Raf-induced MMP9 disrupts tissue architecture of human breast cells in three-dimensional culture and is necessary for tumor growth in vivo

Abstract

Organization into polarized three-dimensional structures defines whether epithelial cells are normal or malignant. In a model of morphogenesis, we show that inhibiting key signaling pathways in human breast cancer cells leads to “phenotypic reversion” of the malignant cells. Using architecture as an endpoint, we report that, in all cases, signaling through Raf/MEK/ERK disrupted tissue polarity via matrix metalloproteinase9 (MMP9) activity. Induction of Raf or activation of an engineered, functionally inducible MMP9 in nonmalignant cells led to loss of tissue polarity, and reinitiated proliferation. Conversely, inhibition of Raf or MMP9 with small molecule inhibitors or shRNAs restored the ability of cancer cells to form polarized quiescent structures. Silencing MMP9 expression also reduced tumor growth dramatically in a murine xenograft model. LC-MS/MS analysis comparing conditioned medium from nonmalignant cells with or without active MMP9 revealed laminin 111 (LM1) as an important target of MMP9. LM1 has been implicated in acinar morphogenesis; thus, its degradation by MMP9 provides a mechanism for loss of tissue polarity and reinitiation of growth associated with MMP9 activity. These findings underscore the importance of the dynamic reciprocity between the extracellular matrix integrity, tissue polarity, and Raf/MEK/ERK and MMP9 activities, providing an axis for either tissue homeostasis or malignant progression.

Human matrix metalloproteinases: An ubiquitarian class of enzymes involved in several pathological processes

“…Several molecular pathways, once activated, regulate MMP-9 expression: MMP-9 is controlled by the Raf/MEK/ERK cascade and the transcription factors SLUG and NF-jB induce MMP-9 expression (Beliveau et al, 2010;Sun et al, 2008;Moon et al, 2007). Furthermore, the transcriptional stability is maintained through the p38 MAPK/MAPK2 signaling and, interestingly tissue transglutaminase 2 is reported to promote MMP-9 expression (Cai et al, 2011;Kumar et al, 2010;Li et al, 2011).…”

“…Consistently with the identification of cis-acting promoter elements, transcriptional activation of MMP-9 is regulated by a multitude of factors which render highly complex the plethora of stimuli that finely tune its biological activity (Lauricella- Lefebvre et al, 1993;Munaut et al, 1999). MMP-9 is induced by adhesion molecules, mainly activated integrins, cytokines and growth factors (Beliveau et al, 2010;Zhao and Benveniste, 2008). As an example, cell binding to vitronectin or fibronectin trigger MMP-9 expression, IL-1b, TNF-a or TGF-b induce MMP-9 in a autocrine and paracrine fashion, EGF release or the aberrant activation of the EGFR result in over-expression of MMP-9 (Coulson-Thomas et al, 2010;Ehrenfeld et al, 2011;Ikari et al, 2011;Lin et al, 2009;Yu and Stamenkovic, 2000).…”

Raf kinases in cancer–roles and therapeutic opportunities

“…Additional regulators of tissue architecture are matrix metalloproteases, which are often overexpressed in cancer (Kessenbrock et al, 2010). Increased expression of one of these enzymes, matrix metalloprotease-9, by the Raf/MEK/ERK pathway in threedimensional breast tissue cultures leads to a remodeling of the microenvironment, which induces loss of tissue polarity and re-initiation of proliferation (Beliveau et al, 2010).…”

Molecular Mechanisms of Glioma Cell Motility

“…MMP-2 and MMP-9 convert latent pro-migratory transforming growth factor (TGF)-β into its active form, which in turn induces MMP-2 in a feedback loop (see the section "The Role of TGF-β in Glioma Cell Motility" (45-47)). MMP-9 expression or activity can be regulated by: activation of signal transducer and activator of transcription (STAT)3; epidermal growth factor (EGF); FN; vitronectin (VN); interleukin (IL)-1β; tumor necrosis factor (TNF)-a; and TGF-β (47)(48)(49)(50)(51)(52). Furthermore, glioma cells exploit MMP-14 that is expressed by surrounding microglia cells (53).…”