Journal: Molecular Cancer

Article Title: Genome-wide in vivo CRISPR screen identifies TGFβ3 as actionable biomarker of palbociclib resistance in triple negative breast cancer

doi: 10.1186/s12943-024-02029-4

Figure Lengend Snippet: TGFβ3 potentiates palbociclib anti-tumor effect in vivo. a mRNA expression levels of TGFB1, TGFB2 and TGFB3 in SUM159PT following TGFB3 -specific overexpression using CRISPR activation (CRISPR/dCas9 SAM) ( n = 3). Data are represented as mean ± standard deviation (SD). Significance was calculated using two-sided, unpaired t-test, p -value * < 0.05. b Mice from control (lentiSAMv2) or TGFB3 -overexpressing (TGFB3g2 SAM) groups ( n = 13) were each randomized into treatment groups (vehicle, n = 6; palbociclib, n = 7). I.p. injections of the vehicle treatment or a low dose of palbociclib (10 mg/kg) were administered until study endpoint. Data are represented as mean ± SD. c Reduction in tumor growth presented for each group treated with palbociclib, lentiSAMv2 or TGFB3g2 SAM, as compared to the same groups treated with the vehicle. Data are represented as mean, at each timepoint. d left Tumor volumes of individual mice in each group at study endpoint. right Tumor weights of individual mice in each group at study endpoint. Midlines at median. Significance was calculated using ordinary, one-way ANOVA with Tukey’s multiple comparisons test, p -value * < 0.05, ** < 0.01, *** < 0.001. e Average mRNA expression levels of TGFB3 in tumors derived from the vehicle-treated control mice ( n = 6) and the TGFB3 -overexpressing mice ( n = 6). Data are represented as mean ± SD. Significance was calculated using two-sided, unpaired t-test, p -value * < 0.05, ** < 0.01, *** < 0.001. f Protein levels of TGFB3 (60 kDa) in tumors derived from the vehicle-treated control mice ( n = 6) and the TGFB3 -overexpressing mice ( n = 6). g Spontaneous metastasis to the lungs was assessed. Lung nodules were counted and compared in lungs derived from the vehicle-treated control mice ( n = 7) and the TGFB3 -overexpressing mice ( n = 6). Data represent metastatic nodule count per pair of lungs per mouse. Midlines at median. Significance was calculated using nonparametric Mann–Whitney U-test, p -value * < 0.05, ** < 0.01, *** < 0.001. h The effect of TGFB3 CRISPR-mediated knockout on lung colonization was assessed. Data represent metastatic nodule count per pair of lungs per mouse. Midlines at median. i Schematic representation of the use of recTGFβ3 in combination with palbociclib. MDA-MB-231 TNBC cells were transplanted into the mammary fat pads of NSG mice. Tumors were palpable before mice were randomized into treatment groups: vehicle, n = 9; recTGFβ3, n = 8; palbociclib, n = 8, combo (recTGFβ3 + palbociclib), n = 9. j Average tumor volume was measured over time. Data are represented as mean ± SD. k Tumor volumes of individual mice in each group at study endpoint. Midlines at median. Significance was calculated using ordinary, one-way ANOVA with Tukey’s multiple comparisons test, p -value * < 0.05. l Quantification of Ki67-positive cells stained by immunohistochemistry in tumor tissues from all four groups. Data are represented as mean ± SD ( n = 3–4). Significance was calculated using two-sided, unpaired t-test, p -value * < 0.05. m Representative images of Ki67 staining in two tumors per group

Article Snippet: Recombinant human TGFβ3 ligand (PeproTech, Inc, cat. no. 100-36E) was dissolved in 10 mM citric acid buffer with 0.1% BSA.

Techniques: In Vivo, Expressing, Over Expression, CRISPR, Activation Assay, Standard Deviation, Control, Derivative Assay, MANN-WHITNEY, Knock-Out, Staining, Immunohistochemistry

