JAK2-binding long noncoding RNA promotes breast cancer brain metastasis

Abstract

Conventional therapies for breast cancer brain metastases (BCBMs) have been largely ineffective because of chemoresistance and impermeability of the blood-brain barrier. A comprehensive understanding of the underlying mechanism that allows breast cancer cells to infiltrate the brain is necessary to circumvent treatment resistance of BCBMs. Here, we determined that expression of a long noncoding RNA (lncRNA) that we have named lncRNA associated with BCBM (Lnc-BM) is prognostic of the progression of brain metastasis in breast cancer patients. In preclinical murine models, elevated Lnc-BM expression drove BCBM, while depletion of Lnc-BM with nanoparticle-encapsulated siRNAs effectively treated BCBM. Lnc-BM increased JAK2 kinase activity to mediate oncostatin M- and IL-6-triggered STAT3 phosphorylation. In breast cancer cells, Lnc-BM promoted STAT3-dependent expression of ICAM1 and CCL2, which mediated vascular co-option and recruitment of macrophages in the brain, respectively. Recruited macrophages in turn produced oncostatin M and IL-6, thereby further activating the Lnc-BM/JAK2/STAT3 pathway and enhancing BCBM. Collectively, our results show that Lnc-BM and JAK2 promote BCBMs by mediating communication between breast cancer cells and the brain microenvironment. Moreover, these results suggest targeting Lnc-BM as a potential strategy for fighting this difficult disease.

Keywords: Breast cancer; Cell Biology; Oncology.

Figure 1. Lnc-BM correlates with breast cancer and BCBM.

(A and B) LncRNA profiling in 231-Par and 231-Br cells. (C) RNAscope detection of Lnc-BM expression in human breast cancer and adjacent normal tissues. Left panel: Representative images. Right panel: Statistical analysis; 29 normal breast tissues (NBT), 118 breast cancer tissues, unpaired Student’s t test. Scale bars: 100 μm. (D–F) TissueScan Cancer Panels were analyzed by RT-qPCR for Lnc-BM expression in human breast cancer and adjacent normal tissues (1-way ANOVA). (G) Kaplan-Meier recurrence-free survival (RFS) analysis of Lnc-BM expression in breast cancer patients (n = 49 and 72 tissues, respectively, log rank test). (H) Determination of Lnc-BM expression in primary breast cancers with recurrence to local or distant sites by RT-qPCR. ΔCt: The Ct value of GAPDH was subtracted from the Ct value of Lnc-BM; ΔΔCt: the median of ΔCt of Lnc-BM from all samples was subtracted from the ΔCt value of each sample (1-way ANOVA). (I) RNAscope detection of Lnc-BM expression in BCBM tissues (n = 14 tissues). Scale bars: 100 μm. Data are mean ± SEM, *P < 0.05.

Figure 2. Lnc-BM is required and sufficient to promote BCBM.

(A) In vitro BBB transmigration activity of the 231-Br cells harboring control or Lnc-BM shRNAs. The number of transmigrated cells relative to the control cells is plotted (n = 3 independent experiments, unpaired Student’s t test). Scale bars: 100 μm. (B) Bioluminescence imaging (BLI) (n = 5 animals) of mouse, 4 weeks after intracardiac injection of 231-Br cells harboring indicated shRNAs (1-way ANOVA). Scale bars: 200 μm. White arrows: brain blood vessels. (C) Representative images and statistical analysis of H&E staining of mouse brain tumor burden at 35 days after intracardiac injection of 231-Br cells harboring indicated shRNAs (1-way ANOVA). Scale bars: 200 μm. n = 5 animals per group, 3 sections per brain. Micromet., micrometastatic lesions; Macromet., macrometastatic lesions. (D) Representative images of BLI (n = 8 animals), brain ex vivo bright field and ex vivo GFP, and statistical analysis of brain area photo flux (right panel) 5 weeks after intraarterial injection of HCC1954-Br cells stably expressing indicated shRNAs (1-way ANOVA). White arrow: brain metastatic lesions. Scale bars: 3 mm. (E) Kaplan-Meier plot of survival in the experiment of D (n = 8 animals per group, log rank test). Data are mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. Inhibition of Lnc-BM represses BCBM.

(A) Flowchart of the experiments. (B and C) Representative BLI images (B) and quantification of BLI in the head region (C) of mice 4 weeks after intracardiac injection of 231-Br cells followed by i.v. administration of indicated nanoparticle-siRNAs (NP-siRNAs) (n = 10 animals, 1-way ANOVA). (D) Kaplan-Meier plot of survival in the experiment of B (n = 10 animals, log rank test). Data are mean ± SEM, **P < 0.01, ***P < 0.001.

