SENP1 promotes hypoxia-induced cancer stemness by HIF-1α deSUMOylation and SENP1/HIF-1α positive feedback loop

Abstract

Objective: We investigated the effect and mechanism of hypoxic microenvironment and hypoxia-inducible factors (HIFs) on hepatocellular carcinoma (HCC) cancer stemness.

Design: HCC cancer stemness was analysed by self-renewal ability, chemoresistance, expression of stemness-related genes and cancer stem cell (CSC) marker-positive cell population. Specific small ubiquitin-like modifier (SUMO) proteases 1 (SENP1) mRNA level was examined with quantitative PCR in human paired HCCs. Immunoprecipitation was used to examine the binding of proteins and chromatin immunoprecipitation assay to detect the binding of HIFs with hypoxia response element sequence. In vivo characterisation was performed in immunocompromised mice and stem cell frequency was analysed.

Results: We showed that hypoxia enhanced the stemness of HCC cells and hepatocarcinogenesis through enhancing HIF-1α deSUMOylation by SENP1 and increasing stabilisation and transcriptional activity of HIF-1α. Furthermore, we demonstrated that SENP1 is a direct target of HIF-1/2α and a previously unrecognised positive feedback loop exists between SENP1 and HIF-1α.

Conclusions: Taken together, our findings suggest the significance of this positive feedback loop between HIF-1α and SENP1 in contributing to the increased cancer stemness in HCC and hepatocarcinogenesis under hypoxia. Drugs that specifically target SENP1 may offer a potential novel therapeutic approach for HCC.

Keywords: HEPATOCELLULAR CARCINOMA; MOLECULAR BIOLOGY; MOLECULAR PATHOLOGY; SIGNALING.

Conflict of interest statement

Competing interests: None declared.

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Figures

Figure 1

The effect of hypoxia and hypoxia-inducible factor (HIF)-1/2α on the stemness of MHCC-97L cells. (A–C) The in vitro cell abilities for self-renewal (A), migration (B) and chemoresistance (C) to doxorubicin and sorafenib were enhanced under hypoxic condition (1% O2) in MHCC-97L cells and the knockdown of HIF-1α or HIF-2α suppressed these hypoxia-induced effects. (D) Hypoxia-induced increase of mRNA expression of stemness-related genes (Oct3/4, Nanog, BMI-1 and Notch1) was inhibited in HIF-1α or HIF-2α knockdown MHCC-97L cells. (E) CD24+ and CD133+ cell populations were increased in hypoxia-treated MHCC-97L cells, but the knockdown of HIF-1α or HIF-2α blunted the effects. (F) CD24+ population was analysed by FACS in sorted CD24+ (left) and CD24− (right) cells from MHCC-97L cells after cultured for indicated time under hypoxia or normoxia. (*p<0.05, **p<0.01, ***p<0.001, compared with the negative control in normoxia (20% O2); #p<0.05, ##p<0.01, ###p<0.001, compared with the negative control in hypoxia (1% O2)).

Figure 2

Upregulation of specific SUMO proteases 1 (SENP1) in hepatocellular carcinoma (HCC) tissues and the correlation between SENP1 and hypoxia-inducible factor (HIF)-α target genes. (A and B) SENP1 was consistently and significantly upregulated among the six SENP family members examined in our RNA-sequencing data on 16 pairs of human HCCs and their corresponding non-tumourous livers (NT-Ls) and data from The Cancer Genome Atlas (TCGA) of National Cancer Institute, USA (202 HCC tumours of all aetiologies including ‘unspecified’ aetiologies). (C) Using quantitative PCR on a larger cohort of 107 pairs of HCC tumour and corresponding NT-Ls, the similar result of SENP1 upregulation in HCC tumours was observed. (D) Correlation analysis of relative mRNA levels of SENP1 and VEGFa, VEGFb, LOX, LOXL2, PLOD2 or CD24 using our RNA-sequencing data on 16 pairs of human HCCs and their corresponding NT-Ls.

Figure 3

Effect of specific SUMO proteases 1 (SENP1) overexpression on the stemness of hepatocellular carcinoma (HCC) cells. (A–E) The effects of overexpression of SENP1, SENP1mut and non-target control (NTC) on the stemness of HCC cells shown by in vitro abilities of self-renewal (A), migration (B), CD24+ cell population (C) and mRNA expression of stemness-related genes (D) and chemoresistance (E), in hypoxic condition. (F) Limiting dilution xenograft formation of Huh-7 cells with NTC or SENP1 overexpression. (*p<0.05, **p<0.01, compared with the negative control in normoxia (20% O2), #p<0.05; ##p<0.01, compared with the negative control in hypoxia (1% O2)).

