| Background and Aims:Neoadjuvant chemotherapy(NAC) has been investigated as an important therapeutic strategy for bulky or locally advanced squamous cervical cancer(SCC). Squamous cell carcinoma of the NAC sensitivity to chemotherapy treatment is a key factor in the success of SCC. The PI3 K / Akt(phosphoinositide 3-kinase-Akt signaling pathway) gene polymorphism may affect the sensitivity of tumor cells to chemotherapeutic drugs. In the present work we analysis the genetic polymorphisms in the PI3K/Akt pathway and different NAC sensitivity proteins in SCC samples from Chinese Han patients of Gansu Province and its surrounding region, in order to search for predictive polymorphism loci in PI3K/Akt pathway and proteins of linked to NAC resistance/sensitivity. Methods:1. NAC was applied to 259 patients who were diagnosed with SCC including FIGO(the Clinical International Federation of Gynecology and Obstetrics) stage IB2–IIB tumours, and each of the patients received more than two cycles of NAC. The short-term(~15 days of treatment) response to NAC was estimated by the change in tumour size, which was evaluated by the Response Evaluation Criteria in Solid Tumours criteria. Overall, 168 patients were classified as NAC-responders, and 91 patients were considered NAC non-responders. Peripheral blood specimens for genetic analysis were collected from each patient. Genotyping was performed using i Plex Gold Genotyping Assay and Sequenom Mass Array. In this study, we determined the association of the 17 tag single nucleotide polymorphisms in PIK3 CA, Akt1, Akt2 and PTEN genes with chemosensitivity in SCC patients treated with platinum-based NAC.2. Of the ten patients with FIGO stage IB-IIA were included in this analysis. Each patient received more than two cycles of NAC. The NAC regimen was paclitaxel followed by cisplatin. Using Response Evaluation Criteria in Solid Tumors(RECIST) criteria, patients with complete or partial responses were classified as NAC responders, and patients with stable or progressive disease were considered NAC non-responders. Five patients in the NAC responders, and five in the NAC non-responders were selected. Two-dimensional gel electrophoresis(2-DE) accompanied by matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry(MALDI-TOF-MS) was used to analyze and identify differentially expressed proteins. The Image Master 2D Platinum software was used for gel image based quantification and statistical analysis. The web portal for the String database was used for protein–protein interaction network analysis, and the David database was used to analysis the protein function. The three highest expressed proteins were validated by Western blotting. Results:1. The patients with SCC carrying heterozygous AG at rs3729679 in PIK3 CA had a significantly increased risk of chemoresistance compared with those carrying AA genotype(P=0.022). And the patients carrying heterozygous TC at rs12494623 in PIK3 CA demonstrated a poorer treatment response compared with those carrying a CC genotype(P=0.018). The patients carrying heterozygous CG at rs2498786 in Akt1 demonstrated higher treatment response compared with those carrying a GG genotype(P=0.036). Furthermore, the patients had a higher frequency of the rs10416620 and rs62107593 G alleles in the Akt2 gene(P=0.037) and the rs34716810 A allele(P=0.037) had significantly decreased risk of chemoresistance. And a marginal association of the Akt2: rs892120 G allele(P=0.045) was observed in the NAC-responders. The CC genotype at rs17431184(P=0.061) in PTEN showed a trend for decreasing risk of chemoresistance. To better understand the relationship between 17 t SNPs in PTEN, Akt1, Akt2 and PIK3 CA genes and chemosensitivity, we stratified our data based on whether patients were treated with cisplatin or carboplatin-based treatment regimens. The results showed that a heterozygous genotype within two loci in the PIK3 CA gene was strongly associated with chemoresistance in cisplatin combination chemotherapy(rs3729679: P=0.010 and rs12494623: P=0.008). Haplotype analysis for the polymorphisms in Akt1 showed that the GGCC haplotype was a risk effect for NAC non-response(P=0.018). The low frequency of haplotype GGGGAG in Akt2 has protective effect, lower the risk of drug resistance in patients with SCC(P = 0.043).2. Proteins from NAC-responder and NAC non-responder group generated a master expression profile map. Differential expression was defined as greater than 1.5-fold or-1.5-fold change in expression with Student t-test(P value) of 0.05 or less between NAC-responder and NAC non-responder group by using Image Master 2D Platinum software. Seventy two spots were found differentially expressed, 37 showed up regulation while 35 showed down regulation in the NAC non-responders versus NAC-responders. The higher multiples of differentially expressed and clear twelve differential protein spots were excised from the stained gel and identified by MALDI-TOF-MS. Seven proteins were found to be up-regulated in the NAC non-responders, including Tryptase beta-2, Stathmin1, Annexin A1, 14-3-3 σ, SCAA2, Hsp27 and Hsp70. Five proteins were found to be down-regulated in the NAC non-responder group, such as Guanine nucleotide-binding protein subunit beta-2-like 1, albumin, POTE ankyrin domain family member E, Cap G and PKM2. A system biology approach was used to analyze these proteins to major pathway involved in phosphorylation, acetylation and apoptosis. To validate the results of the 2-DE analysis, three proteins, Hsp70, stathmin1 and PKM2 were selected based on high-fold changes. Contrasted with the NAC non-responder group, the Western blot analysis revealed that Hsp70 and stathmin1 decreased, whereas PKM2 was highly expressed in all the investigated NAC-responders tissues in SCC patients, thus confirming the 2-DE analysis results. Conclusions:1. In this study, the genetic polymorphisms in Akt1, Akt2 and PIK3 CA genes were associated with chemosensitivity in SCC patients treated with platinum-based NAC. The loci of rs2498786, rs892120, rs10416620, rs34716810, rs62107593, rs3729679 and rs12494623 genetic polymorphisms in the PI3K/Akt pathway may predict platinum-based chemotherapy response in SCC patients.2. In different cisplatin-based NAC efficacy SCC patients, PKM2 and Tryptase beta-2 may probably affect tumor cell growth and drugs metabolism by modulating intracellular glycolysis; Hsp70, Stathmin 1 and Guanine nucleotide-binding protein subunit beta-2-like 1 by phosphoprotein and acetylation and annexin A1, Hsp27 and 14-3-3 σ joint action to inhibit apoptosis, involved in squamous cell carcinoma of cisplatin-based NAC resistance. The three highest expressed proteins of Hsp70, stathmin1 and PKM2 might be potential tissue biomarkers to predict the efficacy of chemotherapy for SCC patients. |
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