1. Cancer Biology

Three-dimensional growth of breast cancer cells potentiates the anti-tumor effects of unacylated ghrelin and AZP-531

  1. CheukMan C Au
  2. John B Furness
  3. Kara Britt
  4. Sofya Oshchepkova
  5. Heta Ladumor
  6. Kai Ying Soo
  7. Brid Callaghan
  8. Celine Gerard
  9. Giorgio Inghirami
  10. Vivek Mittal
  11. Yufeng Wang
  12. Xin Yun Huang
  13. Jason A Spector
  14. Eleni Andreopoulou
  15. Paul Zumbo
  16. Doron Betel
  17. Lukas Dow
  18. Kristy A Brown  Is a corresponding author
  1. Department of Medicine, Weill Cornell Medicine, United States
  2. Centre for Cancer Research, Hudson Institute for Medical Research, Australia
  3. Department of Molecular and Translational Sciences, Monash University, Australia
  4. Department of Anatomy and Neuroscience, University of Melbourne, Australia
  5. Peter MacCallum Cancer Centre, Australia
  6. Sir Peter MacCallum Department of Oncology, University of Melbourne, Australia
  7. Weill Cornell Medicine - Qatar, Qatar
  8. Department of Pathology, Weill Cornell Medical College, United States
  9. Department of Cardiothoracic Surgery, Department of Cell and Developmental Biology, Neuberger Berman Lung Cancer Center, Weill Cornell Medicine, United States
  10. Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University, United States
  11. Department of Surgery, Weill Cornell Medicine, United States
  12. Applied Bioinformatics Core, Weill Cornell Medical College, United States
  13. Institute for Computational Biomedicine, Weill Cornell Medical College, United States
https://doi.org/10.7554/eLife.56913
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Cite this article
  1. CheukMan C Au
  2. John B Furness
  3. Kara Britt
  4. Sofya Oshchepkova
  5. Heta Ladumor
  6. Kai Ying Soo
  7. Brid Callaghan
  8. Celine Gerard
  9. Giorgio Inghirami
  10. Vivek Mittal
  11. Yufeng Wang
  12. Xin Yun Huang
  13. Jason A Spector
  14. Eleni Andreopoulou
  15. Paul Zumbo
  16. Doron Betel
  17. Lukas Dow
  18. Kristy A Brown
(2020)
Three-dimensional growth of breast cancer cells potentiates the anti-tumor effects of unacylated ghrelin and AZP-531
eLife 9:e56913.
https://doi.org/10.7554/eLife.56913
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  3. Download .RIS
7 figures, 2 tables and 2 additional files

Figures

Figure 1 with 1 supplement
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Unacylated ghrelin inhibits the 3D growth of breast cancer cells.

(a, c) Unacylated ghrelin (UAG; 100 pM) inhibits the growth of a panel of breast cancer cell lines under serum-stimulated conditions (six replicates/group) or (b) ER+ breast cancer cell lines in the presence of estradiol (10 nM; six replicates/group). (c) UAG (100 pM) suppresses cell growth of basal-like and mesenchymal-like TNBC breast cancer cell lines that are WT for BRAF and KRAS (6–9 replicates/group). Effects of UAG are abrogated in (d) BRAF-transfected MCF7 cells, and (e) BRAF- and (f) KRAS-mutated colon cancer cells (6–12 replicates/group). Loss of mutated alleles of BRAF or KRAS sensitizes cells to the effect of UAG. Data represent mean ± SEM. Experiments were repeated at least twice. UAG: unacylated ghrelin; VC: vehicle control; FBS: fetal bovine serum; E2: estradiol.

Figure 1—source data 1

Unacylated ghrelin inhibits the 3D growth of breast cancer cells.

https://cdn.elifesciences.org/articles/56913/elife-56913-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
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Unacylated ghrelin inhibits the growth of breast cancer cells in 3D.

