The 40 S ribosomal S6 kinase 1 (S6K1) acts downstream of mTOR (mammalian target of rapamycin) and is sensitive to inhibition by rapamycin. The chromosomal region 17q23 containing the RPS6KB1 gene is frequently amplified in breast cancer cells, leading to S6K1 overexpression. The role of S6K1 in disease development and progression is supported by the observation that S6K1 overexpression is associated with poor prognosis in breast cancer patients. However, the identity of mammary cell-specific S6K1 targets is not well understood. In this study, we report that overexpression of S6K1 endows breast cancer cells with a proliferative advantage in low serum conditions and enhanced sensitivity to rapamycin. We investigate the molecular mechanism behind this observation to show that S6K1 regulates estrogen receptor ␣ (ER␣) by phosphorylating it on serine 167, leading to transcriptional activation of ER␣. By contributing to the activation of ER␣, S6K1 promotes ER␣-mediated cell proliferation and may be a target of therapeutic intervention in breast cancer. mTOR (mammalian target of rapamycin) is a conserved protein kinase that is a key regulator of cell growth and proliferation in response to extracellular cues, including nutrient availability and growth stimuli. Rapamycin is a naturally derived inhibitor of mTOR that was revealed to be an inhibitor of cell proliferation, as manifested by its potent immunosuppressive properties and activity against solid tumors (1). The 40 S ribosomal S6 kinase 1 (S6K1) 2 is one of the best characterized downstream targets of mTOR. Rapamycin treatment results in rapid dephosphorylation and inactivation of S6K1 (2). S6K1 is an important regulator of cell size control, protein translation, and cell proliferation (3). The 40 S ribosomal protein S6 is the best characterized target of S6K1 (4). Other targets have been reported, some of which include the apoptotic protein Bad, the eukaryotic elongation factor 2 kinase, the eukaryotic translation initiation factor 4B (eIF4B), the RNA-binding protein SKAR, and the translational inhibitor PDCD4 (5-9). It appears that S6K1 regulates its targets to increase the biosynthetic capacity of the cell that is necessary for cell division (9 -11).Data suggest that S6K1 is implicated in breast cancer. S6K1 is encoded by the RPS6KB1 gene localized to the chromosomal region 17q23. Region 17q23 is amplified in several breast cancer cell lines and in ϳ30% of primary tumors (12), whereas S6K1 is overexpressed in the majority of cell lines and primary tumors with this amplification (13-17). Furthermore, the role of S6K1 in disease development and progression is supported by the observation that RPS6KB1 amplification and S6K1 overexpression are associated with poor prognosis in breast cancer patients (13,18).Interestingly, whereas RPS6KB1 is amplified in several cancer types, high level (multicopy) amplification of RPS6KB1 is limited to breast cancer (16). This suggests that S6K1 may have a specific role in regulating the growth of breast cancer cells. Overexpression...

Keywords: p70 S6 kinase 1 p90 ribosomal S6 kinase Mammalian target of rapamycin Rapamycin Estrogen receptor a Breast cancer a b s t r a c tResistance to anti-estrogen therapy is a major clinical concern in treatment of breast cancer. Estrogen-independent phosphorylation of estrogen receptor a, specifically on Ser167, is one of the contributing causes to development of resistance, and a prognostic marker for the disease. Here, we dissect the signaling pathways responsible for Ser167 phosphorylation. We report that the mTOR/ S6K1 and MAPK/RSK contribute non-overlapping inputs into ERa activation via Ser167 phosphorylation. This cooperation may be targeted in breast cancer treatment by a combination of mTOR and MAPK inhibitors.

