Krüppel-like factor 4 promotes high-mobility group box 1-induced chemotherapy resistance in osteosarcoma cells.
Bottom Line: Osteosarcoma is the most common primary malignant bone tumor, and the frequent acquisition of chemoresistance is often an obstacle to achieving favorable outcomes during chemotherapy.In this study, quantitative real-time PCR and western blot analysis revealed that KLF4 expression was significantly increased in response to cisplatin, methotrexate and doxorubicin treatment in osteosarcoma cells, and knockdown of KLF4 increased sensitivity to these anticancer drugs by decreasing cellular clonogenic ability and increasing apoptosis.Moreover, our data suggest that KLF4-regulated drug resistance might, at least partially, positively regulate high-mobility group box 1 (HMGB1), which was found to be a significant contributor to chemoresistance in osteosarcoma cells in our previous study.
Affiliation: Department of Orthopaedics, The 2nd Xiangya Hospital, Central South University, Changsha, China.Show MeSH
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Mentions: To determine whether KLF4 directly regulates HMGB1 expression, we firstly performed promoter reporter assays to identify the KLF4‐responsive region on the HMGB1 promoter. MG‐63 cells were co‐transfected with pcDNA3.1‐KLF4 expression plasmid and pGL3 GFP reporter with five different fragments of the promoter region of HMGB1 (Fig. 5a). As shown in Figure 5b, progressive deletion of the promoter sequence from −1899 to −456 did not affect the HMGB1 promoter activity, but deletion between −456 and −240 significantly attenuated the promoter activity. The data indicate that the region between −456 and −240 is critical for responding to KLF4. In addition, EMSA also revealed that KLF4 could directly bind to the region between −456 and −240 on the HMGB1 promoter. As shown in Figure 6a, preincubation of nuclear extracts from Cis‐treated MG‐63 cells with MHGB1 probes resulted in DNA/protein complex formation, which was competed on with the addition of unlabeled (cold) probes (Fig. 6a, lanes 2–3), whereas preincubation with mutant unlabeled probes had no affect on DNA/protein complex formation (Fig. 6a, lane 4). Moreover, addition of KLF4 antibody resulted in the formation of a shift band (Fig. 6a, lane 5). These data suggest that KLF4 is able to bind to the KLF4‐responsive core region on the HMGB1 promoter. Finally, ChIP assays combined with quantitative PCR (ChIP‐qPCR) were performed in MG‐63 and SaOS‐2 cells treated with 50 uM Cis for 48 h and it was found that KLF4 could directly bind to the KLF4‐responsive core region on the HMGB1 promoter in MG‐63 and SaOS‐2 cells in vivo. As shown in Figure 6b, immunoprecipitation of the chromatin/protein complex with KLF4 resulted in the enrichment of KLF4 protein at the HMGB1 promoter region near the region between −456 and −240. The promoter sequence of HMGB1 was also analyzed using Matinspector Professional (www.genomatix.de) and TESS (www.cbil.upenn.edu), The KLF4‐binding site was existed at region −379 to −335 bp upstream the TSS of HMGB1. Based on our recent study and previous work,16 here we provide a model of KLF4‐induced chemotherapy in osteosarcoma cells, in which increased expression of KLF4 during chemotherapy activates HMGB1, thus further promoting autophagosome maturation and autophagy (Fig. 7). Therefore, KLF4/HMGB1 interaction is a potential therapeutic target for use in osteosarcoma.
Affiliation: Department of Orthopaedics, The 2nd Xiangya Hospital, Central South University, Changsha, China.