Circadian regulator BMAL1::CLOCK promotes cell proliferation in hepatocellular carcinoma by controlling apoptosis and cell cycle

Abstract

Hepatocellular carcinoma (HCC) remains a global health challenge whose incidence is growing worldwide. Previous evidence strongly supported the notion that the circadian clock controls physiological homeostasis of the liver and plays a key role in hepatocarcinogenesis. Despite the progress, cellular and molecular mechanisms underpinning this HCC-clock crosstalk remain unknown. Addressing this knowledge gap, we show here that although the human HCC cells Hep3B, HepG2, and Huh7 displayed variations in circadian rhythm profiles, all cells relied on the master circadian clock transcription factors, BMAL1 and CLOCK, for sustained cell growth. Down-regulating Bmal1 or Clock in the HCC cells induced apoptosis and arrested cell cycle at the G₂/M phase. Mechanistically, we found that inhibiting Bmal1/Clock induced dysregulation of the cell cycle regulators Wee1 and p21 which cooperatively contribute to tumor cell death. Bmal1/Clock knockdown caused downregulation of Wee1 that led to apoptosis activation and upregulation of p21 which arrested the cell cycle at the G₂/M phase. Collectively, our results suggest that the circadian clock regulators BMAL1 and CLOCK promote HCC cell proliferation by controlling Wee1 and p21 levels, thereby preventing apoptosis and cell cycle arrest. Our findings shed light on cellular impact of the clock proteins for maintaining HCC oncogenesis and provide proof-of-principle for developing cancer therapy based on modulation of the circadian clock.

Keywords: apoptosis; cell cycle; circadian clock; hepatocellular carcinoma.

Fig. 1.

The core clock genes Bmal1 and Clock are required for HCC cell growth independent of circadian oscillation. (A–C) Representative bioluminescence of Bmal1-Luc reporter in Hep3B (A), HepG2 (B), and Huh7 (C) cells synchronized by 150 nM dexamethasone (n = 3). (D–F) Relative cell numbers of Hep3B (D), HepG2 (E), and Huh7 (F) transfected with scramble, Bmal1, or Clock siRNA. Data are represented as mean ± SD (n = 4). Statistical significance was determined by two-way ANOVA with Tukey multiple comparison test (***P < 0.001). (G–I) Transcript level of genes in Hep3B (G), HepG2 (H), and Huh7 (I) transfected with scramble, Bmal1, or Clock siRNA was determined by RT-qPCR. Displayed are the means ± SD (n = 3 cell culture wells) normalized to Rplp0 expression levels. Statistical significance was determined by a two-tailed Student’s t test (***P < 0.001). (J and K) NU/J nude mice were subcutaneously injected with 3 × 10⁶ Hep3B cells transduced with indicated shRNAs. Xenograft tumors were imaged (J) and weighed (K). Displayed are the means ± SD (n = 3). Statistical significance was determined by Student’s t test (***P < 0.001).

Fig. 2.

Down-regulating Bmal1/Clock activates apoptosis and G₂/M phase cell cycle arrest. (A) Representative flow cytometry analysis of FITC-Annexin V/PI staining in Hep3B cells transfected with scramble, Bmal1, or Clock siRNA. (B) Quantification of FITC-Annexin V/PI-positive cells presented in (A). Displayed are the means ± SD (n = 3). Statistical significance was determined by Student’s t test (***P < 0.001). (C) Representative cell cycle analysis of Hep3B cells following transfection with scramble, Bmal1, or Clock siRNA. (D) Quantification of cell cycle phases presented in (C). Displayed are the means ± SD (n = 3). Statistical significance was determined by Student’s t test (**P < 0.01, ***P < 0.001). (E) Control and Bmal1 knockout mouse livers were harvested at 4-h intervals throughout 24 h. Transcript level of genes was analyzed by using RT-qPCR. Displayed are the means ± SD (n = 3 or 4) normalized to non-oscillating Rplp0 expression levels. P-values determined by two-tailed Student’s t test were displayed (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 3.