#4062 Background: The 40S ribosomal S6 kinase 1 (S6K1) acts downstream of the mammalian Target of Rapamycin (mTOR), and is sensitive to inhibition by rapamycin. Chromosomal region 17q23 containing the RPS6KB1 gene is frequently amplified in breast cancer cells, and S6K1 is overexpressed in about 30% of cases. Overexpression of S6K1 correlates with increased rapamycin sensitivity. The role of S6K1 in disease development and progression is supported by the observation that S6K1 overexpression is associated with poor prognosis in breast cancer patients. However, the reason for high-level amplification of the RPS6KB1 gene specifically in breast cancer, and not other cancer types, and the identity of breast cell-specific S6K1 targets is not well understood. In this study, we report that S6K1 regulates growth of breast cancer cells by phosphorylating Estrogen Receptor α (ERα), and co-expression of S6K1 and ERα in breast cancer cells renders them sensitive to combination therapy with rapamycin and tamoxifen.
 Materials and Methods: Using immunoblot analysis we determined expression levels of S6K1 in breast cancer cell lines, and the phosphorylation status of ERα on Ser167 after serum or mitogen stimulation. We compared the effect of low-serum media, and rapamycin and/or 4-hydroxytamoxifen (4-HT) addition on breast cancer cell proliferation using neutral red uptake assay.
 Results: We determined that overexpression of S6K1 in breast cancer cells confers proliferative advantage in low serum, contributing to neoplastic transformation. Next, we wished to identify targets of S6K1 in control of proliferation of breast cancer cells. One of the proteins we identified as a target of S6K1 is ERα. ERα is frequently co-overexpressed with S6K1 in breast cancer cells, which could be an indication of co-selection, and ERα contains a putative S6K1 phosphorylation motif RERLAS167. We confirmed that ERα phosphorylation may be mediated by S6K1. We shown that in S6K1-overexpressing MCF7 cells, serum-stimulated ERα phosphorylation is rapamycin-sensitive. Moreover, ERα phosphorylation stimulated by insulin and tumor-promoting phorbol esters is also rapamycin-sensitive. S6K1 can phosphorylate ERα in an in vitro kinase assay. Lastly, shRNA-mediated suppression of S6K1 expression in MCF7 cells reduces ERα phosphorylation. Subsequently, we examined whether co-overexpression of S6K1 and ERα in breast cancer cells sensitizes them to combination therapy with rapamycin and tamoxifen. We analyzed proliferation of cells treated with rapamycin and/or 4-HT. We observed that in S6K1- and ERα-overexpressing cells, treatment with rapamycin and 4-HT resulted in greater inhibition of cell growth.
 Discussion: Our data suggest that one of the targets of S6K1 signaling in breast cancer cells is ERα, and together, these proteins promote breast cancer cell proliferation. The ultimate objective would be to determine whether combined inhibition of S6K1 and the estrogen receptor could be used as targeted therapy for patients with double positive expression of S6K1 and ER. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4062.

Purpose of the Study: Phosphorylation of estrogen receptor α (ERα) Ser167 is associated with development of endocrine resistance, and is a prognostic marker for the disease. Hereby we dissect the signaling pathways responsible for Ser167 phosphorylation. Experimental Procedures: MCF7 cells were grown in standard media. For experiments, cells were deprived of serum for 24h, and were pre-treated with 20ng/mL rapamycin for 30 min and/or 10 µM U0126 for 2h, and then stimulated with 100nM insulin or 100ng/mL PMA. Cell extracts were prepared, and proteins were resolved on SDS-PAGE, followed by immunoblots. For the siRNA studies, double-stranded RNAs for RSK1 and RSK2 were transfected using Lipofectamine2000 (Invitrogen). At 72h post-transfection, cells were lysed using 1X Passive Lysis Buffer (Promega). For luciferase reporter assays, cells were transfected with plasmids encoding for firefly luciferase under control of three ERE, and control renilla luciferase. At 48h post-transfection, cells were harvested and relative luciferase activity was measured using the Dual Luciferase Reporter Assay System and Glomax 20/20 luminometer (Promega). Data: mTOR/S6K1 and MAPK/RSK contribute non-overlapping inputs into ERα activation via Ser167 phosphorylation. The degree of input of each pathway depends on the nature of the activating signal. In unsynchronized cells that are growing in the presence of serum or in cells acutely stimulated with insulin, which preferentially activated the mTOR/S6K1 pathway, we observed that the MAPK/RSK pathway has a minor contribution to Ser167 phosphorylation. However, the MAPK/RSK contribution is more evident following acute stimulation with PMA, which activates both the mTOR/S6K1 and MAPK/RSK pathways. In response to PMA we observed significant rapamycin-insensitive Ser167 phosphorylation, which was U0126-sensitive. Using RNAi against RSK1/2 isoforms we demonstrated that this rapamycin-insensitive phosphorylation is mediated by RSK. In addition to being sensitive to different stimuli, the mTOR and MAPK pathways are also activated with different kinetics. S6K1 and RSK perform a non-redundant role with regard to ERα Ser167 phosphorylation, and are responsible for a biphasic phosphorylation pattern. The early-activated RSK acts in a priming role, while the late-responding S6K1 is responsible for the maintenance of high-level phosphorylation. Estrogen receptor is an important clinical target in treatment of breast cancer. However, resistance to anti-estrogen therapy often develops. One of the mechanisms of resistance is through mitogenic estrogen-independent activation of ERα. Thus, a question arises whether dual inhibition of the mTOR and MAPK pathways may result in a better clinical response. We investigated the effect of a combination of rapamycin and U0126 on transcriptional activity mediated by ERα. We demonstrated that the combination produces a greater inhibition of ERα activity than each agent alone. This strategy may be effective for combating resistance to anti-estrogen or endocrine therapy. Conclusions: The data presented here reveal that the effect of rapamycin therapy in breast cancer could be augmented by combination with MEK inhibitors, which would target two major pathways leading to ERα activation and stimulation of breast cancer cell proliferation. Citation Information: Cancer Res 2009;69(23 Suppl):A31.