Krüppel-like factor 8 promotes triple negative breast cancer angiogenesis and metastasis through matrix metalloproteinases

Krüppel-like factor 8 (KLF8) is a transcription factor known to promote breast cancer. KLF8 upregulates matrix metalloproteinases (MMPs) that degrade the extracellular matrix (ECM) and are associated with enhanced angiogenesis. However, KLF8's role in BC angiogenesis through MMPs has not been elucidated. We hypothesize that KLF8's upregulation of MMPs promotes an angiogenic switch in triple-negative breast cancer (TNBC), the most aggressive breast cancer subtype, by degrading the ECM to create physical space and increase the bioavailability of pro-angiogenic factors. Inducible KLF8 overexpression (MCF10A-Ras) and knockdown (MDA-MB-231) cell models were used to study the function of KLF8 in vitro . We employed the broad-spectrum matrix metalloproteinase (MMP) inhibitor GM6001 to confirm dependency, endothelial tube formation and HUVEC recruitment assays to evaluate angiogenesis potential, Quantitative real-time PCR (qRT-PCR) and Vascular Endothelial Growth Factor (VEGF)-A ELISA to measure growth factor expression and active factor release. Findings were then validated using the in vivo xenograft tumor growth and tail vein lung metastasis assays, angiogenesis in excised tumors was quantitatively evaluated using microvessel density staining (cluster of differentiation 31 (CD31)), with MMP rescue experiments used to establish molecular pathway. Our results show that KLF8 overexpression promotes endothelial cell migration and tube formation in an MMP-dependent manner. The KLF8-MMP-VEGF axis is established using VEGFA ELISA that shows KLF8-dependent accumulation of active VEGF in conditioned medium. This effect was eliminated by MMP inhibition, Notably, KLF8 does affect VEGF mRNA expression. In vivo experiments validate these results showing that KLF8 overexpression markedly increases angiogenesis, xenograft tumor growth, and lung metastasis. On the other hand, MMP-9 or MMP-14 rescue in the knockdown cells restores the effects of KLF8 knockdown. Poor distant metastasis-free survival and elevated angiogenic markers are associated with high KLF8 expression in human breast cancer cohorts. In conclusion, our work demonstrates KLF8's role in promoting TNBC metastasis by activating MMP9 and MMP14, which in turn enhances the angiogenic microenvironment through the release of soluble VEGF. This work advances our understanding of KLF8's carcinogenic function and suggests that KLF8-MMP signaling could be a promising antiangiogenic therapeutic target for TNBC.