LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells

Zhu, Pingping; Wang, Yanying; Wu, Jiayi; Huang, Guanling; Liu, Benyu; Ye, Buqing; Du, Ying; Gao, Guangxia; Tian, Yong; He, Lei; Fan, Zusen

doi:10.1038/ncomms13608

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Published: 01 December 2016

LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells

Pingping Zhu¹^na1,
Yanying Wang¹^na1,
Jiayi Wu^1,2,
Guanling Huang^1,2,
Benyu Liu^1,2,
Buqing Ye¹,
Ying Du¹,
Guangxia Gao^1,2,
Yong Tian³,
Lei He⁴ &
…
Zusen Fan^1,2

Nature Communications volume 7, Article number: 13608 (2016) Cite this article

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Abstract

Liver cancer stem cells (CSCs) may contribute to the high rate of recurrence and heterogeneity of hepatocellular carcinoma (HCC). However, the biology of hepatic CSCs remains largely undefined. Through analysis of transcriptome microarray data, we identify a long noncoding RNA (lncRNA) called lncBRM, which is highly expressed in liver CSCs and HCC tumours. LncBRM is required for the self-renewal maintenance of liver CSCs and tumour initiation. In liver CSCs, lncBRM associates with BRM to initiate the BRG1/BRM switch and the BRG1-embedded BAF complex triggers activation of YAP1 signalling. Moreover, expression levels of lncBRM together with YAP1 signalling targets are positively correlated with tumour severity of HCC patients. Therefore, lncBRM and YAP1 signalling may serve as biomarkers for diagnosis and potential drug targets for HCC.

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Introduction

Hepatocellular carcinoma (HCC) is the most prevalent subtype of liver cancer and ranks the third leading cause of cancer-related deaths¹. Liver transplantation and surgical resection are the first-line treatment for HCC. Even after surgical resection, the 5-year survival rate of HCC patients remains poor, owing to high recurrence rates. The high rate of recurrence and heterogeneity are the two major features of HCC². Cancer stem cells (CSCs) have been defined to be a small subset of cancer cells within the tumour bulk, exhibiting self-renewal and differentiation capacities³. CSCs may well contribute to tumour initiation, metastasis, recurrence, as well as drug resistance^3,4,5. Liver CSCs can be enriched by some defined surface markers^6,7,8. Several recent studies reported that Wnt/β-Catenin, Notch, Hedgehog, transforming growth factor-β, and phosphatase and tensin homologue signalling pathways are implicated in the regulation of liver CSC self-renewal^9,10,11. However, the biology of liver CSCs remains largely elusive.

Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nucleotides without protein-coding potentials¹². Accumulating evidence shows that lncRNAs are involved in physiological and pathological progresses, including embryonic development, organ formation, X chromatin inactivation, tumorigenesis and so on refs 12, 13, 14, 15. LncRNAs can recruit transcription factors and remodelling complexes to modulate gene expression¹¹ and they can also interact with messenger RNAs and regulate the stability of mRNAs. Several recent studies demonstrated that lncRNAs can associate with some important proteins and modulate their functions^16,17,18. LncRNAs have been reported to be implicated in tumour formation and metastasis^16,17,19. However, how lncRNAs regulate the self-renewal of liver CSCs remains largely unknown.

Yes-associated protein (Yap) and transcriptional co-activator with PDZ-binding domain motif (Taz) are transcriptional cofactors that shuttle between the cytoplasm to the nucleus where they interact with TEAD (TEA domain family member) transcription factors to activate downstream gene expression^20,21. Accumulating evidence links the activity of Yap and Taz to tumorigenesis and chemoresistance^22,23,24. However, how YAP1 signalling is activated in liver CSCs remains unknown.

Here we define a highly transcribed lncRNA in liver CSCs that we call lncBRM (lncRNA for association with Brahma (BRM), gene symbol LINCR-0003), which associates with BRM and modulates the BRG1/BRM switch in the BRG1-associated factor (BAF) complex, leading to activation of YAP1 signalling and promotion of liver CSC self-renewal.

Results

LncBRM is highly expressed in HCC tumours and liver CSCs

Surface markers CD133 (ref. 25) and CD13 (ref. 6) have been widely used as liver CSC markers, respectively. We recently sorted a small subpopulation from HCC cell lines and HCC samples with these two combined makers and defined this subset of CD13⁺CD133⁺ cells as liver CSCs^11,25. We performed transcriptome microarray analysis of CD13⁺CD133⁺ (liver CSCs) and CD13⁻CD133⁻ (non-CSCs) cells and identified 286 differentially expressed lncRNAs in liver CSCs compared with that in non-CSCs¹¹. We previously showed that an uncharacterized lncRNA lncTCF7 regulates the maintenance of liver CSCs through recruitment of the SWI/SNF complex to activate Wnt signalling. Among the differentially expressed lncRNAs in liver CSCs, we chose top ten highly expressed lncRNAs and silenced these lncRNAs in HCC cell lines for in vitro oncosphere formation assays. We noticed that lncBRM depletion most dramatically inhibited oncosphere formation (Fig. 1a). This result was further validated by serial sphere formation assays (Supplementary Fig. 1A,B). In addition, we deleted lncBRM in Hep3B and Huh7 cells by CRISPR/Cas9 technology and found that lncBRM knockout (KO) surely impaired serial sphere formation (Supplementary Fig. 1C,D). Notably, lncBRM knockdown did not affect the expression of its nearby genes (Supplementary Fig. 1E,F), suggesting that lncBRM exerts its function in trans.