Unacylated ghrelin (UAG; 100pM) inhibits the growth of (a) MCF7 and (b) MDA-MB-468 cells grown in 3D in matrigel or collagen, but not in 2D. (c) UAG had no effect on MDA-MB-231 cell growth in 2D or 3D. Dose-dependent effects of UAG and doxorubicin on (d) MCF7, (e) MDA-MB-468 and (f) MDA-MB-231 cell number in 3D culture. (g) Acyl ghrelin (AG) and UAG (100 pM) inhibit breast cancer cell growth in serum-stimulated conditions. (h) Binding of Cy3-labeled UAG to MCF7, MDA-MB-468 and MDA-MB-231 cells over time (representative images). Scale bar represents 20 μm. Data represent mean ± SEM with three replicates/group. Experiments repeated at least twice. UAG: unacylated ghrelin; AG: acylated ghrelin; VC: vehicle control; FBS: fetal bovine serum.

Figure 1—figure supplement 1—source data 1

Unacylated ghrelin inhibits the growth of breast cancer cells in 3D.

https://cdn.elifesciences.org/articles/56913/elife-56913-fig1-figsupp1-data1-v1.xlsx
Figure 2 with 1 supplement
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Unacylated ghrelin suppresses breast cancer cell growth via Gαi-dependent inhibition of cAMP formation.

(a) Unacylated ghrelin (UAG; 10–1000 pM) inhibits the forskolin-stimulated production of cAMP in MCF7 cells (3–4 replicates/group). (b) UAG (100 pM) stimulates activation of Gαi (three replicates/group). (c) UAG (100 pM) suppresses the growth of CRISPR GNAI1 and GNAI3 KO cells, but not GNAI2 KO MCF7 cells, suggesting Gαi2-coupled GPCR-mediated effects. Suppression of (d) estradiol- or (e) serum-stimulated breast cancer cell growth with UAG (100 pM) is prevented in the presence of Gαi inhibitor, pertussis toxin (20 ng/ml, 200 ng/ml; three replicates/group). (f) PKA inhibitor (KT5720), adenylyl cyclase inhibitor (SQ22536) or cAMP antagonist (cAMPS-RP) suppress the serum-stimulated growth of MCF7 cells (three replicates/group). (g) UAG (100 pM) inhibits the forskolin-stimulated growth of MCF7 and MDA-MB-468 cells, but not MDA-MB-231 (three replicates/group). (h) U0126 (MEK inhibitor) inhibits the forskolin-stimulated growth of MCF7, MDA-MB-468 and MDA-MB-231 cells (three replicates/group). (i) A model summarizing the putative mechanism of action of UAG in breast cancer cells and compounds used to dissect mechanism of action. Data represent mean ± SEM. Experiments were repeated at least twice. UAG: unacylated ghrelin; VC: vehicle control; FBS: fetal bovine serum; E2: estradiol; PT: pertussis toxin; M: melatonin; FSK: forskolin.

Figure 2—source data 1

Unacylated ghrelin suppresses breast cancer cell growth via Gαi-dependent inhibition of cAMP formation.

https://cdn.elifesciences.org/articles/56913/elife-56913-fig2-data1-v1.xlsx
Figure 2—source data 2

Unacylated ghrelin suppresses breast cancer cell growth via Gαi-dependent inhibition of cAMP formation.

https://cdn.elifesciences.org/articles/56913/elife-56913-fig2-data2-v1.pdf
Figure 2—figure supplement 1
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Unacylated ghrelin suppresses breast cancer cell growth via Gαi-dependent mechanisms.

(a) Unacylated ghrelin (UAG) has no effect on the release of intracellular Ca2+ in MCF7 cells. (b) Loss of GNAI2 (blue outline) using CRISPR and three validated gRNAs is associated with loss of response to UAG (100 pM), while KO of GNAI1 or GNAI3 had no effect (green and red outline, respectively). (c) Suppression of estradiol-stimulated ZR75 cell growth by UAG (100 pM) is prevented by Gαi inhibitor, pertussis toxin (20 ng/ml, 200 ng/ml). (d) PKA (KT5720) or adenylyl cyclase (SQ22536) inhibitors, or cAMP antagonist (cAMPS-RP) suppress the growth of MDA-MB-468 cells. (e) UAG (100 pM) inhibits the forskolin-stimulated growth of ZR75 cells. Data represent mean ± SEM with three replicates/group. Experiments were repeated at least twice. UAG: unacylated ghrelin; FBS: fetal bovine serum; E2: estradiol; FSK: forskolin.

Figure 2—figure supplement 1—source data 1

Unacylated ghrelin suppresses breast cancer cell growth via Gαi-dependent mechanisms.

https://cdn.elifesciences.org/articles/56913/elife-56913-fig2-figsupp1-data1-v1.xlsx
Figure 3
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Unacylated ghrelin (UAG) inhibits MAPK and Akt signaling.

