Exploration Of New Therapeutic Targets For The Treatment Of Non-small Cell Lung Cancer

Lung cancer was the leading cause of cancer death in China and throughout the world[1,2]. Combinations of various therapeutic approaches including surgery, radiotherapy, chemotherapy, and molecular targeted therapy are presently the main treatments performed. Oncologists perform these treatments comprehensively according to pathology, stage, genetic status, and performance status （PS） of patients.Substantial progress has been made in the treatment of malignancies in China over the past three decades. This improvement has been due in part to knowledge and information being transmitted more quickly, which allows oncologists in China to acquire advanced knowledge promptly. Furthermore, some clinical practice guidelines for lung cancer, such as’Diagnosis and Treatment Procedures for Primary Lung Cancer’issued and published by China’s Ministry of Health and the National Comprehensive Cancer Network （NCCN）（Chinese version） have been implemented in China in recent years. In addition, more and more clinical trials have been conducted in China, which also promoted progress in the treatment of lung cancer. But huge population, span geography, diverse socioeconomic backgrounds produce wide disparities in lung cancer care in China.Patient characteristics and treatments were retrospectively investigated using the outpatient database of the Guangdong Lung Cancer Institute （GLCI） in China from October2004to January2013.Data from the relatively complete information of2535outpatients with lung cancer were collected to analyse clinical characteristics. These patients were spread over29provinces and165cities in China. The mean patient age was58years. Female patients accounted for39.5%（1002/2535）, and48.2%（1222/2535） of patients were never-smokers. The most common histological diagnosis was adenocarcinoma （1592/2535,62.8%） followed by squamous carcinoma （358/2535,14.1%）. These results demonstrate that carcinogenic factors other than smoking may play an important role in the development of lung cancer. The great majority of patients with stage I to II NSCLC underwent surgical treatment. A total of14.6%（21/144） and32.4%（80/247） of patients with stage IA and IB NSCLC underwent perioperative chemotherapy;A lot of patients with stage IIIA NSCLC received surgery. But relatively low ratios of patients received the combination of radiotherapy and chemotherapy. A total of449patients with advanced NSCLC received second-line chemotherapy.205（45.7%） patients received non-recommended regimens. Of these,83.9%（172/205） of patients underwent platinum-based doublet chemotherapy. A total of128patients with NSCLC received third-line chemotherapy. Nearly half of the patients （49.2%,63/128） received platinum-based doublets. Several studies showed that treatment based on guidelines can improve survival of patients with lung cancer [3-5]. Treatment disparity was defined as treatment not follow the Chinese Lung Cancer Guideline, including perioperative chemotherapy application for resectable tumors or not; radiotherapy and chemotherapy application for locally advanced tumors or not; molecular targeted therapy based on mutation status or not and so on. The difference of treatment disparities was not statistically significant in gender （P=0.203）. However, the treatment disparities were significantly larger in patients younger than the age of65compared with patients older than the age of65（P<0.001）. Patients from developed cities underwent more standard treatment compared with the ones from other regions （P=0.046）. There were significant treatment disparities between GLCI and other hospitals（P<0.001）.18.2%（197/1080） and50.6%（482/952） treatment compliances in GLCI and other hospitals with guideline-recommended treatment, respectively. Suvival data from727patients with NSCLC showed that2-year OS and5-year OS of patients with NSCLC was38%and16%, respectively. The survival rate of patient with lung cancer is still low. We need to find new therapeutic targetsfor lung cancer.NSCLC accounts for approximately85%of lung cancer cases with most patients being diagnosed with locally advanced and distant metastatic disease. Although a combination of various therapeutic approaches including surgery, chemotherapy, and radiotherapy can be used to treat NSCLC, the prognosis has reached a plateau currently. Generally speaking, the response rate （RR） of standard first-line platinum-based combination chemotherapy is approximately17%-32%, progression-free survival （PFS） of3-6months and overall survival （OS） of7-11months [6-9]. To improve clinical prognosis for patients with lung cancer, several targeted therapies are being developed, particularly in patients with specific molecular features, such as EGFR （Epidermal Growth Factor Receptor） mutation, KRAS mutation and ALK （AnaplasticLymphoma Kinase） fusions[10,11]. But52%of patients with NSCLC do not have these molecular features, especially in squamous cell carcinoma （SCC）[12]. There are two strategies for exploration of new therapeutic targets for the treatment of NSCLC. One is to find new driver genes; the other one is to find targeted drugs for those driver genes that have not targeted drugs.Transcriptome sequencing reveals gene expression and splicing differences, discovers millions of genetic variations and gene fusions. In the fisrtpart of our research, we will profile the fusion genes in a large-scale manner in SCC by whole-transcriptome sequencing technology.KRAS mutation represents a unique molecular subtype involving in approximately20%of NSCLC in western countries and5.4%in China [13,18,19]. Recent studies indicate that KRAS mutation associated with shortened survival and was an independently prognostic factor in NSCLC [13,20,21]. However, no available drug can block KRAS directly.Therefore, it is important to find effective treatments for these subtype patients. KRAS mutant leads to the activation of the MAPK （RAS/RAF/MEK/ERK） signaling pathway, which leads to uncontrolled cell proliferation and antiapoptosis. Several studies are evaluating other potential therapeutic targets in MAPK signaling pathway that function downstream of KRAS. AZD6244（Selumetinib） is a second-generation MEK1/2inhibitor that has