NUPR1, a new target in liver cancer: implication in controlling cell growth, migration, invasion and sorafenib resistance

Abstract

Sorafenib, an oral multikinase inhibitor, is the only approved agent for the treatment of advanced hepatocellular carcinoma (HCC). However, its benefits are modest, and as its mechanisms of action remain elusive, a better understanding of its anticancer effects is needed. Based on our previous study results, we investigated here the implication of the nuclear protein 1 (NUPR1) in HCC and its role in sorafenib treatment. NUPR1 is a stress-inducible protein that is overexpressed in various malignancies, but its role in HCC is not yet fully understood. We found that NUPR1 expression was significantly higher in primary human HCC samples than in the normal liver. Knockdown of NUPR1 significantly increased cell sensitivity to sorafenib and inhibited the cell growth, migration and invasion of HCC cells, both in vitro and in vivo. Moreover, NUPR1 silencing influenced the expression of RELB and IER3 genes. Unsurprisingly, RELB and IER3 knockdown also inhibited HCC cell viability, growth and migration. Using gene expression profiling of HCC cells following stable NUPR1 knockdown, we found that genes functionally involved in cell death and survival, cellular response to therapies, lipid metabolism, cell growth and proliferation, molecular transport and cellular movement were mostly suppressed. Network analysis of dynamic gene expression identified NF-κB and ERK as downregulated gene nodes, and several HCC-related oncogenes were also suppressed. We identified Runt-related transcription factor 2 (RUNX2) gene as a NUPR1-regulated gene and demonstrated that RUNX2 gene silencing inhibits HCC cell viability, growth, migration and increased cell sensitivity to sorafenib. We propose that the NUPR1/RELB/IER3/RUNX2 pathway has a pivotal role in hepatocarcinogenesis. The identification of the NUPR1/RELB/IER3/RUNX2 pathway as a potential therapeutic target may contribute to the development of new treatment strategies for HCC management.

Figure 1

Expression of ER stress genes after sorafenib treatment in HCC cells. (a and c) Dose- and (b and d) time-dependent effects of sorafenib treatment on ER stress gene expression in HCC cell lines determined by qPCR (a and b) and semiquantitative-PCR (c and d). In panels (a and c), HCC cells were treated with the indicated concentrations of sorafenib, and total RNA was extracted after 24 h of treatment. In panels (b and d), HepG2 and Huh7 cells were treated with 7.5 μM sorafenib, and total RNA was extracted at different times of treatment. U=unspliced XBP1 mRNA; S=spliced XBP1 mRNA. In panels (a and b), relative expression was calculated as the ratio of drug-treated samples versus control (DMSO) and corrected by the quantified expression level of β-actin. The results shown are the means±S.D. of three experiments, each performed in triplicate

Figure 2

Expression of NUPR1 in HCC cells. (a) NUPR1 protein and (b) mRNA expression in HCC cells in basal condition. (c) Immunofluorescence analysis of NUPR1 protein expression after treatment for 3 h with the indicated concentrations of sorafenib in HCC cells. (d) HCC cells were treated with the indicated concentrations of sorafenib, and total RNA was extracted after 24 h of treatment. (e) HepG2 and Huh7 cells were treated with 7.5 μM sorafenib, and total RNA was extracted at different times of treatment

Figure 3

NUPR1 expression in human HCC samples. (a) NUPR1 protein expression levels were examined by immunohistochemistry in the NL (A) and HCC (B) tissues. Magnification= × 20, insert magnification= × 40. Scale bar=100 μm. (b) NUPR1 gene expression analysis in 17 HCC tissues and 5 surrounding non-tumor cirrhotic tissues (LC) performed by qPCR. Data are indicated as NUPR1 fold change (relative quantitation, RQ) compared with control (RQ=1 calculated as the mean of NUPR1 Ct in NL tissues). Data are expressed as mean±S.D. NS=non-significant

Figure 4

NUPR1 regulates cell viability, growth, migration and invasion of HCC cells. (a) Cell viability of HCC cells transfected with siNUPR1 (siNUPR1 #1) and siNC was assessed by MTS assay after treatment with the indicated concentrations of sorafenib for 48 h. Data are expressed as the percentage of control cells and are the means±S.D. of three separate experiments, each performed in triplicate. *P<0.05, **P<0.01. (b) Representative images of clonogenic assay of HCC cells transfected with siNUPR1 (siNUPR1 #1) and siNC. The experiment continued for 14 days. Surviving colonies were stained and counted. Data are expressed as the percentage of colonies and are the means±S.D. of three separate experiments, each performed in duplicate. (c) Representative images of wound-healing assay after NUPR1 siRNA-mediated gene silencing (shNUPR1 #1) in PLC/PRF/5. The experiment was conducted for 24 h. Data are reported as the percentage of cell migration and represent the average±S.D. of three experiments, each performed in duplicate. *P<0.05. (d) Representative images of transwell migration assay of Hep3B shNUPR1 (shNUPR1 #1) or Hep3B pSilencer cells. Data are reported as the percentage of migrated cells compared with control (siNC) and are the means±S.D. of three separate experiments, each performed in duplicate (*P<0.05). (e) Matrigel invasion assay in Hep3B shNUPR1 cells (shNUPR #1) compared with pSilencer as control. Data are reported as the percentage of invaded cells compared with control (pSilencer) and are the means±S.D. of three separate experiments, each performed in duplicate. *P<0.05

