Smurf1 silencing restores PTEN expression that ameliorates progression of human glioblastoma and sensitizes tumor cells to mTORC1/C2 inhibitor Torin1

Abstract

Amplification of ubiquitin E3 ligase Smurf1 promotes degradation of PTEN leading to hyperactivation of the Akt/mTORC1 pathway. However, inhibitors of this pathway have not hitherto yielded promising results in clinical studies because of strong drug resistance. Here, we investigated Smurf1 expression in various glioblastoma (GB) cell lines and patient tissues. The therapeutic efficacy of Smurf1 silencing and Torin1 treatment was assessed in GB cells and orthotopic mouse model. We found Smurf1 loss elevates PTEN levels that interrupt the epidermal growth factor receptor pathway activity. Cotreatment with Smurf1 silencing and mTORC1/C2 inhibitor Torin1 remarkably decreased phosphorylation of Akt, and mTORC1 downstream targets 4EBP1 and S6K resulting in synergistic inhibitory effects. Smurf1 knockdown in orthotopic GB mouse model impaired tumor growth and enhanced cytotoxicity of Torin1. Together, these findings suggest a rational combination of Smurf1 inhibition and Torin1 as a promising new avenue to circumvent PI3K/Akt pathway-driven tumor progression and drug resistance.

Keywords: Molecular biology; Oncology.

Graphical abstract

Figure 1

Smurf1 is elevated in GB cells (A) Immunohistochemistry (IHC) was performed for Smurf1 protein expression in GB patient tissues and in the normal temporal lobe. Tissues were first sectioned, and then sections were probed with primary antibodies against Smurf1. Target protein expression was evaluated via indirect detection using a labeled secondary antibody. After staining with hematoxylin, the antigen-antibody complex was visualized under a bright-field microscope. In IHC stained images brown tint shows positive immunoreactivity for Smurf1 antigen. Scale bars, 50 μm. (B) Different tumor cells, including PTEN-wt (LN229, U343), and PTEN-mut (U251, LNZ308, U87, U118, and #19005) GB cells were grown under standard culture conditions described in methods. For expression analysis, cells were lysed and whole-cell lysates were examined through Western blotting for the expression of EGFR, p-Akt^S473, Akt, PTEN, Smurf1, and β-actin proteins. Results shown here represent three independent experiments.

Figure 2

Depletion of Smurf1 decreased GB cell viability (A) PTEN-wt (LN229 and U343) and PTEN-mut (U118, U251, LNZ308, and U87) GB cell lines were stably transfected with si-Control or si-Smurf1. Western blotting was employed to detect target proteins p-Akt, Akt, Smurf1, and β-actin. Five independent experiments showed similar protein expressions. (B) The expression of Smurf1 in Smurf1 shRNA-transduced LN229 and LNZ308 cells was examined by Western blotting. Blots show that shSmurf1 effectively knocked down Smurf1. (C) Anti-proliferative effect of Smurf1 silencing was measured through clonogenic assay. Crystal violet-stained cells represent proliferation and colony formation following shPLKO and shSmurf1 transfection in the LN229 cell line. Smurf1 loss significantly reduces the colony formation capability of LN229 cells (∗∗∗, P < 0.001). Data shown here are means ± SEM of five independent experiments. (D) Anti-proliferative effect of Smurf1 silencing was measured through clonogenic assay. Crystal violet-stained cells represent proliferation and colony formation following shPLKO and shSmurf1 transfection in the LNZ308 cell line. No significant impact on colony formation potential of LNZ308 cells was observed after Smurf1 knockdown (NS, P > 0.05). Data shown here are means ± SEM of five independent experiments. (E) Comparison of the tyrosine phosphorylation pattern. Western blot of phospho-4G10 in control and Smurf1 shRNA-transduced U343 and LN229 cell lines confirmed that Smurf1 loss causes decreased phosphotyrosine levels. (F) Analysis of protein expressions in shSmurf1 transfected LN229 cells. Blots show that Smurf1 knockdown in LN229 cells is associated with decline in the expressions of p-Akt and p-p70S6K, which are key regulatory proteins of PI3K/Akt pathway.

Figure 3

Smurf1 knockdown reduced PTEN dependent EGF signaling (A–D) GB cell lines: (A) PTEN-wt LN229 cells; (B) PTEN-wt U343 cells; (C) PTEN-mut LNZ308 cells; and (D) PTEN-mut U251 cells were stably transfected with shPLKO or shSmurf1, followed by starvation in serum and growth factor free medium for 48 h and stimulation for 5 min with 100 ng/mL EGF. Immunoblotting is performed on the whole cell lysates to check Smurf1, Akt, and p-Akt levels. In PTEN-wt cells, Smurf1 loss significantly altered the level of p-Akt under analysis; however, PTEN-mut did not show a notable response. All data are presented from three independent experiments, and similar results were obtained.

