sl0101 and Breast-Neoplasms

A series of novel indolin-2-ones inhibitors against p90 ribosomal S6 protein kinase 2 (RSK2) were designed and synthesized and their structure-activity relationship (SAR) was studied. The most potent inhibitor, compound 3s, exhibited potent inhibition against RSK2 with an IC50 value of 0.5 μM and presented a satisfactory selectivity against 23 kinases. The interactions of these inhibitors with RSK2 were investigated based on the proposed binding poses with molecular docking simulation. Four compounds and six compounds exhibited moderate anti-proliferation activities against PC 3 cells and MCF-7 cells, respectively.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Humans; Indoles; Male; Molecular Docking Simulation; Prostatic Neoplasms; Protein Kinase Inhibitors; Ribosomal Protein S6 Kinases, 90-kDa; Structure-Activity Relationship

Y-box binding protein-1 (YB-1) is the first reported oncogenic transcription factor to induce the tumor-initiating cell (TIC) surface marker CD44 in triple-negative breast cancer (TNBC) cells. In order for CD44 to be induced, YB-1 must be phosphorylated at S102 by p90 ribosomal S6 kinase (RSK). We therefore questioned whether RSK might be a tractable molecular target to eliminate TICs. In support of this idea, injection of MDA-MB-231 cells expressing Flag-YB-1 into mice increased tumor growth as well as enhanced CD44 expression. Despite enrichment for TICs, these cells were sensitive to RSK inhibition when treated ex vivo with BI-D1870. Targeting RSK2 with small interfering RNA (siRNA) or small molecule RSK kinase inhibitors (SL0101 and BI-D1870) blocked TNBC monolayer cell growth by ∼100%. In a diverse panel of breast tumor cell line models RSK2 siRNA predominantly targeted models of TNBC. RSK2 inhibition decreased CD44 promoter activity, CD44 mRNA, protein expression, and mammosphere formation. CD44(+) cells had higher P-RSK(S221/227) , P-YB-1(S102) , and mitotic activity relative to CD44(-) cells. Importantly, RSK2 inhibition specifically suppressed the growth of TICs and triggered cell death. Moreover, silencing RSK2 delayed tumor initiation in mice. In patients, RSK2 mRNA was associated with poor disease-free survival in a cohort of 244 women with breast cancer that had not received adjuvant treatment, and its expression was highest in the basal-like breast cancer subtype. Taking this further, we report that P-RSK(S221/227) is present in primary TNBCs and correlates with P-YB-1(S102) as well as CD44. In conclusion, RSK2 inhibition provides a novel therapeutic avenue for TNBC and holds the promise of eliminating TICs.

Topics: Animals; Apoptosis; Benzopyrans; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Female; Flow Cytometry; Fluorescent Antibody Technique; Humans; Hyaluronan Receptors; Mice; Mice, Inbred NOD; Mice, SCID; Monosaccharides; Promoter Regions, Genetic; Pteridines; Real-Time Polymerase Chain Reaction; Ribosomal Protein S6 Kinases, 90-kDa; Y-Box-Binding Protein 1

Basal-like breast cancers (BLBC) frequently overexpress the epidermal growth factor receptor (EGFR) and subsequently have high levels of signaling through the MAP kinase pathway, which is thought to contribute to their aggressive behavior. While we have previously reported the expression of Y-box binding protein-1 (YB-1) in 73% of BLBC, it is unclear whether it can be regulated by a component of the MAP kinase signaling pathway. Phosphorylation of YB-1 at the serine 102 residue is required for transcriptional activation of growth-enhancing genes, such as EGFR. Using Motifscan we identified p90 ribosomal S6 kinase (RSK) as a potential candidate for activating YB-1.. Inhibition of RSK1 and RSK2 was achieved using siRNA and the small molecule SL0101. RSK1, RSK2, activated RSK and kinase-dead RSK were expressed in HCC1937 cells. Kinase assays were performed to illustrate direct phosphorylation of YB-1 by RSK. The impact of inhibiting RSK on YB-1 function was measured by luciferase assays and chromatin immunoprecipitation.. Using an in vitro kinase assay, RSK1 and RSK2 were shown to directly phosphorylate YB-1. Interestingly, they were more effective activators of YB-1 than AKT or another novel YB-1 kinase, PKC alpha. Phosphorylation of YB-1 (serine 102 residue) is blocked by inhibition of the MAP kinase pathway or by perturbing RSK1/RSK2 with siRNA or SL0101. In immortalized breast epithelial cells where RSK is active yet AKT is not, YB-1 is phosphorylated. Supporting this observation, RSK2-/- mouse embryo fibroblasts lose the ability to phosphorylate YB-1 in response to epidermal growth factor. This subsequently interfered with the ability of YB-1 to regulate the expression of EGFR. The RSK inhibitor SL0101 decreased the ability of YB-1 to bind the promoter, transactivate and ultimately reduce EGFR expression. In concordance with these results the expression of constitutively active RSK1 increased YB-1 phosphorylation, yet the kinase-dead RSK did not.. We therefore conclude that RSK1/RSK2 are novel activators of YB-1, able to phosphorylate the serine 102 residue. This provides a newly described mechanism whereby YB-1 is activated in breast cancer. This implicates the EGFR/RSK/YB-1 pathway as an important component of BLBC, providing an important opportunity for therapeutic intervention.

