Cancer-preceding Gene Expression Changes in Mouse Colon Mucosa

Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in the Western world and interactions between genetic and environmental factors, including diet, are suggested to play a critical role in its etiology. Yet, the mechanisms by which diet impacts colorectal tumorigenesis remain largely unknown. Colorectal cancer evolves as a multistep process, which requires a series of genetic and epigenetic alterations in growth regulatory genes. The process is accelerated in individuals with inherited cancer predisposition such as Lynch syndrome (LS) which is one of the most common inherited cancer susceptibility syndromes and caused by inherited mutation in one of the DNA mismatch repair (MMR) genes. CRC is thought to develop via the so called adenoma-carcinoma sequence. However, the early events that occur in colon mucosa prior to polyp formation remain unknown. The research presented here investigates the gene expression changes arising in histologically normal colonic mucosa as putative cancer-preceding events available for early detection. This was achieved by pursuing a long-term feeding experiment in the mouse model for human Lynch syndrome (Mlh1+/-), and the wild type (Mlh1+/+) littermates, fed with either Western-style (WD) diet or healthy AIN-93G control diet. Carcinomas developed mainly in WD fed mice. WD also accelerated the progression of carcinogenesis. In the first study, the expression of 94 growth-regulatory genes previously linked to human CRC was studied for 5 weeks and 12 months old mice. Promoter CpG island methylation status was also studied for the genes which showed reduced expression. In mice fed for 12 months with WD, proximal colon mucosa, the predominant site of cancer formation in LS, exhibited a significant expression decrease in several tumor suppressor genes. Furthermore, a reduced mRNA expression was accompanied by an increased CpG dinucleotide promoter methylation of the respective genes suggesting a cause for the mRNA down regulation. The strongest expression decrease together with a significant increase in its promoter methylation was seen in Dkk1, an antagonist of the canonical Wnt signaling pathway. Furthermore, the inactivation of Dkk1 seemed to predispose to neoplasias in the proximal colon. The findings suggest that the inactivation of Dkk1 is a prominent early marker for colon oncogenesis. In study 2 the aim was to comprehensively clarify the role of Mlh1 expression during colon tumorigenesis, which is usually associated with Lynch syndrome and MSI. Here, the same mouse model and diets were used to study cancer-preceding expression changes in the colon mucosa of 12 and 18-month-old mice. The Mlh1 protein expression and MSI status were studied in the colon carcinomas, and the effect of inherited predisposition (Mlh1+/) and Western-style diet on those. CRC development always includes a lack of genomic integrity and the different types of genomic instabilities, such as chromosomal instability and MSI are thought to reflect distinct cancer initiating mechanisms. In the present study neither wildtype Mlh1+/+ nor heterozygote Mlh1+/- mice lacked the Mlh1 protein or showed MSI in their CRCs, while Mlh1 RNA expression was already significantly decreased in their normal mucosa. Instead, CRC mice showed a distinct expression profile with shortage of Mlh1 and several other chromosomal segregation gene-specific transcripts in mucosa and