Research ArticleCancer

Therapeutic targeting of casein kinase 1δ in breast cancer

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Science Translational Medicine  16 Dec 2015:
Vol. 7, Issue 318, pp. 318ra202
DOI: 10.1126/scitranslmed.aac8773
  • Fig. 1. CK1δ is a clinically relevant and effective target for select breast cancer subtypes.

    (A) CK1δ mRNA expression in invasive ductal breast carcinomas (IDC) versus adjacent normal tissue (***P = 1 × 6.78−15). (B) Expression of CK1α1, CK1δ, and CK1ε across PAM50 breast cancer subtypes based on RNA sequencing (RNA-seq) data (n = 972 tumor samples, 113 solid tissue normal). Log2-normalized read count [RNA-seq by expectation-maximization (RSEM)] is shown. (C) CSNK1D DNA copy number analysis in invasive breast carcinomas clustered according to CK1δ expression (n = 303). Gene-level copy number estimates (GISTIC2 threshold) of −2 (dark blue), −1 (light blue), 0 (white), 1 (light red), 2 (dark red), representing homozygous deletion, single copy deletion, diploid normal copy, low-level copy number amplification, or high-level copy number amplification are shown. (D) Scatter plot of CSNK1D log2 mRNA expression versus log2 copy number values (972 breast cancer patients). (E) CK1δ and CK1ε protein expression inindicated breast cancer cell lines and MCF10A mammary epithelial cells. (F) Chemical structure of SR-3029. (G) Antiproliferative potency of SR-3029 in the indicated breast cancer cell lines. Data are plotted as % proliferation versus vehicle (n = 6). (H) Clonogenic growth and survival of the indicated cells in the presence of SR-3029 or vehicle (n = 3; P = 0.0008). (I) Percent apoptosis by propidium iodide (PI)/annexin V fluorescence-activated cell sorting (FACS) after 72 hours of treatment with indicated doses of SR-3029 (n = 3; left to right, ***P = 0.0007 and 0.0001). (J) Left: Clonogenic growth of MDA-MB-231 cells overexpressing CK1δ or green fluorescent protein (GFP) ± SR-3029 (n= 3; ***P = 0. 001, **P = 0.0035). Right: Western blot confirming CK1δ overexpression ±30 nM SR-3029 at 48 hours. (K) Relative growth (left: n = 3; siδ1, P= 0.01; siδ2, P = 0.003) and percent cell death by trypan blue dye exclusion (right: n = 3; siδ1, P = 0.01; siδ2, P = 0.027) 5 days after transfection of MDA-MB-231 with nontargeting (NT) or CK1δ small interfering RNAs (siRNAs). (L) Quantitative real-time PCR (qPCR) data and immunoblot confirming knockdown of CK1δ but not CK1ε (n = 3; ***P < 0.0001). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AU, arbitrary units.

  • Fig. 2. Inhibition of CK1δ/CK1ε impairs orthotopic breast tumor growth in vivo.

    (A) Effects of CK1δ/CK1ε inhibitors on growth and establishment of MDA-MB-231-luc tumors monitored by luminescence intensity over time. Mice were treated once per day with SR-3029 or vehicle (10:10:80, dimethyl sulfoxide (DMSO)/Tween 80/water) at 20 mg/kg by intraperitoneal injection. Arrow indicates start of treatment (n = 8 for each cohort; **P = 0.01). (B and C) Tumor size by luminescence (B) and comparison of gross tumor size (C) at day 55. Representative tumors are shown. (D) Growth curves of indicated TNBC tumor models in mice treated with vehicle (black line) or SR-3029 (blue line) as above. Arrows indicate timing of first dose (n = 8 to 10 for each cohort; **P = 0.01, ***P = 0.0008). (E) Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining on serial sections of vehicle- and SR-3029–treated MDA-MB-231 tumors (representative images are shown) (scale bars, 200 μm). (F) Kaplan-Meier survival curves corresponding to studies shown in (D) (P value calculated by log-rank test). (G) Body weight of mice treated daily with SR-3029 (blue line) or vehicle (black line) was monitored for 8 weeks (n = 8 to 10). DAPI, 4′,6-diamidino-2-phenylindole.

  • Fig. 3. Silencing or inhibition of CK1δ provokes breast tumor regression and blocks growth of PDX breast models.

