Inhibitors targeting the PI3K pathway have the potential to be effective anticancer agents and, as such, are being developed at a rapid pace. However, previous experience with targeted therapies predicts that patients who initially respond invariably relapse due to acquisition of drug resistance. To anticipate mechanisms of resistance to PI3K inhibitors, we have screened a library of kinase ORFs and have identified a number of kinases that circumvent PI3K inhibitor sensitivity. Validated candidates included potent activators of PI3K and ERK signaling pathways, such as ERBB2 and IGF1R, as well as downstream effectors AKT1 and AKT3. In addition, we have identified the RSK family members RSK3 and RSK4 as repressors of PI3K inhibitor function. Functional studies have implicated RSKs in the regulation of diverse cellular processes, including transcription, translation, survival, cell-cycle progression, and migration, through phosphorylation of targets including CREB, GSK3, TSC2, rpS6, raptor, eIF4B, BAD, and p27, among others (26
). The RSKs have all been linked with tumorigenesis, albeit in different contexts. RSK1 and RSK2 have been reported as overexpressed in breast and prostate cancer, while RSK3 has been proposed to be a tumor suppressor in ovarian cancer (55
). RSK4 has previously been characterized as essential for p53-dependent proliferation arrest as well as stress- and oncogene-induced senescence (28
). Interestingly, the RSK4 isoform exhibits constitutively high activity, is upregulated in MMTV-Myc mouse breast tumors, is aberrantly expressed in breast cancer, and has been implicated in sunitinib resistance (58
). Here, we demonstrate that RSK3 and RSK4 can also mediate resistance to PI3K inhibitors in breast cancer cells both in vitro and in vivo.
Our observations strongly support a role for retention of rpS6 and eIF4B phosphorylation in the resistance phenotype of RSK-overexpressing cells, in agreement with a previous report noting retention of rpS6 phosphorylation in breast cancer cell lines exhibiting intrinsic resistance to PI3K inhibition (9
). Previous studies have suggested that RSKs directly phosphorylate rpS6 at Ser235/236
and eIF4B at Ser422
. The former promotes binding of rpS6 to the 7-methylguanosine cap complex and enables cap-dependent translation to proceed, while the latter is critical for eIF4B binding to the cap complex and enhanced helicase activity of eIF4A and increased cellular translation (64
). In agreement with these results, we observed that RSK4-overexpressing cells exhibited elevated levels of overall translation, which are maintained in the presence of PI3K inhibitors (Figure F). These results are also consistent with a previous report implicating upregulation of cap-dependent translation by eIF4E amplification in promoting resistance to BEZ235 (67
As RSKs are directly regulated by RAF/MEK/ERK signaling, we hypothesized that inhibition of this pathway would overcome the resistance phenotype of RSK-overexpressing cells and reverse all associated cellular phenotypes. We observed that addition of MEK or RSK inhibitors restored responsiveness of RSK-expressing cells to PI3K inhibitors by all parameters analyzed, including translation, S6 phosphorylation, cell viability, and in vivo tumor formation (Figures – and Supplemental Figures 5–8). Importantly, this reversal of phenotype was specific for RSKs, as AKT1-overexpressing cells remained refractory to PI3K inhibition even with the addition of MEK or RSK inhibitors.
One potential limitation of this study is the fact that we were unable to examine RSK inhibition, either through chemical inhibition or knockdown of RSK4, in relevant xenograft models. This is primarily due to the technical difficulty of the experiments and the lack of suitable chemical reagents currently available. Significantly, however, in both in vitro and in vivo experiments, MEK inhibitors inhibited RSK phosphorylation (Figure E, Supplemental Figure 6A, and data not shown), indicating that the MEK inhibitors used in our animal models effectively inhibited RSK activity. Collectively, our data suggest that RSK overexpression renders tumors insensitive to PI3K inhibition, which can be overcome by inhibiting the MEK/ERK/RSK pathway.
The observations presented here support the notion that breast cancer cells upregulate overall protein translation and cell proliferation through overlapping but parallel pathways, the PI3K/mTOR and ERK/RSK pathways (Figure F). Interestingly, another significant outlier in our screen, the protooncogene PIM2, regulates key effectors of cap-dependent translation, including eIF4E, 4EBP1, and S6K, independently of the PI3K/mTOR pathway, supporting the notion that combined pharmacological inhibition of multiple translational regulators should be explored (Figure F) (68
A number of reports have recently shown that an elevated ERK activation signal, either through intrinsic KRAS mutations or through the activation of compensatory feedback loops observed following PI3K inhibition, limits the effectiveness of PI3K inhibitors in the clinic (13
). Early clinical trials assessing the effectiveness of PI3K and MEK inhibitors have demonstrated some evidence of efficacy in certain tumor types (69
). However, initial reports seem to suggest that the use of MEK inhibitors in the clinic results in undesired toxicities, limiting the effectiveness of this compound (70
). Importantly, our studies suggest that targeted RSK inhibition is as effective as MEK inhibition when used in combination with PI3K inhibitors, resulting in similar degrees of decreased proliferation and augmented apoptosis. As RSK-specific inhibitors target only a single effector arm of MAPK signaling, they may provide a therapeutic window circumventing many of the potential toxicities associated with current MEK-PI3K inhibitor combination strategies. Moreover, we anticipate that use of this combination will also be indicated in the treatment of tumors that exhibit evidence of MEK/ERK–driven signaling.