Pancreatic ductal adenocarcinoma (PDAC), the most common histological subtype of pancreatic cancer, has one of the highest case fatality rates of all known solid malignancies. Over the past decade, ...several landmark studies have established mutations in
and
as the predominant drivers of PDAC pathogenesis and therapeutic resistance, though treatment options for PDACs and other tumors with these mutations remain extremely limited. Hampered by late tumor discovery and diagnosis, clinicians are often faced with using aggressive and non-specific chemotherapies to treat advanced disease. Clinically meaningful responses to targeted therapy are often limited to the minority of patients with susceptible PDACs, and immunotherapies have routinely encountered roadblocks in effective activation of tumor-infiltrating immune cells. Alternative RNA splicing (ARS) has recently gained traction in the PDAC literature as a field from which we may better understand and treat complex mechanisms of PDAC initiation, progression, and therapeutic resistance. Here, we review PDAC pathogenesis as it relates to fundamental ARS biology, with an extension to implications for PDAC patient clinical management.
Pancreatic adenosquamous carcinoma (PASC) is an aggressive cancer whose mutational origins are poorly understood. An early study reported high-frequency somatic mutations affecting UPF1, a ...nonsense-mediated mRNA decay (NMD) factor, in PASC, but subsequent studies did not observe these lesions. The corresponding controversy about whether
mutations are important contributors to PASC has been exacerbated by a paucity of functional studies. Here, we modeled two
mutations in human and mouse cells to find no significant effects on pancreatic cancer growth, acquisition of adenosquamous features,
splicing, UPF1 protein, or NMD efficiency. We subsequently discovered that 45% of
mutations reportedly present in PASCs are identical to standing genetic variants in the human population, suggesting that they may be non-pathogenic inherited variants rather than pathogenic mutations. Our data suggest that
is not a common functional driver of PASC and motivate further attempts to understand the genetic origins of these malignancies.
Actin-based tubular connections between cells have been observed in many cell types. Termed "tunneling nanotubes (TNTs)," "membrane nanotubes," "tumor microtubes (TMTs)," or "cytonemes," these ...protrusions interconnect cells in dynamic networks. Structural features in these protrusions vary between cellular systems, including tubule diameter and the presence of microtubules. We find tubular protrusions, which we classify as TMTs, in a pancreatic cancer cell line, Dartmouth-Hitchcock Pancreatic Cancer (DHPC)-018. TMTs are present in DHPC-018-derived tumors in mice, as well as in a mouse model of pancreatic cancer and a subset of primary human tumors. DHPC-018 TMTs have heterogeneous diameter (0.39-5.85 µm, median 1.92 µm) and contain actin filaments, microtubules, and cytokeratin 19-based intermediate filaments. TMTs do not allow intercellular transfer of cytoplasmic GFP. Actin filaments are cortical within the protrusion, as opposed to TNTs, in which filaments run down the center. TMTs are dynamic in length, but are long lived (median >60 min). Inhibition of actin polymerization, but not microtubules, results in TMT loss. Extracellular calcium is necessary for TMT maintenance. A second class of tubular protrusion, which we term cell-substrate protrusion, has similar width range and cytoskeletal features but makes contact with the substratum as opposed to another cell. Similar to previous work on TNTs, we find two assembly mechanisms for TMTs, which we term "pull-away" and "search-and-capture." Inhibition of Arp2/3 complex inhibits TMT assembly by both mechanisms. This work demonstrates that the actin architecture of TMTs in pancreatic cancer cells is fundamentally different from that of TNTs and demonstrates the role of Arp2/3 complex in TMT assembly.
Translation initiation is orchestrated by the cap binding and 43S preinitiation complexes (PIC). Eukaryotic initiation factor 1A (EIF1A) is essential for recruitment of the ternary complex and for ...assembling the 43S PIC. Recurrent
mutations in papillary thyroid cancers are mutually exclusive with other drivers, including
.
mutations are enriched in advanced thyroid cancers, where they display a striking co-occurrence with
, which cooperates to induce tumorigenesis in mice and isogenic cell lines. The C-terminal
mutation is the most prevalent in advanced thyroid cancer. EIF1AX-A113splice variants stabilize the PIC and induce ATF4, a sensor of cellular stress, which is co-opted to suppress EIF2α phosphorylation, enabling a general increase in protein synthesis. RAS stabilizes c-MYC, an effect augmented by EIF1AX-A113splice. ATF4 and c-MYC induce expression of amino acid transporters and enhance sensitivity of mTOR to amino acid supply. These mutually reinforcing events generate therapeutic vulnerabilities to MEK, BRD4, and mTOR kinase inhibitors. SIGNIFICANCE: Mutations of
component of the translation PIC, co-occur with
in advanced thyroid cancers and promote tumorigenesis.
drives an ATF4-induced dephosphorylation of EIF2α, resulting in increased protein synthesis. ATF4 also cooperates with c-MYC to sensitize mTOR to amino acid supply, thus generating vulnerability to mTOR kinase inhibitors.
