Somatic Variation and Genomic Instability Generated by Retrotransposons

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Johns Hopkins University
Genomic instability is strongly linked to the development and malignancy of cancer and by studying premalignant conditions we can gain a better understanding of the sources of genomic instability and improve cancer prevention and treatment. The genome is very unstable in both premalignant and malignant conditions; however, it is unknown as to what extent different types of instability contribute. Retrotransposition is an active source of genomic instability in the human genome and has the potential to change DNA structure and RNA expression. Retrotransposons are repetitive sequences that can “copy and paste” into the genome at new sites within an individual cell, and hundreds are known to be active in the human genome. Despite the enormous influence of retrotransposons on the genome composition of many organisms, the degree to which they contribute to somatic genomic instability is unknown. Because retrotransposition has been observed in many gastrointestinal epithelial cancer types, we focused on L1 mobilization as a source of instability in cancer. We hypothesized that L1 retrotransposition is active in esophageal squamous cell carcinoma (SCC), esophageal adenocarcinoma (EAC), and EAC’s precursor Barrett’s esophagus (BE). To test our hypothesis, we evaluated 5 patients with benign BE, 5 patients with BE and concomitant esophageal adenocarcinoma (EAC), 10 additional patients with EAC, and 9 patients with SCC to determine the level of L1 activity in these diseases. Following L1-seq, we confirmed 160 somatic insertions by PCR in 17 of 29 individuals. We observed clonal amplification of several insertions which appeared to originate in normal esophagus (NE) or BE and were later clonally expanded in BE, in EAC, or in SCC. Additionally, we observed evidence of clonality within the EAC cases: specifically, 22 of 25 EAC-only insertions were present identically in distinct regions available from the same tumor, suggesting they occurred in the founding tumor cell of these lesions. Our data show that somatic retrotransposition occurs early in many patients with BE and EAC, and indicate that early events occurring in histologically normal esophageal cells may be clonally expanded in esophageal adenocarcinogenesis. Additionally, we evaluated L1 ORF1 protein expression in 9 of the carcinoma cases for which formalin-fixed paraffin embedded tissue was available. Using immunohistochemistry, we detected expression of ORF1p in all tumors evaluated. Interestingly, we also observed dim ORF1p expression in the normal esophagus of all 4 patients for whom additional blocks of normal esophagus containing squamous epithelium was available. To determine if ORF1p expression is a hallmark of unaffected tissues, we obtained both skin and esophageal biopsies from two unaffected individuals. In both biopsies, ORF1p expression was evident in the squamous cell epithelium. ORF1p may be expressed in many normal epithelial tissues which could account for the high incidence of somatic retrotransposition events in epithelial cancers. Thus, our data show that L1 is weakly expressed in normal esophagus and retrotransposition can occur in normal tissue during the development of esophageal adenocarcinoma and squamous cell carcinoma. Due to the pervasive activity of retrotransposons in epithelial cancer, it is likely that somatic insertions may play a role in some tumorigenesis.
Retrotransposons, Cancer