Transcription factor Late SV40 Factor (LSF) functions as an oncogene in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a highly aggressive cancer with no currently available effective treatment. Understanding of the molecular mechanism of HCC development and progression is imperative for developing novel, effective, and targeted therapies for this lethal disease. In this article, we document that the cellular transcription factor Late SV40 Factor (LSF) plays an important role in HCC pathogenesis. LSF protein was significantly overexpressed in human HCC cells compared to normal hepatocytes. In 109 HCC patients, LSF protein was overexpressed in >90% cases, compared to normal liver, and LSF expression level showed significant correlation with the stages and grades of the disease. Forced overexpression of LSF in less aggressive HCC cells resulted in highly aggressive, angiogenic, and multiorgan metastatic tumors in nude mice. Conversely, inhibition of LSF significantly abrogated growth and metastasis of highly aggressive HCC cells in nude mice. Microarray studies revealed that as a transcription factor, LSF modulated specific genes regulating invasion, angiogenesis, chemoresistance, and senescence. The expression of osteopontin (OPN), a gene regulating every step in tumor progression and metastasis, was robustly up-regulated by LSF. It was documented that LSF transcriptionally up-regulates OPN, and loss-of-function studies demonstrated that OPN plays an important role in mediating the oncogenic functions of LSF. Together, these data establish a regulatory role of LSF in cancer, particularly HCC pathogenesis, and validate LSF as a viable target for therapeutic intervention.

Fig. 1.

LSF is overexpressed in HCC. (A) LSF expression was detected by Western blot in the indicated cells. β-tubulin was used as loading control. (B) Analysis of LSF expression in tissue microarray by immunohistochemistry. (C) FISH was performed on human HCC samples for LSF and D12Z3 (probe targeting pericentromeric region of chromosome 12). Red, LSF; green, D12Z3; arrow, cell displaying four signals for each of the probes, indicating four copies of these regions of chromosome 12.

Fig. 2.

LSF overexpression increases proliferation, anchorage-independent growth, and invasion of HepG3 cells. (A) Control-8 (Cont-8) and Control-13 (Cont-13) clones are neomycin-resistant clones, and LSF-1 and LSF-17 clones are LSF-overexpressing clones of HepG3 cells. Western blot analysis was performed to detect LSF and β-tubulin expression in these cells. (B) LSF WT-Luc, luciferase reporter plasmid preceded by four tandem LSF-binding sites; LSF-MT-Luc, luciferase reporter plasmid preceded by mutated LSF-binding sites. The indicated cells were transfected with either empty pGL3-basic vector or LSF WT-Luc or LSF MT-Luc along with renilla luciferase expression vector. Luciferase assay was performed 2 days later, and firefly luciferase activity was normalized by renilla luciferase activity. (C) Cell viability of the indicated cells at the indicated time points was measured by standard MTT assay. (D) Colony formation assay for the indicated cells. Colony number per 250 cells is shown. (E) Soft agar assay for the indicated cells. For D and E, the colonies were scored 2 weeks after plating. (F) Matrigel invasion assay using the indicated clones. (Inset) Invading cells. For B–F, the data represents mean ± SEM.

Fig. 3.

Dominant negative LSF (LSFdn) inhibits proliferation, anchorage-independent growth, and invasion by QGY-7703 cells. (A) Control-1 (Cont-1) and Control-7 (Cont-7) clones are neomycin-resistant clones, and LSFdn-8 (dn-8) and LSFdn-15 (dn-15) clones are dominant negative LSF-overexpressing clones of QGY-7703 cells. Western blot analysis was performed to detect LSF and β-tubulin expression in these cells. (B) The indicated cells were transfected with either empty pGL3-basic vector or LSF WT-Luc or LSF MT-Luc along with renilla luciferase expression vector. Luciferase assay was performed 2 days later, and firefly luciferase activity was normalized by renilla luciferase activity. (C) Cell viability of the indicate cells at the indicated time points was measured by standard MTT assay. (D) Colony formation assay for the indicated cells. Colony number per 250 cells is shown. (E) Soft agar assay for the indicated cells. For (D) and (E), the colonies were scored 2 weeks after plating. (F) Matrigel invasion assay using the indicated clones. (Inset) Invading cells. For B–F, the data represents mean ± SEM.

Fig. 4.

