ARTICLE https://doi.org/10.1038/s42003-021-02405-6 OPEN Bromodomain-containing protein BRPF1 is a therapeutic target for liver cancer Carol Lai-Hung Cheng1,2, Felice Hoi-Ching Tsang1,2, Lai Wei1,2, Mengnuo Chen1,2, Don Wai-Ching Chin1,2, Jialing Shen1,2, Cheuk-Ting Law1,2, Derek Lee1,2, Carmen Chak-Lui Wong 1,2, Irene Oi-Lin Ng 1,2 & ✉ Chun-Ming Wong 1,2 Epigenetic deregulation plays an essential role in hepatocellular carcinoma (HCC) progres- sion. Bromodomains are epigenetic “readers” of histone acetylation. Recently, bromodomain inhibitors have exhibited promising therapeutic potential for cancer treatment. Using tran- scriptome sequencing, we identified BRPF1 (bromodomain and PHD finger containing 1) as the 1234567890():,; most significantly upregulated gene among the 43 bromodomain-containing genes in human HCC. BRPF1 upregulation was significantly associated with poor patient survival. Gene ablation or pharmacological inactivation of BRPF1 significantly attenuated HCC cell growth in vitro and in vivo. BRPF1 was involved in cell cycle progression, senescence and cancer stemness. Transcriptome sequencing revealed that BRPF1 is a master regulator controlling the expression of multiple key oncogenes, including E2F2 and EZH2. We demonstrated that BRPF1 activated E2F2 and EZH2 expression by facilitating promoter H3K14 acetylation through MOZ/MORF complex. In conclusion, BRPF1 is frequently upregulated in human HCCs. Targeting BRPF1 may be an approach for HCC treatment. 1 State Key Laboratory of Liver Research, The University of Hong Kong, Pok Fu Lam, Hong Kong. 2 Department of Pathology, Li Ka Shing Faculty of Medicine, ✉ The University of Hong Kong, Pok Fu Lam, Hong Kong. email: [email protected] COMMUNICATIONS BIOLOGY | (2021) 4:888 | https://doi.org/10.1038/s42003-021-02405-6 | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-021-02405-6 epatocellular carcinoma (HCC) is the most common type leukemogenesis associated with MOZ-TIF2 fusion23. Yet, the of liver cancer, accounting for ~80% of primary liver pathological and functional role of BRPF1 in cancer, especially H 1 cancers . HCC is the sixth most common cancer and the liver carcinogenesis, is still largely unknown. fifth deadliest cancer worldwide2,3. HCC is more commonly seen 3 in men and has a higher incidence rate in developing countries . Results HCC development is driven by the accumulation of genetic and Deregulation of bromodomain-containing genes in human epigenetic alterations. The major causes of HCC are hepatitis B fl HCC. Lysine acetylation is an abundant posttranslational mod- (HBV) and hepatitis C (HCV) viral infections, cirrhosis, a atoxin ification in histone and nonhistone proteins. As an unique reader B1 ingestion, excessive alcohol consumption, and nonalcoholic of protein acetylation, bromodomain-containing proteins are fatty liver disease (NAFLD)4. Though the risk factors for HCC are fi important for recognizing and responding to acetylation marks to relatively well de ned, the diagnosis and prognosis of HCC are regulate various cellular processes24,25. We hypothesized that poor. Symptoms usually appear at the late stage of HCC, and 5 deregulation of bromodomain-containing proteins might con- metastasis is commonly observed in HCC patients . tribute to liver carcinogenesis. We therefore compared the Liver transplantation or surgical resection is not applicable for 6 expression levels of 43 human bromodomain-containing genes patients diagnosed at the late stage of HCC . Molecularly targeted (as listed in the ChromoHub database26) in 16 pairs of HBV- therapy is the major method for the treatment of advanced HCC. associated primary HCC samples and their corresponding non- Sorafenib has long been the only FDA-approved molecularly 7 tumor (NT) liver samples by transcriptome sequencing (Fig. 1a, targeted therapy for HCC . Recently, lenvatinib, regorafenib, and b). We found that about half of bromodomain-containing genes cabozantinib have also been approved for HCC treatment. These were highly upregulated in primary HCC. The results showed drugs are all multi-tyrosine kinase inhibitors, which are not that upregulation of bromodomain-containing genes is a com- fi 8–10 speci c and extend the survival rate only by a few months . mon phenomenon in human HCC, implying that the demand for Drug resistance to sorafenib is commonly observed in HCC reading acetylation marks is increased for HCC cell growth. patients11. Immunotherapy maybe a promising new direction for HCC treatment. However, only a subset of HCC patients are 11 Frequent upregulation of BRPF1 in human HCC. In the above responsive to anti-PD1 monoclonal antibodies . Therefore, the fi discovery of potential therapeutic target and the development of transcriptome analysis, we