Browsing by Subject "Colorectal cancer"

Colorectal cancer (CRC) kills more than half a million people a year worldwide. Usually the disease develops over several years via multiple steps which involve both genetic and epigenetic alterations. CRC is often diagnosed at late stage, when the cancer has already metastasized, and the prognosis is relatively poor. Several studies suggest that the first changes towards colorectal cancer occur and can be detected in histologically normal tissue before the appearance of any detectable lesion. The precancerous cells harbouring those changes may form a field of tissue, which is predisposed to malignant transformation. The study of pre-cancerous tissue might reveal the earliest changes in CRC development, which can be used as biomarkers for early detection and prevention of CRC. The aim of this thesis was to revise and investigate whether the aberrant expression of the six chromosomal segregation genes, Bub1, Mis18a, Pms2, Rad9a, Tpx2, and Mlh1, would signal carcinogenesis in mouse colon mucosa. Altogether fourteen mice, of which six had a proximal colon carcinoma, were selected for the study. The expression analysis was performed to histologically normal colon mucosa collected from the proximal and distal colon of each mice in order to investigate whether the possible pre-cancerous changes are found exclusively in the close proximity to the carcinoma. The expression was quantified with reverse transcription quantitative polymerase chain reaction (RTqPCR). No statistically significant gene expression differences were found between the carcinoma and control mice, indicating that the studied mice did not display cancer-preceding expression changes of the six studied genes in the carcinoma adjacent histologically normal colon mucosa. The results differed from the previously reported results, where the expressions of the six genes were found to be downregulated in the carcinoma adjacent mucosa. Here, the sample size was presumably not large enough to reveal statistically significant clustering of the expression patterns. However, Bub1 seemed to have a downregulated trend in the carcinoma adjacent mucosa, which supports the previously suggested role of Bub1 alterations in CRC initiation.

Suolistosyövät ovat läntisen maailman kolmanneksi yleisimpiä syöpiä. Alati kehittyvistä hoitomuodoista huolimatta edelleenkin joka kolmas suolistosyövistä leviää muihin elimiin, ja merkittävä osa potilaista menehtyy. Yleisesti tiedetään, että syöpäsolut vaativat jakautuakseen useita erilaisia mutaatioita, joiden ansiosta ne pääsevät karkaamaan elimistön kontrollista. Suolistosyövissä tyypillinen mutaatio on solunsisäistä Wnt-signalointireittiä säätelevässä APC-geenissä. APC:n periytyvä mutaatio aiheuttaa Familial Adenomatous Polyposis (FAP)-nimisen sairauden, jossa potilaan suolistoon kehittyy jopa satoja adenoomia. Nämä etenevät hoitamattomana syöväksi lähes 100% todennäköisyydellä. Tämän tutkielman tarkoituksena oli selvittää tarkemmin suolistosyövän kantasolujen ja niitä säätelevien, Wnt-signalointia vahvistavien kasvutekijöiden, R-spondiinien (RSPO), toimintaa, erityisesti kasvainten kehityksen alkuvaiheissa. Tätä tarkoitusta varten tuotettiin AAV-vektori, jonka avulla voitiin käynnistää systeeminen R-spondiinituotanto suolistosyövän Apc-mutantissa hiirimallissa. R-spondiinien tiedetään edistävän suoliston terveiden kantasolujen toimintaa, ja niiden on ajateltu olevan merkittävässä roolissa myös suolistosyöpien kehityksessä. AAV-RSPO1-vektori aikaansaikin villityypin hiirissä odotetusti suolen kantasolujen toiminnan kiihtymisen. Kuitenkin hiiren suolistokasvaimissa vaikutus oli päinvastainen; RSPO1-käsittely hidasti ApcMin-hiirten adenoomien kasvua, kiihdytti apoptoosia spesifisti adenoomasoluissa sekä pidensi kasvaimista kärsivien hiirten elinikää verrokkeihin nähden merkittävästi. Mekanistisissa tutkimuksissa kävi ilmi, että AAV-RSPO1-käsittely aktivoi TGFB-signalointireitin, jonka tiedetään aktivoivan solunsisäistä apoptoosikoneistoa yksinomaan kasvainsoluissa, ja siten rajoittavan niiden kasvua. Tämän seurauksena suolen terveet solut saavuttivat kilpailuedun kasvainsoluihin nähden, ja syrjäyttivät kasvainsoluja, jolloin adenoomista päästiin eroon lähes kokonaan. Näiden löydösten perusteella voisikin olla mahdollista kehittää uusia hoitomuotoja suolistosyöpiin, erityisesti FAP:tä sairastaville potilaille.

Colorectal cancer (CRC), which refers to the cancer of the colon and the rectum currently ranks as the second leading cause of cancer related death worldwide and as the third most common form of cancer in both males and females. The latest reports show that approximately 10% of all new cancer cases globally are diagnosed as CRC annually. Initiation of sporadic CRC is commonly caused by somatic mutations causing the loss of function of the tumor suppressor gene APC. This leads to aberrant activation of the canonical Wnt signalling pathway. The ApcMin/+ mice model the progression of CRC as they carry a constitutive heterozygous nonsense mutation in Apc allele and develop intestinal adenomas. TCF/LEF transcription factor family members are best known as the main downstream effectors of canonical Wnt signalling. In the presence of nuclear β-catenin, TCF/LEF proteins bind to it through their β-catenin-binding domain and activate the transcription of Wnt target genes. The TCF7 gene encodes several isoforms of TCF1 protein, which are traditionally divided into long and short isoforms, transcribed from different promoters. Previously, it has been shown that Tcf7 deletion (Tcf7-/-) in ApcMin/+ mice increases the formation of adenomas. The aim of my study is to better understand the role of Tcf7 and its isoforms in CRC tumorigenesis. To study the Tcf7 deletion in intestinal adenoma development, ApcMin/+; Tcf7mut/mut; Villin CreERT2 mouse strain was established. The expression of the full-length isoforms (p45) is constitutively prevented in the Tcf7mut/mut mice. Moreover, tamoxifen administration to these mice led to the deletion of all isoforms in the intestinal epithelium. The number of intestinal tumors, their sizes and the survival of the Tcf7 deleted ApcMin/+ mice were analyzed and compared to ApcMin/+ mice. Intestinal tissues of the mice were collected after euthanasia. The tissue samples were preserved in paraffin, and later cut into sections for IHC, stained and imaged. The deletion of Tcf7 was confirmed at the RNA level by qPCR, and at the protein level by immunohistochemistry (IHC). IHC and single-cell RNA sequencing was used to further analyze the effect of Tcf7 deletion in mouse intestinal adenomas. The deletion of all Tcf7 isoforms or only the p45 isoforms in ApcMin/+ mice increased robustly the numbers of intestinal tumors. IHC analysis of the intestinal adenomas showed that the deletion of p45 isoforms was sufficient to cause a dramatic decrease in total Tcf1 expression in the adenoma cells. These results were supported by the qPCR results. In summary, our results lead us to believe that the deletion of p45 isoforms causes an acceleration of tumorigenesis in the adenoma model. Without the Apc mutation, the mice did not develop intestinal adenomas. Interestingly, the expression of the Wnt-target gene Prox1 in intestinal adenomas was decreased when Tcf7 was deleted. We next aim to optimize our protocol for single cell dissociation of adenomas and re-run the single-cell RNA sequencing analysis for further analysis of the mechanisms behind the increased tumorigenesis.