published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Epithelial ovarian cancer (EOC) is difficult to detect in early stage disease, resulting in a high mortality rate. The molecular events underlying EOC development remain largely unknown. Chromosome 3 exhibits frequent deletions and rearrangements in EOC by cytogenetic analysis. In addition, loss of heterozygosity (LOH) mapping of matched ovarian tumour and constitutional DNA samples exhibits specific regions of chromosome 3 loss involving distinct regions: 3p25-p26, 3p24 and a region proximal to 3p14. Thus, chromosome 3p loss points to the location of tumour suppressor genes (TSG) implicated in tumourigenesis, based on Knudson's 'two-hit' model and the paradigm of the classical TSG. The dissertation hypothesis states at least one TSG implicated in EOC is located on chromosome 3p. A novel complementation approach based on the transfer of normal chromosome 3 fragments into OV-90, a tumourigenic EOC cell line harbouring LOH of the 3p arm, was used to generate functional evidence for chromosome 3p TSGs. Three hybrids exhibited complete suppression of tumourigenic potential based on the inability to form colonies in soft agarose, spheroids in cell culture, and tumours in nude mice xenograft models. While all hybrids had acquired various chromosome 3 regions, they all shared in common a 3p12-pcen interval, suggesting at least one common gene may have affected the suppression of tumourigenicity in the OV-90-derived hybrids. Twelve known/hypothetical genes mapping to 3p12-pcen region were characterized based on gene expression and mutation analysis following a classical model for TSG inactivation. To establish the relevance to EOC, gene expression of candidates was investigated in primary cultures of normal ovarian surface epithelial cells and both malignant serous and benign serous tumour samples. The gene expression and genetic analysis identified seven TSG candidates, none of which appeared to be mutated or transcriptionally silenced based on classical mechanisms of TSG inactivation in OV-90, thus suppression of tumourigenicity may have resulted from the functional complementation of one more haploinsufficient 3p12-pcen genes. Several genes (GBE1, VGLL3, ZNF654 ) appeared underexpressed in malignant tumours and these findings suggest the intriguing possibility that more than one 3p12-pcen gene was involved in the suppression of tumourigenicity in OV-90, and by extension, EOC.

The Hippo-LATS signaling pathway originally identified in Drosophila is conserved in mammalian systems and serves essential roles in mediating size control as well as tumorigenesis. In humans, the core kinase cassette consisting of adaptor proteins WW45 and MOB1, and Ser/Thr kinases MST1/2 and LATS1/2 signal by phosphorylating and inactivating transcriptional co-activators YAP and TAZ, causing cell growth arrest. As the central kinases within the Hippo-LATS pathway, examining the cellular and molecular phenotypes of LATS1 and LATS2 (LATS) will provide insight into the role of this pathway in tumorigenesis. By simultaneously knocking down both LATS1 and LATS2, genes that were differentially expressed were identified through a whole human genome microarray screen. The multitude of genes identified including CYR61, MYLK, CDKN1A, SLIT2, and TP53INP1 not only provide further evidence for the role of LATS in cell proliferation and apoptosis, but also implicate LATS in novel functions such as cell motility. Loss of LATS1 and/or LATS2 enhances cell migration whereas overexpression of LATS1 dramatically inhibits cell migration in multiple cell lines. The ability of LATS to regulate cell migration occurs through two potential mechanisms. Firstly, LATS functions through its kinase substrates YAP and/or TAZ, or alternatively, LATS1 directly binds actin and inhibits actin polyermization. Thus, through loss of functions studies, we identified a novel role for LATS in regulating cell migration as well as novel mechanisms of LATS function. As an important signaling molecule within the cell, LATS and the Hippo-LATS pathway are tightly regulated. Using clues from the Drosophila pathway, we examined how the previously uncharacterized gene, hEx, functions within this pathway. Importantly, this thesis characterizes hEx as a putative tumor suppressor showing that it can inhibit cell proliferation, sensitize cancer cells to Taxol treatment as well as inhibit tumor growth in nude mice. However, unlike Drosophila expanded, hEx functions independently of the Hippo-LATS pathway, suggesting that the mammalian signaling pathway is more complicated. The research findings from this thesis enhance our knowledge of the Hippo-LATS pathway in tumorigenesis by elucidating new functions and mechanisms of LATS functions as well as by exploring how upstream components function in relation to this pathway. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2011-09-23 10:06:54.687

