SLFN11 inhibits hepatocellular carcinoma tumorigenesis and metastasis by targeting RPS4X via mTOR pathway

Abstract

Hepatocellular carcinoma (HCC) remains one of the most refractory malignancies worldwide. Schlafen family member 11 (SLFN11) has been reported to play an important role in inhibiting the production of human immunodeficiency virus 1 (HIV-1). However, whether SLFN11 also inhibits hepatitis B virus (HBV), and affects HBV-induced HCC remain to be systematically investigated. Methods: qRT-PCR, western blot and immunohistochemical (IHC) staining were conducted to investigate the potential role and prognostic value of SLFN11 in HCC. Then SLFN11 was stably overexpressed or knocked down in HCC cell lines. To further explore the potential biological function of SLFN11 in HCC, cell counting kit-8 (CCK-8) assays, colony formation assays, wound healing assays and transwell cell migration and invasion assays were performed in vitro. Meanwhile, HCC subcutaneous xenograft tumor models were established for in vivo assays. Subsequently, immunoprecipitation (IP) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analyses were applied to understand the molecular mechanisms of SLFN11 in HCC. Co-IP, immunofluorescence and IHC staining were used to analyze the relationship between ribosomal protein S4 X-linked (RPS4X) and SLFN11. Finally, the therapeutic potential of SLFN11 with mTOR pathway inhibitor INK128 on inhibiting HCC growth and metastasis was evaluated in vitro and in vivo orthotopic xenograft mouse models. Results: We demonstrate that SLFN11 expression is decreased in HCC, which is associated with shorter overall survival and higher recurrence rates in patients. In addition, we show that low SLFN11 expression is associated with aggressive clinicopathologic characteristics. Moreover, overexpression of SLFN11 inhibits HCC cell proliferation, migration, and invasion, facilitates apoptosis in vitro, and impedes HCC growth and metastasis in vivo, all of which are attenuated by SLFN11 knockdown. Mechanistically, SLFN11 physically associates with RPS4X and blocks the mTOR signaling pathway. In orthotopic mouse models, overexpression of SLFN11 or inhibition of mTOR pathway inhibitor by INK128 reverses HCC progression and metastasis. Conclusions: SLFN11 may serve as a powerful prognostic biomarker and putative tumor suppressor by suppressing the mTOR signaling pathway via RPS4X in HCC. Our study may therefore offer a novel therapeutic strategy for treating HCC patients with the mTOR pathway inhibitor INK128.

Keywords: Schlafen family member 11; hepatocellular carcinoma; mTOR inhibitor; prognostic biomarker; ribosomal protein S4 X-linked.

Figure 1

SLFN11 is downregulated in human HCC and correlates with poor prognosis. (A) qRT-PCR analysis of SLFN11 mRNA expression in 116-paired tumor and nontumor liver tissues. (B, C) Western blot of SLFN11 protein expression in 12-paired nontumor (N) and tumor (T) liver tissues. (D) mRNA and protein expression level of SLFN11 in a normal liver cell line (L-02)and four HCC cell lines (HCCLM3, Hep3B, SMMC-7721, and PLC/PRF/5). (E) Representative IHC staining images indicating low and high expression of SLFN11 in HCC tissue microarray. Scale bars = 200 μm or 20 μm, respectively. (F) Kaplan-Meier curves for overall survival and recurrence-free survival based on SLFN11 expression in the Fudan LCI cohort 1. *** P < 0.001.

Figure 2

SLFN11 inhibits cell proliferation, migration, invasion, and facilitates apoptosis in vitro. (A) The overexpressing and knockdown efficiency of SLFN11 was verified by qRT-PCR and Western blot assays in HCCLM3 and SMMC-7721 cells. (B) Effects of SLFN11 overexpression and knockdown on cell proliferation by CCK-8 in HCCLM3 and SMMC-7721 cells. (C) Effects of SLFN11 overexpression and knockdown on cell proliferation by colony formation assays in HCCLM3 and SMMC-7721 cells. (D) Effects of SLFN11 overexpression and knockdown on cell migratory abilities by wound healing assays in HCCLM3 and SMMC-7721 cells. (E) Effects of SLFN11 overexpression and knockdown on cell migratory and invasive capacities by transwell assays in HCCLM3 and SMMC-7721 cells. (F) Effects of SLFN11 overexpression and knockdown on cell apoptosis by flow cytometry in HCCLM3 and SMMC-7721 cells. ** P < 0.01, *** P < 0.001.

