Hypoxia-inducible factors mediate coordinated RhoA-ROCK1 expression and signaling in breast cancer cells

Significance Breast cancers often contain regions of reduced O2 availability, leading to increased activity of hypoxia-inducible factors (HIFs). Here, we demonstrate that HIFs activate transcription of the Rho family member RHOA and Rho kinase 1 (ROCK1) genes, leading to cytoskeletal changes that underlie the invasive cancer cell phenotype. ROCK1 is a kinase that regulates myosin light-chain activity, leading to actin-myosin contraction, which is the basis for cell movement. Coordinately increased levels of RhoA and ROCK1 mRNA in human breast cancers predicted patient mortality. These results demonstrate that a microenvironmental stimulus, hypoxia, can activate a critical signal transduction pathway, independent of genomic alterations, to drive cancer progression. Overexpression of Rho kinase 1 (ROCK1) and the G protein RhoA is implicated in breast cancer progression, but oncogenic mutations are rare, and the molecular mechanisms that underlie increased ROCK1 and RhoA expression have not been determined. RhoA-bound ROCK1 phosphorylates myosin light chain (MLC), which is required for actin-myosin contractility. RhoA also activates focal adhesion kinase (FAK) signaling. Together, these pathways are critical determinants of the motile and invasive phenotype of cancer cells. We report that hypoxia-inducible factors coordinately activate RhoA and ROCK1 expression and signaling in breast cancer cells, leading to cell and matrix contraction, focal adhesion formation, and motility through phosphorylation of MLC and FAK. Thus, intratumoral hypoxia acts as an oncogenic stimulus by triggering hypoxia-inducible factor → RhoA → ROCK1 → MLC → FAK signaling in breast cancer cells.

[1]  G. Semenza,et al.  Analysis of Hypoxia-Inducible Factor 1α Expression and its Effects on Invasion and Metastasis , 2007 .

[2]  G. Semenza,et al.  Hypoxia-inducible factor 1 is a master regulator of breast cancer metastatic niche formation , 2011, Proceedings of the National Academy of Sciences.

[3]  A. Levchenko,et al.  Hypoxia-inducible factor-dependent breast cancer-mesenchymal stem cell bidirectional signaling promotes metastasis. , 2012, The Journal of clinical investigation.

[4]  S. Narumiya,et al.  Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. , 1999, Science.

[5]  N. Boyd,et al.  Mammographic density. Potential mechanisms of breast cancer risk associated with mammographic density: hypotheses based on epidemiological evidence , 2008, Breast Cancer Research.

[6]  Kozo Kaibuchi,et al.  Regulation of Myosin Phosphatase by Rho and Rho-Associated Kinase (Rho-Kinase) , 1996, Science.

[7]  G. Semenza,et al.  Hypoxia Response Elements in the Aldolase A, Enolase 1, and Lactate Dehydrogenase A Gene Promoters Contain Essential Binding Sites for Hypoxia-inducible Factor 1* , 1996, The Journal of Biological Chemistry.

[8]  Jayanta Debnath,et al.  Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. , 2003, Methods.

[9]  Christian A. Rees,et al.  Molecular portraits of human breast tumours , 2000, Nature.

[10]  Lukas D. Osborne,et al.  HIF1α and HIF2α independently activate SRC to promote melanoma metastases. , 2013, The Journal of clinical investigation.

[11]  G. Semenza,et al.  Inhibitors of hypoxia-inducible factor 1 block breast cancer metastatic niche formation and lung metastasis , 2012, Journal of Molecular Medicine.

[12]  C. Geng,et al.  Over Expression of RhoA is Associated with Progression in Invasive Breast Duct Carcinoma , 2010, The breast journal.

[13]  Steven J. M. Jones,et al.  Comprehensive molecular portraits of human breast tumours , 2013 .

[14]  G. Semenza,et al.  Collagen prolyl hydroxylases are essential for breast cancer metastasis. , 2013, Cancer research.

[15]  L. Holmberg,et al.  Gene expression profiling spares early breast cancer patients from adjuvant therapy: derived and validated in two population-based cohorts , 2005, Breast Cancer Research.

[16]  P. Bonnier,et al.  Overexpression of hypoxia‐inducible factor HIF‐1α predicts early relapse in breast cancer: Retrospective study in a series of 745 patients , 2005, International journal of cancer.

[17]  D. Schlaepfer,et al.  Intrinsic focal adhesion kinase activity controls orthotopic breast carcinoma metastasis via the regulation of urokinase plasminogen activator expression in a syngeneic tumor model , 2006, Oncogene.

[18]  Alan Hall,et al.  Rho GTPases: biochemistry and biology. , 2005, Annual review of cell and developmental biology.

[19]  Mikala Egeblad,et al.  Matrix Crosslinking Forces Tumor Progression by Enhancing Integrin Signaling , 2009, Cell.

[20]  Andy J. Minn,et al.  Genes that mediate breast cancer metastasis to lung , 2005, Nature.

[21]  Denis Wirtz,et al.  Predicting how cells spread and migrate , 2013, Cell Adhesion & Migration.

[22]  Yoshiharu Matsuura,et al.  Phosphorylation and Activation of Myosin by Rho-associated Kinase (Rho-kinase)* , 1996, The Journal of Biological Chemistry.

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

[24]  C. Rueden,et al.  Bmc Medicine Collagen Density Promotes Mammary Tumor Initiation and Progression , 2022 .

[25]  P. V. van Diest,et al.  Levels of hypoxia‐inducible factor‐1α independently predict prognosis in patients with lymph node negative breast carcinoma , 2003, Cancer.

[26]  A. Chambers,et al.  MDA-MB-435 and M14 cell lines: identical but not M14 melanoma? , 2009, Cancer research.

