Fig 1: 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 2: TSPAN8 promotes tumorigenesis in mice. a Flow cytometry was used to separate TS+ from TS− tumor cells derived from three human breast cancer patients. These cells were subcutaneously injected (103, 104, 105, 106 cells per mouse) into NOD/SCID mice. Tumor formation ability and stem cell frequency were analyzed. b, c MDA-MB-231 cells with or without TSPAN8 overexpression or depletion were subcutaneously injected (1000, 5000, 10,000, 50,000 cells per mouse) into 6-week-old female nude mice. Tumor formation ability and stem cell frequency were analyzed (b). Kaplan–Meier survival analysis was performed (c). P values were calculated by log-rank test (n = 15 mice, P < 0.001). d–g Nude mice were subcutaneously injected with 1 × 107 Luc-MDA-MB-231 cells with or without depletion or overexpression of TSPAN8 in the presence or absence of ATXN3 shRNA expression. Tumor volumes were measured every week (d). Representative image of mice with xenograft tumors (e) and IHC staining of tumor samples (scale bar = 20 μm) (f) are shown. The expression levels of the indicated proteins in these tumors were examined by immunoblotting analyses (g). Each bar represents the mean ± SD. **P < 0.01, ***P < 0.001, 2-way ANOVA
Fig 3: TSPAN8 expression is upregulated in breast CSCs. a Microarray analyses of expression of 33 members of tetraspanins family expressed in CSCs (spheres) or their corresponding non-CSCs (adherent cells) derived from three independent breast cancer patients. b Freshly isolated primary human tissues derived from two independent breast cancer patients were grown as adherent cells or spheres. Immunoblotting analyses of TSPAN8, TSPAN15 and TSPAN1 were performed. c Representative confocal images for TSPAN8 (green), ALDHA1 (red), and nuclei (blue) of adherent cells or spheres from two breast cancer patients were shown. d Quantification of the relative fluorescence intensity of TSPAN8 and ALDHA1 in spheres or adherent cells. Two-tailed Student’s t test was used for statistical analysis. ***P < 0.001, **P < 0.01, *P < 0.05. e Immunoblotting analyses of TSPAN8, NANOG, OCT4, and ALDHA1 expression in breast cancer cells from three patients were performed. TS−: TSPAN8-negative breast cancer cells, TS+: TSPAN8-positive breast cancer cells. f Flow cytometry analysis of the ratios of CD44+/CD24− cells in TS+ breast cancer cells or TS− breast cancer cells derived from three human breast cancer patients. g Semi-quantitative analyses of IHC staining of TSPAN8 in tissue sections of 90 breast cancer patients with different pathological molecular subtypes. ***P < 0.001, **P < 0.01, *P < 0.05 and N.S., not significant (P > 0.05) by repeated measures with Student’s t test. LuA = luminal A subtype, LuB = luminal B subtype, Her2 = Her2 amplified subtype, TNCB = triple-negative subtype. h Kaplan–Meier of survival of 90 patients with breast tumors (two groups stratified by TSPAN8 expression level. Differences between the groups were shown by a log-rank test. i–k Immunohistochemistry analyses of TSPAN8 expression in specimens of breast cancer patients with NAC-S (neo-adjuvant chemotherapy sensitive) and NAC-R (neo-adjuvant chemotherapy resistant) characteristics (scale bar = 50 μm, n = 20). l–n Immunohistochemistry analyses of TSPAN8 expression in specimens of breast cancer patients with Pre-NAC (before neo-adjuvant chemotherapy) and Post-NAC (after neo-adjuvant chemotherapy) (scale bar = 50 μm, n = 20). Note that some dots overlapped
Fig 4: 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 5: TSPAN8 correlates with PTCH1, SHH, and ATXN3 expression. a Representative IHC images of TSPAN8 in breast tumors (n = 20) and their matched adjacent tissues. b Representative IHC images of the expression levels of SHH, PTCH1, and ATXN3 in breast tumors. c–f Positive correlation of TSPAN8 expression with SHH (c), PTCH1 (d), and ATXN3 (e) expression and positive correlation between PTCH1 and ATXN3 expression (f) are shown. *P < 0.05, **P < 0.01, ***P < 0.001. The P values and correlation coefficient were analyzed as indicated. g Kaplan–Meier plot of survival from 90 breast cancer patients is shown. A log-rank test was used to calculate the difference between the two groups. h A mechanism of TSPAN8-enhanced Sonic Hedgehog signaling pathway. The interaction between TSPAN8 and PTCH1 leads to recruitment of ATXN3 deubiquitinating enzyme to the SHH-PTCH1 complex and subsequent deubiquitylation of PTCH1 and inhibition of proteasome-mediated degradation of SHH and PTCH1. Stabilized SHH and PTCH1 promote the binding of protein kinase GRK2 to SMO, phosphorylation and translocation of SMO to cilia, GLI1 activation for downstream gene expression, cancer cell stemness, as well as tumor formation in mice
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