Regorafenib (Stivarga) pharmacologically targets epithelial-mesenchymal transition in colorectal cancer

Abstract

Epithelial-to-mesenchymal transition (EMT) is well-known to evoke cancer invasion/metastasis, leading to a high frequency of mortality in patients with metastatic colorectal cancer (mCRC). Protein tyrosine phosphatase (PTPase)-targeted therapy has been identified as a novel cancer therapeutic. Previously, we proved that sorafenib with anti-EMT potency prevents TGF-β1-induced EMT/invasion by directly activating SH2-domain-containing phosphatase 1 (SHP-1)-dependent p-STAT3Tyr705 suppression in hepatocellular carcinoma. Regorafenib has a closely related chemical structure as sorafenib and is approved for the pharmacotherapy of mCRC. Herein, we evaluate whether regorafenib activates PTPase SHP-1 in the same way as sorafenib to abolish EMT-related invasion/metastasis in CRC. Notably, regorafenib exerted potent anti-EMT activity to curb TGF-β1-induced EMT/invasion in vitro as well inhibited lung metastatic outgrowth of SW480 mesenchymal cells in vivo. Mechanistically, regorafenib-enhanced SHP-1 activity significantly impeded TGF-β1-induced EMT/invasion via low p-STAT3Tyr705 level as proved by a SHP-1 inhibitor or siRNA-mediated SHP-1 depletion. Conversely, overexpression of SHP-1 further enhanced the inhibitory effects of regorafenib on TGF-β1-induced p-STAT3Tyr705 and EMT/invasion. Regorafenib directly activates SHP-1 by potently relieving the autoinhibited N-SH2 domain of SHP-1 to inhibit TGF-β1-induced p-STAT3Tyr705 and EMT/invasion. Importantly, the clinical evidence indicated that SHP-1 was positively correlated with E-cadherin and that significantly determined the overall survival of CRC patients. This result further confirms our in vitro data that SHP-1 is a negative regulatory PTPase in EMT regulation and serves as a pharmacological target for mCRC therapy. Collectively, activating PTPase SHP-1 by regorafenib focusing on its anti-EMT activity might be a useful pharmacotherapy for mCRC.

Keywords: CRC; EMT; SHP-1; STAT3; regorafenib.

Figure 1. The expression of SHP-1 and E-cadherin correlates with overall survival of CRC patients

A. Representative immunohistochemical patterns revealed that clinical CRC patient#1 with strong positive expression of SHP-1 had strong positive expression of E-cadherin. Patient#2 with weak positive expression of SHP-1 had weak positive expression of E-cadherin. B. Patients with weak expression of both SHP-1 and E-cadherin had a more significant reduction in median OS than others. (N.A., not available)

Figure 2. The inhibition of mesenchymal characteristics by regorafenib is dependent on p-STAT3Tyr705 down-regulation

A. Western blotting of p-STAT3^Tyr705, STAT3, mesenchymal (vimentin and fibronectin) and epithelial (E-cadherin) markers in SW480 cells 24 h after treatment with dose-escalation of regorafenib. GAPDH was used as a loading control. The invasive capability of cells was measured. The results are shown as mean ± SD of three independent experiments (*, P < 0.05). Data are presented as a percentage relative to control SW480 cells. B. Western blotting was used to detect the expression of p-STAT3^Tyr705, STAT3, vimentin, fibronectin and E-cadherin in Hct-116 cells 24 h after either co-treatment with TGF-β1 (10 ng/ml) and/or the indicated dose of regorafenib (1, 2.5, 5 μM). GAPDH was used as a loading control. Invasion assay was performed in cells. The results are shown as mean ± SD of three independent experiments (*, P < 0.05). Data are presented as the percentage relative to control Hct-116 cells. C. Western blotting of p-STAT3^Tyr705, STAT3, vimentin, fibronectin and E-cadherin in STAT3-overexpressing Hct-116 cells 24 h after either co-treatment with TGF-β1 (10 ng/ml) and/or regorafenib (2.5 μM). GAPDH was used as a loading control. Invasion assay was performed in these cells. The results are shown as mean ± SD of three independent experiments (*, P < 0.05). Data are presented as the percentage relative to control Hct-116 cells. D. Immunofluorescence microscopy analysis of rhodamine phalloidin-stained F-actin, DAPI-stained nuclei, vimentin and E-cadherin in the cells shown in (C). Image analysis of vimentin and E-cadherin was quantified by two independent experiments. (*, P < 0.05)

