Inhibition of rho-associated kinase signaling prevents breast cancer metastasis to human bone.

Rho-associated kinase (ROCK) signaling plays a fundamental role in regulating cell morphology, adhesion, and motility. Aberrant expression of ROCK is related to tumor metastases and poor clinical outcome. Here, we show that ROCK expression is increased in metastatic human mammary tumors and breast cancer cell lines compared with nonmetastatic tumors and cell lines. Overexpression of ROCK confers a metastatic phenotype on the nonmetastatic MCF-7 cell line. Inhibition of ROCK activity, by either a specific ROCK inhibitor (Y27632) or ROCK-targeted small interfering RNAs, reduces cell migration and proliferation in vitro and metastasis to bone in vivo using a novel "human breast cancer metastasis to human bone" mouse model. Expression of the c-Myc-regulated miR-17-92 cluster is shown to be elevated in metastatic breast cancer cells compared with nonmetastatic cells and diminished by Y27632 treatment. Furthermore, blockade of miR-17 is shown to decrease breast cancer cell invasion/migration in vitro and metastasis in vivo. Together, these findings suggest that augmented ROCK signaling contributes to breast cancer metastasis. The effects of ROCK on tumor cell invasion/motility and growth may derive from regulating cytoskeletal actin-myosin contraction and modulating the c-Myc pathway, including c-Myc-dependent microRNAs. Inhibition of ROCK or the pathway it stimulates, therefore, may represent a novel approach for treatment of breast cancer metastases.

[1]  W. E. Gye,et al.  CANCER RESEARCH , 1923, British medical journal.

[2]  David L Kaplan,et al.  Tissue-engineered bone serves as a target for metastasis of human breast cancer in a mouse model. , 2007, Cancer research.

[3]  B. Trock,et al.  C-myc amplification in breast cancer: a meta-analysis of its occurrence and prognostic relevance , 2000, British Journal of Cancer.

[4]  C. Cordon-Cardo,et al.  A multigenic program mediating breast cancer metastasis to bone. , 2003, Cancer cell.

[5]  Michael A McDevitt,et al.  Alterations of the HBP1 transcriptional repressor are associated with invasive breast cancer. , 2007, Cancer research.

[6]  X. Castellsagué,et al.  Synergistic cooperation between c-Myc and Bcl-2 in lymph node progression of T1 human breast carcinomas , 1999, Breast Cancer Research and Treatment.

[7]  X. Bustelo,et al.  Transcriptomal profiling of the cellular transformation induced by Rho subfamily GTPases , 2007, Oncogene.

[8]  Hiroyuki Tagawa,et al.  MicroRNA-17-92 down-regulates expression of distinct targets in different B-cell lymphoma subtypes. , 2008, Blood.

[9]  N. Andrews,et al.  Deficiency of heme-regulated eIF2α kinase decreases hepcidin expression and splenic iron in HFE−/− mice , 2008, Haematologica.

[10]  Shuh Narumiya,et al.  An essential part for Rho–associated kinase in the transcellular invasion of tumor cells , 1999, Nature Medicine.

[11]  D. Chung,et al.  Hypoxic Regulation of Vascular Endothelial Growth Factor through the Induction of Phosphatidylinositol 3-Kinase/Rho/ROCK and c-Myc* , 2006, Journal of Biological Chemistry.

[12]  Anne J. Ridley,et al.  ROCKs: multifunctional kinases in cell behaviour , 2003, Nature Reviews Molecular Cell Biology.

[13]  A. Biankin,et al.  The estrogen and c-Myc target gene HSPC111 is over-expressed in breast cancer and associated with poor patient outcome , 2008, Breast Cancer Research.

[14]  M. Pickering,et al.  miR-17 and miR-20a temper an E2F1-induced G1 checkpoint to regulate cell cycle progression , 2009, Oncogene.

[15]  L. Penn,et al.  Reflecting on 25 years with MYC , 2008, Nature Reviews Cancer.

[16]  S. Elledge,et al.  The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases , 1993, Cell.

[17]  J. George,et al.  MicroRNA‐134 Modulates the Differentiation of Mouse Embryonic Stem Cells, Where It Causes Post‐Transcriptional Attenuation of Nanog and LRH1 , 2008, Stem cells.

[18]  S. Lowe,et al.  A microRNA polycistron as a potential human oncogene , 2005, Nature.

[19]  J. Mendell.  miRiad Roles for the miR-17-92 Cluster in Development and Disease , 2008, Cell.

[20]  C. Schlotter,et al.  C-myc, not HER-2/neu, can predict recurrence and mortality of patients with node-negative breast cancer , 2003, Breast Cancer Research.

[21]  O. Bernard,et al.  A role for LIM kinase in cancer invasion , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[22]  S. Narumiya,et al.  Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. , 2000, Molecular pharmacology.

