Journal: Nature Communications

Article Title: Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer

doi: 10.1038/s41467-021-25422-9

Figure Lengend Snippet: The effect of fulvestrant (Fulv, 100 nM), CDK4/6 inhibitor (CDK4/6i, palbociclib, 200 nM in the MCF-7 and T47D cell models; 5 µM in the ZR-75-1 cell model) and AKT inhibitor (AKTi, capivasertib, 500 nM in the MCF-7 cell model; 200 nM in the T47D model; 150 nM in the ZR-75-1 cell model), as single agents or in double and triple combinations, was assessed in all cell lines by crystal violet growth assay ( A , B , E , F , I and J ) and CellTiter-Blue viability assay ( C , D , G , H , K , and L ) performed over 6 days. Outgrowth of resistant colonies was investigated in MCF-7 ( M and N ) and T47D models ( O and P ) by weekly evaluation of the percentage of 48 wells at 50% or greater confluence (positive wells) over 12 weeks. Experiments were conducted in three biological replicates and data are shown as mean ± SEM. Asterisks indicate significant differences in the one-way ANOVA test at day 6 (*0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, and **** p < 0.0001).

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in ethanol 96%, AKTi capivasertib (HY-15431, MedchemExpress), CDK4/6i ribociclib succinate hydrate (HY-15777C, MedchemExpress), CDK4/6i abemaciclib (HY-16297A, MedchemExpress), and dual PI3K/mTORi gedatolisib (#HY-10681, MedchemExpress) were dissolved in DMSO (Sigma-Aldrich) and CDK4/6i palbociclib isothiocyanate (HY-A0065, MedchemExpress) was dissolved in water.

Techniques: Growth Assay, Viability Assay

Journal: Nature Communications

Article Title: Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer

doi: 10.1038/s41467-021-25422-9

Figure Lengend Snippet: Apoptotic levels were determined by evaluating the presence of cytoplasmic nucleosomes in MCF-7 ( A ), T47D ( B ), and ZR-75-1 ( C ) cells using an ELISA cell death detection assay. Data are shown with error bars representing mean ± SEM of three biological replicates. Asterisks indicate significant differences in the one-way ANOVA test (*0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, and **** p < 0.0001). D Western blot analysis of apoptosis markers in the three fulvestrant-resistant breast cancer models. Densitometry analysis of Western blot bands of cleaved-PARP was performed using ImageJ software. Data are shown as the area under the curve (AUC) normalized to loading control. E Western blot analysis of key signal transduction proteins in the three fulvestrant-resistant breast cancer models. F Western blotting analysis of p-AKT (S473) and total AKT expression in the three fulvestrant-resistant breast cancer models. Densitometry analysis of Western blot bands of p-AKT (S473) and total AKT was performed using ImageJ software. Data are shown as the area under the curve (AUC) normalized to loading control. GAPDH was used as a loading control for Western blotting analysis. For all Western blots, a representative of two biological replicates is shown. Both apoptosis assay and harvesting protein for Western blotting analysis were performed 3 days after treatment with fulvestrant (Fulv, 100 nM), CDK4/6 inhibitor (CDK4/6i, palbociclib, 200 nM in the MCF-7 and T47D cell models; 5 µM in the ZR-75-1 cell model) and AKT inhibitor (AKTi, capivasertib, 500 nM in the MCF-7 cell model; 200 nM in the T47D model; 150 nM in the ZR-75-1 cell model).

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in ethanol 96%, AKTi capivasertib (HY-15431, MedchemExpress), CDK4/6i ribociclib succinate hydrate (HY-15777C, MedchemExpress), CDK4/6i abemaciclib (HY-16297A, MedchemExpress), and dual PI3K/mTORi gedatolisib (#HY-10681, MedchemExpress) were dissolved in DMSO (Sigma-Aldrich) and CDK4/6i palbociclib isothiocyanate (HY-A0065, MedchemExpress) was dissolved in water.