(a) Unacylated ghrelin (UAG; 100 pM) inhibits ERK activity (EYFP FRET) in EKAR-transfected MCF7 cells (10 replicates/group). Data were normalized to vector ECFP signal. Scale bar represent 50 μm. Western blotting demonstrates that UAG causes a decrease in the (b) phosphorylation of ERK1/2 and downstream MAPK target p90RSK and (c) expression of cMYC in MCF7 cells. UAG also causes a decrease in (d) the phosphorylation of Akt and its downstream target, p70S6K, as well as (e) FoxO3a nuclear localization FoxO3a-RFP-transfected cells, an effect that is attenuated in cells treated with PI3K inhibitor LY294002 (five replicates/group). Data represent mean ± SEM. Experiment were repeated at least twice. UAG: unacylated ghrelin; FBS: fetal bovine serum.

Figure 3—source data 1

Unacylated ghrelin suppresses EKAR and FoxO3 nuclear localization.

https://cdn.elifesciences.org/articles/56913/elife-56913-fig3-data1-v1.xlsx
Figure 3—source data 2

Unacylated ghrelin inhibits MAPK and Akt signaling.

https://cdn.elifesciences.org/articles/56913/elife-56913-fig3-data2-v1.pdf
Figure 4 with 1 supplement
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Unacylated ghrelin causes cell cycle arrest and apoptosis.

Unacylated ghrelin (UAG) significantly inhibits the proliferation of (a) MCF7, (b) ZR75 and (c) MDA-MB-468 in the presence of estradiol (10 nM) or serum (3–6 replicates/group). Representative images showing EdU incorporation (green). Hoechst nuclear stain; blue. Scale bar represent 100 μm. Effects are mediated via induction of G1-phase cell cycle arrest (RFP+) and a reduction in the number of cells in S/G2/M-phase (GFP+) and G1/S transition (YFP+) in (d) MCF7 and (e) MDA-MB-468 cells (four replicates/group). (Hoechst nuclear stain; blue). Scale bar represents 100 μm. (f) UAG stimulates cell death in MCF7 and MDA-MB-468 cells, but not MDA-MB-231 cells (three replicates/group). Western blot results demonstrating that UAG inhibits CDK4/cyclin D3, pRB (Ser 795) and BCL2, and stimulates BAX in (g) MCF7 and (h) MDA-MB-468 cells. Data represent mean ± SEM. Experiments were repeated at least twice. UAG: unacylated ghrelin; VC: vehicle control; FBS: fetal bovine serum; E2: estradiol.

Figure 4—source data 1

Unacylated ghrelin causes cell cycle arrest and apoptosis.

https://cdn.elifesciences.org/articles/56913/elife-56913-fig4-data1-v1.xlsx
Figure 4—source data 2

Unacylated ghrelin causes cell cycle arrest and apoptosis.

https://cdn.elifesciences.org/articles/56913/elife-56913-fig4-data2-v1.pdf
Figure 4—figure supplement 1
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Unacylated ghrelin causes cell cycle arrest and apoptosis.

(a) Unacylated ghrelin (UAG) significantly inhibits the proliferation of tamoxifen-resistant LCC2 cells in the presence of estradiol (10 nM). Representative images showing EdU incorporation (green). Hoechst nuclear stain (blue). Scale bar represents 100 μm. (b) No effect of UAG on cell cycle was observed in MDA-MB-231 cell using FUCCI cell cycle system. Flow cytometry analysis demonstrating that UAG induces G1-phase cell cycle arrest and apoptosis in (c, d) MCF7, (e, f) MDA-MB-468 and (g, h) ZR75 cells. Data represent mean ± SEM with three replicates/group. Experiments were repeated at least twice. UAG: unacylated ghrelin; FBS: fetal bovine serum; E2: estradiol.

Figure 4—figure supplement 1—source data 1

Unacylated ghrelin causes cell cycle arrest and apoptosis.

https://cdn.elifesciences.org/articles/56913/elife-56913-fig4-figsupp1-data1-v1.xlsx
Figure 5 with 2 supplements
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Unacylated ghrelin inhibits tumor growth in xenograft models and patient-derived tumor cells.

