PDF file, 707K, S1. Comparison of alpha3beta1 and alpha6beta4 integrin expression in 4T1 cell lines. S2. BLI of spontaneous and experimental metastasis (color) S3. Certain large primary mammary fat pad tumors exhibited substantial centralized zones of necrosis. S4. Silencing alpha3 integrin leads to fewer pulmonary metastases following mammary fat pad injection. S5. RNAi-mediated silencing of RhoC has no impact on 4T1 cell metastatic colonization. S6. Cox-2, matrix metalloproteinase, VEGF, and transendothelial migration assays. S7. MDA-MB-231 breast cancer cells do not secrete laminin-332 or laminin-511 or depend on alpha3 integrin in short term proliferation assays.
ARTICLE ABSTRACT
Significant evidence implicates α3β1 integrin in promoting breast cancer tumorigenesis and metastasis-associated cell behaviors in vitro and in vivo. However, the extent to which α3β1 is actually required for breast cancer metastasis remains to be determined. We used RNA interference to silence α3 integrin expression by approximately 70% in 4T1 murine mammary carcinoma cells, a model of aggressive, metastatic breast cancer. Loss of α3 integrin reduced adhesion, spreading, and proliferation on laminin isoforms, and modestly reduced the growth of orthotopically implanted cells. However, spontaneous metastasis to lung was strikingly curtailed. Experimental lung colonization after tail vein injection revealed a similar loss of metastatic capacity for the α3-silenced (α3si) cells, suggesting that critical, α3-dependent events at the metastatic site could account for much of α3β1′s contribution to metastasis in this model. Reexpressing α3 in the α3si cells reversed the loss of metastatic capacity, and silencing another target, the small GTPase RhoC, had no effect, supporting the specificity of the effect of silencing α3. Parental, α3si, and α3-rescued cells, all secreted abundant laminin α5 (LAMA5), an α3β1 integrin ligand, suggesting that loss of α3 integrin might disrupt an autocrine loop that could function to sustain metastatic growth. Analysis of human breast cancer cases revealed reduced survival in cases where α3 integrin and LAMA5 are both overexpressed.Implications: α3 integrin or downstream effectors may be potential therapeutic targets in disseminated breast cancers, especially when laminin α5 or other α3 integrin ligands are also over-expressed. Mol Cancer Res; 12(1); 143–54. ©2013 AACR.
