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lnc-β-Catm elicits EZH2-dependent β-catenin stabilization and sustains liver CSC self-renewal

Abstract

Liver cancer stem cells (CSCs) may contribute to the high rate of recurrence and heterogeneity of hepatocellular carcinoma (HCC); however, the molecular mechanisms underlying their self-renewal and differentiation remain largely unknown. Through analysis of transcriptome microarray data, we identified a long noncoding RNA (lncRNA) called lnc-β-Catm, which is highly expressed in human HCC tumors and liver CSCs. We found that lnc-β-Catm is required for self-renewal of liver CSCs and tumor propagation in mice. lnc-β-Catm associates with β-catenin and the methyltransferase EZH2, thereby promoting β-catenin methylation. Methylation suppresses the ubiquitination of β-catenin and promotes its stability, thus leading to activation of Wnt–β-catenin signaling. Accordingly, the expression of lnc-β-Catm, EZH2 and Wnt–β-catenin targets is positively correlated with cancer severity and prognosis of people with HCC.

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Figure 1: Lnc-β-Catm is highly expressed in HCC tumors and liver CSCs.
Figure 2: Lnc-β-Catm is required for self-renewal of liver CSCs.
Figure 3: Lnc-β-Catm interacts with β-catenin and EZH2 in liver CSCs.
Figure 4: EZH2 methylates β-catenin at K49 in liver CSCs.
Figure 5: β-catenin methylation suppresses phosphorylation and ubiquitination of β-catenin.
Figure 6: Lnc-β-Catm initiates Wnt signaling by promoting β-catenin stability.
Figure 7: lnc-β-Catm, EZH2 and Wnt–β-catenin targets are related to HCC severity.

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Acknowledgements

We thank J. Jia for technical support. This work was supported by the National Natural Science Foundation of China (91419308 to Z.F., 31530093 to Z.F., 81330047 to Z.F. and 81402459 to Y.W.); the State Projects of Essential Drug Research and Development (2012ZX09103301-041 to Z.F.); the 973 Program of the MOST of China (2015CB553705 to Z.F.); the Strategic Priority Research Programs of the Chinese Academy of Sciences (XDA01010407 to Z.F.); and the Beijing Natural Science Foundation (7162125 to Y.W.).

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Authors and Affiliations

Authors

Contributions

P.Z. designed and performed experiments, analyzed data and wrote the paper; Y.W. performed experiments and analyzed data; G.H. and J.W. performed some experiments; L.H. provided HCC samples and analyzed data; B.Y., B.L. and Y.D. analyzed data. Z.F. initiated the study and organized, designed and wrote the paper.

Corresponding author

Correspondence to Zusen Fan.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Characterization and high expression of Lnc-β-Catm in liver CSCs.

(a) Heatmap of differently expressed lncRNAs in Liver CSCs (CD13+CD133+) and non-CSCs (CD13-CD133-) according to transcriptome analyses. (b) 3’ and 5’ RACE for full length of lnc-β-Catm. The length of lnc-β-Catm was 2281 nucleotides verified by sequencing. Black arrowhead denotes the full length of lnc-β-Catm. (c) Histogram of lnc-β-Catm coding potential analyzed by CPC (left panel), CPAT (middle panel) and PhyloCSF (right panel). HOX transcript antisense RNA (Hotair), X inactivation-specific transcript (XIST) and lncTCF7 serve as control non-coding RNAs. β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serve as control coding genes. For CPC and phyloCSF, scores above 0 indicates coding potential, whereas scores below 0 represent no coding potential. CPAT scores indicate the possibility of coding. (d) Anti-Myc and anti-β-actin Western blots. Samples were shown in left panels. Full length of lnc-β-Catm was cloned into an eukaryotic expression vector pcDNA4-His-Myc B with transcription initiating codon ATG in three expression patterns. T, thymine. (e) Scatter plots (means±s.d.) of nomorlized lnc-β-Catm expression levels. eHCC, early HCC; aHCC, advanced HCC. (f, g) Histogram of lnc-β-Catm expression levels in liver CSCs (CD13+CD133+) and non-CSCs (CD13-CD133-) (f), or in oncospheres and non-spheres (g). Error bars, s.d. (n = 4 cell cultures). Two tailed Student’s t-test was used for statistical analysis, **, P < 0.01; ***, P<0.001. (h) Western blots (upper panel) and realtime PCR (lower panel) of Nucleocytoplasmic separation fractions. Error bars, s.d. (n = 4 cell cultures). U1 RNA serves as a positive control for nuclear location. EEA1, endosome antigen 1; H3, histone 3. For b, d, h, uncropped blots and gels can be found in Supplementary Data Set 1.

