Conclusions
What are risk factors and symptoms of LADC?
Radon is an important risk factor of lung cancer as well as Tobacco smoke, which puts people at the most risk. It's important to get a Radon detector. This is especially important for older people who are prone to cellular damage.
What are current treatments for LADC?
LADC treatment includes immunotherapy, chemotherapy, surgery and specific inhibition of cancer driving genes, in some cases EGFR. In these cases, Tyrosine Kinase Inhibitors can be used to reduce cell proliferation and hopefully tumor growth. However, there are cases where TKI treatment doesn't result in effective inhibition of the EGFR signaling pathway.
|
What causes tumor growth?
Epithelial transitions can result in pluripotent mesenchymal cells that can transform into various cell types, including cancerous phenotypes. Understanding the role of this process in LADC is important to understand the reasons behind tyrosine kinase inhibition and EGFR signaling in cell proliferation and migration.
|
What is the gap in knowledge?
Aim 1
The goal of the first aims is to successfully mutate the tyrosine kinase domain so that its catalytic activity is altered. Sequencing of mutated tissue will discover whether the desired result has been achieved. Studying cell proliferation and migration will demonstrate the function of EGFR is tyrosine-independent pathways.
|
Ensemble and ClustalOmega will be used to align known homologs of the EGFR gene and I will identify unique Amino acids in the Tyrosine kinase domain that might be important for cell proliferation and cell adhesion in the lung.
CRISPR/Cas9 plasmid delivery to mouse eggs will mutate the corresponding DNA segments in mouse eggs7, followed by DNA sequencing of wildtype and transfected mice.
To measure cell the rate of proliferation and migration in the lung, I would use a cell counting kit and a wound healing (scratch) assay, respectively.
Aim 2
The second aims is meant to study tyrosine-kinase independent function when exposed to various chemicals, including tyrosine kinase inhibitors. Altering EGFR's function in the absence of fully function tyrosine kinase domains will hopefully affect cell procreation and migration in a way that contrasts aim 1.
|
Samples will be subject to a variety of chemicals such as hydroxylamine that cleave cysteine- palmitoylation bonds
Reverse chemical genomic screening will quantify exogenous ligand to EGFR between wildtype and transfected mice. Mutant epithelial and control sample tissues will be subjected to these chemicals.
Once again, cultures will be measured for proliferation and cell migration by scratch assay and cell counting.
Aim 3
Collect samples exposed to small molecules and those without. Comparing mutant tissue gene expression with wild type may implicate other domains of EGFR in signaling besides its main catalytic activity.
I will perform a microarray on wildtype and transfected mice mouse cells treated with chemicals from the screen.
Afterwards, Gene Ontology analysis using PANTHER can enrich gene expression with GO terms relating to cell growth patterns such as cell proliferation, migration and protein palmitoylation which are all implicated in epithelial transformations.
What are future directions for LADC research?
There are many ways to study mutations in EGFR as outlined in the flowchart bellow. This experiment mostly focused on cell viability and protein function, however clinical studies are necessary to understand Lung Adenocarcinoma. In vivo Zebrafish models with xenograft human tissue are an emerging method, also included in the graphic.
Files
Presentation Draft 1:
presentation_rough_draft_1.pptx.zip |
Presentation Draft 2:
final_presentation_rough_draft_2_kye_nichols.pptx.zip |
Final Presentation:
kyenicholsfinalpresentation2020.pptx.zip |
Presentation Figures
kyenicholspresentation-2020.pdf |
References
Sun, Pan, et al, Frequency of Driver Mutations in Lung Adenocarcinoma from Female Never-Smokers Varies with Histologic Subtypes and Age at Diagnosis, 2012. Retrieved from: https://clincancerres.aacrjournals.org/content/18/7/1947
1.) Zhang, Natasha B. Leighl, et. al (2019). Emerging therapies for non-small cell lung cancer [Journal of Hematology and Oncology Article], pg 1. Retrieved from: https://jhoonline.biomedcentral.com/articles/10.1186/s13045-019-0731-8
2.) Kern, Tjaden, et al., Inhibitors of the epidermal growth factor receptor in apple juice extract. (2005). Retrieved from: https://www.ncbi.nlm.nih.gov/pubmed/15759309
3.) Sven Bogdan, Klämbt, Epidermal growth factor receptor signaling (2001), pg 3. Retrieved from: https://www.cell.com/current-biology/comments/S0960-9822(01)00167-1 4.) Thomas, Weihua, Rethink of EGFR in Cancer With Its Kinase Independent Function on Board (August, 2019). Retrieved from: https://www.frontiersin.org/articles/10.3389/fonc.2019.00800/full
4.) Tian, Shi, et. al (2018). Different subtypes of EGFR exon19 mutation can affect prognosis of patients with non-small cell lung adenocarcinoma [Pubmed Article]. Retrieved from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6211626/
