ARTICLE
Received 23 Apr 2016 | Accepted 20 Jul 2016 | Published 25 Nov 2016
DOI: 10.1038/ncomms12692 OPEN
A feed-forward loop between lncARSR and YAP activity promotes expansion of renal tumour-initiating cells
Le Qu1,2,*, Zhenjie Wu2,*, Yaoming Li3,4,*, Zhipeng Xu2,*, Bing Liu2, Feng Liu5, Yi Bao2, Dengshuang Wu2, Jiayi Liu2, Anbang Wang2, Xiaoyuan Chu6, Yinghao Sun3, Cheng Chen6, Zhengyu Zhang1 & Linhui Wang2
Renal tumour-initiating cells (T-ICs) contribute to tumorigenesis, progression and drug resistance of renal cell carcinoma (RCC). However, the underlying mechanism for the propagation of renal T-ICs remains unclear. Here we show that long non-coding RNA lncARSR is upregulated in primary renal T-ICs and associated with a poor prognosis of clear cell RCCs (ccRCC). Knockdown of lncARSR attenuates the self-renewal, tumorigenicity and metastasis of renal T-ICs. Conversely, forced lncARSR expression enhances T-IC properties of RCC cells. Mechanistically, the binding of lncARSR to YAP impedes LATS1-induced YAP phosphorylation and facilitates YAP nuclear translocation. Reciprocally, YAP/TEAD promotes lncARSR transcription, thus forming a feed-forward circuit. The correlation between lncARSR and YAP is validated in a ccRCC cohort, where the combination of these two parameters exhibits improved prognostic accuracy. Our ndings indicate that lncARSR plays a critical role in renal T-ICs propagation and may serve as a prognostic biomarker and potential therapeutic target.
1 Department of Urology, Jinling Hospital, Nanjing University Clinical School of Medicine, Nanjing 210002, China. 2 Department of Urology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China. 3 Department of Urology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China. 4 Department of Urology, Daping Hospital, Third Military Medical University, Chongqing, China. 5 Obstetrics and Gynecology of Navy PLA General Hospital, Beijing 100048, China. 6 Department of Medical Oncology, Jinling Hospital, Nanjing University Clinical School of Medicine, Nanjing 210002, China. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to C.C. (email: mailto:[email protected]
Web End [email protected] ) or to Z.Z. (email: mailto:[email protected]
Web End [email protected] ) or to L.W. (email: mailto:[email protected]
Web End [email protected] ).
NATURE COMMUNICATIONS | 7:12692 | DOI: 10.1038/ncomms12692 | http://www.nature.com/naturecommunications
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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12692
Renal cell carcinoma (RCC) is the most common kidney cancer in adults1 and a challenging disease with poor prognosis2. Increasing appreciation of cell heterogeneity
within clear cell renal cell carcinoma (ccRCC)3 has focused attention on a distinct subpopulation of cells called tumour-initiating cells (T-ICs) or cancer stem cells (CSCs)4 in ccRCC. T-ICs exhibit extended self-renewal potential and tumour-initiating ability5. Tumours that harbour an abundant T-IC population or have high expression of stemness-related genes may signal a poor clinical outcome in RCC patients6,7. Therefore, identication of the underlying mechanisms governing renal T-ICs propagation may lead to the discovery of promising therapeutic strategies for RCC patients.
Long non-coding RNA (lncRNA) is a subgroup of transcripts with more than 200 nt and limited coding potential. lncRNAs modulate biological process via diverse mechanisms8, including mobilizing transcriptional co-regulators or chromatin-modifying complex9,10 at transcription level, and interacting with RNAs1113 and protein complex14,15 or modifying signal proteins16,17 at post-transcription level. Several lncRNAs have been reported to regulate the self-renewal of T-ICs especially liver T-ICs1820. Nevertheless, the role of lncRNA in the regulation of renal T-ICs remains unknown.lncARSR (lncRNA Activated in RCC with Sunitinib Resistance, ENST00000424980) was a newly identied lncRNA to promote the sunitinib resistance of RCC in our previous study21. Accumulating evidence indicated that T-ICs surviving from drug therapy and giving rise to tumour regrowth might be a major culprit for therapeutic resistance2225. Indeed, the expression signature of stem cell2628 or targets of Nanog, Oct4, Sox2 and c-Myc (NOSM) in human ESCs2931 were signicantly enriched in our mRNA prole of sunitinib-resistant RCC cells (GSE69535) (Supplementary Fig. 1a), prompting us to explore the role of lncARSR in renal T-ICs.
In this study, we rst nd that lncARSR is highly expressed in primary renal T-ICs and predicts poor prognosis. Next, by using loss-of-function analysis in T-ICs and gain-of-function analysis in RCC cells, we demonstrate that lncARSR promotes the self-renewal capacity, tumorigenicity and metastasis of renal T-ICs. Further mechanism study reveals that lncARSR interacts with Yes-associated protein (YAP) to block its phosphorylation by LATS1, facilitating YAP nuclear translocation. Interestingly, we nd that YAP in turn promotes the transcription of lncARSR, forming a feed-forward loop. Clinical investigation also conrms the correlation between lncARSR and YAP, and demonstrates the value of combining lncARSR and YAP to improve the prognostic accuracy for RCC patients. Altogether, we discover that lncARSR promotes the expansion of renal T-ICs via interacting with YAP.
ResultslncARSR is upregulated in T-ICs and predicts poor prognosis. CD105 and CD133 are well-accepted renal T-IC markers32. In tumour cells isolated from primary ccRCC tissues, pearson correlation analysis revealed that lncARSR levels were positively correlated with the expression of CD105 and CD133 (Fig. 1a). To determine the expression of lncARSR in renal T-ICs, we enriched T-ICs by ow cytometry sorting or sphere formation (Supplementary Fig. 1b,c). As shown in Fig. 1b, lncARSR levels were upregulated in sorted CD105 or CD133 primary ccRCC cells. Compared with adherent cells, lncARSR expression was increased in RCC spheres derived from human primary ccRCC cells. Notably, lncARSR level was reduced to origin level when the spheres were reattached (Fig. 1c,d). Several RCC cell lines showed the similar results (Supplementary Fig. 1d,e). These data indicated that lncARSR was preferentially upregulated in renal T-ICs.
