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Impact of different K-ras mutations on tumor progression and drug sensitivity in non-small-cell lung cancer

Worldwide, lung cancer is one of the most common cause of cancer incidence and mortality. Even though there was some improvement in survival during the past decades, only 20% of patients present potentially resectable early-stage disease and the 5-year survival rate remains poor at approximately 16%. Mutations in the K-ras gene occur frequently in NSCLC and they mostly involve codons 12 and 13, causing aminoacid substitutions that present different frequencies compared to other tumor types, in particular colon cancer, in which K-ras mutations play a key role, especially as regards the outcome of drug therapies. Considering these characteristics, the laboratory wants to investigate whether different aminoacids in the same position of K-ras proteins could determine a different regulation of the genes targeted by Ras-Raf-MAPK pathway and a different impact on drug resistance in lung cancer. Clones overexpressing K-ras bearing the three most common mutations found in NSCLC patients (G12C, G12D and G12V) were generated from the human NSCLC cell line NCI-H1299. These clones were tested for their sensitivity toward a panel of agents commonly used in the treatment of NSCLC patients and the most interesting finding was represented by the lower cisplatin sensitivity of the G12C clone. So, the aim of this project consists in better understanding the molecular basis of this resistance, given that preliminary results obtained to date did not highlight any important differences in the resistance mechanisms among clones. We decided to investigate the levels of cisplatin-DNA adducts after cisplatin treatment and we highlighted that, after 24 hours from treatment start, Pt-DNA adduct levels were significantly lower in the G12C cisplatin-resistant clone compared to the others. Afterwards, we performed ɣH2AX immunofluorescence in order to investigate whether the extent of DNA damage inflicted by a fixed cisplatin concentration was comparable among K-ras mutant clones. ɣH2AX was observed in the WT, G12D, and G12V clones after 16, 24, and 48 hours from treatment, while in the G12C resistant clone ɣH2AX was almost undetectable at every time point of the experiment. Altogether, these new findings suggested that cisplatin damage was quickly resolved before DSBs formation in the resistant clone. Therefore, we decided to investigate the possible role of the nucleotide excision repair system in cisplatin resistance, but no differences were observed. Then, we hypothesized that the K-ras G12C mutation could induce the removal of platinum mono-adducts from DNA before crosslink formation, and, when we decided to evaluate the cell viability of all clones following combined treatment with cisplatin and methoxyamine, a known base excision repair inhibitor, we observed that the cell viability in the G12C clone was similar to that observed in other clones. Therefore, we hypothesized that the presence of the G12C mutation might alter BER function. Recent studies identified overexpression of BER genes possible mechanism of cisplatin resistance in tumors. We found that the DNA polymerase β gene was present in the G12C clone at higher levels compared to other clones. Unfortunately, when we tried to confirm the role played by Polβ in the response to cisplatin by analyzing the cell viability of all clones following transfection with a siRNA that blocks Polβ expression and a concomitant treatment with cisplatin, we did not see any significant differences in cell viability. However, western blot analysis of Polβ protein levels showed that protein levels were unmodified 48 hours after transfection, in spite of substantial downregulation of Polβ mRNA. This observation suggests that the protein has a long half-life and that blocking its mRNA for 48 hours is not enough to achieve significant protein depletion. In conclusion, we currently hypothesize that the K-ras G12C mutation could induce the removal of platinum mono-adducts from DNA before crosslink formation and we think that the molecular basis of this phenomenon are founded in a different functionality of the BER, even if we are still unable to define which kind of alterations are present. In the meantime, in order to confirm and extend the data generated in NCI-H1299 to another cellular model, we selected the NCI-H1437 cell line to generate three other different clones that present the three most common mutations of K-ras. What is important is that these new clones will have basal levels of K-ras, unlike the clones generated with NCI-H1299 that show overexpressed K-ras. Induction of site-specific mutation in this cell line could be obtained thanks to Zinc Finger Nucleases (ZFNs) technology, based on the creation of a donor plasmid with the desired mutation. This technology allows the insertion of the desired mutation directly into the K-ras locus, leading to the generation of mutated cells with characteristics more closely resembling to those present in human tumors.