Techniques: Enzyme-linked Immunosorbent Assay, Detection Assay, Western Blot, Software, Control, Transduction, Expressing, Apoptosis Assay

Journal: Nature Communications

Article Title: Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer

doi: 10.1038/s41467-021-25422-9

Figure Lengend Snippet: Tumor growth curves of MCF-7 ( A ) and 182R-1 ( B ) tumors following treatment with fulvestrant (Fulv, 100 mg/Kg bodyweight; N = 7) or vehicle (castor oil, N = 8) administered subcutaneously once a week. Treatment was initiated when tumors reached 50 mm 3 and continued for 5 weeks. C Mice were sacrificed on week 5 and MCF-7 and 182R-1 tumors were excised. Tumor growth curves of 182R-1 tumors treated with CDK4/6 inhibitor (CDK4/6i, palbociclib, 50 mg/Kg bodyweight; N = 8) alone ( D ), in combination with fulvestrant (100 mg/Kg bodyweight; N = 7 and N = 10) ( D and E ), or in combination with both AKT inhibitor (AKTi, capivasertib 100 mg/Kg bodyweight) and fulvestrant (100 mg/Kg bodyweight; N = 6 and N = 10) ( D and E ), or vehicle (castor oil and 25% w/v HPB cyclodextrin; N = 8) ( D ). CDK4/6i and AKTi were administered by oral gavage once daily for 5 days a week when tumors reached 50 mm 3 ( D ) or 250 mm 3 ( E ), and treatment was continued for up to 8 weeks. Data are shown as mean tumor volume ± SEM. Asterisks indicate a significant difference in ANOVA one-way test ( D ) or two-tailed t -test ( A , B and E ) at the endpoint (*0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001 and **** p < 0.0001).

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in ethanol 96%, AKTi capivasertib (HY-15431, MedchemExpress), CDK4/6i ribociclib succinate hydrate (HY-15777C, MedchemExpress), CDK4/6i abemaciclib (HY-16297A, MedchemExpress), and dual PI3K/mTORi gedatolisib (#HY-10681, MedchemExpress) were dissolved in DMSO (Sigma-Aldrich) and CDK4/6i palbociclib isothiocyanate (HY-A0065, MedchemExpress) was dissolved in water.

Techniques: Two Tailed Test

Journal: Nature Communications

Article Title: Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer

doi: 10.1038/s41467-021-25422-9

Figure Lengend Snippet: The effect of fulvestrant (Fulv, 100 nM), CDK4/6 inhibitor (CDK4/6i, palbociclib, 200 nM) and AKT inhibitor (AKTi, capivasertib, 250–500 nM in the MCF-7 cell model; 100 nM in the T47D cell model), as single agents or in the double and triple combination, was assessed in all cell lines by crystal violet growth assay ( A , B , E , and F ) and CellTiter-Blue viability assay ( C , D , G , and H ) performed over 6 days. Outgrowth of resistant colonies was investigated in MPF-R ( I ) and TPF-R ( J ) cells by weekly evaluation of the percentage of 48 wells at 50% or greater confluence (positive wells) over 12 weeks. Experiments were conducted in three biological replicates and data are shown as mean ± SEM. Asterisks indicate significant differences in one-way ANOVA tests at day 6 (*0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, and **** p < 0.0001).

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in ethanol 96%, AKTi capivasertib (HY-15431, MedchemExpress), CDK4/6i ribociclib succinate hydrate (HY-15777C, MedchemExpress), CDK4/6i abemaciclib (HY-16297A, MedchemExpress), and dual PI3K/mTORi gedatolisib (#HY-10681, MedchemExpress) were dissolved in DMSO (Sigma-Aldrich) and CDK4/6i palbociclib isothiocyanate (HY-A0065, MedchemExpress) was dissolved in water.