To investigate the clinical signicance of lncARSR, we determined lncARSR expression in a total of 310 ccRCC tissues from two independent cohorts. The average level of lncARSR was higher in ccRCC tumours than adjacent non-tumour tissues determined by quantitative PCR with reverse transcription (qRTPCR) and in situ hybridization (ISH) (Fig. 1e,f; Supplementary Fig. 1f and Supplementary Table 1). Notably, lncARSR expression was elevated in poorly differentiated ccRCC tumours compared with well-differentiated tumours (Supplementary Fig. 1g), prompting a putative role of lncARSR in renal T-ICs. Correlation regression analysis revealed that high lncARSR expression in ccRCC tissues was associated with aggressive clinical features (Supplementary Tables 2 and 3). Moreover, patients with higher lncARSR levels exhibited worse overall survival and shorter time to recurrence (Fig. 1g,h). Multivariate analysis manifested that high lncARSR level was an independent predictor for poor prognosis of ccRCC patients (Supplementary Tables 47).
lncARSR is required for the maintenance of renal T-ICs. To explore the potential role of lncARSR in renal T-ICs, we suppressed lncARSR expression utilizing two independent lenti-virus-based short hairpin RNAs (shRNAs) in primary ccRCC cells and cell lines (Supplementary Fig. 2a). Flow cytometry analysis showed that knockdown of lncARSR decreased the proportion of CD105 or CD133 cells (Fig. 2a). Primary ccRCC spheres with lncARSR knockdown exhibited impaired self-renewal capacity on serial passage and decreased expression of pluripotent transcription factors (Fig. 2bd). Similar results were also observed in RCC cell lines (Supplementary Fig. 2b,c), indicating that knockdown of lncARSR attenuated the self-renewal capacity of renal T-ICs.
To further determine the effect of lncARSR on the tumor-igenicity of renal T-ICs, sphere-derived shlncARSR or shGFP cells were inoculated into nude mice. In vivo limiting dilution assay revealed that suppression of lncARSR signicantly reduced tumour incidence and T-IC frequency (Fig. 2e and Supplementary Table 8) consistent with the cell culture studies. Moreover, RCC cells derived from the shlncARSR-xenografts showed impaired ability to form secondary tumours by serial passage compared to control xenografts (tumour incidence: shGFP, 4/4; shlncARSR-1, 0/4; shlncARSR-2, 0/4) (Supplementary Fig. 2d), indicating that interference of lncARSR impaired the tumour formation ability of renal T-ICs. Furthermore, cells from dissociated shlncARSR spheres formed decreased number and size of pulmonary metastatic lesions in mice (Fig. 2f,g). Collectively, these results demonstrated that lncARSR played a critical role in the maintenance of renal T-ICs.
lncARSR promotes renal T-ICs expansion. Next, we over-expressed lncARSR in RCC cells by lentivirus delivery (Supplementary Fig. 3a). lncARSR-overexpressing cells harboured expanded proportion of CD105 or CD133 cells (Fig. 3a).
Primary ccRCC cells with lncARSR overexpression exhibited enhanced sphere-forming ability and increased expression of pluripotent transcription factors (Fig. 3bd). In vitro limiting dilution assay revealed that lncARSR overexpression increased the T-IC frequency of primary ccRCC cells (Fig. 3e). Similar results were also achieved in RCC cell lines (Supplementary Fig. 3bd). Moreover, lncARSR overexpression yielded an enhanced tumorigenicity and pulmonary metastasis of RCC cells in vivo (Fig. 3fh and Supplementary Table 9). These data indicated that lncARSR facilitates the expansion of renal T-ICs.
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Figure 1 | lncARSR is upregulated in T-ICs and predicts poor prognosis. (a) The correlation between the transcription level of lncARSR and CD105 (left) or CD133 (right) in primary ccRCC cells (n 22) was determined by qRTPCR analysis. Data were normalized to b-actin as DCt and analysed by
Spearmans correlation analysis. (b) qRTPCR analysis of lncARSR in MACS sorted CD105 (left) or CD133 (right) primary ccRCC cells relative to negative cells. Data are represented as means.d.; *Po0.05 and ***Po0.001; two-tailed Students t-test. (c) qRTPCR analysis of lncARSR in primary ccRCC adherent, spheres and re-adherent cells. Data are represented as means.d.; **Po0.01 and ***Po0.001; two-tailed Students t-test. (d) FISH analysis of lncARSR in primary ccRCC #4 adherent and sphere cells using biotin-labelled LNA probe. The nuclei were stained with DAPI. Scale bar, 20 mm. (e) lncARSR expression in human ccRCC tissues and adjacent tissues determined by qRTPCR analysis in cohort 1 (n 105, Po0.001, MannWhitney
U-test). The lncARSR expression was normalized to b-actin (DCt) and compared with the maximum DCt. Data are presented as DDCt. The horizontal
lines in the box plots represent the median, the boxes represent the interquartile range and the whiskers represent the 2.5th and 97.5th percentiles. (f) lncARSR expression in human ccRCC tissues and adjacent tissues determined by RNA in situ hybridization (ISH) analysis in cohort 2 (n 205,
Po0.001, MannWhitney U-test). The lncARSR expression was normalized to the minimum signal of lncARSR positive staining. The horizontal lines in the box plots represent the median, the boxes represent the interquartile range, and the whiskers represent the 2.5th and 97.5th percentiles. (g) KaplanMeier analysis of overall survival (left, Po0.001, log-rank test) or recurrence rate (right, Po0.001, log-rank test) of ccRCC patients in low and high lncARSR groups in cohort 1. (h) KaplanMeier analysis of overall survival (left, Po0.001, log-rank test) or recurrence rate (right, Po0.001, log-rank test) of ccRCC patients in low and high lncARSR groups in cohort 2.
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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12692
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Figure 2 | lncARSR is required for the maintenance of renal T-ICs. (a) Flow cytometric analysis of the proportion of CD105 (left) or CD133 (right) cells in lncARSR-knockdown and control RCC cells (n 3). Data are represented as means.d.; *Po0.05 and **Po0.01; two-tailed Students t-test.
(b) Spheres formation assay of lncARSR-knockdown and control primary ccRCC cells (n 3). The number of primary, secondary and tertiary passaged
spheres was counted after 7 days. (c) Western blot analysis of Oct4 and Sox2 in lncARSR-knockdown and control primary ccRCC spheres. GAPDH acted as a loading control. (d) qRTPCR analysis of indicated mRNAs in lncARSR knockdown and control primary ccRCC spheres. Data are represented as means.d.; *Po0.05 and **Po0.01; two-tailed Students t-test. (e) In vivo limiting dilution assay of lncARSR knockdown and control sphere-derived RCC cells. Tumours were observed over 2 months; n 6 for each group. (f,g) Representative microscopic images of pulmonary metastatic lesions at 12 weeks
after the injection of indicated sphere-derived RCC cells into the tail vein of nude mice (upper). Red arrows indicate lung metastatic tumours. Scale bar, 200 mm. The number (lower left) and diameter (lower right) of lung metastatic tumours in each group (n 8) were calculated. Data are represented as
means.d.; **Po0.01 and ***Po0.001; two-tailed Students t-test.
lncARSR physically interacts with YAP. Our previous data demonstrated that lncARSR was mainly localized in cytoplasm and could act as competing endogenous RNA to sequestrate miRNA21. However, knockdown of Dicer had little effect on lncARSR-induced upregulation of stemness genes (Supplementary Fig. 4a,b),
excluding the role of miRNA in lncARSR-induced T-IC property.
