Effects Of Overexpression Wild-type Or Gain-of-Function Mutation SHP-2Tyrosine Phosphatase On Malignant Biological Behaviors Of Breast Tumor Cells And Its Molecular Mechanism Study

Breast cancer is one of the common and frequent encountered diseases. Itsmorbidity and mortality are increasing year by year. And now breast cancer is on thetop of female malignant tumors list. Most important reasons for patients death aremetastasis and recurrence of tumor. There is little well-pleasing remedy for such ofpatients. More and more researches reported that Gain-of-Function Mutation(GOF)and abnormal expression of SHP-2play important roles in tumor occurrence andmetastasis. But, the relationship of SHP-2to malignant progression of breast cancerand the role of the SHP-2pathway in the biology of human breast cancer cells remainunknown.SHP-2is a member of PTPs family, which is a bona fide oncogene, has beenproven to express ubiquitously in mammalian tissue. SHP-2and other protein tyrosinephosphatases (PTPs) regulate several cellular processes and contribute totumorigenesis. SHP-2plays a largely positive role in numerous cell signalingpathways and essential for hematopoiesis and lymphopoiesis. As compared withnormal hematopoietic progenitor cells, the level of SHP-2expression in leukemiacells is remarkably elevated in adult human leukemia. Besides, the overexpression ofSHP-2has been found in almost all the breast cancer cells line. Substantive papershave reported that germline mutations of PTPN11(encodes SHP-2in humans) presentin nearly50%Noonan Syndrome (NS), myeloid leukemia, especially the childhoodleukemia, and some solid tumors. SHP-2was obviously up-regulated in breast cancertissues with lymph nodes metastasis suggesting that SHP-2may play essential roles inbreast cancer development forbidden. Until now, there was little knowledge about therelation between SHP-2and breast cancer. 【Methods】To explore the effects of GOF and overexpression of SHP-2on breast cancercell lines MDA-MB231, we constructed human SHP-2eukaryotic expression vectorsuccessfully, genetiein-resistant cell lines were screened after the recombinantplasmids were transfected into MDA-MB231cells. The detected the effect of SHP-2on cell growth, cloned forming ability, apoptosis, invasive and migration ability, thestructure and function of tight junction, then analysis the mechanism of effectsabove.The results showed as follows.【Results】1、The recombinant plasmid pcDNA3.1SHP-2-WT and pcDNA3.1SHP-2D61G/+eukaryotic expression vector were constructed successfully, followed by sequencingafter enzyme digestion identification; Screen and identification of SHP-2expressionclone by transfection.Western blot screen three clones with SHP-2stable exressionwhich named pcDNA3.1、pcDNA3.1SHP-2-WT and pcDNA3.1SHP-2D61G/+breastcancer and NIH3T3cells.2、The effects of SHP-2GOP and overexpression wild-type on MDA-MB231cellgrowth: MTS results show the control and vector groups have similar cellproliferation speed and GOP or overexpression wild-type group have a supernalspeed. Plate ans soft agar clone forming assay shows overexpression group has moreclones than control and vector groups. Above all, SHP-2transfection may increasecell two and three clone forming ability.3、The effects of SHP-2on breast cancer cell adhesion and apoptosis: Celladhesion assay in vitro shows the rate of cell adhesion in GOF or overexpressiongroup is higher than control and vector groups. Annexin-V/PE stain results show thatthe rate of apoptosis in over expression group is also lower than control and vectorgroups. Above all, SHP-2may accrescence capability of cell adhesion and counteractapoptosis.4、The effects of SHP-2on breast cancer cell metastatic ability: Wound healing assay found that cell migration rate was higher in SHP-2expression cells.Transwellassay showed that invasive rate were more in SHP-2expression cells. Above all,SHP-2may increase the cell migration and invasive ability.5、 The effects of SHP-2on MDA-MB231cell invasive ability in mice:Tumorigenicity assay showed the tumor weight of pcDNA3.1SHP-2-WT andpcDNA3.1SHP-2D61G/+MDA-MB-231group were ascendancy than control group(P<0.01). GOF group node distant organ metastasis were found in tumors.6、 The mechanisms of SHP-2’ s effects on MDA-MB231cell biologicalbehaviour:Western blot assay showed that the level of phospho-AKT/ERK was higherin SHP-2overexpression wild-type cells. Above all, the catalytic activity of SHP-2isimportant for Ras/Erk activation since SHP-2can up-regulate directly Erk activity andPI3K activity.【Conclusion】(1) The recombinant plasmid pcDNA3.1SHP-2-WTand pcDNA3.1SHP-2D61G/+eukaryoticexpression vector were constructed successfully.(2) GOF and overexpression wild-type of SHP-2can promote MDA-MB231celladhesion, proliferation, migration,invasive and tumorigenesis ability.(3) The effects of SHP-2on MDA-MB231biological phenotype are relateed withthe activation of Ras-Raf-MAPK/PI3K-AKT signal pathway.