Browsing by Subject "colorectal cancer"
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(2021)MLH1 is a gene that codes for one of the four mismatch repair (MMR) proteins alongside MSH2, MSH6, and PMS2. The main function of the MMR proteins is to recognize base mismatches and insertion-deletion loops formed during DNA replication and aid in their excision. Inherited heterozygous pathogenic variants in any of the four MMR genes lead to Lynch syndrome, an inherited cancer syndrome that predisposes to multiple different cancer types, most notably colorectal cancer. Loss of the expression of an MMR gene causes MMR-deficiency, which leads to microsatellite instability, the accumulation of mutations in microsatellite regions of the DNA. The higher mutational burden caused by MMR-deficiency is thought to be the main driving force of genomic instability and tumorigenesis in MMR-deficient cells. In addition to MMR, MLH1 and the MMR machinery have roles in other anticarcinogenic cellular processes, such as DNA damage signaling and DNA double-strand break repair. Recently, MLH1 has also been shown to have a significant role in regulating mitochondrial metabolism and oxidative stress responses. The identification of MMR-proficient tumors in Lynch syndrome patients begs the question whether the lower amount of functional MLH1 observed in MLH1 mutation carriers could cause problems with these functions and pose alternative routes to tumorigenesis. In line with this, it has been shown that the role of MLH1 in cell cycle regulation in DNA damage signaling is notably more sensitive to decreased amount of the protein compared to its role in MMR. The main goal of the thesis was to study the effects of decreased MLH1 expression on gene expression, cellular functions, and possible alternative tumorigenic pathways. In order to achieve this, the coding transcriptome of human fibroblast cell lines expressing MLH1 at different levels was sequenced and the resulting data analyzed. The study revealed that decreased MLH1 expression affects cellular functions associated with mitochondrial function and oxidative stress responses in cells with functional MMR. Particularly NRF2-controlled cytoprotective defence systems were observed to be downregulated. Decreased MLH1 expression was also observed to affect several cellular functions associated with reorganization of the cytoskeleton and interactions with the extracellular matrix. These results strengthen the recently made notions that MLH1 has a role in controlling the function of mitochondria and in mitigating oxidative stress, and that these two functions are connected. The study also brings to light new information on the possible role of MLH1 in controlling the organization of the cytoskeleton, which has previously received little attention. Dysfunction of mitochondria, increased oxidative stress, and reorganization of the cytoskeleton, as a result of decreased MLH1 expression, could pose events that facilitate malignant transformation of cells prior to the total loss of MMR function.
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(2022)Colorectal cancer (CRC) is one of the most common types of cancers, encompassing approximately 10 % of all cancer cases worldwide. Regulation of cell proliferation and cell fate decisions is crucial for maintaining cellular homeostasis and preventing CRC initiation, a process in which the Wingless (Wnt)/β-catenin signalling pathway is known to play an important role. For instance, somatic mutations occurring in the Apc gene lead to aberrant activation of the Wnt/β-catenin pathway, which further leads to the accumulation of β-catenin into the nucleus where the TCF/LEF transcription factors, including TCF1, TCF3, TCF4, and LEF1, bind β-catenin to activate downstream Wnt target genes and promote CRC development. TCF1 is encoded by the gene Tcf7 that can be alternatively spliced to produce long (p45) and short (p33) isoforms whose function in CRC development has remained poorly understood. Previously, deletion of Tcf7 has been found to increase intestinal adenoma formation in mice with one mutated Apc allele (ApcMin/+), which are predisposed to development of multiple intestinal adenomas. To study how heterozygous deletion of the p45 isoforms affects intestinal adenoma formation and the cellular transcriptome, we have studied ApcMin/+ mice, which have a heterozygous mutation of Tcf7 gene encoding only the p33 isoform (AmTp45∆/+). In our study, we used immunohistochemistry and RT-qPCR together with a single-cell RNA sequencing (scRNA-seq) analysis. Heterozygous deletion of the p45 isoforms in the ApcMin/+ mice dramatically increased the numbers of intestinal tumours, spleen size and its white pulp areas but it had no effect on cell proliferation or expression of the Wnt-target gene Prox1 in intestinal adenomas. In addition, ApcMin/+ mice with both heterozygous p45 and Lef1 deletions developed significantly more intestinal tumours. Without the Apc mutation, neither of these mouse models developed intestinal adenomas or spleen abnormalities. RT-qPCR analysis showed decreased expression of Tcf7 in the small intestine of the AmTp45∆/+ mice. scRNA-seq analysis revealed that the AmTp45∆/+ mice downregulated various Wnt antagonists and stem cell markers and upregulated several genes that function in different metabolic pathways. Overall, these results support the concept that Tcf7 functions in synergy with Apc to repress intestinal malignancy. Our results establish a basis for comparison of the relative importance and specific functions of the TCF/LEF1 family members in CRC development.
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