Pharmacological Targeting SHP-1-STAT3 Signaling Is a Promising Therapeutic Approach for the Treatment of Colorectal Cancer

Abstract

STAT3 activation is associated with poor prognosis in human colorectal cancer (CRC). Our previous data demonstrated that regorafenib (Stivarga) is a pharmacological agonist of SH2 domain-containing phosphatase 1 (SHP-1) that enhances SHP-1 activity and induces apoptosis by targeting STAT3 signals in CRC. This study aimed to find a therapeutic drug that is more effective than regorafenib for CRC treatment. Here, we showed that SC-43 was more effective than regorafenib at inducing apoptosis in vitro and suppressing tumorigenesis in vivo. SC-43 significantly increased SHP-1 activity, downregulated p-STAT3(Tyr705) level, and induced apoptosis in CRC cells. An SHP-1 inhibitor or knockdown of SHP-1 by siRNA both significantly rescued the SC-43-induced apoptosis and decreased p-STAT3(Tyr705) level. Conversely, SHP-1 overexpression increased the effects of SC-43 on apoptosis and p-STAT3(Tyr705) level. These data suggest that SC-43-induced apoptosis mediated through the loss of p-STAT3(Tyr705) was dependent on SHP-1 function. Importantly, SC-43-enhanced SHP-1 activity was because of the docking potential of SC-43, which relieved the autoinhibited N-SH2 domain of SHP-1 and inhibited p-STAT3(Tyr705) signals. Importantly, we observed that a significant negative correlation existed between SHP-1 and p-STAT3(Tyr705)expression in CRC patients (P = .038). Patients with strong SHP-1 and weak p-STAT3(Tyr705) expression had significantly higher overall survival compared with patients with weak SHP-1 and strong p-STAT3(Tyr705) expression (P = .029). In conclusion, SHP-1 is suitable to be a useful prognostic marker and a pharmacological target for CRC treatment. Targeting SHP-1-STAT3 signaling by SC-43 may serve as a promising pharmacotherapy for CRC.

Figure 1

SC-43, a derivative of regorafenib, induces more potent apoptosis of CRC cells than regorafenib. (A) Chemical structures of regorafenib and SC-43. (B) MTT assay was performed to measure the cell viability in parental colon cancer cell lines (Hct-15, Hct-116, DLD1, HT-29, and SW480) 2 days after treatment with regorafenib or SC-43 in a dose-dependent manner. The results are shown as mean ± SD of three independent experiments, made in triplicate. *P < .05. (C) Sub-G1 analysis was performed in the cells 2 days after treatment with regorafenib or SC-43 in a dose-dependent manner. The results are shown as mean ± SD of three independent experiments, made in triplicate. *P < .05. (D) Apoptotic cell death was measured in the cells 24 hours after treatment with dose escalation of regorafenib or SC-43. The results are shown as mean ± SD of three independent experiments, made in triplicate. *P < .05.

Figure 2

SC-43 triggers apoptosis via p-STAT3^Tyr705 downregulation. (A) Western blotting of p-STAT3^Tyr705, the cleaved fragments of PARP, and the survival markers (cyclin D1, Mcl-1, and survivin) in cells 24 hours after treatment with dose escalation of SC-43. β-Actin was used as a loading control. The cleaved fragments of PARP are indicated by arrows. (B) Overexpression of STAT3 in Hct-116 and Hct-15 cells rescued apoptosis induced by treatment with 5 μM SC-43 for 24 hours. Data from apoptosis assay are shown as mean ± SD of three independent experiments, made in triplicate. *P < .05.

Figure 3

SC-43 enhances SHP-1 tyrosine phosphatase activity by targeting the autoinhibited SH2 domain of SHP-1 to suppress p-STAT3^Tyr705signaling and further induce apoptosis. (A, upper panels) SHP-1 tyrosine phosphatase activity was measured in the cells (Hct-116, DLD1, HT-29, and Hct-15) after treatment with or without 5 μM SC-43 for 24 hours. (Lower panels) SC-43 increased the SHP-1 tyrosine phosphatase activity in immunoprecipitation-SHP-1–containing cell extract. The results are shown as mean ± SD of three independent experiments, made in triplicate.*P < .05. (B) SC-43 induced a decrease in p-STAT3^Tyr705. Apoptosis occurring in Hct-15 cells was dependent on SHP-1 as assessed by using 20 nM of SHP-1 phosphatase-specific inhibitor (PTPIII) and 25 nM of siRNA specifically depleted SHP-1 as shown in the left panels and middle panels, respectively. (Right panels) Overexpression of SHP-1 in Hct-15 cells treated with 5 μM SC-43 increased the effects of SC-43 on p-STAT3^Tyr705 and apoptosis. Data from apoptosis assay are shown as mean ± SD of three independent experiments, made in triplicate.*P < .05. (C, upper panels) A schematic representation of wild-type and mutant SHP-1 (dN1 with a deletion of the N-terminal inhibitory domain, and D61A, a single point mutation). (Lower panels) Two days after Hct-116 cells were treated with 5 μM SC-43, SHP-1 activity was potently increased in wild-type SHP-1–expressing but not in dN1 or D61A mutant SHP-1–expressing cells. Data from SHP-1 activity are shown as mean ± SD of three independent experiments, made in triplicate (*P < .05; nonsignificant). (D, upper panels) Western blotting of p-STAT3^Tyr705 and SHP-1 in the wild-type and mutant-type (dN1 And D61A) SHP-1–transfected Hct-116 cells 2 days after treatment with or without SC-43 at 5 μM. (Lower panels) Apoptosis was detected in these cells as shown in upper panels of (D). Data from apoptosis assay are shown as mean ± SD of three independent experiments, made in triplicate (*P < .05; nonsignificant).

Figure 4

SC-43 shows more effective tumor inhibition than regorafenib in a CRC subcutaneous tumor model. (A) Tumor sizes were measured at the times indicated after mice received regorafenib (10 mg/kg per day), SC-43 (10 mg/kg per day), or vehicle. Points, mean (n = 7); bars, SEM. *P < .05, **P < .01. (B) Tumor weight was measured on day 25 after tumor excision. Columns, mean; bars, SD. *P < .05, **P < .01. (C, upper panels) The Levels of p-STAT3^Tyr705 and STAT3 were measured by Western blotting on day 25 after excision of vehicle-, regorafenib-, and SC-43–treated tumors. β-Actin was used as a loading control. (Lower panels) SHP-1 activity was measured in vehicle-, regorafenib-, and sc-43–treated tumors on day 25 after tumor excision. Columns, mean; bars, SD. *P < .05, **P < .01. (D) SC-43 induced potent apoptosis in CRC which was mediated through SC-43–enhanced SHP-1 activity. SC-43 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 was the major trigger of apoptosis.

Figure 5

Overall survival of patients with CRC relative to the expression of SHP-1 and p-STAT3^Tyr705. Patients with strong SHP-1and weak p-STAT3 expression had better OS than others.