Journal: Genes & diseases

Article Title: ITGA2 promotes expression of ACLY and CCND1 in enhancing breast cancer stemness and metastasis.

doi: 10.1016/j.gendis.2020.01.015

Figure Lengend Snippet: Figure 7 ITGA2 pathway is associated with outcomes of the patients with breast cancer. (A-C) Breast Cancer KaplaneMeier Plotter analyses of RFS correlated with CCND1 expression (a, Log rank P Z 0.043, n Z 701), and ACLY expression (b, Log rank P Z 0.001, n Z 407), and OS associated with miR-206 expression (c, Log rank P Z 0.002, n Z 159) in ER-breast cancer with best cut off. (D-F) KM plots of RFS correlation with ITGA2 expression in ER- (Log rank p Z 0.0406, n Z 189) (D), grade 3 ER- (Log rank P Z 0.048, n Z 347) (E), and all grade 3 (Log rank P Z 0.037, n Z 444) (F) breast cancer, with best cut off selected in Breast Cancer KaplaneMeier Plotter analyses. (G) Breast cancer miner plot of any-event (AE) free survival association with ITGA2 expression in TNBC. Log rank P Z 0.02, n Z 1258, median cut off. (H) Box plot for differential ACLY and CCND1 expression levels between clustered and single CTCs of breast cancer patients and PDXs (GSE109761 and GSE111065).16,31 Wilcox t test P Z 0.004 and 0.024 for ACLY and CCND1 comparisons, respectively. (I) Schematic CD49b signaling pathway induced by extracellular matrix factors such as collagen I fibers that result in the phosphorylation of FAK and upregulation of CCND1 and ACLY levels to promote proliferation, stemness, and metastasis of TNBC.

Article Snippet: Antibodies used: Cd49b (Rabbit pAb, Thermo Fisher Scientific PA5-26061), pFAK, total FAK (Rabbit, Cell Signaling, mAb 8556 and pAB 3285), b-actin (Mouse mAb, Abcam ab8224).

Techniques: Expressing, Phospho-proteomics

Journal: Biochimica et biophysica acta. General subjects

Article Title: Extracellular matrix stiffness regulates degradation of MST2 via SCF βTrCP .

doi: 10.1016/j.bbagen.2022.130238

Figure Lengend Snippet: Fig. 4. Perturbation of ILK and the actomyosin cytoskeleton regulates MST2 levels: A) Immunoblots of MCF10A cells treated with an Integrin-Linked Kinase (ILK) inhibitor (CPD022) for 1, 3 and 6 h. AKT phosphorylation was detected to assess the efficiency of ILK inhibition. β-actin was used as loading control. B) Immunoblot for MST2 of MCF10A cells treated with FAK inhibitor (FAKi 14) for 1, 3 and 6 h. FAK phosphorylation was detected to monitor its inhibition. β-actin was used as loading control. C) Immunoblots of MCF10A cells treated with CPD022 or MLN4294 in combination with MnCl2 for 6 h. Both inhibition of CUL1 neddylation and ILK reversed the effect of integrin hyperactivation. β-actin was used as loading controls. Note that the band corresponding to CUL1 neddylation (indicated by an arrow) disappeared in the presence of MLN4924 indicating that the inhibition was efficient. D) Co-IP revealed that the interaction of endogenous MST2 and βTrCP decreases after ILK inhibition, despite integrin hyperactivation. MCF10A cells were treated with DMSO (ct), CPD022, MnCl2 or CPD022 concomitantly with MnCl2 for 6 h. Co-IP was performed with an MST2 antibody or a control IgG, followed by incubation with protein A/G-coated beads. βTrCP and MST2 were only detected in the MST2 immunoprecipitants. MST2 was detected to confirm the Co-IP. Arrows indicate bands corresponding to IgG that were detected by the secondary antibody. E) Immunoblot for MST2 of MCF10A cells treated with a myosin ATPase activity inhibitor (Blebbistatin, 2.5 μM) for 1, 3 and 6 h. β-actin was used as loading control. (A–E) Fold changes of protein levels and protein phosphorylation (phosphorylated/total) are shown under their respective immunoblots. F) Schematic depicting the pathway of MST2 degradation induced by ECM stiffness. In a stiff ECM, hyperactive integrin signaling results in ILK activation and actomyosin contraction leading to ubiquitylation of MST2 by SCFβTrCP and consequent MST2 degradation via proteasome 26S. This schematic was generated with Biorender.

Article Snippet: To screen the pathways mediating MST2 degradation, MCF10A cells were treated for 1, 3 and 6 h with the inhibitors for ILK (1 μM CPD022, Calbiochem, #407331), FAK (5 μM FAK inhibitor 14, Tocris # 3414), PI3K (30 μM LY294002, Gibco # PHZ1144), AKT (20 μM MK2206, Cayman Biochemicals #1032350-13-2) and myosin ATPase activity inhibitor (5 μM Blebbistatin, Tocris #1852).

Techniques: Western Blot, Phospho-proteomics, Inhibition, Control, Co-Immunoprecipitation Assay, Incubation, Activity Assay, Activation Assay, Generated