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A ROR1–HER3–lncRNA signalling axis modulates the Hippo–YAP pathway to regulate bone metastasis

Nature Cell Biology volume 19, pages 106–119 (2017)Cite this article

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Abstract

Bone metastases remain a serious health concern because of limited therapeutic options. Here, we report that crosstalk between ROR1–HER3 and the Hippo–YAP pathway promotes breast cancer bone metastasis in a long noncoding RNA-dependent fashion. Mechanistically, the orphan receptor tyrosine kinase ROR1 phosphorylates HER3 at a previously unidentified site Tyr1307, following neuregulin stimulation, independently of other ErbB family members. p-HER3 Tyr1307 recruits the LLGL2–MAYA–NSUN6 RNA–protein complex to methylate Hippo/MST1 at Lys59. This methylation leads to MST1 inactivation and activation of YAP target genes in tumour cells, which elicits osteoclast differentiation and bone metastasis. Furthermore, increased ROR1, p-HER3 Tyr1307 and MAYA levels correlate with tumour metastasis and unfavourable outcomes. Our data provide insights into the mechanistic regulation and linkage of the ROR1–HER3 and Hippo–YAP pathway in a cancer-specific context, and also imply valuable therapeutic targets for bone metastasis and possible therapy-resistant tumours.

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Figure 1: ROR1 promotes the colonization and growth of breast cancer cells within the bone.
Figure 2: ROR1-dependent phosphorylation of HER3 at Tyr1307 correlates with breast cancer clinical parameters.
Figure 3: Crosstalk between ROR1–HER3 and the Hippo–YAP pathway.
Figure 4: LncRNA MAYA is required for activation of YAP.
Figure 5: Characterization of MAYA–LLGL2–NSUN6 associations.
Figure 6: LncRNA-mediated, NSUN6-dependent methylation inhibits the kinase activity of MST1.
Figure 7: The ROR1/HER3–LLGL2/MAYA/NSUN6 signalling axis regulates YAP activity.
Figure 8: MAYA serves as a promising therapeutic target for bone metastasis.

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Acknowledgements

We are grateful to J. Massague and X. Zhang for providing the MDA-MB-231 LM2 and BoM-1833 cell lines and to D. Yu for providing the MDA-MB-231-BRN and BT474-BRN cells. We thank D. Aten for assistance with figure presentation. This work was supported by National Institutes of Health Pathway to Independence Award (R00CA166527) and Cancer Prevention Research Institute of Texas First-time Faculty Recruitment Award (R1218) grants to L.Q.Y. and National Institutes of Health Pathway to Independence Award (R00DK094981) to C.R.L.

Author information

Author notes

  1. Shouyu Wang & Aifu Lin

    Present address: Present addresses: Department of Molecular Cell Biology and Toxicology, School of Public Health, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China (S.W.); College of Life Sciences, Zhejiang University, Hangzhou 310058, China (A.L.).,

  2. Chunlai Li and Shouyu Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA

    Chunlai Li, Shouyu Wang, Zhen Xing, Aifu Lin, Ke Liang, Qingsong Hu, Jun Yao, Peter K. Park, Mien-Chie Hung, Chunru Lin & Liuqing Yang

  2. Department of Genitourinary Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA

    Jian Song & Gary E. Gallick

  3. Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA

    Zhongyuan Chen & Han Liang

  4. Department of Statistics, Rice University, Houston, Texas 77030, USA

    Zhongyuan Chen

  5. Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA

    David H. Hawke & Han Liang

  6. Department of Molecular Cell Biology and Toxicology, School of Public Health, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China

    Jianwei Zhou

  7. Department of Oncology, Yixing People’s Hospital, 75 Zhenguan Road, Yixing 214200, China

    Yan Zhou

  8. Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA

    Shuxing Zhang

  9. The Graduate School of Biomedical Sciences, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA

    Mien-Chie Hung, Chunru Lin & Liuqing Yang

  10. Center for Molecular Medicine and Graduate Institute of Cancer Biology, China Medical University, Taichung 404, Taiwan

    Mien-Chie Hung

  11. Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston McGovern Medical School, Houston, Texas 77030, USA

    Leng Han

  12. Center for RNA Interference and Non-Coding RNAs, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA

    Liuqing Yang

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  1. Chunlai Li
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Contributions

C.L.L. and S.W. devised and performed most experiments. K.L., J.S. and G.E.G. helped with mouse intracardiac injections. A.L., Z.X. and Q.H. helped with biochemistry studies. D.H.H. performed mass spectrometry analysis. Clinical specimens were ascertained and processed by J.Z. and Y.Z. The histological staining and corresponding analysis were performed by K.L. P.K.P. assisted with manuscript drafting. J.Y., L.H., Z.C. and H.L. performed bioinformatics analysis. S.Z. and M.-C.H. contributed to discussion and data interpretation. L.Y. and C.R.L. initiated and supervised the project and wrote the paper with input from all authors.

