Browsing by Subject "RalA"

The Ras family of proteins, composed of H, N, and KRas, function as small GTPases that act as "molecular switches" relaying signals from the cell membrane to the rest of the cell in a highly regulated manner. However, Ras signaling is aberrantly activated in a majority of human cancers either through an activating mutation or by activation or overexpression of upstream or downstream elements in the Ras pathway, endowing cells with many tumorigenic phenotypes. Ras is known to promote tumorigenesis through activation of cell intrinsic signaling including the Raf, PI3K, and RalGEF pathways. In regards to the latter, RalGEFs activate two other small GTPases, RalA and RalB. The role of these two proteins in Ras-mediated cancer was poorly understood. I thus assessed the requirement of RalA and RalB in tumor metastasis discovering that both proteins promote this critical step in cancer.

Ras does not, however, function solely by intrinsic cell signaling. Indeed, it was recently shown that oncogenic Ras signaling induces secretion of cytokines, a category of small molecules involved in cell to cell communication and inflammatory response. Moreover, the release of these cytokines was shown to promote tumorigenesis in an extrinsic fashion by increasing tumor vasculature, or angiogenesis. I noted that one of these cytokines hCXCL-8 (IL-8) belonged to the ELR+ CXC family of cytokines, suggesting that the entire family of ELR+ CXC cytokines may promote Ras driven tumorigenesis. Indeed, I found that expression of oncogenic Ras led to secretion of all ELR+ CXC chemokines in oncogenic Ras driven tumor cell lines, a mouse tumor, a human tumor, and were sometimes elevated in the serum of pancreatic cancer patients, the cancer most associated with oncogenic Ras mutations. Moreover, knockdown of one of these chemokines, hCXCL-1, in pancreatic cancer cells or genetic ablation of the receptor for these cytokines in mice, reduced Ras driven tumorigenesis. Taken together, these results suggest that oncogenic Ras also promotes tumorigenesis through a cell extrinsic pathway by secretion of ELR+ CXC chemokines.

The genes encoding the Ras family of small GTPases are mutated to yield constitutively active GTP-bound oncoproteins in one-third of all human cancers. In many other cancers lacking Ras mutations, Ras is activated by other means. One common example of such activation is found in breast cancer, in which epidermal growth factor receptor (EGFR) family receptor tyrosine kinases, including EGFR and HER2 (ErbB-2/Neu), are frequently amplified and overexpressed, which in turn activates Ras. In human cells, activation of the Ral guanine nucleotide exchange factor, or RalGEF, effector pathway is necessary for Ras-mediated tumorigenesis and metastasis. RalGEFs activate the two highly similar Ral GTPases, RalA and RalB. While RalA has been shown to be required for Ras-mediated tumorigenesis, RalB is important for tumor metastasis. Activated Ral GTPases bind to and activate a limited number of effector proteins, including RalBP1, Sec5, and Exo84, to affect numerous diverse activities of the cell. This dissertation research sought to determine which of these well-characterized Ral effector proteins were required for oncogenic mutant Ras-induceded tumorigenesis and metastasis of human cells, as well as to examine the role of RalA in breast cancer cells that can activate Ras through EGFR and HER2 overexpression.

RNA interference-mediated loss-of-function analysis demonstrated that Sec5 and Exo84 are required for oncogenic Ras-mediated tumorigenesis, and, at least in part, metastasis. Additionally, both gain-of-function and inhibition studies showed that RalA activation is induced by EGFR and HER2 in breast cancer cell lines stimulated with EGF. Furthermore, stable suppression of RalA expression inhibited tumorigenic growth of breast cancer cells, and RalA activation was shown to be higher in a majority of mammary adenocarcinomas versus matched patient normal mammary tissue. These studies provide new insights into the importance of RalA activation in breast cancer, as well as the molecules downstream of RalA and RalB that may be responsible for mediating their effects on tumorigenesis and metastasis.