Antoaneta Belcheva

Gut Microbial Metabolism Drives Transformation of Msh2-Deficient Colon Epithelial Cells

The etiology of colorectal cancer (CRC) has been linked to deficiencies in mismatch repair and adenomatous polyposis coli (APC) proteins, diet, inflammatory processes, and gut microbiota. However, the mechanism through which the microbiota synergizes with these etiologic factors to promote CRC is not clear. We report that altering the microbiota composition reduces CRC in APC(Min/+)MSH2(-/-) mice, and that a diet reduced in carbohydrates phenocopies this effect. Gut microbes did not induce CRC in these mice through an inflammatory response or the production of DNA mutagens but rather by providing carbohydrate-derived metabolites such as butyrate that fuel hyperproliferation of MSH2(-/-) colon epithelial cells. Further, we provide evidence that the mismatch repair pathway has a role in regulating β-catenin activity and modulating the differentiation of transit-amplifying cells in the colon. These data thereby provide an explanation for the interaction between microbiota, diet, and mismatch repair deficiency in CRC induction. PAPERCLIP:

The Multifaceted Role of the Intestinal Microbiota in Colon Cancer

In recent years, our understanding of the mechanisms underlying colorectal carcinogenesis has vastly expanded. Underlying inflammation within the intestine, diet, and most recently, the gut microbiota, have been demonstrated to influence the development of colorectal cancer. However, since cancer is ultimately a genetic disease, these factors are thought to create genotoxic stress within the intestinal environment to promote genetic and epigenetic alterations leading to cancer. In this review, we will focus on how gut microbes intersect with inflammation, diet, and host genetics to influence the development of colon cancer.

The mitochondrial protein NLRX1 controls the balance between extrinsic and intrinsic apoptosis.

Background: NLRX1 is a member of the Nod-like receptor family that localizes to the mitochondrial matrix and whose function remains poorly understood. Results: NLRX1 plays a key role in regulating apoptotic cell death and is important in the control of tumorigenesis. Conclusion: NLRX1 regulates the balance between extrinsic and intrinsic apoptosis in cancer cells. Significance: Targeting NLRX1 function could be an interesting strategy against cancer. NLRX1 is a mitochondrial Nod-like receptor (NLR) protein whose function remains enigmatic. Here, we observed that NLRX1 expression was glucose-regulated and blunted by SV40 transformation. In transformed but not primary murine embryonic fibroblasts, NLRX1 expression mediated resistance to an extrinsic apoptotic signal, whereas conferring susceptibility to intrinsic apoptotic signals, such as glycolysis inhibition, increased cytosolic calcium and endoplasmic reticulum stress. In a murine model of colorectal cancer induced by azoxymethane, NLRX1−/− mice developed fewer tumors than wild type mice. In contrast, in a colitis-associated cancer model combining azoxymethane and dextran sulfate sodium, NLRX1−/− mice developed a more severe pathology likely due to the increased sensitivity to dextran sulfate sodium colitis. Together, these results identify NLRX1 as a critical mitochondrial protein implicated in the regulation of apoptosis in cancer cells. The unique capacity of NLRX1 to regulate the cellular sensitivity toward intrinsic versus extrinsic apoptotic signals suggests a critical role for this protein in numerous physiological processes and pathological conditions.

Negative Supercoiling Creates Single-Stranded Patches of DNA That Are Substrates for AID–Mediated Mutagenesis

Antibody diversification necessitates targeted mutation of regions within the immunoglobulin locus by activation-induced cytidine deaminase (AID). While AID is known to act on single-stranded DNA (ssDNA), the source, structure, and distribution of these substrates in vivo remain unclear. Using the technique of in situ bisulfite treatment, we characterized these substrates—which we found to be unique to actively transcribed genes—as short ssDNA regions, that are equally distributed on both DNA strands. We found that the frequencies of these ssDNA patches act as accurate predictors of AID activity at reporter genes in hypermutating and class switching B cells as well as in Escherichia coli. Importantly, these ssDNA patches rely on transcription, and we report that transcription-induced negative supercoiling enhances both ssDNA tract formation and AID mutagenesis. In addition, RNaseH1 expression does not impact the formation of these ssDNA tracts indicating that these structures are distinct from R-loops. These data emphasize the notion that these transcription-generated ssDNA tracts are one of many in vivo substrates for AID.