To dissect the mechanism underlying the promotive role of lncARSR in renal T-IC, we performed RNA pull-down assay to seek lncARSR-interacting proteins (Fig. 4a). The band specically
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Figure 3 | lncARSR promotes renal T-ICs expansion. (a) Flow cytometric analysis of the proportion of CD105 (left) or CD133 (right) cells in lncARSR-overexpressing and control RCC cells (n 3). Data are represented as means.d.; *Po0.05 and **Po0.01; two-tailed Students t-test. (b) Spheres
formation assay of lncARSR-overexpressing and control primary ccRCC cells (n 3). The number of spheres was counted after 7 days. (c) Western blot
analysis of Oct4 and Sox2 in lncARSR-overexpressing and control primary ccRCC cells. (d) qRTPCR analysis of indicated mRNAs in lncARSR-overexpressing and control primary ccRCC cells. Data are represented as means.d.; **Po0.01; two-tailed Students t-test. (e) In vitro limiting dilution assay of lncARSR-overexpressing and control primary ccRCC cells. The results were shown as natural logarithm of the proportion of T-ICs. (f) In vivo limiting dilution assay of lncARSR-overexpressing and control RCC cells. Tumours were observed over 2 months; n 6 for each group. (g,h) Representative
microscopic images of pulmonary metastatic lesions at 12 weeks after the injection of indicated RCC cells into the tail vein of nude mice (upper). Red arrows indicate lung metastatic tumours (left). Scale bar, 200 mm. The number (lower left) and diameter (lower right) of lung metastatic tumours in each group (n 8) were calculated. Data are represented as means.d.; **Po0.01; two-tailed Students t-test.
pulled down by lncARSR was subject to mass spectrometry analysis (Supplementary Data 1). Nine proteins that localized in cytoplasm with corresponding molecular mass were selected and subjected to loss-of-function analysis (Supplementary Fig. 4c). Four out of the nine candidate proteins were required for the sphere-forming ability of RCC cells (Supplementary Fig. 4d), but only YAP was further reproducibly detected by independent RNA pull-down assays (Fig. 4b,c and Supplementary Fig. 4e). RNA immunoprecipitation (RIP) assay conrmed the interaction between YAP and lncARSR in RCC spheres (Fig. 4d). Consistently, lncARSR co-localized with YAP in the cytoplasm by RNA uorescence in situ hybridization (RNA FISH) and
immunouorescence (Fig. 4e), which was validated by quantitative co-localization analysis (Pearsons correlation R 0.658382)33. Furthermore, RNA pull-down assay with
truncated lncARSR mutants demonstrated that the 50 segment of lncARSR (nucleotides 1310) was responsible for its interaction with YAP (Fig. 4f). RNA electrophoretic mobility shift assay (EMSA) revealed that RNAYAP complex was efciently competed by unlabelled lncARSR probes (nucleotides 1310) and super-shifted by anti-YAP antibody (Fig. 4g), further conrming the binding of the 50 segment of lncARSR to YAP.
Moreover, data from EMSA suggested a 1:1 stoichiometry between the lncARSR and YAP. RNA folding analysis34 of
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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12692
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Figure 4 | lncARSR physically interacts with YAP. (a) RNA pull-down assays were performed with cytoplasmic lysates of RCC spheres using full-length lncARSR and antisense RNA probes, followed by mass spectrum. Red arrows indicate target band. (b) MS/MS proles of target band (corresponding peptide sequences of YAP) retrieved by lncARSR. (c) Western blot analysis of YAP in RNA pull-down precipitates retrieved by biotin-labelled lncARSR or antisense RNA from the cytoplasmic lysates of RCC spheres. (d) RIP assay of the enrichment of YAP on lncARSR relative to IgG in the cytoplasmic lysates of adherent and spheres RCC cells (n 3). b-action acted as a loading control. Data are represented as means.d.; **Po0.01; two-tailed Students t-test.
(e) RNA FISH analysis of lncARSR (red) and immunouorescence detection of YAP (green) in A498 spheres. The rightmost graph shows the co-localized signals between the red signal (lncARSR) and the green signal (YAP). Pearsons correlation: R 0.658382. Scale bar, 10 mm. (f) Upper: gel electrophoresis
of in vitro transcribed biotin-labelled RNA of full-length and truncated lncARSR. Lower: western blot analysis of YAP in RNA pull-down precipitates retrieved by different fragments of lncARSR probe. (g) RNA EMSA assays of the interaction of different fragments of lncARSR and YAP. Biotin-labelled RNA containing nucleotides 1310 or 282591 bp of lncARSR acted as probes. Non-labelled RNA probes acted as competitors. Anti-YAP antibody acted for super-shift experiment. Shift indicates the shift in mobility induced by the biotin-labelled RNA probe and YAP complex. Super-shift indicates the super-shift in mobility induced by the biotin-labelled RNA probe, YAP and anti-YAP antibody complex.
lncARSR 50-segment predicted a stable stem-loop structure (Supplementary Fig. 4f), which might provide the spatial conformations for its binding with YAP.
YAP is responsible for lncARSR-mediated T-IC properties. YAP, a transcription co-activator in Hippo signalling35,36, has been reported to play critical roles in T-ICs expansion in various cancers37,38. Indeed, the expression of YAP was nuclear-enriched in renal T-ICs (Fig. 5a). Knockdown of YAP impaired the proportion of CD105 or CD133 cells and attenuated the
sphere-forming capacity and the expression of pluripotent transcription factors in RCC spheres (Fig. 5b,c and Supplementary Fig. 5a,b). Conversely, introduction of constitutively activated YAP (YAP-5SA)39 increased the T-IC properties of RCC cells (Fig. 5d,e and Supplementary Fig. 5c,d), indicating a critical role of YAP in the maintenance and expansion of renal T-ICs.
Notably, introduction of YAP-5SA restored the self-renewal capacity and the expression of pluripotent transcription factors in lncARSR-knockdown RCC spheres (Fig. 5f,g). While knockdown of YAP eliminated the discrepant T-IC properties triggered by
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150
100
50
** NS
shGFP
Ctrl+shGFP
Ctrl+shYAP
IncARSR+shGFP
IncARSR+shGFP
8
Relative mRNA
expression
1
0
Tumour-free mice (%)
Ctrl
1.0
IncARSR
Number of
shYAP
0.5
0.0
103 104 105 106
shGFP
0 shYAP
shGFP
shYAP
shGFP
shYAP
k
Lung metastasesCtrl IncARSR
30 ** **
NS NS
shGFP
Number of tumour
metastases per lung
Diameter of largest
tumour metastases per lung
600
400
200
10
Ctrl shGFP
0 Ctrl shYAP
IncARSR shYAP
IncARSR shGFP
shYAP
Ctrl shGFP
0 Ctrl shYAP
IncARSR shYAP
IncARSR shGFP
Figure 5 | YAP is responsible for lncARSR-mediated T-IC properties. (a) Western blot analysis of YAP in subcellular fractions of RCC adherent and sphere cells. GAPDH and Histone H3 acted as cytoplasm and nucleus marker, respectively. (b) Spheres formation assay of YAP-knockdown and control RCC spheres (n 3). The number of spheres was counted after 7 days. (c) qRTPCR analysis of indicated mRNAs in YAP-knockdown and control RCC
spheres (n 3). (d) Spheres formation assay of YAP-5SA-overexpressing and control RCC cells (n 3). The number of spheres was counted after 7 days.
