Critical role of DEK and its regulation in tumorigenesis and metastasis of hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality globally. Therefore, it is quite essential to identify novel HCC-related molecules for the discovery of new prognostic markers and therapeutic targets. As an oncogene, DEK plays an important role in cell processes and participates in a variety of cellular metabolic functions, and its altered expression is associated with several human malignancies. However, the functional significance of DEK and the involved complex biological events in HCC development and progression are poorly understood. Here, combing the results from clinical specimens and cultured cell lines, we uncover a critical oncogenic role of DEK, which is highly expressed in HCC cells. DEK protein encompasses two isoforms (isoforms 1 and 2) and isoform 1 is the most frequently expressed DEK isoform in HCC cells. DEK depletion by using shRNA inhibited the cell proliferation and migration in vitro and suppressed tumorigenesis and metastasis in mouse models. Consistently, DEK overexpression regardless of which isoform produced the opposite effects. Further studies showed that DEK induced cell proliferation through upregulating cell cycle related CDK signaling, and promoted cell migration and EMT, at least in part, through the repression of β-catenin/E-cadherin axis. Interestingly, isoform 1 induced cell proliferation more efficiently than isoform 2, however, no functional differences existed between these two isoforms in cell migration. Together, our study indicates that DEK expression is required for tumorigenesis and metastasis of HCC, providing molecular insights for DEK-related pathogenesis and a basis for developing new strategies against HCC.

Keywords: DEK; HCC; isoform; metastasis; migration.

Figure 1. DEK expression is elevated in HCC tissues and cell lines

A. Gene expression data obtained from GSE25097 dataset were used to analyze DEK expression in tumor and adjacent samples. Units for Y-axis are absolute expression value from microarray data. B. RT-qPCR was used to determine the DEK expression in HCC cells and liver normal cells. C. Tumor and adjacent non-tumor tissue pairs from patients with HCC were collected and RT-qPCR was performed to measure the DEK expression. D. Kaplan-Meier analysis was performed to determine the correlation between the DEK expression and overall survival of HCC patients. Units for Y-axis are survival percentages of HCC patients. Data are represented as mean ± SEM, Results of RT-qPCR presented represent the mean of triplicate experiments ± SEM. *P<0.05; **P<0.01.

Figure 2. DEK knockdown inhibits cell proliferation and migration in SMMC7721 cells

A. Western blotting was performed to detect the knockdown effect by shRNA against DEK (shDEK-1 and shDEK-2). The non-target shRNA-expressing cells (shCTRL) were the knockdown control cells. B. MTS assay was performed to determine the cell proliferation when DEK was knocked down in SMMC7721 cells. C. Clonogenic assay was performed to measure the capacity of colony formation when DEK was depleted. 1×10³ SMMC7721 cells were seeded in 6-well-plate to form colonies in 3 weeks and colonies with no less than 50 cells/colony were counted. Quantitation of colony number was shown in the right panel. D. Morphology showing knockdown of DEK in SMMC7721 cells reverts typically mesenchymal morphology to epithelial characteristics. E. Wound-healing assay was employed to determine the migration of SMMC7721 cells in response to DEK depletion. Cells were monitored within 24 hours to evaluate the rate of migration into the scratched area. Results presented represent the mean of triplicate experiments ± SEM. *P<0.05; **P<0.01.

Figure 3. Knockdown of DEK suppresses tumor growth and metastasis in vivo

A. Representative images showing xenograft tumors at day 42 post subcutaneous injection (n=5). B, C. Tumor sizes measured and depicted as tumor volume (B) or tumor weight (C). D. Representative images showing metastatic liver nodules in nude mice by splenic-vein injection of DEK knockdown SMMC7721 cells and the knockdown control cells. The arrows indicate the metastatic tumor on the surface of the liver. E. The numbers of nodules were quantified on nude mice livers (n=5). F. H&E staining was performed on serial sections of metastatic tumors and normal liver (×200). The arrows show the tumor foci in mouse liver section. Data are represented as mean ± SEM. **P<0.01.

Figure 4. DEK overexpression promotes cell proliferation and migration in MHCC97L cells

A. Western blotting was performed to determine the effect of DEK overexpression. B. MTS assay was performed to determine the cell proliferation when DEK isoform 1 (Iso1) or isoform 2 (Iso2) was ectopically expressed in MHCC97L cells. Cells overexpressing empty vector (EV) were the control cells. C. Clonogenic assay was performed to measure the capacity of colony formation when DEK was overexpressed. 1×10³ MHCC97L cells were seeded in 6-well-plate to form colonies in 2 weeks and colonies with no less than 50 cells/colony were counted. Quantitation of colony number was shown in the right panel. D. Morphology showing DEK overexpression facilitates EMT in MHCC97L cells E. Wound-healing assay was employed to determine the migration of MHCC97L cells in response to DEK overexpression. Cells were monitored within 24 hours to evaluate the rate of migration into the scratched area. Results presented represent the mean of triplicate experiments ± SEM. *P<0.05; **P<0.01.

Figure 5. DEK promotes cell proliferation through the regulation of cell cycle related genes

A, B. Flow cytometry analysis was performed to determine the cell cycle progression when DEK was depleted (A) or overexpressed (B). C, D. RT-qPCR was performed to determine the expression of CDK1, CDK2, CDK4 and PCNA genes when DEK was depleted (C) or overexpressed (D). Results presented represent the mean of triplicate experiments ± SEM. *P<0.05; **P<0.01.

Figure 6. DEK negatively regulates E-cadherin through enhancing active β-catenin

A, B. RT-qPCR was performed to determine the E-cadherin expression at mRNA levels when DEK was depleted (A) or overexpressed (B) in HCC cells. C, D. Western blotting was performed to measure the protein levels of E-cadherin and enhances active β-catenin when DEK was depleted (C) or overexpressed (D) in HCC cells. Data are represented as mean ± SEM. *P<0.05; **P<0.01.