Topics: Animals; Benzopyrans; Blotting, Western; Breast Neoplasms; Cell Proliferation; Cells, Cultured; Chromatin Immunoprecipitation; Electrophoretic Mobility Shift Assay; Embryo, Mammalian; ErbB Receptors; Female; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Immunoprecipitation; Luciferases; MAP Kinase Signaling System; Mice; Monosaccharides; Neoplasms, Basal Cell; Phosphorylation; Promoter Regions, Genetic; Protein Kinase C-alpha; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 90-kDa; RNA, Small Interfering; Serine; Y-Box-Binding Protein 1

We have previously reported the isolation of kaempferol 3-O-(3'',4''-di-O-acetyl-alpha-l-rhamnopyranoside) from Forsteronia refracta [Xu, Y.-M.; Smith, J. A.; Lannigan, D. A.; Hecht, S. M. Biorg. Med. Chem.2006, 14, 3974-3977.]. This flavonoid glycoside, termed SL0101, is a specific inhibitor of p90 ribosomal S6 kinase (RSK) with a dissociation constant of 1 microM. In intact cells, however, the EC50 for inhibition of RSK activity is 50 microM, which suggests that the efficacy of SL0101 could be limited by cellular uptake. Therefore, we investigated the possibility of developing a more potent RSK inhibitor by synthesizing SL0101 analogs with increased hydrophobic character. The total syntheses of kaempferol 3-O-(3'',4''-di-O-butyryl-alpha-L-rhamnopyranoside) (Bu-SL0101) and kaempferol 3-O-(2'',3'',4''-tri-O-acetyl-alpha-L-rhamnopyranoside) (3Ac-SL0101) were performed. The IC50 for inhibition of RSK activity in in vitro kinase assays for the analogs was similar to that obtained for SL0101. 3Ac-SL0101 demonstrated the same remarkable specificity for inhibiting RSK activity in intact cells as SL0101; however, Bu-SL0101 was not completely specific. 3Ac-SL0101 was approximately 2-fold more potent at inhibiting MCF-7 cell proliferation compared to SL0101 and preferentially decreased MCF-7 cell growth, as compared to the growth of the normal human breast line, MCF-10A. Thus the discovery of 3Ac-SL0101 as a more potent RSK-specific inhibitor than SL0101 should facilitate the development of RSK inhibitors as anti-cancer chemotherapeutic agents.

Topics: Antineoplastic Agents; Benzopyrans; Breast Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Molecular Structure; Monosaccharides; Rhamnose; Ribosomal Protein S6 Kinases, 90-kDa

p90 ribosomal S6 kinase (RSK) is an important downstream effector of mitogen-activated protein kinase, but its biological functions are not well understood. We have now identified the first small-molecule, RSK-specific inhibitor, which we isolated from the tropical plant Forsteronia refracta. We have named this novel inhibitor SL0101. SL0101 shows remarkable specificity for RSK. The major determinant of SL0101-binding specificity is the unique ATP-interacting sequence in the amino-terminal kinase domain of RSK. SL0101 inhibits proliferation of the human breast cancer cell line MCF-7, producing a cell cycle block in G(1) phase with an efficacy paralleling its ability to inhibit RSK in intact cells. RNA interference of RSK expression confirmed that RSK regulates MCF-7 proliferation. Interestingly, SL0101 does not alter proliferation of a normal human breast cell line MCF-10A, although SL0101 inhibits RSK in these cells. We show that RSK is overexpressed in approximately 50% of human breast cancer tissue samples, suggesting that regulation of RSK has been compromised. Thus, we show that RSK has an unexpected role in proliferation of transformed cells and may be a useful new target for chemotherapeutic agents. SL0101 will provide a powerful new tool to dissect the molecular functions of RSK in cancer cells.

Topics: Amino Acid Sequence; Benzopyrans; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Humans; Molecular Sequence Data; Monosaccharides; Phosphorylation; Plant Extracts; Protein Kinase Inhibitors; Protein Structure, Tertiary; Ribosomal Protein S6 Kinases, 90-kDa; Substrate Specificity