Robert T. Cormier

A transposon-based genetic screen in mice identifies genes altered in colorectal cancer

Human colorectal cancers (CRCs) display a large number of genetic and epigenetic alterations, some of which are causally involved in tumorigenesis (drivers) and others that have little functional impact (passengers). To help distinguish between these two classes of alterations, we used a transposon-based genetic screen in mice to identify candidate genes for CRC. Mice harboring mutagenic Sleeping Beauty (SB) transposons were crossed with mice expressing SB transposase in gastrointestinal tract epithelium. Most of the offspring developed intestinal lesions, including intraepithelial neoplasia, adenomas, and adenocarcinomas. Analysis of over 16,000 transposon insertions identified 77 candidate CRC genes, 60 of which are mutated and/or dysregulated in human CRC and thus are most likely to drive tumorigenesis. These genes include APC, PTEN, and SMAD4. The screen also identified 17 candidate genes that had not previously been implicated in CRC, including POLI, PTPRK, and RSPO2.

Interaction of Muc2 and Apc on Wnt Signaling and in Intestinal Tumorigenesis: Potential Role of Chronic Inflammation

Somatic mutations of the adenomatous polyposis coli (APC) gene are initiating events in the majority of sporadic colon cancers. A common characteristic of such tumors is reduction in the number of goblet cells that produce the mucin MUC2, the principal component of intestinal mucus. Consistent with these observations, we showed that Muc2 deficiency results in the spontaneous development of tumors along the entire gastrointestinal tract, independently of deregulated Wnt signaling. To dissect the complex interaction between Muc2 and Apc in intestinal tumorigenesis and to elucidate the mechanisms of tumor formation in Muc2(-/-) mice, we crossed the Muc2(-/-) mouse with two mouse models, Apc(1638N/+) and Apc(Min/+), each of which carries an inactivated Apc allele. The introduction of mutant Muc2 into Apc(1638N/+) and Apc(Min/+) mice greatly increased transformation induced by the Apc mutation and significantly shifted tumor development toward the colon as a function of Muc2 gene dosage. Furthermore, we showed that in compound double mutant mice, deregulation of Wnt signaling was the dominant mechanism of tumor formation. The increased tumor burden in the distal colon of Muc2/Apc double mutant mice was similar to the phenotype observed in Apc(Min/+) mice that are challenged to mount an inflammatory response, and consistent with this, gene expression profiles of epithelial cells from flat mucosa of Muc2-deficient mice suggested that Muc2 deficiency was associated with low levels of subclinical chronic inflammation. We hypothesize that Muc2(-/-) tumors develop through an inflammation-related pathway that is distinct from and can complement mechanisms of tumorigenesis in Apc(+/-) mice.

Intestinal‐specific PPARγ deficiency enhances tumorigenesis in ApcMin/+ mice

Multiple investigations of the effects of peroxisome proliferator‐activated receptor γ (PPARγ) ligands on colon cancer have produced contradictory results. While some studies demonstrated increased numbers of colonic polyps in ApcMin/+ mice treated with various thiazolidinedione (TZD) PPARγ ligands, others reported amelioration of tumor multiplicity and progression in both ApcMin/+ mice and in mice with chemically‐induced colon cancer. Here, we addressed the role of PPARγ in murine intestinal tumorigenesis using gene knockout methodology. We found that either heterozygous or homozygous intestinal‐specific PPARγ deficiency enhanced the number of ApcMin/+ tumors in both the small intestine and colon, especially in the colon, where PPARγ deficiency also modulated tumor incidence. Gender significantly affected tumor multiplicity independent of PPARγ genotype. Female ApcMin/+ mice developed more tumors in the small intestine and more tumors overall, whereas male ApcMin/+ mice developed more tumors in the colon. Nevertheless, intestinal PPARγ deficiency enhanced tumorigenesis irrespective of gender. Our results suggest that PPARγ functions as a tumor resistance factor in the mouse intestine and warrant further investigation of the PPARγ‐dependent and independent actions of TZDs in cancer.

