Background: Very recently a gene marker panel that allows the mutational analysis of APC, CTNNB1, B-RAF and K-RAS was conceived. The aim of the present study was to use the 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signalling pathways to determine the percentage of sporadic colorectal carcinomas (CRC) carrying at least one of the four above-mentioned genes in a mutated form alone and/or in combination with microsatellite instability (MSI) and to compare the sensitivity of the gene marker panel used in this study with that of gene marker panels previously reported in the scientific literature. Methods: CTNNB1 and B-RAF were screened by PCR-single-strand conformation polymorphism analysis and K-RAS gene mutations by restriction fragment length polymorphism analysis. For the mutational analysis of the APC gene mutation cluster region (codons 1243–1567) direct DNA sequencing was performed. The U.S. National Cancer Institute microsatellite panel (BAT25, BAT26, D2S123, D5S346 and D17S250) was used for MSI analysis. Results: It could be shown that about 80% of early stage CRC (UICC stages I and II) and over 90% of CRC in the UICC stage IV carried at least one mutated gene and/or showed MSI. No significant increase in the gene mutation frequencies could be determined when comparing tumours in the UICC stage I with those in UICC stage IV. Conclusions: When compared with previously published gene marker panels the 4-gene marker panel used in the present study shows an excellent performance, allowing to detect genetic alterations in 80–90% of human sporadic CRC samples analyzed.

Osteosarcoma is the most common bone malignancy in children and adolescents with poorly understood aetiology. Recently, disease susceptibility and aetiology in several cancers have been associated with genomic copy-number (CN) change. We therefore studied the contribution of CN change in osteosarcoma. We report that individuals with osteosarcoma have increased germline structural variation compared to controls. These CN variants (CNVs) preferentially localize to genes implicated in control of osteoblast differentiation, bone mineralization and ossification. We propose that germline CNVs contribute to osteosarcoma susceptibility through deregulation of developmental processes controlled by genes contained within CNVs. Further supporting the notion that germline CNVs in individuals with osteosarcoma are pathogenic, we demonstrate that CNVs are associated with poor patient survival. Finally, we characterize two germline CNVs, at chromosome 1q43 and 2p11.2, which are overrepresented in osteosarcoma patients and propose that they contribute to osteosarcoma susceptibility through effect on neighbouring genes, which could be involved in control of microtubule dynamics and tumour suppression. We further characterize two regions in the tumour genome of osteosarcoma patients that harbour recurrent CN alterations (CNAs). These include deletions at chromosome 3q13.31 and vi ii amplifications at chromosome 7p14.1, which are the most altered regions in osteosarcoma and contest the view that CNAs in osteosarcoma are non-recurrent. Both chromosome 3q13.31 and 7p14.1 CNAs involve genes implicated in carcinogenesis, including LASMP at 3q13.31 and TARP at 7p14.1, while 3q13.31 CNAs also involve two non-coding RNAs. We further show that expression of 3q13.31 genes correlates with the presence of 3q13.31 CNAs. We report that chromosome 3q13.31 and 7p14.1 CNAs are also common in other cancers, identifying these loci as candidates with a global role in carcinogenesis. Supporting the notion that 3q13.31 deletions play a role in osteosarcomagenesis, we find that depletion of 3q13.31 genes promotes proliferation of osteoblasts by regulation of apoptotic and cell-cycle transcripts and also VEGF receptor 1 and that genetic deletions of 3q13.31 are associated with poor survival of osteosarcoma patients. In summary, our study implicates germline and somatic CN changes in osteosarcoma and represents a model approach for elucidation of elements contributing to disease susceptibility and aetiology in human cancer.