Figure 4. Lnc-BM is required for brain metastatic cancer cell vascular co-option.

(A) 231-Br cells harboring indicated shRNAs were subjected to cancer cell adhesion assay. The number of cancer cells attached and spread along HUVECs relative to total cancer cells was plotted (n = 3 independent experiments, unpaired Student’s t test). Scale bars: 200 μm. (B) Immunofluorescence (IF) with indicated antibodies in brain metastasis–bearing mice 35 days after intracardiac injection of 231-Br cells harboring indicated shRNAs (n = 3 independent experiments, paired Student’s t test). Scale bars: 50 μm. (C) Representative confocal images of cancer cell vascular co-option and quantification of infiltrated depth (right panel) of brain slices cocultured with 231-Br cells harboring indicated shRNAs (n = 3 independent experiments, unpaired Student’s t test) . Scale bars: 50 μm. White arrows: infiltrated cells. Data are mean ± SEM, *P < 0.05, **P < 0.01.

Figure 5. Lnc-BM promotes cancer cell adhesion to brain endothelium.

Representative images (A–C) and statistical analysis (D) of cancer cell adhesion assay of 231-Br cells harboring indicated sgRNAs cocultured with HBECs (A), HUVECs (B), or HMECs (C). Scr, scramble. Scale bars: 200 μm. Data are mean ± SEM, n = 3 independent experiments. NS, P > 0.05; **P < 0.01; 1-way ANOVA.

Figure 6. Lnc-BM impedes cancer cells from FasL-induced apoptosis.

(A and B) IF staining of cleaved caspase-3 in brain slices cocultured with 231-Br cells harboring indicated shRNAs (A) or in brain metastatic lesions (B, related to Figure 2C). Left panels: Representative images. Right panel: Statistical analysis (n = 3 independent experiments, unpaired Student’s t test). Scale bars: 100 μm. (C) FACS analyses of 231-Br cells transfected with indicated shRNAs followed by sFasL (200 ng/ml) treatment for 24 hours (n = 3 independent experiments, unpaired Student’s t test). (D and E) IF staining of cleaved caspase-3 in brain slices cocultured with 231-Br cells harboring scramble or Lnc-BM sgRNAs, followed by transfection of indicated expression constructs. (D) Representative images. (E) Statistical analysis (n = 3 independent experiments, unpaired Student’s t test). White arrows: apoptotic cells. Scale bars: 200 μm. Data are mean ± SEM; NS, P > 0.05; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7. Lnc-BM–mediated signaling pathway triggered by OSM is required for BCBM.

(A) Summary of protein identification score revealed by lncRNA pull-down followed by mass spectrometry analysis using sense (Sen.) or antisense (As.) transcripts of indicated lncRNAs. (B and C) IB detection using indicated antibodies in 231-Br cells treated with the indicated cytokines for 30 minutes (B) or in 231-Br and 231-Par cells treated with OSM (50 ng/ml) for the indicated times. *Unspecific band. (D) IHC staining of OSMR in human breast cancer (n = 112 tissues) and adjacent normal tissues (NBT; n = 26 tissues). Scale bars: 100 μm. (E) Kaplan-Meier RFS analysis of OSMR expression in breast cancer patients detected by IHC (n = 35 and 57 tissues, respectively, log rank test). (F) IB detection using indicated antibodies in 231-Br cells harboring indicated shRNAs treated with OSM for the indicated times. *Unspecific band.

Figure 8. JAK2 is required for BCBM.

(A) BLI image (n = 7 and 8 animals) of mice 4 weeks after intracardiac injection of indicated cells. Left panel: Representative images. Right panel: Statistical analysis (1-way ANOVA). Scale bars: 200 μm. White arrows: mouse brain blood vessel. (B) Kaplan-Meier plot of brain-metastasis-free survival in the experiment of (A) (n = 7 and 8 animals, respectively, log rank test). (C) IHC staining of p-JAK2 in human breast cancer (n = 110 tissues) and adjacent normal tissues (NBT) (n = 28 tissues). Scale bars: 100 μm. (D) Kaplan-Meier RFS analysis of p-JAK2 in breast cancer patients detected by IHC (n = 42 and 43 tissues, respectively, log rank test). Data are mean ± SEM, ***P < 0.001.

Figure 9. Lnc-BM associates with JAK2 in vitro and in vivo.

