Michael A. McIlhatton

Deficiencies in Mouse Myh and Ogg1 Result in Tumor Predisposition and G to T Mutations in Codon 12 of the K-Ras Oncogene in Lung Tumors

Oxidative DNA damage is unavoidably and continuously generated by oxidant byproducts of normal cellular metabolism. The DNA damage repair genes, mutY and mutM, prevent G to T mutations caused by reactive oxygen species in Escherichia coli, but it has remained debatable whether deficiencies in their mammalian counterparts, Myh and Ogg1, are directly involved in tumorigenesis. Here, we demonstrate that deficiencies in Myh and Ogg1 predispose 65.7% of mice to tumors, predominantly lung and ovarian tumors, and lymphomas. Remarkably, subsequent analyses identified G to T mutations in 75% of the lung tumors at an activating hot spot, codon 12, of the K-ras oncogene, but none in their adjacent normal tissues. Moreover, malignant lung tumors were increased with combined heterozygosity of Msh2, a mismatch repair gene involved in oxidative DNA damage repair as well. Thus, oxidative DNA damage appears to play a causal role in tumorigenesis, and codon 12 of K-ras is likely to be an important downstream target in lung tumorigenesis. The multiple oxidative repair genes are required to prevent mutagenesis and tumor formation. The mice described here provide a valuable model for studying the mechanisms of oxidative DNA damage in tumorigenesis and investigating preventive or therapeutic approaches.

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.

Altered expression of the septin gene, SEPT9, in ovarian neoplasia.

The septin family of genes has been implicated in a variety of cellular processes including cytokinesis, membrane transport and fusion, exocytosis, and apoptosis. One member of the septin family maps to chromosome 17q25.3, a region commonly deleted in sporadic ovarian and breast tumours, and has also been identified as a fusion partner of MLL in acute myeloid leukaemias. The present study demonstrates that the pattern of expression of multiple splice variants of this septin gene is altered in ovarian tumours and cell lines. In particular, expression of the zeta transcript is detectable in the majority of tumours and cell lines, but not in a range of non‐malignant adult and fetal tissues. Zeta expression is accompanied by loss of the ubiquitous beta transcript. Somatic mutations of the gene were not detected in ovarian tumours, but it was demonstrated that beta expression in tumour cell lines can be reactivated by 5‐azacytidine treatment, suggesting a role for methylation in the control of expression of this gene. Copyright

Genomic organization, complex splicing pattern and expression of a human septin gene on chromosome 17q25.3

The Ov/Br septin gene, which is also a fusion partner of MLL in acute myeloid leukaemia, is a member of a family of novel GTP binding proteins that have been implicated in cytokinesis and exocytosis. In this study, we describe the genomic and transcriptional organization of this gene, detailing seventeen exons distributed over 240 kb of sequence. Extensive database analyses identified orthologous rodent cDNAs that corresponded to new, unidentified 5′ splice variants of the Ov/Br septin gene, increasing the total number of such variants to six. We report that splicing events, occurring at non-canonical sites within the body of the 3′ terminal exon, remove either 1801 bp or 1849 bp of non-coding sequence and facilitate access to a secondary open reading frame of 44 amino acids maintained near the end of the 3′ UTR. These events constitute a novel coding arrangement and represent the first report of such a design being implemented by a eukaryotic gene. The various Ov/Br proteins either differ minimally at their amino and carboxy termini or are equivalent to truncated versions of larger isoforms. Northern analysis with an Ov/Br septin 3′ UTR probe reveals three transcripts of 4.4, 4 and 3 kb, the latter being restricted to a sub-set of the tissues tested. Investigation of the identified Ov/Br septin isoforms by RT–PCR confirms a complex transcriptional pattern, with several isoforms showing tissue-specific distribution. To date, none of the other human septins have demonstrated such transcriptional complexity.

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.

