PMC

Integrin β5 promotes tumor growth and angiogenesis by facilitating matrix adhesion and signaling via Src-FAK and Ras-ERK pathways that may operate independently in carcinoma cells.

(A) RT-PCR of integrins αv, β1, β3, β5, and β6 in a panel of non-tumorigenic mammary epithelial and breast carcinoma cells treated with TGF-β1 (2–5ng/ml for 24h). In brackets is indicated the number of PCR cycles performed.(B) Immunoblot analysis of integrin β1 and integrin β5 in a panel of non-tumorigenic mammary epithelial and breast carcinoma cells. α-Tubulin is used as loading control. Anchorage-independent growth analysis of MDA-MB-231 (C) and MCF7 (D) treated with 100nM siRNA to integrin β5. Immunoblot analysis shows the effective suppression of integrin β5 expression.

(A) Immunoblot analysis of whole-cell protein extracts from control, shB5 and shB5-over-expressing integrin β5 MDA-MB-231 cells treated with TGF-β1 (2ng/ml) for 24h (upper panel) or 2h (lower panel). Membranes were probed with antibodies for FAK, phospho-FAK-Tyr861, phospho-paxillin-Ty31, paxillin, ERK1/2, phospho-ERK1/2, phospho-Smad2, and GAPDH as loading control. (B) Immunoblot analysis of whole-cell protein extracts from control and siRNA-ITGB5-treated BT549, MCF7, and T47D cells. Membranes were probed with antibodies for integrin β5, phospho-FAK-Tyr861, FAK, ERK 1/2, phospho-ERK1/2 and a-tubulin. (C) MDA-MB-231 (upper panel) and MCF10A (lower panel) cells were treated with the EGFR inhibitor AG1478 for 2h at the indicated concentrations. Immunoblot analysis was performed on whole-cell extracts probing for FAK, phospho-FAK-Tyr397, phospho-FAK-Tyr861, ERK1/2, and phospho-ERK1/2. (D) Immunoblot analysis of whole-cell protein extracts from control and shB5 cells, treated with EGF (100ng/ml) for 2h. Where indicated, a 1h pre-treatment with AG1478 (5μM) was performed. Membranes were probed for phospho-EGFR-Tyr845, total EGFR, phospho-ERK1/2, and total ERK1/2. (E) Immunoblotting of whole-cell protein extracts from control cells, treated with 100ng/ml EGF for 2h. Where indicated, a 1h pre-treatment with 100nM Dasatinib was done. Membranes were probed for phospho-EGFR-Tyr845 and EGFR.

(A) Anchorage-independent growth analysis of MDA-MB-231 cells treated with the FAK inhibitor PF573228, the Src inhibitor Dasatinib, or the MEK inhibitor U0126 at the indicated concentration. Statistical analysis by Student t-Test is provided (* p< 0.05; ** p<0.001). (B) MDA-MB-231 cells were treated with the FAK inhibitor PF573228 or the MEK inhibitor U0126 for 24h at the indicated concentrations. Immunoblotting was performed on whole-cell extracts probing for FAK, phospho-FAK-Tyr397, phospho-FAK-Tyr861, ERK1/2, and phospho-ERK1/2. (C) Immunoblot analysis of whole-cell protein extracts from control and siRNA to FAK treated MDA-MB-231 cells that were treated with 2ng/ml TGF-β1 for 2h and 24h. Membranes were probed with antibodies for FAK, phospho-Smad2, Smad2, phospho-ERK1/2, and GAPDH as loading control. (D) MDA-MB-231 cells were treated with the Src inhibitor Dasatinib at 100nM for the indicated times. Immunoblot analysis was performed on whole-cell extracts probing for FAK, phospho-FAK-Tyr397, phospho-FAK-Tyr861, phospho-FAK-Tyr925, phospho-paxillin-Tyr31, paxillin, ERK1/2, phospho-ERK1/2 and α-tubulin.

