Biochemical effects of inherited MMR gene mutations and diet on colon cancer risk

Colorectal cancer (CRC) is one of the leading causes of death in developed countries. Although, a small fraction of cancers are caused by inherited genetic predisposition most of the CRCs are sporadic. In CRC, cancer risk is associated with lifestyle factors and aging. Even in dominantly inherited CRC predisposition such as in Lynch syndrome (LS), which is linked to germline mu- tations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2, cancer develops as a result of accumulation of genetic and epigenetic changes. After diagnosing an LS family, to be able to offer contiguous pre-symptomatic surveillance and predictive gene counseling to mutation carriers in a family, the pathogenicity assessment of a mutation is needed. Dependent on the type and the site of a germline mutation, inherited cancer risk may vary from high to low and especially in the latter case cancer risk may be strongly affected by lifestyle factors such as diet. Epidemiological studies on humans and previous studies on mice have shown that especially a Western-style diet (WD) may predispose colon mucosa to CRC. However, the mechanisms, which mediate the effects of diet on tumorigenesis are largely unknown. Since both genetic and lifestyle factors have been shown to predispose to cancer, this the- sis analyzed biochemical defects caused by inherited MMR gene mutations and Western diet exposure. Different MMR gene mutations may compromise MMR function through various biochemical defects. Here, we studied 18 inherited non-truncating mutations in MSH2, the second most frequently mutated gene among Lynch syndrome patients. We assessed protein stability, DNA binding, and ATP mediated DNA release abilities of the MSH2 variants. The majority of variants in the amino terminal region including the connector and lever domains p.V161D, p.G162R, p.G164R, p.L173P, p.L187P, p.C333Y, p.D603N) affected protein stabil- ity. Variations in the ATPase domain (p.A636P, p.G674A, p.C697F, p.I745-I746del, p.E749K) totally abolished either mismatch binding or release. Four protein variants (p.T33P, p.A272 V, p.G322D, p.V923E) expressed slightly reduced mismatch binding and/or release efficiencies compared to wild-type (WT) MSH2 protein, while two variants (p.N127S, p.A834T) were in- distinguishable from WT. To define the effects of Western-style diet, we analyzed protein expression changes in histolog- ically normal colon mucosa of wild type (Mlh1+/+) and CRC predisposed mice (Mlh1+/-) after a long-term feeding experiment with WD and AIN-93G control diet. Using network analysis and data mining we also determined which of the affected proteins might be putative play- ers in early CRC development. Our results pinpoint changes in a complex protein interaction network involved in ATP synthesis coupled proton transport, oxidoreduction coenzyme and nicotinamide nucleotide metabolic processes, which are important in the generation of reactive oxygen species (ROS) and cellular protection against ROS toxicity. Additionally, we detected SELENBP1 and LGALS4, which are implied in neoplastic processes. Our studies show that mutations in the MMR gene affect the biochemistry of MMR, can have an effect on the phenotype of the mutation carriers and in the latest study suggest that the high sensitivity to Western diet may be linked to haplo-insufficiency caused by a loss of function mutation in the Mlh1+/- mice.Colorectal cancer (CRC) is one of the leading causes of death in developed countries. Although, a small fraction of cancers are caused by inherited genetic predisposition most of the CRCs are sporadic. In CRC, cancer risk is associated with lifestyle factors and aging. Even in dominantly inherited CRC predisposition such as in Lynch syndrome (LS), which is linked to germline mu- tations in the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2, cancer develops as a result of accumulation of genetic and epigenetic changes. After diagnosing an LS family, to be able to offer contiguous pre-symptomatic surveillance and predictive gene counseling to mutation carriers in a family, the pathogenicity assessment of a mutation is needed. Dependent on the type and the site of a germline mutation, inherited cancer risk may vary from high to low and especially in the latter case cancer risk may be strongly affected by lifestyle factors such as diet. Epidemiological studies on humans and previous studies on mice have shown that especially a Western-style diet (WD) may predispose colon mucosa to CRC. However, the mechanisms, which mediate the effects of diet on tumorigenesis are largely unknown. Since both genetic and lifestyle factors have been shown to predispose to cancer, this the- sis analyzed biochemical defects caused by inherited MMR gene mutations and Western diet exposure. Different MMR gene mutations may compromise MMR function through various biochemical defects. Here, we studied 18 inherited non-truncating mutations in MSH2, the second most frequently mutated gene among Lynch syndrome patients. We assessed protein stability, DNA binding, and ATP mediated DNA release abilities of the MSH2 variants. The majority of variants in the amino terminal region including the connector and lever domains p.V161D, p.G162R, p.G164R, p.L173P, p.L187P, p.C333Y, p.D603N) affected protein stabil- ity. Variations in the ATPase domain (p.A636P, p.G674A, p.C697F, p.I745-I746del, p.E749K) totally abolished either mismatch binding or release. Four protein variants (p.T33P, p.A272 V, p.G322D, p.V923E) expressed slightly reduced mismatch binding and/or release efficiencies compared to wild-type (WT) MSH2 protein, while two variants (p.N127S, p.A834T) were in- distinguishable from WT. To define the effects of Western-style diet, we analyzed protein expression changes in histolog- ically normal colon mucosa of wild type (Mlh1+/+) and CRC predisposed mice (Mlh1+/-) after a long-term feeding experiment with WD and AIN-93G control diet. Using network analysis and data mining we also determined which of the affected proteins might be putative play- ers in early CRC development. Our results pinpoint changes in a complex protein interaction network involved in ATP synthesis coupled proton transport, oxidoreduction coenzyme and nicotinamide nucleotide metabolic processes, which are important in the generation of reactive oxygen species (ROS) and cellular protection against ROS toxicity. Additionally, we detected SELENBP1 and LGALS4, which are implied in neoplastic processes. Our studies show that mutations in the MMR gene affect the biochemistry of MMR, can have an effect on the phenotype of the mutation carriers and in the latest study suggest that the high sensitivity to Western diet may be linked to haplo-insufficiency caused by a loss of function mutation in the Mlh1+/- mice