(e) qRTPCR analysis of indicated mRNAs in YAP-5SA-overexpressing and control RCC cells (n 3). (f) Spheres formation assay of A498 spheres
transfected with indicated plasmids (n 3). The number of spheres was counted after 7 days (left). Representative images of spheres are shown (right).
Scale bar, 200 mm. (g) qRTPCR analysis of Oct4 and Sox2 in A498 spheres transfected with indicated plasmids after 48 h (n 3). (h) Spheres formation
assay of A498 cells transfected with indicated plasmids (n 3). The number of spheres was counted after 7 days (left). Representative images of spheres
are shown (right). Scale bar, 200 mm. (i) qRTPCR analysis of Oct4 and Sox2 in A498 cells transfected with indicated plasmids after 48 h (n 3).
(bi) Data are represented as means.d.; **Po0.01; two-tailed Students t-test. (j) In vivo limiting dilution assay of indicated RCC cells. Tumours were observed over 2 months; n 6 for each group. (k) Representative microscopic images of pulmonary metastatic lesions at 12 weeks after the injection of
indicated RCC cells into the tail vein of nude mice (upper). Red arrows indicate lung metastatic tumours (left). Scale bar, 200 mm. The number (middle) and diameter (right) of lung metastatic tumours in each group (n 8) were calculated. Data are represented as means.d.; **Po0.01; two-tailed
Students t-test.
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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12692
lncARSR overexpression (Fig. 5h,i) or depletion (Supplementary Fig. 5e,f), indicating that YAP is required for lncARSR-mediated renal T-IC maintenance and expansion. More importantly, YAP depletion abrogated the lncARSR-enhanced tumorigenicity and pulmonary metastasis of RCC cells in vivo (Fig. 5j,k and Supplementary Table 10). Together, these data demonstrate that YAP is responsible for lncARSR-promoted renal T-IC expansion.
lncARSR blocks LATS1-mediated YAP phosphorylation. To determine how lncARSR regulated YAP activity, we constructed truncated YAP mutants to unravel its binding sites with lncARSR. RIP assay revealed that the WW1/2 domain of YAP (residues 155263) was required for its interaction with lncARSR (Fig. 6a), which was conrmed by RNA pull-down assay (Fig. 6b). The WW1/2 domain of YAP has been reported to interact with LATS1 (large tumour suppressor kinase 1), which could induce YAP phosphorylation and subsequent cytoplasmic retention40. Therefore, we speculated that lncARSR might block LATS1YAP interaction to promote YAP nuclear translocation. Immuno-precipitation assay showed that lncARSR knockdown facilitated the interaction between LATS1 and YAP in RCC spheres (Fig. 6c and Supplementary Fig. 6a,b), and overexpression of lncARSR or its 50-segment attenuated LATS1YAP interaction in adherent cells (Fig. 6d and Supplementary Fig. 6c). As a result, YAP phosphorylation was increased by lncARSR knockdown and suppressed on overexpression of lncARSR or its 50-segment (Supplementary Fig. 6d,e). Furthermore, in vitro kinase assay showed that lncARSR or its 50-segment could protect YAP from phosphorylation by puried active LATS1 (Fig. 6e). As expected, YAP nuclear translocation was enhanced on lncARSR overexpression and attenuated upon lncARSR knockdown (Fig. 6f,g; Supplementary Fig. 6f,g). YAP translocates into the nucleus mainly serving as a transcription co-activator. As shown in Fig. 6h and Supplementary Fig. 6h, overexpression of lncARSR or its 50-segment enhanced YAP luciferase reporter41 activity and the expression of YAP downstream genes. Notably, knockdown of LATS restored the self-renewal capacity and the expression of pluripotent transcription factors in lncARSR-knockdown spheres (Fig. 6i,j and Supplementary Fig. 6i). Together, these results demonstrated that lncARSRYAP interaction prevented the phosphorylation of YAP by LATS1 and facilitated YAP nuclear translocation to promote renal T-IC properties.
YAP/TEAD complex transactivates lncARSR. Regulatory feed-forward loops have been reported to involve in tumour initiation and progression42,43. As shown in Fig. 7a, lncARSR levels were repressed on YAP deletion in RCC spheres. While lncARSR levels were enhanced by introducing YAP-5SA but not YAP-5SADC39, a YAP mutant lacking transactivation domain (Fig. 7b and Supplementary Fig. 7a), indicating that YAP might modulate lncARSR transcription. It has been reported that YAP transactivates target genes mainly through interacting with TEA domain family (TEAD)44. Intriguingly, overexpression of YAP-5SA-S94A (mutant without TEAD-binding capacity)44 or Verteporn treatment45, a drug selectively disrupts YAP-TEAD binding, failed to promote lncARSR transcription (Fig. 7c). Interference of TEAD family members revealed that TEAD1, TEAD3 and TEAD4 but not TEAD2 were required for YAP-induced lncARSR expression and the upregulation of lncARSR in RCC spheres (Fig. 7d and Supplementary Fig. 7b,c). Notably, bioinformatics analysis predicted two conserved TEAD-binding (TB) sites in lncARSR promoter region (Fig. 7e, upper panel). Chromatin immunoprecipitation (ChIP) assay demonstrated that YAP was enriched on the predicted TB sites in lncARSR promoter, and the enrichment was signicantly upregulated in
spheres (Fig. 7e, lower panel). Furthermore, ChIPre-ChIP assays conrmed the co-occupancy of YAP and TEAD1 on lncARSR promoter (Fig. 7f,g). Collectively, these data indicated that YAP/TEAD complex bound to lncARSR promoter and promoted lncARSR transcription, therefore forming a positive feedback circuit in renal T-ICs.
Combining lncARSR and YAP exhibits improved prognostic value. To verify the correlation between lncARSR and YAP activity in clinical samples, we examined the RNA levels of lncARSR and YAP target genes in 52 human ccRCC tumour specimens. As shown in Fig. 8a, the levels of lncARSR were positively correlated with the levels of YAP target genes CTGF, BIRC5 and CYR61. Moreover, we performed immunochemistry staining of YAP in ccRCC tissue microarray, which had been subjected to ISH analysis of lncARSR. Expression of lncARSR was positively correlated with nuclear accumulation of YAP in ccRCC tissues (Fig. 8b and Supplementary Table 11 and Supplementary Fig. 8a). The ratio of patients with nuclear YAP enrichment was increased in high lncARSR group compared with that in low lncARSR group (Fig. 8c). Although either high lncARSR (Fig. 1h) or nuclear YAP in ccRCC predicts a poor prognosis (Fig. 8d), ccRCC patients with both elevated lncARSR level and nuclear YAP expression displayed even worse prognosis (Fig. 8e), indicating a better prognostic value of combining the two parameters in comparison with lncARSR or YAP alone.