Supplementary Figure 2 Lnc-β-Catm enhances self-renewal of liver CSCs.

(a) Relative lnc-β-Catm expression levels of lnc-β-Catm and control cells. Error bars, s.d. (n = 3 cell cultures). Two tailed Student’s t-test was used for statistical analysis, **, P < 0.01; ***, P<0.001. (b, c) Serial sphere formation (b) and tumor propagation (c) with lnc-β-Catm overexpressing and control cells. Error bars, s.d. (n = 3 cell cultures for b, n = 5 mice for c). Two tailed Student’s t-test was used for statistical analysis, *, P < 0.05; **, P<0.01; ***, P<0.001. (d) Tumor-free mice ratios after 3 months’ tumor formation with lnc-β-Catm overexpressing (oeLnc) and control (oeVec) cells. n = 6 mice for each group.

Supplementary Figure 3 Lnc-β-Catm associates with β-catenin and EZH2.

(a) Relative mRNA expression levels in lnc-β-Catm silenced and control cells (lower panels), 16 nearby genes of lnc-β-Catm locus (less than 2 Mb) (upper panels) were analyzed. Error bars, s.d. (n = 4 cell cultures). Two tailed Student’s t-test was used for statistical analysis, **, P < 0.01. (b, c) MS-MS profiles of β-catenin (b) and EZH2 (c), corresponding peptide sequences are listed on the top of the corresponding graphs. (d) Different regions of lnc-β-Catm (left panel) were labeled with biotin and incubated with PLC sphere lysates, followed by RNA pulldown assays (right panel). (e) Stem-loop structures of full length (1-2281 nt), segment #6 (1181-1437 nt) and segment #9 (1938-2281 nt) of lnc-β-Catm. Predictions were based on minimum free energy (MFE) and partition function. Color scales denote confidence of predictions for each base with shades of red indicating strong confidence (http://rna.tbi.univie.ac.at/). (f) Anti-β-catenin and anti-EZH2 Western blots. Samples were derived from co-immunoprecipitation (co-IP) with PLC spheres. (g, h) Anti-β-catenin, anti-EZH2, anti-β-actin (loading control) and anti-Oct4 (serum treated control) Western blots. Samples were immunoprecipitates from spheres (S) and non-spheres (N) (g), or serum treated spheres (h). (i) Intensity profiles along the diagonal from upper left to lower right. Green profiles indicate lnc-β-Catm gray value (intensity), red profiles indicate β-catenin intensity, and blue EZH2. (j) Flag-β-catenin truncates (upper panels) overexpressing spheres were established, followed by co-IP assays and Western blots (lower panels). (k) Anti-Myc and anti-His Western blots (right panels) with EZH2 truncates overepressing spheres, as in j. (l, m) Histogram of lnc-β-Catm enrichment after RNA immunoprecipitation assays. β-catenin truncates (l) and EZH2 truncates (m) overexpressing PLC spheres were used. Error bars, s.d. (n = 4 independent experiments). Throughout figure, uncropped blots and gels can be found in Supplementary Data Set 1.

Supplementary Figure 4 Characterization of β-catenin methylation.

(a) Anti-Methylated lysine, anti-β-catenin, anti-H3 and anti-EEA1 Western blots. Samples were nuclear (N) and cytoplasmic (C) fractions of the indicated spheres. EEA1, endosome antigen 1; H3, histone 3. (b) Western blots for β-catenin methylation signals in HCC tumor tissues (T) and peri-tumor tissues (P). (c) Methylation observation of β-catenin in peri-tumor and tumor tissues. β-catenin (green), methylated lysine (red), and EZH2 (blue). Scale bars, 20 μm. Uncropped blots in a and b can be found in Supplementary Data Set 1.

Supplementary Figure 5 β-catenin methylation promotes its stability.

(a) Anti-phosphorylated β-catenin (p-β-catenin), anti-β-catenin, anti-EZH2 and anti-β-actin (control) Western blots using EZH2 overexpressing (oeEZH2) and control (oeVec) spheres. (b) Anti-K48 linkage ubiquitylation (K48-Ub), anti-β-catenin and anti-β-actin (control) Western blots. EZH2 inhibitors (GSK126 and GSK343) treated and control (DMSO) spheres were used for β-catenin immunoprecipitation. (c) Anti-β-catenin and anti-β-actin (control) Western blots of methylated and non-methylated β-catenin supplemented with sphere lysates (left panels). Relative β-catenin levels in the right panel. Error bars, s.d. (n = 3 cell cultures). Throughout figure, uncropped Western blot results can be found in Supplementary Data Set 1.