Tumor volume in response to treatment with 50 μg/kg (blue), 100 μg/kg (red) or 200 μg/kg (purple) UAG in mice xenografted with (a) MCF7 (six replicates/group), (b) ZR75 (five replicates/group), or allografted with (c) J110 (five replicates/group) cells. Representative tumor (below) with scale bar representing 10 mm. UAG significantly increases the number of cells with apoptotic nuclei in (d) MCF7, (e) ZR75 and (f) J110 xenografts. (g) UAG (100 pM) significantly inhibits the growth of patient-derived ER+ breast cancer cells and 4013-TG3 TNBC cells, but not 3204-TG6 TNBC cells. (h) Heatmap representing baseline differential expression of MAPK-target genes in responsive vs. non-responsive patient-derived cells. Data represent mean ± SEM. UAG: unacylated ghrelin; VC: vehicle control; FBS: fetal bovine serum.

Figure 5—source data 1

Unacylated ghrelin and cyclic analog AZP-531 inhibit tumor growth in xenograft models and patient-derived tumor cells.

https://cdn.elifesciences.org/articles/56913/elife-56913-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
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Unacylated ghrelin and cyclic analog AZP-531 inhibit tumor growth in xenograft models and patient-derived tumor cells.

Representative images of tumors in response to treatment with 50 μg/kg or 100 μg/kg unacylated ghrelin (UAG) in nude mice xenografted with (a) MCF7 (six replicates/group) and (b) ZR75 (five replicates/group) cells. Effect of UAG on tumor volume of J110 (five replicates/group) (c) allografts in FVB mice or (d) xenografts in nude mice compared to vehicle control. (e) UAG (100 pM) significantly inhibits the growth of patient-derived ER+ breast cancer, 2147-TG5 TNBC, but has no effect on the growth of 3887-TG7 TNBC. Data represent mean ± SEM. Representative images of tumors in response to treatment with 200 μg/kg AZP-531 in (f) NSG mice xenografted with MDA-MB-468 cells (8–9 replicates/group) or (g) FVB mice allografted with J110 cells (five replicates/group). Scale bars represent 10 mm. UAG: unacylated ghrelin; VC: vehicle control.

Figure 5—figure supplement 1—source data 1

Unacylated ghrelin and cyclic analog AZP-531 inhibit tumor growth in xenograft models andpatient-derived tumor cells.

https://cdn.elifesciences.org/articles/56913/elife-56913-fig5-figsupp1-data1-v1.xlsx
Figure 5—figure supplement 2
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Expression pattern of MAPK-target genes in responder and non-responder TNBC patient-derived breast cancer cases.

Red and blue colors indicate high and low gene expression, respectively.

Figure 6
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Unacylated ghrelin analog, AZP-531, inhibits breast cancer cell growth in vitro, ex vivo and in vivo.

AZP-531 causes the dose-dependent inhibition of (a) MCF7 and (b) MDA-MB-468 and (c) patient-derived TNBC breast cancer cell growth in 3D, compared with chemotherapeutic agent doxorubicin (three replicates/group). (d) Unacylated ghrelin (UAG; 10–1000 pM) and AZP-531 (AZP; 10–1000 pM) inhibits the forskolin-stimulated production of cAMP in MCF7 cells (3–6 replicates/group). Data represent mean ± SEM. Experiments were repeated at least twice. Tumor volume in response to treatment with 200 μg/kg AZP-531 (purple) in mice xenografted with (e) MDA-MB-468 (8–9 replicates/group) or allografted with (f) J110 (five replicates/group) cells. Representative images (below) with scale bars representing 10 mm.

Figure 6—source data 1

Unacylated ghrelin analog, AZP-531, inhibits breast cancer cell growth in vitro, ex vivo and in vivo.

https://cdn.elifesciences.org/articles/56913/elife-56913-fig6-data1-v1.xlsx
Author response image 1
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Tables

