Role of phosphoinositide 3-kinase in the aggressive tumor growth of HT1080 human fibrosarcoma cells.
We have developed a model system of human fibrosarcoma cell lines that do or do not possess and express an oncogenic mutant allele of N-ras. HT1080 cells contain an endogenous mutant allele of N-ras, whereas the derivative MCH603 cell line contains only wild-type N-ras. In an earlier study (S. Gupta et al., Mol. Cell. Biol. 20:9294-9306, 2000), we had shown that HT1080 cells produce rapidly growing, aggressive tumors in athymic nude mice, whereas MCH603 cells produced more slowly growing tumors and was termed weakly tumorigenic. An extensive analysis of the Ras signaling pathways (Raf, Rac1, and RhoA) provided evidence for a potential novel pathway that was critical for the aggressive tumorigenic phenotype and could be activated by elevated levels of constitutively active MEK. In this study we examined the role of phosphoinositide 3-kinase (PI 3-kinase) in the regulation of the transformed and aggressive tumorigenic phenotypes expressed in HT1080 cells. Both HT1080 (mutant N-ras) and MCH603 (wild-type N-ras) have similar levels of constitutively active Akt, a downstream target of activated PI 3-kinase. We find that both cell lines constitutively express platelet-derived growth factor (PDGF) and PDGF receptors. Transfection with tumor suppressor PTEN cDNA into HT1080 and constitutively active PI 3-kinase-CAAX cDNA into MCH603 cells, respectively, resulted in several interesting and novel observations. Activation of the PI 3-kinase/Akt pathway, including NF-kappaB, is not required for the aggressive tumorigenic phenotype in HT1080 cells. Activation of NF-kappaB is complex: in MCH603 cells it is mediated by Akt, whereas in HT1080 cells activation also involves other pathway(s) that are activated by mutant Ras. A threshold level of activation of PI 3-kinase is required in MCH603 cells before stimulatory cross talk to the RhoA, Rac1, and Raf pathways occurs, without a corresponding activation of Ras. The increased levels of activation seen were similar to those observed in HT1080 cells, except for Raf and MEK, which were more active than HT1080 levels. This cross talk results in conversion to the aggressive tumorigenic phenotype. This latter observation is consistent with our previous observation that overstimulation of the activity of endogenous members of Ras signaling pathways, activated MEK in particular, is a prerequisite for aggressive tumorigenic growth.
Gupta
S
S
Department of Microbiology and Molecular Genetics, College of Medicine, University of California-Irvine, Irvine, CA 92697-4025, USA.
Stuffrein
S
S
Plattner
R
R
Tencati
M
M
Gray
C
C
Whang
Y E
YE
Stanbridge
E J
EJ
D000199:Actins / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005354:Fibrosarcoma / Q000503:physiopathology*; D006801:Humans; D020672:I-kappa B Proteins; D051379:Mice; D008819:Mice, Nude; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D020574:Proto-Oncogene Proteins c-sis / Q000378:metabolism; D014407:Tumor Cells, Cultured; D051025:bcl-Associated Death Protein
D000199:Actins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B; D019869:Phosphatidylinositol 3-Kinases; D010982:Platelet-Derived Growth Factor; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2; D020574:Proto-Oncogene Proteins c-sis; D051025:bcl-Associated Death Protein; C494819:BAD protein, human; C494820:Bad protein, mouse; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; C075278:platelet-derived growth factor A; C494918:AKT1 protein, human