Supplementary Figure 6 Lnc-β-Catm promotes Wnt signaling by increasing β-catenin stability.

(a) Relative expression levels of Wnt-β-catenin target genes in lnc-β-Catm silenced and control spheres. (b) Relative β-catenin protein levels in lnc-β-Catm depleted spheres and control spheres. Error bars, s.d. (n=3 cell cultures). Two tailed Student’s t-test was used for statistical analysis, **, P < 0.01; ***, P < 0.001. (c) Anti-methylated lysine and anti-β-catenin (immunoprecipitation control) Western blots. Lnc-β-Catm overexpressing (oeLnc) and control (oeVec) HCC primary spheres were used. (d, e) Realtime PCR (d) and Western blots (e) of Wnt-β-catenin target genes in lnc-β-Catm KO cells (lnc-β-Catm KO) and rescued cells (lnc-β-Catm KO+rescueLnc). Error bars, s.d. (n=3 cell cultures). (f-i) Anti-methylated lysine, anti-β-catenin, anti-ubiquitination and anti-actin (control) Western blots for β-catenin methylation (f), phosphorylation (g), ubiquitination (h) and stability (i). Samples were lnc-β-Catm KO cells, rescued cells and control cells. For i, relative β-catenin protein levels were calculated and shown in the right panel. Error bars, s.d. (n = 3 cell cultures). Two tailed Student’s t-test was used for statistical analysis, *, P < 0.05; **, P < 0.01; ***, P < 0.001. (j, k) Sphere formation (j) and xenograft tumor growth (k). Samples were lnc-β-Catm silenced cells rescued with Wnt-β-catenin target genes (c-Myc, Ccnd1, and Pttg1). Scale bar, 500 μm. Error bars, s.d. (n = 3 independent experiments). Two tailed Student’s t-test was used for statistical analysis, *, P < 0.05. (i) Sphere formation of lnc-β-Catm overexpressed spheres supplemented with Wnt-β-catenin inhibitor WIKI4. Typical images were shown in left panels and sphere formation ratios were calculated (right panels). Error bars, s.d. (n = 3 independent experiments). Two tailed Student’s t-test was used for statistical analysis, *, P < 0.05; **, P < 0.01. ns, not significant. Throughout figure, uncropped blots and gels can be found in Supplementary Data Set 1.

Supplementary Figure 7 Lnc-β-Catm plays a predominant role in HCC and liver CSCs.

(a, b) Expression levels of EZH2 and Wnt-β-catenin target genes in HCC tumors (a) and metastasis patients (b) derived from Wang’s cohort. Data are shown as box-and-whisker plots. Whiskers: 5th and 95th percentiles; Horizontal lines: median levels; Boxes: interquartile range (IQR); upper and lower edges: 75th and 25th percentiles. (c) Kaplan-Meier survival analysis of Wnt-β-catenin target genes. HCC samples were divided into 2 groups according to the indicated gene expression levels. (d) Sphere formation of lnc-β-Catm and lncTCF7 silenced and control HCC primary cells. 31 HCC primary cells were used. *, **, ***, lncRNA shRNA versus control shRNA. #, ##, lnc-β-Catm shRNA versus lncTCF7 shRNA. Error bars, s.d. (n = 3 cell cultures). Two tailed Student’s t-test was used for statistical analysis, *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, P < 0.05; ##, P < 0.01. (e, f) Confocal observation with CD133 antibody (e), Oct4 antibody and c-Myc antibody (f). Control, lnc-β-Catm silenced and lncTCF7 silenced spheres were used. Scale bar, 20 μm.

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Supplementary Figures 1–7 and Supplementary Tables 1–3 (PDF 2331 kb)

Supplementary Data Set 1

Uncropped blots and gels (PDF 2659 kb)

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Zhu, P., Wang, Y., Huang, G. et al. lnc-β-Catm elicits EZH2-dependent β-catenin stabilization and sustains liver CSC self-renewal. Nat Struct Mol Biol 23, 631–639 (2016). https://doi.org/10.1038/nsmb.3235

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