Journal: Nature Communications

Article Title: Autophagy inhibition elicits emergence from metastatic dormancy by inducing and stabilizing Pfkfb3 expression

doi: 10.1038/s41467-019-11640-9

Figure Lengend Snippet: Aberrant Pfkfb3 expression correlates with emergence from metastatic dormancy. a Laser capture microdissection of cresyl violet-stained 12-μm-thick murine lung tumors from Fig. at ×1.25 magnification. b Heatmap derived from microarray analysis with threefold differences between each evaluated group ( n = 3 mice/group). c Ingenuity Pathway Analysis (IPA) hierarchical heatmap derived from Downstream Effects Analysis. Z -score represents activation of each biological function where blue represents decreased function and orange represents increased function. Fold change cutoff of 3. d IPA-derived pathway analysis identified pathways significantly altered (threefold change) between dormant D2.OR and metastatic D2.A1 derived pathways. e KEGG Pathway analysis reveals major pathways altered between dormant D2.OR and metastatic D2.A1 cells (threefold change cutoff). f Spider plot depicts the number of genes altered more than threefold between comparison groups in genes belonging to the identified metabolic pathways. g Pfkfb3 expression in samples analyzed via Affymetrix GeneChip Mouse 2.0 ST microarrays

Article Snippet: For co-immunoprecipitation assays, the cells were transiently transfected either singly or in multiple combinations with recombinant GFP-tagged p62/SQSTM1 (pDestEGFP-p62 (ref. )), FLAG-tagged Pfkfb3 (pWZL-Neo-Myr-Flag-DEST Addgene plasmid # 15300), or HA-tagged ubiquitin (pcDNA-HA-ubiquitin).

Techniques: Expressing, Laser Capture Microdissection, Staining, Derivative Assay, Microarray, Activation Assay

Journal: Nature Communications

Article Title: Autophagy inhibition elicits emergence from metastatic dormancy by inducing and stabilizing Pfkfb3 expression

doi: 10.1038/s41467-019-11640-9

Figure Lengend Snippet: Aberrant Pfkfb3 expression correlated with disease progression and reduced relapse-free survival in human breast cancers. a Expression of Pfkfb3 mRNA in unprimed and primed D2.OR and D2.A1 cells as indicated. Data are the mean (±SD; n = 3) Pfkfb3 transcript levels normalized to D2.OR 2D. ** P ≤ 0.01 and **** P ≤ 0.0001. This experiment was independently repeated for a total of three experiments, all with similar results. b Analysis of Pfkfb3 protein in unprimed D2.A1 and D2.OR cells. c mRNA expression across the 4T1 progression series. Data are the mean (±SD; n = 3) Pfkfb3 transcript levels normalized to 67NR cells. ** P ≤ 0.01). This experiment was independently repeated for a total of three experiments, all with similar results. d Transformed NME cells harbor higher Pfkfb3 expression than NMuMG cells. Data are the mean (±SD; n = 3) Pfkfb3 transcript levels normalized to NMuMG cells. ** P ≤ 0.01. This experiment was independently repeated for a total of three experiments, all with similar results. e High expression of Pfkfb3 is common in PDX models compared to normal HMECs. Data are the mean (±SD; n = 3) Pfkfb3 transcript levels normalized to HMECs. ** P ≤ 0.01 and *** P ≤ 0.001. This experiment was independently repeated for a total of three experiments, all with similar results. f PFKFB3 mRNA expression by RT-PCR in human tumor cell lines. Data are the mean (±SD; n = 3) Pfkfb3 transcript levels normalized to HMECs. ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. This experiment was independently repeated for a total of three experiments, all with similar results. g PFKFB3 protein expression by immunoblot with β-actin used as a loading control. h Relapse-free survival analysis of 618 basal and HER2+ patients stratified by PFKFB3 expression levels. The expression range of the probe was between 225–5404 and the cutoff value for high PFKFB3 expression was 921 ( https://www.kmplot/analysis.com )

Article Snippet: For co-immunoprecipitation assays, the cells were transiently transfected either singly or in multiple combinations with recombinant GFP-tagged p62/SQSTM1 (pDestEGFP-p62 (ref. )), FLAG-tagged Pfkfb3 (pWZL-Neo-Myr-Flag-DEST Addgene plasmid # 15300), or HA-tagged ubiquitin (pcDNA-HA-ubiquitin).

