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

We first interrogated the circadian rhythms in three widely used human HCC cells, Hep3B, HepG2, and Huh7, finding each displayed a distinct pattern (Fig. 1 A–C), with the Hep3B cells oscillating most robustly and the Huh7 cells not technically cycling according to circadian parameters (SI Appendix, Fig. S1↗). To study the functional roles of the core circadian clock in the HCC cells, we targeted Bmal1 and Clock by introducing siRNA-mediated gene knockdown. Despite the heterogeneous intrinsic circadian oscillations, targeting either Bmal1 or Clock potently impaired the proliferation of all three HCC cell lines (Fig. 1 D–I). We validated the clock machinery dependency in vivo with Hep3B xenografts by showing that shRNA-mediated Bmal1/Clock knockdown strongly inhibited tumor growth (Fig. 1 J and K). Therefore, the master circadian clock transcription factor heterodimer BMAL1::CLOCK plays a critical role in HCC cell proliferation, irrespective of the robustness of circadian oscillations or other genetic backgrounds, and is relevant to impaired tumor growth in vivo.

To elucidate the molecular mechanisms underlying BMAL1::CLOCK-regulated HCC proliferation, we determined cellular effects of Bmal1 and Clock knockdown. Flow-cytometric measurement of Annexin V and propidium iodide (PI) staining as well as the cleavage of CASPASE 3 revealed the activation of bonafide apoptosis upon Bmal1 or Clock knockdown (Fig. 2 A and B and SI Appendix, Fig. S2↗). Bmal1/Clock-targeted HCC cells displayed a decreased G₁ fraction and a concomitant increase in the G₂/M fraction relative to cells transduced with a non-targeting control siRNA (Fig. 2 C and D), indicating inhibited cell cycle progression. To determine if similar regulation occurs in normal tissues, we performed RT-qPCR characterization of the Bmal1 knockout mouse liver, which identified extensively disrupted transcription of cell cycle genes compared to the wild-type liver; specifically, Wee1 and Ccnb1 were down-regulated and p21 and Myc were up-regulated (Fig. 2E). Therefore, BMAL1::CLOCK may control transcription of the cell cycle regulators (20).

We were accordingly prompted to investigate whether BMAL1::CLOCK promotes the growth of HCC cells by modulating cell cycle progression and division. We first inspected the mechanism underpinning clock-regulated cell cycle progression. As cell cycle progression requires well-orchestrated transcriptional control of the cell cycle regulators (21), we characterized the impact of the altered transcription of cell cycle genes in response to Bmal1 depletion (Fig. 2E). siRNA-mediated Wee1 downregulation induced G₁ phase accumulation (Fig. 3 A and B), which is compatible with its functional roles in activating the G₂/M checkpoint (22). In contrast, the knockdown of Ccnb1, a G₂/M-specific cyclin, reduced the fraction of cells in G₁ phase and increased the proportion of cells in the G₂/M phase (Fig. 3 A and B). Overexpression of p21, a cyclin-dependent kinase inhibitor that promotes cell cycle arrest (23), increased the fraction of cells arrested in G₂/M phase (Fig. 3 C and D). Consistent with previous studies (24), induction of Myc expression increased cell population arrested in the G₂/M phase (Fig. 3 C and D). Therefore, the G₂/M phase cell cycle arrest resulting from Bmal1/Clock knockdown (Fig. 2 C and D) is likely associated with transcriptional downregulation of Ccnb1 and upregulation of p21 and Myc.

Cell cycle checkpoints regulate the rate of cell division. Next, we assessed whether the Ccnb1, Myc, and p21-associated G₂/M phase cell cycle arrest in response to Bmal1/Clock downregulation reduced cell proliferation. Knockdown of Ccnb1 alone had a minor impact on HCC cell growth (Fig. 4 A–F). Also, upregulation of the proto-oncogene Myc is unlikely to lead to HCC proliferation inhibition. Therefore, the G₂/M phase arrest does not necessarily result in reduced cell proliferation per se, in concordance with previous reports that depending on molecular context, either inhibition or activation of the G₂/M checkpoint can induce cell death (25).

Bmal1 knockout increased the basal level of p21 and reversed its peak expression from ZT0 to ZT12 (Fig. 2E). Gréchez-Cassiau and co-workers reported that the cyclic expression of p21 was controlled by the RORE-binding clock transcription factors REV-ERBs and RORs and up-regulated p21 was responsible for the decreased hepatocyte proliferation rate in the Bmal1 knockout mouse liver (26). We found that concurrent knockdown of p21 partially rescued HCC growth inhibition induced by Bmal1/Clock knockdown (Fig. 4 G and H). p21 overexpression did not substantially change the activity of apoptosis in Hep3B cells (SI Appendix, Fig. S3↗), in line with previous reports (27). Collectively, our findings indicate that BMAL1::CLOCK negatively regulates the RORE-containing cell cycle regulator p21 to maintain proliferation rate of HCC cells.

