[CANCER RESEARCH 64, 4585– 4592, July 1, 2004] Involvement of Ras Activation in Human Breast Cancer Cell Signaling, Invasion, and Anoikis Lynn B. Eckert, 1 Gretchen A. Repasky, 1 Aylin S. U ¨ lku ¨, 1,2 Aidan McFall, 1 Hong Zhou, 1,3 Carolyn I. Sartor, 1,3 and Channing J. Der 1,2 1 Lineberger Comprehensive Cancer Center, 2 Department of Pharmacology, and 3 Department of Radiation Oncology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina ABSTRACT Although mutated forms of ras are not associated with the majority of breast cancers (<5%), there is considerable experimental evidence that hyperactive Ras can promote breast cancer growth and development. Therefore, we determined whether Ras and Ras-responsive signaling pathways were activated persistently in nine widely studied human breast cancer cell lines. Although only two of the lines harbor mutationally activated ras, we found that five of nine breast cancer cell lines showed elevated active Ras-GTP levels that may be due, in part, to HER2 activa- tion. Unexpectedly, activation of two key Ras effector pathways, the extracellular signal-regulated kinase (ERK) mitogen-activated protein ki- nase and phosphatidylinositol 3-kinase/AKT signaling pathways, was not always associated with Ras activation. Ras activation also did not correlate with invasion or the expression of proteins associated with tumor cell invasion (estrogen receptor  and cyclooxygenase 2). We then examined the role of Ras signaling in mediating resistance to matrix deprivation- induced apoptosis (anoikis). Surprisingly, we found that ERK and phos- phatidylinositol 3-kinase/AKT activation did not have significant roles in conferring anoikis resistance. Taken together, these observations show that Ras signaling exhibits significant cell context variations and that other effector pathways may be important for Ras-mediated oncogenesis, as well as for anoikis resistance, in breast cancer. Additionally, because ERK and AKT activation are not strictly associated with Ras activation, pharmacological inhibitors of these two signaling pathways may not be the best approach for inhibition of aberrant Ras function in breast cancer treatment. INTRODUCTION The three human ras genes (H-, N-, and K-Ras) encode regulated GTP/GDP on-off switches that relay extracellular signals to cytoplas- mic signaling networks (1–3). In normal cells, extracellular stimuli that act on diverse cell surface receptors that include receptor tyrosine kinases (e.g., HER2) cause transient activation of Ras. Ras, in turn, associates with and activates multiple downstream effectors that stim- ulate cytoplasmic signaling cascades that regulate cell proliferation, survival, and differentiation. The best-characterized downstream effector targets of Ras are the Raf serine/threonine kinases (4). Ras binds to and promotes Raf activation, and activated Raf, in turn, phosphorylates and activates the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)1 and MEK2 dual specificity kinases, which then phosphorylate and activate the ERK1 and ERK2 mitogen-activated protein kinases. The recent identification of mutationally activated B-raf alleles in melanoma, colon, and other types of cancers support the importance of aberrant RafMEKERK signaling in Ras- me- diated cancer progression and maintenance (5, 6). The second best characterized Ras effectors are the phosphatidyli- nositol 3'-kinases (PI3K; Refs. 7, 8). PI3K lipid kinases regulate phosphoinositide lipid metabolism, in particular the conversion of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5- trisphosphate. An important consequence of phosphatidylinositol 3,4,5-trisphosphate production is the activation of the AKT serine/ threonine kinase. Experimental studies have demonstrated the impor- tance of PI3K/AKT signaling in Ras-induced transformation of some but not all cell types (9 –11). The loss of PTEN tumor suppressor function, a negative regulator of PI3K, supports the importance of this signaling pathway in Ras-mediated human cancer development (12). Aside from Raf and PI3K, other effectors important in promoting Ras transformation include guanine nucleotide exchange factors for the Ral and Rac small GTPases (13–15). Additional Ras effectors have been identified that promote apoptosis (Nore1 and RASSF1), and consequently, antagonize tumorigenesis (8). Altogether, 20 functionally distinct Ras effectors have been identified, and effector utilization by Ras is complex and remains poorly understood. Addi- tional complexity is added by the significant cell context variations observed for Ras effector activation and utilization. For example, although the majority of human pancreatic cancers harbor mutated ras alleles, persistent ERK activation is not associated consistently with Ras activation (16). In a mouse model for pancreatic cancers driven by Ras activation, the tumors that arise possess activated Ras, yet the ERK and AKT pathways are not persistently activated (17). These and other examples emphasize that the signaling outcomes of Ras activa- tion are variable and not always predictable. The high frequency of ras mutations seen in some neoplasms (e.g., 90% of pancreatic cancers) supports a critical role for aberrant Ras activation in the progression and maintenance of these cancers (18). In contrast, ras mutations are rarely seen in some neoplasms, such as breast cancers (5%). However, despite this low frequency, there is considerable experimental evidence that aberrant Ras activation and signaling may promote breast cancer development (19). Instead of direct mutational activation in breast cancers, Ras may be activated by persistent upstream signaling. In particular, the HER2 [ErbB2/epider- mal growth factor receptor (EGFR)2/Neu] receptor tyrosine kinase is overexpressed and persistently activated in approximately 20 –25% of human breast cancers (20, 21). Persistent HER2 signaling promotes oncogenic transformation, in part, by activation of Ras (22–24). However, in light of cell context differences in Ras signaling, it is not clear what signaling pathways are stimulated by HER2-induced Ras activation. One critical step in tumor cell progression is the acquisition of anchorage-independent growth potential (25). Normal breast epithe- lial cells deprived of matrix attachment undergo apoptosis, or anoikis (26 –28), whereas tumor cells or oncogene-transformed cells have escaped this requirement and continue proliferate (29). The signaling mechanisms that prevent anoikis have been evaluated in experimental model cell systems. For example, Ras activation has been shown to block anoikis in untransformed MCF-10A human breast epithelial Received 2/5/04; revised 4/3/04; accepted 5/4/04. Grant support: NIH Grants CA42978 and CA69577 (C. Der), and P50CA58223 National Cancer Institute Breast Cancer Specialized Programs of Research Excellence and Breast Cancer Research Foundation K08CA83753 (C. Sartor). G. Repasky was supported as a Merck Fellow of the Life Sciences Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Channing J. Der, University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, CB# 7295, Department of Pharmacology, Chapel Hill, NC 27599-7295. Phone: (919) 966-5634; Fax: (919) 966-0162; E-mail: cjder@med.unc.edu. 4585 Research. on January 8, 2016. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from