Journal: NPJ Precision Oncology

Article Title: IL-6 predicts CDK4/6 inhibitor resistance, identifying STAT3 as a target in HR + /HER2-negative metastatic breast cancer

doi: 10.1038/s41698-025-01041-1

Figure Lengend Snippet: A Relative gene expression of IL-6 by quantitative (q)RT-PCR analysis in MCF7 parental (Par) and resistant (Res) cell lines after shRNA knockdown of IL-6 by one of two clones (shIL-6 #1 and shIL-6 #2) or control scrambled shRNA (shSCR). Cells were grown in culture for 3 days. B Relative gene expression of STAT3 by qRT-PCR analysis. C Secreted IL-6 levels measured by ELISA and normalized based on the number of cells (relative concentration [conc]). D Western blot analysis of pSTAT3 (Y705) and total STAT3 after 3 days in culture. E Dose-response curves of treatment with 0.01–12 μM palbociclib for 6 days followed by 6 days of recovery compared with MCF7 Par shSCR. Number of cells/growth was assessed using the crystal violet assay. Data were normalized to DMSO (100%), plotted, and analyzed using nonlinear regression on GraphPad Prism 9. Dashed line and bar graphs depict half-maximal inhibitory concentration (IC 50 ) values. F Dose-response curves after 24 h of estrogen deprivation followed by re-addition of 10 nM beta-estradiol and treatment with varying concentrations (0.01–12 μM) of fulvestrant for 2 days compared with MCF7 Par shSCR. Number of cells/growth was assessed using the crystal violet assay as in panel E. G Dose-response curves with 0.01–12 μM TTI-101 for 3 days followed by 9 days of recovery. Number of cells/growth was assessed using the crystal violet assay as in panel ( E ). H qRT-PCR analysis of estrogen responsive genes—ER and PgR. I qRT-PCR analysis of epithelial-mesenchymal transition (EMT) markers N-cadherin and Vimentin. J qRT-PCR analysis of transcription factors related to breast cancer stem cell-like markers CD44 and ALDH1. K Western blot analysis of estrogen receptor (ERa), PgR, EMT markers, and G1/S transition proteins. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Palbociclib isethionate (MedChemExpress) was diluted in 0.5 W/V% methyl cellulose 400 (FUJIFILM Wako Chemicals), TTI-101 was diluted in 60:40 Labrasol:PEG400 with brief sonication for about 2 min, and vehicle was a 50:50 mixture of 0.5 W/V% methyl cellulose 400 and 60:40 Labrasol:PEG400.

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, shRNA, Knockdown, Clone Assay, Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, Crystal Violet Assay

Journal: NPJ Precision Oncology

Article Title: IL-6 predicts CDK4/6 inhibitor resistance, identifying STAT3 as a target in HR + /HER2-negative metastatic breast cancer

doi: 10.1038/s41698-025-01041-1

Figure Lengend Snippet: A , B Dose-response curves in MCF7 and T47D parental (Par) control scrambled shRNA (shSCR), shIL-6 #1, and shIL-6 #2 cells, depicting the effect of treatment with 0.01–12 μM palbociclib for 6 days followed by 6 days of recovery ( A ) and after 24 h of estrogen deprivation followed by re-addition of 10 nM beta-estradiol and treatment with varying concentrations (0.01–12 μM) of fulvestrant for 2 days ( B ). Dashed line depicts half-maximal inhibitor concentration (IC 50 ) values. C , D Bar graphs depicting the average IC 50 values in MCF7 and T47D parental shSCR, shIL-6 #1, and shIL-6 #2 cells treated with palbociclib ( C ) and fulvestrant ( D ) as indicated in A , B. E , F Doubling time every 3 days for 21 days showing the effect of shRNA knockdown of IL-6 on the proliferation of MCF7 and T47D parental cell lines. G , H MCF7 parental ( G ) and T47D ( H ) shSCR, shIL-6 #1, and shIL-6 #2 cells were treated with 1 μM palbociclib every 3 days for 21 days with measurements taken every 3 days as indicated in G for cell growth and IL-6 secreted protein levels (ELISA). I Representative images of senescence-associated ß-galactosidase (SA-βgal) staining in MCF7 parental shSCR, shIL-6 #1, and shIL-6 #2 cells treated with or without 1 μM palbociclib every 3 days for 21 days. Quantification of SA-βgal+ cells was calculated every 3 days relative to number of cells. J Representative images of SA-βgal staining in MCF7 resistant (Res) and T47D Res shSCR, shIL-6 #1, and shIL-6 #2 cells treated with or without 1 μM palbociclib every 3 days for 6 days. Quantification of SA-βgal+ cells was calculated at day 6 relative to number of cells. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Palbociclib isethionate (MedChemExpress) was diluted in 0.5 W/V% methyl cellulose 400 (FUJIFILM Wako Chemicals), TTI-101 was diluted in 60:40 Labrasol:PEG400 with brief sonication for about 2 min, and vehicle was a 50:50 mixture of 0.5 W/V% methyl cellulose 400 and 60:40 Labrasol:PEG400.