LncBRM located on human chromosome 5 between the ACTBL2 and PLK2 genes (Supplementary Fig. 1G). LncBRM consisted of six exons, containing 1,321 nucleotides with a modestly conserved locus according to Phylop analysis (Supplementary Fig. 1G). The full length of lncBRM was further amplified by rapid-amplification of complementary DNA ends approaches and validated by sequencing (Supplementary Fig. 1H). In addition, lncBRM had no protein-coding potential (Supplementary Fig. 1I,J). LncBRM was highly expressed in HCC primary tumour tissues through northern blotting (Fig. 1b) and it was also highly transcribed in advanced HCC samples through quantitative reverse transcriptase–PCR (RT–qPCR; Fig. 1c), further validated by in situ hybridization (Fig. 1d). These results indicate that lncBRM is highly expressed in HCC tumour tissues.

Considering high expression levels of lncBRM in liver CSCs based on transcriptome data, we next wanted to confirm lncBRM expression in liver CSCs from HCC primary samples. We observed that lncBRM was indeed highly expressed in liver CSCs derived from six HCC primary samples (Fig. 1e). Of 33 primary samples we tested, we selected out these 6 HCC samples with high expression levels of lncBRM (1, 8, 9, 13, 17 and 20; Supplementary Table 1) and used these 6 HCC samples for our following studies. We then incubated HCC primary cells and cell lines for sphere formation, followed by enrichment of oncosphere cells (Sphere) and non-sphere cells (Non-sphere) for further examination. We found that lncBRM was also highly expressed in oncosphere cells derived from HCC primary samples and cell lines (Fig. 1e,f), which was further validated by RNA fluorescence in situ hybridization (RNA-FISH; Fig. 1g). In addition, lncBRM was mainly located in the nucleus (Fig. 1g). Similar observations were obtained by nucleocytoplasmic fractionation of HCC cells (Fig. 1h). Altogether, lncBRM is highly expressed in HCC tumour tissues and liver CSCs.

LncBRM is required for the self-renewal of liver CSCs

We next wanted to determine the role of lncBRM in the self-renewal maintenance of liver CSCs. We silenced lncBRM in 6 HCC primary tumour cells (Fig. 2a and Supplementary Fig. 2A), followed by sphere formation assays. For short hairpin RNA (shRNA) designs, we searched for 12 shRNA candidates against lncBRM and selected out 2 most efficient silencing shRNAs (termed shLncBRM1 and shLncBRM2) for our following studies. We noticed that lncBRM depletion markedly repressed sphere formation (Fig. 2a). Reduced self-renewal capacity was further verified in lncBRM-silenced cells through serial sphere formation assays (Fig. 2b). We established stably silenced lncBRM and scrambled RNA (shCtrl)-treated HCC primary tumour cells and injected 1 × 10⁶ lncBRM-silenced and shCtrl cells into BALB/c nude mice, followed by measurement of tumour volumes every 4 days. LncBRM-depleted cells significantly reduced tumour propagation compared with shCtrl-treated cells (Fig. 2c), which was further validated by in vivo serial xenograft passage assays (Fig. 2d). Furthermore, lncBRM silencing displayed much weaker tumour initiation and tumorigenic cell frequency as measured by a limiting dilution xenograft analysis (Fig. 2e and Supplementary Table 2A). To validate the results by lncBRM knockdown, we also established lncBRM KO cells by using a CRISPR/Cas9 approach. LncBRM KO displayed impaired self-renewal capacities of liver CSCs (Supplementary Fig. 2B,C), which were really in agreement with those of lncBRM knockdown results. Therefore, these results indicate that lncBRM depletion abrogates the stemness of liver CSCs.

**Figure 2: *LncBRM* is required for the self-renewal maintenance of liver CSCs.**

We then generated lncBRM stably overexpressed HCC primary tumour cells using lentivirus (Supplementary Fig. 2D), followed by examination of sphere formation and self-renewal capacities. We observed that lncBRM overexpression augmented in vitro oncosphere formation and self-renewal (Fig. 2f,g), as well as promoted xenograft tumour propagation and tumorigenic cell frequency (Fig. 2h, Supplementary Fig. 2E,F and Supplementary Table 2B). Collectively, lncBRM promotes the self-renewal of liver CSCs and in vivo tumour propagation.

LncBRM modulates the BRG1/BRM switch

To determine associated proteins of lncBRM, we performed RNA pulldown assays in oncosphere cell lysates with biotin-labelled lncBRM. One overtly differential band around 170 kDa appeared by silver staining (Fig. 3a) and identified to be BRM by mass spectrometry (Supplementary Fig. 3A). In fact, lncBRM could precipitate BRM in liver oncosphere cell lysates by RNA immunoprecipitation, but not other BAF complex components (Fig. 3b and Supplementary Fig. 3B). Through domain mapping, we defined that 3 segment (607∼951) of lncBRM interacted with BRM (Fig. 3c). In addition, stable stem-loop structure of the binding fragment was predicted by RNA folding analysis (Supplementary Fig. 3C). The interaction of lncBRM with BRM was further validated by an RNA electrical mobility shift assay (EMSA; Fig. 3d). The co-localization of lncBRM and BRM in non-sphere and oncosphere cells was visualized by immunofluorescence staining (Fig. 3e) and mainly distributed in the nucleus. These data indicate that lncBRM associates with BRM in the nuclei of liver CSCs.

