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

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.

[1]  M. Bissell.  The differentiated state of normal and malignant cells or how to define a "normal" cell in culture. , 1981, International review of cytology.

[2]  Primary hepatocyte culture: substratum requirements and production of matrix components. , 1981, Federation proceedings.

[3]  Z. Werb,et al.  Secretion of metalloproteinases by stimulated capillary endothelial cells. II. Expression of collagenase and stromelysin activities is regulated by endogenous inhibitors. , 1986, The Journal of biological chemistry.

[4]  M J Banda,et al.  Secretion of metalloproteinases by stimulated capillary endothelial cells. I. Production of procollagenase and prostromelysin exceeds expression of proteolytic activity. , 1986, The Journal of biological chemistry.

[5]  M J Bissell,et al.  Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane. , 1989, Development.

[6]  M. Bissell,et al.  Expression of extracellular matrix components is regulated by substratum , 1990, The Journal of cell biology.

[7]  M. Bissell,et al.  Control of mammary epithelial differentiation: basement membrane induces tissue-specific gene expression in the absence of cell-cell interaction and morphological polarity , 1991, The Journal of cell biology.

[8]  M. Bissell,et al.  A novel transcriptional enhancer is involved in the prolactin- and extracellular matrix-dependent regulation of beta-casein gene expression. , 1992, Molecular biology of the cell.

[9]  M. Bissell,et al.  Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[10]  M. McMahon,et al.  Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase , 1993, Molecular and cellular biology.

[11]  O. Petersen,et al.  Trisomy 7p and malignant transformation of human breast epithelial cells following epidermal growth factor withdrawal. , 1996, Cancer research.

[12]  C. Larabell,et al.  Reversion of the Malignant Phenotype of Human Breast Cells in Three-Dimensional Culture and In Vivo by Integrin Blocking Antibodies , 1997, The Journal of cell biology.

[13]  M. Bissell,et al.  Characterization of BCE-1, a Transcriptional Enhancer Regulated by Prolactin and Extracellular Matrix and Modulated by the State of Histone Acetylation , 1998, Molecular and Cellular Biology.

[14]  C. Larabell,et al.  Reciprocal interactions between beta1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: a different perspective in epithelial biology. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[15]  I. Ellis,et al.  Pathological prognostic factors in breast cancer. , 1999, Critical reviews in oncology/hematology.

[16]  C. Anderson,et al.  E7-transduced human breast epithelial cells show partial differentiation in three-dimensional culture. , 1999, Cancer research.

[17]  M. Bissell,et al.  Division of Labor among the α6β4 Integrin, β1 Integrins, and an E3 Laminin Receptor to Signal Morphogenesis and β-Casein Expression in Mammary Epithelial Cells , 1999 .

[18]  S. Zucker,et al.  Critical appraisal of the use of matrix metalloproteinase inhibitors in cancer treatment , 2000, Oncogene.

[19]  Shigeyoshi Itohara,et al.  Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis , 2000, Nature Cell Biology.

[20]  D. Hanahan,et al.  MMP-9 Supplied by Bone Marrow–Derived Cells Contributes to Skin Carcinogenesis , 2000, Cell.

[21]  M. Bissell,et al.  ErbB2, but not ErbB1, reinitiates proliferation and induces luminal repopulation in epithelial acini , 2001, Nature Cell Biology.

[22]  H. Avraham,et al.  The Invasive Phenotype in HMT-3522 Cells Requires Increased EGF Receptor Signaling Through Both PI 3-Kinase and ERK 1,2 Pathways , 2002, Cell communication & adhesion.

[23]  B. Fingleton,et al.  Matrix Metalloproteinase Inhibitors and Cancer—Trials and Tribulations , 2002, Science.

[24]  Jayanta Debnath,et al.  The Role of Apoptosis in Creating and Maintaining Luminal Space within Normal and Oncogene-Expressing Mammary Acini , 2002, Cell.

[25]  Mina J Bissell,et al.  Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition. , 2010, Journal of cell science.

[26]  Deepika Walpita,et al.  Studying actin-dependent processes in tissue culture , 2002, Nature Reviews Molecular Cell Biology.

[27]  Sophie Lelièvre,et al.  beta4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium. , 2002, Cancer cell.

[28]  Z. Werb,et al.  New functions for the matrix metalloproteinases in cancer progression , 2002, Nature Reviews Cancer.

[29]  M. Crépin,et al.  FGF-2 and TPA induce matrix metalloproteinase-9 secretion in MCF-7 cells through PKC activation of the Ras/ERK pathway. , 2002, Biochemical and biophysical research communications.

[30]  I. Ellis,et al.  Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. , 2002, Histopathology.

[31]  R. Hansen,et al.  Phenotypic reversion or death of cancer cells by altering signaling pathways in three-dimensional contexts. , 2002, Journal of the National Cancer Institute.

[32]  K. Mann,et al.  A critical review of the methods for cleavage of fusion proteins with thrombin and factor Xa. , 2003, Protein expression and purification.

[33]  R. Lupu,et al.  Blockage of heregulin expression inhibits tumorigenicity and metastasis of breast cancer , 2003, Oncogene.

[34]  B. Bauvois,et al.  Matrix Metalloproteinase-9 Silencing by RNA Interference Triggers the Migratory-adhesive Switch in Ewing's Sarcoma Cells* , 2003, Journal of Biological Chemistry.