Techniques: Expressing, Transformation Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Nature Communications

Article Title: Autophagy inhibition elicits emergence from metastatic dormancy by inducing and stabilizing Pfkfb3 expression

doi: 10.1038/s41467-019-11640-9

Figure Lengend Snippet: Aberrant Pfkfb3 expression drives emergence from metastatic dormancy and increased BCSC frequency. a D2.OR and D2.A1 cells were incubated in the absence or presence of the Pfkfb3 inhibitor, 3PO (0–500 μM), and differences in cell viability were quantified by CellTiter-Glo Assay. Data are the mean (±SD; n = 3). ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. This experiment was independently repeated for a total of three experiments, all with similar results. b Pfkfb3 overexpression drives D2.OR organoid growth in 3D cultures, while Pfkfb3-deficiency suppresses the growth of D2.A1 organoids in 3D culture, c , d Parental and Pfkfb3-deficient D2.A1 cells (1 × 10 6 cells/injection) were inoculated into the lateral tail veins of BALB/c mice ( n = 5). Pulmonary tumor growth was monitored by BLI and normalized to Day 0 readings. IHC images (×20 magnification) show loss of Pfkfb3 knockdown in tumors excised on Day 72. e Parental and Pfkfb3-overexpressing D2.OR cells or f parental and Pfkfb3-deficient D2.A1 cells were monitored for mammosphere forming ability by ELDA ( n = 8). Stem cell frequency was evaluated using the ELDA: Extreme Limiting Dilution Analysis ( http://bioinf.wehi.edu.au/software/elda/ ). These experiments were independently repeated for a total of three experiments, all with similar results. g Evaluating CD49f/CD24 expression levels by flow cytometry in parental and Pfkfb3-overexpressing D2.OR cells and in parental and Pfkfb3-deficient D2.A1 cells

Article Snippet: For co-immunoprecipitation assays, the cells were transiently transfected either singly or in multiple combinations with recombinant GFP-tagged p62/SQSTM1 (pDestEGFP-p62 (ref. )), FLAG-tagged Pfkfb3 (pWZL-Neo-Myr-Flag-DEST Addgene plasmid # 15300), or HA-tagged ubiquitin (pcDNA-HA-ubiquitin).

Techniques: Expressing, Incubation, Glo Assay, Over Expression, Injection, Software, Flow Cytometry

Journal: Nature Communications

Article Title: Autophagy inhibition elicits emergence from metastatic dormancy by inducing and stabilizing Pfkfb3 expression

doi: 10.1038/s41467-019-11640-9

Figure Lengend Snippet: Pfkfb3 expression is inversely related to the levels of autophagy in breast cancer cells. a Dormant D2.OR cells derived from Fig. express significantly more autophagy-associated as compared to their metastatic D2.A1 counterparts. Data are gene expression levels in D2.A1 cells normalized to D2.OR cells using a two-fold cutoff ( http://www.heatmapper.ca/expression/ ). LC3B puncta accumulation (scale bar = 69 μM) ( b ) were quantified ( c , d ) using ImageJ particle analysis. D2.OR cells are more autophagic as compared to D2.A1 cells as determined by immunoblotting cell lysates ( e ) and IHC from mammary fat pad tumors (×10 magnification) harvested from Fig. ( f ) for p62 and LC3B, and by autophagic flux using mCherry-GFP-LC3-expressing D2.OR cells propagated in 2D- or 3D cultures ( g ; data are the mean ± SEM; n = 4 for 2D-cultures; n = 6 for 3D cultures, **** P ≤ 0.0001). h One hundred and thirty breast cancer tumor slices were stained for PFKFB3 expression. Representative IHC images of PFKFB3 staining in (left) ER+/PR+/HER2+ and (right) ER+/PR+/HER2+ breast cancer samples. Scale bar (×4 and ×20 magnifications) = 100 μM. i In all, 80% (104/130) cores exhibit at least moderate (PFKFB3 intensity >2+) PFKFB3 staining and >98% (128/130) cores are PFKFB3 positive. j PFKFB3 staining intensity scores by breast cancer subtype. k Representative images of high PFKFB3 and low ATG3 IHC staining. l Immunohistochemical staining of breast cancer tumor cells with high PFKFB3 expression (3+ staining intensity; n = 49 samples) were evaluated for autophagy expression. Scale bar (×4 and ×20 magnifications) = 100 μM

