Manipulation of DNA Repair Proficiency in Mouse Models of Colorectal Cancer

Technical and biological innovations have enabled the development of more sophisticated and focused murine models that increasingly recapitulate the complex pathologies of human diseases, in particular cancer. Mouse models provide excellent in vivo systems for deciphering the intricacies of cancer biology within the context of precise experimental settings. They present biologically relevant, adaptable platforms that are amenable to continual improvement and refinement. We discuss how recent advances in our understanding of tumorigenesis and the underlying deficiencies of DNA repair mechanisms that drive it have been informed by using genetically engineered mice to create defined, well-characterized models of human colorectal cancer. In particular, we focus on how mechanisms of DNA repair can be manipulated precisely to create in vivo models whereby the underlying processes of tumorigenesis are accelerated or attenuated, dependent on the composite alleles carried by the mouse model. Such models have evolved to the stage where they now reflect the initiation and progression of sporadic cancers. The review is focused on mouse models of colorectal cancer and how insights from these models have been instrumental in shaping our understanding of the processes and potential therapies for this disease

Regulation of BLM Nucleolar Localization

Defects in coordinated ribosomal RNA (rRNA) transcription in the nucleolus cause cellular and organismal growth deficiencies. Bloom’s syndrome, an autosomal recessive human disorder caused by mutated recQ-like helicase BLM, presents with growth defects suggestive of underlying defects in rRNA transcription. Our previous studies showed that BLM facilitates rRNA transcription and interacts with RNA polymerase I and topoisomerase I (TOP1) in the nucleolus. The mechanisms regulating localization of BLM to the nucleolus are unknown. In this study, we identify the TOP1-interaction region of BLM by co-immunoprecipitation of in vitro transcribed and translated BLM segments and show that this region includes the highly conserved nuclear localization sequence (NLS) of BLM. Biochemical and nucleolar co-localization studies using site-specific mutants show that two serines within the NLS (S1342 and S1345) are critical for nucleolar localization of BLM but do not affect the functional interaction of BLM with TOP1. Mutagenesis of both serines to aspartic acid (phospho-mimetic), but not alanine (phospho-dead), results in approximately 80% reduction in nucleolar localization of BLM while retaining the biochemical functions and nuclear localization of BLM. Our studies suggest a role for this region in regulating nucleolar localization of BLM via modification of the two serines within the NLS

Mutational Mechanisms That Activate Wnt Signaling and Predict Outcomes in Colorectal Cancer Patients

APC biallelic loss-of-function mutations are the most prevalent genetic changes in colorectal tumors, but it is unknown whether these mutations phenocopy gain-of-function mutations in the CTNNB1 gene encoding β-catenin that also activate canonical WNT signaling. Here we demonstrate that these two mutational mechanisms are not equivalent. Furthermore, we show how differences in gene expression produced by these different mechanisms can stratify outcomes in more advanced human colorectal cancers. Gene expression profiling in Apc-mutant and Ctnnb1-mutant mouse colon adenomas identified candidate genes for subsequent evaluation of human TCGA (The Cancer Genome Atlas) data for colorectal cancer outcomes. Transcriptional patterns exhibited evidence of activated canonical Wnt signaling in both types of adenomas, with Apc-mutant adenomas also exhibiting unique changes in pathways related to proliferation, cytoskeletal organization, and apoptosis. Apc-mutant adenomas were characterized by increased expression of the glial nexin Serpine2, the human ortholog, which was increased in advanced human colorectal tumors. Our results support the hypothesis that APC-mutant colorectal tumors are transcriptionally distinct from APC-wild-type colorectal tumors with canonical WNT signaling activated by other mechanisms, with possible implications for stratification and prognosis

hMSH2 Recruits ATR to DNA Damage Sites for Activation during DNA Damage-induced Apoptosis*

DNA damage response (DDR) activates a complex signaling network that triggers DNA repair, cell cycle arrest, and/or cell death. Depending on the type and severity of DNA lesion, DDR is controlled by “master” regulators including ATM and ATR protein kinases. Cisplatin, a major chemotherapy drug that cross-links DNA, induces ATR-dependent DDR, resulting in apoptosis. However, it is unclear how ATR is activated. To identify the key regulators of ATR, we analyzed the proteins that associate with ATR after cisplatin treatment by blue native-PAGE and co-immunoprecipitation. The mismatch repair protein hMSH2 was found to be a major ATR-binding protein. Functionally, ATR activation and its recruitment to nuclear foci during cisplatin treatment were attenuated, and DNA damage signaling, involving Chk2, p53, and PUMA-α, was suppressed in hMSH2-deficient cells. ATR activation induced by the DNA methylating agent N-methyl-N-nitrosourea was also shown to be hMSH2-dependent. Intriguingly, hMSH2-mediated ATR recruitment and activation appeared independent of replication protein A, Rad17, and the Rad9-Hus1-Rad1 protein complex. Together the results support a hMSH2-dependent pathway of ATR activation and downstream Chk2/p53 signaling

Modulation of mismatch repair and genomic stability by miR-155

Inactivation of mismatch repair (MMR) is the cause of the common cancer predisposition disorder Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer (HNPCC), as well as 10–40% of sporadic colorectal, endometrial, ovarian, gastric, and urothelial cancers. Elevated mutation rates (mutator phenotype), including simple repeat instability [microsatellite instability (MSI)] are a signature of MMR defects. MicroRNAs (miRs) have been implicated in the control of critical cellular pathways involved in development and cancer. Here we show that overexpression of miR-155 significantly down-regulates the core MMR proteins, hMSH2, hMSH6, and hMLH1, inducing a mutator phenotype and MSI. An inverse correlation between the expression of miR-155 and the expression of MLH1 or MSH2 proteins was found in human colorectal cancer. Finally, a number of MSI tumors with unknown cause of MMR inactivation displayed miR-155 overexpression. These data provide support for miR-155 modulation of MMR as a mechanism of cancer pathogenesis