5.) Ohashi, Rai, et al., Induction of lung adenocarcinoma in transgenic mice expressing activated EGFR driven by the SP‐C promoter (2008). Retrieved from: https://www.ncbi.nlm.nih.gov/pubmed/18564139
6.) Martin, Chemical approaches for profiling dynamic palmitoylation (2013). Retrieved from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3693551/
7.) Cancer Treatment Centers of America, Lung cancer types, pg 2. Retrieved from: https://www.cancercenter.com/cancer-types/lung-cancer/types
8.) Uramoto, Iwata, et. al (2010). Epithelial–Mesenchymal Transition in EGFR-TKI Acquired Resistant Lung Adenocarcinoma [Pubmed Article]. Retrieved from: https://www.ncbi.nlm.nih.gov/pubmed/20682976
9.) Sawabata, Circulating tumor cells in lung cancer: cluster circulating tumor cells as hybrid epithelial-mesenchymal transition/mesenchymal-epithelial transition (2017). Retrieved from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5723813/
10.) Lin, Wang, et al., EGFR-TKI resistance in NSCLC patients: mechanisms and strategies (2014). Retrieved from: https://www.nature.com/articles/onc2012158
11.) Tabeta, Hoebe, et al, Velvet, a Dominant Egfr Mutation That Causes Wavy Hair and Defective Eyelid Development in Mice (2003). Retrieved from: https://pdfs.semanticscholar.org/7eee/c89406e69357fa8ccf3874696448de616699.pdf?_ga=2.21516347.1433771477.1588236341-1015871252.1588236341
12.) Sugio, Uratamoto, Ono, et al., (2006). Mutation within the tyrosine kinase domain of EGFR gene specifically occur in lung adenocarcinoma patient with a low exposure of tobacco smoking [Pubmed Article]. Retreivedfrom: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3216424/
13.) Daniel B. Costa, Kim-Son H. Nguyen, et. Al, (2009). Effects of erlotinib in EGFR mutated non-small cell lung cancers with resistance to gefitinib [Pubmed Article]. Retrieved from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2596582/pdf/nihms-59709.pdf
https://www.nature.com/articles/s41419-017-0063-y
1.) Zhang, Natasha B. Leighl, et. al (2019). Emerging therapies for non-small cell lung cancer [Journal of Hematology and Oncology Article], pg 1. Retrieved from: https://jhoonline.biomedcentral.com/articles/10.1186/s13045-019-0731-8
2.) Kern, Tjaden, et al., Inhibitors of the epidermal growth factor receptor in apple juice extract. (2005). Retrieved from: https://www.ncbi.nlm.nih.gov/pubmed/15759309
3.) Sven Bogdan, Klämbt, Epidermal growth factor receptor signaling (2001), pg 3. Retrieved from: https://www.cell.com/current-biology/comments/S0960-9822(01)00167-1 4.) Thomas, Weihua, Rethink of EGFR in Cancer With Its Kinase Independent Function on Board (August, 2019). Retrieved from: https://www.frontiersin.org/articles/10.3389/fonc.2019.00800/full
4.) Tian, Shi, et. al (2018). Different subtypes of EGFR exon19 mutation can affect prognosis of patients with non-small cell lung adenocarcinoma [Pubmed Article]. Retrieved from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6211626/
5.) Ohashi, Rai, et al., Induction of lung adenocarcinoma in transgenic mice expressing activated EGFR driven by the SP‐C promoter (2008). Retrieved from: https://www.ncbi.nlm.nih.gov/pubmed/18564139
6.) Martin, Chemical approaches for profiling dynamic palmitoylation (2013). Retrieved from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3693551/
7.) Cancer Treatment Centers of America, Lung cancer types, pg 2. Retrieved from: https://www.cancercenter.com/cancer-types/lung-cancer/types
8.) Uramoto, Iwata, et. al (2010). Epithelial–Mesenchymal Transition in EGFR-TKI Acquired Resistant Lung Adenocarcinoma [Pubmed Article]. Retrieved from: https://www.ncbi.nlm.nih.gov/pubmed/20682976
9.) Sawabata, Circulating tumor cells in lung cancer: cluster circulating tumor cells as hybrid epithelial-mesenchymal transition/mesenchymal-epithelial transition (2017). Retrieved from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5723813/
10.) Lin, Wang, et al., EGFR-TKI resistance in NSCLC patients: mechanisms and strategies (2014). Retrieved from: https://www.nature.com/articles/onc2012158
11.) Tabeta, Hoebe, et al, Velvet, a Dominant Egfr Mutation That Causes Wavy Hair and Defective Eyelid Development in Mice (2003). Retrieved from: https://pdfs.semanticscholar.org/7eee/c89406e69357fa8ccf3874696448de616699.pdf?_ga=2.21516347.1433771477.1588236341-1015871252.1588236341
12.) Sugio, Uratamoto, Ono, et al., (2006). Mutation within the tyrosine kinase domain of EGFR gene specifically occur in lung adenocarcinoma patient with a low exposure of tobacco smoking [Pubmed Article]. Retreivedfrom: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3216424/
13.) Daniel B. Costa, Kim-Son H. Nguyen, et. Al, (2009). Effects of erlotinib in EGFR mutated non-small cell lung cancers with resistance to gefitinib [Pubmed Article]. Retrieved from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2596582/pdf/nihms-59709.pdf
https://www.nature.com/articles/s41419-017-0063-y