Gut microbial metabolism and colon cancer: Can manipulations of the microbiota be useful in the management of gastrointestinal health?

The gut microbiota is an important component of the human body and its immune-modulating and metabolic activities are critical to maintain good health. Gut microbes, however, are sensitive to changes in diet, exposure to antibiotics, or infections, all of which cause transient disruptions in the microbial composition, a phenomenon known as dysbiosis. It is now recognized that microbial dysbiosis is at the root of many gastrointestinal disorders. However, the mechanisms through which bacterial dysbiosis initiates disease are not fully understood. Microbially-derived metabolites and their role in disease have also attracted significant attention. Identification of cancer-associated bacteria and understanding the contributions of microbial metabolism in health and disease are exciting but challenging areas that will allow defining microbial biomarkers for predicting gastrointestinal disorders. Understanding the complex interactions between gut microbiota, diet, host immune system and host genetics will be critical to developing more personalized therapies and approaches to treat disease.

The mismatch repair pathway functions normally at a non-AID target in germinal center B cells.

Deficiency in Msh2, a component of the mismatch repair (MMR) system, leads to an approximately 10-fold increase in the mutation frequency in most tissues. By contrast, Msh2 deficiency in germinal center (GC) B cells decreases the mutation frequency at the IgH V region as a dU:dG mismatch produced by AID initiates modifications by MMR, resulting in mutations at nearby A:T base pairs. This raises the possibility that GC B cells express a factor that converts MMR into a globally mutagenic pathway. To test this notion, we investigated whether MMR corrects mutations in GC B cells at a gene that is not mutated by AID. Strikingly, we found that GC B cells accumulate 5 times more mutations at a reporter gene than during the development of the mouse. Notably, the mutation frequency at this reporter gene was approximately 10 times greater in Msh2(-/-) compared with wild-type GC B cells cells. In contrast to the V region, the increased level of mutations at A:T base pairs in GC B cells was not caused by MMR. These results show that in GC B cells, (1) MMR functions normally at an AID-insensitive gene and (2) the frequency of background mutagenesis is greater in GC B cells than in their precursor follicular B cells.

MicroRNAs at the epicenter of intestinal homeostasis.

Maintaining intestinal homeostasis is a key prerequisite for a healthy gut. Recent evidence points out that microRNAs (miRNAs) act at the epicenter of the signaling networks regulating this process. The fine balance in the interaction between gut microbiota, intestinal epithelial cells, and the host immune system is achieved by constant transmission of signals and their precise regulation. Gut microbes extensively communicate with the host immune system and modulate host gene expression. On the other hand, sensing of gut microbiota by the immune cells provides appropriate tolerant responses that facilitate the symbiotic relationships. While the role of many regulatory proteins, receptors and their signaling pathways in the regulation of the intestinal homeostasis is well documented, the involvement of non‐coding RNA molecules in this process has just emerged. This review discusses the most recent knowledge about the contribution of miRNAs in the regulation of the intestinal homeostasis.

Gut microbiota and colon cancer: the carbohydrate link

Understanding the complex pathophysiology of colorectal cancer and the interaction between host genetics, the gut microbiome, and diet has attracted significant attention in the last few years. The discovery that gut microbial metabolites may dictate the course of colorectal cancer progression supports the development of microbial-targeted strategies.

