EMBO/SourceData huggingface dataset:
Figure 1A. LRP6 level is reduced during serum starvation. HEK293 cells were incubated in serum-free medium for the indicated hours. Cells were lysed and the cell lysates were analyzed by immunoblotting.B. Serum starvation reduced the amount of LRP6. HEK293 cells were incubated in serum-free medium for 3 h. Then cells were additionally incubated for 1 h after addition of 10% serum. Cells were lysed and the cell lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β-actin and p-YAP/YAP of three independent immunoblots was quantified (biological replicates, middle and right panel, respectively). N.S.: non-significant, **P < 0.01 and ***P < 0.001. Error bars indicate standard deviation of biological triplicate measurements. Student's t-test was used for statistical analysis.C. Loss of LRP6 in serum starvation was seen in different cell lines. Cell lines were incubated in serum-free medium for 2 h. Cells were lysed, and the cell lysates were analyzed by immunoblotting. S.E., short exposure; L.E., long exposure.D. Glucose starvation decreased LRP6. HEK293 cells were incubated in glucose-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.E. The decrease of LRP6 by serum starvation was mediated by the lysosomal degradation pathway. HEK293 cells were incubated in serum-free medium for 4 h with DMSO or Bafilomycin A1 (100 nM). Cells were lysed and the cell lysates were analyzed by immunoblotting.Loss of LRP6 by serum starvation was mediated in a clathrin HEK293 cells were transfected with clathrin siRNA and incubated in serum-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.G. Loss of LRP6 by serum starvation was mediated in a caveolin-1-dependent manner. HEK293 cells were transfected with caveolin siRNA and incubated in serum-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.H. Lipid components of serum were essential for maintaining LRP6. HEK293 cells were incubated in serum-free medium with A2058 BSA (Sigma) or Fraction V BSA for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β-actin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. N.S.: non-significant and *P < 0.05. Student's t-test was used for statistical analysis.
Figure 3A. Knockdown of LRP6 activated LATS1. HEK293T cells were transfected with siRNA for GFP and LRP6. After transfection, cells were lysed and the cell lysates were analyzed by immunoblotting.B. Overexpression of LRP6 reduced the amount of the active form of LATS1 that was induced by serum starvation. HEK293T cells were transfected with empty vector (-) or VSVG-LRP6. 1 d after transfection, cells were incubated in serum-free medium for 4 h. After incubation, cells were lysed, and the cell lysates were analyzed by immunoblotting.C. Merlin and LATS1/2 were necessary for the LRP6-knockdown-mediated YAP phosphorylation. HEK293T cells were transfected with siRNA for GFP, LRP6, Merlin, and LATS1/2. Cells were lysed and the cell lysates were analyzed by immunoblotting.D. Merlin and LATS1/2 were necessary for the LRP6-knockdown-mediated decrease of YAP reporter activity. HEK293T cells were seeded and transfected with siRNA for GFP, LRP6, Merlin and LATS1/2. After 1 d, these cells were transfected again with pRL-TK and 8XGTIIC luciferase reporter constructs. One day after transfection, cells were lysed and luciferase activity was measured. Error bars indicate standard deviation of biological triplicate measurements. **P< 0.01 and N.S: Non-significant. Student's t-test was used for statistical analysis.E. Merlin and LATS1/2 were necessary for the LRP6 knockdown-mediated-cytoplasmic localization of YAP. HEK293A cells were transfected with siRNA for GFP, LRP6, Merlin, and LATS1/2, and immunofluorescence analysis was performed. The figure shows representative images of multiple areas. DAPI was used for nucleus staining. Scale bars: 10 μm. Quantification of cells that have nuclear YAP is shown in Fig EV3B.F. YAP phosphorylation by loss of LRP6 was dependent on the interaction between Merlin and LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6, and Merlin, and after 1 d were transfected again with HA-Merlin and HA-Merlin∆FERM. Cells were lysed and the cell lysates were analyzed by immunoblotting. The arrow indicates endogenous Merlin and HA-Merlin, and the arrowhead indicates HA-Merlin∆FERM.G. MST1/2 were necessary for the LRP6-knockdown-mediated activation of LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6 and MST1/2. Cells were lysed and the cell lysates were analyzed by immunoblotting.H. MAP4K4/6/7 were necessary for the LRP6 knockdown mediated activation of LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6, and MAP4K4/6/7. Cells were lysed and the cell lysates were analyzed by immunoblotting.
