The long noncoding RNA lncZic2 drives the self-renewal of liver tumor–initiating cells via the protein kinase C substrates MARCKS and MARCKSL1

Zhenzhen Chen,Yating Liu,Lintong Yao,Shiying Guo,Yanfeng Gao,Pingping Zhu

doi:10.1074/jbc.ra117.001321

Abstract

Liver tumor-initiating cells (TICs) form small subsets of cells in hepatocellular tumors and account for tumorigenesis, metastasis, recurrence, and drug resistance. Recently, we found that the transcription factor Zic family member 2 (ZIC2) is highly expressed in liver TICs and required for their self-renewal. However, the molecular mechanisms underlying self-renewal of liver TICs remain unclear. Here, using expression profiling and CRISPR-interference assays with clinical samples of human liver cancers, we identified a long noncoding RNA (lncRNA), lncZic2, that is located near the ZIC2 locus and was highly expressed in liver cancer and liver TICs. We found that lncZic2 is required for the self-renewal of liver TICs in a ZIC2-independent manner. lncZic2 drove the expression of myristoylated alanine-rich protein kinase C substrate (MARCKS) and MARCKS-like 1 (MARCKSL1), whose expression levels were increased during liver tumorigenesis and liver TIC self-renewal. Mechanistically, lncZic2 interacted with BRM/SWI2-related gene 1 (BRG1) and recruited this transcriptional regulator to the promoters of the MARCKS and MARCKSL1 gene, which activated expression of these genes. Moreover, we noted that depletion of lncZic2 and BRG1 decreases MARCKS and MARCKSL1 expression and diminishes liver TIC levels. In conclusion, lncZic2 is required for the self-renewal of liver TICs by up-regulating MARCKS and MARCKSL1 gene expression via the transcription factor BRG1. Our findings suggest that the lncZic2-BRG1-MARCKS/MARCKSL1 signaling cascade might be a potential target for eliminating liver TICs in the management of liver cancer.

Highlights

Liver tumor–initiating cells (TICs) form small subsets of cells in hepatocellular tumors and account for tumorigenesis, metastasis, recurrence, and drug resistance
We found that lncZic2 is required for the self-renewal of liver TICs in a Zic family member 2 (ZIC2)-independent manner. lncZic2 drove the expression of myristoylated alanine-rich protein kinase C substrate (MARCKS) and MARCKSlike 1 (MARCKSL1), whose expression levels were increased during liver tumorigenesis and liver TIC self-renewal
LncZic2 is required for the self-renewal of liver TICs by up-regulating MARCKS and MARCKSL1 gene expression via the transcription factor BRM/SWI2-related gene 1 (BRG1)

Summary

To whom correspondence may be addressed

Hotmail.com. 3 The abbreviations used are: HCC, hepatocellular carcinoma; TIC, tumor-initiating cell; lncRNA, long non-coding RNA; CRISPRi, CRISPR-interference; PKC, protein kinase C; MARCKS, myristoylated alanine-rich protein kinase C substrate; EMSA, electrophoretic mobility shift assay; FISH, fluorescence in situ hybridization. We found that the transcription factor ZIC2 is highly expressed in liver cancer and liver TICs [12]. ZIC2 is required for the self-renewal of liver TICs through OCT4 expression. We identified that a long noncoding RNA, lncSox, is highly expressed in liver cancer and liver TICs [23]. Samples was extracted, and lncZic expression levels were examined by real-time PCR. C and D, lncZic expression levels in CD133ϩ liver TICs (C) and stem-like oncospheres (D) were examined through real-time PCR. LncZic average expression levels in liver non-TICs and nonspheres were normalized as 1. We focused on lncZic and found that lncZic was highly expressed in liver cancer and liver TICs. Interestingly, lncZic is not involved in the transcription regulation of ZIC2 but regulates the expression of MARCKS and MARCKSL1. Through interacting with BRG1, lncZic is required for the interaction between BRG1 and the MARCKS/ MARCKSL1 promoter. lncZic2–BRG1–MARCKS/MARCKSL1 can be targeted for liver TIC clearance

Results

Discussion

Experimental procedures