    (A) Left: qPCR data confirming efficient knockdown of CK1δ but not CK1ε after 72 hours of treatment of MDA-MB-231–shCK1δ–expressing cells with Dox (0.3 μg/ml) (n = 3; ***P = 0.0003). Right: Corresponding CK1δ protein expression. (B) Cells expressing Dox-inducible CK1δ or nontargeting shRNA were treated with Dox (1 μg/ml) for 72 hours and transfected with vectors expressing CK1δ or GFP complementary DNA resistant to shRNA. After a further 72 hours, percent cell death was measured by trypan blue dye exclusion in MDA-MB-231–shCK1δ (black) and cells expressing nontargeting shRNAs (white) (n = 4; ***P = 0.0001). (C) Growth of orthotopic MDA-MB-231–shCK1δ tumors in mice ± Dox (200 mg/kg; administered ad libitum in chow), as monitored by caliper measurements (n = 8 for each cohort; **P = 0.006). Arrow indicates addition of Dox. (D) CK1δ knockdown in tumor tissue isolated from three independent mice, 7 days after Dox administration began. (E) Immunoblot comparing CK1δ expression in extracts of normal human breast or three independent BCM-4013 PDX tumors. (F) Growth curves of BCM-4013 PDX tumors in mice treated with vehicle (black line) or SR-3029 (blue line). Arrow indicates timing of first dose (n = 12 for each cohort; ***P = 0.0002). (G) Kaplan-Meier survival curve corresponding to studies shown in (F) (P value calculated using log-rank test). (H) TUNEL staining on serial sections of vehicle- and SR-3029–treated BCM-4013 tumors (representative images are shown) (scale bars, 200 μm).

  • Fig. 4. Modulation of the Wnt/β-catenin pathway is a biomarker for CK1δ activity and inhibition.

    (A) Wnt pathway genes significantly enriched in CK1δ-overexpressing human breast tumors (fold change, >2; P < 0.05) (red, CK1δ gene). (B) Effect of SR-3029 (+) or vehicle (−) treatment (18 hours, 30 nM) on nuclear versus cytoplasmic β-catenin in the indicated breast cancer cell lines. (C) Expression of active β-catenin (ABC) in MDA-MB-231 cells after 18 hours of treatment with SR-3029 or vehicle or after transfection with CK1δ siRNAs (harvested at 48 hours). (D) Inhibition of TCF-dependent luciferase activity in MDA-MB-231 cells treated with increasing doses of SR-3029 for 6 hours or after 5 days of treatment with Dox (1 μg/ml) to activate expression of indicated shRNAs (n = 3; *P = 0.013, ***P = 0.0002). (E and F) Effect of CK1δ inhibition (left, 24 hours of treatment with 30 or 100 nM SR-3029) or knockdown (right, 48 hours after transfection) on expression of indicated proteins (E) and mRNAs by immunoblot or qPCR (F), respectively (n = 3). (G) Expression of indicated mRNAs 24 hours after treatment with 100 nM SR-3029 (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001; SFRP1, P = 0.0003; WNT3, P = 0.001; WNT9A, P = 0.0007; MYC, P = 0.0271; CCND1, P = 0.0054; CD44, P = 0.0071). (H) TCF-dependent luciferase activity in HEK293T cells ± CK1δ shRNA expression induced with Dox (1 μg/ml) for 72 hours followed by ±3 hours of treatment with WNT3A (1 μg/ml; left to right, ***P = 1 × 7.23−5 and 1 × 5.57−6). (I) HEK293T cells stably expressing the TCF-dependent luciferase reporter were transfected with a control vector or a constitutively active (nuclear) mutant of β-catenin (S33Y) and incubated overnight ± SR-3029 before addition of recombinant WNT3A for 3 hours [representative of three independent experiments is shown; ***P = 0.0004 (left) and 0.0002 (right)]. (J) Immunostaining for active β-catenin expression (scale bars, 200 μm). (K) Relative growth 4days after infection of indicated cell lines with lentiviruses expressing either nontargeting or β-catenin shRNAs (n = 3; left to right, *P = 0.05, ***P = 0.0009, ***P = 0.001, ***P = 0.001) and corresponding Western blots (right). TCF/LEF, Tcell factor/lymphoid enhancer factor; RLU, relative luminescence units.

  • Fig. 5. CK1δ is a necessary and sufficient driver of Wnt/β-catenin signaling in human breast cancer.