(A) RIP-qPCR detection of the indicated RNAs retrieved using indicated antibodies in 231-Br cells with OSM treatment for the indicated times (n = 3 independent experiments, paired Student’s t test). (B) In vitro RNA pull-down coupled with dot-blot assay using indicated RNA transcripts and recombinant proteins. Right panel: Annotation for each dot. (C and D) Graphic illustration of MS2-TRAP assay (C, top panel). IB (C, bottom panel) and RT-qPCR detection of MS2-Lnc-BM (D) of anti-GST immunoprecipitates in cells transfected with indicated expression constructs, followed by OSM stimulation (n = 3 independent experiments, paired Student’s t test). (E) Saturation curve K_D determination of interaction between indicated lncRNAs and recombinant GST-tagged JAK2 (n = 3 independent experiments). (F) Top panel: Graphic illustration of Lnc-BM–JAK2 interaction. Bottom panel: IB detection of streptavidin pull-down using His-tagged JH2 WT/mutants and biotinylated Lnc-BM. (G) Competition binding assay K_D determination of interaction between Lnc-BM FL/mutants and recombinant JH2 WT/mutants, with unlabeled Lnc-BM titrated from 10 μM to 0.1 nM (n = 3 independent experiments). Data are mean ± SEM; NS, P > 0.05; *P < 0.05, ***P < 0.001.

Figure 10. Lnc-BM modulates kinase activity of JAK2.

(A) Kinase assay using anti-JAK2 immunoprecipitates retrieved from 231-Br cells transfected with indicated siRNAs followed by OSM treatment. (B and C) In vitro kinase assay using recombinant JAK2 JH1/JH2 domain (B) or JAK2 JH1 domain (C), GST-STAT3, and indicated RNA transcripts in the presence and absence of ATP. (D) Graphic illustration of Lnc-BM–mediated JAK2 JH1/JH2 domain conformational alteration from autoinhibition to activation. (E) FLAG-tag pull-down followed by IB detection using antibodies in HEK-293T cells transfected with indicated vectors. (F) Anti-JAK2 immunoprecipitates were detected using indicated antibodies in 231-Br cells transfected with indicated siRNA. (G) Kinase assay using anti-FLAG immunoprecipitates in γ-2A cells transfected with SFB-JAK2 (aa 482–809), in the presence of indicated RNA transcripts and ATP.

Figure 11. ICAM1 facilitates breast cancer cell vascular co-option and invasion.

(A) Heatmap representation of the expression of STAT3 target genes in indicated cells treated with 50 ng/ml OSM or vehicle for 4 hours. The colors represent the fold changes of gene expression induced by OSM over vehicle. (B) GSEA of STAT3 target gene signature in A. NES, normalized enrichment score. (C) Top panel: Representative images of p-STAT3 and Lnc-BM staining in brain metastatic tissues of breast cancers. Bottom panel: Pearson’s correlation analysis (n = 14 tissues). Scale bars: 100 μm. (D and E) Trans-BBB assay (D) or cancer cell adhesion assay (E) was performed in Lnc-BM–deficient 231-Br cells stably overexpressing ICAM1 and MMP9 (n = 3 independent experiments, paired Student’s t test). B. vec., blank vector. Scale bars: 100 μm (D), 200 μm (E). Data are mean ± SEM; NS, P > 0.05; *P < 0.05, **P < 0.01.

Figure 12. Lnc-BM promotes recruitment of macrophages into the metastatic niche to form positive-feedback loop.

(A) RT-qPCR analysis of IL6ST-associated cytokine expression in BV2 cells incubated with conditioned media from indicated cells (n = 3 independent experiments, paired Student’s t test). (B and C) IHC detection of CD11b and Ly6G (B) or IF staining of IBA1 (C) in brain metastatic lesions of 231-Br cells harboring indicated shRNAs (related to Figure 2C; n = 5 animals per group, 3 sections per brain). B, brain tissue; Met, metastatic cancer cells. Scale bars: 200 μm (B), 100 μM (C). (D) ELISA detection of CCL2 concentration in conditioned media of 231-Br cells harboring indicated shRNAs treated with OSM (50 ng/ml) or IL-6 (50 ng/ml) for 12 hours (n = 3 independent experiments, paired Student’s t test). (E and F) RIP quantitative real-time PCR detection of Lnc-BM retrieved by indicated antibodies (E) or IB detection using indicated antibodies (F) in 231-Br cells treated with conditioned media from primed U937 cells in addition to indicated human antibodies (n = 3 independent experiments, paired Student’s t test; E). (G) Model of the action of Lnc-BM in mediating BCBM. Data are mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.