A Sleeping Beauty transposon-mediated screen identifies murine susceptibility genes for adenomatous polyposis coli (Apc)-dependent intestinal tumorigenesis

It is proposed that a progressive series of mutations and epigenetic events leads to human colorectal cancer (CRC) and metastasis. Furthermore, data from resequencing of the coding regions of human CRC suggests that a relatively large number of mutations occur in individual human CRC, most at low frequency. The functional role of these low-frequency mutations in CRC, and specifically how they may cooperate with high-frequency mutations, is not well understood. One of the most common rate-limiting mutations in human CRC occurs in the adenomatous polyposis coli (APC) gene. To identify mutations that cooperate with mutant APC, we performed a forward genetic screen in mice carrying a mutant allele of Apc (ApcMin) using Sleeping Beauty (SB) transposon-mediated mutagenesis. ApcMin SB-mutagenized mice developed three times as many polyps as mice with the ApcMin allele alone. Analysis of transposon common insertion sites (CIS) identified the Apc locus as a major target of SB-induced mutagenesis, suggesting that SB insertions provide an efficient route to biallelic Apc inactivation. We also identified an additional 32 CIS genes/loci that may represent modifiers of the ApcMin phenotype. Five CIS genes tested for their role in proliferation caused a significant change in cell viability when message levels were reduced in human CRC cells. These findings demonstrate the utility of using transposon mutagenesis to identify low-frequency and cooperating cancer genes; this approach will aid in the development of combinatorial therapies targeting this deadly disease.

The roles of sPLA2-IIA (Pla2g2a) in cancer of the small and large intestine.

The mouse secretory phospholipase A2 group IIA (sPLA2-IIA) gene Pla2g2a has been identified as a susceptibility gene for cancer of the small and large intestine. Interestingly, unlike most previously identified tumor susceptibility genes, Pla2g2a does not behave like a classical oncogene or tumor suppressor gene. Hence, identification of its biological functions in tumor development may shed new light on general mechanisms that modulate colon cancer risk. So far, sPLA2-IIA has been proposed to play a role in anti-bacterial defense, inflammation and eicosanoid generation, in clearance of apoptotic cells, and in the Wnt signaling pathway. More recently, comparison of RNA expression profiles of colon from Pla2g2a-transgenic to Pla2g2a-deficient mice confirmed and even extended sPLA2-IIAs diverse biological effects. In this review we aim to summarize current knowledge about the various links of sPLA2-IIA to cancer of the gastro-intestinal tract, and propose several models to illustrate its putative biological effects on tumor development.

The role of KCNQ1 in mouse and human gastrointestinal cancers.

Kcnq1, which encodes for the pore-forming α-subunit of a voltage-gated potassium channel, was identified as a gastrointestinal (GI) tract cancer susceptibility gene in multiple Sleeping Beauty DNA transposon-based forward genetic screens in mice. To confirm that Kcnq1 has a functional role in GI tract cancer, we created ApcMin mice that carried a targeted deletion mutation in Kcnq1. Results demonstrated that Kcnq1 is a tumor suppressor gene as Kcnq1 mutant mice developed significantly more intestinal tumors, especially in the proximal small intestine and colon, and some of these tumors progressed to become aggressive adenocarcinomas. Gross tissue abnormalities were also observed in the rectum, pancreas and stomach. Colon organoid formation was significantly increased in organoids created from Kcnq1 mutant mice compared with wild-type littermate controls, suggesting a role for Kcnq1 in the regulation of the intestinal crypt stem cell compartment. To identify gene expression changes due to loss of Kcnq1, we carried out microarray studies in the colon and proximal small intestine. We identified altered genes involved in innate immune responses, goblet and Paneth cell function, ion channels, intestinal stem cells, epidermal growth factor receptor and other growth regulatory signaling pathways. We also found genes implicated in inflammation and in cellular detoxification. Pathway analysis using Ingenuity Pathway Analysis and Gene Set Enrichment Analysis confirmed the importance of these gene clusters and further identified significant overlap with genes regulated by MUC2 and CFTR, two important regulators of intestinal homeostasis. To investigate the role of KCNQ1 in human colorectal cancer (CRC), we measured protein levels of KCNQ1 by immunohistochemistry in tissue microarrays containing samples from CRC patients with liver metastases who had undergone hepatic resection. Results showed that low expression of KCNQ1 expression was significantly associated with poor overall survival.