The Caenorhabditis elegans Eph RTK, VAB-1, has known roles in neuronal and epidermal morphogenesis as well as oocyte maturation through interaction with its ephrin ligands. In humans, Eph receptors are involved in nervous and vascular system development and have been implicated in cancer formation and progression. DAF-18, a C. elegans ortholog of the human tumour suppressor gene, PTEN, has been identified as an interacting partner with the Eph RTK, VAB-1. Mutations in human PTEN have been associated with numerous cancers and in the worm, DAF-18 is a well studied member of the DAF-2/Insulin receptor-like signaling pathway which has roles in dauer formation, thermotolerance and adult longevity. Our lab has previously shown that VAB-1/EphR binds DAF-18. To further investigate the significance of this interaction as well as offer additional function to the proteins involved, I have shown that VAB-1/EphR is a negative regulator of DAF-18/PTEN at the protein level. Western blotting reveals that endogenous expression of DAF-18/PTEN is low in wild-type animals and expression is increased in a vab-1/ephR mutant. Additionally, VAB-1/EphR and DAF-18/PTEN are expressed in head neurons, oocytes and the germline precursor cells, Z2/Z3. vab-1/ephR mutants show increases longevity and sensitivity to dauer conditions which is consistent with increased DAF-18/PTEN activity. Lastly, daf-18(ok480) is able to suppress the oocyte maturation phenotype and increased MAPK expression displayed by vab-1(dx31) animals, providing genetic evidence of an interaction. By identifying the tissues where these proteins are co-expressed and substantiating the interaction with multiple analyses, novel roles may be proposed for each: VAB-1/EphR in DAF-2/Insulin signaling and DAF-18/PTEN in oocyte maturation downstream of VAB-1/EphR signaling. This work provides further understanding of how an organism coordinates complex developmental processes and reiterates the notion that cellular signaling is a complex network of interacting players. As many signaling pathways are evolutionarily conserved, my research in C. elegans may provide a mechanism on how Eph RTKs and PTEN are regulated in more complex organisms, including humans. / Thesis (Master, Biology) -- Queen's University, 2009-02-27 17:09:10.582

Osteosarcoma is the most common bone malignancy in children and adolescents with poorly understood aetiology. Recently, disease susceptibility and aetiology in several cancers have been associated with genomic copy-number (CN) change. We therefore studied the contribution of CN change in osteosarcoma. We report that individuals with osteosarcoma have increased germline structural variation compared to controls. These CN variants (CNVs) preferentially localize to genes implicated in control of osteoblast differentiation, bone mineralization and ossification. We propose that germline CNVs contribute to osteosarcoma susceptibility through deregulation of developmental processes controlled by genes contained within CNVs. Further supporting the notion that germline CNVs in individuals with osteosarcoma are pathogenic, we demonstrate that CNVs are associated with poor patient survival. Finally, we characterize two germline CNVs, at chromosome 1q43 and 2p11.2, which are overrepresented in osteosarcoma patients and propose that they contribute to osteosarcoma susceptibility through effect on neighbouring genes, which could be involved in control of microtubule dynamics and tumour suppression. We further characterize two regions in the tumour genome of osteosarcoma patients that harbour recurrent CN alterations (CNAs). These include deletions at chromosome 3q13.31 and vi ii amplifications at chromosome 7p14.1, which are the most altered regions in osteosarcoma and contest the view that CNAs in osteosarcoma are non-recurrent. Both chromosome 3q13.31 and 7p14.1 CNAs involve genes implicated in carcinogenesis, including LASMP at 3q13.31 and TARP at 7p14.1, while 3q13.31 CNAs also involve two non-coding RNAs. We further show that expression of 3q13.31 genes correlates with the presence of 3q13.31 CNAs. We report that chromosome 3q13.31 and 7p14.1 CNAs are also common in other cancers, identifying these loci as candidates with a global role in carcinogenesis. Supporting the notion that 3q13.31 deletions play a role in osteosarcomagenesis, we find that depletion of 3q13.31 genes promotes proliferation of osteoblasts by regulation of apoptotic and cell-cycle transcripts and also VEGF receptor 1 and that genetic deletions of 3q13.31 are associated with poor survival of osteosarcoma patients. In summary, our study implicates germline and somatic CN changes in osteosarcoma and represents a model approach for elucidation of elements contributing to disease susceptibility and aetiology in human cancer.

Philosophiae Doctor - PhD / Retinoblastoma Binding Protein 6 (RBBP6) is a 250 kDa multi-domain protein that has been implicated in diverse cellular processes including apoptosis, mRNA processing and cell cycle regulation. Many of these functions are likely to be related to its interaction with tumour suppressor proteins p53 and the Retinoblastoma protein (pRb), and the oncogenic Y-Box Binding Protein-1 (YB-1). RBBP6 inhibits the binding of p53 to DNA and enhances the HDM2-mediated ubiquitination and proteasomal degradation of p53. Disruption of RBBP6 leads to an embryonic lethal phenotype in mice as a result of widespread p53-mediated apoptosis. RBBP6 promotes ubiquitination and degradation of YB-1, leading to its proteasomal degradation in vivo.The first part of this thesis describes in vitro investigations of the interaction betweenbacterially-expressed human p53 and fragments of human RBBP6 previously identified as interacting with p53, in an attempt to further localise the region of interaction on both proteins. GST-pull down assays and immunoprecipitation assays confirmed the interaction, and localised it to the core DNA binding domain of p53 and a region corresponding to residues 1422-1668 of RBBP6. However in Nuclear Magnetic Resonance (NMR) chemical shift perturbation assays no evidence was found for the interaction. NMR showed the relevant region of RBBP6 to be unfolded,and no evidence was found for interaction-induced folding. The R273H mutant of the p53 core domain did not abolish the interaction, in contrast to reports that the corresponding murine mutation (R270C) did abolish the interaction.The second part of this thesis describes in vitro investigations of the ubiquitination of YB-1 by RBBP6. A fragment corresponding to the first 335 residues of RBBP6,denoted R3, was expressed in bacteria and found to be soluble. Contrary to expectation, in a fully in vitro assay R3 was not able to ubiquitinate YB-1. However,following addition of human cell lysate, YB-1 was degraded in an R3-dependent and proteasome-dependent manner, indicating that R3 is required for ubiquitination and proteasomal degradation of YB-1. However R3 is not sufficient, with one or more factors being supplied by the cell lysate. In view of the pro-tumourigenic effects of YB-1 in many human cancers, these results lay the foundation for an understanding of the regulatory effect of RBBP6 on YB-1 and its potential role in anti-tumour therapy.

