24?h post transfection, cells were harvested for western blot analysis (f and g), RT-qPCR (h) and glucose consumption measurement (i)
24?h post transfection, cells were harvested for western blot analysis (f and g), RT-qPCR (h) and glucose consumption measurement (i). disrupting the interaction between FBP1 and TRIM28 in pancreatic cancer cells. Moreover, we demonstrated that FBP1 promoted c-Myc degradation through disrupting the ERK-c-Myc axis. Conclusions FBP1 modulates the sensitivity of pancreatic cancer cells to BET inhibitors by decreasing the expression of c-Myc. These findings highlight FBP1 could be used as a therapeutic niche for patient-tailored therapies. Electronic supplementary material The online version of this article (10.1186/s13046-018-0888-y) contains supplementary material, which is available to authorized users. value 0.05 was considered statistically significant. All the values are expressed as the means SD. Results FBP1 is responsible for modulating the BET inhibitor sensitivity in PDAC The is a well-known tumor suppressor gene that exhibits low expression or loss of expression in many types of MSX-130 solid tumors [20C22]. Given the importance of the inhibition of cancer progression by FBP1 and the unclear underlying molecular mechanism for this, we performed a drug screening assay in FBP1 knockdown or overexpressing pancreatic cancer cells (PANC-1) and compared the IC50 values of each small molecule with that of the controls (Fig.?1a). We found that the IC50 values of JQ1, the most studied BET inhibitor , in FBP1 knockdown group was higher than control group (Fig. ?(Fig.1a1a and ?andb).b). In contrast, the IC50 value of JQ1 in FBP1 overexpression group was lower than that of the control (Fig. ?(Fig.1a1a and ?andb).b). These data suggest that FBP1 is involved in regulating JQ1 sensitivity in pancreatic cancer (Fig. ?(Fig.1b),1b), using gemcitabine as a positive control (Fig. ?(Fig.1a),1a), consistent with previous findings showing that FBP1 loss is responsible for gemcitabine resistance in pancreatic cancer . In order to verify the role of FBP1 in sensitizing PDAC cells to JQ1-induced apoptotic death, PANC-1 cells were treated with JQ1 MSX-130 alone or in combination with FBP1-targeted shRNAs. The knockdown of FBP1 not only increased the pancreatic cancer cells viability (Fig. ?(Fig.1c1c and ?andf),f), but also promoted PANC-1 cell resistant to JQ1 drug via decreasing the cleaved PARP expression and caspase-3 activity (Fig. ?(Fig.1c1c-?-1f).1f). Together, our data indicate that FBP1 loss plays a vital role in BET inhibitors resistance in PDAC cells. Open in a separate window Fig. 1 FBP1 is responsible for modulating the MSX-130 BET inhibitor sensitivity in PDAC. a, PANC-1 cells were infected with lentivirus expressing control, FBP1-specific shRNAs. After 48?h infection, shControl cells were transfected with pcDNA3.1 or Flag-FBP1 constructs. All cells were treated with different doses of indicated chemicals 24?h post-transfection. The cell Adipor2 viability was measured by MTS assay. Heat map showing the IC50 ratio (log2 (IC50 ratio)) between shControl versus shControl, knockdown FBP1 versus shcontrol or overexpression FBP1 versus control treated with indicated chemicals. b, PANC-1 cells were infected with lentivirus expressing control, FBP1-specific shRNAs. After 48?h infection, shControl cells were transfected with pcDNA3.1 MSX-130 or Flag-FBP1 constructs. Cells were treated with different doses of JQ1 24?h post-transfection. The cell viability was measured by MTS assay. Data shown are mean values SD from six replicates. c-f, PANC-1 cells were infected with lentivirus expressing control or FBP1-specific shRNAs. After 72?h infection, cells were harvested for MTS assay (c), western blotting (d), caspase 3 activity assay (e) and colony formation assay (f). All data are shown as mean values SD (values are also shown To investigate the clinical relevance of FBP1 in regulating c-Myc protein levels in MSX-130 pancreatic cancer patients, we assessed both c-Myc and FBP1 protein levels in 8 non-tumor and tumor-paired human pancreatic cancer specimens (Fig. ?(Fig.4e).4e). We found that c-Myc expression was up-regulated in pancreatic cancer tissues compared with adjacent normal pancreatic tissue (Fig. ?(Fig.4e4e and ?andf).f). In contrast, the protein level of FBP1 was lower in pancreatic cancer tissues compared to adjacent normal control tissues (Fig. ?(Fig.4e4e and ?andF).F). Intriguingly, there was no correlation between and at the mRNA level (Fig. ?(Fig.4g).4g). Meanwhile, we used a tissue microarray of human pancreatic cancer specimens obtained from a cohort of patients (mRNA level was not overtly correlated with that of (Fig. ?(Fig.4g).4g). Our data suggested that FBP1 regulated c-Myc expression by influencing its post-translational modifications. We systematically investigated whether FBP1 regulate the stability of c-Myc protein in PDAC cells. The overexpression of Flag-FBP1 decreased the protein level of c-Myc in PANC-1 cells, and this effect of FBP1 was completely blocked by the treatment of the proteasome inhibitor MG132 (Fig.?5a and ?andb).b). Moreover, the knockdown of endogenous FBP1 prolonged.