**Figure 3: *LncBRM* associates with BRM to initiate the BRG1/BRM switch.**

The SWI/SNF complex is an evolutionally conserved multi-subunit chromatin remodelling complex, which uses ATPase subunit BRM or BRG1 to provide ATP for remodelling nucleosomes^26,27. Mammals contain two yeast BRM homologues BRM and BRG1, which are widely expressed and amino acid sequences are 75% identical. Nonetheless, these subunits are mutually exclusive, as each SWI/SNF complex possesses BRM or BRG1, forming a BRM-embedded or BRG1-embedded BAF complex. However, the physiological roles of the BRM-embedded or BRG1-embedded BAF complex have not been defined yet. We noticed that anti-BRM antibody but not anti-BRG1 antibody could precipitate lncBRM in oncosphere cell lysates (Fig. 3f), validating the interaction between BRM and lncBRM. We observed that BRM-embedded BAF complex was dramatically declined in oncosphere cells (Fig. 3g), but BRG1-embedded BAF complex predominantly existed in these cells, suggesting a BRG1/BRM switch in liver CSCs. The anti-BRM (catalogue number 11966) and anti-BRG1 (catalogue number 21634-1-AP) were specifically recognized their respective proteins.

We then incubated lncBRM transcripts with oncosphere lysates for co-immunoprecipitation assays. We observed that lncBRM increased assembled BRG1-embedded BAF complex (Fig. 3h), but not BRM-embedded complex. Moreover, lncBRM depletion impaired the BRG1-embedded BAF complex in oncosphere cells (Fig. 3i). To further verify this result, we used lncBRM-silenced sphere lysates to conduct size fractionation assays as previously described²⁵. BRG1 and BRM constitute the BRG1- and BRM-embedded BAF complex, respectively, which can be detected by sucrose density gradient centrifugation. We noticed that lncBRM depletion indeed caused more free BRG1 in oncespheres (Fig. 3j), suggesting impaired assembly of the BRG1-biased BAF complex. In contrast, lncBRM overexpression led to more free BRM in oncespheres (Fig. 3k), suggesting impaired assembly of the BRM-biased BAF complex. Finally, we found that lncBRM depletion did not significantly impact BRM stability or ATPase activity (Supplementary Fig. 3D,E). Collectively, lncBRM associates with BRM to sequester BRM away, to initiate assembly of BRG1-biased BAF complex in liver CSCs, resulting in the BRG1/BRM switch.

BRG1 initiates activation of YAP1 signalling in liver CSCs

We next wanted to explore the function of BRG1-embedded BAF complex in the self-renewal maintenance of liver CSCs. We generated BRG1 and BRM KO cells using CRISPR/Cas9 approaches as previously described²⁵. We detected expression levels of major self-renewal-related pathways in BRG1 KO and BRM KO spheres. We found that BRG1 deletion dramatically abrogated YAP1 signalling (Fig. 4a). By contrast, BRM KO remarkably activated YAP1 signalling. These results were further validated by immunoblotting (Fig. 4b), suggesting that BRG1-embedded BAF complex could be involved in the activation of YAP1 signalling.

**Figure 4: BRG1-embedded BAF complex initiates YAP1 signalling in liver CSCs.**

Through domain mapping, we observed that BRG1 bound to YAP1 promoter (Fig. 4c) and validated by luciferase assays (Fig. 4d). However, BRM had no such binding activity. The interaction of BRG1 with YAP1 promoter was further verified by EMSA assays (Fig. 4e). We noticed that BRG1 deletion significantly suppressed YAP1 promoter activation by DNase I sensitivity assays (Fig. 4f). By contrast, BRM deletion enhanced YAP1 promoter activation. In parallel, H3K4me3 antibody did not enriched at YAP1 promoter regions in BRG1 KO oncospheres (Fig. 4g). By contrast, it could precipitate the YAP1 promoter regions in BRM KO cells, suggesting the activation of YAP1 promoter. We next deleted the BRG1-binding region of YAP1 promoter (YAP1PKO) using a CRISPR/Cas9 approach. In YAP1PKO cells, BRG1 depletion has no effect on activation of YAP1 signalling (Fig. 4h). However, BRG1 depletion did suppress the YAP1 signalling in wild-type cells. These data indicate that BRG1 induces the activation of YAP1 signalling via binding its promoter.

BRG1 drives YAP1 expression through a KLF4-dependent manner

To further determine how YAP1 expression was activated by BRG1-embedded BAF complex, we analysed the BRG1-binding regions of YAP1 promoter. We noticed that the BRG1-binding region of YAP1 promoter contained a specific KLF4 binding sequence. KLF4, together with other three Yamanaka pluripotency factors, is able to reprogramme adult fibroblasts into induced pluripotent cells^28,29. KLF4 can inhibit somatic genes in an early phase and subsequently initiate pluripotency genes in a late phase during cellular reprogramming^30,31. Of note, KLF4 interacted with BRG1 but not BRM in oncospheres (Fig. 5a). Furthermore, KLF4 bound to the same BRG1-binding region of YAP1 promoter (Fig. 5b), which was verified by EMSA assays (Fig. 5c). We next performed chromatin immunoprecipitation (ChIP) with anti-KLF4 antibody through oncosphere lysates, followed by size fractionation with detection by immunoblotting. We observed that KLF4 co-eluted with BRG1-embedded BAF complex components and Yap1 promoter (Fig. 5d), suggesting KLF4 recruits the BRG1-embedded BAF complex to Yap1 promoter. In addition, KLF4 deletion abolished the binding of BRG1 with YAP1 promoter (Fig. 5e) and subsequently impaired YAP1 transcription (Fig. 5f). Importantly, KLF4 rescue in KLF4-depleted HCC cells could restore YAP1 activation (Supplementary Fig. 4A). These results indicate that KLF4 is involved in the YAP1 activation.

