Protein kinase A gating of a pseudopodial-located RhoA/ROCK/p38/NHE1 signal module regulates invasion in breast cancer cell lines.
Metastasis results from a sequence of selective events often involving interactions with elements of the tumor-specific physiological microenvironment. The low-serum component of this microenvironment confers increased motility and invasion in breast cancer cells by activating the Na+/H+ exchanger isoform 1 (NHE1). The present study was undertaken to characterize the signal transduction mechanisms underlying this serum deprivation-dependent activation of both the NHE1 and the concomitant invasive characteristics such as leading edge pseudopodia development and penetration of matrigel in breast cancer cell lines representing different stages of metastatic progression. Using pharmacological and genetic manipulation together with transport and kinase activity assays, we observe that the activation of the NHE1 and subsequent invasion by serum deprivation in metastatic human breast cells is coordinated by a sequential RhoA/p160ROCK/p38MAPK signaling pathway gated by direct protein kinase A phosphorylation and inhibition of RhoA. Fluorescence resonance energy transfer imaging of RhoA activity and immunofluorescence analysis of phospho-RhoA and NHE1 show that serum deprivation dynamically remodels the cell, forming long, leading edge pseudopodia and that this signal module is preferentially compartmentalized in these leading edge pseudopodia, suggesting a tight topographic relation of the signaling module to an invasion-specific cell structure.
Cardone
Rosa A
RA
Department of General and Environmental Physiology, University of Bari, 70126 Bari, Italy.
Bagorda
Anna
A
Bellizzi
Antonia
A
Busco
Giovanni
G
Guerra
Lorenzo
L
Paradiso
Angelo
A
Casavola
Valeria
V
Zaccolo
Manuela
M
Reshkin
Stephan J
SJ
D001943:Breast Neoplasms / Q000473:pathology*; D027682:Cation Transport Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D003094:Collagen / Q000378:metabolism; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D018450:Disease Progression; D015536:Down-Regulation; D004338:Drug Combinations; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D006801:Humans; D006863:Hydrogen-Ion Concentration; D047908:Intracellular Signaling Peptides and Proteins; D007797:Laminin / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011509:Proteoglycans / Q000378:metabolism; D011554:Pseudopodia / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D013347:Subcellular Fractions; D013997:Time Factors; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D027682:Cation Transport Proteins; D016895:Culture Media, Serum-Free; D004338:Drug Combinations; D047908:Intracellular Signaling Peptides and Proteins; D007797:Laminin; D008565:Membrane Proteins; D011509:Proteoglycans; C490998:SLC9A1 protein, human; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; C059485:matrigel; D012694:Serine; D003094:Collagen; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020742:rhoA GTP-Binding Protein