    (A) Cell growth (left) and apoptosis (right) measured after 72 hours ± SR-3029 in MDA-MB-231 cells transfected with an empty vector or β-catenin–S33Y (n = 4; *P = 0.05). (B) MDA-MB-231–shCK1δ cells treated with Dox (1 μg/ml; 4 days) were transfected with an empty vector or β-catenin–S33Y, and cell number was measured after 72 hours (n = 3; ***P = 0.001). (C) Expression of nuclear and cytoplasmic β-catenin in MCF7 cells engineered to overexpress CK1δ or GFP. Bottom: Quantification of nuclear β-catenin expression normalized to histone H4 (n = 3; *P = 0.02). (D) Immunostaining for active β-catenin in MCF7 cells overexpressing CK1δ or GFP (scale bars, 200 μm). (E) qPCR analysis of β-catenin targets in MCF7-CK1δ versus MCF7-GFP cells (n = 3; **P = 0.01, ***P = 0.001). (F) Immunoblot confirming CK1δ overexpression and increased cyclin D1. (G) Effect of SR-3029 on clonogenic growth of MCF7 cells overexpressing CK1δ versus GFP (n = 6; left to right, **P = 0.01, **P = 0.002). (H) Growth of MCF7-CK1δ and MCF7-GFP cells 4 days after infection with β-catenin shRNA lentiviruses (n = 3, left to right, ***P = 0.0006, **P = 0.004, **P = 0.001). Right: Immunoblot showing CK1δ overexpression and knockdown of β-catenin.

  • Fig. 6. CK1δ is a driver of Wnt/β-catenin signaling in vivo.

    (A and B) Expression of nuclear and cytoplasmic β-catenin (A) and the indicated mRNAs (B), in MDA-MB-231 tumors from mice treated with SR-3029 (20 mg/kg) versus vehicle daily for 7 days (n = 4; *P = 0.05, **P = 0.01, ***P = 0.001). (C) Effects of SR-3029 on tumor cyclin D1 protein expression at day 7. Right: Panel shows quantification (n = 3; **P = 0.01). (D) Frequency of CSNK1D copy number amplifications in renal papillary cell carcinoma (n = 172) and bladder cancer tumors (n = 220; TCGA). (E) Correlation of CSNK1D DNA copy number and CK1δ expression in renal papillary cell carcinoma (n = 172) and bladder cancer (n = 220). (F) −Log10 P values showing significant overlap between Wnt/β-catenin pathway genes and CK1δ signature lists (P < 0.05; fold change, >1.5) for indicated cancer types (red line is threshold of significance, P = 0.05).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/318/318ra202/DC1

    Fig. S1. CK1δ, but not CK1ε, is overexpressed in human breast cancer and associated with CSNK1D gene amplification.

    Fig. S2. CK1δ is overexpressed in human breast cancer specimens.

    Fig. S3. Silencing of CK1δ and CK1ε, but not CK1ε alone, impairs growth and survival of MDA-MB-231 breast cancer cells.

    Fig. S4. Selective inhibition of FLT3 has no effect on proliferation of MDA-MB-231 breast cancer cells.

    Fig. S5. Treatment with SR-3029 impairs growth of triple-negative and HER2+ breast tumor models.

    Fig. S6. Long-term daily dosing with SR-3029 has no adverse effects on major organ histology.

    Fig. S7. Selective silencing of CK1δ is sufficient to impair growth of MDA-MB-231 breast cancer cells.

    Fig. S8. Wnt/β-catenin pathway genes are a hallmark of CK1δ-expressing breast cancers.

    Fig. S9. Constitutively active β-catenin N90 rescues the growth inhibitory effects of CK1δ inhibition and silencing.

    Fig. S10. CK1δ expression in MCF7 breast cancer cells sensitizes them to SR-3029.

    Fig. S11. Cyclin D1 expression is suppressed in TNBC MDA-MB-231–derived tumors after knockdown of CK1δ.

    Fig. S12. Inhibition of CK1δ/CK1ε has no effect on intestinal homeostasis.

    Fig. S13. Canonical Wnt signaling genes are correlated with CK1δ expression in human renal and bladder cancer.

    Table S1. TCGA RNA-seq and GISTIC2 copy number data and correlation analysis for breast cancer (BRCA) tumors.

    Table S2. TCGA RNA-seq and GISTIC2 copy number data for basal-like Pam50 breast tumors.

    Table S3. TCGA RNA-seq and GISTIC2 copy number data for HER2-positive Pam50 breast tumors.

    Table S4. TCGA RNA-seq and GISTIC2 copy number data for luminal A Pam50 breast tumors.

    Table S5. TCGA RNA-seq and GISTIC2 copy number data for luminal B Pam50 breast tumors.

    Table S6. Potency of the CK1δ/CK1ε inhibitor SR-3029 for human breast cancer subtypes.