.
Little is known about the distal excretory component of the urinary tract in Danio rerio (zebrafish). This component is affected by many human diseases and disorders of development. Here, we have ...undertaken multi-level analyses to determine the structure and composition of the distal urinary tract in the zebrafish. In silico searches identified uroplakin 1a (ukp1a), uroplakin 2 (upk2) and uroplakin 3b (upk3b) genes in the zebrafish genome (orthologues to genes that encode urothelium-specific proteins in humans). In situ hybridization demonstrated ukp1a expression in the zebrafish pronephros and cloaca from 96 h post-fertilization. Haematoxylin and Eosin staining of adult zebrafish demonstrated two mesonephric ducts uniting into a urinary bladder that leads to a distinct urethral opening. Immunohistochemistry identified Uroplakin 1a, Uroplakin 2 and GATA3 expression in zebrafish urinary bladder cell layers that match human urothelial expression. Fluorescent dye injections demonstrated zebrafish urinary bladder function, including urine storage and intermittent micturition, and a urethral orifice separate from the larger anal canal and rectum. Our findings reveal homology between the urinary tracts of zebrafish and humans, and offer the former as a model system to study disease.
Notch signaling regulates cell fate decisions in a wide variety of adult and embryonic tissues. Here we show that Notch pathway components and Notch target genes are upregulated in invasive ...pancreatic cancer, as well as in pancreatic cancer precursors from both mouse and human. In mouse pancreas, ectopic Notch activation results in accumulation of nestin-positive precursor cells and expansion of metaplastic ductal epithelium, previously identified as a precursor lesion for pancreatic cancer. Notch is also activated as a direct consequence of EGF receptor activation in exocrine pancreas and is required for TGFα-induced changes in epithelial differentiation. These findings suggest that Notch mediates the tumor-initiating effects of TGFα by expanding a population of undifferentiated precursor cells.
Pancreatic adenocarcinoma (PDAC) epitomizes a deadly cancer driven by abnormal KRAS signaling. Here, we show that the eIF4A RNA helicase is required for translation of key KRAS signaling molecules ...and that pharmacological inhibition of eIF4A has single-agent activity against murine and human PDAC models at safe dose levels. EIF4A was uniquely required for the translation of mRNAs with long and highly structured 5' untranslated regions, including those with multiple G-quadruplex elements. Computational analyses identified these features in mRNAs encoding KRAS and key downstream molecules. Transcriptome-scale ribosome footprinting accurately identified eIF4A-dependent mRNAs in PDAC, including critical KRAS signaling molecules such as PI3K, RALA, RAC2, MET, MYC, and YAP1. These findings contrast with a recent study that relied on an older method, polysome fractionation, and implicated redox-related genes as eIF4A clients. Together, our findings highlight the power of ribosome footprinting in conjunction with deep RNA sequencing in accurately decoding translational control mechanisms and define the therapeutic mechanism of eIF4A inhibitors in PDAC. SIGNIFICANCE: These findings document the coordinate, eIF4A-dependent translation of RAS-related oncogenic signaling molecules and demonstrate therapeutic efficacy of eIF4A blockade in pancreatic adenocarcinoma.
Invasive pancreatic cancer is thought to develop through a series of noninvasive duct lesions known as pancreatic intraepithelial neoplasia (PanIN). We used cDNA microarrays interrogating 15,000 ...transcripts to identify 49 genes that were differentially expressed in microdissected early PanIN lesions (PanIN-1B/2) compared with microdissected normal duct epithelium. In this analysis, a cluster of extrapancreatic foregut markers, including pepsinogen C, MUC6, KLF4, and TFF1, was found to be up-regulated in PanIN. Up-regulation of these genes was further validated using combinations of real-time reverse transcription-PCR, in situ hybridization, and immunohistochemistry in a total of 150 early PanIN lesions from 81 patients. Identification of these gastrointestinal transcripts in human PanIN prompted assessment of other foregut markers by both semiquantitative and real-time reverse transcription-PCR, revealing similar up-regulation of Sox-2, Gastrin, HoxA5, GATA4/5/6, Villin and Forkhead 6 (Foxl1). In contrast to frequent expression of multiple gastric epithelial markers, the intestinal markers intestinal fatty acid binding protein, CDX1 and CDX2 were rarely expressed either in PanIN lesions or in invasive pancreatic cancer. Hedgehog pathway activation induced by transfection of immortalized human pancreatic ductal epithelial cells with Gli1 resulted in up-regulation of the majority of foregut markers seen in early PanIN lesions. These data show frequent up-regulation of foregut markers in early PanIN lesions and suggest that PanIN development may involve Hedgehog-mediated conversion to a gastric epithelial differentiation program.
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