Ccnb1 knockdown and p21 or Myc overexpression cause G₂/M phase arrest. (A) Representative cell cycle analysis of Hep3B cells following transfection with scramble, Wee1, or Ccnb1 siRNA. (B) Quantification of cell cycle phases presented in (A). Displayed are the means ± SD (n = 3). Statistical significance was determined by Student’s t test (***P < 0.001; ns, not significant). (C) Representative cell cycle analysis of Hep3B cells following lentiviral transduction of EGFP, p21, or Myc gene. (D) Quantification of cell cycle phases presented in (C). Displayed are the means ± SD (n = 3). Statistical significance was determined by Student’s t test (***P < 0.001).

Fig. 4.

Bmal1/Clock controls HCC cell growth by suppressing p21. (A–C) Relative cell numbers of Hep3B (A), HepG2 (B), and Huh7 (C) transfected with scramble or Ccnb1 siRNA. Data are represented as mean ± SD (n = 4). Statistical significance was determined by two-way ANOVA with Tukey multiple comparison test (ns, not significant). (D–F) Transcript level of genes in Hep3B (D), HepG2 (E), and Huh7 (F) transfected with scramble or Ccnb1 siRNA was determined by RT-qPCR. Displayed are the means ± SD (n = 3 cell culture wells) normalized to Rplp0 expression levels. Statistical significance was determined by a two-tailed Student’s t test (***P < 0.001). (G) Relative cell numbers of Hep3B transfected with scramble, Bmal1, Clock, or p21 siRNA. Data are represented as mean ± SD (n = 4). Statistical significance was determined by two-way ANOVA with Tukey multiple comparison test (***P < 0.001). (H) Transcript level of genes in Hep3B transfected with scramble, Bmal1, Clock, or p21 siRNA was determined by RT-qPCR. Displayed are the means ± SD (n = 3 cell culture wells) normalized to Rplp0 expression levels. Statistical significance was determined by a two-tailed Student’s t test (**P < 0.01, ***P < 0.001; ns, not significant).

Fig. 5.

Bmal1/Clock controls HCC cell growth by activating Wee1. (A–C) Relative cell numbers of Hep3B (A), HepG2 (B), and Huh7 (C) transfected with scramble, Bmal1, Clock, or Wee1 siRNA. Data are represented as mean ± SD (n = 4). Statistical significance was determined by two-way ANOVA with Tukey multiple comparison test (***P < 0.001). (D–F) Transcript level of genes in Hep3B (D), HepG2 (E), and Huh7 (F) transfected with scramble or Wee1 siRNA were determined by RT-qPCR. Displayed are the means ± SD (n = 3 cell culture wells) normalized to Rplp0 expression levels. Statistical significance was determined by a two-tailed Student’s t test (***P < 0.001). (G) Representative flow cytometry analysis of FITC-Annexin V/PI staining in Hep3B cells transfected with scramble or Wee1 siRNA. (H) Quantification of FITC-Annexin V/PI-positive cells presented in (G). Displayed are the means ± SD (n = 3). Statistical significance was determined by Student’s t test (***P < 0.001). (I) Transcript level of genes in Hep3B stably overexpressing GFP or Wee1 was determined by RT-qPCR. Displayed are the means ± SD (n = 3 cell culture wells) normalized to Rplp0 expression levels. Statistical significance was determined by a two-tailed Student’s t test (***P < 0.001; ns, not significant). (J) Relative cell numbers of Hep3B stably overexpressing GFP or Wee1 followed by transfection with Bmal1 or Clock siRNA. Cell numbers were normalized to Hep3B cells transfected with scramble siRNA of the same time point. Data are represented as mean ± SD (n = 4). Statistical significance was determined by two-way ANOVA with Tukey multiple comparison tests (***P < 0.001). (K) IGV genome tracks showing BMAL1 enrichment at the Wee1 gene loci in the mouse liver and Hep3B cells, based on normalized ChIP-seq read coverage. Track heights are indicated. (L) Mechanism of BMAL1::CLOCK-regulated HCC oncogenesis. BMAL1::CLOCK binds the E-box element on chromatin and activates the transcription of Rev-erbs and Wee1. REV-ERBs suppresses the expression of p21 through binding RORE element. The inhibition of cell cycle arrest and apoptosis, which are activated by p21 downregulation and Wee1 upregulation, respectively, collaboratively contribute to pro-proliferative activity of BMAL1::CLOCK.