Hyaluronan-CD44 interaction promotes c-Src-mediated twist signaling, microRNA-10b expression, and RhoA/RhoC up-regulation, leading to Rho-kinase-associated cytoskeleton activation and breast tumor cell invasion.
Dysregulation of microRNAs is observed in many cancers, including breast cancer. In particular, miR-10b appears to play an important role in tumor cell invasion and breast cancer progression. In this study, we investigated hyaluronan (HA)-induced CD44 (a primary HA receptor) interaction with c-Src kinase and the transcriptional factor, Twist, in breast tumor cells (MDA-MB-231 cells). Our results indicate that HA binding to CD44 promotes c-Src kinase activation, which, in turn, increases Twist phosphorylation, leading to the nuclear translocation of Twist and transcriptional activation. Further analyses reveal that miR-10b is controlled by an upstream promoter containing the Twist binding site(s), whereas ChIP assays demonstrate that stimulation of miR-10b expression by HA/CD44-activated c-Src is Twist-dependent in breast tumor cells. This process results in the reduction of a tumor suppressor protein (HOXD10), RhoA/RhoC up-regulation, Rho-kinase (ROK) activation, and breast tumor cell invasion. Treatment of MDA-MB-231 cells with PP2 (a c-Src inhibitor) or Twist-specific siRNAs effectively blocks HA-mediated Twist signaling events, abrogates miR-10b production, and increases HOXD10 expression. Subsequently, this c-Src/Twist signaling inhibition causes down-regulation of RhoA/RhoC expression and impairment of ROK-regulated cytoskeleton function (e.g. tumor cell invasion). To further evaluate the role of miR-10b in RhoGTPase signaling, MDA-MB-231 cells were also transfected with a specific anti-miR-10b inhibitor in order to silence miR-10b expression and block its target functions. Our results demonstrate that anti-miR-10b inhibitor not only enhances HOXD10 expression but also abrogates HA/CD44-mediated tumor cell behaviors in breast tumor cells. Taken together, these findings indicate that the HA-induced CD44 interaction with c-Src-activated Twist plays a pivotal role in miR-10b production, leading to the down-regulation of tumor suppressor protein (HOXD10), RhoGTPase-ROK activation, and tumor cell invasion. All of these events are critical prerequisite steps for the acquisition of metastatic properties by human breast cancer cells.
Bourguignon
Lilly Y W
LY
Department of Medicine, Endocrine Unit (111N), University of California at San Francisco and Veterans Affairs Medical Center, San Francisco, California 94121, USA. lilly.bourguignon@ucsf.edu
Wong
Gabriel
G
Earle
Christine
C
Krueger
Katherine
K
Spevak
Christina C
CC
D000276:Adjuvants, Immunologic / Q000378:metabolism; D017209:Apoptosis; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D000081247:CSK Tyrosine-Protein Kinase; D002448:Cell Adhesion; D002465:Cell Movement; D049109:Cell Proliferation; D047369:Chromatin Immunoprecipitation; D003599:Cytoskeleton / Q000378:metabolism*; D005260:Female; D005455:Fluorescent Antibody Technique; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D051879:Twist-Related Protein 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D054460:rho-Associated Kinases / Q000235:genetics / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism; D000075024:rhoC GTP-Binding Protein; D019061:src-Family Kinases
D000276:Adjuvants, Immunologic; C497483:CD44 protein, human; D018398:Homeodomain Proteins; D018960:Hyaluronan Receptors; C547183:MIRN10 microRNA, human; D035683:MicroRNAs; D009687:Nuclear Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C103173:TWIST1 protein, human; D014157:Transcription Factors; D051879:Twist-Related Protein 1; C066737:RHOA protein, human; C072064:HOXD10 protein, human; D006820:Hyaluronic Acid; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D054460:rho-Associated Kinases; C066690:RHOC protein, human; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D000075024:rhoC GTP-Binding Protein