TSPAN12 Precedes Tumor Proliferation by Cell Cycle Control in Ovarian Cancer

Abstract

TSPAN12, a member of the tetraspanin family, has been highly connected with the pathogenesis of cancer. Its biological function, however, especially in ovarian cancer (OC), has not been well elucidated. In this study, The Cancer Genome Atlas (TCGA) dataset analysis revealed that upregulation of TSPAN12 gene expression was significantly correlated with patient survival, suggesting that TSPAN12 might be a potential prognostic marker for OC. Further exploration showed that TSPAN12 overexpression accelerated proliferation and colony formation of OVCAR3 and SKOV3 OC cells. Knockdown of TSPAN12 expression in A2780 and SKOV3 cells decreased both proliferation and colony formation. Western blot analysis showed that several cyclins and cyclin-dependent kinases (CDK) (e.g., Cyclin A2, Cyclin D1, Cyclin E2, CDK2, and CDK4) were significantly involved in the regulation of cell cycle downstream of TSPAN12. Moreover, TSPAN12 accelerated mitotic progression by controlling cell cycle. Thus, our data demonstrated that TSPAN12 could be a novel molecular target for the treatment of OC.

Keywords: TSPAN12; cell cycle; ovarian cancer; proliferation.

Fig. 1. TSPAN12 expression is significantly correlated with poor prognosis in OC patients

(A–C) Three datasets were downloaded from TCGA database. Patients were automatically categorized into two groups with high and low expression level of TSPAN12 gene by the online software. Median survival of each group were shown in days. Overall survival of OC patients from each dataset was analyzed with log-rank method after extraction of available clinical data from online Kaplan–Meier Plotter tool. P < 0.05 was considered significant.

Fig. 2. TSPAN12 promotes cell proliferation in OC cells

(A–D) Stable A2780 and SKOV3 cells with TSPAN12 knockout were seeded in 12-well plates (5 × 10³ cells/well). Cells were stained by crystal violet (0.5% w/v) on day 1 to 5, and then the absorbance of crystal violet dissolved in DMSO was recorded at 490 nm. Two individual knockout clones were employed in evaluating the proliferation effect of TSPAN12 knockout in OC cells. (E and F) Overexpression of TSPAN12 on the proliferation of OC cells were examined following the above protocol after treatment with or without CDK inhibitor, AT7519 (Inh, 100 nM) for 72 h at concentration of 100 nM. ***P < 0.001; ****P < 0.0001. Results are representative of three distinct experiments.

Fig. 3. TSPAN12 promotes colony formation in OC cells

(A–D) Stable A2780 and SKOV3 cells from two single clones with TSPAN12 knockout were seeded in 6-well plates (5 × 10² cells/well) in parallel with control group. Two weeks after inoculation, cell colonies were stained by crystal violet (0.5% w/v), and colony numbers were counted. (E and F) Colony formation assay in OVCAR3 and SKOV3 cells with TSPAN12 overexpression were performed following the above protocol (1 × 10³ cells/well for inoculation) after treatment with or without CDK inhibitor, AT7519 (Inh, 100 nM) for 72 h. *P < 0.05; **P < 0.01; ****P < 0.0001. Results are representative of three independent experiments.

Fig. 4. TSPAN12 gene knockout inhibits cell growth in subcutaneous and orthotopic xenograft mouse model

(A and C) In subcutaneous model, 5 × 10⁶ A2780-sgCtrl cells were subcutaneously inoculated into the left flank area of five-week-old female BALB/c nude mice with A2780-sgTSPAN12 cells into the right flank (mice were numbered from #1 to #8). (B and D) Subcutaneous xenograft tumor mass and tumor weight were measured of A2780-sgTSPAN12 mice and the controls. (E) Orthotopic mice were established by surgically implantation of tumor pieces into mouse ovary after the subcutaneous tumors have reached the diameter of 1 cm. (F) Tumors in orthotopic xenograft mice were extracted and measured after euthanization. ***P < 0.001.

Fig. 5. TSPAN12 regulates cell cycle in OC cells

(A–H) Cells were treated with serum-free medium for 24 h and then collected at time 0 h, 4 h, 8 h, and 16 h after releasing. Cell cycle progression in A2780, SKOV3 with TSPAN12 knockout were examined by flow cytometry analysis after PI staining. (I–L) OVCAR3 and SKOV3 stable cells with TSPAN12 overexpression were first treated with or without CDK inhibitor, AT7519 (Inh, 100 nM) for 72 h (100 nM), and then evaluated by flow cytometry analysis according to the above protocol. P values between groups were listed above each bar chart. Results are representative of three independent experiments.

Fig. 5. TSPAN12 regulates cell cycle in OC cells

(A–H) Cells were treated with serum-free medium for 24 h and then collected at time 0 h, 4 h, 8 h, and 16 h after releasing. Cell cycle progression in A2780, SKOV3 with TSPAN12 knockout were examined by flow cytometry analysis after PI staining. (I–L) OVCAR3 and SKOV3 stable cells with TSPAN12 overexpression were first treated with or without CDK inhibitor, AT7519 (Inh, 100 nM) for 72 h (100 nM), and then evaluated by flow cytometry analysis according to the above protocol. P values between groups were listed above each bar chart. Results are representative of three independent experiments.

Fig. 6. Regulation of OC progression by TSPAN12 is highly associated with cyclin and CDK proteins

(A–D) Stable A2780, SKOV3 cells with TSPAN12 silencing were analyzed by western blot analysis of Cyclin A2, Cyclin E2, Cyclin D1, CDK2, and CDK4. (E-H) OVCAR3 and SKOV3 cells with TSPAN12 overexpression were analyzed by western blot analysis after being treated with or without CDK inhibitor, AT7519 (Inh, 100 nM) for 72 h. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are presented as the mean ± standard deviation (SD) of three independent experiments.