Retrotransposon insertions in the clonal evolution of pancreatic ductal adenocarcinoma - PubMed (original) (raw)

doi: 10.1038/nm.3919. Epub 2015 Aug 10.

Jared P Steranka 1, Alvin Makohon-Moore 1 2, Allison Moyer 1, Peilin Shen 1, Reema Sharma 1, Zachary A Kohutek 3, Cheng Ran Huang 4, Daniel Ahn 1, Paolo Mita 5 6, Martin S Taylor 6, Norman J Barker 1, Ralph H Hruban 1 7 8, Christine A Iacobuzio-Donahue 1 2 7 8, Jef D Boeke 4 5 6 7 9, Kathleen H Burns 1 4 6 7

Affiliations

Retrotransposon insertions in the clonal evolution of pancreatic ductal adenocarcinoma

Nemanja Rodić et al. Nat Med. 2015 Sep.

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed after the disease has metastasized; it is among the most lethal forms of cancer. We recently described aberrant expression of an open reading frame 1 protein, ORF1p, encoded by long interspersed element-1 (LINE-1; L1) retrotransposon, in PDAC. To test whether LINE-1 expression leads to somatic insertions of this mobile DNA, we used a targeted method to sequence LINE-1 insertion sites in matched PDAC and normal samples. We found evidence of 465 somatic LINE-1 insertions in 20 PDAC genomes, which were absent from corresponding normal samples. In cases in which matched normal tissue, primary PDAC and metastatic disease sites were available, insertions were found in primary and metastatic tissues in differing proportions. Two adenocarcinomas secondarily involving the pancreas, but originating in the stomach and duodenum, acquired insertions with a similar discordance between primary and metastatic sites. Together, our findings show that LINE-1 contributes to the genetic evolution of PDAC and suggest that somatic insertions are acquired discontinuously in gastrointestinal neoplasms.

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Figures

Figure 1

Figure 1

Experimental approach. (a) Normal (N); primary (P) tumor samples; and metastatic (M) samples were collected. Sections were evaluated histologically to assess viability and composition prior to DNA extraction. (b) Transposon Insertion Profiling (TIP)-seq. A full-length (6kb) LINE-1 is diagrammed (blue). TSD, target site duplication; UTR, untranslated region; ORF, open reading frame. PCR amplicons initiate from an oligonucleotide complementary to the 3' UTR and extend to a restriction enzyme site. These are sheared and sequenced. Alignment to the reference genome shows read pileups (peaks, lower left). There are no alignments 5′ of the insertion; reads align to the plus (blue) and minus (orange) strands 3' of the insertion. Peaks found in neoplastic samples and not matched normal samples indicate potential somatic insertions. Reads spanning the polyadenylated tail and unique DNA indicate the point of insertion. Insertions are validated by spanning PCR (lower right). The gel shows four lanes with a marker in the leftmost; amplicons from normal (N), primary (P) tumor, and metastasis (M) are shown. The “empty” or pre-insertion site is amplified in all samples, whereas the insertion is recovered from primary and metastatic disease sites. (c) Structure of LINE-1 insertions. LINE-1 is frequently 5' truncated (upper left). Insertions can have inverted 5′ ends (upper right). The diagram shows a sequence breakpoint (//); the 3′ LINE-1 sequence is in the regular orientation (rightward arrow), and 5′ LINE-1 sequence is in the opposing orientation (leftward arrow). Finally, 3′ transduction events incorporate DNA downstream of the parental LINE-1 at the new insertion site.

Figure 2

Figure 2

Somatic LINE-1 insertions in PDAC. (a) Genomic positions are arrayed clockwise in a Circos plot. Positions of all somatic LINE-1 insertion sites are marked with radial hashes color- coded by case. Insertions found in the primary tumor are shown in the outermost wheel; insertions found in the metastasis are found in the middle wheel. Those shared by the two sites of disease are marked innermost. (b) Image b. shows insertions from a single case. Twenty-nine (29) LINE-1 insertions were identified in the primary tumor, and 34 in the metastatic sample. Twenty-three (23) were shared between the two sites of disease. (c) LINE-1 ORF1p protein expression levels as scored by immunohistochemistry (0-3) corresponded to LINE-1 insertion events detected in primary PDAC tumors though not to statistical significance (p=0.07, ANOVA). (d) Survival after diagnosis was inversely related to somatically acquired LINE-1 insertions found in the primary tumor (p=0.025). Each hash on the y-axis indicates one month. (e) Survival after diagnosis was inversely related to LINE-1 ORF1p immunoreactivity (p=0.03).