Corresponding authors

Correspondence to Chunru Lin or Liuqing Yang.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 ROR1 promotes proliferation and mobility of breast cancer cells.

(a) Oncomine box plot showing ROR1 copy numbers in human ERPR-/HER2- triple negative breast cancer (TNBC) and non-TNBC subtypes (n = 73 and 495 tumors respectively, one-way ANOVA). (b) Oncomine box plots showing ROR1 expression levels in a series of human TNBC (n = 211, 178, 46, 26, 22 and 39 tumors, one-way ANOVA) and non-TNBC subtypes (n = 1,340, 320, 250, 89, 44 and 129 tumors, one-way ANOVA). (c) Oncomine box plots showing ROR1 expression levels in human Non-small Cell Lung Carcinoma (n = 33, 63, 10, 26, and 132 tumors, one-way ANOVA) and Small cell lung carcinoma (n = 9 and 6 tumors, one-way ANOVA) or squamous cell lung carcinoma cohorts (n = 154, 75, 10, 26, and 21 tumors, one-way ANOVA). (d and e) Immunoblotting (IB) of indicated proteins in parental/ROR1 KO MDA-MB-231 (d) and BoM-1833 (e) single cell clones. (f and g) Cell proliferation assay of individual (f) and pooled (g) clones of ROR1 KO MDA-MB-231 cells. (h) IB detection of indicated proteins in ROR1 KO cells expressing WT ROR1 or K506A mutant. (i) Cell invasion assay of ROR1 KO MDA-MB-231 cells with expression of indicated plasmid (left, Scale bars, 200 μm; right, quantification). (j and k) Cell migration (j) and invasion (k) assay of pooled clones of ROR1 KO MDA-MB-231 cells with overexpression of indicated plasmid (Scale bars, 200 μm). (l) TRAP staining showing the osteoclast differentiation in the presence of M-CSF only, M-CSF + RANKL, or combined M-CSF + RANKL and conditioned media (CM) from ROR1 KO BoM-1833 cells or CM from ROR1 KO BoM-1833 cells KO supplemented with PBS or recombinant CTGF (50 ng ml−1) (Scale bars, 200 μm). For a-c, the boxes show the median ± 1 quartile, with whiskers extending to the most extreme data point within 1.5 interquartile ranges from the box boundaries. For f, g and i-k, mean ± s.e.m. were derived from n = 3 independent experiments (n.s., P > 0.05, ∗P < 0.05 and ∗∗P < 0.01, ∗∗∗P < 0.001, two-tailed paired Student’s t-test). Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9 Statistics source data for i-l are in Supplementary Table 8.

Supplementary Figure 2 Identification of ROR1-associated proteins, characterization of ROR1 phosphorylation and examination of the role of p-HER3 (Tyr1307) in breast and lung cancer.

(a) A list of top ROR1-associated proteins identified by protein pull-down and MS in MDA-MB-231 cells. (b) IB detection of p-HER3 (Tyr1307) in MDA-MB-231 cells treated with or without NRG1 using antibodies pre-incubated with indicated blocking peptides. Antibodies generated from two independent rabbits were tested. (c and d) Immunoprecipitation (IP) and IB detection of indicated proteins in MDA-MB-231 cells transfected with indicated plasmids (c) or treated with indicated small compound inhibitors (d) followed by NRG1 treatment. (e) IP and IB detection of indicated proteins in 32D cells transfected with indicated expression vectors followed by NRG1 stimulation. (f) IB detection of indicated proteins in parental/HER3 KO MDA-MB-231 single cell clones. (g and h) Cell proliferation assay of individual (g) and pooled (h) clones of HER3 KO MDA-MB-231 cells. (i) Cell invasion assay of individual clones of HER3 KO MDA-MB-231 cells with overexpression of indicated plasmid (left, Scale bars, 200 μm; right, quantification). (j and k) Cell migration (j) and invasion (k) assay of pooled clones of HER3 KO MDA-MB-231 cells (left, Scale bars, 200 μm; right, quantification). (l) Immunohistochemical (IHC) staining of p-HER3 (Tyr1307) in normal lung tissues, lung adenocarcinomas, non-metastatic (TnN0M0) and metastatic (TnN > 0M ≥ 0) lung adenocarcinomas (left, Scale bars, 40 μm; right, n = 75, 75, 43 and 32 lung tissues/adenocarcinomas respectively, median, one-way ANOVA) (m) Kaplan-Meier survival analysis of p-HER3 (Tyr1307) low and high lung adenocarcinoma patients (n = 20 and 55 patients respectively, log rank test). For g–k, mean ± s.e.m. were derived from n = 3 independent experiments (∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001, two-tailed paired Student’s t-test). Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9 Statistics source data for i-l are in Supplementary Table 8.

