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, 15 (11), 2598-2608

Development of a RSK Inhibitor as a Novel Therapy for Triple-Negative Breast Cancer

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Development of a RSK Inhibitor as a Novel Therapy for Triple-Negative Breast Cancer

Katarzyna A Ludwik et al. Mol Cancer Ther.

Abstract

Metastatic breast cancer is an incurable disease and identification of novel therapeutic opportunities is vital. Triple-negative breast cancer (TNBC) frequently metastasizes and high levels of activated p90RSK (RSK), a downstream MEK-ERK1/2 effector, are found in TNBC. We demonstrate, using direct pharmacologic and genetic inhibition of RSK1/2, that these kinases contribute to the TNBC metastatic process in vivo Kinase profiling showed that RSK1 and RSK2 are the predominant kinases targeted by the new inhibitor, which is based on the natural product SL0101. Further evidence for selectivity was provided by the observations that silencing RSK1 and RSK2 eliminated the ability of the analogue to further inhibit survival or proliferation of a TNBC cell line. In vivo, the new derivative was as effective as the FDA-approved MEK inhibitor trametinib in reducing the establishment of metastatic foci. Importantly, inhibition of RSK1/2 did not result in activation of AKT, which is known to limit the efficacy of MEK inhibitors in the clinic. Our results demonstrate that RSK is a major contributor to the TNBC metastatic program and provide preclinical proof-of-concept for the efficacy of the novel SL0101 analogue in vivo Mol Cancer Ther; 15(11); 2598-608. ©2016 AACR.

Conflict of interest statement

No conflicts.

Figures

Figure 1
Figure 1. C6″-n-propyl cyclitol SL0101 (1b) shows improved potency compared to the parent compound
(A) Structure and IC50 for selected SL0101 analogues. (B) Efficacy of (1a) and (1b) in inhibiting proliferation of MCF-7 and MCF-10A cells. Symbol, mean ± S.D. (n ≥ 2, triplicate; *p < 0.01 compared to vehicle). (C) The in vitro stability of (1b) (25 μM) is increased in comparison to (1a) (100 μM). Bar, mean (n=2, quadruplicate; *p < 0.0001). (D) Analysis of lysates from MCF-7 cells pre-treated with (1a), (1b) or DMSO for 2h and treated with or without 500 nM PMA (20 min). (E) Representative images of MCF-7 cells treated as in D. Scale bar = 10 μm. Bar graph showing the decrease in pS6. (n ≥ 30 cells). (F) Representation of (1b) specificity in a kinase screen indicating % inhibition at 10 μM compared to RSK2. (G) Bioluminescence images of NSG mice at day 50 after IC injection with MCF-7-Luc cells. (H) Representative paraffin-embedded tibia sections from mice in (G) treated with (40 mg/kg) or vehicle 2h prior to euthanasia. Scale bar = 40 μm. Bar graph showing the decrease in pS6. (n= 6 sections/mouse).
Figure 2
Figure 2. Active RSK in TNBC
(A) Activated RSK levels are increased in TNBC. Bar, median ± quartile (n ≥ 5 field/tissue sample). (B) Representative paraffin-embedded sections of normal breast and TNBC tissue stained for the cytokeratins 8 (K8), 14 (K14) and phospho-Thr359/phospho-Ser363 RSK (pRSK). Scale bar = 20 μm. (C) Analysis of TNBC cell lysates normalized using the housekeeping protein, RAN. (D) Quantitation of the levels of pRSK normalized to RAN. (n=3) (E) Comparison of pRSK relative to total RSK1 and RSK2 for various TNBC lines. To control for antibody sensitivity the levels of RSK1 and RSK2 were determined using purified, recombinant protein.
Figure 3
Figure 3. RSK is required for TNBC proliferation, survival and motility
(A) IC50s for (1b) in MCF-7 and TNBC lines. Bar, median ± range (n ≥ 2, ≥ quadruplicate). (B) Correlation of IC50 for inhibition of proliferation by (1b) of TNBC lines versus activated RSK normalized to total RSK2 levels. (C) Analysis of lysates from MDA-MB-231 cells transduced with scramble (scrbl) or double transduced with RSK1/2 targeting shRNAs. Bar: non-relevant lanes removed. ns:nonspecific (D) Efficacy of (1b) in inhibiting proliferation of MDA-MB-231 cells transduced as in (C). Symbol, mean ± S.D. (n ≥ 2, triplicate; *p < 0.03 compared to vehicle). (E) Bar graph showing (1b) IC50 for MDA-MB-231 proliferation in 2D and 3D. Bar, median ± range (n ≥ 2, ≥ quadruplicate). (F) IC50s for (1b) for survival of TNBC lines. Bar, median ± range (n ≥ 2, triplicate). (G) Efficacy of (1b) in inhibiting survival of MDA-MB-231 cells transduced as in (B). Symbol, mean ± S.D (n ≥ 2, triplicate; *p < 0.01 compared to vehicle). Scatter plots showing efficacy of (1a) and (1b) in inhibiting motility of (H) MDA-MB-231, (I) HCC70 and (J) HDQ-P1. Each circle represents a cell trace. Bar, median (n ≥ 2, 30 cells/treatment).
Figure 4
Figure 4. RSK1 and RSK2 contribute to the metastatic phenotype
(A) Representative bioluminescence images of NSG mice injected IC with MDA-MB-231-Luc cells transduced with scramble (scrbl), RSK1- or RSK2-targeting shRNAs (t=19 d). (B) RSK1 and RSK2 decreased the metastatic burden in mice from (A) (t=19 d). (n = 8 mice/group). (C) Kaplan-Meier curves from (A). (n= 8 mice/group; test = log-rank). (D) The number of metastatic foci is constant in mice from (A). Symbol, mean. (E) Total bioluminescence in mice from (A) is decreased with RSK1 or RSK2 silencing. Each line represents a mouse; the data are fold change over day 6. (F) Loss of RSK1 or RSK2 decreased the number of metastatic foci in numerous organs in mice from (A) (t=19 d). Bar, mean ± SD (n = 8 mice/group). (G) Representative ex vivo bioluminescence images of livers of mice from (A). (H) Ex vivo analysis confirms that silencing RSK1 or RSK2 decreased the number of metastatic foci in the livers from (A). Bar, median ± quartile (n=4 mice/group).
Figure 5
Figure 5. Pharmacological inhibition of metastatic colonization by (1b)
(A) Bioluminescence images of NSG mice injected IC with HDQ-P1-Luc cells at t=1h and 24h after injection. At 2h after injection mice were treated with vehicle, (1b) (40 mg/kg) IP Q12h or trametinib (tram) (2 mg/kg) IP Q24h. Inhibition of RSK or MEK decreases total metastatic burden (B) and the number of metastatic foci in individual organs (t=24h) (C). Bar, mean ± SD (n=4 mice/group, *p<0.05). (D) Inhibition of RSK or MEK decrease total metastatic burden (t=6d). (n=4 mice/group). Representative ex vivo bioluminescence images of livers (E) and adrenal glands (G) (t=6d). Ex vivo analysis confirms that inhibiting RSK or MEK activity decreased the metastatic burden in livers (F) and adrenals (H). (n=4 mice/group).
Figure 6
Figure 6. (1b) does not activate AKT
Analysis of lysates from (A) MDA-MB-231 and (B) HDQ-P1 cells treated with vehicle, trametinib (1 μM) or (1b) (25 μM) for 2h. Bar = separate gels.

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