Experiments were independently performed three times To further verify the inhibitory effect of LOXL4, we constructed cell lines that stably expressed LOXL4
April 22, 2022
Experiments were independently performed three times To further verify the inhibitory effect of LOXL4, we constructed cell lines that stably expressed LOXL4. death. Furthermore, the nude mouse xenograft model showed that the 5-aza-CR-dependent LOXL4-p53 axis reduces tumor growth. A positive correlation between LOXL4 expression and overall survival in liver cancer patients with Asenapine maleate wild-type p53 tumors was observed. In conclusion, we found that 5-aza-CR-induced LOXL4 upregulation reactivates wild-type p53 and triggers cell death, which blocks liver cancer development. mutations [9-11], which means at least half of liver cancer patients possess tumors with WT, but compromised p53. Therefore, reactivating compromised p53 would be a potential target for liver cancer therapy. Lysyl oxidase-like 4 (LOXL4) is one of five paralogues in the lysyl oxidase (LOX) family, which includes LOX and LOXL1C4 . Asenapine maleate The major function of the LOX family is covalent cross-linking of collagens and/or elastin in the extracellular matrix (ECM). Aberrant expression and activity of these proteins have been reported in several cancer types [12-14]. However, the role of LOXL4 in tumor biology remains enigmatic. A few studies have suggested that it promotes Asenapine maleate tumor proliferation and/or metastasis in head and neck squamous cell carcinoma and gastric cancer [15, 16]. However, in bladder and breast cancer, LOXL4 might function as a tumor suppressor because its loss promotes cancer cell proliferation and metastasis [16, 17]. We speculate that LOXL4 executes its progressive or repressive roles in different tumors depending on tumor cell context and tumor stages. Currently, how LOXL4 functions in liver cancer is not understood. Here, we found that LOXL4 is a novel regulator that contributes to p53 activation in liver cancer. 5-azacytidine treatment upregulated expression, leading to LOXL4 binding with p53, which increased p53 phosphorylation at serine 15 and resulted in p53 activation. Disruption of the LOXL4-p53 axis promoted tumor cell proliferation, whereas enhanced LOXL4-p53 interaction strongly reduced tumor cell growth both in vitro and in vivo. Together, our results illustrate that 5-azacytidine-dependent derepression functionally contributes to the activation of compromised p53, which offers a promising therapeutic strategy for liver cancer. Results A genome-wide CRISPR screen identified LOXL4 as a novel regulator of 5-aza-CR-dependent cell death 5-azacytidine (5-aza-CR) is a small molecule that induces DNA damage and is primarily used in clinic for treatment of myelodysplastic syndrome [18, 19]. To measure the effect of 5-aza-CR on liver cancer cells, we tested four cell lines (HepG2, SK-Hep1, Hep3B, and Huh7) using Hoechst and propidium iodide (PI) double staining. As shown in Fig.?1a, a low dose (1?M) of 5-aza-CR-induced substantial cell death in HepG2 and SK-Hep1 cells, while an even higher dose (5?M) caused no obvious damage to either Asenapine maleate Hep3B or Huh7 cells. Next, we measured cell survival across different time points. As shown in Fig.?1b, the survival rates of HepG2 and SK-Hep1 cells were close to zero, while Huh7 and Hep3B cells exhibited greater than 60% survival after 32?h of treatment. Furthermore, 5-aza-CR treatment induced both apoptosis and necrosis in HepG2 and SK-Hep1 cells, but not in Hep3B and Huh7 cells (Fig. S1). Open in a separate window Fig. 1 A genome-wide CRISPR screen identified LOXL4 as a novel regulator of Asenapine maleate 5-aza-CR-dependent cell death. a Live and dead cell imaging after Hoechst 33324 and propidium iodide (PI) double staining. Cells were treated with or without 5-aza-CR (1 or 5?M) for 24?h and then double stained for 0.5?h. Scale bar: 100?m. Experiments were independently performed three times. b Survival rates of HepG2, Huh7, Hep3B, and SK-Hep1 cells in response to 5-aza-CR treatment. Cells were treated with 5-aza-CR (5?M) for different lengths of time: 0, 4, 8, 16, and 32?h, followed by trypan blue staining. The survival rates of living cells were calculated using Life Tech (Invitrogen) CountnessR. Data were from three independent experiments performed in triplicate; error bars represent SEM. c Workflow of lenti-CRISPR/cas9 screening for genes required for 5-aza-CR-induced cell death. The five key steps included in this BCL2L workflow are as follows: (1) lentiviral library infection of SK-Hep1 cells for 2 days; (2) selection of efficiently infected cells using puromycin (5?g/mL); (3) 5-aza-CR (5?M) treatment for 2 days; (4) extraction and sequencing of the genomes of surviving cells using an Illumina sequencer with a HiSeq instrument followed by analysis with BaseSpace Sequence Hub; (5) validation of candidate genes by evaluating the survival rate over 50% after 5-aza-CR (5?M) treatment for 2 days. d Enriched genes. The sequencing results were analyzed and summarized in Gene Ontology terms using David GO. e The top 10 genes contributing to 5-aza-CR-induced cell death. The genes were selected based on a survival rate of over 50% after 5-aza-CR (5?M) treatment for.