Research ArticleCancer

ABL kinases promote breast cancer osteolytic metastasis by modulating tumor-bone interactions through TAZ and STAT5 signaling

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Science Signaling  02 Feb 2016:
Vol. 9, Issue 413, pp. ra12
DOI: 10.1126/scisignal.aad3210
  • Fig. 1 Increased expression of ABL genes in invasive breast cancer is associated with metastasis.

    (A) ABL2 copy number in 813 normal samples compared with 789 invasive breast tumor samples in the TCGA database. (B) ABL2 mRNA abundance in 61 normal samples compared with 532 invasive breast tumor samples in the TCGA database. Results shown in (A) and (B) are based on the data generated by the TCGA Research Network (http://cancergenome.nih.gov/); whiskers represent 1st and 99th percentile. (C and D) Kaplan-Meier representation of the probability of cumulative overall distant metastasis–free survival (DMFS) in 2830 breast cancer cases (C) or 482 basal breast cancer cases (D) according to ABL2 expression. (E) Kaplan-Meier representation of the probability of cumulative overall distant metastasis-free survival in 279 HER2-enriched breast cancer cases according to ABL1 expression. (F) Kaplan-Meier representation of the probability of cumulative bone metastasis-free survival (BMFS) in 42 breast cancer cases according to ABL1 expression. P values (log-rank test) and hazard ratios (HR) are shown in the graph.

  • Fig. 2 Knockdown of ABL kinases decreases breast cancer bone metastasis.

    (A) Experimental design. (B) Survival of mice after intracardiac injection of 1833 (1 × 105) breast cancer cells transduced with control shRNA (Scr) or shRNAs against ABL1 and ABL2 (shAA); n = 10 mice per group. (C to F) Bioluminescent images (C and E) of bone metastasis from representative mice at day 22 after inoculation with 1833 cells (n = 10 mice per group) or day 35 after inoculation with SCP28 cells (n = 8 mice per group). Quantification of bone metastases (D and F). (G and H) Representative H&E staining (G) and quantification of H&E-stained tumor area of bone lesions. Arrows indicate tumor. n = 3 mice per group. Scale bar, 200 μm. Met, metastatic. (I and J) Representative x-ray and μCT reconstruction (I) and quantification of bone volume (BV)/total volume (TV) from μCT analysis of the mouse tibias (J). n = 3 mice per group. (K) Representative immunoblots of 1833 cells transfected with control shRNA, shRNA against ABL1 (shABL1), ABL2 (shABL2), and shRNA#2 against both ABL1 and ABL2 (shAA#2) and ABL1/ABL2 knockdown cells with overexpression of mouse Abl1/Abl2 (shAA + mAbl1/Abl2). n = 3 blots. p, phosphorylated. (L) Bioluminescent images of bone metastases from representative mice at day 18 after inoculation. n = 8 mice per group. (M) Quantification of (L). *P < 0.05, one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test.

  • Fig. 3 Allosteric inhibition of ABL kinases decreases breast cancer bone metastasis.

    (A) Experimental design. (B) Survival of mice after intracardiac injection of 1833 (1 × 105) breast cancer cells and treatment with either dimethyl sulfoxide (DMSO) control or the allosteric ABL inhibitor GNF5. n = 10 mice per group. (C) Bioluminescent images of representative mice at day 22 after inoculation. (D) Quantification of bone metastases. n = 10 mice per group. (E and F) Representative H&E staining (E) and quantification (F) of stained tumor area of bone lesions. Arrows indicate tumor. n = 3 mice per group. Scale bar, 200 μm. (G and H) Representative x-ray and μCT reconstruction (G) of mouse tibias and quantification (H) of bone volume/total volume. n = 3 mice per group.

  • Fig. 4 ABL kinases are required for tumor survival and tumor-induced osteolysis in the bone microenvironment.

    (A and B) Control or ABL1/ABL2 knockdown 1833 cells (1 × 105) were injected directly into the tibias of the mice. Representative bioluminescent images (A) taken at day 21 after inoculation and quantification (B) of bone lesions are shown. n = 5 mice per group. (C and D) Representative H&E staining (C) and quantification (D) of H&E-stained tumor area of mouse tibias from each group. n = 3 mice per group. Scale bar, 500 μm. (E and F) Representative 3D μCT reconstruction of mouse tibias (E) and quantification (F) of bone volume/total volume from μCT analysis. n = 3 mice. (G and H) Representative images (G) of TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling) staining of cells treated with TRAIL and the indicated shRNA and quantification of the percent of TUNEL-positive cells (H). n = 3 biological replicates. Scale bar, 100 μm. (I) Immunoblotting was performed using the indicated antibodies on whole-cell lysates from cells incubated or not with TRAIL. n = 3 blots.

