TSSC3 represses self-renewal of osteosarcoma stem cells and Nanog expression by inhibiting the Src/Akt pathway

Abstract

Osteosarcoma is the most common type of bone cancer, and the second leading cause of cancer-related death in children and young adults. Osteosarcoma stem cells are essential for osteosarcoma initiation, metastasis, chemoresistance and recurrence. In the present study, we report that: 1) higher TSSC3 expression indicates a better prognosis for osteosarcoma patients, and; 2) overexpression of TSSC3 significantly decreases sphere-forming capacity, tumor initiation, stemness-related surface markers and Nanog expression in osteosarcoma cells. We also discovered that higher Nanog expression correlates to a worse prognosis for osteosarcoma patients, and overexpression of Nanog increases the stem-related phenotype in osteosarcoma cells. Knockdown of Nanog suppresses these phenotypes. Inhibition of Nanog expression and self-renewal of osteosarcoma cells by TSSC3 overexpression appears to be mediated through inactivation of the Src/Akt pathway. In the clinical setting, expression of TSSC3, p-Src and Nanog is associated with recurrence, metastasis and surgical intervention. Lower TSSC3 expression, higher Nanog expression or higher p-Src expression indicate a poor prognosis for osteosarcoma patients. Overall, our study demonstrates that TSSC3 inhibits the stem-like phenotype and Nanog expression by inactivation of the Src/Akt pathway; this emphasizes the importance of Nanog in osteosarcoma stem cells.

Keywords: Nanog; SRC; TSSC3; cancer stem cells; osteosarcoma.

Figure 1. Higher expression of TSSC3 indicates improved prognosis for osteosarcoma patients, represses a stem-like phenotype of OS cells and decreases Nanog expression

(A) Representative images of IHC staining of low (left panel) and high (right panel) TSSC3 expressing cells. (B) Kaplan-Meier curve showing that higher expression of TSSC3 is significantly associated with a better prognosis (P < 0.05). (C) TSSC3 expression in monolayer MTH or SaOS2 cells is higher than that of MTH or SaOS2 sphere cells, respectively (Bars, mean±SEM, *P < 0.05. Upper panel, RT-PCR; lower panel, Western blot). (D) The number of CD133, CD117, and Stro-1 positive MTH (left) or SaOS2 cells (right) significantly decreased after overexpression of TSSC3 (Bars, mean±SEM, *P < 0.05). (E) Efficiency of tumor sphere formation by Lv-TSSC3 MTH or Lv-TSSC3 SaOS2 cells is lower than that of Lv-empty MTH and SaOS2 cells, respectively (Bars, mean±SEM, *P < 0.05). (F) Nanog expression is reduced after TSSC3 overexpression in MTH (left) or SaOS2 (right) cells; meanwhile, Oct4 and Sox2 expression levels were slightly decreased. (G) Nanog expression is suppressed after overexpression of TSSC3 (Bars, mean±SEM, *P < 0.05). (H) There are significantly fewer xenografts generated by Lv-TSSC3 MTH than by Lv-empty MTH cells (N=4; P = 0.0028).

Figure 2. Higher expression of Nanog is associated with a worse prognosis for OS patients and significantly enhances the stem-like phenotype of OS cells

(A) Representative IHC staining images of low (left panel) and high (right panel) Nanog expression. (B) Kaplan-Meier curve showing that higher expression of Nanog is significantly related to a poor prognosis (P < 0.05). (C) Efficiency of tumor sphere formation by Lv-TSSC3/Lv-Nanog MTH (left) or SaOS2 cells (right) is higher than in Lv-TSSC3/Lv-empty cells, respectively (Bars, mean±SEM, *P < 0.05). (D) The percentage of CD133, CD117 and Stro-1 positive Lv-TSSC3 MTH (left) or SaOS2 cells (right) is significantly increased after Nanog overexpression (Bars, mean±SEM, *P < 0.05). (E) The IC₅₀ values of Lv-TSSC3 MTH and SaOS2 cells under cisplatin treatmentis are higher after Nanog overexpression. Migration (F) and invasion (G) capacity is enhanced in Lv-TSSC3 MTH and SaOS2 cells after Nanog overexpression (Bars, mean±SEM, *P < 0.05). (H) There are significantly more xenografts generated by MTH and SaOS2 cells after Nanog overexpression (N=5; P = 0.0001; P = 0.004).

