Generation And Phenotype Analysis Of The Transgenic Mouse Model Of OTUD3, A Novel Deubiquitinating Enzyme For The Tumor Suppressor PTEN

Physiological function of phosphatase and tensin homologue(PTEN) was not discovered until 1997 identified as a novel tumour suppressor gene on chromosome 10 at the 10q23 locus. During 18 years of studies, researchers have found its importance on the suppression of occurrence and development of the tumor. PTEN usually has a high mutation rate or a low expression rate in a wide variety of tumors like glioma, prostate cancer, breast cancer and lung cancer. While it’s genetic mutation rate is high in diseases susceptibility to tumor like Cowden-syndrome and Bannayan-Zonana syndrome. In addition, the heterozygous mice with the loss of PTEN gene developed spontaneous breast cancer, thyroid tumor, endometrioma, et al, and homozygous mice lacking PTEN induced the programmed cell death for cell proliferation too rapidly and the individuals dead in embryonic 9.5 days. Recent studies on PTEN transgenic mice indicated that with PTEN overexpression, the mice got a higher resistance to tumors. All the evidence suggested that PTEN is an important tumor suppressor gene. PTEN got its suppressor function through its sub cell location both in cytoplasm and nucleus function. In the cytoplasm, by using its lipid phosphatase activity, PTEN reverses the phosphorylation of PI3 K, inhibits PI3K-Akt and m TOR signaling pathways, and thus exhibits as a tumor suppressor. In the nucleus, PTEN functions mainly by maintaining genome stability.Recent studies showed that the suppressor ability of PTEN rely on the dosage of protein level. Little changes on PTEN protein level is closely related with the occurrence of tumor. When PTEN protein level dropped to 80%, the incidence of breast cancer increased as high as 40%. These facts were enough to illustrate PTEN dose change of tumor susceptibility has important influence. Although the post-translation modification of PTEN consisted of phosphorylation, acetylation, ubiquitin, ubiquitin-like modification, the protein stability is mainly regulated by ubiquitin modification. Ubiquitin modification is a stable balance system in the organism. Ubiquitin modification and deubiquitin modification combined to regulate the stability of the protein. The PTEN ubiquitin ligases were discovered reported in the literature and five proteins(NEDD4-1, WWP2, XIAP, CHIP, TRIM27 / RFP) were identified. But the deubiquitinase had been uncovered till we carried out the work on screening of deubiquitinase which regulated the stability and protein balance of PTEN. We screened almost 90 molecules in the human deubiquitinase library. OTU Domain Containing 3(OTUD3) was found and defined as the deubiquitinase which could remove ubiquitin molecule, regulating the stability of the protein enzymes to ubiquitin. And experiments at cell level also showed that OTUD3 could maintain the stability of PTEN, thus inhibiting the PI3K-Akt pathway, suppressing cell proliferation, migration, and reducing the incidence to tumors in nude mice. Since OTUD3 could stabilized PTEN protein via its deubiquitinase activity, acting as a tumor suppressor in molecular and cell experiments, whether it has a similar function under physiological conditions remained unknown.Since we wanted to figure out the answer for the question” whether OTUD3 can physiologically stabilize PTEN and function as a tumor suppressor? ”, we developed the OTUD3 and PTEN transgenic mice via bacterial artificial chromosome(BAC) strategy which had high efficiency on protecting the inserted fragment. The transgenic mouse model was a good example for the application of genetic engineering technology. By extracting and culturing the mouse embryonic fibroblasts(MEFs), we built up the transgenic mice lines, analyzed the phenotype of transgenic mice and carrying out a series of experiments. The result suggested that there existed interaction between OTUD3 and PTEN proteins, and the transgenic mice got a higher expression level of PTEN since the OTUD3 level was higher than the wild type ones. This kind of interaction was occurred by OTUD3 acting as the deubiquitinase to remove ubiquitin from PTEN, as a result stabilize the PTEN protein. We then design the experiments to find out that whether OTUD3 was involved in the inhibition of PI3K-Akt signaling pathways through the stability level of PTEN protein in primitive cells.On the animal’s overall phenotype, By observing the gradient expression level of these two mice model, we found that: both the OTUD3 and PTEN transgenic mice were smaller in size than the wild type ones. With the increment of OTUD3 and PTEN expression level, the phenotype became more obvious. This phenomenon told us OTUD3 overexpression played a role on the metabolism in vivo, regulating the PTEN protein level and eventually showed up on the body size.MMTV-Py V MT transgenic mice develop breast cancer spontaneously, which is regarded as a classical mouse model in research on breast cancer. In order to study OTUD3 as a tumor suppressor in animal level, we mated the MMTV-Py V MT mice with OTUD3 transgenic mice, developing a genetically engineered mice double mixed model. Using this model, we tracked the function of OTUD3 on the occurrence and development of breast cancer. Our data indicated that OTUD3 overexpression could suppress the tumor. No matter on the time of tumor formation or on the number and size, the breast cancer occurrence and development had been significantly inhibited. This evidence proved the importance of OTUD3 as a regulator on the stability of the PTEN. OTUD3 plays an important role as a tumor suppressor.Our research also explored the relationship between OTUD3 and PTEN in different breast cancer cell lines. The comparison showed a related OTUD3 and PTEN expression level in PTEN plus cell lines, which indicated the ubiquitous of the OTUD3 regulation on PTEN protein other than few cell lines. In addition, we observed OTUD3 is abnormally low expression in high metastatic breast cancer cell lines,suggesting OTUD3 might be involved in the process of breast cancer metastasis or drug resistance. We conducted our experiments via immunohistochemical methods. Results showed a synergistic effect between OTUD3 and USP13 on the regulatory on PTEN protein. When both the two deubiquitinase were low, PTEN protein got a lowest expression level, and vice versa. When either of them was high, PTEN protein level was medium.Our study about OTUD3 transgenic mouse model and its MEFs proofed OTUD3 has ability to remove poly-ubiquitination and stabilize the protein level of PTEN under physiological condition. The overall phenotype study of OTUD3 and PTEN transgenic mice also showed: OTUD3 transgenic mice exhibits a similar phenotype with PTEN transgenic mice, which through stabilizing PTEN protein levels in mice. Moreover, by introducing the MMTV-Py V MT breast cancer spontaneously developing mouse model and constructing double transgenic mouse model with OTUD3 transgenic mouse model, we illustrated, by regulating the protein level of PTEN, OTUD3 shows ability to suppress the occurrence and development of breast cancer. Integrating these results, we had provided some important genetic evidences which can answer the question that whether OTUD3 is a physiological deubiquitinase of PTEN? In addition, the studies exploring the relationship between OTUD3 and PTEN in different breast cancer cell lines and the relationship between OTUD3, USP13 and PTEN in breast cancer tissues also widened our knowledge on OTUD3 functions as a tumor suppressor in vivo.