MAGE-A9Regulates The Resistance In Estrogen Receptors Positive (ER+) Breast Cancers Via Efp-mediated14-3-3σ Degradation

Objective: Endocrine therapy is one of the most effective comprehensivetreatments for estrogen receptor positive(ER+) breast cancer patients.However, about40%of the ER+breast cancer patients are resistant toendocrine therapy and it has become a severe obstacle for the treatment ofbreast cancer. In breast cancer, the downstream target genes play importantroles in mediating estrogenic activity, and one of them is estrogen-responsivefinger protein gene (Efp). It has been shown that Efp is E3ubiquitin ligasecontaining RING (Really interesting new gene) domain, and it mediates theubiquitination and degradation of14-3-3σ, a negative regulator of cell cycle,which underlying the drug resistance to endocrine treatment for ER-positivebreast cancer patients. Melanoma-associated antigen (MAGE) family proteinsinteract with RING domain containing protein including E3ubiquitin ligase,and regulate their ubiquitination activity. In this study, we investigate theinfluence of MAGE-A9on Efp-mediated14-3-3σ ubiquitylation anddegradation pathway, study the role of MAGE-A9in endocrine therapyresistance in Estrogen receptors positive (ER+) breast cancers, and providesome new evidence and experiment data for breast cancer therapy.Methods:1Immunohistochemical staining was used to detect the proteinexpression of MAGE-A9, Efp and14-3-3σ in60ER+breast cancer tissues,and the correlation among the expressions of MAGE-A9, Efp and14-3-3σwasanalyzed.2Gene overexpression, RT-PCR and Western-Blot were used to detectthe influence of exogenous MAGE-A9on Efp-mediated14-3-3σ degradation.3Immunoprecipitation were used to detect the protein-protein interaction between MAGE-A9and Efp.4PCR and gene recombination technique were used to construct theMAGE-A9expression plasmids, and their effects on the proliferation of breastcancer cells was investigated.Results:1In60ER+breast cancer patients,27patients are MAGE-A9proteinexpression positive (45.0%),41patients are Efp protein expression positive(68.3%), and9patients are14-3-3σ protein expression position (15.0%). Efpprotein expression was detected in19of27(70.1%) patients with theMAGE-A9protein expression positive and in22of33(66.7%) patients withthe MAGE-A9protein expression nagetive, indicating that Efp proteinexpression is not related with MAGE-A9protein expression (χ~2=0.094, P=0.759).14-3-3σ protein expression was detected in0of27(0%) patientswith the MAGE-A9protein expression positive and in9of33(27.3%)patients with the MAGE-A9protein expression negative, implying that14-3-3σ protein expression was negatively regulated by MAGE-A9(χ~2=6.656,P=0.01).14-3-3σ protein expression was detected in2of41(4.90%) patients with the Efp protein expression positive and in7of19(36.8%) patients with the Efp protein expression negative, suggesting that theexpression of14-3-3σ is also negatively regulated by Efp protein expression(χ~2=8.0748,P=0.005).2In MCF-7cells,14-3-3σ mRNA expressions in the groups with with14-3-3σ transfection alone,14-3-3σ and Efp cotransfection and14-3-3σ,MAGE-A9and Efp cotransfection were not significantly different (P>0.05).But14-3-3σ protein expression in the group with14-3-3σ and Efpcotransfection and14-3-3σ, MAGE-A9and Efp cotransfection werecotransfection was significantly lower than the group with14-3-3σtransfection alone(P<0.05).3Immunoprecipitation experiment showed that there is physicalinteraction between MAGE-A9and Efp. 4Four Myc-His-pcDNA3.1(-)MAGE-A9deletion mutants expressionplasmid was successfully constructed, includingMyc-His-pcDNA3.1(-)MAGE-A9(1-29),Myc-His-pcDNA3.1(-)MAGE-A9(1-114),Myc-His-pcDNA3.1(-)MAGE-A9(115-285) andMyc-His-pcDNA3.1(-)MAGE-A9(286-315).5RT-PCR and Western-Blot results showed that four MAGE-A9deletionmutants expressed well in MCF-7cells.6Colony formation assay showed that after transfection and G418selection, the clone forming rate of MCF-7cells expressing control plasmid,Myc-His-pcDNA3.1(-)MAGE-A9(1-29),Myc-His-pcDNA3.1(-)MAGE-A9(1-114),Myc-His-pcDNA3.1(-)MAGE-A9(115-285) andMyc-His-pcDNA3.1(-)MAGE-A9(286-315).were (30.00±0.93)%,(20.35±0.89)%,(23.00±0.90)%,(72.50±1.99)%and(22±1.03)%, indicating that Myc-His-pcDNA3.1(-)MAGE-A9(115-285)promoted colony formation (P<0.05).7MTT assay showed that after transfection with control plasmid,Myc-His-pcDNA3.1(-)MAGE-A9(1-29),Myc-His-pcDNA3.1(-)MAGE-A9(1-114),Myc-His-pcDNA3.1(-)MAGE-A9(115-285) andMyc-His-pcDNA3.1(-)MAGE-A9(286-315), and cultured for24h and48h, thecell survival rates of MCF-7cells were100.00%and100.00%,102.85%and108.50%,98.72%and95.51%,147.86%and228.61%, and98.74%and100.00%, confirming that Myc-His-pcDNA3.1(-)MAGE-A9(115-285)increased cell survival (P<0.01).Conclusion:1In ER+breast cancer tissues,14-3-3σ protein level is negativelyregulated by MAGE-A9and Efp.2MAGE-A9interactes with Efp to promote Efp-mediated degradation of14-3-3σ. 3Myc-His-pcDNA3.1(-)MAGE-A9(115-285) deletion mutantsignificantly increases the proliferation of MCF-7cells.