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Epigenetic Loss of Heterozygosity of Apc and an Inflammation-Associated Mutational Signature Detected in Lrig1+/−-Driven Murine Colonic Adenomas Jessica L

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Epigenetic Loss of Heterozygosity of Apc and an Inflammation-Associated Mutational Signature Detected in Lrig1+/−-Driven Murine Colonic Adenomas Jessica L Preston and Stiffler BMC Cancer (2020) 20:126 https://doi.org/10.1186/s12885-020-6616-y RESEARCH ARTICLE Open Access Epigenetic loss of heterozygosity of Apc and an inflammation-associated mutational signature detected in Lrig1+/−-driven murine colonic adenomas Jessica L. Preston* and Nicholas Stiffler Abstract Background: The loss of a single copy of adenomatous polyposis coli (Apc) in leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1)-expressing colonic progenitor cells induces rapid growth of adenomas in mice with high penetrance and multiplicity. The tumors lack functional APC, and a genetic loss of heterozygosity of Apc was previously observed. Methods: To identify genomic features of early tumorigenesis, and to profile intertumoral genetic heterogeneity, tumor exome DNA (n = 9 tumors) and mRNA (n = 5 tumors) sequences were compared with matched nontumoral colon tissue. Putative somatic mutations were called after stringent variant filtering. Somatic signatures of mutational processes were determined and splicing patterns were observed. Results: The adenomas were found to be genetically heterogeneous and unexpectedly hypermutated, displaying a strong bias toward G:C > A:T mutations. A genetic loss of heterozygosity of Apc was not observed, however, an epigenetic loss of heterozygosity was apparent in the tumor transcriptomes. Complex splicing patterns characterized by a loss of intron retention were observed uniformly across tumors. Conclusion: This study demonstrates that early tumors originating from intestinal stem cells with reduced Lrig1 and Apc expression are highly mutated and genetically heterogeneous, with an inflammation-associated mutational signature and complex splicing patterns that are uniform across tumors. Keywords: Lrig1, Lgr5, Colorectal cancer, Intestinal stem cells, Adenoma, Mutations Background driven, and CMS4 tumors are mesenchymal with VEGF Human colorectal cancer (CRC) is the second leading activation. However, this classification system oversim- cause of cancer death in the US, and ~ 25% of patients plifies the diversity and interrelatedness of cancer cell with CRC are incurable at the time of diagnosis [1, 2]. subtypes. The specific steps required to counteract the Genetic heterogeneity is inherent to this disease, provid- critical aspects of CRC progression remain poorly under- ing tumor cells with the ability to rapidly adapt and re- stood despite decades of research. sist treatments [3, 4]. Currently CRC is clinically The cell-of-origin of CRC derives from a population of segregated into four broad subcategories based on the rapidly-dividing stem cells located at the base of the co- expression of various biomarker molecules called con- lonic epithelial crypts, which are identifiable based on sensus molecular subtypes (CMS1–4) [5]. Generally, the expression of Leucine-rich repeat containing G CMS1 tumors display microsatellite instability and im- protein-coupled receptor 5 (Lgr5). Lgr5 is the down- mune activation, CMS2 tumors are epithelial and display stream target of R-spondin in the canonical Wnt/β-ca- WNT pathway activation, CMS3 tumors are KRAS- tenin pathway. Mutations in the tumor-suppressor gene adenomatous polyposis coli (Apc) and other members of * Correspondence: [email protected] the canonical Wnt pathway are the hallmark of CRC [6– Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Preston and Stiffler BMC Cancer (2020) 20:126 Page 2 of 10 9]. Loss of heterozygosity (LOH) of Apc tends to occur genes, and can therefore sometimes reveal sources of during the early stages of human CRC tumorigenesis. mutation and mechanisms of tumorigenesis. For ex- The standard mouse model used for CRC research, ample, C > A transversion point mutations typically ApcMin/+, contains a truncating point mutation in one occur in low frequencies compared to C > T transition copy of Apc. It is believed that most ApcMin/+ tumors point mutations. However, lung and esophageal tumors have lost Apc function through a spontaneous genetic caused by tobacco tar often contain an abundance of LOH [10]. ApcMin/+ mice exhibit tumor formation pre- C > A transversions due to the conjugation of nitrosa- dominately in the small intestine rather than in the distal mines to glutathione, which forms guanine