Oncogenic Ras induces IR resistance in many tumor types, including pancreatic cancer, where over 90% of tumors carry oncogenic mutations in K-Ras. Recent work indicates that K-Ras-mediated resistance to IR in pancreatic cancer cells is in part dependent upon the Ras effector PI3K (9
), but does not require Raf (1
). However, the role of the Ral small GTPases, which are also important Ras effectors, is unknown. As such, we focused our studies on Ral. We found that knockdown of either RalA or RalB sensitized MIA PaCa-2 cells to IR. Although RalA and RalB are 82% identical, they have distinct functions in pancreatic cancer cells (20
). Thus, it was perhaps unexpected that both RalA and RalB sensitized cells to IR. However, it appears that RalA and RalB do not function via completely overlapping mechanisms, as knockdown of both proteins simultaneously did not further increase sensitization.
The molecular mechanisms of Ras-regulated IR response are poorly understood. Ras-induced resistance has been proposed to function through modulation of cell cycle checkpoints, as oncogenic H-Ras causes resistance and prolongation of G2 phase in primary rat embryo cells (32
). Additionally, oncogenic H-Ras expression enhances repair of gamma-, UV- and cisplatin-induced DNA damage in NIH 3T3 cells (35
). The ability to repair DSBs is widely considered the most critical factor controlling IR sensitivity (37
). As such, we used γH2AX as a marker of IR-induced DSBs. As expected, γH2AX expression increased shortly after IR regardless of Ral expression levels. In control cells, γH2AX levels declined by 3 h after IR while RalA and RalB knockdown resulted in high γH2AX levels for at least 6 h. RalB knockdown resulted in a slightly weaker defect than did RalA knockdown. RalA upregulation in RalB knockdown cells may explain this difference. It would also be interesting to determine whether there are differential Ral isoform effects on S-phase distribution following radiation, which could further impact total cellular γH2AX. To our knowledge, this is the first study to show a role for Ral proteins in repair of IR-induced DNA damage. Interestingly, in immortalized fibroblasts and an osteosarcoma-derived p53-deficient cell line, oncogenic H-Ras also inhibits the G2-M checkpoint in a manner dependent on Ral proteins but not RalBP1 (38
). This is consistent with our findings that RalBP1 does not mediate Ral-dependent changes in IR responses in MIA PaCa-2 cells, which are also p53-deficient. Ral proteins also drive activation of cyclin D1 and cell cycle progression from G1-S in a RalBP1-independent manner (40
). Thus, Ral proteins are involved in two distinct cell cycle checkpoints. It will be of interest in future studies to determine if modulation of these checkpoints is utilized by Ral to render cells radioresistant.
Given previous reports indicating that RalB functions as an anti-apoptotic factor, we analyzed PARP cleavage to measure caspase-3-dependent apoptosis (18
). We found that control and RalA knockdown cells exhibited similar PARP cleavage kinetics post-IR. Since RalA knockdown decreases clonogenic survival and perturbs DSB resolution, we conclude that RalA likely contributes to cell death primarily via a caspase-independent mechanism, such as post-mitotic catastrophe (41
). In constrast to control and RalA knockdown, RalB knockdown caused more rapid induction of apoptosis. This is consistent with published data indicating that RalB normally inhibits apoptosis (18
). RalB engages Sec5, which in turn binds and activates TBK1 kinase to promote survival (31
). As Sec5 knockdown did not cause IR sensitivity, it is possible that RalB-dependent apoptosis is not a major contributor to IR sensitivity of MIA PaCa-2 cells. Alternatively, RalB-dependent apoptosis may occur via a Sec5-independent mechanism and constitute a significant component of IR sensitivity.
RalBP1 is a multi-domain protein with diverse functions (17
). It can protect cells by transporting toxic metabolites (45
). RalBP1 is critical for IR response in mice and mouse embryonic fibroblasts (24
). Thus, RalBP1 was an excellent candidate for mediation of Ral-dependent IR resistance, and we were surprised that RalBP1 knockdown had no significant effect on clonogenic growth of MIA PaCa-2 cells post-IR. It is unlikely that knockdown was insufficient to reduce clonogenic survival in this context, since RalBP1 protein was reduced by ~90%. Instead, our findings likely differ from previous reports on RalPB1 due to differences in cell type or cell context. In future analyses, it will be interesting to see whether tumor type- or cell type-specific genetic alterations can predict a requirement for Ral in radiation responsiveness.
We next considered a Ral effector that is part of the exocyst, a protein complex involved in vesicle targeting. The exocyst is important for processes including establishing and maintaining cell polarity. It has been suggested that exocyst-induced polarity changes alter cell proliferation and survival (25
). Ral directly regulates exocyst formation via interaction with two components, Sec5 and Exo84 (47
). We thought it plausible that Ral proteins direct specific exocyst-mediated protein and membrane movements that control IR response. However, our clonogenic survival assays indicated that Sec5 knockdown does not alter IR response of MIA PaCa-2 cells.
Other Ral effectors have been identified, although their roles in oncogenesis remain poorly characterized. Although not a classical Ral effector pathway, in that interaction does not depend on GTP binding, Ral activation of phospholipase D1 (PLD1) has been implicated in cytokinesis and other cellular processes (48
). Ral proteins also transmit signals using ZONAB, a Y-box transcription factor that is regulated by association with Ral. ZONAB is unlikely to mediate Ral-dependent changes in IR sensitivity because the interaction between RalA and ZONAB occurs only when cells are grown at high density (50
), whereas RalA and RalB knockdowns significantly alter IR response of cells grown in isolation.
In conclusion, our results indicate that both RalA and RalB make significant contribution to K-Ras dependent IR resistance of MIA PaCa-2 pancreatic cancer cells. Sensitization due to knockdown of RalA or RalB is, at least in part, due to decreased ability to repair DNA damage. RalB knockdown also may cause sensitization by increasing susceptibility to apoptosis. Neither canonical effector, RalBP1 nor Sec5, has a significant individual role in post-IR survival. Thus, it will be interesting to determine whether irradiation causes Ral proteins to engage atypical or novel effectors.