**Figure 5: KLF4 binds *YAP1* promoter and recruits the BRG1-embedded BAF complex to initiate YAP1 expression.**

We next established YAP1 promoter mutant (Mut) cells by replacement of the KLF4-binding region of YAP1 promoter with a CRISPR/Cas9 approach. We observed that the YAP1 promoter mutant abrogated the interaction of KLF4 with YAP1 promoter (Supplementary Fig. 4B). H3K4me3 and H3K27Ac enrichment at gene promoters denotes the activation of genes. Consistently, the YAP1 promoter mutant reduced enrichment of BRG1 and H3K4me3 (Supplementary Fig. 4B). Moreover, overexpression of BRG1 in wild-type cells promoted YAP1 promoter activation, whereas BRG1 overexpression in YAP1 promoter mutant cells failed to initiate YAP1 promoter activation (Fig. 5g). In parallel, overexpression of lncBRM achieved similar results (Fig. 5h). We also showed that lncBRM recruited the BRG1-embedded BAF complex to YAP1 promoter (Supplementary Fig. 4C,D). These observations were further validated by luciferase assay (Supplementary Fig. 4E), ChIP assay (Supplementary Fig. 4F) and western blotting (Supplementary Fig. 4G). These data indicate that lncBRM and BRG1 induces YAP1 expression in a KLF4-dependent manner. Collectively, YAP1 promoter enriches KLF4 and KLF4 recruits the BRG1-embedded BAF complex, to initiate the activation of YAP1 transcription.

YAP1 is required for the self-renewal of liver CSCs

We further determined the physiological role of YAP1 signalling in the regulation of liver CSCs. Of note, YAP1 overexpression in non-sphere cells significantly restored sphere formation and enhanced self-renewal potential by serial passage assays (Fig. 6a). Conversely, the YAP1-specific inhibitor Verteporfin treatment in non-sphere cells abrogated sphere formation and impaired self-renewal capacity (Fig. 6a). Consequently, YAP1 overexpression in non-CSC cells augmented expression levels of stemness surface markers and pluripotency factors (Supplementary Fig. 5A). By contrast, Verteporfin treatment showed opposite results. Therefore, YAP1 signalling is required for the self-renewal maintenance of liver CSCs.

**Figure 6: *LncBRM*-mediated YAP1 signalling is required for self-renewal of liver CSCs and tumour propagation.**

We next deleted YAP1 gene in HCC primary tumour cells using CRISPR/Cas9 technology (Supplementary Fig. 5B). We found that YAP1 KO displayed impaired sphere formation (Fig. 6b) and reduced tumour-initiating capacity (Fig. 6c and Supplementary Table 2C). Consequently, YAP1-deficient cells decreased xenograft propagation (Fig. 6d). Conversely, YAP1-overexpressing cells enhanced sphere formation and xenograft propagation (Supplementary Fig. 5C and data not shown). Thus, YAP1 is essential for the self-renewal maintenance of liver CSCs and tumour initiation.

We next examined the role of YAP1 signalling target genes in liver CSCs. YM155 has been reported to be an inhibitor of BIRC5 expression³², whereas Siomycin A is a specific inhibitor against FOXM1. Of note, YM155 or Siomycin A treatment significantly suppressed oncosphere formation (Fig. 6e). Similarly, BIRC5 or FOXM1 depletion also reduced sphere formation (Supplementary Fig. 5D,E), indicating YAP1 signalling is required for promoting self-renewal of liver CSCs. Finally, Verteporfin treatment abolished enhanced sphere formation induced by lncBRM overexpression (Supplementary Fig. 5F). Consistently, YAP1 could rescue sphere formation reduced by lncBRM knockdown (Fig. 6f). Altogether, lncBRM-mediated YAP1 signalling is required for the self-renewal of liver CSCs and tumour propagation.

BRG1 and YAP1 targets are positively related to HCC severity

We analysed the expression levels of BRG1/BRM and Yap signalling target genes using online available data sets. We noticed that BRG1 was highly expressed in HCC tumours (Fig. 7a), whereas BRM was lowly expressed in HCC tumour tissues derived from Wang’s cohort (GSE14520; ref. 33). In addition, YAP1 signalling target genes were also highly expressed in HCC tumours (Fig. 7b). Moreover, increased BRG1 levels and decreased BRM levels were positively correlated with severity of HCC patients (GSE14520; Fig. 7c). In parallel, expression levels of YAP1 signalling targets were also related to HCC severity (Fig. 7d). Of note, increased BRG1 levels and decreased BRM levels were also positively related to metastasis of HCC patients (Fig. 7e) and expression levels of YAP1 targets showed the same trend as BRG1 (Fig. 7f). We validated these observations by examination of HCC samples with RT–qPCR (Fig. 7g,h), immunoblotting (Fig. 7i) and immunohistochemical staining (Fig. 7j). Similar observations were also achieved by analysis of other two cohorts (Zhang’s cohort (GSE25097) and Wang’s cohort (GSE54238); Supplementary Fig. 6A–E). However, for tumour stage, metastasis and survival analyses, Wang’s cohort (GSE54238) and Zhang’s cohort (GSE25097) were not suitable for these analyses because of limited clinical information (GSE25097) or sample numbers (GSE54238). In addition, several Yap1 target genes we focused on (Ankrd1, Birc5 and so on) were not available in Wang’s cohort (GSE54238) and Zhang’s cohort (GSE25097) due to microarray platforms. More importantly, expression levels of YAP1 target genes were consistent with clinical prognosis of HCC patients (GSE14520; Supplementary Fig. 6F). Collectively, expression levels of BRG1 and YAP1 target genes are well related to cancer severity and prognosis of HCC patients.