[35]  Z. Shao,et al.  Prognostic value of matrix metalloproteinases (MMP-2 and MMP-9) in patients with lymph node-negative breast carcinoma , 2004, Breast Cancer Research and Treatment.

[36]  D. Radisky,et al.  Polarity and proliferation are controlled by distinct signaling pathways downstream of PI3-kinase in breast epithelial tumor cells , 2004, The Journal of cell biology.

[37]  R. Suzuki,et al.  Experimental metastasis is suppressed in MMP-9-deficient mice , 1999, Clinical & Experimental Metastasis.

[38]  S. White,et al.  Loss of Dicer fowls up centromeres , 2004, Nature Cell Biology.

[39]  M J Bissell,et al.  Microenvironmental Regulators of Tissue Structure and Function Also Regulate Tumor Induction and Progression : The Role of Extracellular Matrix and Its Degrading Enzymes , 2022 .

[40]  D. Albertson,et al.  Rac1b and reactive oxygen species mediate MMP-3-induced EMT and genomic instability , 2005, Nature.

[41]  D. Jeoung,et al.  A Splice Variant of CD99 Increases Motility and MMP-9 Expression of Human Breast Cancer Cells through the AKT-, ERK-, and JNK-dependent AP-1 Activation Signaling Pathways* , 2006, Journal of Biological Chemistry.

[42]  Ximei Wu,et al.  Methyl-3-indolylacetate inhibits cancer cell invasion by targeting the MEK1/2-ERK1/2 signaling pathway , 2006, Molecular Cancer Therapeutics.

[43]  M. Bissell,et al.  Gene expression signature in organized and growth-arrested mammary acini predicts good outcome in breast cancer. , 2006, Cancer research.

[44]  J. Quigley,et al.  Matrix metalloproteinases and tumor metastasis , 2006, Cancer and Metastasis Reviews.

[45]  E. Golemis,et al.  Interdependence of cell attachment and cell cycle signaling. , 2006, Current opinion in cell biology.

[46]  Christopher M. Overall,et al.  Validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy , 2006, Nature Reviews Cancer.

[47]  Christopher M Overall,et al.  Tumour microenvironment - opinion: validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy. , 2006, Nature reviews. Cancer.

[48]  Heath B Acuff,et al.  Matrix metalloproteinase-9 from bone marrow-derived cells contributes to survival but not growth of tumor cells in the lung microenvironment. , 2006, Cancer research.

[49]  M. Bissell,et al.  Rap1 integrates tissue polarity, lumen formation, and tumorigenic potential in human breast epithelial cells. , 2007, Cancer research.

[50]  P. Brown,et al.  Transcriptional modulation of genes encoding structural characteristics of differentiating enterocytes during development of a polarized epithelium in vitro. , 2007, Molecular biology of the cell.

[51]  F. Vizoso,et al.  Study of matrix metalloproteinases and their inhibitors in breast cancer , 2007, British Journal of Cancer.

[52]  M. Bissell,et al.  Targeting TACE-dependent EGFR ligand shedding in breast cancer. , 2007, The Journal of clinical investigation.

[53]  Genee Y. Lee,et al.  Three-dimensional culture models of normal and malignant breast epithelial cells , 2007, Nature Methods.

[54]  Inke Näthke,et al.  Cell polarity in development and cancer , 2007, Nature Cell Biology.

[55]  J. Pouysségur,et al.  The ERK1/2 mitogen-activated protein kinase pathway as a master regulator of the G1- to S-phase transition , 2007, Oncogene.

[56]  Andrew J. Ewald,et al.  Matrix metalloproteinases and the regulation of tissue remodelling , 2007, Nature Reviews Molecular Cell Biology.

[57]  M. Bloomston,et al.  Matrix metalloproteinases and their role in pancreatic cancer: A review of preclinical studies and clinical trials , 2002, Annals of Surgical Oncology.

[58]  Susan M. Kilroy,et al.  Tumor-Specific Urinary Matrix Metalloproteinase Fingerprinting: Identification of High Molecular Weight Urinary Matrix Metalloproteinase Species , 2008, Clinical Cancer Research.

[59]  Xìao-chun Xu,et al.  Prognostic significance of MMP‐9 and TIMP‐1 serum and tissue expression in breast cancer , 2008, International journal of cancer.

[60]  T. Hunter,et al.  PI-3 kinase activity is necessary for ERK1/2-induced disruption of mammary epithelial architecture , 2009, Breast Cancer Research.

[61]  Roy A Jensen,et al.  A human breast cell model of preinvasive to invasive transition. , 2008, Cancer research.

[62]  Jordi Alcaraz,et al.  Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia , 2008, The EMBO journal.

[63]  B. Fingleton,et al.  Effect of ablation or inhibition of stromal matrix metalloproteinase-9 on lung metastasis in a breast cancer model is dependent on genetic background. , 2008, Cancer research.

[64]  Mina J. Bissell,et al.  Sustained activation of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific function , 2009, The Journal of cell biology.

[65]  M. Bissell,et al.  Endothelial cell migration and vascular endothelial growth factor expression are the result of loss of breast tissue polarity. , 2009, Cancer research.