Nitric Oxide-Donating Aspirin Derivatives Suppress Microsatellite Instability in Mismatch Repair-Deficient and Hereditary Nonpolyposis Colorectal Cancer Cells

Nitric oxide-donating nonsteroidal anti-inflammatory drugs (NO-NSAIDs) are an emergent class of pharmaceutical derivatives with promising utility as cancer chemopreventive agents. Aspirin and sulindac have been shown to be effective in selecting for cells with reduced microsatellite instability (MSI) that is inherent in mismatch repair (MMR)-deficient hereditary nonpolyposis colorectal cancer (HNPCC) cells. The effect of NO-NSAIDs on MSI in MMR-deficient HNPCC cells is unknown. Here, we have examined genetically defined MMR-deficient murine embryo fibroblasts, murine colonocytes, and isogenic human HNPCC tumor cell lines treated with acetylsalicylic acid (aspirin; ASA) and three isomeric derivatives of NO-aspirin (NO-ASA). The MSI profiles were determined and compared with the Bethesda Criteria. We found that the ASA- and NO-ASA-treated MMR-deficient cell lines displayed a dose-dependent suppression of MSI that appeared as early as 8 weeks and gradually increased to include up to 67% of the microsatellite sequences examined after 19 to 20 weeks of continuous treatment. Residual resistance to microsatellite stabilization was largely confined to mononucleotide repeat sequences. Control (MMR-proficient) cells showed no changes in microsatellite status with or without treatment. The relative dose-dependent stabilization selection was: ortho-NO-ASA approximately para-NO-ASA > meta-NO-ASA >> ASA. Moreover, the doses required for stabilization by the ortho- and para-NO-ASA were 300- to 3,000-fold lower than ASA. These results suggest that NO-ASA derivatives may be more effective at suppressing MSI in MMR-deficient cell lines than ASA and should be considered for chemopreventive trials with HNPCC carriers.

Aspirin and Low Dose Nitric Oxide-Donating Aspirin Increase Life Span in a Lynch Syndrome Mouse Model

Nonsteroidal anti–inflammatory drugs (NSAID) appear to be effective cancer chemopreventives. Previous cellular studies showed that aspirin (acetylsalicylic acid: ASA) and nitric oxide–donating ASA (NO-ASA) suppressed microsatellite instability (MSI) in mismatch repair (MMR)-deficient cells linked to the common cancer predisposition syndrome hereditary nonpolyposis colorectal cancer or Lynch syndrome (LS/HNPCC), at doses 300- to 3,000-fold less than ASA. Using a mouse model that develops MMR-deficient intestinal tumors that appear pathologically identical to LS/HNPCC, we show that ASA (400 mg/kg) and low-dose NO-ASA (72 mg/kg) increased life span by 18% to 21%. We also note a trend where ASA treatment resulted in intestinal tumors with reduced high MSI (H-MSI) and increased low MSI (L-MSI) as defined by the Bethesda Criteria. Low-dose NO-ASA had a minimal effect on MSI status. In contrast to previous studies, high-dose NO-ASA (720/1,500 mg/kg) treatments increased tumor burden, decreased life span, and exacerbated MSI uniquely in the LS/HNPCC mouse model. These results suggest that MMR-deficient tissues/mice may be specifically sensitive to intrinsic pharmacokinetic features of this drug. It is likely that long-term treatment with ASA may represent a chemopreventive option for LS/HNPCC patients. Moreover, as low-dose NO-ASA shows equivalent life span increase at 10-fold lower doses than ASA, it may have the potential to significantly reduce the gastropathy associated with long-term ASA treatment. Cancer Prev Res; 4(5); 684–93. ©2011 AACR.

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.Significance: These findings suggest that colon adenomas driven by APC mutations are distinct from those driven by WNT gain-of-function mutations, with implications for identifying at-risk patients with advanced disease based on gene expression patterns. Cancer Res; 78(3); 617-30. ©2017 AACR.

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.