published_or_final_version / Medicine / Master / Doctor of Medicine

published_or_final_version / Pathology / Master / Master of Medical Sciences

Oncogenic mutations in lung cancer further our knowledge about cancer initiation and progression, and may guide personalized treatment. The fact that targeted therapy is most effective in subsets of patients with defined molecular targets indicates the need for classification of clinically-related molecular tumor phenotypes based on the presence of oncogenic mutations, including EGFR mutations and EML4-ALK rearrangements. The identification of EGFR mutations, in up to half of lung adenocarcinomas in Asians, could predict clinical sensitivity to tyrosine kinase inhibitor (TKI). However, testing for mutations is not always possible due to tumor tissue availability. The therapeutic decision sometimes remains a clinical one especially for elderly lung cancer patients but no known mutation status. We studied the survival outcomes of targeted therapy versus conventional chemotherapy in elderly patients with lung cancer when we did not yet have routine EGFR mutation testing and demonstrated comparable survival outcomes in targeted therapy compared to chemotherapy, implying that survival with targeted therapy could be better if the treatment population could be selected with EGFR mutations. Though testing for EGFR mutation in tumor biopsy have later become routine practice and remains the accepted reference for therapeutic decision, the detection of EGFR mutations in plasma DNA with high diagnostic performance will be useful adjunct for diagnostic and therapeutic monitoring. Among patients with EGFR mutations in tumor biopsy, the concurrent detection of EGFR mutation in plasma DNA was found to confer a less favorable prognosis in terms of overall survival than those patients with EGFR mutations in tumor biopsy but the corresponding mutation was not detected in plasma. Other oncogenic mutations with therapeutic implications in lung tumors are yet to be fully explored, like ALK, KRAS, ROS1 or NTRK1 mutations. It is not exactly the tumor – but the mutations in the tumor that need to be explored with reference to clinical behavior. Even with EGFR mutation with well-established clinical implications, further exploration into its mechanistic functions will help in understanding of drug resistance. Lung cancer cell lines established from patients with known mutation profiles could be useful tools for studying the biology of known molecular targets as well as for therapeutic testing. Four new lung adenocarcinoma and one mesothelioma cell lines were established from patients with different clinical characteristics and oncogenic mutation profiles. These cell lines with defined mutation profiles will provide tools for exploration of lung cancer and mesothelioma biology with respect to molecular therapeutic targets. The Large Tumor Suppressor 2 (LATS2) gene was a differentially expressed gene between EGFR mutant and wildtype lung adenocarcinomas. The differential LATS2 expression levels were predictive of survival in patients with resected lung AD and may modulate tumor growth via different signaling pathways in EGFR mutant and wild-type tumors. The identification of oncogenic mutations has led to a new paradigm of targeted therapy in lung cancer. Further improvements in outcome of lung cancer management will stem from research into the biology of oncogenic mutations and their clinico-pathological correlations, which would fuel parallel development of clinically efficacious targeted therapies. / published_or_final_version / Medicine / Master / Doctor of Medicine

Non-small cell lung cancer (NSCLC) causes most of the cancer deaths worldwide. Tyrosine kinase inhibitors (TKIs), like erlotinib and crizotinib, are commonly used as specific treatments targeting epidermal growth factor receptor (EGFR)-mutated and anaplastic lymphoma kinase (ALK)-rearranged NSCLC. Autophagy is a highly conserved cellular process in response to stress. Tumor microenvironment (TME) is composed of both tumor cells and stromal cells. This study aimed to investigate whether autophagy could confer intrinsic and acquired resistance to TKIs in NSCLC, and its role in the presence of TME or in animal models. In the first part of this study, the effect of EGFR TKI or ALK TKI on sensitive NSCLC cells to generate autophagy was investigated, and manipulation of autophagy in these cell lines was performed. Autophagy inhibition was shown to enhance apoptotic effect of TKIs in sensitive NSCLC cells. This part provided strong evidence that TKIs and autophagy inhibitor chloroquine (CQ) work synergistically in sensitive NSCLC cells. Autophagy induction by erlotinib treatment was observed in a HCC827 (lung adenocarcinoma, EGFR exon 19 del) xenograft model, which was in line with the in vitro observation. Correspondingly, the combination of erlotinib (12.5 mg/kg) with CQ (50 mg/kg) in the HCC827 xenograft model achieved greater tumor growth suppression, compared with single drug treatments. In the second part of this study, a model of TME was established to allow study of autophagy under such circumstances. An activated TME with cytokine production, autophagy induction and epithelial-to-mesenchymal transition (EMT) was generated by co-culturing NSCLC cells and human fibroblasts. Sensitivity to TKI under TME was not affected, and combination of chloroquine with TKI under TME remained synergistic compared with single treatments. In the third part of this study, erlotinib-resistant (ER) HCC827 cells were acquired by stepwise exposure to increasing concentrations of erlotinib in cell culture. Common acquired resistance mechanisms to EGFR TKI (EGFR T790M or c-MET amplification) were excluded in this ER HCC827 model, except EMT. Autophagy status in ER HCC827 cells was studied and autophagy manipulation was performed. It was found that CQ and erlotinib worked synergistically to induce cell death even in ER HCC827 cells. In an ER HCC827 xenograft model, significant degree of autophagy and EMT was evident. Interestingly, combining erlotinib (25 mg/kg) with CQ (50 mg/kg) showed better inhibitory effect on tumor growth compared with single treatments. In summary, TKIs induced both apoptosis and autophagy in EGFR-mutated and ALK-rearranged NSCLC cells. Autophagy inhibition by CQ enhanced TKI-induced cell death in sensitive cells. The presence of TME did not confer TKI resistance. Autophagy was highly activated in EGFR-mutated NSCLC cells with acquired resistance to erlotinib. Combination of CQ with erlotinib remained synergistic in the presence of TME and acquired resistance, both in vitro and in vivo. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Community Medicine / Master / Master of Public Health