showed activity in NSCLC with KRAS mutations in vitro and in vivo[14,22,23]. Pemetrexed is a multitargeted antifolate inhibitor with well tolerability.A study showed that KRAS mutations tumors sensitive to pemetrexed, possibly by upregulation of mir-181c that can downregulate KRAS gene expression. Another study found that the combination of pemetrexed and sorafenib has a synergistic effect. Tumors exposed to pemetrexed resulted in an increased phospho-MAPK level, which was inhibited by subsequent exposure to sorafenib. In the secondsection of our research, we will explore the effect of the combination treatment with selumetinib and pemetrexed in NSCLC harboring KRAS mutations.Chapter oneStudy on the gene fusions in non-small cell lung cancerMethodThrough isolation of high-quality RNA from10cases of squamous cell carcinoma, high-throughput data of whole transcriptome sequencingwas generated on the platform of Illumina HiSeq2000. According to the human reference genome assembly （hg19）, large-scale of alignment will be performed to identify potential gene rearrangements, which will be further validated by specific RT-PCR and capillary sequencing.Result1.The Q30of these10cases was more than85%, and the mapped bases were more than5Gb;2. There were a lot of SNV in these10samples （mean12.15/Mb）. The frequency of transition and transversion was the same in smokers and non-smokers. The most common were C:G-T:A and A:T-G:C;3.There was no difference of expression level in smokers and non-smokers;4.Based on the result of Defuse software, we found a lot of gene fusions in these samples. Of these,50gene fusions were found in4or moren than4samples;5. A total of132gene fusions were found in these samples according to TopHat software.70.5%of gene fusions were intrachromosomal rearrangements. Based on three evidences, we chose26gene fusionsand verified20gene fusions using PCR and DNA sequencing;6. In the20validated gene fusions, five of them （ECE2-PSMD2, ARHGAP42-TMEM123, WWTR1-AGTR1, TNFSF10-FXR1, FAM38A-APRT） were in-frame fusion, and the others were out of frame fusion. We found that ECE2-PSMD2fusion exsited in the normal tissue of GLCI₆.7. We did not detect these five gene fusions in20tumor samples of lung squamous cell carcinoma using RT-PCR and DNA sequencing.Conclusion1. The data from10squamous cell carcinoma using transcriptome sequencing had a high quality. The Q30was more than85%;2. There were a lot of SNV in these10samples （mean12.15/Mb）. The SNV was the same in smokers and non-smokers. The most common were C:G-T:A and A:T-G:C. It was different from adenocarcinoma;3. Based on the result of Defuse software, we found a lot of gene fusions and50gene fusions were found in4or moren than4samples;4. A total of132gene fusions were found using TopHat software.70.5%of gene fusions were intrachromosomal rearrangements. We verified20gene fusions using PCR and DNA sequencing;5. ECE2-PSMD2fusion exsited in the normal tissue of GLCI6.Chapter twoAZD6244plus Pemetrexed combination therapy in KRAS mutant NSCLC cell linesMethodWe picked up one colorectal carcinoma cell line and ten non-small cell lung cancer cell lines with KRAS mutation cultured with5%dialyzed FBS. In MTS assay, we treated cells with different concentration pemetrexed in combination with1 micromole AZD6244or pemetrexed followed AZD6244or AZD6244followed pemetrexed. And then we verified the results in other KRAS mutant NSCLC cell lines. Second, we detected the protein levels of TS, pRB, pERK in concurrent treatment and sequential treatment. Third, differences of cell proliferation in pemetrexed single agent, AZD6244single agent, or sequencial groups were tested by cell clone formation assay.Statistical analyses were performed by SPSS13.0software. Measurement data were presented by meanstandard±deviation. At first, data were test by normal distribution and homoscedasticity test. Cell proliferation differences between different groups were tested by factorial analysis, pairwise comparisons used with LSD method. Tambane’s T2were used for unequal variances. P＜0.05was considered significant difference.Result1. The effect of pemetrexed followed AZD6244and concurrent treatment was better than single agent in Calu-6and H2009cell lines. According to the growth curve, pemetrexed followed by AZD6244had synergistic effect.2. We did MTS in other KRAS mutant cell lines. We found there were synergistic effects in H2009, H157, A549, H1792, and Calu-1cell lines and no synergistic effect in H460, Calu-6, A427, H2887, and H23cell lines.3. AZD6244can down-regulate protein level of TS, pRB and pERK.48hour is the best treatment time.To further determine the molecular mechanisms of synergistic effect using pemetrexed followed AZD6244in KRAS mutant NSCLC cell lines, we examined the TS, pERK, pRB expression in Oh,6h,16h,24h,48h and72h after these cell lines treated with AZD6244. The results indicated that after treatment with AZD6244for48hours, TS, pERK, pRB expression was down regulated.We detected the TS, pERK, pRB expression after KRAS mutant NSCLC cell lines treated with pemetrexed. The results indicated that after treatment with pemetrexed for48hours, TS, pERK, pRB expression was up regulated. In addition, protein levels of TS, pERK, and pRB were upregulated with increasing concentration of Pemetrexed and downregulated with increasing concentration of AZD6244. The Bcl-2and MCL-1expression was down regulated in the group of sequential treatment.4. There was no synergistic effect of sequencial treatment in cell clone formation assay.Conclusion1. The effect of pemetrexed followed AZD6244and concurrent treatment was better than single agent in Calu-6and H2009cell lines.2. In other cell lines, we found there were synergistic effects in H2009, H157, A549, H1792, and Calu-1cell lines and no synergistic effect in H460, Calu-6, A427, H2887, and H23cell lines3. AZD6244can down-regulate protein level of TS, pRB and pERK.48hour is the best treatment time. Protein levels of TS, pERK, and pRB were upregulated with increasing concentration of pemetrexed and downregulated with increasing concentration of AZD6244. The Bcl-2and MCL-1expression was down regulated in the group of sequential treatment.4. There was no synergistic effect of sequencial treatment in cell clone formation assay.