rat brain hnRNP A2 was shown to have two nucleic acid binding sites. In the presence of heparin one site binds single-stranded oligodeoxyribonucleotides irrespective of sequence but not the corresponding oligoribonucleotides. Both the hnRNP A2-binding cis-acting element for the cytoplasmic RNA trafficking element, A2RE, and the ssDNA telomere repeat match a consensus sequence for binding to a second sequence-specific site identified by mutational analysis. hnRNP A2 protected the telomeric repeat sequence, but not the complementary sequence, against DNase digestion: the glycine-rich domain was found to be necessary, but not sufficient, for protection. The N-terminal RRM (RNA recognition motif) and tandem RRMs of hnRNP A2 also bind the single-stranded, template-containing segment of telomerase RNA. hnRNP A2 colocalizes with telomeric chromatin in the subset of PML bodies that are a hallmark of ALT cells, reinforcing the evidence for hnRNPs having a role in telomere maintenance. Our results support a model in which hnRNP A2 acts as a molecular adapter between single-stranded telomeric repeats, or telomerase RNA, and another segment of ssDNA.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binds a 21-nucleotide myelin basic protein mRNA response element, the A2RE, and A2RE-like sequences in other localized mRNAs, and is a trans-acting factor in oligodendrocyte cytoplasmic RNA trafficking. Recombinant human hnRNPs A1 and A2 were used in a biosensor to explore interactions with A2RE and the cognate oligodeoxyribonucleotide. Both proteins have a single site that bound oligonucleotides with markedly different sequences but did not bind in the presence of heparin. Both also possess a second, specific site that bound only A2RE and was unaffected by heparin. hnRNP A2 bound A2RE in the latter site with a K d near 50 nM, whereas the K d for hnRNP A1 was above 10 M. UV cross-linking assays led to a similar conclusion. Mutant A2RE sequences, that in earlier qualitative studies appeared not to bind hnRNP A2 or support RNA trafficking in oligodendrocytes, had dissociation constants above 5 M for this protein. The two concatenated RNA recognition motifs (RRMs), but not the individual RRMs, mimicked the binding behavior of hnRNP A2. These data highlight the specificity of the interaction of A2RE with these hnRNPs and suggest that the sequence-specific A2RE-binding site on hnRNP A2 is formed by both RRMs acting in cis.The family of more than 20 heterogeneous nuclear ribonucleoproteins (hnRNPs) 1 appears to play diverse roles in the post-transcriptional processing of hnRNA and subsequent packaging, transport, and translation of mRNA (1-4). hnRNPs A1, A2, B1, B2, C1, and C2 are the major components of 40S "core particles," studied primarily in HeLa cells, which are thought to package nascent ssRNA in the nucleus in a histonelike fashion (5-10). The hnRNP A/B proteins have a modular structure with two N-terminal RNA recognition motifs (RRMs) followed by a glycine-rich region ( Fig. 1) (11). The three-dimensional structures of the tandem RRMs of hnRNP A1, which are assumed to be the principal mediators of the RNA-protein interactions, have been determined (12, 13). Although the three-dimensional structures of other hnRNPs of the A/B family have not been determined, their RRMs share sufficient sequence identity with those of hnRNP A1 to suggest that they share the same tertiary fold: a four-stranded antiparallel ␤-sheet flanked by two helices. This module adopts a comparable fold in the RRMs of more distantly related proteins, such as U1A (14 -17).Although a number of the hnRNPs have a general role in intranuclear packaging of RNA, some also manifest sequencespecific binding (1, 4, 18 -22). In addition to being a major component of core particles (23), hnRNP A2 binds a telomeric sequence (24, 25, 43) and a small RNA segment known to be necessary and sufficient for transport of the message encoding myelin basic protein in the cytoplasm of oligodendrocytes, the A2RE (26 -28). The binding of hnRNP A2 to A2RE has been shown, by mutational and antisense approaches, to be essential for cytoplasmic RNA trafficking in oligodendrocytes (29).Although hnRNP A2 has been shown to ...

Highlights d HNF4A loss upregulates GSK3b and drives a squamous-like metabolic profile d GSK3b targeting inhibits glycolysis in squamous patientderived cell lines (PDCLs) d A subset of squamous PDCLs acquires GSK3b drug tolerance d ATAC-seq analysis reveals an accessible WNT gene program in drug-tolerant PDCLs

Largely owing to widespread deployment of microarray analysis, many of the transcriptional events associated with invasive cell migration are becoming clear. However, the transcriptional drives to invasive migration are likely modified by alternative splicing of pre-mRNAs to produce functionally distinct patterns of protein expression. Heterogenous nuclear ribonucleoprotein (hnRNP A2) is a known regulator of alternative splicing that is upregulated in a number of invasive cancer types. Here, we report that although siRNA of hnRNP A2 had little influence on the ability of cells to migrate on plastic surfaces, the splicing regulator was clearly required for cells to move effectively on three-dimensional matrices and to invade into plugs of extracellular matrix proteins. We used exon-tiling microarrays to determine that hnRNP A2 controlled approximately six individual splicing events in a three-dimensional matrix-dependent fashion, one of which influenced invasive migration. Here, we show that alternative splicing of an exon in the 5′ untranslated region of a gene termed TP53INP2 is a key event downstream of hnRNP A2 that is necessary for cells to invade the extracellular matrix. Furthermore, we report that the consequences of altered TP53INP2 splicing on invasion are likely mediated via alterations in Golgi complex integrity during migration on three-dimensional matrices. [Cancer Res 2009;69(24):9219-27]

A-Raf kinase can inhibit apoptosis by binding to the proapoptotic mammalian sterile 20-like kinase (MST2). This function relies on expression of hnRNP H, which ensures the correct splicing of a-raf mRNA needed to produce full-length A-Raf protein. Here, we showed that expression of hnRNP H and production of full-length ARaf is positively controlled by c-Myc. Low c-Myc reduces hnRNP H expression and switches a-raf splicing to produce A-Raf short , a truncated protein. Importantly, A-Raf short fails to regulate MST2 but retains the Ras-binding domain such that it functions as a dominant negative mutant suppressing Ras activation and transformation. Human colon and head and neck cancers exhibit high hnRNP H and high c-Myc levels resulting in enhanced ARaf expression and reduced expression of A-Raf short . Conversely, in normal cells and tissues in which c-Myc and hnRNP H are low, A-Raf short suppresses extracellular signal regulated kinase activation such that it may act as a safeguard against oncogenic transformation. Our findings offered a new paradigm to understand how c-Myc coordinates diverse cell functions by directly affecting alternate splicing of key signaling components. Cancer Res; 71(13); 4664-74. Ó2011 AACR.

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