Localization of uPAR and MMP-9 in lipid rafts is critical for migration, invasion and angiogenesis in human breast cancer cells.

BACKGROUND: uPAR and MMP-9, which play critical roles in tumor cell invasion, migration and angiogenesis, have been shown to be associated with lipid rafts. METHODS: To investigate whether cholesterol could regulate uPAR and MMP-9 in breast carcinoma, we used MßCD (methyl beta cyclodextrin, which extracts cholesterol from lipid rafts) to disrupt lipid rafts and studied its effect on breast cancer cell migration, invasion, angiogenesis and signaling. RESULTS: Morphological evidence showed the association of uPAR with lipid rafts in breast carcinoma cells. MßCD treatment significantly reduced the colocalization of uPAR and MMP-9 with lipid raft markers and also significantly reduced uPAR and MMP-9 at both the protein and mRNA levels. Spheroid migration and invasion assays showed inhibition of breast carcinoma cell migration and invasion after MßCD treatment. In vitro angiogenesis studies showed a significant decrease in the angiogenic potential of cells pretreated with MßCD. MßCD treatment significantly reduced the levels of MMP-9 and uPAR in raft fractions of MDA-MB-231 and ZR 751 cells. Phosphorylated forms of Src, FAK, Cav, Akt and ERK were significantly inhibited upon MßCD treatment. Increased levels of soluble uPAR were observed upon MßCD treatment. Cholesterol supplementation restored uPAR expression to basal levels in breast carcinoma cell lines. Increased colocalization of uPAR with the lysosomal marker LAMP1 was observed in MßCD-treated cells when compared with untreated cells. CONCLUSION: Taken together, our results suggest that cholesterol levels in lipid rafts are critical for the migration, invasion, and angiogenesis of breast carcinoma cells and could be a critical regulatory factor in these cancer cell processes mediated by uPAR and MMP-9.

Stat3-siRNA induces Fas-mediated apoptosis in vitro and in vivo in breast cancer.

Stat3, a member of the signal transducer and activator of transcription family, has the potential to mediate cell survival, growth and differentiation. Stat3 is constitutively activated in numerous cancers, including >50% of breast cancers. Previous studies demonstrated that constitutively activated Stat3 plays an important role in breast cancer development and progression by promoting cell proliferation and inhibiting apoptosis. The present study was designed to investigate the potential use of RNA interference (RNAi) to block Stat3 expression and activation, as well as the subsequent effect on human breast cancer cell growth. Our studies show that knockdown of STAT3 expression by siRNA reduced expression of Bcl-xL and survivin in MDA-MB-231 cells, and also led to Fas mediated intrinsic apoptotic pathway by activating caspases -8, -9, -3 and PARP1 cleavage. In nude mice, pRNAi-Stat3 significantly suppressed tumor growth compared with controls. It also suppressed Stat3 expression, and downregulated BcL-xL and upregulated Fas, Fas-L and cleaved caspase-3 expression within the tumor, which significantly induced apoptosis and led to tumor suppression. Thus, targeting Stat3 signaling using siRNA may serve as a novel therapeutic strategy for the treatment of breast cancers expressing constitutively activated Stat3.