Supplementary Figure 3 Identification and characterization of p-LLGL2 (Tyr499) and MST1 (Lys59me2).

(a) Summary of top LLGL2-associated proteins identified by LLGL2 pull-down followed by MS in MDA-MB-231 cells. (b and e) Annotated MS/MS spectrum assigned to the LLGL2 peptide VGSFDP[p]YSDDPR, at 717.782 Da. (b) and the STK4/MST1 peptide ETGQIVAI[dime] KQVPVESDLQEIIK, at 1233.200 Da. (e). Data acquired from analysis of the tryptic digest by high-sensitivity LC-MS/MS on an Orbitrap Elite high-resolution mass spectrometer. (c and f) IB detection of p-LLGL2 (Tyr499) (c) and MST1 (Lys59me2) (f) in MDA-MB-231 cells treated with or without NRG1, using antibodies pre-incubated with indicated blocking peptides. Antibodies generated from two independent rabbits were tested. (d) IB detection of indicated proteins in cells transfected with indicated expression vectors followed by NRG1 stimulation. Scanning densitometric analysis was performed for p-LLGL2 (Tyr499) blot. (g) IB detection of indicated proteins in MDA-MB-231 cells treated with LPA or NRG1. Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9.

Supplementary Figure 4 Identification of MAYA-associated proteins, characterization of MAYA transcript and its correlation with lung cancer.

(a) Summary of the top MAYA-associated proteins identified by RNA pull-down followed by MS in MDA-MB-231 cells. (b) Determination of MAYA transcript in MDA-MB-231 cells by 5′-and 3′-RACE. R1: repeat 1, R2: repeat 2. (c) Top, northern blot detection of MAYA in pre-made human tissue RNA blots; bottom, RNA gel electrophoresis. (d) Top, northern blot detection of MAYA in MDA-MB-231 cells with indicated treatment; bottom, RNA gel electrophoresis. (e) Examination of MAYA expression in multiple tumor types and their normal counterparts by RNAscope assay. (f) MAYA RNAscope staining intensities in normal lung tissues, lung adenocarcinomas, non-metastatic (TnN0M0) and metastatic (TnN > 0M ≥ 0) lung adenocarcinomas (n = 75, 75, 50 and 25 lung tissues/adenocarcinomas respectively, median, one-way ANOVA) (g) Kaplan-Meier survival analysis of MAYA low and high lung cancer patients (n = 21 and 54 patients respectively, log rank test). (h and i) RT-qPCR detection of MAYA and CTGF expression level in MCF-7 cells transfected with the indicated siRNAs that grown under spare or confluent culture conditions (h) or followed by lysophosphatidic acid (LPA) treatment (1 μM, 2 h) (i). For h and i, mean ± s.e.m. were derived from n = 3 independent experiments (n.s., P > 0.05 and∗∗P < 0.01, two-tailed paired Student’s t-test). Unprocessed original scans of all blots/gels with size marker are shown in Supplementary Fig. 9.

Supplementary Figure 5 Characterization of MAYA-LLGL2 and MAYA-NSUN6 interactions.

(a) Electrophoresis of in vitro transcribed Xef1α (left) or MAYA (right) sense (sen.) and anti-sense (as.) transcripts. (b) Streptavidin RNA pull-down assay, followed by IB detection of indicated proteins, using MAYA sense (sen.), anti-sense (as.) and Xef1α sense RNA. The presence of RNA transcripts was detected by streptavidin-HRP using dot-blot assay. (c) Electrophoresis of in vitro transcribed full-length MAYA or truncated transcripts. (d) RNA REMSA assay was performed using recombinant GST-tagged LLGL2 (aa. 101-200) or FLAG-tagged NSUN6 in the presence of 32P-labeled or cold MAYA RNA probes (nt. 251-290 and nt. 851-890), respectively. (e) Cell fractionation followed by RT-qPCR detecting relative MAYA expression in MDA-MB-231 cells with indicated stimuli (mean ± s.e.m. were derived from n = 3 independent experiments, n.s., P > 0.05, two-tailed paired Student’s t-test). Unprocessed original scans of all blots/gels with size marker are shown in Supplementary Fig. 9. An unprocessed original scans of EMSA image for d is also shown in Supplementary Fig. 9.

