Specificity of Ras Signaling in Breast Cancer

Institution: Scripps Research Institute
Investigator(s): Janis Jackson, M.D. -
Award Cycle: 1999 (Cycle V) Grant #: 5JB-0139 Award: $236,706
Award Type: IDEAS II
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (1999)
In breast cancer, the normal control mechanisms that regulate growth, differentiation and/or cell death become defective. These control mechanisms consist of regulatory proteins on the surface and an inside of cells that send signals to each other via complex protein pathways. Collectively, this is referred to as signal transduction. Breast cancer cells frequently contain regulatory proteins on the external surfaces, such as the Her-2 receptor and EGF receptor, that have become defective or are expressed at abnormally high levels. These defective/over-expressed receptor proteins send continuous signals to a regulatory protein called Ras, and these signals convert Ras from its inactive GDP-bound state to its active GTP-bound state. Breast cancer cells also frequently contain estrogen receptors, and when these receptors bind estrogen, they also send activating signal to Ras. Ras is located on the inner surface of cells, and once it has become activated, it sends activating signals to other regulatory proteins, and thereby initiating downstream signaling cascades that can promote breast cancer cell growth.

Human cells contain four very similar Ras proteins, H-Ras, N-Ras, K-Ras 4A and K-Ras 4B, but it is currently unknown whether each or only one of these Ras proteins contributes to breast cancer cell growth. The purpose of our studies is to determine whether the Her-2, EGF, and/or estrogen receptors send activating signals to each of the Ras proteins or only one of the Ras proteins.

For our experiments we will use breast cancer cells over-expressing the Her-2, EGF, or estrogen receptor. cDNAs encoding each of the four Ras proteins will be introduced into these cells by transfection. To determine whether the Her-2, EGF and/or estrogen receptors send activating signals to each or only one of the four Ras proteins, we will assess the relative amounts of GTP vs.GDP bound to each of the Ras proteins in the transfected cells. In addition, we will determine whether there are any differences in two downstream indicators of Ras activation (activation of MAP kinase and cell growth) in these cells.

Our studies are important, because demonstration that a single Ras protein contributes to breast cancer could provide the rationale for the development of inhibitors specific for that single Ras protein. These inhibitors could potentially help neutralize breast cancer growth in the approximately 30% of human breast cancer containing defective/over-expressed Her-2 or EGF receptors. In addition, these inhibitors might also be helpful in treating the approximately 50+% of breast cancers containing estrogen receptors.

Final Report (2001)
Human cells contain four homologous Ras proteins, but it is unknown whether each of these Ras proteins participates in distinct signal transduction cascades or has different biological functions. To directly address these issues, we assessed the relative ability of constitutively active (G12V) versions of each of the four Ras homologs to activate the effector protein Raf-1 in vivo. In addition, we compared their relative abilities to induce transformed foci, enable anchorage-independent growth, and stimulate cell migration. We found a distinct hierarchy between the four Ras homologs in each of the parameters studied. The hierarchies were as follows: for Raf-1 activation, Ki-Ras 4B > Ki-Ras 4A >>> N-Ras > Ha-Ras; for focus formation, Ha-Ras >/= Ki-Ras 4A >>> N-Ras = Ki-Ras 4B; for anchorage-independent growth, Ki-Ras 4A >/= N-Ras >>> Ki-Ras 4B = Ha-Ras = no growth; and for cell migration, Ki-Ras 4B >>> Ha-Ras > N-Ras = Ki-Ras 4A = no migration. Our results indicate that the four Ras homologs significantly differ in their abilities to activate Raf-1 and induce distinctly different biological responses. These studies, in conjunction with our previous report that demonstrated that the Ras homologs can be differentially activated by upstream guanine nucleotide exchange factors (Jones, M. K., and Jackson, J. H. (1998) J. Biol. Chem. 273, 1782-1787), indicate that each of the four Ras proteins may qualitatively or quantitatively participate in distinct signaling cascades and have significantly different biological roles in vivo. Importantly, these studies also suggest for the first time that the distinct and likely cooperative biological functions of the Ki-ras-encoded Ki-Ras 4A and Ki-Ras 4B proteins may help explain why constitutively activating mutations of Ki-ras, but not N-ras or Ha-ras, are frequently detected in human carcinomas.