Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting PEG10 as a novel therapeutic approach to overcome CDK4/6 inhibitor resistance in breast cancer

doi: 10.1186/s13046-023-02903-x

Figure Lengend Snippet: Characterization of palbociclib-resistant cells with high PEG10 expression and EMT process activation. A Venn diagram representing the number of upregulated genes in MCF7-PR and T47D-PR cells. A gene showing fold change ≥ 2 compared with that in parental cells (MCF7 and T47D) is considered an upregulated gene. B Heat map showing the list of commonly upregulated genes in MCF7-PR and T47D-PR cells. C mRNA expression of PEG10 in the palbociclib-resistant (MCF7-PR and T47D-PR) compared with their corresponding parental (MCF7 and T47D) cells by qRT-PCR. Three independently repeated experiments were performed with similar results. Independent sample t-test: * p < 0.05, ** p < 0.01. D Immunoblots showing PEG10 (RF1) and PEG10 (FR2) protein expression in the palbociclib-resistant (MCF7-PR and T47D-PR) and parental (MCF7 and T47D) cells. E Association of PEG10 expression and palbociclib sensitivity using GDSC database. Palbociclib sensitivity was defined as IC 50 ≤ 3.5 µM. P- value was calculated by independent sample t-test. F A panel of genes associated with EMT process from microarray data analysis in palbociclib-resistant (MCF7-PR and T47D-PR) versus parental (MCF7 and T47D) cells. G mRNA expression of EMT markers in the palbociclib-resistant (MCF7-PR and T47D-PR) cells compared with their corresponding parental (MCF7 and T47D) cells by qRT-PCR. Three independently repeated experiments were performed with similar results. Independent sample t-test: * p < 0.05, ** p < 0.01, *** p < 0.001, Abbreviation: ns, not significant. H Immunoblots showing protein expression of mesenchymal markers (ZEB1 & LAMC2), and epithelial marker (E-cadherin) in MCF7-PR and T47D-PR cells compared with MCF7 and T47D cells, respectively. I Association of ZEB1 expression and palbociclib sensitivity in 11 HR+ breast cancer cell lines from GDSC database. Palbociclib sensitivity was defined as IC 50 ≤ 3.5 µM. P- value was calculated by independent sample t-test

Article Snippet: The section was stained with PEG10 primary antibody (cat# 14412–1-AP, Proteintech, USA) or Ki67 primary antibody (cat# ab92742, abcam, USA) at 1:200 dilution for 12 h at 4 °C.

Techniques: Expressing, Activation Assay, Quantitative RT-PCR, Western Blot, Microarray, Marker

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting PEG10 as a novel therapeutic approach to overcome CDK4/6 inhibitor resistance in breast cancer

doi: 10.1186/s13046-023-02903-x

Figure Lengend Snippet: Ectopic overexpression of PEG10 augments EMT and leads to palbociclib resistance. A Immunoblot demonstrates ectopic overexpression of different PEG10 protein isoforms in MCF7 and T47D cells. Ectopic overexpression of PEG10 elevated the ZEB1 and suppressed the E-cadherin expression. B , C Representative images from a migration and invasion assay after ectopic overexpression of different PEG10 protein isoforms in MCF7 and T47D cells. The area of migratory and invading cells from three different non-overlapping 100 × microscopic fields is expressed as mean ± SD in the right panel. Independent sample t-test: * p < 0.05, ** p < 0.01, *** p < 0.001. D - I Cell viability (MTT) assay of MCF7 and T47D cells before and after ectopic overexpression of PEG10-RF1 and subsequent treatment with the indicated dose of palbociclib, abemaciclib, and ribociclib for 72 h. Three independently repeated experiments were performed with similar results. Independent sample t-test: * p < 0.05, ** p < 0.01, *** p < 0.001, Abbreviation: ns, not significant. J , K Cell cycle distribution of MCF7 and T47D cell line before and after the ectopic overexpression of different PEG10 protein isoforms and subsequent treatment with the IC 50 concentration of palbociclib for 48 h. Three independently repeated experiments were performed with similar results. Independent sample t-test: ** p < 0.01, Abbreviation: ns, not significant

Article Snippet: The section was stained with PEG10 primary antibody (cat# 14412–1-AP, Proteintech, USA) or Ki67 primary antibody (cat# ab92742, abcam, USA) at 1:200 dilution for 12 h at 4 °C.