Article Snippet: For co-immunoprecipitation assays, the cells were transiently transfected either singly or in multiple combinations with recombinant GFP-tagged p62/SQSTM1 (pDestEGFP-p62 (ref. )), FLAG-tagged Pfkfb3 (pWZL-Neo-Myr-Flag-DEST Addgene plasmid # 15300), or HA-tagged ubiquitin (pcDNA-HA-ubiquitin).

Techniques: Expressing, Derivative Assay, Western Blot, Staining, Immunohistochemistry, Immunohistochemical staining

Journal: Nature Communications

Article Title: Autophagy inhibition elicits emergence from metastatic dormancy by inducing and stabilizing Pfkfb3 expression

doi: 10.1038/s41467-019-11640-9

Figure Lengend Snippet: Inactivation of autophagy induces BCSC expression of Pfkfb3 and emergence from metastatic dormancy. Inactivation of autophagy induces Pfkfb3 expression in D2.OR cells. Autophagy was inhibited by administration of chloroquine (CQ; 25 μM; a ), and by shRNA-mediated depletion of either Atg3 ( b ), Atg7 ( c ), or p62 ( d ). Inactivation of either Atg3 ( e , *** P ≤ 0.001), Atg7 ( f , ** P ≤ 0.01), or p62 ( f , ** P ≤ 0.01; **** P ≤ 0.0001) promotes D2.OR organoid outgrowth in 3D cultures. This experiment was independently repeated for a total of 3 experiments, all with similar results. g Dual inhibition of autophagy through Atg3 knockdown and Pfkfb3 by PFK-158 treatment inhibits D2.OR organoid outgrowth in 3D cultures. Data represent mean (±SD) of biological replicates performed in triplicate (ns P > 0.05, ** P ≤ 0.01, and **** P ≤ 0.0001). h CD49f/CD24 flow cytometry evaluation in parental and Atg3-deficient D2.OR cells. i BCSC frequency as determined by an ELDA mammosphere assay. j Quantification of total cell numbers isolated following completion of the primary mammosphere assays depicted in i . Data demonstrate significantly larger spheres associated with Atg3-deficient D2.OR cells as compared with parental D2.OR cells. k Parental (i.e., NT) and Atg3-deficient D2.OR cells were inoculated into the lateral tail veins of BALB/c mice (2 × 10 6 cells/mouse) and pulmonary tumor development was monitored by BLI. Photomicrograph shows macroscopic tumors (black arrows) in 2/5 mice inoculated with Atg3-deficient D2.OR cells. l Microscopic nodule count from lung tumors in k . m Bioluminescence imaging (BLI) of Day 70 D2.OR and D2.OR shAtg3-derived tumors. Red arrows indicate the presence of tumor. Yellow circles indicate BLI signal that does not derive from lung tumors, but instead from nonspecific signals present in tail tumors. A scale bar is included where red represents the maximum of the figure (1000) while violet represents the minimum (50). n Representative IHC images show elevated Pfkfb3 expression in tumor nodules produced by Atg3-deficient D2.OR cells. Scale bar (×0.75 magnification) = 2.5 mm. Scale bar (×10 magnification) = 250 μM

Article Snippet: For co-immunoprecipitation assays, the cells were transiently transfected either singly or in multiple combinations with recombinant GFP-tagged p62/SQSTM1 (pDestEGFP-p62 (ref. )), FLAG-tagged Pfkfb3 (pWZL-Neo-Myr-Flag-DEST Addgene plasmid # 15300), or HA-tagged ubiquitin (pcDNA-HA-ubiquitin).