Figure 4

Effect of specific SUMO proteases 1 (SENP1) knockdown on the stemness of hepatocellular carcinoma (HCC) cells. (A–E) The effects of knockdown of SENP1 (shSENP1-#1 and shSENP1-#4) and non-target control (NTC) on the stemness of HCC cells shown by in vitro abilities of self-renewal (A), migration (B) and chemoresistance (C), CD24+ cell population (D) and mRNA expression of stemness-related genes (E) in hypoxic condition. (F) Limiting dilution xenograft formation of MHCC-97L cells with NTC or SENP1 knockdown. (*p<0.05, **p<0.01, ***p<0.001, as compared with the negative control in normoxia (20% O2); #p<0.05, ##p<0.01, ###p<0.001, compared with the negative control in hypoxia (1% O2)).

Figure 5

Specific SUMO proteases 1 (SENP1) play a role as the direct target gene of hypoxia-inducible factor (HIF)-1/2α in hepatocellular carcinoma (HCC) cells. (A and B) Protein (A) and mRNA (B) levels of SENP1 were increased under hypoxia in HIF-1α-dependent and HIF-2α-dependent manner in MHCC-97L cells. (C) Chromatin immunoprecipitation assay was used to determine the bind of HIF-1α, HIF-2α and HIF-1β to the hypoxia response element (HRE) sequence in SENP1 promoter. (*p2); #p<0.05, ##p<0.01, ###p<0.001, as compared with the negative control in hypoxia (1% O2)).

Figure 6

Increased stability and transcriptional activity of hypoxia-inducible factor (HIF)-1α through deSUMOylation by specific SUMO proteases 1 (SENP1) in hepatocellular carcinoma (HCC) cells under hypoxic condition. (A) Western blot analyses were used to examine the protein level of HIF-1α and HIF-2α in SENP1-knockdown and non-target control (NTC) HCC cells under hypoxia or normoxia. (B) immunoprecipitation (IP) assay was used to detect the SUMOylation of HIF-1/2α by SUMO-1 or SUMO-2/3 in HCC cells. IP was conducted to obtain the complex protein with anti-HIF-1α or anti-HIF-2α antibody in SENP1-knockdown or vehicle-infected SMMC-7721 cells, which were incubated under normoxia or hypoxia for 24 hours. Western blot analyses were then carried out on the whole cell lysates and the IP complex with anti-SUMO-1 or SUMO-2/3 antibody. Inputs are presented in the right-most panel. (C) Fold change of the relative luciferase activity was examined by luciferase-reporter assay in SENP1-knockdown and NTC SMMC-7721 cells which were incubated under normoxia or hypoxia for 24 hours. (D) Fold change of the relative luciferase activity was examined in NTC, SENP1 overexpression (SENP1 OE) and SENP1mut overexpression (SENP1mut OE) Huh-7 cells which were incubated under normoxia or hypoxia for 36 hours. (E) IP assay was used to detect the binding of exogenous HIF-1α or the mutants with SUMO-1 in SMMC-7721 cells in normoxia. First, IP was conducted to obtain the complex protein with anti-HIF-1α antibody in SENP1-knockdown or vehicle-infected SMMC-7721 cells, in which HA-SUMO-1 was cotransfected with RGS-HIF-1α wild type (WT), RGS-HIF-1α K391R, RGS-HIF-1α K477R, or RGS-HIF-1α SM. Western blot analyses for indicated proteins in the whole cell lysates and the IP complex with anti-HA antibody. (F) Fold of the relative luciferase activity was examined in SENP1-knockdown and NTC SMMC-7721 cells (in normoxia), in which HA-SUMO-1 was cotransfected with RGS-HIF-1α WT, RGS-HIF-1α K391R, RGS-HIF-1α K477R or RGS-HIF-1α SM. (*p2); #p<0.05, ##p<0.01, ###p<0.001, compared with the negative control in hypoxia (1% O2)).

Figure 7

Mutation of SUMO sites rescues the loss of hypoxia-induced stemness in specific SUMO proteases 1 (SENP1)-knockdown hepatocellular carcinoma (HCC) cells. The effects of SENP1 knockdown on the stemness of HCC cells with overexpressing hypoxia-inducible factor (HIF)-1α wild type (WT), HIF-1α K391R, HIF-1α K477R or HIF-1α SM. In vitro abilities of self-renewal (A), migration (B) and chemoresistance (C), CD24+ cell population (D) and mRNA expression of stemness-related genes (E) were determined in hypoxic condition. SENP1 knockdown suppressed the effects of HIF-1α WT, while the three HIF-1α mutants partially to almost completely rescued HIF-1α-induced enhancement of HCC cell stemness in shSENP1 HCC cells. (F) A cartoon summarising our findings. HIF-1/2α induced by the hypoxic microenvironment increases the transcription of SENP1 in HCC cells. SENP1 represses the SUMOylation of HIF-1α at K391 and K477 by its SUMO protease activity, thus enhancing the stability and transcriptional activity of HIF-1α. These in turn upregulate the expression of HIF target genes, including SENP1 and stemness-related genes, and contribute to the increased stemness properties. (*p<0.05, **p<0.01, ***p<0.001, as compared with the negative control).