[23]  A. Lalwani,et al.  MYH9-siRNA and MYH9 mutant alleles: expression in cultured cell lines and their effects upon cell structure and function. , 2008, Cell motility and the cytoskeleton.

[24]  R. Weinberg,et al.  Ras modulates Myc activity to repress thrombospondin-1 expression and increase tumor angiogenesis. , 2003, Cancer cell.

[25]  C. Croce,et al.  A microRNA expression signature of human solid tumors defines cancer gene targets , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[26]  M. Olson.  Applications for ROCK kinase inhibition. , 2008, Current opinion in cell biology.

[27]  A. Donfrancesco,et al.  Antagomir-17-5p Abolishes the Growth of Therapy-Resistant Neuroblastoma through p21 and BIM , 2008, PloS one.

[28]  John S. Condeelis,et al.  ROCK- and Myosin-Dependent Matrix Deformation Enables Protease-Independent Tumor-Cell Invasion In Vivo , 2006, Current Biology.

[29]  D. Liao,et al.  Correlation of amplification and overexpression of the c-myc oncogene in high-grade breast cancer: FISH, in situ hybridisation and immunohistochemical analyses , 2004, British Journal of Cancer.

[30]  C. Liang,et al.  In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro , 2007, Nature Protocols.

[31]  G. Watkins,et al.  The expression and prognostic value of ROCK I and ROCK II and their role in human breast cancer. , 2008, International journal of oncology.

[32]  H. Tagawa,et al.  Synergistic action of the microRNA‐17 polycistron and Myc in aggressive cancer development , 2007, Cancer science.

[33]  D. Liao,et al.  c-Myc in breast cancer. , 2000, Endocrine-related cancer.

[34]  Charlotte Kuperwasser,et al.  A mouse model of human breast cancer metastasis to human bone. , 2005, Cancer research.

[35]  Erik Sahai,et al.  The actin cytoskeleton in cancer cell motility , 2009, Clinical & Experimental Metastasis.

[36]  R. Fry,et al.  Haem‐regulated eIF2α kinase is necessary for adaptive gene expression in erythroid precursors under the stress of iron deficiency , 2008, British journal of haematology.

[37]  L. Bourguignon,et al.  Rho-kinase (ROK) promotes CD44v(3,8-10)-ankyrin interaction and tumor cell migration in metastatic breast cancer cells. , 1999, Cell motility and the cytoskeleton.

[38]  Stuart A. Aaronson,et al.  Exogenous Expression of N-Cadherin in Breast Cancer Cells Induces Cell Migration, Invasion, and Metastasis , 2000, The Journal of cell biology.

[39]  Norhanom Abdul Wahab,et al.  Protein expression and molecular analysis of c-myc gene in primary breast carcinomas using immunohistochemistry and differential polymerase chain reaction. , 2002, International journal of molecular medicine.

[40]  G. Kirfel,et al.  ROCK signaling mediates the adoption of different modes of migration and invasion in human mammary epithelial tumor cells. , 2006, Experimental cell research.

[41]  N. Sato,et al.  The Rho-Rock-Myosin Signaling Axis Determines Cell-Cell Integrity of Self-Renewing Pluripotent Stem Cells , 2008, PloS one.

[42]  Hiroyuki Tagawa,et al.  Identification and characterization of a novel gene, C13orf25, as a target for 13q31-q32 amplification in malignant lymphoma. , 2004, Cancer research.

[43]  D. Iliopoulos,et al.  E2F1-regulated microRNAs impair TGFbeta-dependent cell-cycle arrest and apoptosis in gastric cancer. , 2008, Cancer cell.

[44]  E. Sahai,et al.  RHO–GTPases and cancer , 2002, Nature Reviews Cancer.

[45]  D. Carbone,et al.  Abrogation of TGF beta signaling in mammary carcinomas recruits Gr-1+CD11b+ myeloid cells that promote metastasis. , 2008, Cancer cell.

[46]  N. Andrews,et al.  The function of heme-regulated eIF2alpha kinase in murine iron homeostasis and macrophage maturation. , 2007, The Journal of clinical investigation.

[47]  Bo Li,et al.  Involvement of Rho/ROCK signalling in small cell lung cancer migration through human brain microvascular endothelial cells , 2006, FEBS letters.

[48]  Shuh Narumiya,et al.  Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension , 1997, Nature.

[49]  Richard D Bruggeman,et al.  Overexpression of c-Myc and Bcl-2 during progression and distant metastasis of hormone-treated breast cancer. , 2007, Experimental and molecular pathology.

[50]  Andrea Cocito,et al.  Genomic targets of the human c-Myc protein. , 2003, Genes & development.

[51]  Michael Q. Zhang,et al.  A global transcriptional regulatory role for c-Myc in Burkitt's lymphoma cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.