Cancer cells are reliant on WEE1-activated G₂/M checkpoint to cope with DNA damage and avoid apoptosis (28). Analogous to Bmal1/Clock knockdown, we found that down-regulating Wee1 attenuated proliferation and caused apoptosis in HCC cells (Fig. 5 A–H). As depleting Bmal1 significantly reduced Wee1 levels in both mouse liver and Hep3B cells (Fig. 2E and SI Appendix, Fig. S4↗), we asked whether Wee1 downregulation contributed to cell lethality resulting from BMAL1::CLOCK inhibition. Overexpression of Wee1 did not change the expression levels of Bmal1 and Clock (Fig. 5I) but could partially rescue cell growth inhibition caused by BMAL1::CLOCK downregulation (Fig. 5J). Wee1 transcription showed robust circadian oscillation in the mouse liver, peaking at the end of the day, in phase with the classical BMAL1::CLOCK target gene Dbp (Fig. 2E). BMAL1 chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) indicated that BMAL1::CLOCK binds Wee1 genes in both mouse liver and Hep3B cells (Fig. 5K). Therefore, Wee1 inhibits apoptosis and contributes to BMAL1::CLOCK-controlled HCC cell growth.

All animal care and experiments were performed under the institutional protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Southern California. For mouse liver RT-qPCR analysis, mice were housed in a 12-h light/12-h dark (LD) cycle with free access to food and water and age of 10 to 12 wk were used. For the xenograft transplantation model, NU/J nude mice (The Jackson Library #002019) were subcutaneously transplanted in the right flank with 3 × 10⁶ Hep3B cells transduced with a non-targeting control shRNA or shRNA targeting either Bmal1 or Clock. Tumor growth was monitored for 4 wk by measuring tumor diameter and weight at the endpoint.

Hep3B and Huh7 cells were grown in complete DMEM (Life Technologies cat. #11995065) supplemented with 10% FBS and 1% penicillin/streptomycin. HepG2 cells were grown in Ham’s F12 (Corning Cellgro 10-080-CV) supplemented with 10% FBS and 1% penicillin and streptomycin. All cells were grown in a 37 °C incubator at 5% CO₂.

For circadian assays, human liver cancer cells were plated on 35-mm dishes and synchronized as previously described by a dexamethasone shock (10, 15, 16, 45). In brief, cell culture media was replaced with HEPES-buffered phenol-free DMEM media containing 100 nM dexamethasone and 100 μM D-luciferin. Dishes were covered with 40-mm glass coverslips (Fisher Scientific) and sealed with vacuum grease to prevent evaporation. Luminescence signals were monitored every 10 min using the LumiCycle luminometer (Actimetrics) at 37 °C without supplementary CO₂. Results shown are representative of at least three independent experiments.

Cells were plated in 96-well plate at a density of ~1,000 cells per well. Relative ATP level was measured following the instruction of CellTiter-Glo Luminescent Cell Viability Assay (Promega, G7570).

Cells were collected and resuspended in Tris-buffered saline with 10 µg/mL DAPI and 0.1% nonidet P-40 detergent for flow cytometry. Cell cycle phases were analyzed using FlowJo software. Apoptosis was measured using FITC-Annexin V antibody and PI staining following manufacturer’s instruction (Life Technology, V13241). Cells were read using flow cytometry (BD Bioscience), and the results were analyzed via FlowJo.

Liver tissues of 10- to 12-wk-old male mice were harvested at indicated Zeitgeber times. Total RNA was isolated using TRIzol reagent according to manufacturer’s instructions (Life Technologies cat. #15596026) and then reverse transcribed to cDNA using iScript cDNA Synthesis kit (Bio-Rad cat. #1708891). We designed real-time primers spanning the exon–intron junctions using the IDT primer-designing software PrimerQuest (https://www.idtdna.com/PrimerQuest↗). Primer sequences are

RT-qPCR analyses were performed as described previously (15, 16, 45) with CFX384 Real-Time PCR Detection System (Bio-Rad).

Frozen mouse liver tissue was homogenized in RIPA buffer containing 1x EDTA-free protease inhibitor cocktail (Roche) using Omni Tissue Homogenizer (Omni International). The total protein concentration was determined by Bio-Rad Protein Assay and then equalized to 15 g/L. 25 μg of total protein was used for the western blot assay, performed as previously described (15, 16, 45). Antibodies used in the western blots are anti-BMAL1 (Cell signaling, #14020), anti-Wee1 (Cell signaling, #4936), and anti-TUBULIN (Sigma-Aldrich, T0198).

The significance of differences between peak distance, period length, and gene expression was evaluated by unpaired Student’s t test (two-tailed), with significant differences at P < 0.05.

M.Q., G.Z., J.N.R., and S.A.K. designed research; M.Q., G.Z., A.V., and R.W. performed research; M.Q., G.Z., H.Q., H.T., Z.J., W.H., H.-J.L., J.N.R., and S.A.K. analyzed data; H.T., Z.J., W.H., H.-J.L., and J.N.R. provided edits of the paper; and M.Q. and S.A.K. wrote the paper.

S.A.K., discloses his financial interest in Synchronicity Pharma, where he serves on the Board of Directors.

All study data are included in the article and/or SI Appendix↗.