Selective Inhibition of Ras, Phosphoinositide 3 Kinase, and Akt Isoforms Increases the Radiosensitivity of Human Carcinoma Cell Lines In-Ah Kim, 1 Sun-Sik Bae, 2 Annemarie Fernandes, 1 JunMin Wu, 1 Ruth J. Muschel, 3 W. Gillies McKenna, 1 Morris J. Birnbaum, 2 and Eric J. Bernhard 1 1 Department of Radiation Oncology and 2 Howard Hughes Medical Institute, University of Pennsylvania; and 3 Department of Pathology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania Abstract Ras activation promotes the survival of tumor cells after DNA damage. To reverse this survival advantage, Ras signaling has been targeted for inhibition. Other contributors to Ras-mediated DNA damage survival have been identified using pharmacologic inhibition of signaling, but this approach is limited by the specificity of the inhibitors used and their toxicity. To better define components of Ras signaling that could be inhibited in a clinical setting, RNA interference was used to selectively block expression of specific isoforms of Ras, phosphoinositide 3 (PI3) kinase, and Akt. Inhibition of oncogenic Ras expression decreased both phospho-Akt and phospho-p42/44 mitogen- activated protein (MAP) kinase levels and reduced clonogenic survival. Because pharmacologic inhibition of PI3 kinases and Akt radiosensitized cell lines with active Ras signaling, whereas inhibition of the MAP/extracellular signal–regulated kinase (ERK) kinase/ERK pathway did not, we examined the contribu- tion of PI3 kinases and Akts to radiation survival. Selective inhibition the PI3 kinase P110A + p85B isoforms reduced Akt phosphorylation and radiation survival. Similarly, inhibition of Akt-1 reduced tumor cell radiation survival. Inhibition of Akt-2 or Akt-3 had less effect. Retroviral transduction and over- expression of mouse Akt-1 was shown to rescue cells from inhibition of endogenous human Akt-1 expression. This study shows that Ras signaling to the PI3 kinase–Akt pathway is an important contributor to survival, whether Ras activation results from mutation of ras or overexpression of epidermal growth factor receptor. This study further shows that selective inhibition of the PI3 kinase P110A + p85B isoforms or Akt-1 could be a viable approach to sensitizing many tumor cells to cytotoxic therapies. (Cancer Res 2005; 65(17): 7902-10) Introduction Ras activation and survival after DNA damage. Ras activation by mutation is frequent in certain human tumors (1, 2), whereas activation of Ras signaling by growth factor receptor deregulation or other mechanisms is common in other tumor types (3, 4). Activation of Ras signaling has been shown to increase the survival of tumor cells exposed to agents that cause DNA damage. Survival after irradiation of many transformed and tumor cell lines is increased by expression of oncogenic Ras (reviewed in ref. 5). Overexpression of the ras proto-oncogene, or receptor-mediated activation of wild-type Ras signaling, has also been shown to promote radiation survival (6, 7). Ras activation also increases survival after DNA damage induced by chemotherapeutic agents. Early studies in mouse fibroblasts showed that expression of oncogenic H-, K-, or N-Ras conferred resistance to cis -platinum (8). Enhanced survival was shown in breast carcinoma cells expressing oncogenic Ras after exposure to cis -platinum (9), paclitaxel, doxorubicin, and 5-fluorouracil (10), and myeloma cells exposed to doxorubicin (11). Nooter et al. (12) showed enhanced resistance to doxorubicin in a rhabdomyosarcoma cell line transfected with the H-ras oncogene. Jansen et al. (13) showed melanoma xenograft resistance to cis -platinum after transfection with oncogenic N-ras . However, resistance has not been reported by all investigators. Choi et al. (14) found that resistance was imparted by oncogenic H-ras , but not K-ras transfection of rodent cells, and equivocal results or no resistance was seen in some other experimental models (15–17). However, the preponderance of evidence links Ras activation to enhanced survival of cells treated with DNA-damaging agents. Disrupting Ras signaling has been shown to reverse the resistance imparted by Ras activation. Inhibition of Ras expression by antisense oligonucleotides has been reported to radiosensitize cells (18). Blocking posttranslational prenylation, which is required for Ras-mediated transformation, has also been shown to reduce radiation survival in a number of rodent and human tumor models that express oncogenic Ras (19–22). Gana-Weisz et al. (23) showed that disruption of Ras membrane anchorage increased SW480 colon carcinoma and Panc-1 pancreatic carcinoma cell sensitivity to gemcitabine. Both of these cell lines express oncogenic K-Ras. These findings point to Ras signaling as an important factor in the response of tumors to a range of cytotoxic therapies. This hypothesis is supported by the results obtained in several clinical studies that have examined Ras activation and tumor responses. These studies have reported K-ras mutation as an independent prognostic factor pointing to poor progression-free survival in patients with stage III non–small cell lung cancer that were treated with surgery, radiation, and chemotherapy (24); non–small cell lung cancer treated with single agent paclitaxel (25); pancreatic cancer treated with radiotherapy (26); and colon cancer treated with CPT- 11 (27). Not all clinical reports, however, have shown an association between ras mutation and resistance (28–30). Ras activation by epidermal growth factor receptor. Signals transmitted through wild-type Ras proteins by mutated or over- expressed tyrosine kinase receptors can also influence cell survival. The epidermal growth factor receptor (EGFR, also denoted as erbB1 or HER1) plays a pivotal role in the regulation of cell growth and differentiation. This receptor transmits growth regulatory Note: I-A. Kim is currently in the Department of Radiation Oncology, Cancer Research Institute, Seoul National University, Seoul, South Korea. Requests for reprints: Eric J. Bernhard, Department of Radiation Oncology, University of Pennsylvania, 185 John Morgan Building, Philadelphia, PA 19104-6072. E-mail: bernhard@mail.med.upenn.edu. I2005 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-0513 Cancer Res 2005; 65: (17). September 1, 2005 7902 www.aacrjournals.org Research Article Research. on December 12, 2015. © 2005 American Association for Cancer cancerres.aacrjournals.org Downloaded from