Elevated Incidence of Polyp Formation in APCMin/+Msh2−/− Mice Is Independent of Nitric Oxide-Induced DNA Mutations

Gut microbiota has been linked to a number of human diseases including colon cancer. However, the mechanism through which gut bacteria influence colon cancer development and progression remains unclear. Perturbation of the homeostasis between the host immune system and microbiota leads to inflammation and activation of macrophages which produce large amounts of nitric oxide that acts as a genotoxic effector molecule to suppress bacterial growth. However, nitric oxide also has genotoxic effects to host cells by producing mutations that can predispose to colon cancer development. The major DNA lesions caused by nitric oxide are 8oxoG and deamination of deoxycytosine bases. Cellular glycosylases that belong to the base excision repair pathway have been demonstrated to repair these mutations. Recent evidence suggests that the mismatch repair pathway (MMR) might also repair nitric oxide-induced DNA damage. Since deficiency in MMR predisposes to colon cancer, we hypothesized that MMR-deficient colon epithelial cells are incapable of repairing nitric-oxide induced genetic lesions that can promote colon cancer. Indeed, we found that the MMR pathway repairs nitric oxide-induced DNA mutations in cell lines. To test whether nitric oxide promotes colon cancer, we genetically ablated the inducible nitric oxide synthase (iNOS) or inhibited iNOS activity in the APCMin/+Msh2−/− mouse model of colon cancer. However, despite the fact that nitric oxide production was strongly reduced in the colon using both approaches, colon cancer incidence was not affected. These data show that nitric oxide and iNOS do not promote colon cancer in APCMin/+Msh2−/− mice.

Missing mismatch repair: a key to T cell immortality

The mismatch repair (MMR) pathway maintains the integrity of the genome by repairing DNA mismatches or loops caused by insertion or deletion of nucleotides, which can be caused by DNA replication, oxidative damage, cytidine deamination, or chemical mutagens. The MMR pathway also maintains the fidelity of the genome by inhibiting recombination between non-identical sequences, and induces apoptosis in response to high levels of DNA damage [1]. Thus, deficiency in MMR can promote cellular transformation through multiple mechanisms. Tumors that are deficient in MMR are characterized by an instability of short tandem DNA repeated sequences known as microsatellites [2]. Microsatellite instability (MSI) is a hallmark of the hereditary non-polyposis colorectal cancer (HNPCC)/Lynch syndrome, but is also observed in many sporadic cancers including hematological malignances [3–6]. Abnormal chromosomal rearrangements give rise to most of the hematological tumors. In this regard, it has been suggested that MMR protects from recombination between divergent DNA sequences by recognizing and binding DNA mismatches [7]. A compromised MMR pathway might therefore create a genomic environment that allows for the development of hematological cancers by increasing the rate of chromosomal rearrangements during T and B cell development [7]. Recently, it has been shown that the biallelic loss of Msh2, the central protein in MMR, was critical to the development of HNPCCrelated lymphomas [8]. This discovery raised further questions about the specific role of other genes involved in the MMR pathway in hematological tumors associated with MSI and colorectal cancer. In this issue of Leukemia and Lymphoma, Reiss and colleagues [9] provide key insights into the role of MLH1, an important component of the MMR pathway, in the development of T cell lymphoma. Using a gene targeting strategy, the authors developed an Mlh1 exon 4 allele that was flanked with loxP sites. This mouse model was then used to specifically delete MLH1 from the T cell compartment (i.e. Mlh1) in order to examine the development of T cell lymphomas and avoid the overlaying effects of tumorigenesis in other cellular systems. The resulting Mlh1 mice showed a reduced tumor predisposition phenotype compared to Mlh1 mice; only 6% of Mlh1 mice developed lymphoma after 40 weeks of age compared to 26% of Mlh1 mice. In addition, tumors from Mlh1 mice developed primarily from double positive (CD4þ / CD8þ ) T cell lymphocytes. This finding contrasts with the observation that T lymphomas in Mlh1 mice occur at many different stages of development. Taken together, the results presented by Reiss and colleagues provide novel evidence that MMR plays a critical role in preventing genetic events that lead to cellular transformation at very early stages, either during T cell development or at earlier stages of hematopoietic development. These data support a recent report that showed that Msh2 plays a critical tumor suppressive role in early B cell precursors [10]. The question of why the deletion of Mlh1 in thymocytes leads to precursor T cell lymphomas while deletion of Mlh1 in all tissues leads to precursor and mature T cell lymphomas will likely be the subject of further investigation.