**Figure 7: Expression levels of BRG1 and YAP1 targets are positively correlated with severity and prognosis of HCC patients.**

Discussion

CSCs may contribute to cancer relapse and metastasis due to their invasive and drug-resistant capacities³⁴. Recently, several surface markers have been identified to enrich liver CSCs^6,8,11, whose heterogeneous markers may represent different cellular origins. However, the biology of liver CSCs remains largely unknown. We previously isolated a rare subset of CD13⁺CD133⁺ cells from most HCC cell lines and primary samples^11,25, and showed that the CD13⁺CD133⁺ subpopulation harbours robust self-renewal and differentiation abilities, serving as liver CSCs. Based on our transcriptome microarray analysis of liver CSCs, we identified an uncharacterized lncRNA termed lncBRM, which maintains the stemness of liver CSCs in trans. LncBRM associates with BRM to initiate the BRG1/BRM switch and the BRG1-embedded BAF complex triggers activation of YAP1 signalling.

LncRNAs, as a new class, exhibit a wide range of expression levels and distinct cellular localizations, reflecting a large and diverse class of regulators^12,35. LncRNAs can exert their functions through diverse modes, including cotranscriptional regulation, modulation of gene expression, scaffolding of nuclear or cytoplasmic complexes, as well as pairing with other RNAs³⁶. Collectively, lncRNAs can function in cis to regulate expression of neighbouring genes or in trans to carry out many roles by various modes³⁵. Several recent studies reported that lncRNAs are implicated in tumour initiation, invasion and metastasis^{16,17,19,37,38}. However, the exact role of lncRNAs in liver CSCs remains largely undefined. Here we showed that lncBRM is highly expressed in HCC tumours and liver CSCs. LncBRM associates with BRM to make the BRG1/BRM switch and BRG1-embedded BAF complex initiates the activation of YAP1 signalling to sustain the self-renewal of liver CSCs. Of note, lncBRM deletion failed to impact asymmetric division ratios and differentiation tendency. Surely, lncBRM and lncTCF7 had their unique predicted tertiary structures. Of note, lncBRM KO did not affect the expression levels of lncTCF7 and its target gene TCF7 in liver CSCs (Supplementary Fig. 7A). Conversely, lncTCF7 deletion did not have an impact on the expression levels of lncBRM and YAP1 either (Supplementary Fig. 7B). These data suggest that lncBRM and lncTCF7 regulate the stemness of liver CSCs by using different pathways. In addition, we observed that depletion of both lncTCF7 and lncBRM more significantly impaired oncosphere formation than lncTCF7 or lncBRM depletion alone (Supplementary Fig. 7C), suggesting that targeting both lncTCF7 and lncBRM may be better than lncBRM or lncTCF7 alone for potential therapy of HCC patients. Liver CSCs are really heterogeneous and many different types of liver CSCs have been reported by the expression of different CSC markers such as CD90, EpCAM, CD24, CD44 and so on. We found that lncBRM was also highly expressed in CD90⁺, EpCAM⁺, CD24⁺ or CD44⁺ CSCs isolated from HCC primary samples (Supplementary Fig. 7D). In addition, lncBRM silencing in CD90⁺, EpCAM⁺, CD24⁺ or CD44⁺ CSCs indeed impaired CSC self-renewal and oncosphere formation (Supplementary Fig. 7E). These observations were consistent with those of CD13⁺CD133⁺ cells¹¹. In our tested liver CSCs, lncBRM was highly expressed in these CSC cells and required for their self-renewal maintenance. In addition, we observed that the ratios of CD13⁺CD133⁺ populations in HCC primary samples were around 3∼12% and over 90% population percentage in HCC patients (Supplementary Fig. 7F). However, there was no significant relationship between the lncBRM expression levels and the ratios of CD13⁺CD133⁺ populations. Moreover, lncBRM deletion failed to affect asymmetric division ratios and differentiation tendency (Supplementary Fig. 7G,H), suggesting that lncBRM mainly regulates the self-renewal of liver CSCs through lncBRM-mediated YAP1 activation.

Chromatin structure is modulated by two general classes of complexes: those that covalently modify histone tails and those that remodel nucleosomes in an ATP-dependent manner^27,39. SWI/SNF remodelling complexes use the energy of ATP hydrolysis to remodel nucleosomes and to regulate gene transcription. Of note, inactivating mutations of several SWI/SNF subunits have been found in various human tumours^26,40,41; thus, the SWI/SNF complexes have been considered to be tumour suppressors. However, the role of SWI/SNF complexes in cancer remains controversial. In mammals, BAF-containing complexes were termed BAF complexes, which encompass one of two mutually exclusive catalytic ATPase subunits, either BRM (also known as SMARCA2) or BRG1 (also called SMARCA4). Although BRM may have some redundancy with BRG1, these two types of complexes could be largely distinct⁴². BRG1-deficient mice die in early embryonic development, whereas BRM-deficient mice are live and heavier than normal mice^43,44. BRG1 tends to interact with Zinc finger proteins through its unique amino-terminal domain, whereas BRM associates with proteins containing two ankyrin repeats⁴⁵. Herein we showed that BRG1 promotes the self-renewal of liver CSCs, but BRM exerts an opposite effect (data not shown). Mechanistically, lncBRM binds to BRM that is replaced by its homologue BRG1 to form the BRG1-biased BAF complex, which triggers the activation of YAP1 signalling. BRM binding with lncBRM may undergo conformational changes to abolish its binding capacity to the BAF complex. Another possibility is that lncBRM may bind to the domain of BRM that is critical for the assembly of BRM-biased BAF complex. In fact, increased BRG1 with decreased BRM appears in HCC tumours and this trend is related to severity of HCC patients. We show that the BRG1/BRM switch plays a critical role in the regulation of liver CSC self-renewal and HCC oncogenesis. A recent report showed that the interaction of Yki/Yap1 and Brm, the Drosophila homologue of mammalian BRG1, promotes Yki-dependent transcription and tissue growth in Drosophila⁴⁶. Here we demonstrate the specific interaction of KLF4 and BRG1 (but not BRM) in liver CSCs to initiate YAP1 signalling activation.