aberrant mitosis in tumors. The genome wide expression profiling experiment demonstrated that cancer-preceding changes are already seen in histologically normal colon mucosa and a that decreased expression of Mlh1 together with other chromosomal segregation genes may form a field-defect in mucosa and trigger MMR-proficient, chromosomally unstable CRC.Paksusuolen syöpä on toiseksi yleisin syöpäkuolleisuuden aiheuttaja länsimaissa ja sekä geneettiset-tekijät että ympäristötekijät kuten ravinto ovat merkittävässä roolissa sen kehittymisessä. Mekanismit, joilla ravinto vaikuttaa syövän kehitykseen, tunnetaan kuitenkin vielä huonosti. Syövän kehitys on monivaiheinen prosessi, joka vaatii useita geneettisiä ja epigeneettisiä muutoksia kasvunrajoitegeeneissä. Tapahtuma on nopeutunut perinnöllisen syöpäalttiussyndrooman, kuten Lynch syndrooman (LS) kantajilla. Väitöskirjatutkimuksessa selvitettiin paksusuolen normaalilimakalvolla esiintyviä geeninilmentymismuutoksia perinnöllisen suolistosyövän hiirimallia käyttäen. Pitkän ruokintakokeen aikana hiirille syötettiin joko ravintosisällöltään länsimaista ruokavaliota mallintavaa jyrsijädieettiä (WD) tai terveellistä kontrollidieettiä (AIN). Suurin osa kasvaimista kehittyi länsimaista ravintoa mallintavissa hiiriryhmissä. WD myös nopeutti suolistokasvaimien kehitystä. Ensimmäisessä osatyössä tutkittiin kolmen eri riskitekijän, ikääntymisen, perityn alttiuden ja länsimaisen dieetin vaikutusta 94 ihmisen suolistosyövän kehityksessä tärkeän geenin ilmentymiseen. Työssä havaittiin usean tutkitun geenin ilmentymisen merkittävä alentuma paksusuolen normaalilla limakalvolla. Alentuma liittyi monen geenin kohdalla DNA:n metyloitumiseen, jonka tiedetään hiljentävän geenin ilmentymistä. Erityisen vahvaa ilmentymisen alentumaa ja siihen liittyvää DNA:n metyloitumista osoitti ihmisen syövän kehityksessä tärkeä kasvunrajoitegeeni Dkk1 (Dickkopf 1). Toisessa osatyössä selvitettiin tärkeän DNA:n emäspariutumavirheiden korjausgeenin, Mlh1:n (mutL homolog 1) merkitystä suolistosyövän kehityksessä samaa perinnöllisen suolistosyövän hiirimallia käyttäen. MLH1 geenin hiljentyminen aiheuttaa puutteellisen emäspariutumavirheiden ns. mismatch repair (MMR) korjausmekanismin, joka ilmenee perimän toistojaksojen epävakautena. Vallitsevasti periytyvä Lynch syndrooma liittyy puutteelliseen MMR mekanismiin, joka ilmenee MMR-proteiinien, kuten MLH1 puutteena kasvaimissa. Yllättäen, Mlh1 geenin mutaatiolla syövälle altistettujen hiirten suolistokasvaimet ilmensivät Mlh1 proteiinia, eivätkä osoittaneet toistojaksojen epävakautta kuten kasvaimet Lynch syndroomassa. Kuitenkin samojen hiirten suolen normaalilla limakalvolla Mlh1 geenin ilmentyminen oli merkittävästi alentunut. Koko genomin kattava RNA-sekvensointianalyysi osoitti lisäksi suolistosyövän kehittäneiden hiirten normaalilta limakalvolta erityisen ilmentymisprofiilin, joka viittasi ongelmiin kromosomien jakautumisessa. Samojen hiirten kasvaimet osoittivat tutkimuksissa epänormaaleja mitooseja, mikä vahvisti normaalilimakalvon löydöksen. Tulokset antavat viitteitä siitä, että Mlh1 geenin ja useiden muiden kromosomien jakautumiseen liittyvien geenien ilmentymisen alentuma johtaa kromosomaalisesti epävakaan kasvaimen kehitykseen ja on mahdollista havaita jo suolen normaalilta limakalvolta. Väitöstyössä löydettiin suolen normaalilimakalvolta useita geenin ilmentymismuutoksia, joita voidaan mahdollisesti käyttää kohonnutta syöpäriskiä ilmentävinä biomarkkereina