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1.1 Lung Cancer Worldwide, lung cancer is the most common cause of major cancer incidence and mortality in men, whereas in women it is the third most common cause of cancer incidence and the second most common cause of cancer mortality. Even if there was some improvement in survival during the past decades, the survival advances that were realized in other diseases have yet to be achieved in lung cancer, considering that the majority of individuals have advanced-stage disease at presentation and only 20% of patients present potentially resectable early-stage disease. The 5-year survival rate re- mains at approximately 16%, and even with targeted therapy, treatment options have improved sur- vival of only a small percentage of patients, which present mutations in genes that can be currently targeted, like EGFR or ALK. The median age at diagnosis for cancer of the lung and bronchus is 71 years; no cases are diagnosed in patients younger than 20 years (Fig. 1) 1 . pipes, tobacco became widely available in ciga- retteformafterthedevelopmentofcigarettewrap- ping machinery in the mid-1800s. Before World War I, cigarette use in the United States was modest. Wynder and Graham estimated that the average adult smoked fewer than 100 cigarettes per year in 1900. 10 Fifty years later, this number rose to approximately 3500 cigarettes per person peryearandreachedamaximumofapproximately 4400 cigarettes per person per year in the mid- 1960s (Fig. 8). 11 In 1964, the US Public Health Service published a landmark report from the Surgeon General on smoking and its effects on health. 12 That seminal report stated the following important principal findings. (1) Cigarette smoking was associated with a 70% increase in the 62 19.4 336 75.2 21.6 446.2 52.3 17.4 293.8 0 50 100 150 200 250 300 350 400 450 500 All ages < 65 ≥ 65 ALL RACES Both Sexes Males Females Incidence Rate per 100,000 0 100 200 300 400 500 600 Lung Cancer Incidence Rates per 100,000 Age at Diagnosis of Lung Cancer Both Sexes Males Females 1-4 <1 5-9 10-14 15-19 20-24 25-29 30-34 35-39 40-44 45-49 50-54 55-59 60-64 65-69 70-74 75-79 80-84 85+ AB Fig. 5. US age-adjusted lung cancer incidence by gender, age, and race. (A) Separated by age <65 years and age 65 years. (B) Separated by age from <1 to 851 years. Rates are per 100,000 and are age-adjusted to the 2000 US standard population. (Data from Howlader N, Noone AM, Krapcho M, et al, editors. SEER Cancer Statis- tics Review, 1975–2008. Bethesda (MD): National Cancer Institute; 2010. Available at: http://seer.cancer.gov/csr/ 1975_2008/, based on November 2010 SEER data submission, posted to the SEER web site, 2011.) Fig. 6. Stage distribution and 5-year relative survival by stage at time of diagnosis for 2001 to 2007. (A) Stage distribution and (B) 5-year relative survival based on stage at diagnosis of lung cancer. Localized disease defined by confinementto primarysite.Regionalrefersto spreadto regionallymph nodes.Distant refersto whencancer has metastasized. Unknown includes unstaged cancers. Stage distribution is based on summary stage 2000 docu- mentations. (Data from Howlader N, Noone AM, Krapcho M, et al, editors. SEER Cancer Statistics Review, 1975– 2008.Bethesda(MD):NationalCancerInstitute;2010.Availableat:http://seer.cancer.gov/csr/1975_2008/,basedon November 2010 SEER data submission, posted to the SEER web site, 2011.) Lung Cancer Epidemiology 609 Figure 1: Age-adjusted lung cancer incidence by gender and age. Taken from 1 Lung cancer incidence and mortality rates are higher in the developed countries, while in under- developed geographic areas, including Central America and most of Africa, are lower, even if the rates are slowly increasing. The World Health Organization estimates that lung cancer deaths will continue to rise, because of an increase in global tobacco use, especially in Asia. So, despite the efforts to curb tobacco smoking, the availability of new diagnostic and genetic technologies, ad- vancements in surgical techniques and the development of new biologic treatments, the overall situation will continue to worsen, unless people change their habits 1 . Introduction 2

Tesi di Laurea Magistrale

Facoltà: Scienze Matematiche, Fisiche e Naturali

Autore: Stefano Colavecchio Contatta »

Composta da 126 pagine.

 

Questa tesi ha raggiunto 53 click dal 18/03/2014.

Disponibile in PDF, la consultazione è esclusivamente in formato digitale.