Figure 4A. The interaction between overexpressed Merlin and LRP6 was reduced under serum starvation condition. HEK293T cells were transfected with Flag-Merlin and LRP6-EGFP. After transfection, cells were incubated in serum-free medium for overnight with Bafilomycin A1 (20 nM). The cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates.The interaction between endogenous Merlin and LRP6 was reduced under serum starvation. HEK293T cells were incubated in serum-free medium for 4 h with Bafilomycin A1 (100 nM). The cell lysates were immunoprecipitated with anti-LRP6 antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates.The interaction between endogenous Merlin and LRP6 was reduced under serum starvation. HEK293T cells were incubated in serum-free medium for 4 h with Bafilomycin A1 (100 nM). The cell lysates were immunoprecipitated with anti-Merlin antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates.D. The interaction between overexpressed Merlin and LATS1 was increased under serum starvation. HEK293T cells were transfected with Myc-LATS1 and EGFP-Merlin. After transfection, cells were incubated in serum-free medium for overnight and then were lysed. The cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.E. Knockdown of LRP6 enhanced the interaction between overexpressed Merlin and LATS1. HEK293T cells were transfected with Myc-LATS1, EGFP-Merlin and LRP6 siRNA. Cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.F. Knockdown of LRP6 enhances the interaction between endogenous Merlin and LATS1. HEK293T cells were transfected with siRNA for GFP or LRP6. After transfection, cells were lysed and the cell lysates were immunoprecipitated with anti-Merlin antibody and analyzed by immunoblotting. WCL, whole-cell lysates.
Figure 5A. Endogenous LRP6 and OGT interact with each other. HEK293T cells were lysed and the cell lysates were immunoprecipitated with anti-LRP6 antibody and blotted with OGT. WCL, whole-cell lysates.B. LRP6 is O-GlcNAcylated. LRP6-EGFP-transfected HEK293T cells were treated in Thiamet G (30 μM) or OSMI-1 (50 μM) for 6 h with Bafiolmycin A1 (100nM). After treatment, cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.C. LRP6 is O-GlcNAcylated. Cell lysates from HEK293T cells were incubated with sWGA-beads in the absence or presence of free GlcNAc (25 mM) for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates.D. Blocking of O-GlcNAcylation reduced the amount of LRP6. HEK293 cells were treated with OSMI-1 (50 μM) and Bafilomycin A1 (100 nM) for 6 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.E. The O-GlcNAcylation on LRP6 was reduced under serum starvation condition. HEK293T cells were transfected with LRP6-EGFP. Cells were incubated in serum-free medium with Bafilomycin A1 (100 nM) for 4 h. After incubation, cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates. The ratio of O-GlcNAc/EGFP from three independent immunoblots was quantified (biological replicates, bottom panel). *P < 0.05. Error bars indicate standard deviation of biological triplicate measurements. Student's t-test was used for statistical analysis.F. Increase of O-GlcNAcylation enhanced the interaction between ectopically expressed LRP6 and Merlin. LRP6-EGFP and Flag-Merlin transfected HEK293T cells were incubated in serum-free medium with Bafilomycin A1 (20 nM) and Thiamet G (30μM) overnight. Cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.G. Enhancement of global O-GlcNAcylation blocked the serum starvation-mediated increase of interaction between overexpressed LATS1 and Merlin. HEK293T cells were transfected with Myc-LATS1 and Flag-Merlin. After transfection, cells were incubated in serum-free medium with Thiamet G (30μM) and Bafilomycin A1 (20nM) overnight. Cells were lysed and the cell lysates were immunoprecipitated with anti-Myc antibody and analyzed by immunoblotting. WCL, whole-cell lysates.
Figure 6A. Nutrient starvation reduced the level of LRP6 in mouse liver. Mice were starved for 36 h and liver tissues were analyzed by immunoblotting (left panel). The ratio of LRP6/Vinculin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. **P < 0.01. Student's t-test was used for statistical analysis.B. Refeeding of nutrients rescued LRP6 in mouse liver. Mice were starved and then refed for 6 h and liver tissues were analyzed by immunoblotting (left panel). The ratio of LRP6/Vinculin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. *P < 0.05. Student's t-test was used for statistical analysis.C. The level of LRP6 was not reduced during nutrient starvation in mouse brain. Mice were starved and brain tissues were analyzed by immunoblotting.D. LRP6 was O-GlcNAcylated in mouse liver. Liver tissues were incubated with sWGA-beads in the absence or presence of free GlcNAc (25 mM) for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates.E. O-GlcNAcylation of LRP6 was reduced during starvation in mouse liver. LRP6 in liver tissue lysates from both fed and starved mice was equilibrated by loading twice as much lysate from the livers of starved mice on the gel. Liver tissues were incubated in sWGA-beads for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates.
