The Involvement Of PI3K/Akt And MAPK/ERK Pathways In Hypopharyngeal Carcinoma And Olfactory Neuroblastoma

Objective:First,to investigate whether PI3K/Akt and MAPK/ERK signaling pathway play a role in the occurrence and development of hypopharyngeal carcinoma,and to further explore the molecular mechanism of inhibition of the activity of hypopharyngeal cancer cells by GDC-0980 and Refametinib,the inhibitors of the two pathways.Furthermore,the anti-tumor effect and molecular mechanism of the two inhibitors were discussed,which will provide theoretical basis and preliminary experimental basis for the development and application of new anti-hypopharyngeal cancer drugs of two pathway inhibitors.Second,to accumulate experience in the diagnosis and treatment of olfactory neuroblastoma(ONB),and to provide an experimental basis for exploring its pathogenesis,this article also reviewed the characteristics of ONB and explored the expression status of the key proteins on PI3K/Akt and MAPK/ERK signaling pathways.Methods1.The phosphorylation level of Akt and Erk in hypopharyngeal carcinoma and adjacent normal tissues were detected by immunohistochemical method,and the relationship between the phosphorylation level of Akt and Erk with the clinicopathological factors of hypopharyngeal carcinoma were analyzed.2.MTT method was used to detect the antiproliferative activity of GDC-0980 and Refametinib against FaDu cells.PI flow cytometry and annexin-V/PI double staining was used to detect the effects of GDC-0980 and Refametinib on the cell cycle distribution and cell apoptosis in FaDu cells.Western-blot was conducted to evaluate the effect of GDC-0980 and Refametinib on phosphorylation or expression level of cell cycle related protein and the key factors in PI3K/Akt and MAPK/ERK pathways.The effect of GDC-0980 and Refametinib on the expression level of p27 mRNA in FaDu cells were evaluated by qRT-PCR assay.The effects of GDC-0980 and Refametinib on cells migration were evaluated by wound healing assay and Transwell migration assay.3.Chou-Talalay method and CalcuSyn software were used to determine whether combined treatment with GDC-0980 and Refametinib would have an impact on cell death.Soft agar cloning formation assay was used to evaluate the synergistic Antitumor effect.PI staining was employed to analyze the effect of two drugs on cell cycle distribution.Western blot assay and Real-time quantitative PCR analysis were used to evaluate the effect of combination use of two drugs on the protein or mRNA expression level of cycle related proteins.Western blot was used to study the effect of combined medication on the key proteins of PI3K/Akt and MAPK/ERK signal pathways.4.We retrospectively analyzed the clinical data of the cases of ONB who were treated from September of 2007 to August of 2015.All the clinical data regarded age,gender,TNM stage,Kadish stage,Hymas grade,treatment protocol,follow-up and imaging features.To analysis of the relevance between the two signal pathways of PI3K/Akt and MAPK/ERK and ONB,we evaluated the phosphorylation level of Akt,Erk,Stat3 and Stat5 in ONB by immunohistochemistry.Results:1.The phosphorylation level of Akt and Erk in hypopharyngeal carcinoma was significantly higher than that in adjacent normal tissue.There was no apparent correlation between the expressions of the two proteins in hypopharyngeal carcinoma.The phosphorylation level of Akt and Erk in hypopharyngeal carcinoma was not related to age,sex or pathological differentiation.There was no obvious difference in The phosphorylation level of Akt between lymphatic metastasis group and non-metastasis group,but The phosphorylation level of Akt in stage ? and ? was significantly higher than that in stage ? and ?.The phosphorylation level of Akt in stage T3 and T4 was significantly higher than that of T1 and T2;The phosphorylation level of Erk protein was greatly higher in stage ? and IV than in stage ? and ?,and distinctively higher in lymphatic metastasis group than in non-lymph node metastasis group.2.GDC-0980 and Refametinib both inhibited FaDu cell proliferation in dose-dependent manner.GDC-0980 and Refametinib induced FaDu cell cycle arrest in the G1 phase without inducing apoptosis.The expression of cell cycle related protein cyclin D1 and the phosphorylation level of Rb decreased,the protein and mRNA expression level of p27 increased.Dose-dependent reduction of p-PDK1,p-Akt,p-mTOR and p-p70 S6K in PI3K/Akt signaling pathway was observed after GDC-0980 treatment.A significant decline of p-Erk was presented in FaDu cell after Refametinib treatment,but the expression of Erk had no significant changes.GDC-0980 significantly attenuated the migration of FaDu cells,but Refametinib did not affect the migration of FaDu cells.3.The combination use of GDC-0980 and Refametinib showed better tumor inhibitory effect on the proliferation of FaDu cells than either drugs using alone.The concentration of 2×IC50 GDC:IC50 Refa showed the best synergistic effect in three concentration ratios.Soft agar cloning formation assay further confirmed that combined use of two drugs more significantly inhibited the cloning formation cability of FaDu cells.The combination of two drugs resulted in G1 phase arrest in FaDu cells.The expression of cyclin D1 and the phosphorylation of pRb decreased,but the protein and mRNA expression level of p27 increased,and the phosphorylation of Akt,mTOR and Erk decreased and Stat3 increased after combined treatment.4.In the 10 cases,6 were diagnosed as late T stage(T3/T4),6 were at late Kadish stage(C/D)and 3 were at high Hyams grade(?)which indicated a poorer prognosis.The overall survival of male is obviously higher than that of female after a series of combined treatment,P-Akt,p-Erk,p-Stat3 and p-Stat5 was detected in 5 patients(50%),9 patients(90%),7 patients(70%)and 0 patients(0%),respectively.Conclusions:1.Both PI3K/Akt and MAPK/ERK pathways play important role in the development of hypopharyngeal carcinoma.GDC-0980 and Refametinib,the two pathway inhibitors,significantly inhibited the viability of hypopharyngeal cancer cells and induced cell cycle arrest in G1 phase,which may be achieved by suppre,ssing PI3K/Akt and MAPK/ERK signaling pathways.The combination of the two drug,s could be used in the treatment of hypopharyngeal cancer to achieve a stronger inhibitory effect than using single drug alone.2.The high phosphorylation level of Akt,Erk,and Stat3 suggested that PI3K/Akt and MAPK/ERK signaling pathways play important role in the pathogenesis and progression of ONB.