CFTR is a tumor suppressor gene in murine and human intestinal cancer

CFTR, the cystic fibrosis (CF) gene, encodes for the CFTR protein that plays an essential role in anion regulation and tissue homeostasis of various epithelia. In the gastrointestinal (GI) tract CFTR promotes chloride and bicarbonate secretion, playing an essential role in ion and acid–base homeostasis. Cftr has been identified as a candidate driver gene for colorectal cancer (CRC) in several Sleeping Beauty DNA transposon-based forward genetic screens in mice. Further, recent epidemiological and clinical studies indicate that CF patients are at high risk for developing tumors in the colon. To investigate the effects of CFTR dysregulation on GI cancer, we generated ApcMin mice that carried an intestinal-specific knockout of Cftr. Our results indicate that Cftr is a tumor suppressor gene in the intestinal tract as Cftr mutant mice developed significantly more tumors in the colon and the entire small intestine. In Apc+/+ mice aged to ~1 year, Cftr deficiency alone caused the development of intestinal tumors in >60% of mice. Colon organoid formation was significantly increased in organoids created from Cftr mutant mice compared with wild-type controls, suggesting a potential role of Cftr in regulating the intestinal stem cell compartment. Microarray data from the Cftr-deficient colon and the small intestine identified dysregulated genes that belong to groups of immune response, ion channel, intestinal stem cell and other growth signaling regulators. These associated clusters of genes were confirmed by pathway analysis using Ingenuity Pathway Analysis and gene set enrichment analysis (GSEA). We also conducted RNA Seq analysis of tumors from Apc+/+ Cftr knockout mice and identified sets of genes dysregulated in tumors including altered Wnt β-catenin target genes. Finally we analyzed expression of CFTR in early stage human CRC patients stratified by risk of recurrence and found that loss of expression of CFTR was significantly associated with poor disease-free survival.CFTR, the cystic fibrosis (CF) gene, encodes for the CFTR protein that plays an essential role in anion regulation and tissue homeostasis of various epithelia. In the gastrointestinal (GI) tract CFTR promotes chloride and bicarbonate secretion, playing an essential role in ion and acid-base homeostasis. Cftr has been identified as a candidate driver gene for colorectal cancer (CRC) in several Sleeping Beauty DNA transposon-based forward genetic screens in mice. Further, recent epidemiological and clinical studies indicate that CF patients are at high risk for developing tumors in the colon. To investigate the effects of CFTR dysregulation on GI cancer, we generated Apc(Min) mice that carried an intestinal-specific knockout of Cftr. Our results indicate that Cftr is a tumor suppressor gene in the intestinal tract as Cftr mutant mice developed significantly more tumors in the colon and the entire small intestine. In Apc(+/+) mice aged to ~1 year, Cftr deficiency alone caused the development of intestinal tumors in >60% of mice. Colon organoid formation was significantly increased in organoids created from Cftr mutant mice compared with wild-type controls, suggesting a potential role of Cftr in regulating the intestinal stem cell compartment. Microarray data from the Cftr-deficient colon and the small intestine identified dysregulated genes that belong to groups of immune response, ion channel, intestinal stem cell and other growth signaling regulators. These associated clusters of genes were confirmed by pathway analysis using Ingenuity Pathway Analysis and gene set enrichment analysis (GSEA). We also conducted RNA Seq analysis of tumors from Apc(+/+) Cftr knockout mice and identified sets of genes dysregulated in tumors including altered Wnt β-catenin target genes. Finally we analyzed expression of CFTR in early stage human CRC patients stratified by risk of recurrence and found that loss of expression of CFTR was significantly associated with poor disease-free survival.