In aggregate, our results unravelled that lncARSR-promoted renal T-ICs propagation via impeding LATS1YAP interaction to facilitate YAP nuclear translocation, which reciprocally enhanced lncARSR transcription, thus forming a feed-forward circuit in renal T-ICs (Fig. 9).
DiscussionMost cancer therapies fail to eradicate tumours due to the existence of T-ICs. However, the understanding of regulatory mechanisms for T-ICs is limited. In this study, we elaborated the critical role of lncARSR in renal T-ICs and the underlying mechanism. We also demonstrated the value of combining lncARSR and YAP to improve the prognostic accuracy for RCC patients. To our knowledge, this is the rst report for lncRNA in the regulation of renal T-ICs.
Accumulating evidence shows that lncRNAs could serve as molecular scaffolds to facilitate multiple proteins interaction, such as lncRNA-NEAT1, which is an essential molecular scaffold for the formation of paraspeckles46, but lncRNA per se have rarely been reported to exhibit direct regulatory effect. In this study, we found that lncARSR, unlike scaffold lncRNAs, bound specically to the WW1/2 domain of YAP, thus protecting YAP from interaction and phosphorylation by LATS1. Our data characterized lncARSR as a direct signal transducer through acting on the functional domains of signalling protein.
The Hippo signalling pathway is an important kinase cascade in the regulation of organ size control and homeostasis. The disruption of Hippo pathway can lead to tumorigenesis47. Gene expression signature denoting YAP/TAZ activity has been tightly linked with stem cell signatures in breast cancer48. Moreover, YAP is required to sustain self-renewal and tumour initiation of T-ICs in various cancers37,38. However, targeting core Hippo cascade is presently frustrating, because upstream kinases are negative regulators of YAP, making it incapable to target. Herein, our study demonstrates that inhibition of lncARSR impairs YAP activity and attenuates renal T-ICs propagation. Intriguingly, apart from Hippo pathway wild-type cells as used in this study, we found that lncARSR could also enhance the expansion of T-ICs in Hippo pathway mutant cells, such as
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a b
Blot : Flag
IP: YAP LATS1 IgG Input
+
+
+
+ +
+
+
+ + kDa
170 130 72
34
+
Input IncARSR pull-down
TEAD-BD WW1/2
154
SH3-BD
Transactivationdomain PDZ-BD
1
1
1
1
504
504
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499
Flag vector YAP 1504 YAP 1154 YAP 155504
YAP 1263
1154
155504
1263
264504
1-287
288-504
1-499
1504
1154
155504
1263
264504
1287
288504
1499
1 155
504
kDa
1504
263
287
288
YAP 264504
YAP 1287 YAP 288504
IP: YAP LATS1 IgG Input
+
+
+
+ +
+
+
+ + kDa
170 130 72
34
+
95
72
55
43
34
26
264
YAP 1499
0 5 10 15Fold enrichment (Flag/IgG)
20
c
d
Ctrl siRNA IncARSR siRNA-1 IncARSR siRNA-2
LATS1
YAP GAPDH
Ctrl IncARSR (1310)
IncARSR LATS1
YAP
GAPDH
10
8
Relative
+
+
+
+
A498
spheres
A498
e h
LATS1 IncARSR
+ + + + + + + + + ++ + + + + + + kDa
72
72
72
** **
**
*** *
IncARSR (1310) IncARSR (282591)
YAP p-YAPS127
p-YAPS381
YAP
luciferase activity
6 4 2 0
+
Vector
IncARSR (1310)
IncARSR
IncARSR
YAP luc
f
g
IncARSR
YAP
+DAPI
YAP
+DAPI
shGFP shIncARSR-2 shIncARSR-1
Ctrl IncARSR (1310) IncARSR
i j
A498 spheres
A498 spheres
shIncARSR-1 shIncARSR-2
shGFP shIncARSR-1 shIncARSR-2
shGFP
Oct41.5
1.0
0.5
Sox2
NS 200
150
100
Number of
** NS
spheres per 1,000 cells
50
0
NS
Relative mRNA
expression
siRNA
**
**
**
Ctrl siRNA LATS siRNA
LATS siRNA
**
LATS
siRNA
0.0
Ctrl siRNA
Ctrl siRNA
Ctrl
LATS siRNA
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LNA-based in situ hybridization. LNA ISH was performed by using miRCURY LNA microRNA ISH Optimization Kit (Exiqon, Denmark) as previously reported51 with minor modications. Briey, the sections were deparafnized and then deproteinated using proteinase K (15 mg ml 1, Roche) for 10 min at 37 C.
The endogenous peroxidases were inactivated in 1% H2O2 for 30 min, and sections were pre-hybridized at 62 C for 30 min in formamide-free Exiqon ISH buffer (Exiqon, Denmark) and hybridized with DIG-labelled LNA probes for lncARSR (50-AGGTTGTCTGAAGTTGGAGTT-30, 50 nM, Exiqon, Denmark) at 62 C overnight. Slides are then stringently washed, incubated with alkaline phosphataseconjugated anti-DIG Fab fragments (Roche, USA) for 60 min and then detected by NBT/BCIP reagent (Invitrogen, USA). Sections were lastly counterstained with nuclear fast red staining solution (Sigma Chemical Co, USA). High-resolution images were captured with an Aperio Scan Scope AT Turbo (Aperio, USA) equipped with Aperio Image Scope software (Aperio, USA). Assessment of the staining was based on the staining intensity and the percentage of positively stained cells using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., USA). The median signal of lncARSR positive staining was dened as cutoff value.
Fluorescence in situ hybridization. FISH was performed as previously described52. The ISH signals were detected using the tyramide signal amplication system (PerkinElmer, USA) and analysed with a uorescence microscope (IX70, Olympus, Japan).
Immunocytochemistry. RCC cells were plated in 12-well plates at 30% conuence and allowed to grow for 24 h. Then, the cells were xed with 10% paraformalde-hyde solution for 15 min at room temperature, permeabilized with 0.4% Triton X-100 in PBS for 5 min, and then blocked with 1% BSA in PBS for 1 h at 37 C. The blocked cells were incubated with anti-FLAG antibody (1:100, Merck Millipore) and anti-YAP antibody (1:100, Cell Signaling Technology) overnight at 4 C, followed by incubation with Alexa Fluor 488-conjugated anti-mouse IgG antibody and Alexa Fluor 555-conjugated anti-rabbit IgG antibody (1:100, Invitrogen, Carlsbad, CA) for 2 h. Nuclear staining of cells was conducted using 4,6-diamidino-2-phenylindole (DAPI). Representative images were acquired using an uorescence microscope (IX70, Olympus, Japan).
Immunohistochemistry. Parafn-embedded sections were deparafnized and rehydrated, followed by antigen retrieval. After primary and secondary antibody incubation, the slides were incubated with diaminobenzidine (DAB) (Dako, USA) and counterstained with hematoxylin (Sigma Chemical Co, USA).