Physical properties of the extracellular matrix (ECM) and mechanical forces are integral to defining tissue architecture and driving specific cell differentiation programmes in embryonic development⁴⁷. In adulthood, tissue homeostasis remains dependent on physical cues, such that perturbations of ECM stiffness are causal to pathological conditions of multiple organs, contributing to ageing and malignant transformation⁴⁸. Mechanotransduction enables cells to perceive and adapt to external forces and physical constraints. YAP1 and Taz can perceive mechanical signals exerted by ECM rigidity and cell shape as mechanotransducers⁴⁹, which requires Rho GTPase activity and reorganization of cytoskeleton, but is independent of the Hippo/Lats cascade. We found that Lats1 is undetectable and in liver CSCs following mechanical train (data not shown). Moreover, we noticed that BRG1 KO or BRM KO did not have an impact on phosphorylation signals of MST1/2 and LATS1 in liver CSCs. Therefore, lncBRM-mediated YAP1 signalling is independent of the Hippo/Lats cascade in the progression of HCC. As for tumorigenesis, inflammation, changes of microenvironments and stiffness of ECM confine the cell’s adhesive area and result in mechanical strain²¹. We added Matrigel, collagen I or methyl-cellulose in sphere formation media followed by oncosphere formation assays. Addition of these three reagents dramatically promoted HCC primary oncosphere formation and augmented expression of pluripotency factors (data not shown), suggesting mechanical strain enhances the initiation of liver CSC self-renewal. However, how mechanical strain modulates the activation of lncBRM-mediated YAP1 signalling in liver CSCs needs to be further investigated.

In summary, lncBRM is highly expressed in HCC tumours and liver CSCs, which triggers YAP1 signalling to promote self-renewal of liver CSCs and initiate tumour propagation (Supplementary Fig. 7I). Therefore, lncBRM, along with YAP1 signalling targets might be used in diagnosis and prognosis, as well as in the development of novel therapeutic drugs against HCC.

Methods

Cells lines

HCC cell lines Huh7, Hep3B and PLC were obtained from Dr Zeguang Han (Shanghai Jiaotong University School of Medicine, Shanghai, China). HCC cell lines were maintained in DMEM medium supplemented with 10% fetal bovine serum, 100 μg ml⁻¹ penicillin and 100 U ml⁻¹ streptomycin. All cell lines were verified by PCR (SV40gp6 for 293 T cells; HBVgp2 for PLC cells; AFP, ALB, HBVgp2 and A2M for Hep3B; and AFP for Huh7 cells). These cell lines were not contaminated by mycoplasma.

For preparation of HCC primary cells, HCC samples were immediately obtained after resection. Tumour bulk was cut into 1 mm³ with scissors, followed by 40 min digestion at 37 °C with collagenase IV (0.05% collagenase IV, 0.05% proteinase, 0.01% DNase and 5 mM CaCl₂ in HBS), with shaking every 10 min. Next, the medium were passed through a 70 μm cell strainer and centrifuged at 50 g for 1 min. Supernatant fractions were collected and further centrifuged at 150 g for 8 min and HCC cells were enriched in pellets. After treatment with Red Blood Cell Lysis Buffer for red cell elimination, the HCC primary cells were obtained, followed by FACS, sphere formation and other experiments.

Antibodies and regents

Anti-β-actin (catalogue number A1978, 1:5,000), anti-Flag (catalogue number F1804, 1:5,000) and anti-YAP1 (catalogue number Y4770, 1:500) antibodies were purchased from Sigma-Aldrich. Anti-BRG1 (catalogue number 21634-1-AP, 1:500), anti-FOXM1 (catalogue number 13147-1-AP, 1:1,000) and anti-KLF4 (catalogue number 11880-1-AP, 1:500) antibodies were obtained from Proteintech Group. Anti-BRM (catalogue number 11966, 1:1,000), anti-BAF170 (catalogue number 12760, 1:1,000), anti-SNF5 (catalogue number 8745, 1:1,000), anti-ARID1A (catalogue number 12354, 1:1,000), anti-Baf155 (catalogue number 11956, 1:1,000), anti-Histone H3 (catalog 4499, 1:2,000) and anti-Oct4 (catalogue number 2750, 1:5,000) antibodies were from Cell signalling technology. Anti-SOX9 antibody (catalogue number AB5535, 1:500) was obtained from Millipore. Anti-BIRC5 antibody (catalogue number sc-8807, 1:1,000) was purchased from Santa Cruz Biotechnology. Horseradish peroxidase (HRP)-conjugated secondary antibodies (catalogue number sc-2004, sc-2005, 1:500) were from Santa Cruz Biotechnology. Phycoerythrin-conjugated CD133 antibody (catalogue number 130-098-826, 1:200) was from Miltenyi Biotec. Fluorescein isothiocyanate-conjugated CD13 antibody (catalogue number 11-0138, 1:500) was from eBioscience. Alexa488-conjugated donkey anti-mouse IgG (catalogue number R37114, 1:500), Alexa594-conjugated donkey anti-rabbit IgG (catalogue number R37119, 1:500) and Alexa594-conjugated donkey anti-mouse IgG (catalogue number R37115, 1:500) antibodies were from Molecular Probes Life Technologies. Epidermal growth factor (catalogue number E5036-200UG), PEG5000 (catalogue number 175233-46-2) and 4,6-diamidino-2-phenylindole (catalogue number 28718-90-3) were purchased from Sigma-Aldrich; N2 supplement (catalogue number 17502-048) and B27 (catalogue number 17504-044) were from Life Technologies; basic fibroblast growth factor (catalogue number GF446-50UG) was from Millpore. LightShift Chemiluminescent RNA EMSA Kit (catalogue number 20158) and Chemiluminescent Nucleic Acid Detection Module (catalogue number 89880) were from Thermo Scientific. T7 RNA polymerase (catalogue number 10881767001) and Biotin RNA Labeling Mix (catalogue number 11685597910) were from Roche.