Techniques: Expressing, shRNA, Inhibition, Flow Cytometry, Isolation, Imaging, Derivative Assay, Produced

Journal: Nature Communications

Article Title: Autophagy inhibition elicits emergence from metastatic dormancy by inducing and stabilizing Pfkfb3 expression

doi: 10.1038/s41467-019-11640-9

Figure Lengend Snippet: Pfkfb3 is an autophagy substrate that binds to the autophagosome cargo protein p62/SQSTM1. a FLAG-tagged Pfkfb3 was transiently expressed in D2.A1 and D2.OR cells and captured by immunoprecipitation with anti-FLAG antibodies. Afterward, p62/SQSTM1 and Pfkfb3 were visualized by immunoblotting as indicated. b Schematic of wild type and p62/SQSTM1 truncation mutants. c Immunoblot (IB) analysis of FLAG-tagged Pfkfb interaction with wild type and mutant p62/SQSTM1 constructs depicted in panel b . d Cycloheximide (CHX) [25 μM] was administered for 0–24 h as indicated. Pfkfb3 was stabilized in Autophagy Low (i.e., D2.A1 and D2.OR shAtg3) cells but not in Autophagy High (i.e., D2.OR) cells. e MG132 [10 μM] treated for 0–4 h as indicated. Pfkfb3 expression in D2.A1, D2.OR, D2.OR shAtg3, and D2.OR shp62 was evaluated by immunoblotting

Article Snippet: For co-immunoprecipitation assays, the cells were transiently transfected either singly or in multiple combinations with recombinant GFP-tagged p62/SQSTM1 (pDestEGFP-p62 (ref. )), FLAG-tagged Pfkfb3 (pWZL-Neo-Myr-Flag-DEST Addgene plasmid # 15300), or HA-tagged ubiquitin (pcDNA-HA-ubiquitin).

Techniques: Immunoprecipitation, Western Blot, Mutagenesis, Construct, Expressing

Journal: Developmental cell

Article Title: Tumor Suppressor Inactivation of GDF11 Occurs by Precursor Sequestration in Triple-Negative Breast Cancer

doi: 10.1016/j.devcel.2017.10.027

Figure Lengend Snippet: (A) SMAD4 knockdown blocks the ability of GDF11 to inhibit 3D invasion in MDA-MB-231 cells. shLuc was induced as a control shRNA.

Article Snippet: pSLIK_TT 3xFLAG SMAD4 neo , This paper , Addgene #83273.

Techniques: Knockdown, Control, shRNA

Journal: Developmental cell

Article Title: Tumor Suppressor Inactivation of GDF11 Occurs by Precursor Sequestration in Triple-Negative Breast Cancer

doi: 10.1016/j.devcel.2017.10.027

Figure Lengend Snippet: Molecular characterization and use of triple-negative cell lines in this study Origin, phenotype, transcriptomic subtype, and genetic mutations of the indicated cell lines are shown along with the figure subpanels in which they appear.

Article Snippet: pSLIK_TT 3xFLAG SMAD4 neo , This paper , Addgene #83273.

Techniques: Transformation Assay

Journal: Developmental cell

Article Title: Tumor Suppressor Inactivation of GDF11 Occurs by Precursor Sequestration in Triple-Negative Breast Cancer

doi: 10.1016/j.devcel.2017.10.027

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pSLIK_TT 3xFLAG SMAD4 neo , This paper , Addgene #83273.

Techniques: Virus, In Situ, Recombinant, Enzyme-linked Immunosorbent Assay, Mutagenesis, RNA Amplification, Expressing, Real-time Polymerase Chain Reaction, Luciferase, Software