Immunohistochemistry of YAP 1:100, (Cell Signaling Technology) was scored by determining the percentage of positive tumour cells (o25% focal, 2550%
moderate and 51100% diffuse), and their staining intensity (0 negative, 1 weak,
2 moderate and 3 strong, see Supplementary Fig. 7d). Nuclear YAP samples
were dened by at least moderate (2 ) nuclear staining in Z25% of tumour cells.
Cytoplasmic YAP samples were dened by no visible nuclear staining or weak (1 ) nuclear staining.
Plasmids construction. The cDNA of lncARSR was amplied by using primeSTAR HS DNA polymerase (Takara, China) and subcloned into the KpnI and XhoI sites of pcDNA3.1 vector, and termed pcDNA3.1-lncARSR. The full-length lncARSR, 5030 (1310) of lncARSR, and 5030 (282591) of lncARSR were constructed by PCR-based amplication from pcDNA3.1-lncARSR plasmid and subcloned into pSPT19 vector, and termed pSPT19-lncARSR, pSPT19-lncARSR (1310) or pSPT19-lncARSR (282591), respectively. pLenti-lncARSR were constructed by PCR-based amplication from pcDNA3.1-lncARSR plasmid and subcloned into pLenti-III vector (Invitrogen, USA). lncARSR-specic shRNA oligos were synthesized by Sangon Co. Ltd (Shanghai). After annealing, double-strand oligos were inserted to lentiviral pLKO.1-Puro vector (Addgene). To produce lentivirus, HEK-293T cells were co-transfected with the lentiviral vector
Figure 6 | lncARSR blocks LATS1-mediated YAP phosphorylation. (a) Left: schematic diagram of different fragments of YAP was shown. Right: RIP assay of the enrichment of different YAP fragments on lncARSR relative to IgG in A498 spheres transfected with different fragments of YAP plasmids (n 3).
BD, binding domain. (b) Western blot analysis of Flag in whole lysates of A498 spheres (input, left) and RNA pull-down precipitates retrieved by biotin-labelled lncARSR from A498 spheres transfected with different fragments of YAP-Flag plasmids. (c) Coimmunoprecipitation of YAP and LATS1 in lysates of lncARSR-knockdown and control A498 spheres. GAPDH acted as a loading control. (d) Coimmunoprecipitation of YAP and LATS1 in lysates of A498 cells with lncARSR overexpression or 50 segment (nucleotides 1310) overexpression. GAPDH acted as a loading control. (e) In vitro phosphorylation assay with puried active LATS1 showing the effects of different fragments of lncARSR on LATS1-induced YAP phosphorylation. (f) RNA FISH analysis of lncARSR and immunouorescence detection of YAP in lncARSR-knockdown and control A498 spheres. Scale bar, 20 mm. (g) RNA FISH analysis of lncARSR and immunouorescence detection of YAP in A498 cells with lncARSR or 50 segment (nucleotides 1310) overexpression. Scale bar, 20 mm. (h) A498 cells were transfected with YAP/TAZ luciferase reporter plasmid (YAP/TAZ luc), together with increasing concentrations of indicated plasmids, and subjected to luciferase reporter assay (n 3). Data were normalized against Renilla luciferase activity and represented as means.d.; *Po0.05 and **Po0.01; two-
tailed Students t-test. (i) qRTPCR analysis of Oct4 and Sox2 in A498 spheres transfected with indicated siRNA and plasmid after 48 h (n 3). (j) Spheres
formation assay of A498 spheres transfected with indicated siRNA and plasmid (n 3). The number of spheres was counted after 7 days (left).
Representative images of spheres are shown (right). Scale bar, 200 mm. (i,j) Data are represented as means.d.; **Po0.01; two-tailed Students t-test.
ACHN cells (data not show), indicating the involvement of other mechanisms. Thus, lncARSR might serve as a potential therapeutic target for RCCs with aberrant YAP activity in wild-type Hippo signalling.
YAP expression or nuclear accumulation has been reported to correlate with poor patient outcome in several types of cancer49. Consistently, our results showed that either high lncARSR expression or nuclear YAP enrichment correlated with poor prognosis of RCC patients. Accumulating evidence indicates that appropriate combination of different markers might be more accurate than a single marker in prognosis evaluation. Herein, we reported that the combination of high lncARSR expression and nuclear YAP accumulation predicted worse prognosis than either marker alone, suggesting a more accurate combinational marker to evaluate the prognosis of RCC patients.
In conclusion, our ndings provide insight into the lncARSR/ YAP axis as potential therapeutic target against renal T-ICs and powerful predictor for poor prognosis of RCC patients.
Methods
Cell lines and reagents. The human RCC cell lines (A498, 769P, Ketr-3, Caki-1, Caki-2) and human embryonic kidney (HEK-293T) cells were purchased from ATCC (Rockville, MD, USA) and cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA). The primary ccRCC cell line 771 was established from a ccRCC patient and cultured in DMEM containing 10% fetal bovine serum. EGF and bFGF were purchased from Peprotech (USA), B27 and insulin was purchased from Invitrogen (USA) and Verteporn was purchased from R&D (#5305, USA).
RCC patients and clinical specimens. All patient samples were collected from the Department of Urology, Changzheng Hospital with written informed consent. The ethical approval was granted from Committees for Ethical Review in Second Military Medical University. This study involved two cohorts of ccRCC patients, cohort 1, 105 patients and cohort 2, 205 patients. All patients received no previous therapy and were followed until May 2015. All RCC samples are clear cell RCC which has been diagnosed by two pathologists, blinded to the clinical data. lncARSR levels in ccRCC and adjacent tissues were determined by qRTPCR in cohort 1 and by locked nucleic acid (LNA) ISH on tissue microarray slides in cohort 2.
In vivo xenograft. All experiments involving mice were undertaken in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and the approval of the Institutional Animal Care and Use Committee (IACUC) at Second Military Medical University. Four- to six-week-old male athymic BALB/c nude mice (SIPPR-BK Experimental Animal Co. China) were housed and fed in standard pathogen-free conditions.
For in vivo limiting dilution assay, RCC cells were mixed with Matrigel (BD) at a ratio of 1:1 and injected subcutaneously at various cell doses per mouse. Kinetic of tumour formation was evaluated per week for 8 weeks. Frequency of T-ICs was determined using ELDA software (http://bioinf.wehi.edu.au/software/elda/index.html
Web End =http://bioinf.wehi.edu.au/software/elda/ http://bioinf.wehi.edu.au/software/elda/index.html
Web End =index.html ) provided by the Walter and Eliza Hall Institute50.
For lung metastasis model, 2 106 single cells were injected into the tail vein of
nude mice. Mice were killed 12 weeks after inoculation and consecutive sections of the whole lung were subjected to haematoxylin-eosin staining. All of the metastatic lesions in lung were calculated microscopically to evaluate the development of pulmonary metastasis.