Serine beta-lactamase-like protein (LACTB) is a tumour suppressor that modulates mitochondrial lipid metabolism and induces differentiation of breast cancer cells. This is achieved by the LACTB-dependent downregulation of phosphatidylserine- decarboxylase (PISD) which subsequently leads to decreases in the amounts of phosphatidylethanolamines and lysophosphatidylethanolamines in mitochondrial membranes. However, PISD was shown to not be a direct substrate of the LACTB enzyme what leaves the identity of the LACTB substrate an open question. To fill this important gap in the mechanism of the LACTB tumour suppressive pathway, this diploma thesis was focused on finding a physiological substrate of LACTB via Proteomic Identification of protease Cleavage Sites (PICS) assay. For this purpose, the other sub-aims of this project were to isolate recombinant wild-type LACTB and its catalytic mutant, to reveal ideal in vitro conditions for LACTB activity and to find out the requirements needed for LACTB multimerization. My results show that in vitro activity of LACTB is increased in the presence of higher pH and calcium ions. I also show that higher LACTB multimeric forms are bound together via disulfide bonds as they disintegrate after treatment with dithiothreitol. Furthermore, and most importantly, I show...

>Magister Scientiae - MSc / This thesis describes the cloning and recombinant expression of domains from the human RBBP6 protein for future in vitro binding studies with pRb and p53. RBBP6 is a splicing-associated protein that is known to interact with both p53 and the Retinoblastoma gene product (pRb), and has recently been shown to be highly upregulated in oesophageal cancer. The pRb binding domain (RbBD) and the p53 binding domain (p53BD) were each expressed using the glutathione-S-transferase (GST) tag affinity system, and affinity purified using a glutathione-linked agarose column. Purified fusion proteins were cleaved to separate the target protein from GST using PreScission ™ Protease, for which there is a recognition sequence located immediately upstream of the multiple cloning site on the pGEX-6P series of plasmids. The pRb binding and p53 binding domains were further purified using cation exchange chromatography. Mass spectrometry confirmed that the RbBD was expressed as a single species of the expected molecular weight. However preliminary NMR analysis suggested that the domain was not fully folded. A total yield of 8 mg of protein was achieved from 11 of culture, which make it feasible to express 15Nand 12Clabelled samples for NMR. The p53BD was found to be expressed at lower levels and subject to C-terminal degradation, which suggest that the C-terminus is unstructured most likely due to the presence of polylysine tail. Human pRb protein was also successfully expressed and purified using the GST affinity system. Human p53 protein was expressed but was found to be insoluble and attempts to purify it were not pursued. Attempts to confirm the interactions between human RBBP6 and p53 and pRb proteins are on-going but fall outside the scope of this thesis. Expression constructs for the RING and zinc finger domains from human RBBP6 were also cloned into the pGEX system for future structural studies using NMR. Both domains were found to be expressed as soluble fusion proteins in preliminary expression studies.