This thesis investigated the cooperation of the Kras2 oncogene with the tumor suppressor gene Dlc1 in lung tumor development. Dlc1 is a negative regulator of RhoGTPase proteins, which are mainly involved in the regulation of the actin cytoskeleton and cell migration. We hypothesized that loss of Dlc1 expression leads to more aggressive tumors, which should also result in increased incidence of metastasis. All experiments were performed in mice containing a heterozygous oncogenic Kras allele and a heterozygous gene trapped Dlc1 allele (KD) and in mice only carrying the oncogenic Kras allele (K+). Throughout all experiments we have consistently found no significant differences between the two groups in terms of tumor burden (tumor numbers, sizes and areas), metastases or methylation patterns. These results suggest that heterozygous downregulation of Dlc1 is not enough to increase tumor formation and metastasis development in the Kras lung tumors.

This thesis investigated the cooperation of the Kras2 oncogene with the tumor suppressor gene Dlc1 in lung tumor development. Dlc1 is a negative regulator of RhoGTPase proteins, which are mainly involved in the regulation of the actin cytoskeleton and cell migration. We hypothesized that loss of Dlc1 expression leads to more aggressive tumors, which should also result in increased incidence of metastasis. All experiments were performed in mice containing a heterozygous oncogenic Kras allele and a heterozygous gene trapped Dlc1 allele (KD) and in mice only carrying the oncogenic Kras allele (K+). Throughout all experiments we have consistently found no significant differences between the two groups in terms of tumor burden (tumor numbers, sizes and areas), metastases or methylation patterns. These results suggest that heterozygous downregulation of Dlc1 is not enough to increase tumor formation and metastasis development in the Kras lung tumors.

Deletion of the long arm of chromosome 6 is one of the most common genetic alterations in human malignant melanoma. Recently, a reciprocal translocation between chromosomes 6q and 17p was detected in a melanoma cell line, UACC-930, using arm painting probes of 6p and 6q. Reciprocal translocation is seldom observed in solid tumors. Upon further characterization of the translocation marker using techniques such as Southern blotting, genomic library screening and DNA sequencing, a complex rearrangement including two inversions of 6q and a translocation between the inverted 6q and 17p, [der(6)inv(6)(q21q22)(q22q27)t(6;17)(q27;p13)], was detected. An NCBI blast search revealed 3 genes being interrupted by the breakpoints: prenyl diphosphate synthase subunit 2 (PDSS2) at 6q21, Parkin at 6q27 and p53 at 17p13. Down-regulation of PDSS2 was commonly observed in 59/87 (67.8%) primary melanomas, which was significantly higher than that in benign nevi (7/66, 10.6%, p<0.001), indicating the tumor-suppressive potential of PDSS2 in melanoma development. To characterize the function of PDSS2 in tumorigenesis, PDSS2 was stably transfected into a highly tumorigenic melanoma cell line, UACC-903. The tumor-suppressive function of PDSS2 was demonstrated by both in vitro and in vivo assays. The results showed that PDSS2 could inhibit tumor cell growth, decrease the colony-forming ability in soft agar, and totally abrogate the tumorigenicity of UACC-903 in nude mice. PDSS2 is the first enzyme involved in the CoQ10 biosynthesis pathway. Other studies have demonstrated PDSS2 mutations can cause severe CoQ10 deficiency and markedly reduced ATP production because of respiratory chain dysfunction. Interestingly, proteomics analysis revealed 7 out of 11 identified proteins (HSPA8, GAPDHS, TPI1, HSPA5, PGK1, ENO1, and ATP5B) differentially expressed in PDSS2-overexpressing cells were related to energy metabolism. Further studies are required to determine how PDSS2 could alter the energy supply in tumor cells. Taken together, these results support the proposal that PDSS2 is a novel tumor suppressor gene which may play an important role in the development of malignant melanoma via altering tumor metabolism.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Thesis (Ph.D.). / Written for the Dept. of Anatomy and Cell Biology. Title from title page of PDF (viewed 2008/05/12). Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "August 2007" Includes bibliographical references.

Thesis (PH.D.)--Michigan State University. Cell and Molecular Biology, 2008. / Title from PDF t.p. (viewed on Aug. 11, 2009) Includes bibliographical references. Also issued in print.