RSK has been shown by numerous groups to be a key regulator of breast cancer proliferation. Our lab was the first to report the importance of RSK in breast cancer proliferation together with the discovery of the first RSK-specific inhibitor, SL0101 [14
]. We found that RSK2 is over-expressed in 50% of human breast cancer tissues, compared to normal tissue [14
]. Since then many other groups have reported similar findings in breast and other tumor models. Law et al. recently showed that inhibition of the insulin-like growth factor 1 receptor (IGF-1R)/insulin receptor (IR) reduced proliferation and RSK activity in tamoxifen-resistent MCF-7 cells [13
]. In human breast tumors high levels of the phosphorylated RSK substrate ribosomal protein S6 (rpS6), were directly correlated with IGF-1R/IR levels, and were associated with poor patient survival [13
]. Thus, high levels of activated RSK promote tumorigenesis. Furthermore, ~56% of human breast tumors showed phosphorylated rpS6 indicating that RSK may be highly active. The ability of RSK to phosphorylate rpS6 has been controversial but recent studies have shown that rpS6 phosphorylation at Ser-235/236 is MAPK-dependent and has been attributed to RSK [25
]. RpS6 is one of many RSK substrates associated with proliferation and tumorigenesis ().
The ability of RSK to regulate survival, anchorage-independent growth, and transformation in breast cancer was recently confirmed by Xian et al [15
]. Their findings indicate that Fibroblast Growth Factor Receptor-1 (FGFR1)-mediated transformation of MCF-10A cells is dependent on RSK. FGFR1 is upregulated in many invasive lobular carcinomas (ILC), which, while ER+, may not respond very well to the ER antagonist, tamoxifen [26
]. Inhibitors of FGFR1 can suppress growth of the ILC cell line, MDA-MB-134, suggesting that FGFR1 signaling is essential for proliferation of these tumors. Knockdown of RSK1 and inhibition of RSK, with the specific small molecule inhibitor chloromethylketone (CMK), reduced proliferation, suppressed colony formation in soft agar, and decreased survival of FGFR1-expressing cells. Together these data strongly support the hypothesis that the RSKs are important in breast cancer etiology.
RSK4 is a putative tumor suppressor in breast cancer [19
]. Thakur et al., found that overexpressing RSK4 in MDA-MB-231 cells suppressed colony formation in soft agar, tumor formation, and metastasis. Curiously, RSK4 levels were found to be elevated in MMTV-c-Myc
transgenic mice [27
]. Expression of myc, a cell cycle regulator [29
], is upregulated very quickly following estrogen treatment and is essential for estrogen-mediated proliferation in breast cancer cells [31
]. Mammary tumors that form in c-Myc
transgenic mice are neither invasive nor metastatic and it is hypothesized that c-myc overexpression upregulates RSK4, which then suppresses aggressive expansion [27
]. Consistent with this hypothesis, c-myc was shown to stimulate RSK4 promoter activity in a luciferase reporter assay [27
]. Our knowledge of RSK4 remains limited. RSK4 may have tumor suppressor functions in some cancer types, but the paucity of data on this kinase suggests that further studies are necessary before specific conclusions can be drawn.