Techniques: Growth Assay, Viability Assay

Journal: Nature Communications

Article Title: Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer

doi: 10.1038/s41467-021-25422-9

Figure Lengend Snippet: Apoptotic levels were determined by evaluating the presence of cytoplasmic nucleosomes in MCF-7 ( A ) and T47D ( B ) cell models using an ELISA cell death detection assay. Data are shown with error bars representing mean ± SEM of three biological replicates. Asterisks indicate significant differences in one-way ANOVA tests (*0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, and **** p < 0.0001). C Western blot analysis of apoptosis markers in both models resistant to combined fulvestrant and CDK4/6i. Densitometry analysis of Western blot bands of cleaved-PARP was performed using ImageJ software. Data are shown as the area under the curve (AUC) normalized to loading control. D Western blot analysis of key signal transduction proteins in breast cancer cell models resistant to combined CDK4/6i and fulvestrant therapy. E Western blotting analysis of p-AKT (S473) and total AKT expression in both breast cancer models resistant to combined fulvestrant and CDK4/6i. Densitometry analysis of Western blot bands of p-AKT (S473) and total AKT was performed using ImageJ software. Data are shown as the area under the curve (AUC) normalized to loading control. GAPDH was used as a loading control for Western blotting analysis. For all Western blots, a representative of two biological replicates is shown. Both apoptosis assay and harvesting protein for Western blotting analysis were performed 3 days after treatment with fulvestrant (Fulv, 100 nM), CDK4/6 inhibitor (CDK4/6i, palbociclib, 200 nM), and AKT inhibitor (AKTi, capivasertib, 250 nM in the MCF-7 cell model; 100 nM in the T47D cell model).

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in ethanol 96%, AKTi capivasertib (HY-15431, MedchemExpress), CDK4/6i ribociclib succinate hydrate (HY-15777C, MedchemExpress), CDK4/6i abemaciclib (HY-16297A, MedchemExpress), and dual PI3K/mTORi gedatolisib (#HY-10681, MedchemExpress) were dissolved in DMSO (Sigma-Aldrich) and CDK4/6i palbociclib isothiocyanate (HY-A0065, MedchemExpress) was dissolved in water.

Techniques: Enzyme-linked Immunosorbent Assay, Detection Assay, Western Blot, Software, Control, Transduction, Expressing, Apoptosis Assay

Journal: Nature Communications

Article Title: Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer

doi: 10.1038/s41467-021-25422-9

Figure Lengend Snippet: A Tumor growth curves of orthotopic MPF-R tumors treated with a CDK4/6 inhibitor (CDK4/6i, palbociclib, 50 mg/Kg bodyweight) combined with fulvestrant (Fulv, 100 mg/Kg bodyweight; N = 9) or in combination with both AKT inhibitor (AKTi, capivasertib, 100 mg/Kg body weight) and fulvestrant (Fulv, 100 mg/Kg bodyweight; N = 10), or vehicle (castor oil and 25% w/v HPB cyclodextrin; N = 10). CDK4/6i and AKTi were administered by oral gavage once daily for 5 days a week, whereas fulvestrant was administered subcutaneously once a week. Treatment was initiated when tumors reached 100 mm 3 and continued for up to 7 weeks. Mice from the control group were sacrificed and tumors excised on week 6 due to their large size, while mice from double and triple combination groups were sacrificed and tumors excised on week 7. Data are shown as mean tumor volume ± SEM. Asterisks indicate significant differences in the two-tailed t -test at the endpoint (*0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, and **** p < 0.0001). B Western blot analysis of key signal transduction proteins in 3 tumors of each treatment group excised when mice were sacrificed. GAPDH was used as a loading control. A representative of two independent experiments is shown. C Tumor volumes over time of the parental PDX KCC_P_3837 untreated (blue) and treated with combined CDK4/6i palbociclib (25 mg/kg in 2.5% DMSO, 25% β-cyclodextrin, 5 days per week by oral gavage) and fulvestrant (100 mg/kg in castor oil, once weekly via subcutaneous injection) (orange), and of the derivative PDX KCC_P_3837-FPR resistant to combined palbociclib and fulvestrant (red) at the third passage of continuous exposure to combined palbociclib and fulvestrant. D Kaplan-Meier survival plot of progression of PDX KCC_P_3837-FPR (resistant to combined CDK4/6i and fulvestrant) under treatment with combined CDK4/6i and fulvestrant with or without AKTi capivasertib (100 mg/kg in 2.5% DMSO, 25% β-cyclodextrin, 5 days per week by oral gavage) ( N = 5 and N = 4, respectively). Progression was defined as tumors growing to at least 5 mm in the shortest dimension. A two-sided p- value ( p < 0.05) was calculated using log-rank testing. E , F Evaluation of metastasis area > 2500 µm 2 relative to lung area at the endpoint (6 weeks) using an experimental metastasis model. MPF-R tumors were treated with fulvestrant (100 mg/Kg body weight) combined with CDK4/6i (palbociclib, 25 mg/Kg bodyweight; N = 10), fulvestrant combined with AKTi (capivasertib, 100 mg/Kg bodyweight; N = 10), or triple combination ( N = 9). TPF-R tumors were treated with the same dosage of fulvestrant and CDK4/6i and 50 mg/Kg bodyweight of AKTi ( N = 7 in fulvestrant and CDK4/6i group, N = 10 in fulvestrant and AKTi and N = 10 in triple combination group). CDK4/6i and AKTi were administered by oral gavage once daily for 5 days a week, while fulvestrant was administered subcutaneously once a week. Treatment was initiated 3 days before injection of cells in the tail vein and continued for up to 6 weeks. Data are shown as mean ± SEM. Significant differences were evaluated by the two-tailed Mann-Whitney test. G Representative micrographs of MPF-R and TPF-R tumors in mice lungs of each treatment group showing cytokeratin expression by immunohistochemistry (scale bars, 200 µm).