Figure 5

NUPR1 regulates expression of RELB and IER3 genes, and RELB and IER3 regulate cell viability and the colony-formation capacity of HCC cells. (a) Gene expression analysis by qPCR in HCC cells after NUPR1 gene silencing (siNUPR1 #1). Data are reported as the percentage of gene expression inhibition of each gene and are the means±S.D. of three separate experiments, each performed in triplicate. (b) Gene expression analysis, by qPCR, in Hep3B shNUPR1 (shNUPR1 #1) cells compared with pSilencer, as control. Data are expressed as reported in panel (a). (c) Western blotting analysis of P-ERK1/2 (Thr202/Tyr204) and total ERK1/2 in Hep3B shNUPR1 (shNUPR #1) and in control cells (pSilencer). (d) Gene expression analyses by qPCR after RELB and IER3 gene silencing in Hep3B cells. Data are expressed as reported in panel (a). (e) Cell viability of Hep3B cells transfected with siRELB, siIER3 and siNC was assessed by MTS assay after treatment with the indicated sorafenib concentrations for 48 h. Data are expressed as reported in Figure 4. *P<0.05. (f) Representative images of the clonogenic assay of Hep3B cells transfected with siRELB, siIER3 and siNC. The experiment continued for 14 days. Surviving colonies were stained and counted. Data are expressed as reported in Figure 4. *P<0.05; ns=non-significant

Figure 6

NUPR1 knockdown inhibited tumor growth of Hep3B cells in nude mice. (a) Microphotography of 8 out of the 12 mice inoculated with stable Hep3B cells harboring NUPR1 shRNA (shNUPR1) (right flank) or non-specific shRNA (pSilencer) (left flank). (b) Microphotographs of tumors collected after 4 weeks of injection with Hpe3B pSilencer cells. (c) Tumor growth of Hep3B cells harboring pSilencer or shNUPR1. *P<0.05; **P<0.01

Figure 7

Functional analysis for the data set of differentially expressed genes and network analysis of dynamic gene expression obtained following shRNA-mediated NUPR1 knockdown in Hep3B cells. (a and b) IPA functional pathway analyses of genes differentially expressed (≥ 3-fold) in Hep3B cells upon NUPR1 suppression. Top functions that meet a P-value cutoff of 0.05 are displayed. The orange line represents the cutoff value for significance. (a) Genes that were upregulated and (b) genes downregulated. (c) The five top-scoring networks were merged and are displayed graphically as nodes (genes/gene products) and edges (the biological relationships between the nodes). Intensity of the node color indicates the degree of up regulation (red) or downregulation (green). Nodes are displayed using various shapes that represent the functional class of the gene product (rhomboid=transporter; square=cytokine; diamond=enzyme; vertical oval=transmembrane receptor; horizontal oval=transcription factor; rectangle=nuclear receptor; hexagon=translation factor; circle=other). Edges are displayed with various labels that describe the nature of the relationship between the nodes:→acts on; -— binding only. The length of an edge reflects the evidence supporting the specific node-to-node relationship, as edges supported by articles from the literature are shorter. Dotted edges represent indirect interaction

Figure 8

RUNX2 regulates cell viability, growth, migration and the expression of NUPR1, RELB and IER3 genes in Hep3B cells and is expressed in human HCC samples. (a) Gene expression analysis by qPCR after NUPR1 gene silencing (shNUPR1 #1) in Hep3B cells. (b) Colony assay after RUNX2 siRNA-mediated gene knockdown. Data are expressed as reported in Figure 4. (c) Representative images of transwell migration assay after RUNX2 gene silencing in Hep3B cells. Data are expressed as reported in Figure 4.**P<0.01. (d) Cell viability of Hep3B cells transfected with siRUNX2 (siRUNX2 #1) and siNC was assessed by MTS assay after treatment with the indicated concentrations of sorafenib for 48 h. Data are expressed as reported in Figure 4. (e) Gene expression analysis after RUNX2 gene silencing (siRUNX2 #1) in Hep3B cells performed by qPCR. Data are expressed as reported in Figure 5. (f) RUNX2 gene expression analysis in 12 HCC tissues performed by qPCR. Data are indicated as reported in Figure 3. (g) RUNX2 protein expression levels were examined by immunohistochemistry in the NL (A) and HCC tissues (B). Magnification= × 20, insert magnification= × 40. Scale bar=100 μm