Table 1
Characteristics of breast cancer cell lines and patient-derived breast cancer cells, and responsiveness to unacylated ghrelin.
Breast cancer cell line/Patient sampleKnown mutationsIntrinsic subtypeReceptor statusResponsive to
Unacylated Ghrelin
Cell line
MCF7CDKN2A, PIK3CALuminal AER+/PR+/HER2-Yes
LCC2N/ALuminal AER+/PR+/HER2-Yes
T47DPIK3CA, TP53Luminal AER+/PR+/HER2-Yes
ZR75PTENLuminal BER+/PR+/HER2+Yes
SKBR3TP53HER2+HER2+Yes
MDA-MB-468PTEN, RB1, SMAD4,
TP53		Basal-likeTNBCYes
MDA-MB-157NF1, TP53Mesenchymal-likeTNBCYes
MDA-MB-231BRAF, KRAS, TP53,
CDKN2A, NF2		Mesenchymal-likeTNBCNo
HS578THRAS, TP53Mesenchymal-likeTNBCNo
DU4475BRAF,APC, MAP2K4,
RB1		Basal-likeTNBCNo
Patient samples
ER+ Case 1N/ALuminal AER+Yes
ER+ Case 2N/ALuminal AER+Yes
2147-TG5N/ABasal-likeTNBCYes
4013-TG3N/ABasal-likeTNBCYes
3887-TG7N/AMesenchymal-likeTNBCNo
3204-TG6N/AMesenchymal-likeTNBCNo

  1. Abbreviations: ER, estrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor 2; TNBC, triple negative breast cancer; N/A: not available.

