Recombinant DNA Technology In Cancer Research

Tumor has been the leading cause of the death all around the world and imposed an enormous burden on human society, of which the same situation China has to face. We believe there will be more chances to practice targeted therapies, to help relieve the pain of patients, and improve the overall survival in the near future based on the better understanding of the molecular mechanisms. Known as a disorder of somatic genetics with genomic instability, tumorigenesis is a long evolutionary process with multiple steps including mutations in cancer associated genes, clonal growth of abnormal cells and selection for the winner adapted to the environment. So it is essential to uncover the drivers and the pathways of the process. However, given the facts of the heterogeneity of tumors and the complexity of the genome, it is still a great challenge to decipher genomic information into general understanding of cancers, to explore the potential oncogenic function of candidate genes, and to develop more targeted drugs and methods.In this thesis, three cancer research projects were described:1) To discover more potential tumor suppressor genes (TSG), we designed the project to screen for TSG using piggyBac transposon (PB) mediated insertional mutagenesis system in Blm-deficient mice, which elevates the rate of mitotic recombination by 20-fold. In the somatic cells of these mice, the normal transcription of the genes would be disrupted randomly when PB integrated into them correctly and the genes would lost function completely when loss-of-heterozygosity (LOH) happens. So tumors would be induced when enough TSGs had been trapped. Given the tag sequences of PB, we could clone the insertions sites from the cancer genomes and the common insertional sites (CIS) would be more likely to be cancer related. Here, we generated several PB transgenic mouse lines and found that the frequent transposition leads to the embryonic lethality; the moderate transposition caused developmental and growth abnormality; and even the low-frequency transposition effected the total survival of the mice compared to the control. We tried to analysis the insertional sites through Splinkerette-PCR combined with next generation sequencing and we found the reads number of some sites were enriched indicating clonal proliferation of the cells, which is consistent to other studies. So far, however, we could not find any confirmed TSGs, and maybe more samples needed to be analyzed. The Results we’ve got now told us it was possible to use piggyBac transposon system to find cancer associated genes without gain-of-function design while it would be much better to do the screening in a specific type of tumor.2) To explore the potential role of Cdk5rap3 gene in Hepatocellular Carcinoma (HCC) and the underlying mechanism, we established a conditional knockout mouse (CKO) with specific deletion of Cdk5rap3 in hepatocyte and then we investigated the susceptibility to the DEN-induced HCC in these CKO mice. The results showed that partially deletion of Cdk5rap3 in mouse liver promoted HCC incidence and growth significantly. Unexpectedly, the expression of Cdk5rap3 in cancer cells were upregulated, which meant tumor cells originated from wild type cells rather than cells with Cdk5rap3 deprived. Occasionally, nodules with negative expression of Cdk5rap3 were found. Since the results and clues were limited, we were not sure the formation of the nodules, a primary tumor from liver cell without Cdk5rap3 or metastatic cells that has already been transformed and the gene was deleted after then. Anyway, the results indicated Cdk5rap3 played important role in the formation of HCC and had distinct functions at different phases and environments. In pre-cancer stage, the deletion of Cdk5rap3 in a small number of cells will disturb the balance of liver cells and created a microenvironment suitable for tumorigenesis; During the transformation, the cells will need the higher expression of Cdk5rap3, or instead of oncogenic function, the upregulation was only response to the transformation. In conclusion, the phenomenon observed is interesting and deserves further work.3) Previously, we found a single nucleotide variation (SNV), called Flt3-ITD c.2076T>A, using whole exome sequencing (WES) in a Flt3-ITD knockin mouse model, which is well-known resistant mutation in AML patients. To confirm whether the SNV we discovered in the mice will bring the same drug resistance, we carried out the experiment. The Flt3-ITD cDNA with SNV was cloned and converted into wildtype nucleotide, named Flt3-ITD c.2076T. Then we transduced Ba/F3 cell line with these two versions of Flt3-ITD sequences and the cells were transformed successfully. At last we tested the sensitivity to the different tyrosine kinase inhibitor (TKI), Quazartinib (AC220), Sorafenib and Ponatinib. As we expected, there was a significant difference between the cell lines transduced with Flt3-ITD c.2076T>A and Flt3-ITD c.2076T, of which the former was resistant to Quazartinib and Sorafenib while both of them were sensitive to Ponatinib, which is consistent to the published result. Our result clearly shown us the SNV would give the leukemia cells the feature of drug resistance and the effect in the mice would be the same. So we need to pay attention when we use the model and explain the result of experiments as the SNV should not be ignored. But actually, this had given us a perfect model for drug resistance research in vivo.All in all, the projects above focused on solve some practical problems in cancer research, using both forward and reverse genetic approaches. Through carrying out the experiment, I have learnt lots of different technologies and information.