SNS-023 sensitizes hepatocellular carcinoma to sorafenib by inducing degradation of cancer drivers SIX1 and RPS16

Abstract

Hepatocellular carcinoma (HCC) remains challenging due to the lack of efficient therapy. Promoting degradation of certain cancer drivers has become an innovative therapy. The nuclear transcription factor sine oculis homeobox 1 (SIX1) is a key driver for the progression of HCC. Here, we explored the molecular mechanisms of ubiquitination of SIX1 and whether targeting SIX1 degradation might represent a potential strategy for HCC therapy. Through detecting the ubiquitination level of SIX1 in clinical HCC tissues and analyzing TCGA and GEPIA databases, we found that ubiquitin specific peptidase 1 (USP1), a deubiquitinating enzyme, contributed to the lower ubiquitination and high protein level of SIX1 in HCC tissues. In HepG2 and Hep3B cells, activation of EGFR-AKT signaling pathway promoted the expression of USP1 and the stability of its substrates, including SIX1 and ribosomal protein S16 (RPS16). In contrast, suppression of EGFR with gefitinib or knockdown of USP1 restrained EGF-elevated levels of SIX1 and RPS16. We further revealed that SNS-023 (formerly known as BMS-387032) induced degradation of SIX1 and RPS16, whereas this process was reversed by reactivation of EGFR-AKT pathway or overexpression of USP1. Consequently, inactivation of the EGFR-AKT-USP1 axis with SNS-032 led to cell cycle arrest, apoptosis, and suppression of cell proliferation and migration in HCC. Moreover, we showed that sorafenib combined with SNS-032 or gefitinib synergistically inhibited the growth of Hep3B xenografts in vivo. Overall, we identify that both SIX1 and RPS16 are crucial substrates for the EGFR-AKT-USP1 axis-driven growth of HCC, suggesting a potential anti-HCC strategy from a novel perspective.

Keywords: MK2206; ML323; SNS-023; gefitinib; sorafenib.

Fig. 1. Lower ubiquitination level of SIX1 is associated with USP1 in clinical HCC tissues.

a Co-IP and Western blotting assays were collectively performed to detect ubiquitination levels of SIX1 in HCC tissues and adjacent normal tissues of 6 HCC patients (number #1, #3, #5, #9, #12 and #15). Quantitative data on the ubiquitination levels are shown on the right. Mean ± SD (n = 6). b Western blotting performed to detect differences in the expression levels of SIX1, USP1, and APC in tumor tissues and adjacent normal tissues of 16 HCC patients. c Quantitative data of (b) are shown. Mean ± SD (n = 16). d Correlation analysis of SIX1 with USP1 or APC protein levels based on (c) using Pearson r assay. The data of tumor tissues and adjacent normal tissues are included in the statistics (n = 32). e Comparison of SIX1, USP1 and APC at mRNA expressions in tumor tissues and normal tissues of HCC patients from the TCGA database. f Kaplan–Meier curves from patients with HCC expressing low and high SIX1/APC from the tissue microarray from the TCGA database. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001.

Fig. 2. USP1 contributes to the stabilization of both SIX1 and RPS16 in HCC cells.

a Co-IP and Western blotting assays were performed to determine the interaction of USP1-SIX1. b An immunofluorescence assay was performed using FLAG and SIX1 antibodies in HepG2 and Hep3B cells transfected with FLAG-USP1 plasmids. Scale bars, 25 μm. c Schematic diagram of FLAG-labeled human USP1 full-length and truncated mutant plasmids. d FLAG-labeled human USP1 full-length and truncated mutant plasmids were co-transfected with the HA-SIX1 plasmids into HEK293T cells for 48 h. Co-IP and Western blotting assays were performed to detect the interaction between FLAG and HA. e Co-IP and Western blotting assays were performed to detect ubiquitination levels of SIX1 in HepG2 cells transfected with USP1 si-RNA-1, −2 or control si-RNA for 48 h, and exposed to MG132 (10 μM) for 6 h before harvest. f Co-IP and Western blotting assays were performed using USP1 antibodies to detect the interaction of USP1-SIX1 and USP1-RPS16 in HepG2 cells transfected with SIX1 si-RNAs, RPS16 si-RNAs, or control si-RNAs for 48 h.

Fig. 3. EGFR-AKT signaling pathway mediates the stabilization of SIX1 and RPS16 via upregulating USP1.

a Western blotting of p-EGFR, EGFR, p-AKT, AKT, USP1, SIX1, and RPS16 in HepG2 and Hep3B cells exposed to EGF for 24 h. b Western blotting of p-EGFR, EGFR, p-AKT, AKT, USP1, SIX1, and RPS16 in HepG2 cells exposed to EGF for 6, 12, and 24 h. c An immunofluorescence assay was performed using USP1, SIX1 and RPS16 antibodies in HepG2 cells exposed to EGF for 24 h. Scale bars, 10 μm. Quantitative data are shown below. Mean ± SD (n = 3). d Western blotting of p-EGFR, EGFR, p-AKT, AKT, USP1, SIX1 and RPS16 in HepG2 and Hep3B cells treated with gefitinib or MK-2206 or DMSO for 9 h, and then exposed to EGF for 24 h. e Western blotting of USP1, SIX1 and RPS16 in HepG2 cells transfected with USP1 si-RNAs or control si-RNAs for 24 h, and then exposed to EGF for 12 h. f Co-IP and Western blotting assays were performed to detect the ubiquitination levels of SIX1 and RPS16 in HepG2 cells treated with gefitinib or DMSO for 9 h and then exposed to EGF for 12 h. Cells were exposed to MG132 (10 μM) for 6 h before harvest. ^##P < 0.01, ^###P < 0.001.