Techniques: Over Expression, Western Blot, Expressing, Migration, Invasion Assay, MTT Assay, Concentration Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting PEG10 as a novel therapeutic approach to overcome CDK4/6 inhibitor resistance in breast cancer

doi: 10.1186/s13046-023-02903-x

Figure Lengend Snippet: PEG10 inhibition suppresses EMT and overcomes palbociclib resistance. A , B Immunoblot showed changes in the ZEB1 and E-cadherin expression after the knockdown of PEG10 using PEG10 siRNA and PEG10-ASO in MCF7-PR and T47D-PR cells. C Representative images from a migration and invasion assay of parental (MCF7 and T47D) versus palbociclib-resistant cells (MCF7-PR and T47D-PR), respectively. The area of migratory and invading cells from three different non-overlapping 100 × microscopic fields is expressed as mean ± SD in the right panel. Independent sample t-test: * p < 0.05, ** p < 0.01, *** p < 0.001. D Representative image from a migration and invasion assay of palbociclib-resistant (MCF7-PR and T47D-PR) cells after the transient PEG10 knockdown by PEG10 siRNA. The area of migratory and invading cells from three different non-overlapping 100 × microscopic fields is expressed as mean ± SD in the right panel. Independent sample t-test: * p < 0.05, ** p < 0.01, *** p < 0.001. E Cell cycle analysis using PI staining after PEG10 knockdown in MCF7-PR and T47D-PR cells. The cell cycle was initially synchronized (syn) at G0/G1 with a double-thymidine block and then released and analyzed at the indicated time points. The bar represents the cell population distribution in each phase of the cell cycle. F Cell viability (MTT) assay of MCF7-PR and T47D-PR cells after treatment with PEG10 siRNA or palbociclib and combination of various concentrations of palbociclib and a fixed concentration of siRNA for 72 h. Three independently repeated experiments were performed with similar results. G Cell viability (MTT) assay of MCF7-PR and T47D-PR cells after treatment with PEG10-ASO or palbociclib and combination of various concentrations of palbociclib and a fixed concentration of ASO for 72 h. Three independently repeated experiments were performed with similar results. H Immunoblot showed induction of cleaved caspase-3 in the combination treatment of PEG10 siRNA and palbociclib. I Immunoblot showed ZEB1 inhibition by ZEB1 shRNA for 48 h. J Cell viability (MTT) assay of MCF7-PR and T47D-PR cells after treatment with ZEB1 shRNA or palbociclib and combination of various concentrations of palbociclib and a fixed concentration of ZEB1 shRNA for 72 h. Three independently repeated experiments were performed with similar results

Article Snippet: The section was stained with PEG10 primary antibody (cat# 14412–1-AP, Proteintech, USA) or Ki67 primary antibody (cat# ab92742, abcam, USA) at 1:200 dilution for 12 h at 4 °C.

Techniques: Inhibition, Western Blot, Expressing, Knockdown, Migration, Invasion Assay, Cell Cycle Assay, Staining, Blocking Assay, MTT Assay, Concentration Assay, shRNA

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting PEG10 as a novel therapeutic approach to overcome CDK4/6 inhibitor resistance in breast cancer

doi: 10.1186/s13046-023-02903-x

Figure Lengend Snippet: PEG10 suppresses natural cell cycle inhibitor p21 and EMT process inhibitor SIAH1. A Immunoblot showed changes in the cell cycle-related protein p21 and ubiquitin-related protein SIAH1 expression in parental (MCF7 and T47D) versus palbociclib-resistant (MCF7-PR and T47D-PR) cells. B , C Immunoblot showed p21 and SIAH1 protein expression level after the transient PEG10 knockdown by using PEG10 siRNA or PEG10-ASO. D , E Cell viability (MTT) assay of MCF7-PR and T47D-PR cells before and after ectopic overexpression of p21 and subsequent treatment with the various concentrations of palbociclib for 72 h. Three independently repeated experiments were performed with similar results. Independent sample t-test: * p < 0.05, ** p < 0.01, *** p < 0.001, Abbreviation: ns, not significant. F , G Cell viability (MTT) assay of MCF7-PR and T47D-PR cells before and after ectopic overexpression of SIAH1 and subsequent treatment with the various concentrations of palbociclib for 72 h. Three independently repeated experiments were performed with similar results. Independent sample t-test: * p < 0.05, ** p < 0.01, *** p < 0.001, Abbreviation: ns, not significant. H Immunoblot showed the depletion of cyclins (cyclin E, cyclin D1, and cyclin A) and CDK2 after the ectopic overexpression of p21 in MCF7-PR and T47D-PR cells. I Immunoblot showed the differential expression of cyclins (cyclin E, cyclin D1, and cyclin A) and CDK2 in parental cells (MCF7 and T47D) versus palbociclib-resistant cells (MCF7-PR and T47D-PR), respectively. J Immunoblot showed the depletion of cyclins (cyclin E, cyclin D1, and cyclin A) and CDK2 after PEG10 knockdown by PEG10 siRNA in MCF7-PR and T47D-PR cells. K , L Immunoblot showed the expression of ZEB1 and E-cadherin after the ectopic overexpression of p21 and SIAH1 in MCF7-PR and T47D-PR cells

Article Snippet: The section was stained with PEG10 primary antibody (cat# 14412–1-AP, Proteintech, USA) or Ki67 primary antibody (cat# ab92742, abcam, USA) at 1:200 dilution for 12 h at 4 °C.