EMSA and RNA EMSA assays

BRG1 and KLF4 EMSA assays were performed using standardized EMSA procedure, and KLF4 and BRG1 were obtained using immunoprecipitation from oncospheres⁵⁰. Biotin-labelled YAP1 promoter (−420∼−380) was purchased from Sangon Company (Shanghai, China) and labelled with [r-³²P] dATP according to standard protocol. Probes and precipitated proteins were incubated in EMSA binding buffer and mobility shift assay was performed using gel electrophoresis.

For RNA EMSA, human BRM plasmid was transfected into Huh7 cells, then cell nuclear extracts were isolated from BRM-overexpressed oncospheres. LncBRM-specific probes were labelled with Biotin using Biotin RNA Labeling Mix (Roche). BRM nuclear extracts and biotin-labelled probe were incubated in 1 × REMSA binding buffer supplementing with Glycerol, transfer RNA and dithiothreitol for 30 min according to LightShift Chemiluminescent RNA EMSA Kit (Thermo Scientific). Native PAGE was performed for separating components followed by transferring onto positively charged NC film (Beyotime Biotechnology). After ultraviolet cross-linking, HRP-conjugated streptavidin was added to detect the biotin signalling according to Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific).

RNA pulldown assay

For RNA pulldown assays, biotin-labelled lncBRM transcripts, lncBRM intron sequence and its antisense were obtained using in vitro transcription with T7 RNA polymerase and Biotin RNA Labeling Mix (Roche), followed by 4 h incubation at 37 °C with oncosphere cell lysates. Then streptavidin-conjugated agarose beads were used for centrifugal enrichment. Precipitated components were separated using SDS–PAGE, followed by silver staining⁵¹. Differential bands were cut for mass spectrometry (LTQ Orbitrap XL).

RNA immunoprecipitation

Primary spheres were treated with 1% formaldehyde for 15 min, then dissolved with modified RIPA buffer (150 mM NaCl, 0.5% sodium deoxycholate, 0.2% SDS, 1% NP40, 1%Triton X-100, 1 mM EDTA and 50 mM Tris pH 8.0), supplementing with Protector RNase inhibitor and protease inhibitors (Roche). Samples were sonicated three times on ice, followed by 13,800 g centrifugation for 10 min. Supernatants were incubated with protein A/G beads for 1 h, followed by 4 h incubation with indicted antibodies and subsequent 2 h incubation with protein A/G beads. LncBRM enrichment was examined using RT–qPCR, IgG enrichment served as controls⁵². Primer sequences are shown in the Supplementary Table 3.

Northern blotting

Total RNA was extracted from HCC samples or oncospheres using standard TRIZOL methods, followed by electrophoresis with formaldehyde denaturing agarose gel. Samples were transferred to positively charged NC film (Beyotime Biotechnology) using 20 × SSC buffer (3.0 M NaCl, 0.3 M sodium citrate, pH 7.0). After ultraviolet cross-linking, membrane was incubated with hybrid buffer for 2 h prehybridization, followed by incubation with Biotin-labelled RNA probes generated by in vitro transcription at 65 °C for 20 h. Biotin signals were detected with HRP-conjugated streptavidin according to the introduction of Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific).

RNA–FISH assay

Fluorescence-conjugated lncBRM probes for RNA–FISH were generated according to protocols of Biosearch Technologies. HCC samples or spheres were treated in a non-denaturing condition, followed by hybridization with DNA probe sets. Then primary antibodies were added after RNA hybridization for co-localization of RNA and indicated proteins. All experiments were performed as described in manuals of Biosearch Technologies¹¹. Treated samples were visualized by confocal microscopy (FV1000, Olympus).

Lentivirus generation and infection

For lncBRM and other lncRNA knockdown, we constructed pSiCoR shRNA system. Sequences of shRNAs were designed according to online tools of Clontech Company and cloned into pSiCoR vector. For virus generation, we transfected 293T cells with pSiCoR along with package plasmids (4 mg pSiCoR vector, 1 mg VSVG, 1 mg pMDL g/p RRE and 2 mg RSV-REV were used for 10 cm dish). Hep3B, Huh7 and HCC primary cells were infected by virus supernatants or PEG5000 (Sigma)-enriched precipitates. After purified with puro or green fluorescent protein (GFP), stable cell lines were established. For overexpression, similar strategy was used. shRNA sequences are listed in Supplementary Table 4.

Western blotting

Primary cells and spheres were crushed with RIPA buffer (150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP40, 1 mM EDTA and 50 mM Tris pH 8.0), followed by separation with SDS–PAGE. Then samples were transferred to NC membrane and incubated with primary antibody in 5% milk. After washing with TBST three times, membranes were blotted with HRP-conjugated secondary antibodies for visualization⁵³. The uncropped blots were shown in Supplementary Fig. 8.

Sphere formation assay

One thousand PLC or Hep3B cells were grown in sphere formation medium (DMEM supplemented with 20 ng ml⁻¹ basic fibroblast growth factor, 20 ng ml⁻¹ epidermal growth factor, N2 and B27). Two weeks later, spheres larger than 100 μm were counted and photographs were taken. For HCC samples, 5,000 primary cells were used for sphere formation.