Figure 1Kaplan-Meier analysis of overall survival in breast cancer patients with recurrent lethal disease, classified according to p-JNK expression. p-JNK was determined by immunostaining of breast cancer samples in a tissue microarray; p-JNK low, n = 31; p-JNK high, n = 10. P value and hazard ratio (HR) were calculated by log-rank test.Immunostaining of p-c-Jun (arrows) in patient-matched breast tumor and lung metastasis. Scale bar, 50 μm.Quantification of p-c-Jun positive (p-c-Jun+) cells per tumor area in matched primary tumors and lung metastases. Boxes depict median from 12 patients with upper and lower quartiles.Quantification of p-c-Jun positive (p-c-Jun+) cancer cells within mammary tumors and matched metastases in mice injected with MDA231-LM2 cells.Immunofluorescence analysis of p-c-Jun in micrometastatic (day 7) and macrometastatic (day 21) nodules in a xenograft mouse model injected intravenously with MDA231-LM2 cells. DAPI was used to stain cell nuclei, and cancer cells express GFP as a marker. Scale bar, 50 μm. Quantification of p-c-Jun+ cancer cellsMammary tumor growth curves from mice described in panel G. Each value represents the mean ± SEM. For MDA231-LM2, n = 12 tumors in 6 mice for each experimental group. For SUM159-LM1, n = 16 tumors in 8 mice for each experimental group.Lung metastatic burden in MDA231-LM2 or SUM159-LM1 tumor-bearing mice treated with JNKi. Quantification of lung metastases. FOV, field of view; LS, lung section. Each value represents the mean ± SEM. For metastatic foci per FOV, 8-10 fields were quantified per mouse. MDA231-LM2 tumor-bearing mice: n = 6 mice per group. SUM159-LM1 tumor-bearing mice, vehicle n = 8 mice; JNKi n = 7 mice.Lung metastatic burden in MDA231-LM2 or SUM159-LM1 tumor-bearing mice treated with JNKi. Representative histology examples of metastasis in lungs, detected by expression of human vimentin. Scale bar, 100 μm.Metastatic burden determined by photon flux in lungs of MDA231-LM2-injected NSG mice treated with vehicle and JNKi. Boxes show the median with upper and lower quartiles. Whiskers represent minimum and maximum values. Vehicle control, n = 8; JNKi-treated, n = 8. Examples of bioluminescence in mice
Figure 2Scatter plot elucidating fold change (FC) in gene expression between samples treated with JNKi compared to vehicle (Y-axis) and expression of constitutively active JNK (MKK7-JNK) versus vector control (X-axis) from biological triplicates. Representative examples of genes that are specifically induced by JNK in metastatic breast cancer cells are highlighted in red.GSEA graphs showing (C) enrichment of a mammary stem cell signature (Pece et al, 2010) in MDA231-LM2 cancer cells expressing MKK7-JNK NES, normalized enrichment score.GSEA graphs showing under-representation of the same mammary stem cell signature in cancer cells treated with JNKi. NES, normalized enrichment score.GSEA showing significant enrichment of four distinct signatures of mammary stem cells within data sets from MDA231-LM2 cells either expressing MKK7-JNK or treated with JNKi. Mammary stem cell signatures are indicated as follows: a, up in mammary stem cells b, up in fetal mammary stem cells ; c, up in adult mammary stem cells and d, up in mammary repopulating unitsHeat map of the JNK signature composed of 68 genes that are induced by expression of MKK7-JNK and repressed by JNKi,Proportion of JNK signature-positive tumors in breast cancer patients from the METABRIC data set classified according to PAM50 intrinsic biological subtypes JNK-S, JNK signature as in panel F. Subtype patient numbers: Luminal, n = 1213; HER2, n = 240; Basal-like, n = 331.
Figure 3Gene expression profile of MDA231-LM2 cells grown as oncospheres or as a monolayer. Examples of genes that are both upregulated in spheres and induced by JNK signaling are highlighted in red.GSEA graph showing enrichment of JNK signature in MDA231-LM2 cancer cells grown as oncospheres. NES, normalized enrichment score.Immunoblot of phosphorylated JNK (p-JNK), JNK, phosphorylated c-Jun (p-c-Jun) and c-Jun in oncospheres from SUM159-LM1 cells. Tubulin was included as a loading control.GSEA of a basal-like breast cancer signature induced in MDA231-LM2 oncospheres.Normalized enrichment score (GSEA) of mammary stem cell signatures in oncospheres from MDA231-LM2 cells.Generation of oncospheres in SUM159 cells expressing constitutively active JNK or inactive mutated JNK. Values are mean from 10 wells per group ± SD.Oncosphere formation in SUM159-LM1 cells following treatment with JNKi or vehicle control. Values are mean from 10 wells per condition ± SD. Representative images of spheresOncosphere formation in JNKi-treated cancer cells from breast cancer patients (pleural effusions or ascites). (I) Quantification of oncospheres per well in 4 different patients. Values are mean from 10 wells ± SD. (J) Representative oncospheres from patient samples. Scale bar, 50 μm.Matrigel invasion assay of MDA231-LM2 cells treated with JNKi. Left, examples of invading cancer cells stained with crystal violet (arrows). Scale bar, 50 μm. Right, quantification of invading cells per field of view (FOV) showing mean from 8 fields ± SEM.
