K-ras codon 12 mutation induces higher level of resistance to apoptosis and predisposition to anchorage-independent growth than codon 13 mutation or proto-oncogene overexpression.
The position of the point mutation in the c-K-ras gene appears associated with different degrees of aggressiveness in human colorectal tumors. In addition, colon tumors carrying K-ras codon 12 mutations associate with lower levels of apoptosis than tumors lacking this mutation. To test the hypothesis of a distinct transforming capacity of different K-ras forms in an in vitro system, we generated stable transfectants of NIH3T3 cells expressing a plasmid containing K-ras mutated at codon 12 (K12) or at codon 13 (K13), or overexpressing the K-ras proto-oncogene (Kwt-oe). We evaluated changes in morphology, proliferative capacity, contact inhibition, and predisposition to apoptosis and anchorage-independent growth in K12, K13, and Kwt-oe transformants. In addition, we studied alterations in expression and/or activation of proteins that participate in signal transduction downstream of Ras or are involved in the regulation of apoptosis and cell-cell (E-cadherin and beta-catenin) and cell-substrate (focal adhesion kinase) interactions. We observed that K13 or Kwt-oe transformants died synchronically 24-48 h after reaching confluency. Their death was apoptotic. In contrast, K12 grew, forming bigger colonies with higher cell densities; and before reaching confluency, spontaneously formed spheroids and showed no sign of apoptosis. The enhanced resistance to apoptosis, loss of contact inhibition, and predisposition to anchorage-independent growth in the K12 transformants were associated with higher AKT/protein kinase B activation, bcl-2, E-cadherin, beta-catenin, and focal adhesion kinase overexpression, and RhoA underexpression, whereas the increased sensitivity of K13 or Kwt-oe transformants to apoptosis was associated with increased activation of the c-Jun-NH2-terminal kinase 1 pathway. All transformants showed a similar overactivation of mitogen-activated protein kinases and levels of bax expression similar to the endogenous level. Therefore, in our in vitro model, the localization of the mutation in the K-ras gene predisposes to a different level of aggressiveness in the transforming phenotype. K12 may increase aggressiveness not by altering proliferative pathways, but by the differential regulation of K-Ras downstream pathways that lead to inhibition of apoptosis, enhanced loss of contact inhibition, and increased predisposition to anchorage-independent growth. These results offer a molecular explanation for the increased aggressiveness of the tumors with K-ras codon 12 mutations observed in the clinical setting.
Guerrero
S
S
Laboratori d'Investigació Gastrointestinal, Institut de Recerca, Hospital de Sant Pau, Barcelona, Spain.
Casanova
I
I
Farré
L
L
Mazo
A
A
Capellà
G
G
Mangues
R
R
D016475:3T3 Cells / Q000166:cytology / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D015820:Cadherins / Q000096:biosynthesis / Q000235:genetics; D002448:Cell Adhesion / Q000235:genetics; D002450:Cell Communication / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D003062:Codon; D003598:Cytoskeletal Proteins / Q000096:biosynthesis / Q000235:genetics; D004789:Enzyme Activation; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D015972:Gene Expression Regulation, Neoplastic; D011905:Genes, ras / Q000235:genetics*; D020935:MAP Kinase Signaling System / Q000235:genetics; D051379:Mice; D010641:Phenotype; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D015534:Trans-Activators; D014162:Transfection; D014170:Transformation, Genetic; D051176:beta Catenin; D020742:rhoA GTP-Binding Protein / Q000096:biosynthesis / Q000235:genetics
C495267:CTNNB1 protein, mouse; D015820:Cadherins; D003062:Codon; D003598:Cytoskeletal Proteins; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D015534:Trans-Activators; D051176:beta Catenin; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020742:rhoA GTP-Binding Protein