  • Fig. 5 Depletion of ABL kinases impairs tumor-induced osteoclast activation in part by decreasing IL-6 secretion.

    (A) In vitro osteoclastogenesis assay. (B) TRAP staining of bone marrow cells treated with conditioned medium (CM) from 1833 breast cancer cells. Scale bar, 50 μm. (C) Quantification of TRAP+ cells in (B). (D) Quantification of TRAP+ cells derived from RAW264.7 cells; ns, not significant. (E and F) RANKL (E) and OPG (F) expression was detected by reverse transcription polymerase chain reaction (RT-PCR) of the osteoblast cell line 7F2 treated with conditioned medium harvested from the indicated 1833 cells. (G) Identification of differentially expressed cytokines in the conditioned medium of 1833 cells using a cytokine antibody array. n = 2 biological replicates. (H) ELISA quantification of IL-6 in conditioned medium of 1833 cells. (I) Quantification of TRAP+ bone marrow–derived osteoclasts incubated with the indicated doses of IL-6. *Significantly different from 0; P value was calculated using one-way ANOVA followed by Tukey’s post hoc test. (J) TRAP staining of bone marrow treated with conditioned medium from 1833 cells with or without added IL-6. (K) Quantification of TRAP+ osteoclasts in (J). *P < 0.05; **P < 0.01, two-way ANOVA followed by Tukey’s post hoc test. n = 3 biological replicates unless otherwise indicated.

  • Fig. 6 ABL kinases regulate the expression of genes in the JAK/STAT and Hippo pathway signatures in metastatic breast cancer cells.

    (A) CummeRbund heat map of genes that were differentially expressed in control and single and double ABL1 and ABL2 knockdown cells. (B) GSEA analysis of the indicated gene signatures in ABL1/ABL2 knockdown cells compared with control cells (Scr). NES, normalized enrichment score. (C) Expression of the indicated genes in control, ABL1 or ABL2 single-knockdown, and ABL1/ABL2 double-knockdown cells quantified using Cufflinks CuffDiff. *Significantly different from control cells (P < 0.05 after Benjamini-Hochberg correction for multiple testing). Error bars represent SD. n = 3 biological replicates for (A) and (C).

  • Fig. 7 ABL kinases are required for TAZ and STAT5 signaling in breast cancer cells.

    (A to C) Immunoblots with the indicated antibodies were performed on whole-cell lysates of 1833 and SCP28 cells. (D) Immunoblots were performed on whole-cell lysates (pSTAT5, STAT5, and tubulin) or conditioned medium (MMP1, IL-6, and TNC). (E) Immunoblots were performed on whole-cell lysates (pSTAT5, STAT5, and tubulin) or conditioned medium (MMP1, IL-6, and TNC) of parental 1833 and SCP28 cells. (F) Immunoblotting with the indicated antibodies were performed on whole-cell lysates (pSTAT5, STAT5, and tubulin) or conditioned medium (MMP1, IL-6, and TNC) of 1833 cells. For (A) to (F), n = 3 blots. (G) Bioluminescent images of representative mice at day 25 after intracardiac injection of 1833 cells. (H) quantification of bone metastasis. n = 5 mice per group. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant, one-way ANOVA followed by Tukey’s post hoc test. (I and J) Bioluminescent images (I) and quantification (J) of bone metastasis from representative mice at day 25 after intracardiac injection of 1833 cells transfected with control [nonspecific (NS)] or shRNAs against STAT5 and TAZ (shSTAT5/shTAZ). n = 8 mice per group. (K) Immunoblots were performed on whole-cell lysate. n = 3 blots.

  • Fig. 8 ABL kinases activate the TAZ and STAT5 pathways to promote breast cancer bone metastasis.

    (A) Kaplan-Meier representation of the probability of cumulative overall disease-free survival in TCGA data set with 971 invasive breast cancer patients according to whether the ABL signature (ABL2, TAZ, AXL, CTGF, STAT5A, STAT5B, TNC, IL6, and MMP1) was altered or not. P value was derived by the log-rank test. (B) Model for the role of ABL kinases in the regulation of breast cancer bone metastasis.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/413/ra12/DC1

    Fig. S1. ABL family protein abundance is increased in breast cancer cells with enhanced bone metastatic activity, and ABL1/ABL2 depletion does not affect proliferation in vitro.