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A498-sph A498
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*** ******
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*
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2 0
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TB1 TB2 Neg IncARSR 2,000 1,500 1,000 500 TSS 500 (bps)
A498 771 Adherent Spheres
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TB2
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TB2
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TB1
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TB2 Neg
TB1 TB2 Neg
Figure 7 | YAP/TEAD complex transactivates lncARSR. (a) qRTPCR analysis of YAP and lncARSR in RCC spheres transfected with shYAP or shGFP plasmid after 48 h (n 3). (b) qRTPCR analysis of YAP and lncARSR in RCC cells transfected with YAP-5SA plasmid or YAP-5SADC plasmid after 48 h
(n 3). (c) qRTPCR analysis of YAP and lncARSR in A498 cells transfected with indicated plasmids or treated with Verteporn (1 mM) for 48 h (n 3).
(d) qRTPCR analysis of lncARSR in YAP-5SA-overexpressing A498 cells transfected with indicated siRNA after 48 h (n 3). (e) Upper: putative TEAD-
binding sites on the promoter region of lncARSR. Lower: ChIP assay of the enrichment of YAP on lncARSR promoter relative to IgG in RCC adherent and sphere cells (n 3). A random region without TB sites acted as a negative control (Neg). (f) ChIPre-ChIP assay was performed using anti-Flag-YAP
antibody rst (Flag-YAP). The eluants were then subjected to a second ChIP assay using anti-TEAD1 antibody (Flag-YAP TEAD1) or control IgG antibody
(Flag-YAP IgG) (n 3). (g) ChIP-re-ChIP assay was performed using anti-TEAD1 antibody rst (TEAD1). The eluants were then subjected to a second
ChIP assay using anti-YAP antibody (TEAD1 YAP) or control IgG antibody (TEAD1 IgG) (n 3). (ag) Data are represented as means.d.; *Po0.05,
**Po0.01 and ***Po0.001; two-tailed Students t-test.
described above and packaging vectors psPAX2 and VSVG using jetPEI (PolyPlus Transfection, France).
The plasmid ag-YAP was kindly provided by professor Fen-yong Sun (Department of Clinical Laboratory Medicine, Shanghai Tenth Peoples Hospital of Tongji University, Shanghai, China). The plasmids YAP luciferase reporter (8xGTIIC-luciferase, #34615), pCMV-ag-YAP-5SA (#27371), pCMV-ag-YAP-5SA/S94A (#33103), pLKO. 1-shYAP (#42540), pcDNA-HA-MST2 (#33098) and pcDNA-Lats1 (#41156) were purchased from Addgene. The YAP truncation mutants were constructed by PCR-based amplication from ag-YAP plasmid.
All constructs were conrmed by DNA sequencing. Sequences of primers used for plasmid construction in this study were listed in Supplementary Table 12.
Cell transfection. Transfection of plasmids was performed by using jetPEI (PolyPlus Transfection, France). Transfection of siRNA (100 nM, GenePharma, China) was performed by using Lipofectamine RNAiMAX (Invitrogen, USA). Sequences of siRNA against specic targets were listed in Supplementary Table 13.
RNA isolation and RTPCR analysis. Total RNA was extracted by TRIzol (Invitrogen, USA). Real-time quantitative PCR was performed on triplicate samples in a reaction mix of SYBR Green (Takara, China) by ABI 7900HT Fast Real-Time
PCR System (Applied Biosystems, USA). The expression of indicated genes was normalized to endogenous reference control b-actin by using 2 DDCt method.
Sequences of primers used for qRTPCR in this study were listed in Supplementary
Table 14.
RNA pull-down. RNA pull-down was performed as previously described14,53. Briey, biotin-labelled RNA was transcribed in vitro using Biotin RNA Labelling Mix (Roche, USA) and T7 or SP6 RNA polymerase (Promega, USA), respectively, treated with RNase-free DNase I (Roche, USA) and then puried with RNeasy Mini Kit (Qiagen, Germany). Biotinylated RNA was incubated with cytoplasmic extract of RCC cells at room temperature for 1 h. Washed streptavidin agarose beads (Invitrogen, USA) were added to each binding reaction and incubated at room temperature for 1 h. Precipitates were washed for three times, boiled in SDS buffer and subjected to SDSpolyacrylamide gel electrophoresis. Specic bands were excised and analysed by mass spectrometry or subjected to western blot detection.
Western blot. Cell lysates or retrieved proteins were analysed by immunoblot with primary antibodies and IRDye 800 CW-conjugated secondary antibody (Rockland Immunochemicals, USA). The intensity of the uorescence was scanned
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a
Pearson R=0.496
P<0.001
Pearson R=0.505
P<0.001
16.00
14.00
Pearson R=0.547
P<0.001
17.50
15.00
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CTGF (Ct)
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BIRC5 (Ct)
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8.00
10.00
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IncARSR (Ct)
IncARSR (Ct)
IncARSR (Ct)
Patient #20
b c
Patient #31 Patient #7
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Cyto-YAP Nuc-YAP
IncARSR YAP
Percentage (%)
80
40
0
Low IncARSR
High IncARSR
d
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0.8
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Overall survival
Cyto YAP
n=103
Nuc YAP
n=102
Cum recurrence rate
0.2
0.0
Nuc YAP n=102
0.4
0.4
0.2
0.0
0.00 20.00 40.00Time after surgery (months)
60.00 80.00 100.00
1.0
0.8
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Cyto YAP
n=103
e
1.0
0.8
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0.00 20.00 40.00Time after surgery (months)
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Figure 8 | Combining lncARSR and YAP exhibits improved prognostic value. (a) The correlation between the transcription level of lncARSR and CTGF (left), BIRC5 (middle) and CYR61 (right) in human ccRCC tissues (n 52) was determined by qRTPCR analysis. Data were normalized to b-actin as DCt
and analysed by Spearmans correlation analysis. (b) Representative images of lncARSR ISH and YAP immunohistochemistry using consecutive sections of human ccRCC tissues. Scale bar, 50 mm. (c) Relative percentages of nuclear and cytoplasmic YAP in high (62.7%) and low (23.3%) lncARSR groups in cohort 2 (Po0.001). (d) KaplanMeier analysis of overall survival (left, P 0.005, log-rank test) or recurrence rate (right, P 0.002, log-rank test) of
ccRCC patients with cytoplasmic or nuclear YAP in cohort 2. (e) KaplanMeier analysis of overall survival (left, lncARSRhighYAPhigh versus
lncARSRhighYAPlow, P 0.039, log-rank test; lncARSRlowYAPhigh versus lncARSRlowYAPlow, P 0.093, log-rank test) or recurrence rate (right,
lncARSRhighYAPhigh versus lncARSRhighYAPlow, P 0.014, log-rank test; lncARSRlowYAPhigh versus lncARSRlowYAPlow, P 0.173, log-rank test) of ccRCC
patients with high or low lncARSR and cytoplasmic or nuclear YAP.
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12692 ARTICLE
Low IncARSR High IncARSR
YAP
YAP LATS
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Figure 9 | Schematic diagram of lncARSRYAP signaling circuit in renal T-ICs. lncARSR overexpression impedes LATS-YAP interaction to facilitate the nuclear translocation of YAP, which in turn transactivates lncARSR expression, forming a feed-forward loop to promote the expansion of renal T-ICs.
by Odyssey uorescence scanner system (Li-Cor Biosciences, USA). Primary antibodies used in this study are listed in Supplementary Table 15.
