Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Therapeutic targeting of nemo-like kinase in primary and acquired endocrine-resistant breast cancer

doi: 10.1158/1078-0432.CCR-20-2961

Figure Lengend Snippet: (a) ERE luciferase reporter assay showing the effect of NLK inhibition/overexpression or VX-702 treatment on ER transcriptional activity in BT483 cells under estrogen deprivation (ED) and 0.5uM tamoxifen treatment. (b) VX-702 significantly inhibits the ER transcriptional activity in MCF7-TamR and T47D-TamR cells in the presence E2, or ED plus different doses of 4-OH tamoxifen treatment. a-b, *P<0.05; **P<0.01, *** P<0.001. P-value was calculated by t-test. (c) Co-immunoprecipitation of ERα using anti-ERα antibody and WB using anti-NLK antibody or anti-ERα antibody in BT483 overexpressing endogenous NLK. (d) In vitro kinase assay of recombinant active NLK using ERα as substrate with or without VX-702 treatment. Bar chart presents the quantified band intensity. The error bars in 3a-c represent the standard deviations. (e) Western blot analysis of ER signaling in BT483 cells following NLK silencing and endocrine treatment. BT483 cells were seeded in phenol red-free medium with 5% charcoal-dextran-stripped FBS containing 0.5uM Tamoxifen or vehicle (ethanol) for 48 hours, and then reverse transfected with siCtrl, siNLK#1, siNLK#2 (10nM) for 72 hours. (f) NLK phosphorylates SRC-3. Upper panel, in vitro kinase assay using recombinant NLK and SRC-3 proteins after the indicated treatment. Middle panel, in vitro kinase assay using recombinant NLK and 1–6A mutant SRC-3 protein. Lower panel, in vitro kinase assay using recombinant NLK and SRC-3 protein with mutation at indicated site (1–6A mutation sites: T24A, S505A, S543A, S857A, S860A, or S867A). (g) ER target gene expression changes following NLK inhibition significantly correlates with their changes following tamoxifen treatment in BT483 and T47D TamR cells. Left, log2 ratio of ER target differential expression (DE) following Tamoxifen treatment correlated with siNLK #1/2 treatment in BT483 cells. Here we used the ER target genes (n=76) compiled from TRUST database (70) in the analysis. Middle, log2 ratio of ER target gene DE following Tamoxifen treatment compared to siNLK #1/2 treatment in T47D TamR cells. Here T47D specific ER target genes (n=83) provided by Lin et al are used in the analysis (71). Right, log2 ratios of ER target DE between Tamoxifen and VX-702 treatment in T47D TamR cells. The T47D specific ER target genes (n=83) are used in the analysis. The Pearson correlation coefficients are shown in the figure with all p-values less than 0.001.

Article Snippet: 0.5 μg of Full length ERα Recombinant Protein (Life Technologies, RP-310) or 0.5 μg of recombinant SRC-3 protein (a gift from Dr. Bert W. O’Malley) ( 24 ) was incubated with 0.1 μg of active recombinant full-length NLK protein N15–10G (SignalChem) in the kinase buffer (SignalChem; 5mM MOPS, 2.5mM beta-glycerol-phosphate, pH 7.2, 5 mM MgCl 2 , 1 mM EGTA, 0.4 mM EDTA and 0.25 mM DTT) containing 50 μM ATP and 2 μCi [ 32 P]γATP at 30 °C for 30 minutes.

Techniques: Luciferase, Reporter Assay, Inhibition, Over Expression, Activity Assay, Immunoprecipitation, In Vitro, Kinase Assay, Recombinant, Western Blot, Transfection, Mutagenesis, Expressing