Western Diet Deregulates Bile Acid Homeostasis, Cell Proliferation, and Tumorigenesis in Colon

Western-style diets (WD) high in fat and scarce in fiber and vitamin D increase risks of colorectal cancer. Here, we performed a long-term diet study in mice to follow tumorigenesis and characterize structural and metabolic changes in colon mucosa associated with WD and predisposition to colorectal cancer. WD increased colon tumor numbers, and mucosa proteomic analysis indicated severe deregulation of intracellular bile acid (BA) homeostasis and activation of cell proliferation. WD also in-creased crypt depth and colon cell proliferation. Despite increased luminal BA, colonocytes from WD-fed mice exhibited decreased expression of the BA transporters FABP6, OST beta, and ASBT and decreased concentrations of secondary BA deoxycholic acid and lithocholic acid, indicating reduced activity of the nuclear BA receptor FXR. Overall, our results suggest that WD increases cancer risk by FXR inactivation, leading to BA deregulation and increased colon cell proliferation. (C) 2017 AACR.Peer reviewe

Mlh1 deficiency in normal mouse colon mucosa associates with chromosomally unstable colon cancer

Colorectal cancer (CRC) genome is unstable and different types of instabilities, such as chromosomal instability (CIN) and microsatellite instability (MSI) are thought to reflect distinct cancer initiating mechanisms. Although 85% of sporadic CRC reveal CIN, 15% reveal mismatch repair (MMR) malfunction and MSI, the hallmarks of Lynch syndrome with inherited heterozygous germline mutations in MMR genes. Our study was designed to comprehensively follow genome-wide expression changes and their implications during colon tumorigenesis. We conducted a long-term feeding experiment in the mouse to address expression changes arising in histologically normal colonic mucosa as putative cancer preceding events, and the effect of inherited predisposition (Mlh1(+/-)) and Western-style diet (WD) on those. During the 21-month experiment, carcinomas developed mainly in WD-fed mice and were evenly distributed between genotypes. Unexpectedly, the heterozygote (B6.129-Mlh1tm1Rak) mice did not show MSI in their CRCs. Instead, both wildtype and heterozygote CRC mice showed a distinct mRNA expression profile and shortage of several chromosomal segregation gene-specific transcripts (Mlh1, Bub1, Mis18a, Tpx2, Rad9a, Pms2, Cenpe, Ncapd3, Odf2 and Dclre1b) in their colon mucosa, as well as an increased mitotic activity and abundant numbers of unbalanced/atypical mitoses in tumours. Our genome-wide expression profiling experiment demonstrates that cancer preceding changes are already seen in histologically normal colon mucosa and that decreased expressions of Mlh1 and other chromosomal segregation genes may form a field-defect in mucosa, which trigger MMR-proficient, chromosomally unstable CRC.Peer reviewe

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

Peer reviewe

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

Mlh1 deficiency in normal mouse colon mucosa associates with chromosomally unstable colon cancer

Abstract Colorectal cancer (CRC) genome is unstable and different types of instabilities, such as chromosomal instability (CIN) and microsatellite instability (MSI) are thought to reflect distinct cancer initiating mechanisms. Although 85% of sporadic CRC reveal CIN, 15% reveal mismatch repair (MMR) malfunction and MSI, the hallmarks of Lynch syndrome with inherited heterozygous germline mutations in MMR genes. Our study was designed to comprehensively follow genome-wide expression changes and their implications during colon tumorigenesis. We conducted a long-term feeding experiment in the mouse to address expression changes arising in histologically normal colonic mucosa as putative cancer preceding events, and the effect of inherited predisposition (Mlh1+/−) and Western-style diet (WD) on those. During the 21-month experiment, carcinomas developed mainly in WD-fed mice and were evenly distributed between genotypes. Unexpectedly, the heterozygote (B6.129-Mlh1tm1Rak) mice did not show MSI in their CRCs. Instead, both wildtype and heterozygote CRC mice showed a distinct mRNA expression profile and shortage of several chromosomal segregation gene-specific transcripts (Mlh1, Bub1, Mis18a, Tpx2, Rad9a, Pms2, Cenpe, Ncapd3, Odf2 and Dclre1b) in their colon mucosa, as well as an increased mitotic activity and abundant numbers of unbalanced/atypical mitoses in tumours. Our genome-wide expression profiling experiment demonstrates that cancer preceding changes are already seen in histologically normal colon mucosa and that decreased expressions of Mlh1 and other chromosomal segregation genes may form a field-defect in mucosa, which trigger MMR-proficient, chromosomally unstable CRC

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