Figure 4SPP1 and TNC expression in the indicated breast cancer cells treated with increasing concentrations of JNKi.SPP1 and TNC expression in MDA231-LM2 cells transduced with pLVX-puro lentivirus without a transgene (control) or expressing constitutively-active JNK (MKK7-JNK) or mutated inactive JNK (MKK7-JNK(mut)).SPP1 and TNC expression in pleural effusion (1 and 2) and ascites (3 and 4) samples from four different patients treated with JNKi (4 μM, 48h) in culture. Values are mean ± SD from triplicates. The results from all four patient samples were included to calculate significance of JNKi on gene expression. SPP1, P = 0.0079; TNC, P = 0.0002.Analysis of SPP1 and TNC mRNA levels in SUM159-LM1 cells grown as monolayer or spheres and treated with vehicle or JNKi. For panels A-D, values represent the mean of qPCR triplicates ± SD.ChIP analysis of c-Jun binding to sequences in SPP1 or TNC promoters. Analysis of SPP1 and TNC promoter fragments pulled down by c-Jun and IgG antibodies. Values represent the mean of qPCR triplicates ± SD.Correlation analysis of SPP1 and TNC with JUN expression in dissected metastatic nodules from breast cancer patients (GSE14020). N = 65 metastasis samples.Lung metastasis in Spp1+/- mice implanted with MDA231-LM2 cells expressing control shRNA (control), and Spp1-/- mice implanted with MDA231-LM2 cells independently expressing one of two different SPP1 shRNAs (SPP1-deficient). Ex vivo lung luminescence was analyzed, panel H. Boxes show the median value with upper and lower quartiles. Whiskers represent minimum and maximum values. Control n = 16 mice, SPP1 deficient (1) n = 14 mice and SPP1 deficient (2) n = 15 mice. P values were determined by a two-tailed Mann-Whitney test. (I) Representative histological images of metastatic foci (top) and ex vivo lung bioluminescence (bottom). In histological samples, immunohistochemistry was used to determine metastatic nodules based on expression of human vimentin (arrows). Scale bar, 100 µm.SUM159-LM1 cells expressing either control shRNA (control), or one of two different TNC shRNAs (shTNC 1 and 2), were injected intravenously into NSG mice. (J) Metastatic colonization was quantified by lung bioluminescence. Control and shTNC (1) n = 8 mice per group, shTNC (2) n = 7 mice. P value was determined by a two-tailed Mann-Whitney test. (K) Representative images of metastatic foci (top) and ex vivo lung bioluminescence (bottom). Arrows denote vimentin-expressing cancer cells in metastases.Kaplan-Meier analysis of breast cancer patients (combined GSE2603 and GSE5327 datasets) showing a link between high SPP1 and TNC expression and poor lung metastasis-free survival. Patient samples, SPP1 and TNC low n = 70; SPP1 and TNC high n = 70. HR, Hazard ratio.
Figure 5Gene expression profile of MDA231-LM2 cells treated with paclitaxel (PAX) for 48 hours. Highlighted are examples of JNK signature genes that are induced by PAX treatment.GSEA normalized enrichment scores of four independent mammary stem cell signatures that arise in response to paclitaxel treatment of MDA231-LM2 cells.Immunofluorescence analysis of p-c-Jun+ cancer cells in lung metastases from mice injected intravenously with MDA231-LM2 cells and treated with PAX at week 3 post injection. Lungs were harvested 72h after PAX treatment. Cancer cells express GFP as a marker. DAPI was used for nuclear staining. Scale bar, 50 µm.Quantification of cancer cells expressing p-c-Jun in panel D. Values are mean from three mice ± SEM. Nine metastatic foci were analyzed per mouse.Western blot analyzing phosphorylated JNK (p-JNK), JNK, phosphorylated c-Jun (p-c-Jun) and c-Jun in MDA231-LM2 cells treated with PAX for 48 hours. Tubulin was used as a loading control.SPP1 and TNC expression in SUM159-LM1 cells treated with increasing concentrations of PAX qPCR triplicates ± SD.SPP1 and TNC expression in SUM159-LM1 cells treated with increasing concentrations of doxorubicin (DOXO). qPCR triplicates ± SD.SPP1 and TNC expression in SUM159-LM1 cancer cells treated with 4 μM 5-Fluorouracil or 4 μM methotrexate. Cells were treated for 48 hours. qPCR triplicates ± SD.SPP1 and TNC expression in SUM159-LM1 cells treated with paclitaxel and JNKi. qPCR triplicates ± SD. SPP1 and TNC repression by JNKi, P < 0.05 for both vehicle and PAX-treated cells.
