ABSTRACT There is an increasing incidence of neoplasms in the kidney and a poor prognosis for patients who are diagnosed with advanced or metastatic kidney cancer. Renal cell carcinoma (RCC) constitutes the most prevalent form of kidney neoplasm in the adult population. Although surgery or cryoablation are successful curative treatments for localized RCC, improved diagnostic methods facilitating early detection and characterization of renal tumours may enable more effective use of less invasive treatments, especially for metastases. Currently there are no suitable tumour markers available for diagnostic, prognostic or predictive purposes. By increasing our knowledge of the underlying molecular characteristics of RCC, we may be able to identify molecular pathways involved in tumour growth and metastasis, and this knowledge may expedite the development of targeted therapies, and may identify useful markers of RCC development and progression. This thesis aimed to identify new genes involved in resistance to cancer therapy in RCC and to analyse their incidence and test their function in human RCC tissue, using immunohistochemistry in a large cohort of patients with RCC and paired normal tissue, and in vitro models. The lack of induction of apoptosis in RCCs by conventional cancer therapies such as chemotherapy, immunotherapy or radiation is central to their resistance to treatment. If apoptotic pathways that are activated in successful treatments were identified, they might be used for targets in future therapies. The hypothesis tested in this thesis was that genes or proteins involved in the molecular control of apoptosis, identified from RCC cell culture models and RNA microarray, will be useful for molecular profiling in human RCCs of different subtypes, as markers of those specific subtypes of the RCCs, as indicators of prognosis of the diagnosis, or as targets for future therapy regimens. The broad aims of this project were: To establish a model in which significantly increased apoptosis in RCCs in cell culture could be correlated with alterations in apoptotic pathway genes, to investigate the functional significance of some of those genes; and to maximise information gained from these basic experimental laboratory studies on new apoptosis genes in RCC development and progression by determining their expression patterns in tissue microarrays (TMAs) generated from different subtypes of human RCCs. The specific aims of the project were: 1) To establish an RCC cell culture model with high levels of induced apoptosis for RNA analyses using microarray to identified apoptotic genes that are novel in RCC investigations; 2) To describe the role and functional significance of some of the novel RCC apoptosis pathway genes using molecular investigations, including silencing RNA techniques; and 3) To use TMAs and immunohistochemistry to determine whether RCC subtypes can be distinguished by protein expression profiles of selected new apoptosis pathway genes. The thesis is presented as a literature review (Chapter 1), materials and methods (Chapter 2), four original research segments (Chapters 3 to 6), and finally a segment that summarises the results and presents future directions (Chapter 7). The first of the original research Chapters (Chapter 3) addressed a question “can apoptosis in RCCs be induced in cell culture to such a level that apoptotic pathways may be analysed?” Two RCC cell lines (ACHN and SN12K-1) were treated with IFN-a (500IU/mL), radiotherapy (20 Gy) or dual therapy of these two treatments. Apoptosis was quantified using microscopy and morphological characteristics and verified using enzymatic labelling of cells. The ACHN cell line, treated with the dual therapy and analysed at 24 hours, had a significantly increased level of apoptosis (p<0.05) compared with the non-therapy treated controls, and negligible mitosis. The increased expression or activation of at least some known apoptotic pathway genes (Bcl-2, Bax, caspase-3, 8 and 9, and p53) was verified in this model. The ACHN/dual therapy model was selected for further study. The second of the original research Chapters (Chapter 4) addressed the question “what apoptosis-regulating genes are significantly different in the apoptotic ACHN cells?” An RNA microarray assay (112 apoptosis-related genes) was carried out using RNA extracted from treated and non-treated ACHN cells and analysed for alterations of at least 1.9-fold in transcript levels. 21 genes had upregulated transcript levels in the treated cells, and one had down-regulated transcript levels. A search of the literature revealed three gene families with altered transcript levels in the treated RCCs that were novel: the TNF receptor-associated factor (TRAF), caspase recruitment domain (CARD) and cell death-inducing DFF-45 effector (CIDE) gene families. Representative members of these families were then investigated for protein expression alterations. The results for one particular gene, TRAF1, indicated it might be worthy of further study in modulation experiments (Chapter 6). The next research chapter (Chapter 5) asked the question “since the ACHN cells express TRAF-1 and have increased TRAF-1 with increasing apoptosis, what happens to the levels of mitosis and apoptosis in these cells when TRAF-1 expression is knocked down?” Silencing RNA (siRNA) techniques were used to knock down TRAF-1 in the ACHN RCC cell line using the same model as was described in Chapter 3. Successful knock down was gained after 72 hrs of transfection with a commercially-available siRNA against TRAF-1. These cells were then treated with the radiation, IFN-α or dual therapy treatments. In the cells with TRAF-1 knock down, there was significantly less apoptosis and more mitosis than was seen in the non-transfected cells. These results indicate that TRAF-1 does play a functional role in induction of apoptosis in RCCs and is worth investigating further in targeted therapies. The final of the research Chapters (Chapter 6) looked at the molecular distinctions among ccRCC, papillary, collecting duct, chromophobe and unclassified types of RCC. Molecules that might distinguish one from another would be valuable clinically, and it would be especially valuable if these molecules could also be targeted for therapeutic benefit. By knowing the action of these molecules in the apoptotic pathways used by RCCs when they do regress or die during cancer therapies, we might be able to devise new targeted therapies for RCCs. This research Chapter asked the question “could molecular profiling with an array of apoptotic pathway genes, novel to investigations in RCCs, provide information that would distinguish the subtypes?” TMAs prepared from 121 RCC and paired normal patient samples, where available, were investigated. Most RCC samples were ccRCC. Antibodies against selected members of the TRAF), CARD and CIDE gene families were selected from the RNA microarray data. These genes (TRAF1, TRAF3, TRAF4, inhibitor of caspase-activated DNAase/ICAD and nucleolar protein-3/NOL3) were analysed in TMAs using immunohistochemistry and digital scanning or bright field microscopy and graded scales of intensity and distribution, blinded to sub-type of RCC. After microscopy, scores for subtypes of RCC were compared with their normal tissue. Significant differences were found for TRAF1 and NOL3. The results indicate TRAF1 and NOL3 have potential for improving outcome or diagnosis in RCCs. In summary, there are no effective treatments against metastatic RCC, and no suitable grade or stage-defining biomarker for metastatic RCC subtypes. One of the main reasons for therapy resistance in RCCs is their inability to use or activate apoptotic pathway molecules. By investigating the reasons for RCC resistance to cancer therapies, we may be able to improve both diagnosis and treatment strategies. By defining the mechanisms and pathways of resistance to therapy-induced apoptosis, and developing methods to manipulate the pathways to cell death to defeat therapy resistance, we will have a better chance to develop successful markers of RCC subtypes and also design new and successful RCC therapies.
| Identifer | oai:union.ndltd.org:ADTP/279260 |
| Creators | Retnagowri Rajandram |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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