Figure 3

Figure 3

Retrotransposition (RT) events. (a) Somatic LINE-1 insertions. Stacked bars show insertions found in only the primary tumor (orange, bottom); those shared by the primary and metastatic samples (middle); and those only in the metastasis (red). Confirmed PDAC cases are shown in colors indicated by the legend; two other gastrointestinal adenocarcinomas are shown in lighter hues. (b) RT rate was calculated for each case during two phases of clonal evolution. The progenitor lineage rate (orange, x-axis) is proportional to the number of LINE-1 insertions acquired within a cellular lineage in the primary tumor antedating its seeding a metastatic site. These are shared by the primary and metastatic sites. The progressor lineage rate (red, y-axis) reflects insertions found only in the metastatic sample. PDAC cases are indicated with black dots; other adenocarcinomas are in grey. (c) Somatically acquired LINE-1 in PDAC. Twenty insertions were detected in the primary and metastatic samples for this case and not in normal DNA. Gene names are listed for 8 intronic insertions. These occurred in the progenitor lineage of the primary tumor (leftmost, orange); they were PCR amplified from all metastases. Eight insertions were found by TIP-seq at a metastatic site and not in the primary tumor; nearest gene names are given for intergenic insertions. Four were present in two additional metastases and occurred in a progressor clone of the primary tumor (middle, red). The remaining four were unique to the metastasis profiled by TIP-seq (rightmost) and occurred later in the primary tumor or at that metastatic site. (d) Somatically acquired LINE-1 and single-nucleotide mutations in PDAC. Numbers of events occurring in the progenitor (orange) and progressor (red) lineages are shown; ratios are on the right. (e) LINE-1 insertions in a gastric adenocarcinoma. Sixty insertions were detected in the primary tumor; most (54) were shared with the metastatic site. (f) LINE-1 insertions in a duodenal adenocarcinoma. Twenty-seven insertions were identified in the primary, and 42 in the metastasis. Eighteen were shared between the two sites.

Figure 4

Figure 4

Effects of somatic LINE-1 insertions in PDAC. (a) A landscape plot showing the human genome. Insertion sites are indicated as raised peaks; areas accumulating more independent insertions are reflected by peak height and color. The pattern is distributed; highly recurrently mutated loci do not stand out. Areas accruing the most LINE-1 insertions are marked. Somatic LINE-1 insertions occurred over a 17MB region on 4q32.2 in 6 cases. The nearest gene is follistatin-like 5 isoform (FSTL5), a putative tumor suppressor gene infrequently mutated in pancreatic cancer. None of the insertions were within the FSTL5 transcription unit. A 19MB interval on 5q33.2-5q35.2 showed somatic L1 insertions in 11 cases. An 11MB interval spanning 8q24.21-8q24.23 showed insertions in 6 cases with some cases showing multiple independent insertions. Seven cases had evidence for insertions over a 6MB interval on 13q21.1-13q21.31. Insertions were broadly distributed within each of these regions with no single gene locus recurrently involved. Peaks at 16p11.2 were precisely superimposed but in satellite DNA, could not be PCR validated, and are likely artifact. (b) The schematic illustrates an intronic LINE-1 in apoptotic protease activating factor 1 (APAF1). The L1 is a 443bp insert with an inverted and truncated 5′ end; it is flanked by 16bp TSDs. (c) An immunohistochemistry slide (20x) shows two regions of the primary tumor with differential expression of APAF1 protein. Immunoreactivity is indicated in brown; the counterstain is blue. The tumor shows higher APAF1 expression in one sector (upper right inset, (d) 100x magnification) and reduced immunoreactivity in a less differentiated region (lower right inset, (e) ). Other tumors showed uniform APAF1 expression.

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References

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