Diluted xenograft tumour formation

For tumour propagation analysis, lncBRM or YAP1 silenced tumour cells (1 × 10⁶) were subcutaneously injected into BALB/c mice. Tumour volumes were measured every 4 days. For tumour-initiating capacity assay, 10, 10², 10³, 10⁴ and 10⁵ cells were injected into BALB/c nude mice, respectively. Three months later, tumour formation was counted, followed by calculation of ratios of tumour-free mice and tumour-initiating cells⁹.

ChIP immunobloting assay

ChIP immunobloting assay and size fractionation were performed using oncospheres²⁵. For ChIP assays, oncospheres were treated with 1% formaldehyde for 10 min for crosslinking (+crosslink), crashed with SDS lysis buffer and followed by ultrasonication. Standard ChIP assay was performed using KLF4 antibody. The eluate (500 μl) was layered onto 35 ml 5–30% (V/V) glycerol gradients followed by ultracentrifugation. S Beckman SW28 rotor was used for ultracentrifugation at 55,200 g for 30 h. Then, fractions were carefully collected and the elution gradients were concentrated to 50 μl using centrifugal concentration tubes. Finally, the samples were analysed using western blotting and PCR assays. For BRG1/BRM switch, spheres were crushed with RIPA buffer, followed by glycerol gradient ultracentrifugation.

CRISPR/Cas9 KO system

BRG1, BRM, KLF4, YAP1 and YAP1 promoter-deficient and YAP1 promoter mutant HCC primary cells were established using a CRISPR/Cas9 system^8,25. Briefly, single guide RNA (sgRNA) was designed by online CRISPR Design Tool (http://tools.genome-engineering.org) and cloned into lenti Cas9-EGFPvector (Addgene catalogue number 63592). After confirming the cutting efficiency of sgRNA, lenti Cas9 was transfected into 293T cells with pVSVg (Addgene catalogue number 8454) and psPAX2 (Addgene catalogue number 12260) for 48 h. Supernatants were collected and concentrated with PEG5000 (Sigma Aldrich), then infected primary cells for 5 days. GFP-positive cells (usually >80%) were enriched, followed by KO efficiency examination using western blotting. For YAP1 promoter KO, a pair of sgRNAs that were derived from the left locus and the right locus of BRG1/KLF4-binding region on YAP1 promoter were cloned into puro lentiCRISPRv2 and GFP lentiCRISPRv2. Two a lentivirus were co-transfected and the KO efficiency was confirmed by DNA sequencing. For YAP1 promoter mutation, sgRNA and template DNA were transfected into Huh7 cells, followed by selection and monoclonalization. Genome DNA was extracted and YAP1 promoter established clones were examined by T7 endonuclease I cleavage, and confirmed by DNA sequencing.

Statistical methods

R language and Bioconductor were used to analyse online available data sets and results were shown as box and whisker plot⁵⁴. For CSC ratio analysis, extreme limiting dilution analysis was performed using tumour-free mouse numbers of 10, 10², 10³, 10⁴ and 10⁵ cells⁵⁵. For most trials, two tailed Student’s t-test was used for statistical analysis. P<0.05 was considered as statistically significant.

Study approval

Human HCC samples were obtained from the Department of Hepatobiliary Surgery, PLA General Hospital (Beijing, China) with informed consents from all human participants, according to the Institutional Review Board approval. We numbered HCC primary samples according to receiving date and used the samples without artificial bias. Six-week-old female BALB/c nude mice were purchased from the Animal Center of the Chinese Academy of Medical Sciences (Beijing, China). All experiments involving mice were approved by the institutional committee of Institute of Biophysics, Chinese Academy of Sciences. Studies were conducted in a blinded manner. Animals were randomly assigned to groups. No sample-size estimates were used and no animals or samples were excluded from analyses.

Data availability

The microarray data used in the study (GSE66529, GSE14520, GSE25097 and GSE54238) are available in a public repository from EBI (http://www.ebi.ac.uk/) or NCBI (http://www.ncbi.nlm.nih.gov/gds/?term). All relevant data are available from the authors.

Additional information

How to cite this article: Zhu, P. et al. LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells. Nat. Commun. 7, 13608 doi: 10.1038/ncomms13608 (2016).

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Acknowledgements

We thank Junying Jia for technical support. We thank Jing Li (Cnkingbio Company Ltd, Beijing, China) for technical support. This work was supported by the National Natural Science Foundation of China (91419308, 91640203, 81330047, 31530093, 81272270, 81402459 and 81101531); 973 Program of the MOST of China (2015CB553705); the State Projects of Essential Drug Research and development (2012ZX09103301-041); the Strategic Priority Research Programs of the Chinese Academy of Sciences (XDA01010407 and XDA01020203); Postdoctoral innovation programme of China Postdoctoral Science Foundation.

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Pingping Zhu and Yanying Wang: These authors contributed equally to this work

Authors and Affiliations

Key Laboratory of Infection and Immunity of CAS, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing, 100101, China
Pingping Zhu, Yanying Wang, Jiayi Wu, Guanling Huang, Benyu Liu, Buqing Ye, Ying Du, Guangxia Gao & Zusen Fan
University of Chinese Academy of Sciences, Beijing, 100049, China
Jiayi Wu, Guanling Huang, Benyu Liu, Guangxia Gao & Zusen Fan
Key Laboratory of RNA Biology and Beijing Noncoding RNA Laboratory, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
Yong Tian
Department of Hepatobiliary Surgery, PLA General Hospital, Beijing, 100853, China
Lei He

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Pingping Zhu
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Contributions

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

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Zhu, P., Wang, Y., Wu, J. et al. LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells. Nat Commun 7, 13608 (2016). https://doi.org/10.1038/ncomms13608

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Received: 16 March 2016
Accepted: 18 October 2016
Published: 01 December 2016
DOI: https://doi.org/10.1038/ncomms13608

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