[27]  R. Gillies,et al.  Hypoxia and adaptive landscapes in the evolution of carcinogenesis , 2007, Cancer and Metastasis Reviews.

[28]  D. A. Hanson,et al.  Focal adhesion kinase: in command and control of cell motility , 2005, Nature Reviews Molecular Cell Biology.

[29]  G. Semenza Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics , 2010, Oncogene.

[30]  Steven J. M. Jones,et al.  Comprehensive molecular portraits of human breast tumors , 2012, Nature.

[31]  G. Semenza,et al.  Procollagen Lysyl Hydroxylase 2 Is Essential for Hypoxia-Induced Breast Cancer Metastasis , 2013, Molecular Cancer Research.

[32]  Sergey V Plotnikov,et al.  Guiding cell migration by tugging. , 2013, Current opinion in cell biology.

[33]  Michael Höckel,et al.  Detection and characterization of tumor hypoxia using pO2 histography. , 2007, Antioxidants & redox signaling.

[34]  M. Zavelevich,et al.  Tumor hypoxia and malignant progression. , 2009, Experimental oncology.

[35]  S. Narumiya,et al.  Rho signaling, ROCK and mDia1, in transformation, metastasis and invasion , 2009, Cancer and Metastasis Reviews.

[36]  Manuela Milani,et al.  Hypoxia-Inducible Factor-1α Expression Predicts a Poor Response to Primary Chemoendocrine Therapy and Disease-Free Survival in Primary Human Breast Cancer , 2006, Clinical Cancer Research.

[37]  G. Semenza,et al.  HIF-1-dependent Expression of Angiopoietin-like 4 and L1CAM Mediates Vascular Metastasis of Hypoxic Breast Cancer Cells to the Lungs , 2011, Oncogene.

[38]  Cynthia A. Reinhart-King,et al.  Tensional homeostasis and the malignant phenotype. , 2005, Cancer cell.

[39]  Charles H. Graham,et al.  Hypoxia-driven selection of the metastatic phenotype , 2007, Cancer and Metastasis Reviews.

[40]  M. Fleming,et al.  The Structure of Dimeric ROCK I Reveals the Mechanism for Ligand Selectivity* , 2006, Journal of Biological Chemistry.

[41]  John S. Condeelis,et al.  Identification and Testing of a Gene Expression Signature of Invasive Carcinoma Cells within Primary Mammary Tumors , 2004, Cancer Research.

[42]  W. Kiosses,et al.  Regulation of the small GTP‐binding protein Rho by cell adhesion and the cytoskeleton , 1999, The EMBO journal.

[43]  R. Madan,et al.  Focal adhesion proteins as markers of malignant transformation and prognostic indicators in breast carcinoma. , 2006, Human pathology.

[44]  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.

[45]  D. Camidge,et al.  Safety, pharmacokinetic, and pharmacodynamic phase I dose-escalation trial of PF-00562271, an inhibitor of focal adhesion kinase, in advanced solid tumors. , 2012, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[46]  S. Steinberg,et al.  Multihistology, Target-Driven Pilot Trial of Oral Topotecan as an Inhibitor of Hypoxia-Inducible Factor-1α in Advanced Solid Tumors , 2011, Clinical Cancer Research.

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

[48]  A. Paradiso,et al.  RhoA protein expression in primary breast cancers and matched lymphocytes is associated with progression of the disease. , 2008, International journal of molecular medicine.

[49]  G. Semenza,et al.  Hypoxia-inducible factor 1-dependent expression of platelet-derived growth factor B promotes lymphatic metastasis of hypoxic breast cancer cells , 2012, Proceedings of the National Academy of Sciences.

[50]  D. Lauffenburger,et al.  Targeting tumor cell motility as a strategy against invasion and metastasis. , 2013, Trends in pharmacological sciences.

[51]  Kshitiz,et al.  Erratum: Hypoxia-inducible factor-dependent breast cancer-mesenchymal stem cell bidirectional signaling promotes metastasis (Journal of Clinical Investigation (2013) 123: 1 (189-205) DOI: 10.1172/JCI64993) , 2013 .

[52]  G. Borisy,et al.  Cell Migration: Integrating Signals from Front to Back , 2003, Science.

[53]  M. Schindl,et al.  Overexpression of hypoxia-inducible factor 1alpha is associated with an unfavorable prognosis in lymph node-positive breast cancer. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[54]  D. Faller,et al.  Hypoxia affects tumor cell invasiveness in vitro: the role of hypoxia-activated ligand HAL1/13 (Ku86 autoantigen). , 2000, Cancer letters.

[55]  Linda Holmquist Mengelbier,et al.  Hypoxia-inducible factor-2alpha correlates to distant recurrence and poor outcome in invasive breast cancer. , 2008, Cancer research.

[56]  Sijin Liu,et al.  Inhibition of rho-associated kinase signaling prevents breast cancer metastasis to human bone. , 2009, Cancer research.

[57]  Kevin W. Eliceiri,et al.  Matrix density-induced mechanoregulation of breast cell phenotype, signaling, and gene expression through a FAK-ERK linkage , 2009, Oncogene.

[58]  J. Pouysségur,et al.  Hypoxia and cancer , 2007, Journal of Molecular Medicine.

[59]  M. Wicha,et al.  Mammary epithelial-specific ablation of the focal adhesion kinase suppresses mammary tumorigenesis by affecting mammary cancer stem/progenitor cells. , 2009, Cancer research.

[60]  Stephanie I. Fraley,et al.  A distinctive role for focal adhesion proteins in three-dimensional cell motility , 2010, Nature Cell Biology.

[61]  P. Pandolfi,et al.  Deciphering the transcriptional complex critical for RhoA gene expression and cancer metastasis , 2010, Nature Cell Biology.