mtcc 3031  (ATCC)

Journal: Cell reports

Article Title: Mesenchymal and stem-like prostate cancer linked to therapy-induced lineage plasticity and metastasis

doi: 10.1016/j.celrep.2022.110595

Figure Lengend Snippet: (A) Top 10 gene sets (black dots) with a ssGSEA score, which correlates positively with the MSPC proportion estimate (%) from the three M-CRPC datasets. P53_DN.V1_UP (red dot) was ranked among the top 10 signatures in all three datasets. Parental gene sets are from the C6 Oncogenic Signatures (n = 189) from the MSigDB Collection. (B) Expression of TP53 and selected target genes in LNCaP cells treated with 10 μM enzalutamide for the indicated times. p values by two-way ANOVA and multiple comparisons test by Dunnett’s method are shown. Error bars represent the mean ± SD of triplicate experiments; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (C and D) IB of LNCaP cells treated with enzalutamide for the indicated times (C). Control and drug-treated LNCaP cells exposed or not to UV light (D) and subjected to IB at the indicated times are shown. Representative blots of n = 3 technical replicates are shown. (E) Heatmap of ranked normalized Z scores of E2F1, BRCA1, and TP53 mRNA. Fragments per kilobase of transcript per million mapped reads (FPKM) values from RNA-seq of LNCaP cells treated with 10 μM enzalutamide for the indicated times are shown. (F) Relative levels of TP53 mRNA in LNCaP cells transfected for 2 days with the indicated small interfering RNAs (siRNAs). Error bars represent the mean ± SD of triplicate experiments; p values by one-way ANOVA and multiple comparison by Dunnett’s test are shown; ***p < 0.001. (G) IB of p53 protein in LNCaP cells transfected for 4 days with the indicated siRNAs. Representative blots of n = 3 technical replicates are shown. (H) Control and TP53-silenced LNCaP cells were subjected to IB with the indicated antibodies. Representative blots of n = 3 technical replicates are shown. (I) Cluster Profiler dot plot of the enrichment of EMT (top) and cell lineage signatures (bottom) in control as compared with TP53 -silenced (left) or enzalutamide-treated LNCaP cells (right). (J) Cell growth of control and TP53-silenced LNCaP cells treated with CSS + DHT or CSS + enzalutamide (10 μM) at the indicated times. Error bars represent the mean ± SD of triplicate experiments; p values by one-way ANOVA and multiple comparison by Dunnett’s test are shown; ****p < 0.0001. (K and L) Quantification (left) and representative images (right) of control and TP53 -silenced LNCaP cells subjected to tumorsphere (K) or organoid formation assay (L). Error bars represent the mean ± SD of triplicate experiments, p values are calculated by one-way ANOVA, and multiple comparison by Dunnett’s test is shown; ****p < 0.0001; scale bars represent 500 μm. (M and N) TP53-silenced LNCaP cells (M) and Trp53-silenced Pten-P8 (−/−) cells (N) were subjected to Matrigel invasion assay with or without hrNRG1. Error bars represent the mean ± SD of triplicate experiments; p values by one-way ANOVA and multiple comparison by Dunnett’s test are shown; **p < 0.01; ***p < 0.001; scale bars represent 500 μm. (O and P) Male NGS mice were injected i.c. with 3.0 × 10 5 LNCaP cells expressing the indicated constructs and subjected 6 weeks later to bioluminescent imaging. Representative images (O) and quantification of luciferase counts (P) are shown. Error bars denote mean ± SD of five mice per group. Two independent experiments are shown; p values by one-way ANOVA and multiple comparison by Dunnett’s test are shown; *p < 0.05; **p < 0.01.

Article Snippet: LNCaP, VCaP, DU145, PC3, NCI-H660, Pten -p8 cells were obtained from ATCC, Cells were cultured in Dulbecco’s modified eagle medium (DMEM; Gibco) or RPMI 1640 medium (Gibco) supplemented with fetal calf serum (FBS, 10%; Gibco), and penicillin-streptomycin (Corning).