Figure 3

SLFN11 inhibits HCC progression in vivo, as well as the proteomic analyses of SLFN11 complexes by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). (A) Effects of SLFN11 overexpression on HCC progression by establishment of subcutaneous xenograft mouse models. Tumor growth curves and weight of xenografts derived from HCCLM3-Vector or HCCLM3-SLFN11 cells are shown. (B) Effects of SLFN11 knockdown on HCC progression by establishment of subcutaneous xenograft mouse models. Tumor growth curves and weight of xenografts derived from SMMC-7721-Control or SMMC-7721-shSLFN11 cells are shown. (C) Venn diagram shows the overlapping and unique proteins identified from the complexes related to SLFN11 overexpression and the vector control cells. (D) Functional enrichment analysis of the unique proteins related to SLFN11 with ingenuity pathway analysis (IPA) software. (E) Pathway enrichment analysis of the unique proteins related to SLFN11 with IPA software. (F) The peptide spectrum of RPS4X by LC-MS/MS assay. The N-terminal and C-terminal collision-induced dissociation fragment ions are indicated by b and y, respectively. ** P < 0.01, *** P < 0.001. Abbreviations: QE, Q Exactive.

Figure 4

SLFN11 blocks the mTOR signaling pathway and physically associates with RPS4X. (A, B) Co-immunoprecipitation (Co-IP) assays were conducted in HCCLM3 cells transfected with a vector containing flag-tagged SLFN11 and HCCLM3 cells; IgG was used as control. (C) Confocal microscopy scan of immunofluorescence staining shows that SLFN11 (green) co-localized with RPS4X (red) in the HCCLM3 cells. DAPI was used for nuclear staining. Scale bars = 10 μm. (D) Representative IHC staining of HCC tumors for SLFN11 and RPS4X expression. Scale bars = 200 μm or 20 μm, respectively. (E) Correlations between SLFN11 and RPS4X expression levels in HCC patients in the Fudan LCI cohort 1. P value was calculated by Pearson Chi-squared test; -/+, negative or weak expression; ++/+++, moderate or strong expression. (F) Changes in the mTOR signaling pathway and apoptotic-related proteins were detected by Western blotting for SLFN11-OE in HCCLM3 cells and SLFN11 knockdown in SMMC-7721 cells. (G) Representative IHC images of xenograft tumors from nude mice subcutaneously injected with corresponding transfected HCCLM3 and SMMC-7721 cells stained with RPS4X, Ki-67, p-S6 and p-eIF4E. Scale bars = 200 μm. Histograms (right) show the IHC score ± SD in each group. ** P < 0.01, *** P < 0.001.

Figure 5

RPS4X is an essential factor in SLFN11-mediated inhibition of the mTOR signaling pathway. (A) Western blot of the knockdown efficiency of RPS4X in HCCLM3 cells. (B) Western blot indicates that once RPS4X was knocked down in HCCLM3 cells, regardless of whether SLFN11 was overexpressed, the phosphorylation of S6 and eIF4E were inhibited at almost the same level. (C) Colony formation assays were conducted to study cell proliferation of HCCLM3-VectorControl cells and HCCLM3-SLFN11 cells with or without RPS4X knockdown. (D) Wound healing assays were performed to detect cell migratory abilities of HCCLM3-VectorControl cells and HCCLM3-SLFN11 cells with or without RPS4X knockdown. (E) Transwell assays were used to investigate the cell migratory and invasive capacities of HCCLM3-VectorControl cells and HCCLM3-SLFN11 cells with or without RPS4X knockdown. (F) CCK-8 assays were conducted to determine the cell proliferation of HCCLM3-VectorControl cells and HCCLM3-SLFN11 cells with or without RPS4X knockdown. (G) Cell apoptosis was assessed by flow cytometry in HCCLM3-VectorControl cells and HCCLM3-SLFN11 cells with or without RPS4X knockdown. ** P < 0.01, *** P < 0.001, NS, not significant.

Figure 6

In vivo efficacy of combined SLFN11 expression and mTOR inhibition. (A) Western blots of p-S6, p-eIF4E, and the proteins involved in cell apoptosis in HCCLM3-Vector, HCCLM3-SLFN11, SMMC-7721-Control, and SMMC-7721-shSLFN11 cells that had been treated with or without INK128 (200 nM). (B, C) Tumor volume and weight of orthotopic xenograft models derived from HCCLM3-Vector and HCCLM3-SLFN11 cells treated as indicated. (D, E) Tumor volume and weight of orthotopic xenograft models derived from SMMC-7721-Control and SMMC-7721-shSLFN11 cells treated as indicated. (F, G) Representative IHC images of orthotopic nude mouse tumor tissues for expression of Ki-67, p-S6, and p-eIF4E from HCCLM3-Vector, HCCLM3-SLFN11, SMMC-7721-Control, and SMMC-7721-shSLFN11 cells treated as indicated. Scale bars = 200 μm. Histograms (bottom) show the IHC score ± SD in each group. ** P < 0.01, *** P < 0.001.

Figure 7

SLFN11 inhibits tumor metastasis in vivo. (A, B) Left panel: Representative hematoxylin and eosin (HE) staining images indicate the effects of SLFN11 and INK128 on lung metastasis from orthotopic nude mice in HCCLM3 and SMMC-7721 cells. Scale bars = 1 mm and 200 μm. Right panel: Histograms show metastatic nodules in the lungs with a mean ± SD in each group. ** P < 0.01, *** P < 0.001. (C) A schematic illustrating the role of SLFN11 in regulating HCC tumorigenesis and metastasis.