- Title
- Investigating the genetics of the development of lung cancer
- Creator
- Chimankar, Vrushali Kashinath
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2021
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Background: Lung Cancer (LC) is one of the most commonly diagnosed cancers and is a leading cause of cancer-related death worldwide. Cigarette smoking is the major risk factor responsible for the development of LC. Despite the advances in cancer therapeutics, LC has a poor survival rate of ~15% over five years. The current image-based diagnostic techniques detect LC when the tumour is already at an advanced stage or metastasised. Since we do not have the data on genetic alterations that takes place early in the development of LC (preneoplastic lesions), the currently available biomarker-based diagnostic techniques also fail in early diagnosis. The main problem with obtaining data on genetic alteration for preneoplastic lesions is the difficulty in tissue collection from humans when the tumour is at early stages. However, since mouse models can be manipulated to develop different stages of LC, the tumour tissue can be collected at different stages and analysed to identify genetic alterations responsible for preneoplastic lesions. Hypothesis and Aims: Our laboratory has previously developed a mouse model that develops bronchioalveolar adenoma (BAA) (early stage of LC) in response to cigarette smoke and tobacco carcinogen 4-methylnitrosamino-3-pyridyl-1-butanone (NNK). We hypothesise that this mouse model could be used as a reference to establish clinically relevant mouse models that develop both BAA and bronchioalveolar carcinoma (BAC) (late stage of LC). Performing whole genome sequencing on tumours isolated from a mouse model that develops BAC will enable the identification of genetic alteration responsible for BAC. The validation of these genetic alterations in mouse models that develop BAA will further enable identification. of genetic alteration that occurs early in the development of LC. Methods: The female A/J mice were treated with 2 carcinogens, cigarette smoke (CS) and NNK. The order of cigarette smoke exposure and NNK administration varied based on the mouse models. The carcinogen treatment was followed by an air recovery period. Histological analysis of the lung was assessed by staining lung sections with haematoxylin and eosin to determine the tumour type, tumour incidence and multiplicity. The airway inflammation was assessed by enumerating the inflammatory cells present in the bronchoalveolar lavage fluid that was collected and processed during the endpoint. Lung function was also analysed using the forced oscillation technique to determine the functional changes in the lung in response to CS exposure and NNK administration. For genome analysis, whole-genome sequencing was performed on DNA extracted from a mouse model where NNK treated mice were exposed to CS for 36 weeks, followed by an air recovery period of 27 weeks (3xNNK+36wk CS+27wk air recovery period) using Illumina NovaSeq 6000 platform. The resultant WGS data were analysed using bioinformatics tools. Results: Mouse models where the female A/J mice were treated with 3 doses of NNK followed by 12, 18, 24, and 36 weeks of CS followed by 12, 18, 24 and 27 weeks of air recovery period respectively developed BAA. The mouse model where NNK (3 doses) treated mice were exposed to 24 and 36 weeks of CS followed by 24 and 27 weeks of air recovery period respectively also developed BAC along with BAA. The mouse model where NNK (3 doses) treated mice were exposed to 12 and 36 weeks of CS followed by 12 and 27 weeks of air recovery period showed 100% tumour incidence in 2 experimental groups, one treated with only NNK (NNK/Air) and other treated with both NNK and CS (NNK/CS). The NNK/CS-exposed mice showed a trend of higher tumour multiplicity as compared to the NNK/Air-exposed mice in these mouse models. The WGS analysis identified 38 somatic mutations in 36 different genes that were common in tumours isolated from NNK/CS and Sal/CS-exposed mice. The genes identified in this analysis were found to be mutated in clinical samples of patients with BAC, as seen in the COSMIC database. The analysis of WGS data also revealed the mutational processes associated with tumours induced in NNK/CS and Sal/CS-exposed mice by generating mutational signatures. The mutational signature revealed that NNK was the major contributor to carcinogenesis in NNK/CS-exposed mice. The mouse models where female A/J mice were first exposed to CS followed by NNK administration and air recovery period developed BAA. The mouse model where mice were exposed to 8 weeks of CS followed by 3 doses of NNK and 8 weeks of the air recovery period showed 100% tumour incidence in 2 experimental groups, one treated with only NNK (Air/NNK) and other treated with both CS and NNK (CS/NNK). The tumour incidence was reduced to 25% and 75% in Air/NNK and CS/NNK-exposed mice, respectively, in the mouse model where mice were exposed to 8 weeks of CS followed by 1 dose of NNK and 8 weeks of the air recovery period (8wk CS + 1xNNK + 8wk air recovery period). This model showed a significantly higher tumour multiplicity in CS/NNK-exposed mice as compared to Air/NNK-exposed mice. With an increase in CS exposure to 12 weeks followed by 1 dose of NNK and 12 weeks of air recovery period model, the tumour incidence was increased to 87.5% in Air/NNK and CS/NNK-exposed mice. This model showed a trend of higher tumour multiplicity in Air/NNK as compared to CS/NNK-exposed mice. Conclusion: When the A/J mice were first treated with NNK followed by CS exposure and air recovery period, the mice develop BAA, which further progress to BAC with an increase in CS exposure beyond 24 weeks in NNK/CS-exposed mice. However, the WGS analysis of tumours from the 3xNNK+36wk CS+27wk air recovery period model revealed NNK as the major contributor to carcinogenesis. By exposing the mice first to CS followed by administration of reduced dose of NNK in 8wk CS + 1xNNK + 8wk air recovery period model, the tumour multiplicity was increased in CS/NNK-exposed mice as compared to Air/NNK-exposed mice. This suggests that CS might be the major contributor of carcinogenesis in this model; however, further analysis is required to confirm this.
- Subject
- lung cancer; genetics; mouse model; cigarette smoke
- Identifier
- http://hdl.handle.net/1959.13/1473743
- Identifier
- uon:49099
- Rights
- Copyright 2021 Vrushali Kashinath Chimankar
- Language
- eng
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