Blocking the JAK2/STAT3 and ERK pathways suppresses the proliferation of gastrointestinal cancers by inducing apoptosis

Abstract

Dysregulated crosstalk between different signaling pathways contributes to tumor development, including resistance to cancer therapy. In the present study, we found that the mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor trametinib failed to suppress the proliferation of PANC-1 and MGC803 cells by activating the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway, while the JAK2 inhibitor fedratinib failed to inhibit the growth of the PANC-1 cells upon stimulation of extracellular signal-regulated kinase (ERK) signaling. In particular, the most prominent enhancement of the anti-proliferative effect resulted from the concurrent blockage of the JAK2/STAT3 and ERK signaling pathways. Furthermore, the combination of the two inhibitors resulted in a reduced tumor burden in mice. Our evidence suggests novel crosstalk between JAK2/STAT3 and ERK signaling in gastric cancer (GC) and pancreatic ductal adenocarcinoma (PDAC) cells and provides a therapeutic strategy to overcome potential resistance in gastrointestinal cancer.

Keywords: Apoptosis; Crosstalk; ERK pathway; Gastrointestinal cancers; JAK2/STAT3 pathway.

Fig. 1. Mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor trametinib fails to inhibit the viability of human gastric cancer (GC) cells and pancreatic ductal adeno carcinoma (PDAC) cells sufficiently due to activation of Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling. MGC803 (a), MIA PaCa-2 (b), and PANC-1 (c) cells were exposed to trametinib at the concentrations of 5 and 10 μmol/L for 24, 48, or 72 h. Cell survival was measured using a Cell Counting Kit-8 (CCK-8) assay. (d) Trametinib was added to MGC803, MIA PaCa-2, and PANC-1 cells at different concentrations for 8 h, after which the states of the JAK2/STAT3 and extracellular signal-regulated kinase (ERK) pathways were measured by western blot. (e) MGC803 cells were treated with 10 μmol/L trametinib at the indicated time points, and the tyrosine phosphorylation levels of JAK2 and STAT3 were measured by western blot. The results are presented as mean±standard deviation (SD) from three independent experiments. ^** P<0.01 and ^*** P<0.001 vs. control (no trametinib).

Fig. 2. Janus kinase 2 (JAK2) inhibitor fedratinib causes a loss in the ability to suppress the survivals of MGC803, MIA PaCa-2, and PANC-1 cells via the inducement of extracellular signal-regulated kinase (ERK) signaling. (a) Fedratinib was added to MGC803, MIA PaCa-2, and PANC-1 cells at the indicated concentrations for 24 h. Cell viability was measured using a Cell Counting Kit-8 (CCK-8) assay. (b) MGC803 cells were treated with 5 μmol/L fedratinib at the indicated time points, and the tyrosine phosphorylation levels of EGFR, JAK2, and STAT3 were measured by western blot. (c) Fedratinib was added to MGC803 cells at different concentrations for 4 h, and the tyrosine phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and ERK were examined by western blot. (d) MIA PaCa-2 and PANC-1 cells were treated with fedratinib at different concentrations for 8 h, and the tyrosine phosphorylation levels of STAT3 and ERK were examined by western blot. The results are presented as the mean±standard deviation (SD) from three independent experiments. ^* P<0.05 and ^** P<0.01 vs. control (no fedratinib).

Fig. 3. Combination of trametinib and fedratinib results in an anti-proliferative effect in MGC803, MIA PaCa-2, and PANC-1 cells through a decrease in the tyrosine phosphorylation levels of extracellular signal-regulated kinase (ERK) and Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) simultaneously. MGC803 (a), MIA PaCa-2 (b), and PANC-1 (c) cells were treated with a combination of trametinib (10 μmol/L) and fedratinib (2 μmol/L) for 24 h. (d) Cell lysates were collected for western blot analysis. The results are presented as mean±standard deviation (SD) from three independent experiments. ^* P<0.05 and ^*** P<0.001 vs. control; ^&& P<0.01 and ^&&& P<0.001 vs. 10 μmol/L trametinib treatment; ^## P<0.01 and ^### P<0.001 vs. 2 μmol/L fedratinib treatment.

Fig. 4. Fedratinib alone or in combination with trametinib induces apoptosis in human MGC803 gastric cancer (GC) cells. (a, b) Apoptotic level of MGC803 cells treated with fedratinib at the indicated concentrations for 24 h was examined by FACS analysis. (c) Expression of the apoptotic markers, including total caspase-3, caspase-9, poly(adenosine diphosphate (ADP)-ribose) polymerase (PARP), and their cleaved forms, was detected by western blot in MGC803 cells exposed to fedratinib alone. (d, e) Apoptotic level of MGC803 cells treated with trametinib and fedratinib was examined by FACS analysis. (f) Expression of caspase-3, caspase-9, PARP, and their cleaved forms in MGC803 cells treated with trametinib and fedratinib was tested by western blot. The results are presented as mean±standard deviation (SD) from three independent experiments. ^* P<0.05, ^** P<0.01, and ^*** P<0.001 vs. control; ^&&& P<0.001 vs. 10 μmol/L trametinib treatment; ^# P<0.05 vs. 2 μmol/L fedratinib treatment. PI, propidium iodide; FITC, fluorescein isothiocyanate.

Fig. 5. Fedratinib combined with trametinib suppresses tumor growth and induces apoptosis in MGC803 gastric cancer (GC) cell xenograft tumors in vivo. (a) Mice with tumors formed by subcutaneous injection of MGC803 GC cells received fedratinib and trametinib alone or in combination (n=6). (b) Resected tumor tissues after the in vivo experiment are shown (n=6). (c) Tumor growth curve was documented based on tumor volume on the indicated following days. (d) Weights of tumors exposed to fedratinib and trametinib alone or in combination were examined on the indicated days (n=6). (e) Apoptotic level was analyzed using immunoblotting of cleaved caspase-9 in tumor tissue lysates. The results are presented as mean±standard deviation (SD). ^* P<0.05 and ^** P<0.01 vs. control; ^& P<0.05 vs. 0.75 mg/kg trametinib treatment; ^# P<0.05 and ^## P<0.01 vs. 30 mg/kg fedratinib treatment.