Techniques: Expressing, Control, RNA Sequencing, Transfection, Comparison, Tube Formation Assay, Invasion Assay, Injection, Construct, Imaging, Luciferase

Journal: Cell reports

Article Title: Mesenchymal and stem-like prostate cancer linked to therapy-induced lineage plasticity and metastasis

doi: 10.1016/j.celrep.2022.110595

Figure Lengend Snippet: (A) Heatmap of predicted drug sensitivity and expression of HER1–4 in MSPC samples from the SU2C-PCF dataset. Spearman correlation between the sensitivity scores of HER2/3 inhibitors neratinib and lapatinib and FGF inhibitors AZD4547 and nintedanib (top) and the expression of mRNA encoding HER1–4 (bottom) is shown. Spearman correlation coefficients are shown on the right. (B) Signaling pathways activated by AR blockade and activation of HER2/3 in PTEN mutant PC cells and rationale for combination therapies. (C) Immunoblotting of re-LNCaP-NRG1 cells treated with NRG1 in the presence of NRG1 and the indicated concentrations of neratinib. Representative blots of n = 3 technical replicates are shown. (D) Cell viability dose-response curve of control LNCaP and re-LNCaP-NRG1 cells exposed to neratinib. Mean ± SD of triplicate experiments is shown. Two-tailed Student’s t test of area under the curve (AUC) is shown; ****p < 0.0001. (E) Subcutaneous tumor growth of re-LNCaP-NRG1 cells in castrated male NSG mice treated daily with 10 mg/kg enzalutamide and/or 40 mg/kg neratinib. Error bars represent mean ± SD. Two-tailed Student’s t test of AUC is shown; *p < 0.05; ****p < 0.0001. (F) Treatment schedule and representative MRI images of liver and adrenal gland metastases generated by re-LNCaP-NRG1 injected i.c. in castrated male NSG mice. Mice were treated daily with 10 mg/kg enzalutamide and/or 40 mg/kg neratinib for the period indicated. (G and H) Re-LNCaP-NRG1 cells (G) and DU145 cells (H) were treated with NRG1 for 4 h with or without 100 nM MLN0128, 50 nM MLN1028, or 0.5 μM MK2206 singly or in combinations and subjected to IB with antibodies to total and activated signaling proteins. Representative blots of n = 3 technical replicates are shown. (I and J) Cell viability dose-response curve of re-LNCaP-NRG1 cells (I) or DU145 cells (J) to 50 nM MLN0128 in combination with the indicated concentrations of neratinib. Error bars represent mean ± SD of triplicate experiments. Two-tailed Student’s t test of AUC is shown; ****p < 0.0001. (K and L) DU145 cells were injected i.c. in castrated male NSG mice. Mice were treated 8 months later with 40 mg/kg/day neratinib and/or 0.3 mg/kg/day MNL0128 for 4 weeks. Representative BLIs (K) and Kaplan-Meier survival analysis (L) are shown; *p < 0.05; **p < 0.01.

Article Snippet: LNCaP, VCaP, DU145, PC3, NCI-H660, Pten -p8 cells were obtained from ATCC, Cells were cultured in Dulbecco’s modified eagle medium (DMEM; Gibco) or RPMI 1640 medium (Gibco) supplemented with fetal calf serum (FBS, 10%; Gibco), and penicillin-streptomycin (Corning).

Techniques: Expressing, Protein-Protein interactions, Activation Assay, Mutagenesis, Western Blot, Control, Two Tailed Test, Generated, Injection

Journal: Cell reports

Article Title: Mesenchymal and stem-like prostate cancer linked to therapy-induced lineage plasticity and metastasis

doi: 10.1016/j.celrep.2022.110595

Figure Lengend Snippet:

Article Snippet: LNCaP, VCaP, DU145, PC3, NCI-H660, Pten -p8 cells were obtained from ATCC, Cells were cultured in Dulbecco’s modified eagle medium (DMEM; Gibco) or RPMI 1640 medium (Gibco) supplemented with fetal calf serum (FBS, 10%; Gibco), and penicillin-streptomycin (Corning).

Techniques: Recombinant, CCK-8 Assay, Multiplex Assay, Microarray, Software