Techniques: Control, shRNA, Concentration Assay, Knockdown, Enzyme-linked Immunosorbent Assay, Staining

Journal: NPJ Precision Oncology

Article Title: IL-6 predicts CDK4/6 inhibitor resistance, identifying STAT3 as a target in HR + /HER2-negative metastatic breast cancer

doi: 10.1038/s41698-025-01041-1

Figure Lengend Snippet: A , B Fold change in the tumor volume in female nude mice bearing either PR-1 ( A ) or PR-4 ( B ) after treatment with combination of palbociclib (palbo) with TTI-101, and vehicle up to 74 days (PR-1) and 52 days (PR-4). The Tukey multiple comparisons test determined p values. C , D Relative change in tumor volume at the treatment endpoint for PR-1 ( C ) and PR-4 ( D ) for each individual mouse. E , F Event-free survival analysis of PR-1 ( E ) and PR-4 ( F ) PDX models treated as described in A-B, calculated based on the days on treatment when fold change in tumor volume reached 1.5 (PR-1) or 7 (PR-4). The Mantel-Cox log-rank test determined p values. G , H Quantification (left panel) and representative images (right panel) of Ki67 immunohistochemical staining at the treatment endpoint. The Tukey multiple comparisons test determined p values. I , J White blood cell count (WBC), red blood cell count (RBC), and neutrophils measured from blood collected at the treatment endpoint. Gray area indicates normal ranges for non–tumor-bearing female nude mice. K , L Change in body weight at the indicated days over the course of treatment. M , N Level of TTI-101 accumulation (ng/g) evaluated by LC-MS/MS in tumor lysates collected 4 h after the last drug was administered at the treatment endpoint in PR-1 and PR-4. ND=not detected. O . Quantification (left panel) and representative images (right panel) of pSTAT3 (Y705) immunohistochemical staining at treatment end point with the indicated treatments. Tukey’s multiple comparisons test determined p -values. * p < 0.05. P , Q Luminex analysis of tumor lysates for pSTAT3 and STAT3 at treatment end point with the indicated treatments in PR-1 (P) and PR-4 (Q). MFI = Mean Fluorescent Intensity, * p < 0.05, tumor lysates were normalized to 1ug/uL. R Tumor human IL-21 (hIL-21) levels as measured by the ProcartaPlex Human Immune Monitoring 65-plex Luminex analysis of PR-1 and PR-4 PDX tumor lysate samples collected at the treatment endpoint. S Hierarchical clustering heat map with the z-scores of significant differentially expressed proteins with adjusted p < 0.3 between combination (Palbo+TTI-101) and vehicle from reverse-phase protein array analysis of PR-1 PDX tumor lysates collected at the treatment endpoint. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Palbociclib isethionate (MedChemExpress) was diluted in 0.5 W/V% methyl cellulose 400 (FUJIFILM Wako Chemicals), TTI-101 was diluted in 60:40 Labrasol:PEG400 with brief sonication for about 2 min, and vehicle was a 50:50 mixture of 0.5 W/V% methyl cellulose 400 and 60:40 Labrasol:PEG400.

Techniques: Immunohistochemical staining, Staining, Cell Characterization, Liquid Chromatography with Mass Spectroscopy, Luminex, Protein Array

Journal: Cancer research

Article Title: Kir2.1 Interaction with Stk38 Promotes Invasion and Metastasis of Human Gastric Cancer by Enhancing MEKK2–MEK1/2–ERK1/2 Signaling

doi: 10.1158/0008-5472.CAN-17-3776

Figure Lengend Snippet: Kir2.1 activates MEK1/2–ERK1/2–Snail–EMT pathway independent of Raf. A, Human phosphokinase antibody array assay showing 11 elevated phosphorylation kinases (left) with ERK1/2 kinases at the highest level (right). Data are shown as mean. B, The over-Kir2.1 gastric cancer cells showing enhanced phosphorylation of ERK1/2 and sh-Kir2.1 gastric cancer cells showing attenuated phosphorylation of ERK1/2. JNK and p38, the other two members of MAPKs in gastric cancer cells, were unaffected by Kir2.1 expression status. C, Decreased invasion capability of gastric cancer cells after treatment with PD98059 (10 μmol/L), a MEK1/2-specific inhibitor. Data are shown as mean ± SD (n = 5; NS, not significant; ***, P < 0.0001, ANOVA test). D, Increased E-cadherin but decreased vimemtin, Snail, and pERK1/2 in PD98059 (10 μmol/L)-treated gastric cancer cells. E, Attenuation of the invasion capability of control gastric cancer cells by treatment with LY3009120 (20 μmol/L), a pan-Raf inhibitor, without effect on over-Kir2.1 gastric cancer cells. Data are shown as mean ± SD (n = 5; ***, P < 0.0001, Student t test). F, Attenuation of the level of pMEK1/2 and pERK1/2 in control gastric cancer cells by LY3009120 (20 μmol/L) treatment, without effect on over-Kir2.1 gastric cancer cells.

Article Snippet: To examine the affectation of Kir2.1 agonist and antagonist and signaling inhibitors on the invasiveness of gastric cancer cells, the Matrigel-transwell system were cultured with or without ML133 (20 μmol/L, TOCRIS) or zacopride (10 μmol/L, Sigma-Aldrich) or PD98059 (10 μmol/L, Selleck Chemicals) or LY3009120 (20 μmol/L, Selleck Chemicals).

Techniques: Ab Array, Phospho-proteomics, Expressing, Control