Inhibition of JAK1, 2/STAT3 signaling induces apoptosis, cell cycle arrest, and reduces tumor cell invasion in colorectal cancer cells

Abstract

Abnormalities in the STAT3 pathway are involved in the oncogenesis of several cancers. However, the mechanism by which dysregulated STAT3 signaling contributes to the progression of human colorectal cancer (CRC) has not been elucidated, nor has the role of JAK, the physiological activator of STAT3, been evaluated. To investigate the role of both JAK and STAT3 in CRC progression, we inhibited JAK with AG490 and depleted STAT3 with a SiRNA. Our results demonstrate that STAT3 and both JAK1 and 2 are involved in CRC cell growth, survival, invasion, and migration through regulation of gene expression, such as Bcl-2, p1(6ink4a), p21(waf1/cip1), p27(kip1), E-cadherin, VEGF, and MMPs. Importantly, the FAK is not required for STAT3-mediated regulation, but does function downstream of JAK. In addition, our data show that proteasome-mediated proteolysis promotes dephosphorylation of the JAK2, and consequently, negatively regulates STAT3 signaling in CRC. Moreover, immunohistochemical staining reveals that nuclear staining of phospho-STAT3 mostly presents in adenomas and adenocarcinomas, and a positive correlation is found between phospho-JAK2 immunoreactivity and the differentiation of colorectal adenocarcinomas. Therefore, our findings illustrate the biologic significance of JAK1, 2/STAT3 signaling in CRC progression and provide novel evidence that the JAK/STAT3 pathway may be a new potential target for therapy of CRC.

Figure 1

AG490 and STAT3 siRNA downregulation of JAK1, 2/STAT3 signaling. (A) Western blot analysis revealed that AG490 induced a concentration-dependent decrease in JAK2 and pJAK2 levels at 24 hours in CRC cells. In the same experiment, decreases in JAK1, pJAK1, and pSTAT3 levels were also identified, although the decrease in JAK3, pJAK3, and STAT3 was not statistically significant. (B) At 72 hours posttransfection, Western blot analysis showed that STAT3 siRNA induced downregulation of STAT3 and pSTAT3 in CRC cells. (C) MG132 blocks downregulation of pJAK2 and pSTAT3, induced by AG490. SW1116 and HT29 cells incubated with AG490 for 16 hours were treated with MG132, a proteasome inhibitor. MG132 induced a time-dependent upregulation of pJAK2 and pSTAT3. The data shown are representative of three separate experiments. GAPDH was used for the loading control. Quantification of the target protein bands relative to GAPDH is shown in the right panel.

Figure 2

Disruption of JAK1, 2/STAT3 signaling is associated with modulation of some STAT3 downstream targets. (A) Western blot analysis showed that AG490 induced concentration-dependent alterations of part, but not all, of the STAT3 downstream targets in SW1116 cells at 24 hours after treatment. Bcl-2 was downregulated, simultaneously associated with the upregulation of p16^ink4a, p21^waf1/cip1, and p27^kip1, whereas survivin showed no detectable change. A similar pattern of changes was identified in HT29 cells. (B) STAT3 siRNA induced similar effects. Indeed, downregulation of Bcl-2, and upregulation of p16^ink4a, p21^waf1/cip1, and p27^kip1 were detected at 72 hours after transfection of the cells. GAPDH was used for the loading control. Quantification of the target protein bands relative to GAPDH is shown in the right panel.

Figure 3

The functional role of JAK1, 2/STAT3 signaling on CRC cell growth, cell cycle progression, and apoptosis. (A) Cell viability as determined by the CCK-8 assay of CRC cells treated with AG490 or STAT3 siRNA. SW1116 cells were treated with solvent only (negative controls) or with 50, 100, or 150 µM AG490. The percentage of viable cells was determined as described in the Materials and Methods section. AG490 significantly decreased the number of viable cells. No significant difference was seen in the three dosages of AG490. Treating HT29 cells with AG490 also induced a concentration-dependent decrease in the number of viable cells at 72 hours. In addition, STAT3 siRNA inhibited CRC cell growth. This suppression lasted for 72 hours, and the cell recovered at 96 hours posttransfection. The results represent mean ± SD of three experiments. (B) Cell cycle analysis was performed using SW1116 cells treated with diluent (left) or AG490 (50, 100, or 150 µM) for 24 hours. Compared to the negative control, our results reveal that AG490 induces a concentration-dependent increase in the proportion of cells in the G₁ phase. Moreover, treating SW1116 cells with STAT3 siRNA blocked the cell cycle in the G₁ phase at 72 hours after transfection. (C) CRC cells was treated with 100 µM AG490 for 24 hours or STAT3 siRNA for 72 hours, and cell apoptosis was detected by morphologic changes and flow cytometric analysis.

Figure 4

The functional role of JAK1, 2/STAT3 signaling on the invasive ability of CRC cell. (A) AG490 suppresses the invasion in CRC cells at 24 hours after treatment. The migrated cell numbers were normalized to that of the negative controls. Our data demonstrated that AG490 significantly inhibits the invasiveness of CRC cells (*P < .05). (B) CRC cells transfected with STAT3 siRNA were examined for their invasive capability. At 48 hours posttransfection, the numbers of migrated cells significantly decreased when compared with that of untreated cells (P < .05). (C) Western blot analysis showed that AG490 induced a concentration-dependent upregulation of E-cadherin, simultaneously associated with the downregulation of FAK in CRC cells at 24 hours after treatment. (D) STAT3 siRNA induced an increase in E-cadherin at 72 hours posttransfection of the cells. However, we failed to find the FAK-level changes by STAT3 siRNA transfection. (E) The concentrations of VEGF, MMP2, and MMP9 in AG490-treated SW1116 cells were tested by ELISA. The data are from three individual experiments, and show reduced secretion of MMP2 and VEGF after AG490 treatment (*P < .05). (F) Effects of STAT3 siRNA on the secretions of VEGF, MMP2, and MMP9. At 48 hours posttransfection, the concentrations of VEGF and MMP2 were decreased compared to that of untreated cells (*P < .05). The experiment was performed three times with consistent findings.

Figure 5

Immunohistochemical staining of the tissue microarray. Predominantly, cytoplasmic staining of STAT3 was frequently detected in normal colonic epithelium, adenomas, and primary colon adenocarcinomas. Nevertheless, nuclear staining of pSTAT3 was mostly presented in adenomas and adenocarcinomas. JAK2 and pJAK2 showed predominantly cytoplasm localization. Original magnification, x200.

Figure 6

Model of the possible contributions of JAK1, 2/STAT3 signaling pathway in CRC development. Proteasome-mediated proteolysis promotes dephosphorylation of the JAK2 kinase and, consequently, negatively regulates JAK/STAT3 signaling in CRC. Other unknown mechanisms may also have been involved in this process. Furthermore, JAK1, 2/STAT3 signaling is implicated in many areas of tumor progression, including cell growth, survival, invasion, and migration by regulation of gene expressions, such as Bcl-2, p16^ink4a, p21^waf1/cip1, and p27^kip1, E-cadherin, VEGF, and MMPs. Additionally, FAK may be a component of the JAK pathway and a downstream of JAK, which may exert its oncogenic effects through interaction with other signal transduction pathways.