Figure 3. Knockdown of Nanog expression reduces the stem-like phenotype of OS cells

(A) Tumor spheres formed by shNanog (shNanog1 and shNanog2) MTH (upper three images, the scale bar represents 100 μm) or SaOS2 cells (lower two images, the scale bar represents 100 μm) are smaller in size than those formed by scrambled MTH or SaOS2 cells, respectively. (B) Efficiency of tumor sphere formation by shNanog MTH (left) or SaOS2 cells (right) is lower than that by scrambled MTH and SaOS2 cells, respectively (Bars, mean±SEM, *P < 0.05). Migration ability (C) and invasion capacity (D) are reduced in shNanog MTH and SaOS2 cells as compared to their respective scrambled cells (Bars, mean±SEM, *P < 0.05). (E) The IC₅₀ of shNanog1 (P1) and shNanog2 (P2) MTH and SaOS2 cells is lower than in scrambled cells treated with cisplatin. (F) There are fewer xenografts generated by Lv-shNanog SaOS2 cells than by Lv-empty SaOS2 cells (P = 0.0097); meanwhile, there are fewer generated by Lv-shNanog MTH cells (N=5; P = 0.0664).

Figure 4. TSSC3 inhibition of Nanog expresssion is mediated by Src

(A) Overexpression of TSSC3 inhibits Src/Akt pathway activity in OS cells. RT-PCR (B) and Western blot analysis (C) show that inactivation of Src results in reduced Nanog expression in OS cells (Bars, mean±SEM, *P < 0.05). (D) Knockdown of Src results in reduced Nanog expression in MTH (left) and SaOS2 cells (right; Bars, mean±SEM, *P < 0.05), while overexpression of TSSC3 does not further reduce Nanog expression (Bars, mean±SEM, P > 0.05). (E) The number of CD133, CD117 and Stro-1 positive MTH (left) and SaOS2 cells (right) is significantly decreased after Src knockdown (Bars, mean±SEM, *P < 0.05), while overexpression of TSSC3 only slightly further decreases expression of these markers. (F) Efficiency of tumor sphere formation by Lv-empty/siSrc MTH (left) and SaOS2 cells (right) is lower than that of scrambled MTH and SaOS2 cells (Bars, mean±SEM, *P < 0.05), while overexpression of TSSC3 does not have a significant additional influence on sphere formation. (G) Tumor spheres formed by Lv-empty/siSrc MTH (upper three images, the scale bar is 100 μm) or SaOS2 cells (lower three images, the scale bar is 100 μm) are smaller in size than those formed by scrambled/Lv-empty MTH or SaOS2. Overexpression of TSSC3 does not have a significant influence on the size of tumor spheres in siSrc cell lines.

Figure 5. TSSC3 inhibits Nanog expression through the Akt pathway

RT-PCR (A) and Western blot analysis (B) show that inactivation of Akt results in reduction of Nanog in OS cells (Bars, mean±SEM, *P < 0.05). (C) Overexpression of TSSC3 inactivates the Akt pathway, and decreases Nanog expression in MTH (left) and SaOS2 cells (right). Treatment with IGF1 activates the Akt pathway and increases Nanog expression. (D) Efficiency of tumor sphere formation by Lv-TSSC3 MTH (left) or SaOS2 cells (right) is lower than that of Lv-empty MTH and SaOS2 cells. IGF1 treatment enhances the efficiency (Bars, mean±SEM, *P < 0.05). (E) The number of CD133, CD117 and Stro-1 positive Lv-TSSC3 MTH (left) or SaOS2 cells (right) significantly decreases compared to Lv-empty cells. IGF1 treatment significantly enhances the number of marker positive cells (Bars, mean±SEM, *P < 0.05).

Figure 6. Clinical analysis of IHC staining of osteosarcoma samples

(A) Representative images of IHC staining for low (left) and high (right) p-Src expression levels. (B) Kaplan-Meier curve showing that lower expression of p-Src is significantly associated with an improved prognosis (P < 0.05). (C) Scores of IHC staining for p-Src and p-Akt are positively associated (upper panel, Bars, mean±SEM, *P < 0.05); meanwhile, those for p-Src and Nanog are also positively associated (lower panel, Bars, mean±SEM, *P < 0.05). (D) Schematic summarizing our findings. In OS cells, higher Nanog expression significantly enhances stemness, motility and chemoresistance. TSSC3 inhibits Src activity, resulting in decreased activity of the Akt pathway and leading to a reduction in Nanog expression and stem-like phenotype in OS cells.