ABSTRACT
Radiation has a limited but relevant role in the adjuvant therapy of gastric cancer (GC) patients. Since Chk1 plays a critical function in cellular response to genotoxic agents, we aimed to analyze the role of Chk1 in GC as a biomarker for radiotherapy resistance. We analyzed Chk1 expression in AGS and MKN45 human GC cell lines by RT-QPCR and WB and in a small cohort of human patient's samples. We demonstrated that Chk1 overexpression specifically increases resistance to radiation in GC cells. Accordingly, abrogation of Chk1 activity with UCN-01 and its expression with shChk1 increased sensitivity to bleomycin and radiation. Furthermore, when we assessed Chk1 expression in human samples, we found a correlation between nuclear Chk1 accumulation and a decrease in progression free survival. Moreover, using a luciferase assay we found that Chk1's expression is controlled by p53 and RB/E2F1 at the transcriptional level. Additionally, we present preliminary data suggesting a posttranscriptional regulation mechanism, involving miR-195 and miR-503, which are inversely correlated with expression of Chk1 in radioresistant cells. In conclusion, Chk1/microRNA axis is involved in resistance to radiation in GC, and suggests Chk1 as a potential tool for optimal stratification of patients susceptible to receive adjuvant radiotherapy after surgery.
Subject(s)
Checkpoint Kinase 1/biosynthesis , Chemoradiotherapy , E2F1 Transcription Factor/metabolism , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Retinoblastoma Binding Proteins/metabolism , Stomach Neoplasms/metabolism , Stomach Neoplasms/therapy , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Protein Ligases/metabolism , Bleomycin/pharmacology , Cell Line, Tumor , Checkpoint Kinase 1/genetics , E2F1 Transcription Factor/genetics , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/radiation effects , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/radiation effects , Humans , Retinoblastoma Binding Proteins/genetics , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , Tumor Suppressor Protein p53/genetics , Ubiquitin-Protein Ligases/geneticsABSTRACT
Aneuploidy and chromosomal instability (CIN) are common features of gastric cancer (GC), but their contribution to carcinogenesis and antitumour therapy response is still poorly understood. Failures in the mitotic checkpoint induced by changes in expression levels of the spindle assembly checkpoint (SAC) proteins cause the missegregation of chromosomes in mitosis as well as aneuploidy. To evaluate the possible contribution of SAC to GC, we analyzed the expression levels of proteins of the mitotic checkpoint complex in a cohort of GC cell lines. We found that the central SAC proteins, Mad2 and BubR1, were the more prominently expressed members in disseminated GC cell lines. Silencing of Mad2 and BubR1 in MKN45 and ST2957 cells decreased their cell proliferation, migration and invasion abilities, indicating that Mad2 and BubR1 could contribute to cellular transformation and tumor progression in GC. We next evaluated whether silencing of SAC proteins could affect the response to microtubule poisons. We discovered that paclitaxel treatment increased cell survival in MKN45 cells interfered for Mad2 or BubR1 expression. However, apoptosis (assessed by caspase-3 activation, PARP proteolysis and levels of antiapoptotic Bcl 2-family members), the DNA damage response (assessed by H2Ax phosphorylation) and exit from mitosis (assessed by Cyclin B degradation and Cdk1 regulation) were activated equally between cells, independently of Mad2 or BubR1-protein levels. In contrast, we observed that the silencing of Mad2 or BubR1 in MKN45 cells showed the induction of a senescence-like phenotype accompanied by cell enlargement, increased senescence-associated ß-galactosidase activity and increased IL-6 and IL-8 expression. In addition, the senescent phenotype is highly increased after treatment with PTX, indicating that senescence could prevent tumorigenesis in GC. In conclusion, the results presented here suggest that Mad2 and BubR1 could be used as prognostic markers of tumor progression and new pharmacological targets in the treatment for GC.