Cancer-Predicting Gene Expression Changes in Colonic Mucosa of Western Diet Fed Mlh1(+/-) Mice

Peer reviewe

Validation of the mRNA expression changes that were associated with WD* and/or inherited <i>Mlh1</i> mutation using TaqMan assays.

<p>Relative expression in different study groups (Mlh1^+/- AIN, Mlh1^+/+ WD*, and Mlh1^+/- WD*) is compared to the control group (Mlh1^+/+ AIN). Each sample is a mixture of eight RNA samples from eight different tp1 mice belonging to each mouse group. Data is presented as mean ± SEM (standard error of the mean) (n = 3), *significant difference compared to the control group. Median permutation method, <i>P</i> < 0.05. (A) <i>Mlh1</i> shows the same 50% expression difference between the genotypes as at the starting point of the study. (B) <i>Dkk1</i> is significantly down regulated in the study groups with WD* and/or <i>Mlh1</i> heterozygosity. (C) <i>Slc5a8</i> does not show significant expression differences at tp1. (D) <i>Hoxd1</i> is down regulated in association with WD* in both genotypes; the down regulation being especially strong in the Mlh1^+/+ WD group. (E) <i>Socs1</i> does not show significant expression differences at tp1. (F) <i>Dkk2</i> is down regulated in association with WD* in both genotypes.</p

Methylation clusters and the NMDS analysis of the methylation data.

<p>Using the Chipster’s Dendrogram tool, two distinct clusters, the higher (Group 1) and the lower methylation cluster (Group 2) were observed at tp1. Except for one mouse in <i>Sfrp1</i> and in <i>Socs1</i> (B214 and B225, respectively) the same 11 mice clustered into the higher methylation cluster at CGIs of <i>Dkk1, Slc5a8, Hoxd1</i>, <i>Socs1</i>, and <i>Sfrp1</i>. The neoplasias are marked with superscripts ( ^ahyperplastic polyp, ^badenoma, ^cadenocarcinoma, ^dnot histologically confirmed) and the mice groups with different colors (black; Mlh1^+/+ AIN, brown; Mlh1^+/- AIN, turquoise; Mlh1^+/+ WD*, pink; Mlh1^+/- WD*). Also according to the NMDS analysis (Chipster) the tp0 and tp1 mice segregate into different parts of the plots indicating differences in their methylation levels. Red refers to tp0 mice, green refers to Group 1 mice, blue refers to Group 2 mice, and turquoise refers to mice that did not belong to either group.</p