Silencing SAPCD2 Represses Proliferation and Lung Metastasis of Fibrosarcoma by Activating Hippo Signaling Pathway

Abstract

The primary problem associated with fibrosarcoma is its high potential to metastasize to the lung. Aberrant expression of SAPCD2 has been widely reported to be implicated in the progression and metastasis in multiple cancer types. However, the clinical significance and biological roles of SAPCD2 in fibrosarcoma remain unknown. Here, we reported that SAPCD2 expression was markedly elevated in fibrosarcoma tissues, and its expression was differentially upregulated in fibrosarcoma cell lines compared with that in several primary fibroblast cell lines. Kaplan-Meier survival analysis revealed that SAPCD2 overexpression was significantly correlated with early progression and metastasis, and poor prognosis in fibrosarcoma patients. Our results further showed that silencing SAPCD2 inhibited the proliferation and increased the apoptosis of fibrosarcoma cells in vitro. Importantly, silencing SAPCD2 repressed lung metastasis of fibrosarcoma cells in vivo. Mechanistic investigation further demonstrated that silencing SAPCD2 inhibited the proliferation and lung metastasis of fibrosarcoma cells by activating the Hippo signaling pathway, as evidenced by the finding that constitutively active YAP1, YAP1-S127A, significantly reversed the inhibitory effect of SAPCD2 downregulation on the colony formation and anchorage-independent growth capabilities of fibrosarcoma cells, as well as the stimulatory effect on the apoptotic ratio of fibrosarcoma cells. In conclusion, SAPCD2 promotes the proliferation and lung metastasis of fibrosarcoma cells by regulating the activity of Hippo signaling, and this mechanism represents a potential therapeutic target for the treatment of lung metastatic fibrosarcoma.

Keywords: Hippo signaling; SAPCD2; fibrosarcoma; lung metastasis; proliferation.

Figure 1

SAPCD2 is upregulated in fibrosarcoma tissues and cells. (A) Real-time PCR and Western blotting analysis of SAPCD2 expression in 4 normal fibroblast cell lines, including human dermal fibroblast primary cell (hDFPC), human lung fibroblasts primary cell (hLFPC), human mammary fibroblasts primary cell (hMFPC), Human embryonic lung fibroblast (HFL1), and 2 fibrosarcoma cell lines HT-1080 and SW684. GAPDH was used as endogenous controls in RT-PCR and α-Tubulin was detected as a loading control in the Western blot. Each bar represents the mean values ± SD of three independent experiments. *P < 0.05. (B) Representative images of SAPCD2 expression in benign fibroma, dermatofibrosarcoma protuberans (DFSP), and fibrosarcoma tissues. (C) The number of fibroma, DFSP, and fibrosarcoma tissues stratified by staining index of IHC. (D) Immunohistochemical staining index of SAPCD2 in fibroma, DFSP, and fibrosarcoma tissues. n.s. indicates no significance.

Figure 2

Overexpression of SAPCD2 correlates with advanced clinicopathological features and poor prognosis in fibrosarcoma patients. (A–C) Kaplan-Meier analysis of overall survival (OS) (A), progression-free survivals (PFS) (B), and distant metastasis-free survival (DMFS) (C) in fibrosarcoma patients with low SAPCD2 expression versus high SAPCD2 expression. (D–E) Kaplan-Meier analysis of overall survival (OS) (D) and progression-free survivals (PFS) (E) in fibrosarcoma patients with low SAPCD2 expression versus high SAPCD2 expression from TCGA.

Figure 3

Silencing SAPCD2 inhibits lung metastasis of fibrosarcoma cells in vivo. (A, B) Real-time PCR and Western blotting analysis of SAPCD2 expression in the indicated fibrosarcoma cells. GAPDH was used as endogenous controls in RT-PCR and α-Tubulin was detected as a loading control in the Western blot. Each bar represents the mean values ± SD of three independent experiments. *P < 0.05. (C) Schematic model illustrating the time and route of HT-1080 cell (vec. or SAPCD2-sh#1/2 stable cell lines) administration in a mouse model of lung metastasis. (D) Representative images of the lung metastases formed and hematoxylin-eosin staining from the indicated cells in the mice. The numbers of lung tumor nests in each group was counted under a low power field and are presented as the median values ± quartile values (right panel). *P < 0.05. (E) The quantification of the number of fibrosarcoma cells (per mm³) in the indicated tumor tissues. Error bars represent the mean ± SD values. *P < 0.05. (F) Kaplan-Meier survival curves from the indicated mice groups. (G) Images of excised tumors from the mice at 30 days after injection with the indicated cells. (H) Tumor volumes were measured every 5 days. Each bar represents the median values ± quartile values. (I) Average weight of excised tumors from the indicated mice. Each bar represents the median values ± quartile values. *P < 0.05.

Figure 4

Downregulation of SAPCD2 retards the proliferation and anoikis resistance of fibrosarcoma cells in vitro. (A, B) The effect of silencing SAPCD2 on the proliferation rate in HT-1080 and SW684 cells by CCK-8 assay. Each bar represents the mean values ± SD of three independent experiments. *P < 0.05. (C) The effect of silencing SAPCD2 on the colony-forming ability in HT-1080 and SW684 cells by colony formation assay. (D) The effect of silencing SAPCD2 on the anchorage-independent growth ability in HT-1080 and SW684 cells by anchorage-independent growth assays. Each bar represents the mean values ± SD of three independent experiments. *P < 0.05. (E) The effects of SAPCD2 on apoptotic ratio in the indicated fibrosarcoma cells via annexin V-FITC/PI staining. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05. (F) The effect of SAPCD2 on the invasion ability in the indicated fibrosarcoma cells. Error bars represent the mean ± S.D. of three independent experiments.

Figure 5

SAPCD2 inactivates Hippo signaling pathway. (A) GSEA analysis showed that SAPCD2 expression level was associated with the Hippo signaling. (B) TEAD transcriptional activity was assessed by HOP-Flash luciferase reporter in the indicated cells. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05. (C) Western blot analysis of total p-MST1/2, MST1, p-LATS1, LATS1, p-YAP1, YAP1 expression and nuclear YAP, TAZ expression in the indicated cells. α-Tubulin was detected as a loading control and nuclear protein p84 was used as the nuclear protein marker. (D) Real-time PCR analysis of CTGF, CYR61, SOX9, HOXA1, RPL13A, and PPIA in the indicated cells. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05. (E) Western blotting analysis of SAPCD2 expression in 4 normal fibroblast cell lines, including human dermal fibroblast primary cell (hDFPC), human lung fibroblasts primary cell (hLFPC), human mammary fibroblasts primary cell (hMFPC), Human embryonic lung fibroblast (HFL1), and 2 fibrosarcoma cell lines HT-1080 and SW684. α-Tubulin was detected as a loading control.

Figure 6

SAPCD2 promotes proliferation and anoikis resistance via inactivates Hippo signaling pathway in fibrosarcoma cells. (A) YAP1-S127A reversed the TEAD transcriptional activity repressed by downregulation of SAPCD2 assessed by HOP-Flash luciferase reporter. Each bar represents the mean values ± SD of three independent experiments. *P < 0.05. (B, C) YAP1-S127A reversed the inhibitory effects of SAPCD2 downregulation on colony-forming ability (B) and anchorage-independent growth capability (C) in fibrosarcoma cells. Each bar represents the mean values ± SD of three independent experiments. *P < 0.05. (D) YAP1-S127A reversed the pro-apoptotic role of SAPCD2 downregulation on in fibrosarcoma cells. Each bar represents the mean values ± SD of three independent experiments. *P < 0.05.