    Fig. S2. ABL kinases promote breast cancer cell invasion.

    Fig. S3. Depletion of ABL kinases does not inhibit metastasis of 4175 breast cancer cells, which show tropism to the lung.

    Fig. S4. Treatment of breast cancer cells with imatinib but not GNF5 promotes ERK activation.

    Fig. S5. CXCL12- and IGF-1–mediated survival pathways are independent of ABL kinases.

    Fig. S6. Depletion of ABL kinases in SKBR3 breast cancer cells decreases tumor-induced osteoclast activation.

    Fig. S7. IL-6 affects RANKL and OPG expression in osteoblasts.

    Fig. S8. Quality control and global statistics of RNAseq analysis for transcriptome comparison of control versus ABL1/ABL2 knockdown breast cancer cells.

    Fig. S9. ABL kinases increase TAZ protein abundance and STAT5 phosphorylation.

    Fig. S10. Depletion of ABL kinases reduces the abundance of TAZ in the nucleus.

    Fig. S11. Allosteric inhibition of ABL kinase activity decreases TAZ protein abundance.

    Fig. S12. ABL2 mRNA expression positively correlates with TAZ mRNA expression in invasive breast cancer patients.

    Fig. S13. Depletion of ABL kinases decreases the binding of TAZ to target genes.

    Fig. S14. Depletion of ABL kinases does not affect YAP1 protein abundance, localization, or tyrosine phosphorylation in breast cancer cells.

    Fig. S15. Expression of a constitutively active STAT5 mutant increases mRNA expression of MMP1, IL6, and TNC.

    Fig. S16. Potential interdependence of TAZ and STAT5 in breast cancer cells.

    Table S1. Differentially expressed genes in control and ABL1/ABL2 knockdown breast cancer cells.

  • Supplementary Materials for:

    ABL kinases promote breast cancer osteolytic metastasis by modulating tumor-bone interactions through TAZ and STAT5 signaling

    Jun Wang, Clay Rouse, Jeff S. Jasper, Ann Marie Pendergast*

    *Corresponding author. E-mail: ann.pendergast{at}duke.edu

    This PDF file includes:

    • Fig. S1. ABL family protein abundance is increased in breast cancer cells with enhanced bone metastatic activity, and ABL1/ABL2 depletion does not affect proliferation in vitro.
    • Fig. S2. ABL kinases promote breast cancer cell invasion.
    • Fig. S3. Depletion of ABL kinases does not inhibit metastasis of 4175 breast cancer cells, which show tropism to the lung.
    • Fig. S4. Treatment of breast cancer cells with imatinib but not GNF5 promotes ERK activation.
    • Fig. S5. CXCL12- and IGF-1–mediated survival pathways are independent of ABL kinases.
    • Fig. S6. Depletion of ABL kinases in SKBR3 breast cancer cells decreases tumor-induced osteoclast activation.
    • Fig. S7. IL-6 affects RANKL and OPG expression in osteoblasts.
    • Fig. S8. Quality control and global statistics of RNAseq analysis for transcriptome comparison of control versus ABL1/ABL2 knockdown breast cancer cells.
    • Fig. S9. ABL kinases increase TAZ protein abundance and STAT5 phosphorylation.
    • Fig. S10. Depletion of ABL kinases reduces the abundance of TAZ in the nucleus.
    • Fig. S11. Allosteric inhibition of ABL kinase activity decreases TAZ protein abundance.
    • Fig. S12. ABL2 mRNA expression positively correlates with TAZ mRNA expression in invasive breast cancer patients.
    • Fig. S13. Depletion of ABL kinases decreases the binding of TAZ to target genes.
    • Fig. S14. Depletion of ABL kinases does not affect YAP1 protein abundance, localization, or tyrosine phosphorylation in breast cancer cells.
    • Fig. S15. Expression of a constitutively active STAT5 mutant increases mRNA expression of MMP1, IL6, and TNC.
    • Fig. S16. Potential interdependence of TAZ and STAT5 in breast cancer cells.
    • Table S1. Differentially expressed genes in control and ABL1/ABL2 knockdown breast cancer cells.

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    Citation: J. Wang, C. Rouse, J. S. Jasper, A. M. Pendergast, ABL kinases promote breast cancer osteolytic metastasis by modulating tumor-bone interactions through TAZ and STAT5 signaling. Sci. Signal. 9, ra12 (2016).

    © 2016 American Association for the Advancement of Science

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