Figure 4

Smurf1 labels PTEN for degradation via ubiquitination and suppression of Smurf1 sensitizes GB cells to Torin1 (A) To validate our hypothesis that Smurf1 exerts its oncogenic effects through PTEN regulation, we transfected LN229 (shSmurf1 and shPLKO) cells with si-Control or si-PTEN for 72 h. Immunoblotting was employed to analyze the PTEN silencing efficacy and expressions of target proteins p-Akt and Akt in cell lysates. Blot shows that Smurf1 knockdown had no effect on p-Akt and Akt protein in si-PTEN treated LN229 (shSmurf1 and shPLKO) cells, verifying that Smurf1 effects on tumors are dependent on PTEN. (B) Impact of Smurf1 knockdown on PTEN expression was further verified by treating LN229 (shSmurf1 and shPLKO) cells with protein synthesis inhibitor CHX (100 μg/mL) for 24 h. Western blotting recapitulated similar results, showing that expression of PTEN is increased after Smurf1 loss, even in the presence of CHX. (C) Pull down assay established a direct physical contact between Smurf1 and PTEN. (D) Immunoprecipitation analysis in LN229 cells using mouse antibody IgG and anti-PTEN antibody also confirmed this interaction. (E) To assess the involvement of Smurf1 in PTEN ubiquitylation, LN229 cells were treated with shSmurf1 (knockdown) and HA-Smurf1 (silent mutant against shSmurf1) (overexpression) in the presence of proteasome inhibitor MG132. LN229 controls received Flag-ubiquitin. It is evident from the blot that Smurf1 silencing decreases PTEN ubiquitination, whereas HA-Smurf1 induced overexpression of Smurf1 significantly enhanced PTEN ubiquitination even in the presence of shSmurf1 and MG132. (F) LN229 cells were transfected with shSmurf1 or shPLKO in the presence or absence of mTOR inhibitor Torin1 (500 nM). Western blotting was carried out to study changes in the expression of target proteins, including p-Akt, Akt, p-p70S6K, and p70S6K. (G) Before cell lysis for expression analysis, MTT assay was performed to evaluate the cytotoxicity of treatments on LN229 GB cells. Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n = 3 in each group). (∗∗∗, p < 0.001).

Figure 5

Transfection of wild-type PTEN into mutant GB cells resolved Torin1 cytotoxicity (A) U251-shSmurf1 and U251-shPLKO cells were transfected with PLHCX-3HA-PTEN followed by treatment with/without Torin1 (500 nM). To measure protein expressions, Western blotting was done for p-Akt, Akt, Smurf1, and β-actin. The graph on the right showed the relative p-Akt intensity of lane 5 and lane 6. (B) U343-shSmurf1 and U343-shPLKO cells treated with/without Torin1 (500 nM). To measure protein expressions, Western blotting was done for p-Akt, Akt, p-p70S6K, p70S6K, Smurf1, and β-actin. (C) The number of cells in each group was counted through colorimetric MTT assay before cell collection or lysis. (D) Levels of the 4EBP1 and p-4EBP1 in LN229-shPLKO and LN229-shSmurf1 cells, in the presence or absence of Torin1 treatment, were determined by Western blotting. β-actin was taken as a loading control. (E) LN229 with or without Smurf1 knockdown treated with/without Torin1 (500 nM) or Rapa. (100 nM). To measure protein expressions, Western blotting was done for p-4EBP1, 4EBP1, p-Akt, Akt, Smurf1, and β-actin. (F) Anti-proliferative effect was measured through clonogenic assay. Crystal violet-stained cells represent proliferation and colony formation following shPLKO and shSmurf1 transfection in Rapamycin resistant cells with or without Torin1 (500 nM) or Rapamycin (100 nM) treatment. Rapamycin resistant cell lines were built by treating LN229 cells in a medium gradually supplemented with Rapamycin for 2 weeks; results shown here represent similar data from three independent experiments; mean ± SEM (n = 3). (NS, p > 0.05; ∗, p < 0.05; ∗∗∗, p < 0.001)

Figure 6

Smurf1 knockdown combined with Torin1 treatment inhibited the cell cycle and promoted apoptosis (A) Representative immunofluorescence staining of Ki67 (red) in PTEN-wt GB cells, LN229 (shSmurf1 and shPLKO). The nuclear localization was verified by staining (DAPI; blue). The number of Ki67⁺ cells was counted in each section. ∗∗, P < 0.01. (B) Cell cycle analysis through flow cytometry. The impact of si-Smurf1 and Torin1 treatments on cell cycle progression was measured in PTEN-wt LN229 GB cells. Cell quantification data in each phase of the cell cycle was represented on the right. (C) Apoptotic efficiency of treatments was analyzed in PTEN-wt GB cells LN229 (shSmurf1 and shPLKO) with or without Torin1 treatment. Cells were stained using Annexin V-FITC/PI kit followed by apoptosis analysis through flow cytometry. All data are displayed as means ± SEM (n = 5).

Figure 7

Cotreatment with Smurf1 silencing and Torin1 produced a synergistic inhibitory effect in an orthotopic mouse model (A–C) LN229 (shSmurf1 or shPLKO) tumor cells were intracranially implanted into the female BALB/c nude mice (n = 15; 5 × 10⁵ tumor cells per animal), which were then randomized into two groups. Starting from ten days post-implantation, mice in each group received 5 times/week intraperitoneal injection of vehicle (75% ethanol +25% PBS) or Torin1 (20 mg/kg). IVIS imaging performed after three weeks of treatment shows in vivo tumor growth (n = 15/group) (A); tumor size in mm² (n = 5/group in replicates) (B); and immunofluorescence staining of DAPI, p-4G10 and PTEN (n = 3/group in replicates) (C).

Figure 8

Targeting Smurf1 promotes Torin1 efficacy in PTEN-wild type GB Allosteric inhibitors of mTORC1 rapamycin are largely ineffective in inhibiting mTORC2 activity. In addition, 4EBP1 is rephosphorylated to long-term rapamycin treatment. Torin1 potently inhibits mTORC1 and mTORC2. We tested the enhanced efficacy of Torin1 by suppression of Smurf1 through sustained inhibition of 4EBP1 phosphorylation.