Background: A human ribosomal protein L9 (RPL9) encodes a protein that is a component of the 60S subunit. RPL9 is located on chromosome 4p14 and is approximately 5.5 kb in length and contains 8 exons. The message for human RPL9 is 712 nucleotides long. Some of the functions of RPL9 documented so far include the crucial involvement of the gene product in cell proliferation and protein biosynthesis. Lipoic acid synthetase (LIAS) is a 1.73 kb gene also located at chromosome 4p14. Alternative splicing occurs at these locus and two transcript variants encoding distinct isoforms have been identified but in this study the results represents both isoforms together. The protein encoded by LIAS gene belongs to the biotin and lipoic acid synthetases family and localizes in the mitochondrion. Function of lipoic acid synthetase is not yet well documented. Some studies have attempted to characterise its function by looking at the biological pathways at which LIAS gene product plays a crucial role, for example the biosynthesis of alpha-lipoic acid. Alpha lipoic acid is a natural antioxidant and it is also naturally-occurring enzyme co-factor found in a number of multi-enzyme complexes regulating oxidative metabolism. Motivation for study: RPL9 and LIAS were previously found to be mutated in CHO (Chinese Hamster Ovary) cell lines and these mutant lines had gained resistance to apoptosis. Aim: The main objective of this study was to evaluate the expression pattern of RPL9 and LIAS in lung cancer and to characterise their role in apoptosis and also to determine if the expression pattern of this genes varies between normal and diseased state of the tissue. Methods: In Situ hybridization, quantitative Real Time PCR, TUNEL and Bio-informatics have been employed in order to attain the objectives of this study. Results: In Situ hybridization showed that RPL9 localises in the cytoplasm and it is up-regulated in lung cancer relative to normal lung. LIAS localises in the cytoplasm and it is also up-regulated in lung cancer. The expression of RPL9 was relatively higher than that of LIAS determined by the intensity of localisation. Quantitative real time PCR confirmed the up-regulation of RPL9 and LIAS in lung cancer. RPL9 and LIAS were found to be up-regulated 8 and 4 fold respectively in lung A549 lung adenocarcinoma relative to MRC5 normal lung fibroblast cell lines. TUNEL showed the highest DNA fragmentation in adenocarcinoma, followed by squamous cell lung carcinoma then large cell lung carcinoma which is the same pattern observed in RPL9 and LIAS mRNA localisation by In Situ hybridization. To further characterise the role of RPL9 and LIAS in human, Bio-informatics tools were used and the results revealed that RPL9 is highly conserved through evolution, up-to 100 % identical to chimpanzee and 98 % to mouse. LIAS was found to be 91 % identical to rat and 90 % identical to mouse. It has been documented that the rate of conservation of a gene in evolution is believed to be correlated with its biological importance and its number of protein–protein interactions. Conclusion: All these discoveries coupled with resistance to apoptosis of CHO cell line in which RPL9 and LIAS were found to be mutated following promoter-trap mutagenesis, strongly suggests that RPL9 might be playing a role in cell cycle and apoptosis. RPL9 has been highly conserved through evolution. Manipulation of this gene can lead to greater biological discoveries in cancer research and the elevated expression of RPL9 can be used as a molecular marker for early detection of cancer.

University of Macau / Institute of Chinese Medical Sciences

published_or_final_version / Pathology / Master / Master of Philosophy