adducts [29]. colon as observed in humans. Interestingly, Tanaka, T., Stomach cancers caused by H. pylori infection also con- et al. 2006 reported that dextran sodium sulfate (DSS)- tain a high incidence of C > A transversions, presumably induced inflammation increased the incidence of polyps due to the inflammation-associated reactive oxygen and in the distal colon of ApcMin/+ mice [11]. Similarly, Yang, nitrogen species (ROS and RNS) [30]. K., et al. 2008 found that reduced mucus production in This work sought to understand the genomic ApcMin/+ mice shifted tumor development toward the changes occurring during the early stages of tumori- distal colon. In 2009 Ritchie, K., et al. crossed a glutathi- genesis in rapidly-growing colonic adenomas in Lrig1- one S-transferase Pi (Gstp) null allele into the standard CreERT2/+;Apcfl/+ mice. Exomic DNA and mRNA ApcMin/+ mouse and reported a 6-fold increase in distal sequences from the adenomas were analyzed in order colorectal adenoma incidence and a 50-fold increase in to detect the presence of transcriptomic and genomic adenoma multiplicity compared to ApcMin mice [12]. alterations. Specifically, the genetic heterogeneity The authors also noted that the colons of the (Gstp) null across tumors and somatic signatures of mouse co- ApcMin/+ mice expressed higher levels of inflammatory lonic adenomas tumors were assessed using exome molecules interleukin 4 (IL4), interleukin 6 (IL6), and ni- DNA profiling, and the prevalence of differential gene tric oxide synthase. Taken together, these results indi- expression and splicing defects was assessed using cate an important role for mucus in reducing mRNA-Seq. inflammation-associated tumors in the distal colon [13]. Leucine-rich repeats and immunoglobulin-like do- mains 1 (Lrig1) is a transmembrane feedback regulator Materials and methods of growth factor receptor tyrosine kinases that is Generation of Lrig1-driven colonic adenomas expressed in the Lgr5+ stem cell population present at Generation of Lrig1-CreERT2/+;Apcfl/+ tumors was pre- the base of colonic crypts [14–17]. Lrig1 acts as a viously described [25]. Briefly, Lrig1-CreERT2/+ mice tumor-suppressor gene in several contexts [18–23]. The were crossed to Apc580S/+ mice (Jackson Laboratory, Lrig1-CreERT2/+;Apcfl/+ inducible mouse model of co- Bar Harbor, ME, U.S.A) [31] to generate Lrig1-CreERT2/ lonic adenoma is based on the conditional Cre- +;Apcfl/+ mice [24]. Adult (6- to 8-week-old) Lrig1- recombinase-driven loss of a single copy of Apc under CreERT2/+;Apcfl/+ mice were intraperitoneally injected the control of the Lrig1 promoter [24, 25]. The colonic with 2 mg tamoxifen per mouse (Sigma-Aldrich, St. stem cells of these mice express one single wild type Louis, MO, U.S.A.) in corn oil for 3 consecutive days copy each of the Lrig1 and Apc genes after the engi- and multiple dysplastic colonic adenomas were extracted − neered recombination of Apc in Lrig1+/ -expressing stem 100 days later. For the DNA studies, the control is non- cells. Within 100 days of the loss of one copy of Apc, tumor tissue parts of the same Lrig1-CreERT2/+;Apcfl/+ rapidly growing adenomas appear in the distal colon mouse, referred to as ‘nontumor’ in figures. For the with extremely high tumor penetrance and multiplicity. RNA studies, the control is untreated wild type C57BL/ The Lrig1-CreERT2/+;Apcfl/+ CRC model is very similar 6 J mice, referred to as ‘wild type’ in figures. to the Gstp-null;ApcMin CRC model in terms of tumor onset, penetrance, multiplicity, anatomical location, and mortality. These findings imply that a common mechan- Exomic DNA sequencing ism involving inflammation-induced tumor formation is Exomic DNA sequencing (n = 9 tumors) was performed responsible for tumorigenesis in the distal colon. by HudsonAlpha Labs (Huntsville, AL, U.S.A.) Exome Human tumors often exhibit distinct patterns of muta- capture using NimbleGen v3.0 captured 64 megabase- tions that can provide clues into the origin and mechan- pair (Mbp) baits. A total of six gigabasepairs (Gbp) of ism of tumorigenesis. The ‘somatic signature of exome data were sequenced per sample. Exomic tumor mutations’ of a tumor is based on the specific nucleotide DNA of nine tumors from three mice was sequenced in alterations present and the background sequence context parallel with adjacent normal colon (n = 3) to 12x aver- of the mutations [26–28]. Somatic signatures are influ- age read depth. Adjacent normal colon tissue was used enced by specific carcinogenic agents and DNA repair for the ‘nonutmor’ control. Preston and Stiffler BMC Cancer (2020) 20:126 Page 3 of 10 DNA read alignment
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