Activated Mutant SHP-2Tyrosine Phosphatase Increases The Risk Of MEFs Malignant Transformation And Tumorigenicity Induced By Arsenic

[Background and Objective]Malignant tumor is one of the major diseases that threaten human health.In recent yearsthe incidence is increased and the patients are younger. A number of studies suggest thattumorigenesis is influenced by environmental and genetic factors in multi-factors andlong-term process.Explore tumorigenesis mechanism is of great significance.SHP-2（Src homology2-containing protein tyrosine phosphatase, SHP-2） is thenon-receptor type tyrosine phosphatase, which widespread variety of mammaliantissues and cells, and involved in many physiological processes, such as cell survival,migration, adhesion, as well as cytoskeleton formation. Studies have shown that SHP-2mutants play an important role in the development of Noonan syndrome （accompaningthe abnormal proliferation of myeloid disease）, myeloid proliferative disease, juvenilemyelomonocytic leukemia （JMML） and other leukemia, including solid tumors. Ourgroup has been committed to research SHP-2（encoded by PTPN11） in the occurrenceand development of leukemia and solid tumors. And we found that SHP-2activatingmutations may be involved in the occurrence and development of mouse leukemia andsolid tumors which caused by environmental factors.Neel’s laboratory found that theactivity of SHP-2D61G mutation enhanced to some extent,and they establishedSHP-2G61G/+knockin mouse model,with the characterization of Noonan syndrome,accompaning the abnormal proliferation of myeloid disease,symptoms of spleenenlargement, but without the leukemia and other malignant diseases. We assume that probably because the mice kept in SPF （Specific pathogen free） gradeenvironment, no contact with any environmental pollutants, so it did not happeningleukemia and other malignant diseases. Flow cytometry analysis showed significantlyhigher incidence of leukemogenesis in these mice treated with MNU. But it isimpossible to contact with such a high dose, high purity DNA-damaging agents in reallife. Reference to relevant information and epidemiological data, we treat the MEF（SHP-2+/+） and MEF （SHP-2D61G/+） cells with a heavy metal contamination As₂O₃（Arsenic trioxide）, and observe the role of SHP-2D61G/+in the MEFs malignanttransformation and tumorigenesis induced by As₂O₃.Arsenic is a recognized chemical carcinogen by IARC （International Agency forResearch on Cancer）. Studies have shown that arsenic and its metabolites induce togenerate ROS （Reactive oxygen species）, and then lead DNA damage, abnormalactivation of multiple signaling pathways as well as other mechanisms to promotetumorigenesis. In order to further investigate the role of ROS in MEFs malignanttransformation induced by As₂O₃and SHP-2D61G/+, we use C3G （Cyanidin-3-Glucoside）,as the ROS inhibitor,combined treatment MEFs cells with As₂O₃,and observe cellbiological behavior and nude mice tumorigenesis under ROS is suppressed.[Methods]1Treat MEFs （SHP-2+/+） and MEFs （SHP-2D61G/+） cells with0.5μM As₂O₃and20μMC3G to build the stable MEFs transformed cell model.2Use MTT and Plate Colony formation assay to detect the change of the transformedcells proliferation, use flow cytometry to analysis the change of cell cycle, use soft agarassay to observe the transformed cells non-anchored growth capacity, use adhesionassay and Transwell migration assay to test the adhesion and migration of thetransformed cells, use nude mice experiment to detect tumorigenicity in vivo. 3Treat MEFs （SHP-2+/+） and MEFs （SHP-2D61G/+） cells with0.5μM As₂O₃and20μMC3G, then detect the ROS levels by Flow cytometry.4The MEFs （SHP-2+/+） and MEFs （SHP-2D61G/+） cells under5μM As₂O₃acuteexposure, then use Western blotting to detect the activation of Erk, p38, JNK, Akt.[Result]1Establish the MEFs transformed cell model with As₂O₃and C3G treatment.2MTT and Colony formation assay results display that Low-dose As₂O₃treatmentpromotes cell proliferation and SHP-2activating mutation in this effect plays a positiverole.3Cell cycle test shows Low-dose As₂O₃treatment resulted in a higher proportion ofcells in S phase; while SHP-2activating mutations exacerbate this effect.4Treated with Low-dose As₂O₃promote MEFs non-anchored growth capacity andSHP-2activating mutation plays a promoting role in this process. Soft agar colonyformation assay shows that compared with the control group, the As₂O₃treatment grouphas more and larger colony formation significantly; in the MEF （SHP-2D61G/+） cells thiseffect is more notable.5Adhesion and Transwell migration assay results show that the adhesion and migrationof cells enhanced, which under Low-dose As₂O₃treatment, and SHP-2activatingmutation exacerbate this effect.6Low-dose As₂O₃promote MEFs cell in nude mice tumorigenesis, SHP-2activatingmutation promote this effect.7The malignant transformation of MEFs caused by As₂O₃and SHP-2D61G/+may berelated to the MAPK/ERK, MAPK/JNK, MAPK/P38and PI3K/ATK activated.8As₂O₃acute treatment can stimulate the generation of ROS in MEFs cell, SHP-2activating mutation can promote this effect; C3G can significantly reduce the level ofROS. 9C3G exposure significantly inhibits the As₂O₃-induced MEFs malignanttransformation and nude mice tumorigenesis. With C3G inhibits the ROS levels, theMEFs malignant behavior weakened which accelerated by As₂O₃treatment,such as theproliferation, adhesion, migration, colony formation and non-anchored growth capacity.And nude mice experiment has similar results.[Summary]1Low-dose As₂O₃promote MEFs cell malignant transformation and tumorigenicity,the SHP-2D61G/+play a positive role in this process.2As₂O₃and SHP-2D61G/+promoting the MEFs malignant transformation may be relatedto the MAPK/ERK, MAPK/JNK, MAPK/P38, PI3K/ATK signaling pathway.3As₂O₃and SHP-2D61G/+caused MEFs cells malignant transformation andtumorigenesis, and it can be inhibited to some extent by reducing ROS levels with C3G.