PELP1 Is a Novel Therapeutic Target in Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths in the United States, with a median survival period of approximately 10 months. There is an urgent need for the development of effective targeted therapies for the treatment of HCC. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) signaling is implicated in the progression of many cancers, although its specific contribution to the progression of HCC is not yet well understood. Analysis of The Cancer Genome Atlas HCC gene expression data sets and IHC analysis of HCC tissue microarray revealed that HCC tumors had elevated expression of PELP1 compared with normal tissues, and high expression of PELP1 is associated with unfavorable survival outcomes. Suppression of PELP1 expression using short hairpin RNA significantly reduced the cell viability, clonogenicity, and invasion of HCC cells. Importantly, SMIP34, a first-in-class small-molecule inhibitor targeting PELP1, effectively decreased the cell viability, clonogenic survival, and invasiveness of HCC cells. Gene expression analysis using RNA sequencing revealed that PELP1 knockdown cells exhibited a decrease in c-Myc, E2F, and other oncogenic pathways related to HCC. Mechanistic studies showed that SMIP34 treatment impaired the Rix complex, a critical component of ribosomal biogenesis, in HCC cells. Furthermore, the knockdown or pharmacologic inhibition of PELP1 significantly decelerated the HCC tumor growth in xenograft models. In summary, our study findings indicate that PELP1 could serve as a promising target for therapeutic intervention in HCC.

Significance: HCC is one of the leading causes of cancer fatalities in the United States. Effective targeted therapeutics for HCC are urgently needed. In this study, we show that PELP1 proto-oncogene is crucial to HCC progression and that PELP1 inhibition reduced HCC cell proliferation in vitro and in vivo. Our results imply that PELP1-targeted drugs like SMIP34 may be useful as new therapeutic agents for HCC treatment.

Figure 1

PELP1 expression is upregulated in HCC, and high PELP1 expression is associated with poor survival of patients with HCC. Data obtained from TNMplot shows increased expression of PELP1 in patients with HCC (A). The results from TCGA-UALCAN database show that PELP1 expression is increased with HCC progression (B). The Clinical Proteomic Tumor Analysis Consortium database shows high PELP1 expression in HCC tumor tissues compared with normal tissues (C). Association of protein expression of PELP1 with overall survival of patients with HCC was obtained from The Human Protein Atlas (D). TMA was used to investigate the PELP1 expression in 86 HCC and six normal liver specimens by IHC and quantified (E and F). Scale bar, 100 μm. P values are calculated using the Student t test. ****, p < 0.0001.

Figure 2

PELP1-KD/SMIP34 treatment decreased cell viability, clonogenicity, and invasiveness of HCC cells. PELP1-KD in Huh7 and Hep3B cell lines were confirmed by Western blot (A). Cell viability and clonogenic assays were performed to assess the impact of PELP1 KD on the growth of Huh7 and Hep3B cells (B and C). The effect of PELP1-KD on the invasion of HCC cells was performed using Matrigel invasion chamber assays (D). The effect of SMIP34 on the cell viability of six HCC cells was measured using MTT assay (E). Effect of SMIP34 (5 μmol/L) on clonogenic cell survival was examined using colony formation assay (F). The effect of SMIP34 (10 μmol/L) treatment on HCC cells invasion was performed using Matrigel invasion chamber assay (G). The results show the mean ± SEM, n = 3. P values are calculated using the Student t test and one-way ANOVA, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 3

Analysis of global transcriptional changes in PELP1-KD HCC cells. Volcano plot of differentially expressed genes with PELP1-KD in Hep3B cells is displayed (n = 3; A). PELP1-downregulated pathways were identified using differentially expressed gene signature (B). Gene set enrichment analysis plots show negatively enriched pathways by PELP1-KD (C). Heatmap images of the specific genes affected by PELP1-KD (D). RT-qPCR was used to validate the specific genes that were differentially regulated by PELP1-KD in RNA-seq analysis (E). Data are represented as the mean ± SEM. P values are calculated using two-way ANOVA, ****, p < 0.0001. ES, enrichment score.

Figure 4

SMIP34 treatment blocked PELP1-mediated extranuclear signaling and decreased global protein synthesis. Huh7, SNU398, Hep3B, and SNU423 cells were treated with vehicle (DMSO 0.01%) or SMIP34 (12.5 μmol/L) to examine PELP1 degradation and the stability of the Rix1 complex by Western blotting (A and C). Hep3B, Huh7, and SNU449 cells were treated with vehicle (DMSO 0.01%) or SMIP34 (12.5 μmol/L), and the status of known PELP1 downstream signaling targets were analyzed by Western blotting (B). Hep3B and Huh7 cells were treated with vehicle (DMSO 0.01%) or SMIP34 (12.5 μmol/L), and the status of E2F1 and c-Myc expressions was analyzed by Western blotting (D). Hep3B and Huh7 cells treated with SMIP34 (5, 10, and 15 μm) or Hep3B and Huh7 cells with PELP1-KD were subjected to 30 miniutes of incubation with puromycin (1 μmol/L). The effect of this treatment on global protein synthesis was measured by Western blotting using an anti-puromycin antibody (E and F).

Figure 5

PELP1-KD or SMIP34 treatment suppresses HCC xenograft tumor growth in vivo. Hep3B–control and Hep3B–PELP1-KD model cells were injected subcutaneously into female (A, D, and G) or male SCID mice (B, E, and H). Tumor volumes were assessed at 3 to 5 days of intervals. Tumor volume (A and B), tumor weights (D and E), and IHC analyses of Ki67 and PELP1 (G and H) for tumors are shown. Hep3B xenograft tumor fragments measuring 2 to 3 mm³ were implanted subcutaneously , and following tumor establishment (∼200 mm³), the mice were randomly assigned to receive either vehicle (control) or SMIP34 (20 mg/kg body weight) 5 days a week through i.p. injection (n = 5). Tumor volume (C), tumor weights (F) and IHC analyses of Ki67 and PELP1 (I) for tumors are shown. Scale bar, 100 μm. Data are represented as the mean ± SEM. P values are calculated using Student t test and two-way ANOVA, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Cont sh (Control short hairpin).