Clusterin suppresses invasion and metastasis of testicular seminoma by upregulating COL15a1

Abstract

Seminoma is the most common subtype of testicular germ cell tumor, with an increasing incidence worldwide. Clusterin (CLU) expression was found to be downregulated in testicular seminoma in our previous study. We now expanded the sample size, and further indicated that CLU expression correlates with tumor stage. Tcam-2 cell line was used to investigate the CLU function in testicular seminoma, and CLU was found to inhibit the proliferation and metastasis abilities. Besides, extracellular matrix protein COL15a1 was demonstrated as the downstream of CLU to affect the epithelial-mesenchymal transition (EMT) process via competitively binding to DDR1 with COL1A1 and inhibiting the phosphorylation of PYK2. MEF2A was found to interact with CLU and bind to the promoter of COL15a1 and so upregulate its expression. This is the first study using testicular xenografts in situ to simulate testicular seminoma metastatic and proliferative capacities. In conclusion, CLU acts as a tumor suppressor to inhibit the metastasis of testicular seminoma by interacting with MEF2A to upregulate COL15a1 and blocking the EMT process.

Keywords: COL15a1; EMT; MEF2A; clusterin; seminoma; testicular xenografts in situ.

Graphical abstract

Figure 1

CLU was significantly downregulated in seminoma according to database and clinical tissues (A) Data downloaded from Oncomine were redrawn by GraphPad software. (B) qRT-PCR analyses were performed with 38 seminoma tissues and 12 normal testes to explore the mRNA expression of CLU. (C) Western blot analyses were performed with 10 pairs of seminoma tumor tissues and adjacent normal tissues to explore the protein expression of CLU. (D) IHC analyses were further used to identify the expression difference. Scale bar: 100 μm. (E) Representative stains of seminoma tissues by IHC, 1 (negative), 2 (weak brown), 3 (moderate brown), 4 (strong brown). Data are represented as mean ± SD. Scale bar: 100 μm, ∗∗∗∗p < 0.0001.

Figure 2

CLU curbed Tcam-2 cells proliferation in vitro and in vivo through arresting cell cycle in G1 phase (A) Stable CLU-knockdown and -overexpression cell lines were established successfully. (B) The optical density (OD) values detected in the CCK-8 proliferation assay were performed after 1–4 days seeding, and higher OD values indicate higher cell viability. (C) Colony formation was photographed 2 weeks after seeding and quantified by ImageJ software. (D) Xenografts in mice in vivo and their ki-67 staining (E), volumes (F), and weights (G). (H) Flow cytometric cell cycle analysis of shCLU, oeCLU, and NC cells showed the percentage of cells in G1 or G2+S phase. Data are represented as mean ± SD. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Scale bar: 100 μm.

Figure 3

CLU knockdown enhanced the invasion, migration, and wound healing of Tcam-2 cells (A) Photographs at 0 and 12 h after scratching. (B) Transwell assays of CLU-knockdown and -overexpression cells. The migration assays were performed without Matrigel and photographed 12 h after seeding. The invasion assays were performed with Matrigel and photographed 48 h after seeding. Data are represented as mean ± SD. Scale bar: 20 μm. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 4

The expression of ECM-associated COL15A1 and COL7A1 was significantly changed with CLU knockdown or overexpression (A) The most enriched GO terms based on the RNA-seq data of oeCLU and oe-NC groups. (B) Data downloaded from TCGA were submitted to GSEA and extracellular matrix organization was also significantly enriched in samples with higher CLU expression. (C) 21 and 115 upregulated ECM-associated genes from GO and GSEA terms were overlapped. COL15a1, COL7A1, and VCAN were screened out. (D) The co-expression of CLU with COL15a1, COL7A1, and VCAN based on the database: GEPIA. (E) The verification of co-expression relationship with 50 seminoma tissues. (F and G) mRNA and protein expression of COL15a1, COL7A1, and VCAN in pretreated Tcam-2 cells. Data are represented as mean ± SD. ∗p < 0.05.

Figure 5

EMT pathway was significantly inhibited in CLU-overexpression cells (A) Western analysis of EMT markers (E-cad, N-cad, Vimentin, β-catenin, and MMP3) in CLU-knockdown and -overexpression cells. (B) 3D spheroid invasion of oeCLU, shCLU, and NC cells 24 h and 72 h after Matrigel addition. The aggressive cells are marked with red arrow. Data are represented as mean ± SD. Scale bar: 100 μm. ∗∗∗∗p < 0.0001.

Figure 6

COL15a1 competitively binds to the DDR1 with collagen I to block the phosphorylation of PYK2 and so inhibit the EMT process (A) Tcam-2 cells (EV) and Tcam-2 cells with high level of CLU (oeCLU) were transfected with NC and shCOL15a1 lentivirus. The invasion abilities of pretreated cells were determined by the transwell assay. (B) The expression of indicated proteins was detected by western blot. (C) COL7a1 was also downregulated as above methods, and the transwell assays were followed subsequently. (D) Western blot assays were performed to detect the indicated proteins level. (E) Pretreated cells were coated or non-coated with collagen I. The cell aggregation indicated their scattering ability. (F) The downstream markers of collagen I (pPYK2, pFAK) and collagen XV (pPYK2) were detected by western blot. (G) In vivo imaging of testicular xenografts in situ injected with four groups of pretreated cells. Metastasis occurred in the lung, medial iliac, mediastinal, and inguinal lymph nodes. The number of metastatic foci and the tumor volumes were recorded. (H) Photographs and H&E staining of testicular xenografts in situ. (I) The protein markers of EMT (E-cad, N-cad, and Vimentin) in group 2 primary tumor and group 4 pulmonary metastasis were detected by IHC. Data are represented as mean ± SD. Scale bar: 20 μm.

Figure 7

CLU interacted with MEF2A to upregulate COL15a1 (A) Jaspar database was used to estimate the potential TFs that could bind to the promoter of COL15a1, and ChIP assay was performed to prove this in oeCLU and NC groups. (B) Luciferase reporter assay was performed in shMEF2A cells to detect the luciferase activity. The mRNA expression of COL15a1 was then explored with qRT-PCR in shMEF2A, oeCLU, and oeCLU + shMEF2A groups. (C) The mutated promoter of COL15a1 was constructed. The luciferase activity of MEF2A was then detected in WT and mutant groups with cells transfected by shCLU, shMEF2A, oeCLU + shMEF2A, and NC. (D) Immunoprecipitation assay was performed with total, nucleus, and cytoplasm protein respectively to study the directly interaction between CLU and MEF2A. (E) Co-localized IF staining displayed the distribution of CLU and MEF2A. (F) The analysis of COL15a1 expression in seminoma based on UALCAN database. (G) The mechanism of CLU in inhibiting testicular seminoma metastasis.