(A) Immunoblot analysis of whole-cell protein extracts from control, shRNA-ITGB5 (shB5) and shRNA-ITGB5-over-expressing integrin β5 (shB5-oeB5) MDA-MB-231 cells, probing for integrin β5 and integrin β1. GAPDH probing shows equal loading. (B) Anchorage-independent growth analysis of MDA-MB-231 cells control, expressing shRNA to integrin β5 or over-expressing integrin β5. Statistical analysis by Student t-Test is provided (** p<0.001). (C) Flow cytometry analysis for cell surface presentation of integrins αvβ5 and β1 in control, sh-ITGB5, and sh-ITGB5-over-expressing integrin β5 MDA-MB-231 cells. (D) Adhesion of control, shB5, and shB5-oeB5 MDA-MB-231 cells on vitronectin-, collagen type I-, and fibronectin-coated wells. Adherent cells were fixed and stained with 0.5% methylene blue in water/methanol (50:50), rinsed with ddH₂O, and solubilized with 1% SDS in PBS. The absorbance was read with a fluorimeter. The experiments were performed in six replicates and repeated at least twice. Results are expressed as percentage of adhesion compared to control. Statistical analysis by Student t-Test is provided (** p<0.001). (E) Wound closure assay was performed on collagen type I-coated wells for control, shB5, and shB5-over-expressing integrin β5 MDA-MB-231 cells. Area of open wound is expressed as percentage of the freshly made wound (0 h).

(A) Clonogenic assay of control, shB5, and shB5-over-expressing integrin β5 MDA-MB-231 cells. Cells (500/well) were grown for 12 days, then stained with 0.5% methylene blue in water/methanol 50:50, rinsed in water and the foci in each well were counted. Two representative wells for each cell line are shown. Statistical analysis by Student t-Test is provided (* p<0.05; ** p<0.001). (B) Survival in suspension conditions. Control, shB5, and shB5-over-expressing integrin β5 MDA-MB-231 cells were cultured over a layer of 1% agarose in serum-free media for 72h. Cells were then collected by centrifugation and counted by Trypan blue exclusion. The percentage of alive cells was calculated. Experiments were performed in triplicates and repeated at least twice. (C) Cell cycle analysis of control, shB5, and shB5-over-expressing integrin β5 cells through propidium iodide (PI) staining. Experiments were repeated twice. Control, shB5, and shB5-over-expressing integrin β5 MDA-MB-231 cells were tested for proliferation on plastic (D) or collagen type-I (E). The data presented represents the average and standard deviation of four independent experiments, performed in six replicates.

(A) MDA-MB-231 control, shB5 and shB5-over-expressing integrin β5 cells (1×10⁶ cells/mouse, 9 mice/group) were injected in the mammary fat-pad of 8-week-old SCID female mice. Mice were evaluated every second day for tumor appearance by palpation. (B) Tumor growth was monitored by serial caliper measurements. The means of tumor volume measures of each group of mice during the study are reported. The volumes were calculated using the equation: tumor volume = π/6(length × width²). (C) Three tumors per group were stained for Ki67. Nine representative sections for each tumor were acquired at 400X magnification. 400 nuclei/field were counted and scored for Ki67-positivity. The percentage of Ki67-positive nuclei is reported. (D) Three tumors per group were stained for CD31. Nine representative sections for each tumor were acquired at a 200X magnification and the percentage of CD31-positive area was quantified using Image J software. (E) Quantitative RT-PCR analysis for VEGF-A in total RNA extracted from four tumors/treatment. Levels of VEGF-A mRNA in each samples were normalized on B2M and the fold difference values were calculated as previously described in Materials and Methods. (F) Quantitative RT-PCR analysis for VEGF-A in total RNA extracted from MDA-MB-231 control, shB5 and U0126-treated control cells (5μM). Statistical analysis by Student t-Test is provided (* p<0.05, ** p<0.001).