    Table S7. No toxic effects from long-term daily treatment with SR-3029.

    Table S8. TCGA RNA-seq and GISTIC2 copy number data for renal papillary (KIRP) tumors.

    Table S9. TCGA RNA-seq and GISTIC2 copy number data for bladder cancer (BLCA) tumors.

    Table S10. Renal papillary cell carcinoma correlation analysis.

    Table S11. Bladder cancer correlation analysis.

    Table S12. Wnt signaling genes significantly associated with CK1δ expression in renal papillary cell carcinoma.

    Table S13. Wnt signaling genes significantly associated with CK1δ expression in bladder cancer.

    Table S14. Oligonucleotides used for qPCR.

    Table S15. siRNA sequences.

    Table S16. shRNA sequences.

  • Supplementary Material for:

    Therapeutic targeting of casein kinase 1δ in breast cancer

    Laura H. Rosenberg, Marie Lafitte, Victor Quereda, Wayne Grant, Weimin Chen, Mathieu Bibian, Yoshihiko Noguchi, Mohammad Fallahi, Chunying Yang, Jenny C. Chang, William R. Roush, John L. Cleveland, Derek R. Duckett*

    *Corresponding author. E-mail: ducketdr{at}scripps.edu

    Published 16 December 2015, Sci. Transl. Med. 7, 318ra202 (2015)
    DOI: 10.1126/scitranslmed.aac8773

    This PDF file includes:

    • Fig. S1. CK1δ, but not CK1ε, is overexpressed in human breast cancer and associated with CSNK1D gene amplification.
    • Fig. S2. CK1δ is overexpressed in human breast cancer specimens.
    • Fig. S3. Silencing of CK1δ and CK1ε, but not CK1ε alone, impairs growth and survival of MDA-MB-231 breast cancer cells.
    • Fig. S4. Selective inhibition of FLT3 has no effect on proliferation of MDA-MB-231 breast cancer cells.
    • Fig. S5. Treatment with SR-3029 impairs growth of triple-negative and HER2+ breast tumor models.
    • Fig. S6. Long-term daily dosing with SR-3029 has no adverse effects on major organ histology.
    • Fig. S7. Selective silencing of CK1δ is sufficient to impair growth of MDA-MB-231 breast cancer cells.
    • Fig. S8. Wnt/β-catenin pathway genes are a hallmark of CK1δ-expressing breast cancers.
    • Fig. S9. Constitutively active β-catenin N90 rescues the growth inhibitory effects of CK1δ inhibition and silencing.
    • Fig. S10. CK1δ expression in MCF7 breast cancer cells sensitizes them to SR-3029.
      Fig. S11. Cyclin D1 expression is suppressed in TNBC MDA-MB-231–derived tumors after knockdown of CK1δ.
    • Fig. S12. Inhibition of CK1δ/CK1ε has no effect on intestinal homeostasis.
    • Fig. S13. Canonical Wnt signaling genes are correlated with CK1δ expression in human renal and bladder cancer.
    • Table S1. TCGA RNA-seq and GISTIC2 copy number data and correlation analysis for breast cancer (BRCA) tumors.
    • Table S2. TCGA RNA-seq and GISTIC2 copy number data for basal-like Pam50 breast tumors.
    • Table S3. TCGA RNA-seq and GISTIC2 copy number data for HER2-positive Pam50 breast tumors.
    • Table S4. TCGA RNA-seq and GISTIC2 copy number data for luminal A Pam50 breast tumors.
    • Table S5. TCGA RNA-seq and GISTIC2 copy number data for luminal B Pam50 breast tumors.
    • Table S6. Potency of the CK1δ/CK1ε inhibitor SR-3029 for human breast cancer subtypes.
    • Table S7. No toxic effects from long-term daily treatment with SR-3029.
    • Table S8. TCGA RNA-seq and GISTIC2 copy number data for renal papillary (KIRP) tumors.
    • Table S9. TCGA RNA-seq and GISTIC2 copy number data for bladder cancer (BLCA) tumors.
    • Table S10. Renal papillary cell carcinoma correlation analysis.
    • Table S11. Bladder cancer correlation analysis.
    • Table S12. Wnt signaling genes significantly associated with CK1δ expression in renal papillary cell carcinoma.
    • Table S13. Wnt signaling genes significantly associated with CK1δ expression in bladder cancer.
    • Table S14. Oligonucleotides used for qPCR.
    • Table S15. siRNA sequences.
    • Table S16. shRNA sequences.

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