SOX9 is a critical regulator of TSPAN8-mediated metastasis in pancreatic cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is the deadliest cancer mainly owing to its proclivity to early metastasis and the lack of effective targeted therapeutic drugs. Hence, understanding the molecular mechanisms underlying early invasion and metastasis by PDAC is imperative for improving patient outcomes. The present study identified that upregulation of TSPAN8 expression in PDAC facilitates metastasis in vivo and in vitro. We found SOX9 as a key transcriptional regulator of TSPAN8 expression in response to EGF stimulation. SOX9 modulation was sufficient to positively regulate endogenous expression of TSPAN8, with concomitant in vitro phenotypic changes such as loss of cell-matrix adherence and increased invasion. Moreover, increased SOX9 and TSPAN8 levels were shown to correlate in human pancreatic cancer specimens and downregulated in vitro by EGFR tyrosine kinase inhibitors. High expression of SOX9 and TSPAN8 has been associated with tumor stage, poor prognosis and poor patient survival in PDAC. In conclusion, this study highlights the importance of the EGF-SOX9-TSPAN8 signaling cascade in the control of PDAC invasion and implies that TSPAN8 may be a promising novel therapeutic target for the treatment of PDAC.

Fig. 1. TSPAN8 is highly expressed in PDAC and is associated with progression and poor prognosis.

A–C Immunohistochemical staining (A), TSPAN8 expression (B–C) and tumor size (D) data for 87 human pancreatic cancer specimens were analyzed. Representative images of normal adjacent tissues (NATs) and tumor tissues (TTs) are shown. Scale bars: 200 μm. E TSPAN8 expression in tumor tissues from patients with distant metastasis and without distant metastasis was analyzed. F The survival times of 87 PDAC patients with low (black curve) and high (red curve) TSPAN8 protein levels (low, 47 patients; high, 40 patients) indicated a significant association of the TSPAN8 level with patient survival, as determined by a log-rank test. G–H A total of 10⁶ SW1990 cells with or without expression of HA-TSPAN8 were injected into athymic nude mice. Representative tumor xenografts are shown (G). The number of visible metastatic lesions in the liver was analyzed by a t test (H). I Kaplan–Meier survival analysis was performed. P values were calculated by a log-rank test (N = 6 mice). J–K Immunohistochemical staining for TSPAN8 was performed on 17 PDAC patient specimens of primary tumor tissues and liver metastases (J). Scale bars: 200 μm. TSPAN8 expression in primary tumor tissues and liver metastasis tissues was analyzed by a t test (K). B, D, E, H, K the values are presented as the means ± SDs. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig. 2. TSPAN8 plays a key role in pancreatic tumor cell invasion and migration.

A, B TSPAN8 expression was examined in the indicated cell lines by immunoblot analysis (A) or qPCR (B). C–D A wound-healing assay was performed on BxPC-3 and AsPC-1 cells expressing the control shRNA, TSPAN8 shRNA vector or reconstituted with shRNA-resistant TSPAN8. E–F Boyden chamber Matrigel invasion assays were performed on BxPC-3 and AsPC-1 cells expressing the control shRNA, TSPAN8 shRNA vector or reconstituted with shRNA-resistant TSPAN8. Scale bars: 100 μm. A, B, D and F the experiments were performed in triplicate, and the t test was performed. The values are presented as the means ± SDs. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig. 3. SOX9 is required for EGF-induced TSPAN8 upregulation.

A, B BXPC-3 and SW1990 cells were treated with or without EGF (100 ng/ml) for different times. TSPAN8 mRNA levels were analyzed by qPCR (A), and immunoblot analysis was performed with the indicated antibodies (B). C BxPC-3 and SW1990 cells were treated with or without gefitinib (10 μM) or erlotinib (10 μM) for 1 hr prior to EGF treatment (100 ng/ml) for 2 hr. Relative mRNA levels were analyzed by qPCR. D HPDE6-C7 cells with stable overexpression of different TFs were generated. TSPAN8 mRNA levels were analyzed by qPCR. E HPDE6-C7 cells with stable overexpression of the TSPAN8 promoter and Flag-SOX9 or Flag-JUN were generated. A luciferase activity assay was performed. F, G BxPC-3 and SW1990 cells were treated with GDC-0994 (10 μM), compound 26 (10 μM) and GO6983 (10 μM) for 1 hr prior to EGF treatment (100 ng/ml) for 2 hr. The SOX9 mRNA and protein levels were analyzed by qPCR and immunoblotting with the indicated antibodies, respectively. H, I BxPC-3 and HCC827 cells with or without stable expression of shSOX9 were treated with EGF (100 ng/ml) for 2 hr. The TSPAN8 mRNA and protein levels were analyzed by qPCR (H) and immunoblotting with the indicated antibodies (I). J BxPC-3 and SW1990 cells were treated with or without gefitinib (10 μM) or erlotinib (10 μM) for 1 hr prior to EGF treatment (100 ng/ml) for 2 hr. Protein expression was analyzed by immunoblotting with the indicated antibodies. In A, C–F, the experiments were performed in triplicate, and the t test was performed. The values are presented as the means ± SDs. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig. 4. SOX9 regulates TSPAN8 mRNA expression by directly binding to its promoter.

A 293 T cells with transient expression of Flag-SOX9 and sites in the TSPAN8 promoter were cultured for 48 h for a luciferase activity assay. B Wild-type (wt) and mutant (mut) binding sites for SOX9 in the TSPAN8 promoter region. C A luciferase activity assay was performed in 293 T cells with transient expression of the wt or mut TSPAN8 gene promoter and Flag-SOX9. D ChIP-qPCR analysis was performed. The Y axis shows the value normalized to the input. E EMSA was performed with Flag-SOX9 and probes for the site 1, site 2, mut 1 or mut 2 region of the TSPAN8 promoter. A, C and D, the experiments were performed in triplicate, and the t test was performed. The values are presented as the means ± SDs. **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig. 5. TSPAN8 and SOX9 expression levels are correlated, and high-expression levels of both are related to poor prognosis.

A, B Immunohistochemical staining for TSPAN8, EGFR and SOX9 was performed on 40 human pancreatic cancer specimens. Representative images of TSPAN8 high-expression and TSPAN8 low-expression tumor tissues are shown (A). Scale bars: 200 μm. Pearson correlation analysis was performed to evaluate associations between the expression levels of TSPAN8 and SOX9 or TSPAN8 and EGFR (B). C Correlation analysis of SOX9 and TSPAN8 mRNA expression in pancreatic cancer, hepatocellular carcinoma and breast cancer was performed with data from the TCGA data set. D Kaplan–Meier survival analysis of patients with pancreatic cancer, hepatocellular carcinoma or breast cancer included in the TCGA data set (the groups were stratified by the SOX9 or TSPAN8 expression level) was performed.

Fig. 6. A schematic diagram of the mechanism by which EGF-ERK-SOX9 regulates TSPAN8 to promote tumor metastasis.

SOX9 is upregulated upon EGF stimulation and binds two regions in the promoter of the TSPAN8 gene to upregulate TSPAN8 expression. High TSPAN8 expression leads to enhanced cell invasion. Therefore, in cancer cells, EGF-ERK-SOX9-TSPAN8 signaling enhances tumor metastasis.