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Escherichia coli)One Shot Stbl3
Chemically Competent E. coli		ThermoFisher ScientificCat# C737303
Strain, strain background (Mus-musculus)BALB/c-Foxn1nu/Arc (BALB/c nude) miceAnimal Resources Centre, AustraliaCat# BCNU; RRID:MGI:2161064Female, 6 week old
Strain, strain background (Mus-musculus)FVB/NJArc (FVB/N) miceAnimal Resources Centre, AustraliaCat# 001800; RRID:IMSR_JAX:001800Female, 6 week old
Strain, strain background (Mus-musculus)NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) miceNOD Scid gamma, Jackson laboratoryCat# 005557; RRID:IMSR_JAX:005557Female, 6 week old
Cell line (Homo-sapiens)MCF7ATCCATCC Cat# HTB-22; RRID:CVCL_0031
Cell line (Homo-sapiens)T47DATCCATCC Cat# HTB-133; RRID:CVCL_0553
Cell line (Homo-sapiens)ZR75-1ATCCATCC Cat# CRL-1500; RRID:CVCL_0588
Cell line (Homo-sapiens)MDA-MB-231ATCCATCC Cat# HTB-26; RRID:CVCL_0062
Cell line (Homo-sapiens)MDA-MB-468ATCCATCC Cat# HTB-132; RRID:CVCL_0419
Cell line (Homo-sapiens)SKBR3ATCCATCC Cat# HTB-30; RRID:CVCL_0033
Cell line (Homo-sapiens)Hs578TATCCATCC Cat# HTB-126; RRID:CVCL_0332
Cell line (Homo-sapiens)HEK293TATCCATCC Cat# CRL-1573; RRID:CVCL_0045
Cell line (Homo-sapiens)DU4475ATCCATCC Cat# HTB-123; RRID:CVCL_1183
Cell line (Homo-sapiens)MDA-MB-157ATCCATCC Cat# HTB-24; RRID:CVCL_0618
Cell line (Homo-sapiens)LCC2obtained from Prof. Robert Clarke
Cell line (Homo-sapiens)RKOYun et al., 2009
Cell line (Homo-sapiens)RKO-T29Yun et al., 2009
Cell line (Homo-sapiens)RKO-A19Yun et al., 2009
Cell line (Homo-sapiens)HCT116Yun et al., 2009
Cell line (Homo-sapiens)HCT116-HWTYun et al., 2009
Cell line (Homo-sapiens)HCT116-HMUTYun et al., 2009
Cell line (Homo-sapiens)J110obtained from Dr. Myles Brown
Biological sample (human)Patient-derived breast tumorsobtained from Dr. Giorgio InghiramiSee Materials and methods section
Biological sample (human)Estrogen receptor positive (ER+) breast tumorsobtained from Dr. Eleni AndreopoulouSee Materials and methods section
AntibodyGoat anti-mouse IgG H and L (HRP)AbcamCat# ab205719; RRID:AB_27550491:5000
AntibodyDonkey anti-Rabbit IgG H and L (HRP)AbcamCat# ab7083; RRID:AB_9554161:5000
AntibodyMouse Monoclonal anti-Tubulin, beta, (KMX-1)MilliporeSigmaCat# MAB3408; RRID:AB_946501:10000
AntibodyMouse Monoclonal anti-β-Actin−PeroxidaseSigma-AldrichCat#A3854; RRID:AB_2620111:5000
AntibodyRabbit Monoclonal anti-CDK4 (D9G3E)Cell SignalingCat# 12790; RRID:AB_26311661:1000
AntibodyMouse Monoclonal anti-Cyclin D3 (DCS22)Cell SignalingCat# 2936; RRID:AB_20708011:2000
AntibodyRabbit Polyclonal anti-phospho-Rb (Ser795)Cell SignalingCat# 9301; RRID:AB_330013)1:1000
AntibodyMouse Monoclonal anti-Rb (4H1)Cell SignalingCat# 9309; RRID:AB_8236291:2000
AntibodyRabbit Monoclonal anti-Bcl-2 (D55G8) (Human Specific)Cell SignalingCat# 4223; RRID:AB_19039091:1000
AntibodyRabbit Monoclonal anti-BaxCell SignalingCat# 2772; RRID:AB_106958701:1000
AntibodyRabbit Polyclonal anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204)Cell SignalingCat# 9101;
RRID:AB_331646		1:1000
AntibodyRabbit Monoclonal anti-p44/42 MAPK (Erk1/2) (137F5)Cell SignalingCat# 4695;
RRID:AB_390779		1:1000
AntibodyRabbit Monoclonal anti-phospho-p90RSK
(Ser380) (D3H11)		Cell SignalingCat# 11989; RRID:AB_26876131:1000
AntibodyRabbit Monoclonal anti-RSK1/RSK2/RSK3 (32D7)Cell SignalingCat# 9355; RRID:AB_6599001:1000
AntibodyRabbit Monoclonal
anti-c-Myc (D84C12)		Cell SignalingCat# 5605; RRID:AB_19039381:1000
AntibodyRabbit Polyclonal anti-phospho-Akt (Ser473)Cell SignalingCat# 9271; RRID:AB_3298251:1000
AntibodyRabbit Polyclonal anti-AktCell SignalingCat# 9272; RRID:AB_3298271:1000
AntibodyRabbit Monoclonal anti-phospho-p70 S6 Kinase (Thr389) (108D2)Cell SignalingCat# 9234; RRID:AB_22698031:1000
AntibodyRabbit Monoclonal anti-p70 S6 Kinase (49D7)Cell SignalingCat# 2708; RRID:AB_3907221:1000
Recombinant DNA reagentpPBJ—puro-FRET3-EKAR-nlsobtained from Dr. John Albeck
Recombinant DNA reagentpCMV-hyPBase
transposase vector		obtained from
Dr. John Albeck
Recombinant DNA reagentpMSCV-puro-Foxo3a-H212R-N400-mCherryobtained from Dr. John Albeck
Recombinant DNA reagentlentiCRISPR v2Sanjana et al., 2014Addgene Cat# 52961; RRID:Addgene_52961
Recombinant DNA reagentBRAFV600E plasmidobtained from Dr. Dan Gough
Recombinant DNA reagentpsPAX2This paperAddgene Cat# 12260; RRID:Addgene_12260
