The origins and consequences of UPF1 variants in pancreatic adenosquamous carcinoma

  1. Jacob T Polaski
  2. Dylan B Udy
  3. Luisa F Escobar-Hoyos
  4. Gokce Askan
  5. Steven D Leach
  6. Andrea Ventura
  7. Ram Kannan
  8. Robert K Bradley

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Abstract

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 UPF1 mutations are important contributors to PASC has been exacerbated by a paucity of functional studies. Here, we modeled two UPF1 mutations in human and mouse cells to find no significant effects on pancreatic cancer growth, acquisition of adenosquamous features, UPF1 splicing, UPF1 protein, or NMD efficiency. We subsequently discovered that 45% of UPF1 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 UPF1 is not a common functional driver of PASC and motivate further attempts to understand the genetic origins of these malignancies.

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  1. Version published to 10.7554/elife.62209 on eLife
  2. eLife

    ###Reviewer #3:

    In the manuscript, Polaski et al. compared the reported UPF1 mutations with a collection of three databases and found 42.5% of these mutations are identical to germline genetic variation. However, most of these overlapped mutations are located within introns, and only present in Exome Aggregation Consortium (ExAC) database (Figure 2). This raised some concerns since the ExAC database mainly reportsreport exon variants rather than intron variants, the authors need to provideneed provide other information such as allele frequency to examine whether these intronic mutations are rare or low-frequency variants. Another suggestion is that the authors may cross-reference UPF1 mutations with the recent gnomAD v3 database (Nature 2020), which provided non-coding genetic variants within much better resolution. In addition, most of the other UPF1 exon mutations are indeed novel as they are not present in any databases (Figure 2 - figure Supplement 1). The authors need to provide some additional analysis such as separating these two types of variants (exon/intron variants) and analyzing the frequency of overlapped UPF1 mutations.

  3. eLife

    ###Reviewer #2:

    This paper aims to resolve the disparity between one report (Liu et al., 2014), which described somatic mutations in pancreatic adenosquamous carcinoma (PASC) that did not typify normal pancreatic tissue of the patients, and other reports (Witkiewicz et al., 2015; Fang et al., 2017; Hayashi et al., 2020), which did not find these mutations. The authors show here that many (40%) of the mutations described by Liu et al. typify genetic variations in the human population at large, and they suggest that these mutations are not pathogenic, e.g. are not drivers of PASC, and also not somatic but, rather, are genetic in origin.

    The authors use CRISPR-Cas9 to generate in mouse pancreatic cancer (KPC) cells, which harbor Kras and Tp53 gene mutations as do PASC patients, a Upf1 gene, and thus its product mRNA, lacking exons 10 and 11, as Liu et al. reported not only inhibits NMD by disrupting UPF1 helicase activity but also promotes tumorigenesis. After injection into mice, the authors found no detectable effects on pancreatic cancer growth compared to the injection of control cells.

    The authors acknowledge that mice may differ from humans. Thus next, rather than using mini-UPF1 genes, as did Liu et al., the authors introduced two of the Liu et al. mutations separately into the UPF1 gene of HEK293T cells. In contrast to Liu et al., the authors found modestly increased NMD efficiency and no evidence of UPF1 pre-mRNA mis-splicing. The authors note that this makes sense since these mutations are found in people not as somatic mutations but genetic mutations, and thus would not be expected to inhibit NMD given the importance of NMD to aspects of human development in utero and beyond.

    This is a very well-written paper describing carefully executed experiments that lead the reader to discount three claims made about UPF1 gene mutations in PANC as described by Liu et al., namely, that these mutations: (i) have a somatic origin, (ii) lead to UPF1 pre-mRNA mis-splicing so as to inhibit NMD, and (iii) promote tumorigenesis. The authors are careful not to over-interpret their data.

    Specific comments:

    Page 4, in reference to Figure 1f. It is unexpected that the variations in UPF1 protein levels were "uncorrelated with NMD efficiency". Possibly, this reviewer doesn't understand what the authors mean. Please clarify.

    Additionally, in this regard, it is better to draw conclusions about NMD efficiency by measuring more than just the efficiency with which mRNA from a reporter construct is targeted for NMD. It is recommended that the authors assay the levels of a few (e.g. three) cellular NMD targets, normalized to the level of their pre-mRNA to control for any changes to gene transcription.

  4. eLife

    ###Reviewer #1:

    This manuscript identifies that the UPF1 variants previously reported as frequent somatic mutations in pancreatic adenosquamous carcinoma are actually germline genetic variants with no clear effects on UPF1 splicing, protein splicing, or nonsense mediated decay. Given that the manuscript challenges a striking finding from a prior study that has not been validated in subsequent studies, it is important to publish to correct the literature. At the same time, several points should be clarified to make sure the data are as comprehensive as possible:

    1. In the experiments evaluating the effect of skipping exons 10-11 of UPF1, it is surprising that this genetic perturbation in UPF1 is actually tolerated in these cells as UPF1 is an essential gene in most cancer cell lines (this point also has likely motivated this current study). Also, the Western blots for UPF1 protein are not particularly clear (Supplementary Figure 1c) and the fact that the cells don't perturb the growth of KPC cells does not prove that UPF1 alterations is not tumorigenic. Have the authors checked to see if UPF1 is downregulated and mis-spliced still in the cells following in vivo growth? A simple in vitro competition assay between UPF1 exon 10-11 targeted cells and control sgRNA cells would also be helpful. It would also be helpful to evaluate if NMD is altered in these cells given these issues.

    2. Although it is clear that the authors have used similar minigene assays as were used in the original publication, a more systematic evaluation for potential alteration in NMD with UPF1 variants (via RNA-seq) would be helpful given that this work questions the prior publication.

    3. Do the authors believe that the UPF1 variants reported as mutations initially in PASC are actually SNPs? The terminology describing what these variants are could be a little clearer in the Abstract and Discussion.

  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript. Eric J Wagner (University of Texas Medical Branch) served as the Reviewing Editor.

    ###Summary:

    The authors have sought to address what has become a considerably debated topic of whether mutations in Upf1 are tumorigenic in pancreatic adenosquamous carcinoma. Specifically, the authors introduced Upf1 mutants found in pancreatic tumors into pancreatic adenosquamous carcinoma cells, and found they did not provide significant advantage for tumor progression. Moreover, the authors described how a significant percentage of Upf1 mutants observed in pancreatic carcinoma are also present as variants in the human population, raising further doubts about their potential role as cancer drivers. Altogether, this work provides further evidence as to whether Upf1 disruptive mutations represent driving factors in pancreatic adenosquamous carcinoma.

  6. Version published to 10.1101/2020.08.14.248864v1 on bioRxiv