The Role of Ral GTPases in Human Oncogenic Transformation

Abstract

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.