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in ethanol 96%, AKTi capivasertib (HY-15431, MedchemExpress), CDK4/6i ribociclib succinate hydrate (HY-15777C, MedchemExpress), CDK4/6i abemaciclib (HY-16297A, MedchemExpress), and dual PI3K/mTORi gedatolisib (#HY-10681, MedchemExpress) were dissolved in DMSO (Sigma-Aldrich) and CDK4/6i palbociclib isothiocyanate (HY-A0065, MedchemExpress) was dissolved in water.

Techniques: Control, Two Tailed Test, Western Blot, Transduction, Injection, MANN-WHITNEY, Expressing, Immunohistochemistry

Journal: Nature Communications

Article Title: Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer

doi: 10.1038/s41467-021-25422-9

Figure Lengend Snippet: Clinical and pathological characteristics of ER+ breast cancer patients with advanced disease treated with combined CDK4/6i and endocrine treatment from pilot and validation cohorts according to p-AKT levels.

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in ethanol 96%, AKTi capivasertib (HY-15431, MedchemExpress), CDK4/6i ribociclib succinate hydrate (HY-15777C, MedchemExpress), CDK4/6i abemaciclib (HY-16297A, MedchemExpress), and dual PI3K/mTORi gedatolisib (#HY-10681, MedchemExpress) were dissolved in DMSO (Sigma-Aldrich) and CDK4/6i palbociclib isothiocyanate (HY-A0065, MedchemExpress) was dissolved in water.

Techniques:

Journal: Nature Communications

Article Title: Co-targeting CDK4/6 and AKT with endocrine therapy prevents progression in CDK4/6 inhibitor and endocrine therapy-resistant breast cancer

doi: 10.1038/s41467-021-25422-9

Figure Lengend Snippet: A Kaplan-Meier plots evaluating progression-free survival (PFS) according to p-AKT (S473) levels in ER+ metastatic lesions from a validation cohort of ER+breast cancer patients treated with combined CDK4/6i and endocrine therapy in the advanced setting. A two-sided p -value ( p < 0.05) was calculated using log-rank testing. Representative micrographs of all breast cancer metastasis sections showing low p-AKT expression (H-score < 150, B and C ) or high p-AKT expression (H-score ≥ 150, D and E ; scale bars, 100 µm).

Article Snippet: Fulvestrant (ICI 182,780, Tocris) was dissolved in ethanol 96%, AKTi capivasertib (HY-15431, MedchemExpress), CDK4/6i ribociclib succinate hydrate (HY-15777C, MedchemExpress), CDK4/6i abemaciclib (HY-16297A, MedchemExpress), and dual PI3K/mTORi gedatolisib (#HY-10681, MedchemExpress) were dissolved in DMSO (Sigma-Aldrich) and CDK4/6i palbociclib isothiocyanate (HY-A0065, MedchemExpress) was dissolved in water.

Techniques: Expressing