A Dominantly Acting Murine Allele of Mcm4 Causes Chromosomal Abnormalities and Promotes Tumorigenesis

Here we report the isolation of a murine model for heritable T cell lymphoblastic leukemia/lymphoma (T-ALL) called Spontaneous dominant leukemia (Sdl). Sdl heterozygous mice develop disease with a short latency and high penetrance, while mice homozygous for the mutation die early during embryonic development. Sdl mice exhibit an increase in the frequency of micronucleated reticulocytes, and T-ALLs from Sdl mice harbor small amplifications and deletions, including activating deletions at the Notch1 locus. Using exome sequencing it was determined that Sdl mice harbor a spontaneously acquired mutation in Mcm4 (Mcm4D573H). MCM4 is part of the heterohexameric complex of MCM2–7 that is important for licensing of DNA origins prior to S phase and also serves as the core of the replicative helicase that unwinds DNA at replication forks. Previous studies in murine models have discovered that genetic reductions of MCM complex levels promote tumor formation by causing genomic instability. However, Sdl mice possess normal levels of Mcms, and there is no evidence for loss-of-heterozygosity at the Mcm4 locus in Sdl leukemias. Studies in Saccharomyces cerevisiae indicate that the Sdl mutation produces a biologically inactive helicase. Together, these data support a model in which chromosomal abnormalities in Sdl mice result from the ability of MCM4D573H to incorporate into MCM complexes and render them inactive. Our studies indicate that dominantly acting alleles of MCMs can be compatible with viability but have dramatic oncogenic consequences by causing chromosomal abnormalities.

Runx1 is a tumor suppressor gene in the mouse gastrointestinal tract

The Runx1 transcription factor plays an important role in tissue homeostasis through its effects on stem/progenitor cell populations and differentiation. The effect of Runx1 on epithelial differentiation of the secretory cell lineage of the colon was recently demonstrated. This study aimed to examine the role of Runx1 in tumor development in epithelial cells of the gastrointestinal tract. Conditional knockout mice that lacked Runx1 expression in epithelial cells of the GI tract were generated. These mice were crossed onto the ApcMin background, killed and their intestinal tumor phenotypes were compared with ApcMinRunx1 wild‐type control mice. Apc‐wild‐type Runx1‐mutant mice were also examined for tumor development. Colons from Runx1 knockout and wild‐type mice were used for genome‐wide mRNA expression analyses followed by gene‐specific quantitative RT‐PCR of whole colon and colon epithelium to identify Runx1 target genes. Runx1 deficiency in intestinal epithelial cells significantly enhanced tumorigenesis in ApcMin mice. Notably, epithelial Runx1 deficiency in Apc‐wild‐type mice was sufficient to cause tumor development. Absence of Runx1 was associated with global changes in the expression of genes involved in inflammation and intestinal metabolism, and with gene sets indicative of a metastatic phenotype and poor prognosis. Gene‐specific analysis of Runx1‐deficient colon epithelium revealed increased expression of genes linked to an expansion of the stem/progenitor cell population. These results identify Runx1 as a novel tumor suppressor gene for gastrointestinal tumors and support a role for Runx1 in maintaining the balance between the intestinal stem/progenitor cell population and epithelial differentiation of the GI tract. (Cancer Sci 2012; 103: 593–599)

Expression of Pla2g2a prevents carcinogenesis in Muc2-deficient mice

Goblet cell depletion and down‐regulation of MUC2 expression are observed in a significant percentage of human non‐mucinous colorectal adenocarcinomas. Direct evidence for the role of MUC2 in gastrointestinal tumor formation was demonstrated by a knockout of Muc2 in mice that resulted in the development of adenocarcinomas in the small and large intestine. The secretory phospholipase Pla2g2a is a protein that confers resistance to ApcMin/+‐induced intestinal tumorigenesis. Like Muc2, in the large intestine Pla2g2a is exclusively expressed by the goblet cells and Pla2g2as tumor resistance is also strongest in the large intestine. Possible genetic interactions between Muc2 and Pla2g2a were examined by creating C57BL/6‐Muc2−/–Pla2g2a transgenic mice. Expression of a Pla2g2a transgene reduced tumorigenesis in the large intestine by 90% in male Muc2−/– mice and by nearly 100% in female Muc2−/– mice. Expression of Pla2g2a also inhibited tumor progression. Microarray gene expression studies revealed Pla2g2a target genes that modulate intestinal energy metabolism, differentiation, inflammation, immune responses and proliferation. Overall, results of the present study demonstrate an Apc‐independent role for Pla2g2a in tumor resistance and indicate that Pla2g2a plays an important role, along with Muc2, in protection of the intestinal mucosa. (Cancer Sci 2008; 99: 2113–2119)