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Hepatocellular carcinoma evades RB1-induced senescence by activating the FOXM1–FOXO1 axis

Abstract

Hepatocellular carcinoma (HCC) is one of the deadliest cancers. The retinoblastoma protein (RB1), a regulator of cell proliferation, is functionally inactivated in HCC by CYCLIN D/E-mediated phosphorylation. However, the mechanism of RB1-inactivation is unclear because only small percentages of HCCs exhibit amplification of CYCLIN D/E or mutations in the CDK-inhibitory genes. We show that FOXM1, which is overexpressed and critical for HCC, plays essential roles in inactivating RB1 and suppressing RB1-induced senescence of the HCC cells. Mechanistically, FOXM1 binds RB1 and DNMT3B to repress the expression of FOXO1, leading to a decrease in the levels of the CDK-inhibitors, creating an environment for phosphorylation and inactivation of RB1. Consistent with that, inhibition of FOXM1 causes increased expression of FOXO1 with consequent activation of RB1, leading to senescence of the HCC cells, in vitro and in vivo. Also, repression-deficient mutants of FOXM1 induce senescence that is blocked by depletion of RB1 or FOXO1. We provide evidence that human HCCs rely upon this FOXM1–FOXO1 axis for phosphorylation and inactivation of RB1. The observations demonstrate the existence of a new autoregulatory loop of RB1-inactivation in HCC involving a FOXM1–FOXO1 axis that is required for phosphorylation of RB1 and for aggressive progression of HCC.

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Fig. 1: RB1-phosphorylation coincides with FOXM1 expression.
Fig. 2: FOXM1 induces phosphorylation RB1 involving RB1 and DNMT3B.
Fig. 3: FOXM1 transcriptionally represses FOXO1 expression.
Fig. 4: FOXM1 regulation of the CDK inhibitors is blocked by FOXO1-siRNA.
Fig. 5: The repression mechanism of FOXM1 regulates stemness and invasiveness by counteracting RB1-induced cellular senescence.
Fig. 6: HepG2 cells were transduce with Lv-Luc, Lv-FOXM1-WT, Lv-FOXM1-IT, or Lv-FOXM1-DD followed by the selection of the infected cells with Puromycin at 1 ug/ml concentration for 5 days.
Fig. 7: Inhibition of FOXM1 induces the senescence of HCC cells, in vivo.
Fig. 8: FOXM1 regulates cellular senescence in HCC through FOXO1 repression.

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Acknowledgements

This work was supported by a grant (I01 BX000131) from the Department of Veterans Affair (Biomedical Laboratory Research Development Service), and a grant from the NIH (5 RO1 CA243247) to PR. The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government. EB acknowledges support from NIH grant (RO1 CA211095).

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VC and PR designed research, analyzed data, and wrote the paper. VC performed the experiments. XL, GG, and EB contributed critical reagents.

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Correspondence to Pradip Raychaudhuri.

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Chand, V., Liao, X., Guzman, G. et al. Hepatocellular carcinoma evades RB1-induced senescence by activating the FOXM1–FOXO1 axis. Oncogene 41, 3778–3790 (2022). https://doi.org/10.1038/s41388-022-02394-8

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