Overexpression of LSF increases and inhibition of LSF decreases tumorigenesis of human HCC cells in nude mice. Control-8, LSF-1, and LSF-17 clones of HepG3 cells were s.c. implanted in athymic nude mice. Tumor volume (A) and tumor weight (B) were measured 3 weeks after implantation. Control-1, Control-7, LSFdn-8, and LSFdn-15 clones of QGY-7703 cells were s.c. implanted in athymic nude mice. Tumor volume (C) and tumor weight (D) were measured 3 weeks after implantation. Immunofluorescence analysis of LSF, Ki-67, and CD31 in tumor sections of LSF-1 and LSF-17 clones of HepG3 cells (E) and Control-7 and LSFdn-15 clones of QGY-7703 cells (F).

Fig. 5.

Overexpression of LSF increases and inhibition of LSF decreases metastasis of human HCC cells in nude mice. (A) Control-8 and LSF-17 clones of HepG3 cells were injected i.v. through the tail vein in athymic nude mice. The internal organs were analyzed 4–6 weeks after injection. (B) Kaplan–Meier survival curve of animals injected with either Control-8 or LSF-17 clones of HepG3 cells. *, mice losing ~20% body weight and euthanized (considered as dead). (C) Control-1 and LSFdn-15 clones of QGY-7703 cells were injected i.v. through the tail vein in athymic nude mice. Metastatic tumors were visible externally in mice injected with the Control-1 clone but not with the LSFdn-15 clone. (D) Graphical representation of metastatic lung nodules in the animals injected with Control-8 and LSF-17 clones of HepG3 cells. (Inset) H&E sections of lungs. (E) Graphical representation of metastatic lung nodules in the animals injected with Control-1 and LSFdn-15 clones of QGY-7703 cells. (Inset) H&E sections of lungs. For (D) and (E), the data represent mean ± SEM.

Fig. 6.

OPN expression is transcriptionally induced by LSF. (A and B) Real-time PCR analysis of CFH and OPN mRNA expression in Control-8 and LSF-17 clones of HepG3 cells (A) and Control-1 and LSFdn-15 clones of QGY-7703 cells (B). (C) OPN expression was detected by ELISA in Control-8 and LSF-17 clones of HepG3 cells (Left) and Control-1 and LSFdn-15 clones of QGY-7703 cells (Right). (D) Schematic diagram of OPN promoter-luciferase construct showing the location of LSF binding sites in the promoter and primers designed for ChIP assay. (E) Control-8 and LSF-17 clones of HepG3 cells were transfected with pGL3-basic vector or OPN-Prom-luc along with renilla luciferase expression vector. Luciferase assay was performed 2 days later, and firefly luciferase activity was normalized by renilla luciferase activity. (F) ChIP assay to detect LSF binding to the OPN promoter.

Fig. 7.

Inhibition of OPN abrogates augmentation of proliferation, anchorage-independent growth, and invasion by LSF. LSF-17-OPNsh-6 (OPNsh-6) and LSF-17-OPNsh-18 (OPNsh-18) clones stably express OPN shRNA and were generated in the background of LSF-17 clone of HepG3 cells. LSF-17consh-15 (Consh-15) clone stably expresses control scrambled shRNA and was also generated in LSF-17 background. (A) OPN mRNA expression detected by real-time PCR in the indicated clones. (B) OPN protein expression detected by ELISA in the indicated clones. (C) Cell viability of the indicated cells at the indicated time points were measured by standard MTT assay. (D) Colony formation assay for the indicated cells. (E) Soft agar assay for the indicated cells. (F) Matrigel invasion assay of the indicated clones. The data represents mean ± SEM.

Fig. 8.

Inhibition of OPN abrogates LSF-induced tumorigenesis and metastasis. The indicated clones were s.c. implanted in athymic nude mice. (A) Tumor volume and tumor weight were measured 3 weeks after implantation. (B) Graphical representation of metastatic lung nodules in the animals injected with the LSF-17consh-15 and LSF-17-OPNsh-18 clones via tail vein. The data represent mean ± SEM. (Inset) H&E sections of lungs of animals injected with the indicated clones. (C) Parental HepG3 cells were treated with conditioned media from Control-8 or LSF-17 clone of HepG3 cells and then subjected to Matrigel invasion. (D) Matrigel invasion assay using LSF-17 clone of HepG3 cells in the presence of neutralizing antibodies. Integrin, anti-αvβ3 integrin antibody; CD44, anti-CD44 antibody. The data represent mean ± SEM.