Techniques: Western Blot, Ubiquitin Proteomics, Expressing, Knockdown, MTT Assay, Over Expression, Quantitative Proteomics

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting PEG10 as a novel therapeutic approach to overcome CDK4/6 inhibitor resistance in breast cancer

doi: 10.1186/s13046-023-02903-x

Figure Lengend Snippet: Combined treatment of palbociclib and PEG10-ASO regresses the palbociclib-resistant breast cancer synergistically in a xenograft model. A Experimental design for in vivo efficacy test using acquired palbociclib-resistant xenograft model in BALB/c nude mice. When tumor reached 60–80 mm3, mice were randomized to control-ASO ( n = 5), palbociclib ( n = 5), PEG10-ASO ( n = 5), and combination ( n = 5) treatment groups. Control-ASO and PEG10-ASO were administrated intraperitoneally (i.p.) for 3 weeks (15 mg/kg/day for first 5 days loading, followed by 17 days of maintenance dose of 15 mg/kg, 3 times/week). Palbociclib (100 mg/kg/day) was given by oral gavage for 3 weeks. B Gross harvested tumor. The black dotted circles represent the complete regression of the tumor. C Average tumor sizes of the indicated treatment groups before sacrifice. Independent sample t-test: *** p < 0.001, Abbreviation: ns, not significant. D Average tumor growth of MCF7-PR xenograft tumor treated with indicated drugs. Error bars represent SD of 5 tumors per group. Independent sample t-test: *** p < 0.001, Abbreviation: ns, not significant. E Average body weight of mice with indicated groups. Error bars represent the SD of 5 mice per group. F Immunoblots using xenograft tumors showed the suppression of PEG10 in the PEG10-ASO and combination treatment groups, along with altered ZEB1 and E-cadherin expression. G IHC staining of PEG10 in MCF7-PR xenograft tumors of indicated groups. Staining images were taken at 400 × magnification. The bar graph represented the PEG10 protein expression in five random, non-overlapped fields. Data are presented as mean ± SD. Independent sample t-test: *** p < 0.001, Abbreviation: ns, not significant. H IHC staining of Ki67 in MCF7-PR xenograft tumors of indicated groups. Staining images were taken at 400 × magnification. The bar graph represented the Ki67 cells in five random, non-overlapped fields. Data are presented as mean ± SD. Independent sample t-test: * p < 0.05, *** p < 0.001. I TUNEL assay using xenograft tumors at sacrifice. Staining images in each group are shown and bar graphs represent average apoptotic cells in each group in five random, non-overlapped fields at 400 × magnification. Data are presented as mean ± SD. Independent sample t-test: ** p < 0.01, *** p < 0.001, Abbreviation: ns, not significant

Article Snippet: The section was stained with PEG10 primary antibody (cat# 14412–1-AP, Proteintech, USA) or Ki67 primary antibody (cat# ab92742, abcam, USA) at 1:200 dilution for 12 h at 4 °C.

Techniques: In Vivo, Control, Western Blot, Expressing, Immunohistochemistry, Staining, TUNEL Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting PEG10 as a novel therapeutic approach to overcome CDK4/6 inhibitor resistance in breast cancer

doi: 10.1186/s13046-023-02903-x

Figure Lengend Snippet: PEG10 overexpression is associated with disease recurrence and CDK4/6 inhibitor resistance in breast cancer patients. A Kaplan–Meier survival curves of RFS in HR+ breast cancer according to relative PEG10 mRNA expression from public mRNA microarray data set of GSE 6532. GSE 6532 analysis was performed using self-downloaded processed data. B - D Kaplan–Meier survival curves of RFS in HR+ breast cancer according to relative PEG10 mRNA expression from public mRNA microarray data sets of B GSE 2034, C E-MTAB-365, and D GSE 3494. Analyses of these data sets were performed using online platform Kaplan–Meier plotter ( https://kmplot.com/analysis/ ). E Univariate and multivariate analysis of prognosticators for RFS in GSE 6532. F IHC staining of PEG10 in patients’ tumor specimens. Negative control (placental tissue, without primary antibody) and positive control (placental tissue, with primary antibody) are shown in the left panel. Representative images of negative and positive PEG10 IHC in pre-CDK4/6 inhibitor treatment tissue are shown in the right panel. G Kaplan–Meier survival curves of PFS according to PEG10 expression by IHC in pre-CDK4/6 inhibitor treatment tissue. H Schematic diagram showing the proposed mechanism that explains how PEG10 is associated with CDK4/6 inhibitors resistance. Abbreviations: RFS, recurrence-free survival; HR (in table of figure), hazard ratio; PFS, progression-free survival; Cut-off for PEG10-High versus PEG10-Low is the upper quartile

Article Snippet: The section was stained with PEG10 primary antibody (cat# 14412–1-AP, Proteintech, USA) or Ki67 primary antibody (cat# ab92742, abcam, USA) at 1:200 dilution for 12 h at 4 °C.

Techniques: Over Expression, Expressing, Microarray, Immunohistochemistry, Negative Control, Positive Control