Absence of adhesion triggers differential FAK and SAPKp38 signals in SW620 human colon cancer cells that may inhibit adhesiveness and lead to cell death.
BACKGROUND
Upon adhesion, anchorage-dependent cells transmit survival signals from the matrix into the cell. Loss of anchorage leads to anoikis. Resistance to anoikis may influence tumor progression and metastasis. To better understand the pathways that regulate the choice between adhesion and cell death, we examined FAK, c-Src andMAPKinase activities in SW620 human colon cancer cells.


METHODS
SW620 cell suspensions were first allowed to adhere to collagen I for 30 minutes and adherent cells were subsequently counted. FAK, p38, c-Src and ERK1/2 phosphorylation were assessed by Western blot in adherent cells and in cells prevented from adhesion by plating unto BSA-pacificated dishes. p38 and FAK were inhibited by SB203580 (20 microM) or by specific FAK antisense nucleotides or FAK siRNA, respectively, and adhesion quantitated. Apoptosis (anoikis) after lack of adhesion was measured colorimetrically in control cells and in cells treated with SB203580.


RESULTS
Adhesion to collagen I nearly doubled FAK phosphorylation at Y397, the autophosphorylation site, and decreased p38 activation by 60% (p<0.001) but did not affect FAK phosphorylation at Y576, the c-Src dependent site. Lowering FAK expression with FAK antisense decreased adhesion to collagen I; the larger decrease in FAK expression obtained with the siRNA (43 +/- 2%) resulted in significantly greater inhibition of adhesion not only to collagen I but also to collagen IV and fibronectin. The p38 inhibitor diminished anoikis and enhanced adhesion. Interestingly, the SB compound also significantly inhibited FAK phosphorylation at Y397 (23 +/- 3% in adherent, 30 +/- 4% in non-adherent cells at 30 minutes and 35 +/- 4% in adherent, 46 +/- 14% in non-adherent cells after 6 hours, p<0.05 for each) and greatly enhanced phosphorylation of ERK1/2, a putative anti-apoptotic component of the MAPK cascade.


CONCLUSIONS
In the absence of adhesion, SW620 cells exhibit increased p38 but decreased FAK activation, signals that may promote cell death. Our observations with the p38 inhibitor SB203580 indicate that inside-out signals, from p38 to FAK, may regulate both adhesion and anoikis in SW620 cells. In addition, the data suggest the presence of cross-talk between the pro-apoptotic p38 and anti-apoptotic ERK1/2 pathways.
Walsh
Mary F
MF
Department of Surgery, John D. Dingell VAMC, Detroit, MI 48201-1932, USA.
Thamilselvan
Vijayalakshmi
V
Grotelueschen
Rainer
R
Farhana
Lulu
L
Basson
Marc
M
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D000081247:CSK Tyrosine-Protein Kinase; D002448:Cell Adhesion / Q000187:drug effects; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000201:enzymology / Q000473:pathology / Q000503:physiopathology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000187:drug effects / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases; D019061:src-Family Kinases
D004791:Enzyme Inhibitors; D007093:Imidazoles; D016376:Oligonucleotides, Antisense; D011725:Pyridines; D034741:RNA, Small Interfering; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D019061:src-Family Kinases; C000631346:CSK protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580