Bibliography: leaves 214-236. / xviii, 238 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physiology, 1999

[Truncated abstract] For this thesis, the molecular biology of cancer was approached from two directions. Firstly, an investigation was conducted on the role of growth factor receptor-bound protein 7 (Grb7) in breast cancer. Grb7 is an adapter molecule that binds to a variety of proteins, including the growth factor receptor and proto-oncogene, ErbB2, and mediates signalling to downstream pathways. It has been linked to cell migration and an invasive phenotype, and is of interest as a therapeutic target. To investigate the role of Grb7 in breast cancer, preliminary experiments were performed that, firstly, determined the expression of wild-type Grb7 and a splice variant, Grb7V, in a range of cell lines, and secondly, aided the development of a protocol for treating cells with short interfering RNA (siRNA) against Grb7 and the ErbB ligand, heregulin (HRG), in a cell system appropriate for measuring the functional outcomes. Using this protocol in conjunction with CellTitre (CT) proliferation assays, it was demonstrated that Grb7 does not play a role in the proliferation of either unstimulated or HRG-stimulated SK-BR-3 breast cancer cells. Furthermore, using the protocol in conjunction with Boyden chamber migration assays, it was shown that inhibition of Grb7 expression has a slight stimulatory effect on HRG-stimulated SK-BR-3 cell migration. Thus, Grb7 was found to play only a minor role in the migration of SK-BR-3 cells, suggesting that it is not an ideal anti-cancer target for breast cancers modelled by this cell system. Concurrently, a second investigation was conducted, which similarly sought insight into the molecular biology of cancer, but adopted a more strategic approach. ... These results provide evidence for a biologically significant role for the miR-7-mediated regulation of EGFR expression. A microarray experiment was also performed to identify genes that were down-regulated following treatment with miR-7 compared to NS precursor. Of 248 down-regulated genes, including EGFR, 37 promising new miR-7 target candidates were identified. Functional clustering of down-regulated genes and promising target candidates suggested that miR-7 may have functionally-related targets involved in processes including cell motility and brain-associated functions. This investigation thus yielded a program capable of accurately predicting a miRNA target not predicted by any other target prediction program, verified a previously unknown miRNA:target interaction with functional consequences in cancer cells and provided the first steps towards investigating miR-7-mediated regulation in greater depth. Furthermore, EGFR was, to our knowledge, the first example of a verified miRNA target with target sites that are not conserved across mammals, an observation with important implications for computational target prediction and the evolution of miRNA regulatory systems. In addition, the demonstrated growth inhibitory and cytotoxic effects of miR-7 on lung cancer cells raise the possibility of a miR-7-based therapeutic for the treatment of EGFR-overexpressing tumours.

Indiana University-Purdue University Indianapolis (IUPUI) / ALDH isoenzymes are known to impact the sensitivity of certain neoplastic cells toward cyclophosphamides and its analogs. Despite its bone marrow toxicity, cyclophos-phamide is still used to treat various recalcitrant forms of cancer. When activated, cyclo-phosphamide forms aldophosphamide that can spontaneously form the toxic phospho-ramide mustard, an alkylating agent unless detoxified by ALDH isozymes to the carbox-yphosphamide metabolite. Prior work has demonstrated that the ALDH1A1 and ALDH3A1 isoenzymes can convert aldophosphamide to carboxyphosphamide. This has also been verified by over expression and siRNA knockdown studies. Selective small molecule inhibitors for these ALDH isoenzymes are not currently available. We hypothe-sized that novel and selective small molecule inhibitors of ALDH3A1 would enhance cancer cells’ sensitivity toward cyclophosphamide. If successful, this approach can widen the therapeutic treatment window for cyclophosphamides; permitting lower effective dos-ing regimens with reduced toxicity. An esterase based absorbance assay was optimized in a high throughput setting and 101, 000 compounds were screened and two new selective inhibitors for ALDH3A1, which have IC50 values of 0.2 µM (CB7) and 16 µM (CB29) were discovered. These two compounds compete for aldehyde binding, which was vali-dated both by kinetic and crystallographic studies. Structure activity relationship dataset has helped us determine the basis of potency and selectivity of these compounds towards ALDH3A1 activity. Our data is further supported by mafosfamide (an analog of cyclo-phosphamide) chemosensitivity data, performed on lung adenocarcinoma (A549) and gli-oblastoma (SF767) cell lines. Overall, I have identified two compounds, which inhibit ALDH3A1’s dehydrogenase activity selectively and increases sensitization of ALDH3A1 positive cells to aldophosphamide and its analogs. This may have the potential in improving chemotherapeutic efficacy of cyclophosphamide as well as to help us understand better the role of ALDH3A1 in cells. Future work will focus on testing these compounds on other cancer cell lines that involve ALDH3A1 expression as a mode of chemoresistance.