The growing body of literature implicating RSK in breast cancer supports the hypothesis that RSK is an important therapeutic target. We have found that treatment with the RSK-specific inhibitor, SL0101 (20 μṂ; 48hṛ, reduced proliferation in the immortalized human breast cancer cell line, MCF-7, but did not effect proliferation of the non-tumorigenic breast cell line, MCF-10A (, [14
]). Consistent with these findings, silencing RSK2 also reduced proliferation in MCF-7 cells. The mechanism by which RSK2 regulates proliferation in breast cancer cells is not well understood. However, significant evidence is emerging that indicates RSK regulates several key breast cancer-associated proteins. For example, we have found that RSK2 stimulates the transcriptional activity of estrogen receptor α (ERα) [33
] which is known to be important in the etiology of many breast cancers. Estrogens can stimulate RSK activity, and RSK2 enhances ERα-mediated transcription by phosphorylation and by physical association [33
]. The interaction of ERα and RSK can be disrupted by tamoxifen. This process may be dependent on the ERK1/2 pathway. Additionally, we have found that RSK2 regulates expression of the oncogene, cyclin D1, which is a co-activator of ERα and overexpressed in approximately 50% of human breast tumors [37
]. The importance of cyclin D1 as an oncogene is highlighted by the finding that overexpression of the protein is sufficient to induce formation of mammary tumors in transgenic animals [39
]. Although the ERK1/2 pathway is known to regulate cyclin D1 levels, we identified that cyclin D1 is a key RSK2 target in breast cancer cells [38
]. Consistent with findings in human tissue, we found that MCF-7 cells overexpress cyclin D1 as compared to MCF-10A cells by approximately 5-fold based on normalization to the housekeeping protein, Ran (). SL0101 (50 μṂ; 4hṛ reduced cyclin D1 levels in MCF-7 cells by 70% at the protein level and 40% at the mRNA level (, [38
]). Importantly, SL0101 did not affect cyclin D1 expression in MCF-10A cells () suggesting that RSK regulation of cyclin D1 is confined to transformed cells. SL0101 inhibits the kinase activity of RSK1 and RSK2 in in vitro
kinase assays, but RSK2 is primarily responsible for the regulation of cyclin D1 levels [38
]. We also found forced nuclear localization of RSK2 drives cyclin D1 expression in the absence of activation of any other signal transduction pathway [38
]. These results suggest that nuclear RSK2 is able to act as an oncogene in breast cancer.
RSK regulates proliferation and cyclin D1 levels in breast cancer cell lines
We have also identified a mechanism by which RSK regulates mRNA localization and translation via stress granules in breast cancer cells [38
]. Normal mammary and breast cancer cells form cytoplasmic RNA complexes called stress granules under either oxidative stress or serum-deprivation stress. In general, stress granules form under conditions in which translation initiation has been reduced or inhibited [40
]. These granules recruit selected mRNAs and associated proteins from polyribosomes, for storage, or for triage through processing bodies [41
]. Stress granules are thought to aid cell survival by acting as sites of translational repression and to facilitate post-stress recovery by acting as reservoirs of poly(A)+
RNA. In breast cells subjected to stress, endogenous RSK2 localizes to stress granules and controls recruitment of other key stress granule proteins in the complex [38
]. In response to stress, RSK interacts directly with the essential stress granule protein, TIA-1, driving stress granule formation. This regulation is physiologically important, because loss of RSK2, via specific knockdown or inhibition, prevents stress granule formation and decreases cell survival in response to stress. In nutritionally-stressed breast cancer cells, addition of mitogen triggers the dissolution of stress granules. Once released from sequestration in the granules, RSK2 accumulates in the nucleus, where it induces cyclin D1 expression in transformed cell lines, driving entry into the cell cycle. RSK2 has not previously been implicated as a regulatory component in the stress response and RSK2-mediated regulation of translation and cellular stress are understudied. However, there is now a significant amount of evidence suggesting that the involvement of RSK2 in translation may be crucial for understanding its role in tumor cell survival. In addition, several recent studies suggest that stress granule formation protects tumor cells from chemotherapy and radiation-induced stress [42
]. Interestingly, cyclin D1 mRNA has been found in stress granules [44
] suggesting that this mechanism may effectively protect mRNAs necessary for proliferation in breast cancer. RSK2 is an essential regulator of stress granules in breast cancer cells, and therefore RSK2 inhibition may increase tumor cell death in response to standard treatments.
Another mechanism by which RSK may regulate breast cancer cell proliferation is through phosphorylation of the transcription factor YB-1 at Ser-102 [45
]. YB-1 regulates expression of numerous proteins associated with tumorigenesis via direct interaction with the promoter regions of target genes [46
]. Not only is YB-1 overexpressed in breast and other cancers, but overexpression of YB-1 is sufficient to drive mammary tumor formation in mice [45
]. Conversely, knockdown of YB-1 suppresses tumor cell proliferation [49
]. Phosphorylation of YB-1 at Ser-102 is essential for nuclear translocation of the protein as well as the interaction of YB-1 with target genes [52
]. Therefore, RSK-dependent phosphorylation at Ser-102 may be essential for YB-1 function. Interestingly, YB-1 nuclear localization and ERα regulation appear to be connected [53
]. However, the mechanism and physiological outcome of this relationship are not yet clear. The observation that RSK participates in ERα and YB-1 signaling suggests that the potential connection between YB-1 and ERα may be mediated by RSK.