Journal: Molecular Cancer

Article Title: Genome-wide in vivo CRISPR screen identifies TGFβ3 as actionable biomarker of palbociclib resistance in triple negative breast cancer

doi: 10.1186/s12943-024-02029-4

Figure Lengend Snippet: Combination of recombinant TGFβ3 and palbociclib synergistically inhibits TNBC cell proliferation in vitro. a Synergy between palbociclib and recTGFβ3 dose combinations was calculated based on four reference models (Bliss, HSA, Loewe, ZIP) using SynergyFinder in four TNBC cell lines (MDA-MB-231, SUM159PT, SUM229PE, 159-R). Synergy maps highlight areas of synergistic (red) or antagonistic (green) interactions between given concentrations of either agent. Grey boxes indicate the area of maximum synergy observed. Mean of a minimum of three independent replicate experiments for each cell line ( n ≥ 3). b ‘Overall synergy scores’ and ‘Most synergistic area scores’ presented for each drug matrix shown in a . Data are represented as score ± 95% confidence interval. c Dot plots show overall synergy scores (black) or most synergistic area scores (pink) for each cell line, with each dot representing the score obtained using the indicated reference model. Midlines represent median scores. Outer vertical lines correspond to minimum and maximum scores obtained. A zero ‘0’ score indicates no interaction between the two agents

Article Snippet: Recombinant human TGFβ3 ligand (PeproTech, Inc, cat. no. 100-36E) was dissolved in 10 mM citric acid buffer with 0.1% BSA.

Techniques: Recombinant, In Vitro

Journal: Molecular Cancer

Article Title: Genome-wide in vivo CRISPR screen identifies TGFβ3 as actionable biomarker of palbociclib resistance in triple negative breast cancer

doi: 10.1186/s12943-024-02029-4

Figure Lengend Snippet: TGFβ3 synergizes with palbociclib in a p21-dependent way . a SUM159PT cells were treated with palbociclib (100 nM) for 2 h, 8 h, 16 h and 24 h and protein lysates were assessed for known CDK4/6i resistance markers (CDK4, cyclin D1, cyclin E1, Rb, phospho-Rb (S780)) by immunoblotting. Relative fold changes in protein levels, compared to untreated cells at each timepoint, were calculated ( n = 3). Data are represented as mean ± SD. Significance was calculated using two-sided, unpaired t-test, p -value * < 0.05, ** < 0.01, *** < 0.001. b SUM159PT (159) and 159-R cells were assessed for known CDK4/6i resistance markers, as well as p21, by immunoblotting. c top SUM159PT and 159-R cells were treated with recTGFβ3 (100 pM) for 24 h and resulting changes in known CDK4/6i resistance markers and p21 were measured by immunoblotting. bottom MDA-MB-231 (231) and SUM229PE (229) cells were treated with recTGFβ3 (200 pM) for 24-48 h and resulting changes in p21 were measured by immunoblotting. d SUM159PT cells were transduced with plasmids encoding control (scramble, scr), Smad2-specific, or Smad3-specific short hairpin RNAs (shRNA). Protein levels of p21 and total Smad2/3 were measured by immunoblotting. e SUM159PT cells were transduced with plasmids encoding control (scr) and p21-specific shRNA. Protein levels of p21 were measured by immunoblotting. f SUM159PT scr shRNA-infected or p21 shRNA-infected cells were treated with varying combinations of palbociclib and recTGFβ3 concentrations. Synergy between dose combinations was calculated using SynergyFinder. upper Synergy maps highlight areas of synergistic (red) or antagonistic (green) interactions between given concentrations of either agent. Grey boxes indicate the area of maximum synergy observed between given recTGFβ3 and palbociclib dose combinations. lower ‘Overall synergy scores’ and ‘Most synergistic area scores’ presented for each drug matrix shown above. Data are represented as score ± 95% confidence interval ( n = 3). Percentage variation in synergy score (score obtained in p21 shRNA cells/score obtained in scr shRNA cells) is also shown (red)

Article Snippet: Recombinant human TGFβ3 ligand (PeproTech, Inc, cat. no. 100-36E) was dissolved in 10 mM citric acid buffer with 0.1% BSA.

Techniques: Western Blot, Transduction, Control, shRNA, Infection

Journal: Molecular Cancer

Article Title: Genome-wide in vivo CRISPR screen identifies TGFβ3 as actionable biomarker of palbociclib resistance in triple negative breast cancer