Recombinant DNA reagentVSV-GReya et al., 2003Addgene Cat# 14888;
RRID:Addgene_14888
Sequence-based reagentSee Supplementary file 1 for GNAI sequence guide strands used in this study
Peptide, recombinant proteinRat des-octanoyl ghrelinChina PeptidesCat# Rat des-octanoyl ghrelin
Peptide, recombinant protein[Des-octanoyl]-Ghrelin (rat)Tocris BioscienceCat# 2951
Peptide, recombinant proteinGhrelin (rat)Tocris BioscienceCat# 1465
Peptide, recombinant proteinAZP531MedChem ExpressCat# HY-P0231
Peptide, recombinant proteinCy3-tagged UAGPepmic Co, LtDCat# Cy3-GR-28
Commercial
assay or kit		EarlyTox Live/Dead Assay KitMolecular DevicesCat# P/N R8340
Commercial
assay or kit		Click-iT Plus EdU Alexa Fluor 488 Imaging KitThermoFisher ScientificCat# C10637
Commercial
assay or kit		Cell Line NucleofectorKit VLonzaCat# VCA-1003
Commercial
assay or kit		Lance Ultra cAMP Detection KitPerkin ElmerCat# TRF0262
Commercial
assay or kit		Gαi Activation Assay KitNew East BiosciencesCat# 80301
Commercial
assay or kit		Premo FUCCI Cell Cycle Sensor (BacMam 2.0)ThermoFisher ScientificCat# P36237
Commercial
assay or kit		PureYield Plasmid
Miniprep System		PromegaCat# A1223
Commercial
assay or kit		PureYield Plasmid Maxiprep SystemPromegaCat# A2392
Chemical compound, drugPertussis Toxin from B. pertussis, Lyophilized (Salt-Free)List Biological LaboratoriesCat# 181
Chemical compound, drugU0126Cell SignalingCat# 9903S
Chemical compound, drugLY294002Cell SignalingCat# 9901
Chemical compound, drugKT 5720Tocris BioscienceCat# 1288
Chemical compound, drugSQ 22536Tocris BioscienceCat# 1435
Chemical compound, drugcAMPS-Rp, triethylammonium saltTocris BioscienceCat# 1337
Chemical compound, drugMelatoninTocris BioscienceCat# 3550
Chemical compound, drugDoxorubicin hydrochlorideSigma-AldrichCat# D1515
Chemical compound, drugForskolinSigma-AldrichCat# F3917
Chemical compound, drugβ-EstradiolSigma-AldrichCat# E8875-1G
Software, algorithmPrismGraphPadRRID:SCR_005375http://www.graphpad.com/scientific-software/prism/
Software, algorithmImarisBitplaneRRID:SCR_007370https://imaris.oxinst.com/
Software, algorithmFijiImageJRRID:SCR_002285https://imagej.net/Fiji
Software, algorithmImage LabBIO-RADRRID:SCR_014210http://www.biorad.com/en-us/product/image-lab-software?ID=KRE6P5E8Z;
Software, algorithmZEN softwareZEISSRRID:SCR_013672https://www.zeiss.com/
microscopy/
us/products/microscope-software/zen-lite.html
Software, algorithmIngenuity Pathway AnalysisQiagenRRID:SCR_008653https://www.qiagenbioinformatics.
com/products/ingenuity-pathway-analysis
Software, algorithmFlowJo softwareFlowJoRRID:SCR_008520https://www.flowjo.com/solutions/flowjo
OtherHoechst 33342, Trihydrochloride,
Trihydrate		ThermoFisher ScientificCat# H3570
OtherFITC Annexin VBD PharmingenCat# 556420
OtherPropidium IodineThermoFisher ScientificCat# P1304MP
OtherFura-2, AM, cell permeantThermoFisher ScientificCat# F1221
OtherProbenecid, Water SolubleThermoFisher ScientificCat# P36400
OtherPluronic F-127ThermoFisher ScientificCat# P6867
OtherBsmBINew England BioLabsCat# R0580S
OtherPolyethylenimine,
linear (PEI)		Polysciences, IncCat# 23966–1
OtherPolybreneSanta Cruz BiotechnologyCat# sc-134220
OtherPuromycin
Dihydrochloride		ThermoFisher ScientificCat# A1113803
OtherBlasticidin S HCl (10 mg/mL)ThermoFisher
Scientific		Cat# A1113903
OtherCorning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-free, *LDEV-free,CorningCat# 356231
OtherCollagenObtained from Dr. Jason Spector

Additional files

Supplementary file 1

Oligonucleotide Sequences.

https://cdn.elifesciences.org/articles/56913/elife-56913-supp1-v1.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/56913/elife-56913-transrepform-v1.docx

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  1. CheukMan C Au
  2. John B Furness
  3. Kara Britt
  4. Sofya Oshchepkova
  5. Heta Ladumor
  6. Kai Ying Soo
  7. Brid Callaghan
  8. Celine Gerard
  9. Giorgio Inghirami
  10. Vivek Mittal
  11. Yufeng Wang
  12. Xin Yun Huang
  13. Jason A Spector
  14. Eleni Andreopoulou
  15. Paul Zumbo
  16. Doron Betel
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Three-dimensional growth of breast cancer cells potentiates the anti-tumor effects of unacylated ghrelin and AZP-531
eLife 9:e56913.
https://doi.org/10.7554/eLife.56913