Cooperative transformation of 32D cells by the combined expression of IRS-1 and V-Ha-Ras.
32D cells expressing v-Ha-Ras fail to show a transformed phenotype. Since Ras requires an active IGF-1R for transformation of fibroblasts, we asked whether expression of IRS-1 or Shc (two of the major substrates of the IGF-1R) could co-operate with oncogenic Ras in transforming 32D cells. We find that IRS-1, but not Shc, in combination with v-Ha-Ras generates a fully transformed phenotype in 32D cells. 32D cells expressing both IRS-1 and v-Ha-Ras (32D/IRS1/Ras) survive and proliferate in the absence of IL-3, do not undergo granulocytic differentiation in the presence of G-CSF and form tumors in nu/nu and syngeneic mice. In contrast, 32D cells expressing singly IRS-1 or v-Ha-Ras exhibit only a block in differentiation capacity. Over-expression of Shc proteins, by itself, promotes differentiation of 32D cells. Concomitant expression of IRS-1 and v-Ha-Ras synergistically phosphorylates ERK-1 and ERK-2 whereas a MEK inhibitor rapidly induces death of 32D/IRS1/Ras transformed cells. Furthermore, transformed 32D/IRS1/Ras cells display high levels of PI3-K activation and undergo rapid apoptosis when exposed to PI3-K inhibitors. The data indicate that: (1) a fully transformed phenotype in 32D cells is generated when a block in differentiation (v-Ha-Ras) is coupled with another differentiation block (IRS-1); (2) PI3-K and MAPK activity are required for the survival of transformed cells; (3) the signals generated by IRS-1 and oncogenic Ras converge on ERK and PI3-K resulting in high levels of activation.
Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute, CRS, Roma, Italy.
B
B
Cristofanelli
B
B
Valentinis
S
S
Soddu
M G
MG
Rizzo
A
A
Marchetti
G
G
Bossi
A R
AR
Morena
M
M
Dews
R
R
Baserga
A
A
Sacchi
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002454:Cell Differentiation; D002470:Cell Survival; D002471:Cell Transformation, Neoplastic; D002867:Chromones / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D015870:Gene Expression; D055504:Insulin Receptor Substrate Proteins; D048728:MAP Kinase Kinase Kinase 1; D051379:Mice; D008819:Mice, Nude; D009025:Morpholines / Q000494:pharmacology; D015689:Oncogene Protein p21(ras) / Q000096:biosynthesis / Q000235:genetics*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011817:Rabbits; D014162:Transfection; D014407:Tumor Cells, Cultured; D000077191:Wortmannin
D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D005419:Flavonoids; D055504:Insulin Receptor Substrate Proteins; D048728:MAP Kinase Kinase Kinase 1; D009025:Morpholines; D015689:Oncogene Protein p21(ras); D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D000077191:Wortmannin; C526295:Irs1 protein, mouse; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C482578:Map3k1 protein, mouse; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one