Fig. 4. SNS-032 promotes ubiquitination and degradation of SIX1 and RPS16 in an EGFR-USP1-dependent manner.

a Western blotting of USP1, SIX1 and RPS16 in HepG2 and Hep3B cells exposed to SNS-032 for 24 h or 6, 12, 24 h. b Western blotting of SIX1 level in HepG2 cells treated with SNS-032 (1 μM) or DMSO for 3 h, and then exposed to cycloheximide (CHX) for 0 to 9 h. Quantitative data are shown on the right. Mean ± SD (n = 3). c Western blotting of RPS16 level in HepG2 cells treated with SNS-032 (1 μM) or DMSO for 12 h, and then exposed to cycloheximide (CHX) for 0 to 36 h. Quantitative data are shown on the right. Mean ± SD (n = 3). d Co-IP and Western blotting assays were performed to detect the ubiquitination levels of SIX1 and RPS16 in HepG2 cells treated with SNS-032. Cells were exposed to MG132 (10 μM) for 6 h before harvest. To detect SIX1 ubiquitination, cells were treated with SNS-032 for 6 h. To detect RPS16 ubiquitination, cells were treated with SNS-032 for 18 h. e Western blotting of FLAG, USP1, SIX1 and RPS16 in HepG2 cells transfected with FLAG-USP1 plasmids or Mock (control plasmids) for 24 h, and then exposed to SNS-032 for 24 h. f Western blotting of p-EGFR, EGFR, p-AKT, AKT, USP1, SIX1 and RPS16 in HepG2 and Hep3B cells exposed to SNS-032 with or without EGF for 24 h. ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001.

Fig. 5. EGF reverses the SNS-032-induced suppression of growth and metastasis in HCC cells.

a Cell viability assay of HepG2, Hep3B, and HCCLM3 cells exposed to SNS-032 for 24, 48, and 72 h. Mean ± SD (n = 3). b Colony formation assays were performed in HepG2 and Hep3B cells exposed to SNS-032 for 24 h, followed by re-seeded and cultured in the 6-well plates for 2 weeks. Quantitative data are shown on the right. Mean ± SD (n = 3). c EdU staining assays were performed in HepG2 and Hep3B cells treated with SNS-032 for 24 h. Quantitative data are shown. Mean ± SD (n = 3). d Cell viability assay of HepG2 and Hep3B cells exposed to SNS-032 with or without EGF for 24 h. Mean ± SD (n = 3). e Migration assays were performed in HepG2 and Hep3B cells exposed to SNS-032 with or without EGF for 24 h, followed by re-seeded and cultured in the trans-wells for 2 days. Scale bars, 50 μm. Quantitative data are shown on the right. Mean ± SD (n = 3). f Western blotting of PARP and CC3 (cleaved caspase 3) in HepG2 and Hep3B cells exposed to SNS-032 with or without EGF for 24 h. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001.

Fig. 6. SNS-032 suppresses the growth of HCC in vivo.

a BALB/c nude mice with Hep3B xenografts were treated with SNS-032 (15 mg·kg⁻¹·d⁻¹) (i.p.) or ML323 (40 mg·kg⁻¹·d⁻¹) (i.p.) or vehicle every other day for a total of 21 days. Xenografts grown in the nude mice are shown. b Tumor size, (c) tumor weight, and (d) body weight of nude mice. Mean ± SD (n = 8). e IHC assays of USP1, SIX1, RPS16, Ki67, and CC3 (cleaved caspase 3) were performed in the tissues from xenografts. Representative images per group are shown at 200×. Scale bars, 50 μm. f Quantitative data of (e). Mean ± SD (n = 3). ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001.

Fig. 7. SNS-032 or gefitinib enhances the sensitivity of HCC cells to sorafenib.

a Colony formation assays were performed in HepG2 and Hep3B cells exposed to SNS-032 with or without sorafenib for 24 h, followed by re-seeded and cultured in the 6-well plates for 2 weeks. Quantitative data are shown on the right. Mean ± SD (n = 3). b BALB/c nude mice with Hep3B xenograft were randomly divided into 6 groups: mice were treated with vehicle, sorafenib (20 mg·kg⁻¹·d⁻¹) (p.o.), SNS-032 (15 mg·kg⁻¹·d⁻¹) (i.p.), gefitinib (30 mg·kg⁻¹·d⁻¹) (p.o.), sorafenib + SNS-032, or sorafenib + gefitinib every other day respectively, for a total 21 days. Xenografts grown in nude mice are shown. c Tumor size, (d) tumor weight, and (e) body weight of nude mice. Mean ± SD (n = 8). f A hypothetical model by which SNS-032 targets the EGFR/USP1 axis-mediated stabilization of SIX1 and RPS16 to suppress HCC. ^###P < 0.001, ^####P < 0.0001.