Supplementary Figure 6 Examination of the role of LLGL2 and NSUN6 in breast cancer cells.

(a and b) IB detection of the indicated proteins in LLGL2 KO (a) and NSUN6 KO (b) MDA-MB-231 cells. (c and d) Cell proliferation assay of individual (c) and pooled (d) clones of LLGL2 KO (left panels) and NSUN6 KO (right panels) MDA-MB-231 cells. (e and f) Cell invasion assay of individual clones of LLGL2 KO (e) and NSUN6 KO (f) MDA-MB-231 cells with overexpression of indicated plasmid (left, Scale bars, 200 μm; right, quantification). (g and h) Cell migration (g) and invasion (h) assay of pooled clones of LLGL2 KO and NSUN6 KO MDA-MB-231 cells (left, Scale bars, 200 μm; right, quantification). For c–h, mean ± s.e.m. were derived from n = 3 independent experiments (n.s., P > 0.05, ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001, two-tailed paired Student’s t-test). Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9 Source data for e–h are in Supplementary Table 8.

Supplementary Figure 7 MAYA is required for NRG1-triggered ROR1-HER3-MST1-YAP signaling axis.

(a) IP and IB detection of indicated proteins in ROR1 KO MDA-MB-231cells transfected with indicated expression vectors followed by NRG1 stimulation. (b and c) RT-qPCR detection of indicated genes in ROR1 KO cells transfected with indicated expression vectors followed by NRG1 stimulation. (d) MAYA expression level in MDA-MB-231 cells transfected with indicated individual LNAs targeting MAYA. (e) RT-qPCR detection of MAYA expression in MDA-MB-231 cells transfected with LNAs against MAYA followed by overexpression of indicated plasmids and NRG1 treatment. (f) IB detection of indicated proteins in A549 cells transfected with LNAs against MAYA followed by overexpression of indicated plasmids and NRG1 treatment. (g–i) RT-qPCR detection of indicated gene expression in MDA-MB-231 cells transfected with LNAs against MAYA followed by overexpression of indicated plasmids and NRG1 treatment (100 ng ml−1 for 1 h). (j) Osteoclast differentiation in the presence of M-CSF only, M-CSF + RANKL, or combined M-CSF + RANKL and CM from scramble (Scr) or MAYA LNA-transfected BoM-1833 cells rescued with indicated plasmids (Scale bars, 200 μm) For b–e and g–i, mean ± s.e.m. were derived from n = 3 independent experiments (n.s., P > 0.05, ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001, two-tailed paired Student’s t-test). Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9. Statistics source data for j are in Supplementary Table 8.

Supplementary Figure 8 Targeting MAYA inhibits the migration of lung cancer cells in vitro and tumor bone metastasis in vivo.

(a) RT-qPCR detection of MAYA expression level in a panel of breast cancer cell lines. (b) RT-qPCR detection of MAYA expression level in BoM-1833 cells harboring indicated shRNAs. (c) Cell proliferation assay of BoM-1833 cells harboring indicated shRNAs. (d and e) Cell migration (d) and invasion (e) assays using BoM-1833 cells harboring indicated shRNAs (left, Scale bars, 200 μm; right, quantification). (f) RT-qPCR detection of MAYA expression level in bone tumors of nude mice inoculated with BoM-1833 cells harboring indicated shRNAs. (g) RT-qPCR detection of MAYA expression level in A549-Luc cells harboring indicated shRNAs. (h) Cell proliferation assay of A549-Luc cells harboring indicated shRNAs. (i and j) Cell migration (i) and invasion (j) assays using A549-Luc cells harboring indicated shRNAs (left, Scale bars, 200 μm; right, quantification). (k and l) Representative BLI images (k) and bone colonization (l) of nude mice with intra-cardiac injection of A549-Luc cells harboring indicated shRNAs (n = 5 mice per group). (m) Graphic illustration of the functional role of NRG1-HER3 triggered, MAYA-mediated Hippo signaling suppression and YAP targets activation for promoting bone metastasis. For a–j and l, mean ± s.e.m. were derived from n = 3 independent experiments (∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001, two-tailed paired Student’s t-test). Statistics source data for d,e,i,j and k–l are in Supplementary Table 8.

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Li, C., Wang, S., Xing, Z. et al. A ROR1–HER3–lncRNA signalling axis modulates the Hippo–YAP pathway to regulate bone metastasis. Nat Cell Biol 19, 106–119 (2017). https://doi.org/10.1038/ncb3464

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