A Thesis Submitted to the School of Graduate Studies in the Partial Fulfillment of the Requirements for the Degree Master of Science / Caenorhabditis elegans and one of its close relatives, Caenorhabditis briggsae, are animal models that are commonly used for comparative studies to understand the evolution of developmental mechanisms and gene function. Although the two species appear nearly identical morphologically, comparative genomic analyses have revealed interesting differences between the genomes. Whether such differ- ences contribute to changes in developmental mechanisms and signalling pathways is an active area of research. One of the most well studied phenotypes associated with C. elegans signalling pathways are those that affect the specification of vulval tissue. Within the system of vuval development, mutants that exhibit the Mul- tivulva (Muv) phenotype are important as they show inappropriate divisions of vulva cells, which model tumour formation. Comparing gene function in different species genetic backgrounds can lead to an understanding of how genetic differ- ences contribute to different responses in cancer development. Genetic screens, conducted in our laboratory, yielded several genes whose loss of function results in a Muv phenotype and identified a novel regulator of C. briggsae vulval devel- opment, Cbr-ivp-3. Using the nematode C. briggssae as experimental system, we have characterized the tumour suppressor gene, Cbr-ivp-3, which impacts cell sig- nalling and cell division. I have carried out molecular genetic analyses of ivp-3 in both C. briggsae and C. elegans and have begun to characterize the functional role of Cbr-ivp-3. The findings in this thesis suggest that Cbr-ivp-3 is functioning to negatively regulate EGF/Cbr-lin-3. / Thesis / Master of Science (MSc) / The nematodes Caenorhabditis elegans and Caenorhabditis briggsae, are commonly used for comparative studies to understand the evolution of developmental mechanisms and gene function. Although both species appear morphologically similar, comparative genomic analyses reveal differences between genomes. Comparing gene function in different genetic backgrounds can lead to an understanding of how genetic differences contribute to different responses in cancer development. Genetic screens have yielded several genes whose loss of function results in a Multivulva phenotype, showing inappropriate division of vulva cells, modeling tumor formation. We have carried out molecular genetic analyses of ivp-3, a novel regulator of C. briggsae vulval development, in both species and have found that Cbr-ivp-3 is regulating vulva development by negatively regulating EGF/Cbr-lin-3.

Caenorhabditis elegans and its congener, C. briggsae are excellent animal models for the comparative study of developmental mechanisms and gene function. Gupta lab is using the vulval tissue in these nematodes as a system to investigate conservation and divergence in signal transduction pathways. Genetic screens conducted earlier in our laboratory recovered several mutants that cause multivulva (Muv) phenotype. The Muv genes act as tumor suppressors and negatively regulate the proliferation of vulval precursors. Genetic and molecular work on these genes has revealed that C. briggsae vulva developmental utilizes novel genes representing a new phenotypic class termed ‘Inappropriate Vulva cell Proliferation (IVP)’ (Sharanya et al., 2015). This indicates that the signaling mechanism in C. briggsae specifies vulval cell fates differently from C. elegans. Interestingly, it has been found that Cbr-ivp mutants show higher levels of Cbr-lin-3 (EGF) transcript, indicating that these genes act genetically upstream of Cbr-lin-3, similar to SynMuv family members in C. elegans. Moreover, RNAi knockdown of the Cbr-lin-3 transcript resulted in the suppression of the multivulva phenotype in mutant animals. Similar suppression was also observed when a MAP kinase inhibitor was used in the previous study. In addition, the role of two other novel negative regulators of cell proliferation, Cbr-lin(bh1) and Cbr-lin(bh3) was also investigated. Preliminary findings on these regulators suggested that both Cbr-lin(bh1) and Cbr-lin(bh3) exhibiting a heritable Muv phenotype and are found to be located on Chromosome I and III respectively. Identification of novel genes and further characterization will help us understand the molecular function of genes and their involvement in the regulation of vulval cell differentiation. The findings of my research work will provide a background for future studies to understand the role of novel genes in reproductive system development. Overall, these results provide evidence that although the morphology of vulva is similar in the two nematode species, underlying mechanisms of development appear to have diverged. / Thesis / Master of Science (MSc)

Genomic DNA is constantly exposed to assaults, which if not dealt with, can lead to genomic instability and carcinogenesis. In response to stress including either Ionising Radiation (IR) or replication stress, ATM and ATR promote the activation of cell cycle checkpoints and initiate repair of DNA damage. Recent studies have revealed that ATM signalling can activate LATS1 via a cascade through RASSF1A and MST2. LATS1 is a tumour suppressor, which forms a barrier to carcinogenesis restricting cell proliferation and promoting apoptosis by stabilising a YAP/p73 transcriptional complex, hence upregulating p73 responsive genes. LATS1 is inactivated through promoter hypermethylation in a number of cancer types including breast cancer and soft tissue sarcoma. This research project seeks to define the mechanism through which LATS1 is involved in IR-induced DNA damage signalling. The data presented in this thesis shows that LATS1 controls CDK2 and regulates phosphorylation of S3291 on BRCA2. Cells lacking LATS1 exhibited enhanced accumulation of damage-induced Rad51 foci leading to cell cycle arrest at the G₂/M checkpoint. Furthermore, the data presented here suggests that LATS1 may not be required for homologous recombination. This work supports the hypothesis that LATS1 inhibits CDK2-dependent phosphorylation of BRCA2 at S3291, hence protecting stalled replication forks from nucleolytic degradation.