doi: 10.1186/s12943-024-02029-4

Figure Lengend Snippet: Schematic diagram depicting TGFβ3-palbociclib synergy. a In the basal context, cells maintain a balance between active (green) and p21-bound, inactive (red) CDK/cyclin complexes. In the presence of palbociclib (orange capsule), CDK4/6 kinase activity is inactivated, and p21 (pink box) bound to CDK4 is released and preferentially displaced to CDK2. This inactivates CDK2/cyclin E complexes and leads to overall cell cycle arrest. b upper When cells undergo prolonged exposure to palbociclib, key cell cycle regulators (CDK4, cyclins D and E) are upregulated, while p21 expression is strongly inhibited. Some CDK/cyclin complexes are inactivated (red), but the overall imbalance in active CDK4/cyclinD1 and CDK2/cyclinE1 complexes (green) leads to decreased responsiveness of cells to palbociclib, acquired resistance to the drug, and continued cell cycling. lower When TGFβ3 is added in the presence of palbociclib, p21 expression levels are restored through TGFβ3 signalling. The increase in p21 by TGFβ3 synergizes with palbociclib’s mechanism of action, allowing for the inactivation of all remaining active CDK/cyclin complexes (red), and ultimately leading to cell cycle arrest

Article Snippet: Recombinant human TGFβ3 ligand (PeproTech, Inc, cat. no. 100-36E) was dissolved in 10 mM citric acid buffer with 0.1% BSA.

Techniques: Activity Assay, Expressing

Journal: Human reproduction (Oxford, England)

Article Title: A novel selective progesterone receptor modulator asoprisnil (J867) down-regulates the expression of EGF, IGF-I, TGFbeta3 and their receptors in cultured uterine leiomyoma cells.

doi: 10.1093/humrep/del035

Figure Lengend Snippet: Figure 1. Effects of asoprisnil on the expression of immunoreactive epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I) and transforming growth factor β (TGFβ3) protein in leiomyoma and normal myometrial cells cultured for 48 or 72 h, as assessed by immunocyto- chemistry. The expression of immunoreactive EGF, IGF-I and TGFβ3 protein in leiomyoma and normal myometrial cells cultured for 48 h (upper panel) and for 72 h (lower panel). EGF in leiomyoma cells cultured without any treatment (a, A), EGF in leiomyoma cells cultured in the presence of 10–7 M asoprisnil (d, D), IGF-I in leiomyoma cells cultured without any treatment (b, B), IGF-I in leiomyoma cells cultured in the presence of 10–7 M asoprisnil (e, E), TGFβ3 in leiomyoma cells cultured without any treatment (c, C) and TGFβ3 in leiomyoma cells cultured in the presence of 10–7 M asoprisnil (f, F). EGF in normal myometrial cells cultured without any treatment (g, G), EGF in normal myometrial cells cultured in the presence of 10–7 M asoprisnil (j, J), IGF-I in normal myometrial cells cultured without any treatment (h, H), IGF-I in normal myometrial cells cultured in the presence of 10–7 M asoprisnil (k, K), TGFβ3 in normal myometrial cells cultured without any treatment (i, I) and TGFβ3 in normal myometrial cells cultured in the presence of 10–7 M asoprisnil (l, L). Bars represent 50 μm. Original magnification 40.

Article Snippet: A mouse monoclonal antibody to human EGF (MAB236, R&D Systems), a goat polyclonal antibody to human IGF-I (AF-291-NA, R&D Systems) and a mouse monoclonal antibody to TGFβ3 (MAB643, R&D Systems) were used as the primary antibodies at a dilution of 1:20, 1:50 and 1:50, respectively.

Techniques: Expressing, Cell Culture

Journal: Human reproduction (Oxford, England)

Article Title: A novel selective progesterone receptor modulator asoprisnil (J867) down-regulates the expression of EGF, IGF-I, TGFbeta3 and their receptors in cultured uterine leiomyoma cells.

doi: 10.1093/humrep/del035

Figure Lengend Snippet: Figure 4. Effects of asoprisnil on the number of viable leiomyoma and normal myometrial cells cultured for 72 h in the presence of either recombinant epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I) or transforming growth factor β (TGFβ3), as assessed by 3-(4,5-dimethylthi- azol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Treatment with 100 ng/ml recombinant EGF signifi- cantly increased the number of viable cultured leiomyoma cells compared with untreated control cultures, and the concomitant treatment with 10–

Article Snippet: A mouse monoclonal antibody to human EGF (MAB236, R&D Systems), a goat polyclonal antibody to human IGF-I (AF-291-NA, R&D Systems) and a mouse monoclonal antibody to TGFβ3 (MAB643, R&D Systems) were used as the primary antibodies at a dilution of 1:20, 1:50 and 1:50, respectively.

Techniques: Cell Culture, Recombinant, MTS Assay, Control