RNA immunoprecipitation. RIP was conducted by EZ-Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA). The RNA fraction precipitated by RIP was analysed by real-time quantitative PCR. Total RNAs (input) and IgG controls were assayed simultaneously to demonstrate that the detected signals were the result of RNAs specically binding to indicated antibody.
Chromatin immunoprecipitation. ChIP was conducted by using EZ-ChIP Kit (Millipore, USA). ChIPre-ChIP was conducted as previously described54. Pooled eluants were diluted to a nal SDS concentration of 0.1% and incubated with fresh antibody-bound beads for the second immunoprecipitation. Fold enrichment was quantied using quantitative RTPCR and calculated as a percentage of input chromatin (% Input). Sequences of primers used for ChIPquantitative PCR (qPCR) in this study were listed in Supplementary Table 16.
Electrophoretic mobility shift assay. EMSA was performed using a LightShift Chemiluminescent RNA EMSA Kit (#20158, Thermo Scientic, USA) according to manufacturers instruction. The biotin-labelled RNA transcripts were in vitro transcribed with the Biotin RNA Labeling Mix as described in RNA pull-down assay. The human YAP recombinant protein was purchased from Abcam (ab#132459).
Luciferase reporter assay. A498 cells were co-transfected with YAP/TAZ luciferase reporter and pcDNA3.1-ARSR using Lipofectamine 2000 (Invitrogen). Each group was run in triplicate in 48-well plates. The luciferase activity was detected by Synergy 2 Multidetection Microplate Reader (BioTek Instruments, Inc.) after 48 h of transfection. Renilla luciferase activity was normalized against Firey luciferase activity.
In vitro phosphorylation assay. A498 cells were co-transfected with plasmids MYC-LATS1 and HA-MST2. After 48 h of transfection, cells were lysed with IP buffer and immunoprecipitated with anti-MYC antibody. The immmunoprecipitates were washed three times with IP buffer, and then washed once with kinase buffer (20 mM Tris-HCl (pH 7.5), 15 mM MgCl2, 5 mM EGTA, 100 mM NaCl and 1 mM DTT). One microgram of recombinant human YAP was incubated with the immunoprecipitated LATS1 and biotin-labelled RNA in 25 ml kinase buffer containing 100 mM ATP at 30 C for 1 h with gentle shaking. The reactions were terminated with SDS-loading buffer and YAP phosphorylation was detected by western blot.
Spheres formation assay. One thousand single cells were seeded into 96-well Ultra-Low Attachment Microplates (Corning, USA) in serum-free DMEM/F12 (Invitrogen, USA), supplemented with B27 (1:50, Invitrogen), 20 ng ml 1 EGF (Peprotech), 10 ng ml 1 bFGF (Invitrogen), and 4 mg ml 1 insulin (Sigma)55.
Spheres were photographed and counted 7 days after seeding (primary spheres). To propagate spheres in vitro, spheres were collected by centrifugation and trypsinized with 0.25% trypsin to obtain single cell, and equal number of cells were then seeded into ultra-low attachment plate (secondary spheres).
In vitro limiting dilution assay. RCC cells were seeded into 96-well Ultra-Low Attachment Microplates (Corning, USA) at various cell doses and incubated under spheres forming conditions for 7 days. Based on the frequency of wells with spheres forming, the proportion of tumour-initiating cells was determined using Poisson distribution statistics and the LCalc Version 1.1 software program (Stem Cell Technologies, Inc. Vancouver, Canada).
Flow cytometry. RCC cells were collected and washed with PBS. RCC cells were incubated with indicated antibody or isotype control antibody for 30 min on ice in the dark. Samples were analysed by FACS apparatus MoFlo XDP (Beckman Coulter, USA).
Data analysis. All statistical analyses in this study were performed with SPSS 16.0 software (SPSS Inc., USA). Data were presented as means.d.. The signicance of mean values between two groups was analysed by two-tailed Students t-test. Spearmans correlation analysis was performed to determine the correlation between two variables. Pearson chi-square test acted to analyse the clinical variables. KaplanMeier survival analysis was utilized to compare ccRCC patient survival based on dichotomized lncARSR expression by log-rank test. Cox proportional hazards regression analyses were utilized to analyse the effect of clinical variables on patient survival. A P value o0.05 was considered signicant.
Data availability. The gene expression proling of generated sunitinib-resistant RCC cell lines have been deposited in GEO with the accession code GSE69535. The mass spectrum data that support the ndings of this study are included in Supplementary Data 1. The authors declare that all other relevant data supporting the ndings of this study are available on request.
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Acknowledgements
We thank Fen-yong Sun (Department of Clinical Laboratory Medicine, Shanghai Tenth Peoples Hospital of Tongji University, Shanghai, China) for supporting the plasmid Flag-YAP, Jin Hou (National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, Shanghai, China) for his valuable comments and expert technical support. This work was supported by grants from the National Natural Science Foundation of China (81272817), the Leading Talent Project of Shanghai (2013046) and the Youth Talent Sailing Program of Shanghai Science and Technology Committee (16YF1403600).
Author contributions
L.Q. and C.C. performed the majority of the experiments; Z.W., B.L., X.C. and Z.Z. provided clinical samples; Y.L. and Z.X. performed the in vivo experiments; F.L., Z.X., Y.B. and D.W. provided technical support; J.L. and A.W. performed experiments on tissue microarrays of ccRCC patient samples; L.Q. and L.W. contributed to study concept and design; L.Q. and C.C. drafted the manuscript; L.W. and Y. S. contributed to study supervision.
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How to cite this article: Qu, L. et al. A feed-forward loop between lncARSR and YAP activity promotes expansion of renal tumour-initiating cells. Nat. Commun. 7:12692 doi: 10.1038/ncomms12692 (2016).
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Abstract
Renal tumour-initiating cells (T-ICs) contribute to tumorigenesis, progression and drug resistance of renal cell carcinoma (RCC). However, the underlying mechanism for the propagation of renal T-ICs remains unclear. Here we show that long non-coding RNA lncARSR is upregulated in primary renal T-ICs and associated with a poor prognosis of clear cell RCCs (ccRCC). Knockdown of lncARSR attenuates the self-renewal, tumorigenicity and metastasis of renal T-ICs. Conversely, forced lncARSR expression enhances T-IC properties of RCC cells. Mechanistically, the binding of lncARSR to YAP impedes LATS1-induced YAP phosphorylation and facilitates YAP nuclear translocation. Reciprocally, YAP/TEAD promotes lncARSR transcription, thus forming a feed-forward circuit. The correlation between lncARSR and YAP is validated in a ccRCC cohort, where the combination of these two parameters exhibits improved prognostic accuracy. Our findings indicate that lncARSR plays a critical role in renal T-ICs propagation and may serve as a prognostic biomarker and potential therapeutic target.
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