Figure 6Growth curves of mammary tumors in vehicle- or PAX-treated Spp1+/- mice implanted with the indicated cells expressing control shRNA, and Spp1-/- mice implanted with the indicated cells expressing SPP1 shRNA. MDA231-LM2: control, PAX and SPP1 def. + PAX n = 16 tumors per group, SPP1 def. n = 14 tumors. SUM159-LM1: control, PAX and SPP1 def. + PAX n = 16 tumors per group, SPP1 def. n = 15 tumors.Quantification of lung metastasis from the animals described in panels A and B. The number of metastatic foci per field of view (FOV) or per lung section (LS) was determined. MDA231-LM2 tumor-bearing mice: control, PAX and SPP1 def. + PAX n = 8 mice per group, SPP1 def. n = 7 mice. SUM159-LM1 tumor-bearing mice: control, PAX and SPP1 def. + PAX n = 8 mice per group, SPP1 def. n = 7 mice.Tumor weight in mice at day 40 after implantation of MDA231-LM2 breast cancer cells in a control or SPP1 deficient setting undergoing combination treatment with doxorubicin (Adriamycin) and cyclophosphamide (AC regimen). Control, AC and SPP1 def. n = 16 tumors per group, SPP1 def. + AC n = 14 tumors.Lung metastasis was quantified in mice that were implanted with MDA231-LM2 tumors and subjected to AC chemotherapy. Each value represents the mean ± SEM; n = 5 mice per group. Ten random fields per lung section were analyzed for percentage of metastatic area. Representative histological images of metastatic nodules in lungs of mice from the experiment described in panel E. Metastases were determined by expression of human vimentin (arrows). Scale bar, 100 µm.Mammary tumor growth curves in PAX-treated NSG mice bearing control or TNC knockdown MDA231-LM2 tumors. Values are means ± SEM, n = 16 tumors per group. **** P < 0.0001. Lung metastases quantified in tumor bearing miceRepresentative histology of human vimentin-positive lung metastases (arrows)
Figure 7Mammary tumor growth curves in NSG mice implanted with MDA231-LM2 or SUM159-LM1 cells and treated with paclitaxel as single agent or in combination with JNKi. Each value represents the mean ± SEM. MDA231-LM2: n = 12 tumors per group. SUM159-LM1: vehicle, JNKi and PAX n = 12 tumors per group; PAX + JNKi n = 10 tumors. **P < 0.01, ***P < 0.001.Metastatic burden in lungs of MDA231-LM2 and SUM159-LM1 tumor-bearing mice treated with paclitaxel as single therapy or in combination with JNKi. Each value represents the mean ± SEM. MDA231-LM2 tumor-bearing mice: n = 6 mice per group. SUM159-LM1 tumor-bearing mice: vehicle, JNKi and PAX n = 6 mice per group; PAX+ JNKi n = 5. Ten fields were quantified per lung section. FOV, field of view, LS, lung section. PAX, paclitaxel. Representative histological images of metastatic foci (arrows) in lungs of the MDA231-LM2-implanted mice described in panel C, stained for human vimentin expression in differentially treated groups. Scale bar, 100 μm.Apoptosis was determined by quantification of TUNEL-positive cells per field of view (FOV) in MDA231-LM2 tumors (from the animals described in (C)) treated with JNKi and/or PAX. Values are means from 6 mice per condition ± SEM.GSEA showing enrichment of the JNK signature in tumor samples from breast cancer patients from the TOP trial (GSE16446), stratified according to pathological response to neoadjuvant chemotherapy. NES, normalized enrichment score. pCR, pathological complete response.Kaplan-Meier analyses of chemotherapy-treated breast cancer patients associating JNK signature (JNK-S) with metastasis-free survival (TOP trial data set, JNK-S low n = 54 patients; JNK-S high n = 53 patients (G)) or overall survival (METABRIC data set, JNK-S low n = 135 patients; JNK-S high n = 135 patients (H)). HR, hazard ratio. P values were determined by log-rank test.