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Tumorigenicity studies often employ outbred nude mice, in the absence of direct evidence that this mixed genetic background will negatively affect experimental outcome. Here we show that outbred nude mice carry two different alleles of Pla2g2a, a genetic modifier of intestinal tumorigenesis in mice. Here, we identify previous unreported linked polymorphisms in the promoter, noncoding and coding sequences of Pla2g2a and show that outbred nude mice from different commercial providers are heterogeneous for this polymorphic Pla2g2a allele. This heterogeneity even extends to mice obtained from a single commercial provider, which display mixed Pla2g2a genotypes. Notably, we demonstrated that the polymorphic Pla2g2a allele affects orthotopic xenograft establishment of human colon cancer cells in outbred nude mice. This finding establishes a non-cell-autonomous role for Pla2g2a in suppressing intestinal tumorigenesis. Using in vitro reporter assays and pharmacological inhibitors, we show promoter polymorphisms and nonsense-mediated RNA decay (NMD) as underlying mechanisms that lead to low Pla2g2a mRNA levels in tumor-sensitive mice. Together, this study provides mechanistic insight regarding Pla2g2a polymorphisms and demonstrates a non-cell-autonomous role for Pla2g2a in suppressing tumors. Moreover, our direct demonstration that mixed genetic backgrounds of outbred nude mice can significantly affect baseline tumorigenicity cautions against future use of outbred mice for tumor xenograft studies.

Mutations and structural alterations of the SWI/SNF-like chromatin remodeler ATRX (Alpha Thalassemia/Mental Retardation, X-linked) have been reported at high frequency in a number of adult and pediatric tumors. However, the consequences of ATRX mutations in cancer and their impact on the epigenome remain ill defined. Particularly intriguing are the large N-terminal deletions of ATRX in neuroblastoma that generate in-frame fusion (IFF) proteins devoid of key chromatin interaction domains, while retaining the SWI/SNF-like helicase domain. We hereby demonstrate that ATRX IFF proteins have distinct genomic distribution compared to wild type (WT) ATRX and are absent from H3K9me3-enriched chromatin, yet bound to active promoters. We find REST (RE-1 Silencing Transcription Factor) as one such ATRX IFF target that is activated and promotes silencing of neuronal differentiation genes. Notably, we uncover that ATRX IFF cells display exquisite sensitivity to EZH2 inhibitors (EZH2i) in vitro and in vivo, due in part to derepression of neurogenesis genes, including a subset of REST targets. Examination of the epigenomic landscape of neuroblastoma tumor specimens harboring ATRX IFFs reveals that H3K27me3 occupies a subset of neurogenesis genes that are transcriptionally silenced and sensitive to EZH2i in our cell-based assays. In summary, this study demonstrates that ATRX structural alterations are not loss-of-function as predicted, and supports EZH2i as a key therapeutic strategy for ATRX IFF neuroblastoma.

Adenomatous polyposis coli (APC) is best known for its crucial role in colorectal cancer suppression. Rodent models with various Apc mutations have enabled experimental validation of different Apc functions in tumors and normal tissues. Since the development of the first mouse model with a germline Apc mutation in the early 1990s, 20 other Apc mouse and rat models have been generated. This article compares and contrasts currently available Apc rodent models with particular emphasis on providing potential explanations for their reported variation in three areas: (i) intestinal polyp multiplicity, (ii) intestinal polyp distribution, and (iii) extraintestinal phenotypes. Cancer Res; 73(8); 2389–99. ©2013 AACR.

Mutation of tumor suppressor adenomatous polyposis coli (APC) initiates most colorectal cancers and chronic colitis increases risk. APC is a nucleo-cytoplasmic shuttling protein, best known for antagonizing Wnt signaling by forming a cytoplasmic complex that marks β-catenin for degradation. Using our unique mouse model with compromised nuclear Apc import (Apc(mNLS)), we show that Apc(mNLS/mNLS) mice have increased susceptibility to tumorigenesis induced with azoxymethane (AOM) and dextran sodium sulfate (DSS). The AOM-DSS-induced colon adenoma histopathology, proliferation, apoptosis, stem cell number and β-catenin and Kras mutation spectra were similar in Apc(mNLS/mNLS) and Apc(+/+) mice. However, AOM-DSS-treated Apc(mNLS/mNLS) mice showed more weight loss, more lymphoid follicles and edema, and increased colon shortening than treated Apc(+/+) mice, indicating a colitis predisposition. To test this directly, we induced acute colitis with a 7 day DSS treatment followed by 5 days of recovery. Compared with Apc(+/+) mice, DSS-treated Apc(mNLS/mNLS) mice developed more severe colitis based on clinical grade and histopathology. Apc(mNLS/mNLS) mice also had higher lymphocytic infiltration and reduced expression of stem cell markers, suggesting an increased propensity for chronic inflammation. Moreover, colons from DSS-treated Apc(mNLS/mNLS) mice showed fewer goblet cells and reduced Muc2 expression. Even in untreated Apc(mNLS/mNLS) mice, there were significantly fewer goblet cells in jejuna, and a modest decrease in colonocyte Muc2 expression compared with Apc(+/+) mice. Colonocytes from untreated Apc(mNLS/mNLS) mice also showed increased expression of inflammatory mediators cyclooxygenase-2 (Cox-2) and macrophage inflammatory protein-2 (MIP-2). These findings reveal novel functions for nuclear Apc in goblet cell differentiation and protection against inflammation-induced colon tumorigenesis.