identi ed BRPF1 as the most sig- fi nificantly upregulated bromodomain-containing gene in HCC, new small molecule inhibitors with high speci city for HCC fi treatment are still urgently needed. according to the statistical signi cance (Rank 1) and expression Bromodomain inhibitors represent a novel type of epigenetic change (Rank 10) (Fig. 1b, c). In fact, not just in the in-house drug and hold great promise for cancer therapy. Bromodomains samples, BRPF1 was also upregulated in HCC, colorectal cancer are epigenetic “reader” domains that specifically recognize and kidney cancer from TCGA database (Fig. 1d). The BRPF1 acetylated lysine residues on histones or nonhistone proteins12. upregulation in TCGA HCC cohort was relatively modest com- Lysine acetylation is crucial for gene transcription, nucleosome pared to that in the in-house samples. However, the in-house data assembly, protein–protein interaction and cellular signaling13. comes from Chinese patients associated with HBV-infection, There are 43 bromodomain-containing proteins in humans, while TCGA database contains samples with different aetiological which are divided into eight subgroups depending on their backgrounds and ethnicities. The upregulation of BRPF1 structural similarities. Among these proteins, the members of the expression in HCC tumor samples may be affected by different bromodomain and extra-terminal (BET) family have received aetiological factors. After all, these expression data from TCGA considerable attention14. As registered in ClinicalTrials.gov, there database reinforced our initial observation in the RNA-seq ana- lysis, suggesting that BRPF1 may be essential for cancer devel- are currently 23 ongoing clinical trials of bromodomain inhibitors fi at various phases. These trials largely focus on the therapeutic opment. We found that copy number gain or ampli cation of the effect of BET inhibitors on human cancers, such as acute leuke- BRPF1 gene located on chromosome 3p25 was frequent in human HCC. In addition, HCC with BRPF1 gene copy number gain/ mia and prostate cancer, as well as metabolic disorders. However, fi the implications of other bromodomain-containing proteins in ampli cation had higher BRPF1 mRNA expression than other human carcinogenesis remain to be explored. HCC, suggesting that gain of gene copy number may directly Herein, we identified bromodomain and PHD finger containing contribute to BRPF1 upregulation in human HCC (Fig. 1e). 1(BRPF1) as the most significantly upregulated bromodomain- BRPF1, an epigenetic reader with multiple reader domains containing gene in human HCC. Overexpression of BRPF1 was (Supplementary Fig. S1), is a major component of the MOZ/ related to a poor survival rate in HCC patients. BRPF1 is an MORF acetyltransferase complex (Fig. 1f). We thus speculated epigenetic reader protein containing a bromodomain, two PHD that activation of the BRPF1/MOZ/MORF complex may play an fingers and a PWWP domain. BRPF1 is mostly known as a important role in liver carcinogenesis. component of the MOZ/MORF acetylation complex15. It has been reported that BRPF1 is critical for the formation of this complex as Clinicopathological relevance of BRPF1 upregulation in it links ING5 and MOZ/MORF together. BRPF1 also plays a role human HCC. BRPF1 has two alternative splicing isoforms in regulating the histone acetyltransferase activity of MOZ/ (BRPF1A and BRPF1B) (Fig. 2a). BRPF1B encodes functional MORF15,16. It has been proposed that the bromodomain of BRPF1 BRPF1 protein, while BRPF1A uses an alternative splice site at may contribute to the chromatin binding and target specificity of exon 6, leading to an insertion of 6 extra amino acids. This the MOZ/MORF complex, wherein the catalytic subunit MOZ/ insertion results in a structural change in the bromodomain that MORF catalyzes H3K9, H3K14, and H3K23 acetylation to activate hinders its binding with acetylated lysine residue27, which causes gene transcription16–18. BRPF1 is indispensable for embryonic BRPF1A to become a functionally inactive isoform. To distin- development. Mouse genetic studies have shown that BRPF1 guish these two BRPF1 isoforms, we reanalyzed our RNA-seq raw knockout affects hematopoiesis as well as brain development and data with an isoform-specific algorithm. We found that BRPF1B even causes early lethality19,20. Moreover, mutation of BRPF1 was the predominant isoform in HCC samples, accounting for causes intellectual disability and facial dysmorphisms in ~80% of the BRPF1 transcripts. This isoform-specific analysis also humans21. Recent studies demonstrated that BRPF1 is recurrently confirmed that only the functional BRPF1B isoform was sig- mutated in adult Shh medulloblastoma22 and also critical for nificantly upregulated in human HCC,