Figure 3. Abolishment of the effects of TGF-β1 on p-STAT3Tyr705 and EMT by regorafenib is through the enhancement of SHP-1 activity, resulting in down-regulation of p-STAT3Tyr705

A. Upper panels, SHP-1 activity was measured in SW480 cells treated with dose-escalation of regorafenib for 24 h. Lower panels, SHP-1 activity was measured in the Hct-116 cells either co-treated with TGF-β1 (10 ng/ml) and/or the indicated dose of regorafenib for 24 h. The results are shown as mean ± SD of three independent experiments (*, P < 0.05). Data are presented as the percentage relative to control SW480 or Hct-116 cells. B. Left panels, western blotting of p-STAT3^Tyr705, STAT3, vimentin, fibronectin and E-cadherin in TGF-β1-treated Hct-116 cells 24 h after either co-treatment with or without SHP-1 siRNA (25 nM) and/or regorafenib (2.5 μM). Right panels, western blotting of p-STAT3^Tyr705, STAT3, vimentin, fibronectin and E-cadherin in TGF-β1-treated Hct-116 cells 24 h after either co-treatment with or without SHP-1 inhibitor (20 nM) and/or regorafenib (2.5 μM). GAPDH was used as a loading control. Invasion assay was performed in these cells shown in left and right panels. The results are shown as mean ± SD of three independent experiments (*, P < 0.05; N.S., non-significant). Data are presented as the percentage relative to control Hct-116 cells. C. Western blotting of p-STAT3^Tyr705, STAT3, vimentin, fibronectin and E-cadherin in SHP-1-overexpressed Hct-116 and HT-29 cells 24 h after either co-treatment with TGF-β1 (10 ng/ml) and/or regorafenib (2.5 μM). GAPDH was used as a loading control. The invasive capability was measured in these cells. The results are shown as mean ± SD of three independent experiments (*, P < 0.05, **, P < 0.01). Data are presented as a percentage relative to the control Hct-116 or HT-29 cells. D. SHP-1 activity was performed in cells shown in (C). The results are shown as mean ± SD of three independent experiments (*, P < 0.05, **, P < 0.01). Data are presented as a percentage relative to control Hct-116 or HT-29 cells. E. A mechanistic representation showing that regorafenib prevents TGF-β1-induced EMT mediated through regorafneib-enhanced SHP-1 activity that effectively downregulates p-STAT3^Tyr705 expression.

Figure 4. Regorafenib enhances SHP-1 activity through potently relieving the autoinhibition of SHP-1, which reinforces the suppressive effect on p-STAT3Tyr705 and mesenchymal characteristics

A. Upper panels, a schematic representation of wild-type and mutant-type SHP-1 including D61A and C453S carrying constitutive and dead activity of SHP-1, respectively. Lower panels, SHP-1 activity was assessed in the wild-type and mutant-type (D61A and C453S) SHP-1-transfected SW480 cells 2 days after transfection with these plasmids. The results are shown as mean ± SD of three independent experiments made in triplicate. (*, P < 0.05; N.S., non-significant) B. Left panels, western blotting of SHP-1, p-STAT3^Tyr705, STAT3, and EMT markers in the wild-type and mutant-type (D61A and C453S) SHP-1-transfected SW480 cells 24 h after treatment with or without regorafenib at 2.5 μM. Right panels, the invasive capability was measured in the cells shown in the left panels of (B). The results are shown as mean ± SD of three independent experiments, made in triplicate. (*, P < 0.05; N.S., non-significant) C. Regorafenib suppressed TGF-β1-induced EMT and invasion in CRC which was mediated through regorafenib-enhanced SHP-1 activity. Regorafenib had the potential to dock to the inhibitory N-SH2 domain and the catalytic PTP domain of SHP-1, resulting in the direct relief of autoinhibition of SHP-1. The activity of SHP-1 tyrosine phosphatase specifically increased the susceptibility to p-STAT3^Tyr705, which antagonized the EMT pathway.

Figure 5. Regorafenib suppresses lung metastatic outgrowth of CRC cells in vivo

A. Representative BLI images of animals in each experimental group at the indicated time points after receiving the indicated drug treatment. B. Normalized BLI signals of lung metastatic outgrowth in mice (n = 5) receiving regorafenib (10 mg/kg/day) or vehicle, daily, when metastatic growth was initially seen in the lung after tail vein injection with Luc2-expressing SW480 cells (2 × 10⁶). Points, mean (n = 5); bars, SEM. *, P < 0.05.